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Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)—although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance. [ABSTRACT FROM AUTHOR]

This present study is aimed to investigate the role of microRNA‐365 (miR‐365) in the development of intervertebral disc degeneration (IDD). Nucleus pulposus (NP) cells were transfected by miR‐365 mimic and miR‐365 inhibitor, respectively. Concomitantly, the transfection efficiency and the expression level of miRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Meanwhile, NP cells apoptosis was measured through propidium iodide (PI)‐AnnexinV‐fluorescein isothiocyanate (FITC) apoptosis detection kit. Subsequently, immunofluorescence (IF) staining was performed to assess the expression of collagen II, aggrecan and matrix metalloproteinase 13 (MMP‐13). In addition, bioinformatic prediction and Luciferase reporter assay were used to reveal the target gene of miR‐365. Finally, we isolated the primary NP cells from rats and injected NP‐miR‐365 in rat IDD models. The results showed that overexpression of miR‐365 could effectively inhibit NP cells apoptosis and MMP‐13 expression and upregulate the expression of collagen II and aggrecan. Conversely, suppression of miR‐365 enhanced NP cell apoptosis and elevated MMP‐13 expression, but decreased the expression of collagen II and aggrecan. Moreover, the further data demonstrated that miR‐365 mediated NP cell degradation through targeting ephrin‐A3 (EFNA3). In addition, the cells apoptosis and catabolic markers were increased in NP cells when EFNA3 upregulated. More importantly, the vivo data supported that miR‐365‐NP cells injection ameliorated IDD in rats models. miR‐365 could alleviate the development of IDD by regulating NP cell apoptosis and ECM degradation, which is likely mediated by targeting EFNA3. Therefore, miR‐365 may be a promising therapeutic avenue for treatment IDD through EFNA3. [ABSTRACT FROM AUTHOR]

Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)—although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance.

Background: Ischemic stroke (IS) represents a major cause of morbidity and mortality across the globe. The aberrant expression of miR-365 has been found to be implicated in a wide array of human diseases, including atherosclerosis and cancer. Studies on single-nucleotide polymorphisms (SNPs) in miRNA genes can help gain insight into the susceptibility to the condition. This study aimed to examine the relationship between miR-365 SNPs and the risk of IS. Methods: The study recruited 215 IS patients and 220 controls. The SNPscans genotyping was employed to genotype three polymorphic loci (rs121224, rs30230, and rs178553) of miR-365. The relative expression of miR-365 in peripheral blood mononuclear cells of the patients and controls was determined by using real-time quantitative PCR. Results: The miR-365 rs30230 polymorphism exhibited a significant association with the risk of developing IS (TC vs. CC: adjusted OR = 0.55, 95% CI: 0.33-0.92, P = 0.022; TT vs. CC: adjusted OR = 0.34, 95% CI: 0.14-0.85, P = 0.021; TC +TT vs. CC: adjusted OR = 0.51, 95% CI: 0.31-0.83, P = 0.007; T vs. C: adjusted OR = 0.57, 95% CI: 0.39-0.83, P = 0.004). Haplotype analysis revealed that the C-T-G haplotype was associated with a decreased risk of IS (OR = 0.68, 95% CI: 0.46-1.00, P = 0.047). Furthermore, miR-365 expression was significantly higher in IS patients than in controls (P < 0.001). Interestingly, patients with rs30230 TC or TT genotypes had lower miR-365 levels compared to their counterparts with CC genotypes (P < 0.001). Conclusions: ThemiR-365 rs30230 polymorphismmight bear an association with IS susceptibility in the Chinese population, and the rs30230 TC/TT genotypemight be a protective factor against IS. [ABSTRACT FROM AUTHOR]

Even though circular RNAs (circRNAs), a class of non-coding endogenous RNA, play a crucial role in the progression of osteosarcoma (OS), the specific function of hsa_circ_0000028 (circUSP48) remains unclear. This study aims to elucidate the mechanism by which circUSP48 regulates OS. We employed qRT-PCR and western blot techniques to quantify circDOCK1, miR-186, and DNMT3A levels. Cell proliferation was assessed using the cell counting kit-8 (CCK-8), 5-Ethynyl-20-deoxyuridine (EdU) assay, and colony formation assay. Cell migration and invasion were evaluated through Transwell and cell scratch assays. Furthermore, we performed dual-luciferase reporter, RIP, and RNA pull-down assays to investigate the association between circUSP48, miR-365, and CYR61. In addition, an in vivo xenograft model was utilized to assess the functional role of circUSP48. High levels of circUSP48 and CYR61 were observed in OS tissues and cells, while miR-365 levels were low. Knockdown of circUSP48 suppressed the multiplication, motility, and invasion of OS cells, thereby reducing carcinoma growth. Moreover, inhibition of miR-365 reversed the OS cell-suppressive effect caused by circUSP48 knockdown through direct interaction with circUSP48. Additionally, circUSP48 upregulated the expression of CYR61 by sponging miR-365. The findings suggest that circUSP48 promotes malignant behavior in OS by regulating the expression of CYR61 through miR-365, making it a potential therapeutic target for OS. [ABSTRACT FROM AUTHOR]

Abstract Background This study investigated the role of lncRNA CASC15 in laryngeal squamous cell carcinoma (LSCC). Methods This study included 58 LSCC patients. Both tumor (LSCC) and adjacent (within 3 cm around tumors) non-tumor tissues from 3 different sites of each patient were collected. CCK-8 assay was used to determine cell proliferation. The expression levels of proteins and mRNAs were determined by Western blotting analysis and qRT-PCRs, respectively. Results CASC15 was upregulated in LSCC and high expression levels of CASC15 predicted poor survival. In LSCC tissues, CASC15 was negatively correlated with miR-365 but positively correlated with cyclin D1. In LSCC cells, overexpression of CASC15 resulted in downregulation of miR-365 and upregulation of cyclin D1. Overexpression of miR-365 did not affect the expression of CASC15 but downregulated cyclin D1. Overexpression of Cyclin D1 did not affect the expression of miR-365 and CASC15. Overexpression of CASC15 and cyclin D1 led to promoted, while overexpression of miR-365 led to inhibited LSCC cell proliferation. In addition, overexpression of miR-365 reduced the effects of overexpression of CASC15. Conclusion Therefore, CASC15 upregulates cyclin D1 by downregulating miR-365 in LSCC to promote cell proliferation.

Background: This study investigated the role of lncRNA CASC15 in laryngeal squamous cell carcinoma (LSCC). Methods: This study included 58 LSCC patients. Both tumor (LSCC) and adjacent (within 3 cm around tumors) non-tumor tissues from 3 different sites of each patient were collected. CCK-8 assay was used to determine cell proliferation. The expression levels of proteins and mRNAs were determined by Western blotting analysis and qRT-PCRs, respectively. Results: CASC15 was upregulated in LSCC and high expression levels of CASC15 predicted poor survival. In LSCC tissues, CASC15 was negatively correlated with miR-365 but positively correlated with cyclin D1. In LSCC cells, overexpression of CASC15 resulted in downregulation of miR-365 and upregulation of cyclin D1. Overexpression of miR-365 did not affect the expression of CASC15 but downregulated cyclin D1. Overexpression of Cyclin D1 did not affect the expression of miR-365 and CASC15. Overexpression of CASC15 and cyclin D1 led to promoted, while overexpression of miR-365 led to inhibited LSCC cell proliferation. In addition, overexpression of miR-365 reduced the effects of overexpression of CASC15. Conclusion: Therefore, CASC15 upregulates cyclin D1 by downregulating miR-365 in LSCC to promote cell proliferation. [ABSTRACT FROM AUTHOR]

Purpose: Diabetic nephropathy (DN) is one of the most serious complications of diabetes that leads to decline of renal function. Although numerous studies have revealed that microRNAs (miRNAs) play essential roles in the progression of DN, whether miR-365 is involved remains elusive. Methods: The successful construction of DN model was confirmed by ELSIA, hematoxylin–eosin (HE) and Masson staining assay. The expression of miR-365 was detected through RT-qPCR. The levels of BDNF, p-TrkB, α-smooth muscle actin (SMA), collagen IV (Col.IV), transforming growth factor-β1 (TGF-β1), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were evaluated by western blot, IF or ELISA assays. Luciferase reporter assay was used to detect the interaction between miR-365 and BDNF. Results: The DN mice model was induced by streptozotocin (STZ). Then miR-365 expression was found to upregulate in tissues of DN rat. Furthermore, elevated expression of miR-365 was found in high glucose (HG)-treated HK-2 cells. Silencing of miR-365 suppressed the accumulation of ECM components and secretion of inflammatory cytokines in HK-2 cells. In addition, it was demonstrated that miR-365 could target BDNF. The protein levels of BDNF and p-TrkB were negatively regulated by miR-365 in HK-2 cells. Moreover, inhibition of miR-365 suppressed the levels of SMA, Col.IV, TGF-β1, TNF-α, and IL-6, indicating the renal fibrosis was inhibited by miR-365 knockdown. Conclusion: MiR-365 could regulate BDNF-TrkB signal axis in STZ induced DN fibrosis and renal function. The results of the current study might provide a promising biomarker for the treatment of DN in the future. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression levels of micro-ribonucleic acid (miR)-146a and miR-365 in the plasma of osteoarthritis (OA) patients, to study their expression with the inflammatory factors and the severity of disease in patients and to analyse their diagnostic significance. Materials and Methods: A total of 42 OA patients diagnosed with OA and treated in our hospital from January 2017 to January 2018 were selected as the subjects, and 28 healthy people were enrolled as controls. The expressions of interleukin-1 beta (IL-1ß) and IL-6 in the plasma of OA patients were detected via immunohistochemical staining. Moreover, the knee joint function of OA patients was evaluated by Lysholm score, Western Ontario and McMaster Universities (WOMAC) score and Visual Analogue Scale (VAS) score. The expression levels of plasma miR-146a and miR-365 in OA patients were measured through RT-PCR. Besides, the significance of the expression levels of miR-146a and miR-365 for the diagnosis of OA was analysed by ROC curves. Results: As compared with healthy people, OA patients had elevated expression levels of plasma IL-1ß and IL-6, decreased Lysholm score, increased WOMAC and VAS scores as well as significantly up-regulated levels of plasma miR-146a and miR-365, which were of important significance for diagnosis. Conclusion: The expression levels of plasma miR-146a, miR-365 and inflammatory factors are notably higher, the disease is more severe, and the function of knee joint movement is weaker in OA patients than those in healthy controls. It can be concluded that the levels of both miR-146a and miR-365 can serve as biomarkers of OA diagnosis. [ABSTRACT FROM AUTHOR]

Abstract Background While bone marrow-derived mesenchymal stem cells (BMSC) are established sources for stem cell-based cartilage repair therapy, articular cartilage-derived mesenchymal stem cells from osteoarthritis patients (OA-MSC) are new and potentially attractive candidates. We compared OA-MSC and BMSC in chondrogenic potentials, gene expression, and regulation by miR-365, a mechanical-responsive microRNA in cartilage (Guan et al., FASEB J 25: 4457–4466, 2011). Methods To overcome the limited number of OA-MSC, a newly established human OA-MSC cell line (Jayasuriya et al., Sci Rep 8: 7044, 2018) was utilized for analysis and comparison to BMSC. Chondrogenesis was induced by the chondrogenic medium in monolayer cell culture. After chondrogenic induction, chondrogenesis and mineralization were assessed by Alcian blue and Alizarin red staining respectively. MiRNA and mRNA levels were quantified by real-time PCR while protein levels were determined by western blot analysis at different time points. Immunohistochemistry was performed with cartilage-specific miR-365 over-expression transgenic mice. Results Upon chondrogenic induction, OA-MSC underwent rapid chondrogenesis in comparison to BMSC as shown by Alcian blue staining and activation of ACAN and COL2A1 gene expression. Chondrogenic induction also activated mineralization and the expression of hypertrophic and osteogenic genes in OA-MSC while only hypertrophic genes were activated in BMSC. MiR-365 expression was activated by chondrogenic induction in both OA-MSC and BMSC. Transfection of miR-365 in OA-MSC induced chondrogenic, hypertrophic, and osteogenic genes expression while miR-365 inhibition suppressed the expression of these genes. Over-expression of miR-365 upregulated markers of OA-MSC and hypertrophy and increased OA scores in adult mouse articular cartilage. Conclusions Induction of chondrogenesis can activate mineralization, hypertrophic, and osteogenic genes in OA-MSC. MiR-365 appears to be a master regulator of these differentiation processes in OA-MSC during OA pathogenesis. These findings have important implications for cartilage repair therapy using cartilage derived stem cells from OA patients.

Gallbladder carcinoma (GBC) is one of the most common fatal biliary tract tumors in the world. Its 3-year survival rate is 30% and the recurrence rate remains very high. miR-365 was downregulated in numerous tumors and worked as tumor suppressor gene. However, the role of miR-365 in GBC was unclear. In this study, our results found that the expression of miR-365 in GBC tissues was reduced rather than that in non-cancerous tissues. miR-365 overexpression inhibited the proliferation, metastasis and expansion of GBC CSCs. Mechanically, bioinformatic and luciferase reporter analysis identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR-365. Overexpression of miR-365 in GBC cells reduced the RAC1 mRNA and protein expression. The special RAC1 inhibitor EHop-106 abolished the discrepancy of growth, metastasis and self-renewal ability between miR-365-overexpression GBC cells and their control cells, which further demonstrated that RAC1 was involved in miR-365-disrupted GBC cells growth, metastasis and self-renewal. More importantly, reduced expression of miR-365 was a predictor of poor prognosis of GBC patients. In conclusion, miR-365 inhibited GBC cell growth, metastasis and self-renewal capacity by directly targeting RAC1, and may therefore prove to be a novel prognosis biomarker for GBC patients. [ABSTRACT FROM AUTHOR]

Changjiang Sun,1 Xizhi Zhang,1 Yong Chen,1 Qingqing Jia,1 Jianqi Yang,1 Yusheng Shu2 1Department of Oncology, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, China; 2Department of Thoracic and Cardiovascular Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, China Background: A number of studies have indicated that expression of miRNA-365 (miR-365) is suppressed in various cancers, suggesting its cancer-suppressive role. In the present investigation, we evaluated the regulation and character of miR-365 in human esophageal squamous cell carcinoma (ESCC). Patients and methods: The tumor tissues and adjacent nontumor tissue samples were collected from 30 patients having ESCC, and the expression levels of miR-365 were studied by quantitative real-time polymerase chain reaction (PCR). MTT and cell invasion by Matrigel assay were done to study the effect of miR-365 on proliferation and metastasis of ESCC cells. An in vivo tumor model was generated by inoculating ESCC cells subcutaneously into BALB nude mice. A study of various biomarkers such as quantitative polymerase chain reaction (qPCR), luciferase activity assay, and Western blot was done to confirm the targets of miR-365. Results: In tumor tissues, a significant downregulation of miR-365 was observed versus the nontumor adjacent tissues and ESCC cells versus the selected esophageal endothelial cells. It was observed that higher expression levels of miR-365 inhibited the cell invasion, colony formation, growth in esophageal cancer cell lines in vitro, and tumor development in vivo. The study of biomarkers suggests involvement of phosphoserine aminotransferase 1 (PSAT1) as a favorable target for miR-365, and its abnormal expression inverted the miR-365-arbitrated suppression of invasion, viability, and epithelial–mesenchymal transition in esophageal cancer cells. A negative correlation existed with expression of miR-365 and PSAT1 in human esophageal cancer tissue samples. Conclusion: The study established that miR-365 exhibits tumor-suppressive action via regulating the levels of PSAT1 and leads to invasion and progressiveness of esophageal cancer. Keywords: esophageal cancer, miR-365, PSAT1, tumor

The present study aimed to investigate the expression levels and clinical value of miR-365 and miR-25 in serum of patients with non-small cell lung cancer (NSCLC). Patients (180) diagnosed with NSCLC at the Affiliated Hospital of Guangdong Medical University from July 2011 to December 2013 were used as the experimental group. Volunteers (90) undergoing health examinations were used as the control group. The serum of the patients was collected after fasting in the morning. The expression levels of miR-365 and miR-25 in the serum of patients was assessed by quantitative real-time PCR (qRT-PCR), and the relationship among miR-365, miR-25 and the postoperative survival rate of NSCLC patients was analyzed. The relative expression level of miR-25 of patients with peripheral infiltration was significantly higher than that of patients without peripheral infiltration (P<0.05). There were significant differences in the relative expression level of miR-25 in different pathological grades and TNM stages, as well as with lymph node metastasis (P<0.05). The survival rate of NSCLC patients with high expression of miR-25 was significantly lower than that of NSCLC patients with low expression of miR-25 (P<0.05). The relative expression level of miR-365 of patients with peripheral infiltration was significantly lower than that of patients without peripheral infiltration (P<0.05). There were significant differences in the relative expression level of miR-365 in different pathological grades and TNM stages, as well as with lymph node metastasis (P<0.05). The survival rate of NSCLC patients with high expression of miR-365 was significantly higher than that of NSCLC patients with low expression of miR-365 (P<0.05). In conclusion, the expression levels of miR-25 and miR-365 were different in the serum of NSCLC patients, and they were closely related to certain clinical characteristics such as peripheral infiltration, pathological grade, tumor diameter, TNM stage and lymph node metastasis. Moreover, it was revealed that miR-25 and miR-365 affected the 5-year survival rate of patients. miR-25 and miR-365 could be used as important tumor markers to evaluate the prognosis of NSCLC patients. [ABSTRACT FROM AUTHOR]

OBJECTIVE: MicroRNAs (miRNAs) act as important regulators in human cancers by regulating the gene expression. The dysregulation of miR-365 has been investigated in many cancers. However, the function of miR-365 remains unknown in lung adenocarcinoma. Therefore, the regulatory mechanism of miR-365 was explored in lung adenocarcinoma. PATIENTS AND METHODS: The expression of miR-365 was detected in cell lines and 67 lung adenocarcinoma tissues using qRT-PCR. The Kaplan-Meier analysis was used to determine the association between miR-365 expressions and the survival rate in patients with lung adenocarcinoma. Transwell assay was then performed to investigate the effect of miR-365 on invasion and migration of lung adenocarcinoma cells. RESULTS: Downregulation of miR-365 and upregulation of ETS1 were identified in lung adenocarcinoma. Furthermore, miR-365 reversely regulated ETS1 expression in lung adenocarcinoma. Functionally, the overexpression of miR- 365 inhibited proliferation, migration, and invasion of lung adenocarcinoma cells. However, the upregulation of ETS1 lessened the inhibitory effect of miR-365 in lung adenocarcinoma. In addition, miR-365 inhibited EMT and inactivated AKT/mTOR pathway in lung adenocarcinoma. CONCLUSIONS: MiR-365 inhibits the progression of lung adenocarcinoma by targeting ETS1 and inactivating the AKT/mTOR pathway. [ABSTRACT FROM AUTHOR]

Abstract Background NKX2–1, a key molecule in lung development, is highly expressed in non-small cell lung cancer (NSCLC), particularly in lung adenocarcinoma (ADK), where it is a diagnostic marker. Studies of the prognostic role of NKX2–1 in NSCLC have reported contradictory findings. Two microRNAs (miRNAs) have been associated with NKX2–1: miR-365, which targets NKX2–1; and miR-33a, which is downstream of NKX2–1. We have examined the effect of NKX2–1, miR-365 and miR-33a on survival in a cohort of early-stage NSCLC patients and in sub-groups of patients classified according to the mutational status of TP53, KRAS, and EGFR. Methods mRNA and miRNA expression was determined using TaqMan assays in 110 early-stage NSCLC patients. TP53, KRAS, and EGFR mutations were assessed by Sanger sequencing. Results NKX2–1 expression was upregulated in never-smokers (P = 0.017), ADK (P

miR-365 is a microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers while suppressing these effects in others. Virtually no information is known about the presence or function of miR-365 in oral cancers. Based upon this information, the primary goal of this project was to evaluate the expression of miR-365 in existing oral cancer cell lines. Five commercially available oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, and CAL27) were obtained and cultured. RNA was then screened by PCR using primers specific for miR-365, as well as matrix metalloproteinase (MMP-2) and a downstream cancer stem cell regulator (NKX2.1), and structural and metabolic standards (beta actin, GAPDH). miR-365 was detected among these oral cancers, and some cells also expressed NKX2.1 and MMP-2, which correlated with miR-365 levels. The relative expression of miR-365, NKX2.1, and MMP-2 RNA was higher than expected. Transfection of miR-365 resulted in a significant increase in proliferation, which was not observed in the negative controls. These data appear to confirm miR-365 expression in oral cancers, which may also be correlated with MMP-2 and NKX2.1 expression. Moreover, the fastest growing oral cancers with the highest viability produced the most miR-365. In addition, miR-365 transfected into cells significantly increased growth, even in normal cells. More research is needed to elucidate the pathways responsible for these observations.

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA‐365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus‐mediated miR‐365 was able to attenuate neointimal formation in rat vein grafts. We found that miR‐365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR‐365 promoted smooth muscle‐specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR‐365 in a rat vein graft model significantly reduced grafting‐induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR‐365 in vein grafts. Specially, to increase the efficiency of miR‐365 gene transfection, a 30% poloxamer F‐127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus‐mediated miR‐365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR‐365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019 [ABSTRACT FROM AUTHOR]

Abstract The pathogenesis and the underlying mechanisms of hepatocellular carcinoma (HCC) remain unclear. LncRNA plasmacytoma variant translocation 1 (PVT1) is an oncogene in a variety of cancers. The role of PVT1 in HCC progression is not completely understood. In the present study, we conducted a series of studies to explore the role of PVT1 on autophagy in HCC cells. Our study found PVT1 levels were markedly up-regulated in the corresponding HCC tissues. Importantly, we found that PVT1 could facilitate cell autophagy in HCC cells. Then, we confirmed that the effect of PVT1 promoting autophagy was dependent on regulating ATG3 expression. Further investigations revealed that PVT1 could upregulate autophagy-related gene 3 (ATG3) expression by acting as an endogenous sponge of miR-365, which was an inhibitor gene on ATG3 protein by targeting 3′UTR of ATG3 mRNA. Moreover, rescue assays indicated that the effect of PVT1 on autophagy of HCC cells were dependent on miR-365. In conclusion, our study demonstrated PVT1 might be a key regulator participating in autophagy in HCC cells. We proved that PVT1 could promote autophagy as ceRNA by targeting miR-365. Highlights • Our study found PVT1 levels were markedly up-regulated in the corresponding HCC tissues. • We found that PVT1 could facilitate cell autophagy in HCC cells. • We confirmed that the effect of PVT1 promoting autophagy was dependent on regulating ATG3 expression. • We found that PVT1 could upregulate ATG3 expression by acting as an endogenous sponge of miR-365. [ABSTRACT FROM AUTHOR]

Radioresistance is a major challenge in lung cancer radiotherapy (RT), and consequently, new radiosensitizers are urgently needed. MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. MiR-365 is dysregulated in non-small cell lung cancer (NSCLC) and is able to restrain the development of NSCLC. However, the relationship between miR-365 and radiosensitivities of NSCLC cells remains largely unknown. Here we reveal that overexpression of miR-365 is able to enhance the radiosensitivity of NSCLC cells through targeting CDC25A. We found that the expression level of miR-365 was positively correlated with the radiosensitivity of NSCLC cell lines. Furthermore, our results showed that overexpression of miR-365 could sensitize A549 cells to the irradiation. However, knockdown of miR-365 in H460 cells could act the converse manner. Mechanically, miR-365 was able to directly target 3′UTR of cell division cycle 25A (CDC25A) mRNA and reduce the expression of CDC25A at the levels of mRNA and protein. And we confirmed that miR-365 could increase the radiosensitivity of NSCLC cells by targeting CDC25A using in vitro and in vivo assays. Taken together, restoration of miR-365 expression enhances the radiosensitivity of NSCLC cells by suppressing CDC25A, and miR-365 could be used as a radiosensitizer for NSCLC therapy. • MiR-365 is positively correlated with the radiosensitivity of NSCLC cells. • MiR-365 suppresses CDC25A through directly targeting its 3′UTR in NSCLC cells. • MiR-365 increases the radiosensitivity of NSCLC cells through targeting CDC25A. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is one of the most malignant adenocarcinomas arising from bile duct epithelial cells. However, the molecular mechanism regulating CCA development and progression still needs to be investigated. Here we found that miR-365 was downregulated in CCA tissues compared with adjacent normal tissues. By functional experiments, we found that overexpression of miR-365 significantly inhibited CCA cell proliferation and promoted cellular apoptosis in vitro. Furthermore, administration with miR-365 markedly suppressed the growth of tumor tissues in vivo. Mechanistically, we identified E2F2 as the target gene of miR- 365 in CCA cells. We found that overexpression significantly inhibited the expression of E2F2 in CCA cells, and there was an inverse correlation between the expression levels of E2F2 and miR-365 in CCA tissues. We also found that E2F2 was highly expressed in CCA tissues and cell lines. Restoration of E2F2 in miR-365- overexpressing CCA cells promoted cell viability and reduced cellular apoptosis in CCA. Collectively, our study demonstrated the essential role of miR-365 and its functional mechanism in CCA cells, which provided a new insight on the design of therapeutic targets for CCA treatment. [ABSTRACT FROM AUTHOR]

Introduction: miR-365 is a non-coding microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers, while suppressing these effects in others. Many microRNAs are produced and then exported extracellularly in exosomes, which are small extracellular vesicles ranging from 30 to 100 nm that are found in eukaryotic fluids and facilitate many cellular functions. Exosomes and extracellular vesicles are produced by many cell types, including oral cancer cells—although no study to date has evaluated miR-365 and oral cancer exosomes or extracellular vesicles. Based on this information, our research question was to evaluate whether oral cancers produce exosomes or extracellular vesicles containing miR-365. Materials and Methods: Two commercially available oral cancer cell lines (SCC25 and CAL27) and a normal oral keratinocyte (OKF4) were grown in serum-free media, supplemented with exosome-depleted fetal bovine serum. Extracellular vesicles and exosomes were then isolated using the Invitrogen total exosome RNA and protein isolation kit for processing using the hsa-miR-365a-5p microRNA qPCR assay kit. Results: RNA was successfully isolated from the exosome-depleted supernatant from each cell line—SCC9, SCC15, SCC25, and CAL27 (oral squamous cell carcinomas) and OKF4 (oral epithelial cell line). Relative concentrations of RNA were similar among each cell line, which were not significantly different, p = 0.233. RNA quality was established by A260:A280 absorbance using a NanoDrop, revealing purity ranging 1.73–1.86. Expression of miR-16 was used to confirm the presence of microRNA from the extracted exosomes and extracellular vesicles. The presence of miR-365 was then confirmed and normalized to miR-16 expression, which demonstrated an increased level of miR-365 in both CAL27 and SCC25. In addition, the normalized relative quantity (RQ) for miR-365 exhibited greater variation among SCC25 (1.382–4.363) than CAL27 cells (1.248–1.536). Conclusions: These results confirm that miR-365 is not only expressed in oral cancer cell lines, but also is subsequently exported into exosomes and extracellular vesicles derived from these cultures. These data may help to contextualize the potential for this microRNA to contribute to the phenotypes and behaviors of oral cancers that express this microRNA. Future research will begin to investigate these potential mechanisms and pathways and to determine if miR-365 may be useful as an oral cancer biomarker for salivary or liquid biopsies.

Abstract: Although cadmium (Cd)‐induced hepatoxicity is well established, pronounced knowledge gaps remain existed regarding the inherent cellular signaling that dictates Cd toxicity. Specifically, the molecular basis for determining the equilibrium between prosurvival and proapoptotic signaling remains poorly understood. Thus, it is recently revealed that long non‐coding RNA (lncRNA) MT1DP, a pseudogene in the metallothionein (MT) family, promoted Cd‐induced cell death through activating the RhoC‐CCN1/2‐AKT pathway and modulating MT1H induction. Here, first the dependency of MT1DP induction on MTF1, an important transcriptional factor in driving the mRNA expression of MT1 members is defined. Additionally, a bridge molecule between MT1DP and nuclear factor erythroid 2‐related factor 2 (Nrf2) is established: miR‐365. Mechanistically, MT1DP induction under Cd stress decreases the nuclear factor erythroid 2‐related factor 2 (Nrf2) level to evoke oxidative stress through the elevation of miR‐365, which acted to repress the Nrf2 level via direct binding to its 3'UTR. In contrast to the competing endogenous RNA (ceRNA) mechanism, a new mechanism is proposed: MT1DP elevated the miR‐365 level though stabilizing its RNA via direct binding. Collectively, the combined data demonstrate a crucial role of MT1DP in reducing the Nrf2‐mediated protection of cells, and this is dependent on the interplay with miR‐365. Hence, the study further expands the knowledge of inducible endogenous lncRNA in modulating oxidative stress. [ABSTRACT FROM AUTHOR]

Prevention of fibrosis and urethral scar formation is critical for a successful urethral reconstruction. We have previously shown that epithelial-differentiated adipose-derived stem cells (EASC) seeded bladder acellular matrix grafts (BAMG) can be used for urethral reconstruction. We have also shown that suppression of tissue inhibitor of metalloproteinases-1 (TIMP-1) reduces epithelial-mesenchymal transition in urethral fibroblasts in vitro and in vivo. However, it is unknown whether suppression of TIMP-1 in EASC seeded BAMG may benefit urethral reconstruction through inhibition of fibrosis. Here, we addressed this question. In a rabbit substitution urethroplasty model, we found that E-cadherin + EASC resulted in wider urethral caliber and formation of less urethral scar tissue, compared to non-purified EASC. Bioinformatics study showed that among all TIMP-1-targeting microRNAs (miRNAs), miR-365 is a conserved one in rabbits and humans, and functionally inhibits TIMP-1 protein translation. MiR-365-transduced E-cadherin + EASC seeded BAMG further reduced fibrosis and increased urethral caliber width during urethral reconstruction in rabbits, compared to E-cadherin + EASC seeded BAMG. Together, these data suggest that EASC seeded BAMG method for urethral reconstruction could be further improved through purification of EASC by E-cadherin and through post-transcriptional inhibition of TIMP-1 via miR-365 in EASC. [ABSTRACT FROM AUTHOR]

miRs play critical roles in oxidative stress-related retinopathy pathogenesis. miR-365 was identified in a previously constructed library from glyoxal-treated rat Müller cell. This report explores epigenetic alterations in Müller cells under oxidative stress to develop a novel therapeutic strategy. To examine the miR-365 expression pattern, in situ hybridization and quantitative RT-PCR were performed. Bioinformatical analysis and dual luciferase report assay were applied to identify and confirm target genes. Streptozotocin (STZ)-treated rats were used as the diabetic retinopathy (DR) model. Lentivirus-mediated anti-miR-365 was delivered subretinally and intravitreally into the rats’ eyes. The functional and structural changes were evaluated by electroretinogram (ERG), histologically, and through examination of expression levels of metallopeptidase inhibitor 3 ( Timp3 ), glial fibrillary acidic protein ( Gfap ), recoverin ( Rcvrn ) and vascular endothelia growth factor A ( Vegfa ). Oxidative stress factors and pro-inflammatory cytokines were analyzed. miR-365 expression was confirmed in the glyoxal-treated rat Müller cell line (glyoxal-treated rMC-1). In the retina, miR-365 mainly localized in the inner nuclear layer (INL). The increased miR-365 participated in Müller cell gliosis through oxidative stress aggravation, as observed in glyoxal-treated rMC-1 and DR rats before 6 weeks. Timp3 was a target and negatively regulated by miR-365. When miR-365 was inhibited, Timp3 expression was upregulated, Müller cell gliosis was alleviated, and retinal oxidative stress was attenuated. Visual function was also partially rescued as detected by ERG. miR-365 was found to be highly expressed in the retina and the abnormality of miR-365/ Timp3 pathway is closely related to the pathology, like Müller gliosis, and the visual injury in DR. The mechanism might be through oxidative stress, and miR-365/ Timp3 could be a potential therapeutic target for treating DR. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that microRNAs(miRNAs) are often aberrantly expressed in osteosarcoma (OS) and play critical roles in OS tumorigenesis. Therefore, the discovery of miRNAs may provide a new and powerful tool for understanding the mechanismof OS initiation and development. The aim of this study was to investigate the functional significance of miR-365 and identify its possible mechanism in OS cells. Here, wefound that the expression level of miR-365 is significantly downregulated in OS tissues and cell lines, and its expression isassociated with the clinical stage, distant metastasis, tumor grade, and poor overall survival rate. The overexpression of miR-365 is able to inhibit cell proliferation, migration, and invasion in Saos-2 and MG-63 cells. Moreover, the cysteine-rich angiogenic inducer 61 (CYR61) has been identified as a target of miR-365 in OS cells, and its expression is found to be significantly increased in OS tissues, which is negatively correlated with miR-365. Furthermore, CYR61 overexpression significantly attenuated the suppressive effects of miR-365 on the proliferation, migration, and invasion of Saos-2 and MG-63 cells. Therefore, we consider that miR-365 acts as a tumor suppressor in OS, partly, by targeting CYR61 expression. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug-resistance traits. We also demonstrated a significant higher level of miR-365 in exosomes derived from drug-resistant CML cells compared with those from sensitive ones using microarray and qRT-PCR. The imatinib sensitive CML cells transfected with pre-miR-365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR-365 induced drug resistance by inhibiting expression of pro-apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug-resistant trait in chronic myeloid leukemia cell by delivering miR-365. [ABSTRACT FROM AUTHOR]

Background: NKX2-1, a key molecule in lung development, is highly expressed in non-small cell lung cancer (NSCLC), particularly in lung adenocarcinoma (ADK), where it is a diagnostic marker. Studies of the prognostic role of NKX2-1 in NSCLC have reported contradictory findings. Two microRNAs (miRNAs) have been associated with NKX2-1: miR-365, which targets NKX2-1; and miR-33a, which is downstream of NKX2-1. We have examined the effect of NKX2-1, miR-365 and miR-33a on survival in a cohort of early-stage NSCLC patients and in sub-groups of patients classified according to the mutational status of TP53, KRAS, and EGFR.Methods: mRNA and miRNA expression was determined using TaqMan assays in 110 early-stage NSCLC patients. TP53, KRAS, and EGFR mutations were assessed by Sanger sequencing.Results: NKX2-1 expression was upregulated in never-smokers (P = 0.017), ADK (P < 0.0001) and patients with wild-type TP53 (P = 0.001). A negative correlation between NKX2-1 and miR-365 expression was found (ρ = -0.287; P = 0.003) but there was no correlation between NKX2-1 and miR-33a expression. Overall survival (OS) was longer in patients with high expression of NKX2-1 than in those with low expression (80.8 vs 61.2 months (P = 0.035), while a trend towards longer OS was observed in patients with low miR-365 levels (P = 0.07). The impact of NKX2-1 on OS and DFS was higher in patients with neither TP53 nor KRAS mutations. Higher expression of NKX2-1 was related to higher OS (77.6 vs 54 months; P = 0.017) and DFS (74.6 vs 57.7 months; P = 0.006) compared to low expression. The association between NKX2-1 and OS and DFS was strengthened when the analysis was limited to patients with stage I disease (P = 0.005 and P=0.003 respectively).Conclusions: NKX2-1 expression impacts prognosis in early-stage NSCLC patients, particularly in those with neither TP53 nor KRAS mutations. [ABSTRACT FROM AUTHOR]

Pro-apoptotic BCL2 associated X (BAX) is traditionally thought to be regulated by anti-apoptotic BCL-2 family members, like BCL2-like 1 (BCL-XL), at the protein level. However, the posttranscriptional regulation of BAX is under explored. In this study, we identified BAX as the novel downstream target of miR-365, which is supported by gain- and loss-of-function studies of onco-miR-365. Loss of BAX by either RNA interference or highly-expressed miR-365 in cells of cutaneous squamous cell carcinoma (CSCC) enhanced the tumor resistance against apoptosis, while repressing cell proliferation, migration, and invasiveness. In vivo experiment confirmed that BAX knockdown promotes the growth of CSCC xenografts. Collectively, our results find a miR-365-BAX axis for alleviating the pro-apoptotic effects of BAX, which promotes CSCC development and may facilitate the generation of novel therapeutic regimens to the clinical treatment of CSCC. [ABSTRACT FROM AUTHOR]

Glucocorticoid administration is the leading cause of secondary osteoporosis. In this study, we tested the hypotheses that histone deacetylase 4 (HDAC4) is associated with glucocorticoid-induced bone loss and that HDAC4 dependent bone loss can be ameliorated by miRNA-365. Our previous studies showed that miR-365 mediates mechanical stimulation of chondrocyte proliferation and differentiation by targeting HDAC4. However, it is not clear whether miR-365 has an effect on glucocorticoid-induced osteoporosis. We have shown that, in MC3T3-E1 osteoblasts, dexamethasone (DEX) treatment decreased the expression of miR-365, which is accompanied by the decrease of cell viability in a dose-dependent manner. Transfection of miR-365 ameliorated DEX-induced inhibition of MC3T3-E1 cell viability and alkaline phosphatase activity, and attenuated the suppressive effect of DEX on runt-related transcription factor 2 (Runx2), osteopontin (OPN), and collagen 1a1 (Col1a1) osteogenic gene expression. In addition, miR-365 decreased the expression of HDAC4 mRNA and protein by direct targeting the 3′-untranslated regions (3′-UTR) of HDAC4 mRNA in osteoblasts. MiR-365 increased Runx2 expression and such stimulatory effect could be reversed by HDAC4 over-expression in osteoblasts. Collectively, our findings indicate that miR-365 ameliorates DEX-induced suppression of cell viability and osteogenesis by regulating the expression of HDAC4 in osteoblasts, suggesting miR-365 might be a novel therapeutic agent for treatment of glucocorticoid-induced osteoporosis. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study aims to investigate the role and mechanism of miR-365 in the coronary atherosclerosis (AS). PATIENTS AND METHODS: Blood were collected from AS patients and healthy people, coronary plaque and adjacent arterial tissue were collected from AS patients. QRT-PCR method was used to detect expressions of miR-365 and IL-6 in coronary plaque, blood monocytes, and serum. Western blot was applied to detect IL-6 protein expression in coronary plaque and blood monocytes, and ELISA was used to detect IL-6 protein expression in serum. RESULTS: Compared with the normal group, IL-6 expression was significantly increased in coronary plaque, blood monocytes, and serum both in mRNA level and in protein level, while miR-365 expression was significantly decreased (p<0.05). CONCLUSIONS: IL-6 expression was significantly up-regulated in coronary plaque, blood monocytes, and serum, whereas miR-365 expression was down-regulated. MiR-365 may regulate the pathogenesis and immune response in AS through targeting IL-6. [ABSTRACT FROM AUTHOR]

OBJECTIVE: A role of microRNA- 365 (miR-365) in the carcinogenesis of breast cancer remains undetermined. In the current study, we addressed this question. PATIENTS AND METHODS: We examined the levels of miR-365 in breast cancer tissue, compared to the paired adjacent non-tumor breast tissue from the patients. We also examined the effects of miR-365 modification on the breast cancer growth and sensitivity to Fluorouracil, as well as the underlying mechanisms. RESULTS: The miR-365 levels in breast cancer tissue were significantly lower than those in control normal breast tissues. Transfection with the miR-365 mimic decreased the breast cancer cell growth and increased their sensitivity to Fluorouracil, while transfection with the antisense of miR-365 (as-miR-365) increased breast cancer cell growth and decreased their sensitivity to Fluorouracil. Bioinformatics analyses showed that GALNT4 was a potential target gene of miR-365. The luciferase activities assay and Western blot verified that miR-365 targeted GALNT4 mRNA to modulate its protein levels. CONCLUSIONS: Our study suggests that downregulation of miR-365 may facilitate carcinogenesis of breast cancer cells via GALNT4, and thus miR-365 appears to be a promising target for breast cancer therapy. [ABSTRACT FROM AUTHOR]

Introduction: Hypoxia occurs in the first trimester of placental development and is implicated in the regulation of trophoblast differentiation. Prolonged hypoxic conditions in the placenta are related to the development of preeclampsia. MicroRNAs (miRNAs) are noncoding, single-stranded RNAs that modulate gene expression by targeting messenger RNA. We hypothesized that, under hypoxic conditions, trophoblasts may have a unique miRNA profile that may play a critical role in placental development.Methods: Total RNA was extracted from human trophoblast, HChEpC1b, exposed to normoxia (20% O2) or hypoxia (2% O2) for 24 h, and the miRNA expression profiles were investigated using a microRNA array. Several differential miRNAs were selected and validated using real-time reverse transcription PCR. We identified potential targets of these miRNAs using in silico analysis. We confirmed a potential target protein by western blot analysis and luciferase assays.Results: The expression of miR-365 was significantly upregulated under hypoxic conditions. In silico analysis showed that miR-365 targeted human leukocyte antigen (HLA)-G. Both hypoxic conditions and overexpression of miR-365 inhibited the expression of HLA-G proteins. The overexpression of miR-365 also decreased the activity of the luciferase reporter containing the 3'-untranslated region (UTR) of HLA-G with the predicted miR-365-binding site.Discussion: HLA-G is a non-classical HLA class-Ib molecule that is expressed mainly in extravillous trophoblasts and which plays a key role in maintaining immune tolerance at the maternal-fetal interface. Our results indicate that miR-365 targets the HLA-G 3' UTR to repress its expression. The expression of miR-365 may play an important role in human placental development and in immunoprotection of the semiallogenic embryo. [ABSTRACT FROM AUTHOR]

Mechanical stress plays an important role in the initiation and progression of osteoarthritis. Studies show that excessive mechanical stress can directly damage the cartilage extracellular matrix and shift the balance in chondrocytes to favor catabolic activity over anabolism. However, the underlying mechanism remains unknown. MicroRNAs (miRNAs) are emerging as important regulators in osteoarthritis pathogenesis. We have found that mechanical loading up-regulated microRNA miR-365 in growth plate chondrocytes, which promotes chondrocyte differentiation. Here, we explored the role of the mechanical responsive microRNA miR-365 in pathogenesis of osteoarthritis (OA).We found that miR-365 was up-regulated by cyclic loading and IL-1β stimulation in articular chondrocytes through a mechanism that involved the transcription factor NF-kB. miR-365 expressed significant higher level in rat anterior cruciate ligament (ACL) surgery induced OA cartilage as well as human OA cartilage from primary OA patients and traumatic OA Patients. Overexpression of miR-365 in chondrocytes increases gene expression of matrix degrading enzyme matrix metallopeptidase 13 (MMP13) and collagen type X (Col X). The increase in miR-365 expression in OA cartilage and in response to IL-1 may contribute to the abnormal gene expression pattern characteristic of OA. Inhibition of miR-365 down-regulated IL-1β induced MMP13 and Col X gene expression. We further showed histone deacetylase 4 (HDAC4) is a direct target of miR-365, which mediates mechanical stress and inflammation in OA pathogenesis. Thus, miR-365 is a critical regulator of mechanical stress and pro-inflammatory responses, which contributes cartilage catabolism. Manipulation of the expression of miR-365 in articular chondrocytes by miR-365 inhibitor may be a potent therapeutic target for the prevention and treatment of osteoarthritis. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The role of miR-365 in cancer cells seemed controversial in previous studies. We thereby in this article aimed to define the role of miR-365 in malignant melanoma (MM) pathogenesis. METHODS:We detected miR-365 expression in malignant melanoma cell lines and then investigated the effects of miR-365 on the metastasis and malignancy of melanoma cells. The correlation between miR-365 level and NRP1 (neuropilin1) was further investigated in clinical malignant melanoma specimens. RESULTS: MiR-365 was strongly down-regulated in malignant melanoma (MM) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of MM. We also found that ectopic expression of miR-365 inhibited MM cell proliferation and MM metastasis in vitro and in vivo. We further identified a novel mechanism of miR-365 to suppress MM growth and metastasis. NRP1 was proved to be a direct target of miR-365, using luciferase assay and western blot. NRP1 over-expression in miR-365 expressing cells could rescue invasion and growth defects of miR-365. In addition, miR-365 expression inversely correlated with NRP1 protein levels in MM. CONCLUSION: Our data suggest that miR-365 functions as a tumor suppressor in MM development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for MM. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases are either post-transcriptionally regulated by interacting with cyclins and cyclin-dependent kinase inhibitors or are transcriptionally regulated by transcription factors, but the latter mechanism has not been extensively investigated. Dysregulated transcription factors resulting from aberrantly expressed microRNAs play critical roles in tumor development and progression. Our previous work identified miR-365 as an oncogenic microRNA that promotes the development of cutaneous squamous cell carcinoma via repression of cyclin-dependent kinase 6, while miR-365 also targets nuclear factor I/B. However, the underlying mechanism(s) of the interaction between nuclear factor I/B and cyclin-dependent kinase 6 are unclear. In this work, we demonstrate that miR-365-regulated nuclear factor I/B transcriptionally inhibits cyclin-dependent kinases 6 and 4 by binding to their promoter regions. In vivo and in vitro experiments demonstrate that the loss of nuclear factor I/B after miR-365 expression or treatment with small interfering RNAs results in the upregulation of cyclin-dependent kinases 6 and 4. This upregulation, in turn, enhances the phosphorylation of retinoblastoma protein and tumor progression. Characterizing this transcriptional repression of cyclin-dependent kinases 6 and 4 by nuclear factor I/B contributes to the understanding of the transcriptional regulation of cyclin-dependent kinases by transcription factors and also facilitates the development of new therapeutic regimens to improve the clinical treatment of cutaneous squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24 −/− mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24 −/− progeroid mice using a massively parallel sequencing technology. With data from 19.5 × 10 6 reads from WT MEFs and 16.5 × 10 6 reads from Zmpste24 −/− MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24 −/− MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24 −/− MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24 −/− MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3′ UTR luciferase-reporter activity, this effect was lost with mutations in the putative 3′ UTR target-site. Consistently, expression levels of miR-365 were found to inversely correlate with endogenous Rasd1 levels. These findings suggest that miR-365 is down-regulated in Zmpste24 −/− MEFs and acts as a novel negative regulator of Rasd1. Our comprehensive miRNA data provide a resource to study gene regulatory networks in MEFs. [ABSTRACT FROM AUTHOR]

Highlights: [•] We found microRNA-365 is an important regulator of NKX2-1 in lung oncogenesis. [•] miRNA-365 expression levels were low, NKX2-1 high, in human lung cancers. [•] We identified the miR-365 response element in the NKX3-1 3’UTR. [•] miR-365 decreased NKX2-1 and reduced proliferation of lung cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that control expression of complementary target mRNAs. A growing number of miRNAs has been implicated in the pathogenesis of cardiac diseases, mostly based not on functional data, but on the observation that they are dysregulated in diseased myocardium. Consequently, our knowledge regarding a potential cardiac role of the majority of miRNAs is limited. Here, we report the development of an assay format that allows the simultaneous analysis of several hundred molecules with regard to their phenotypic effect on primary rat cardiomyocytes. Using automated microscopy and an edge detection algorithm, this assay achieved high reproducibility and a robust assessment of cardiomyocyte size as a key parameter. Screening a library of synthetic miRNAs revealed several miRNAs previously not recognized as pro- or anti-hypertrophic. Out of these, we selected nine miRNAs and confirmed the pro-hypertrophic potential of miR-22, miR-30c, miR-30d, miR-212, miR-365 and the anti-hypertrophic potential of miR-27a, miR-27b and miR-133a. Quantitative analysis of the expression level of pro-hypertrophic miRNAs in primary cardiomyocytes indicated a rather low level of correlation of the phenotypic effects of individual miRNAs and their expression level. This assay allows the automated determination of cell size in primary cardiomyocytes and permitted the identification of a set of miRNAs capable of regulating cardiomyocyte hypertrophy. Elucidating their mechanism of action should provide insight into mechanisms underlying the cardiomyocyte hypertrophic response. This article is part of a Special Issue entitled ‘Possible Editorial’. [Copyright &y& Elsevier]

Metformin is an oral biguanide used to treat diabetes. Recent study showed it may interfere was related to cancer progression and has a positive effect on cancer prevention and treatment, which attracts a new hot research topic. Here we show that Metformin suppressed the proliferation but induced apoptosis of gastric cells. Notably, Metformin enhanced gastriccell apoptosis via modulating AMPK signaling. Furthermore, Metformin and miR-365 synergistically promote the apoptosis of gastric cancer cells by miR-365-PTEN-AMPK axis. Our study unraveled a novel signaling axis in the regulation in gastric cancer, which could be amplified by the application of metformin. The new effect of metformin potentiates its novel therapeutic application in the future. The data generated during this study are included in this article and its supplementary information files are available from the corresponding author on reasonable request. • MiR365 induces gastric cancer cell apoptosis, by inhibiting PTEN expression via interact on 3′UTR of PTEN. • Metformin regulates AMPK phosphorylation by downregulating PTEN expression in gastric cancer cell apoptosis. • Metformin and miR-365 synergistically promote the apoptosis of gastric cancer cells by miR-365-PTEN-AMPK axis. [ABSTRACT FROM AUTHOR]

• MicroRNA-365 is downregulated in human myeloma cells. • MicorRNA-365 targets homeobox A9 in human myeloma cells. • Overexpression of microRNA-365 induces apoptosis in myeloma cells. • Overexpression of microRNA-365 inhibits the invasion of myeloma cells. • MicroRNA-365 inhibits the xenografted tumor growth in vivo. The aberrant micro-RNA (miR) expression has been reported to play a vital role in proliferation and tumorigenesis and of several human cancers. MicroRNA-365 (miR-365) has been shown to exhibit tumor-suppressive or oncogenic role in several human cancers. Nonetheless, little is known about its growth regulatory role in human multiple myeloma. The present study characterized the regulatory control exercised by miR-365 in multiple myeloma. The results showed significant (P < 0.05) upregulation of miR-365 in myeloma tissues and cell lines. Overexpression of miR-365 significantly (P < 0.05) suppressed the proliferation and inhibition of miR-365 promoted the proliferation of the human myeloma cells. The tumor-suppressive effects of miR-365 were found to be the result of apoptosis in the IM-9 myeloma cells. The miR-365 overexpression also suppressed the invasion of the IM-9 myeloma cells. The homeobox gene, HOXA9 was identified as the molecular regulatory target of miR-365 in human myeloma. The overexpression of miR-365 was shown to cause suppression of HOXA9. The silencing of HOXA9 could also suppress the growth of the IM-9 myeloma cells while as the overexpression of HOXA9 could abolish the tumor-suppressive effects of miR-365. The in vivo study revealed that miR-365 inhibits the growth of the xenografted tumors. Nonetheless, the inhibition of miR-365 promotes the growth of the xenografted tumors. To sum up, the current study suggests the tumor-suppressive effects of miR-365 in human myeloma and highlights the applicability of miR-365 as vital therapeutic target against this fatal malignancy. [ABSTRACT FROM AUTHOR]

miR-365 is a microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers while suppressing these effects in others. Virtually no information is known about the presence or function of miR-365 in oral cancers. Based upon this information, the primary goal of this project was to evaluate the expression of miR-365 in existing oral cancer cell lines. Five commercially available oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, and CAL27) were obtained and cultured. RNA was then screened by PCR using primers specific for miR-365, as well as matrix metalloproteinase (MMP-2) and a downstream cancer stem cell regulator (NKX2.1), and structural and metabolic standards (beta actin, GAPDH). miR-365 was detected among these oral cancers, and some cells also expressed NKX2.1 and MMP-2, which correlated with miR-365 levels. The relative expression of miR-365, NKX2.1, and MMP-2 RNA was higher than expected. Transfection of miR-365 resulted in a significant increase in proliferation, which was not observed in the negative controls. These data appear to confirm miR-365 expression in oral cancers, which may also be correlated with MMP-2 and NKX2.1 expression. Moreover, the fastest growing oral cancers with the highest viability produced the most miR-365. In addition, miR-365 transfected into cells significantly increased growth, even in normal cells. More research is needed to elucidate the pathways responsible for these observations. [ABSTRACT FROM AUTHOR]

Introduction: miR-365 is a non-coding microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers, while suppressing these effects in others. Many microRNAs are produced and then exported extracellularly in exosomes, which are small extracellular vesicles ranging from 30 to 100 nm that are found in eukaryotic fluids and facilitate many cellular functions. Exosomes and extracellular vesicles are produced by many cell types, including oral cancer cells—although no study to date has evaluated miR-365 and oral cancer exosomes or extracellular vesicles. Based on this information, our research question was to evaluate whether oral cancers produce exosomes or extracellular vesicles containing miR-365. Materials and Methods: Two commercially available oral cancer cell lines (SCC25 and CAL27) and a normal oral keratinocyte (OKF4) were grown in serum-free media, supplemented with exosome-depleted fetal bovine serum. Extracellular vesicles and exosomes were then isolated using the Invitrogen total exosome RNA and protein isolation kit for processing using the hsa-miR-365a-5p microRNA qPCR assay kit. Results: RNA was successfully isolated from the exosome-depleted supernatant from each cell line—SCC9, SCC15, SCC25, and CAL27 (oral squamous cell carcinomas) and OKF4 (oral epithelial cell line). Relative concentrations of RNA were similar among each cell line, which were not significantly different, p = 0.233. RNA quality was established by A260:A280 absorbance using a NanoDrop, revealing purity ranging 1.73–1.86. Expression of miR-16 was used to confirm the presence of microRNA from the extracted exosomes and extracellular vesicles. The presence of miR-365 was then confirmed and normalized to miR-16 expression, which demonstrated an increased level of miR-365 in both CAL27 and SCC25. In addition, the normalized relative quantity (RQ) for miR-365 exhibited greater variation among SCC25 (1.382–4.363) than CAL27 cells (1.248–1.536). Conclusions: These results confirm that miR-365 is not only expressed in oral cancer cell lines, but also is subsequently exported into exosomes and extracellular vesicles derived from these cultures. These data may help to contextualize the potential for this microRNA to contribute to the phenotypes and behaviors of oral cancers that express this microRNA. Future research will begin to investigate these potential mechanisms and pathways and to determine if miR-365 may be useful as an oral cancer biomarker for salivary or liquid biopsies. [ABSTRACT FROM AUTHOR]

End plate chondrocyte degeneration is a major cause of intervertebral disc degeneration. Mechanical biophysical forces, including intermittent cyclic mechanical tension (ICMT), exacerbate end plate chondrocyte degeneration. However, the underlying molecular mechanism of mechanical stretch-induced end plate chondrocyte degeneration is still unclear. This study sought to determine whether microRNAs (miRNAs) respond to mechanical stretch and play a role in regulating mechanically-induced end plate chondrocyte degeneration. We identified miR-365 as a mechanoresponsive miRNA in primary human end plate chondrocytes after ICMT application by miRNA microarray analysis. The expression of miR-365 was down-regulated in the disc samples obtained from patients with disc degeneration. We also found that the miR-365 stimulates chondrocyte proliferation but does not promote end plate chondrocyte death. Using bioinformatic analyses and subsequent confirmation by real-time RT-PCR, we identified multiple candidate target genes of miR-365 that responded to in vitro mechanical stimulation; among them, HDAC4 was fully characterized. Mutation of putative miR-365 binding sites in HDAC4 mRNA abolished miR-365 mediated repression of HDAC4 3′-untranslated region (3′UTR) luciferase reporter activity, suggesting that miR-365 binds to the HDAC4 3′UTR. Overexpression of miR-365 significantly decreased the HDAC4 protein level, suggesting that miR-365 acts as an endogenous attenuator of HDAC4 in human end plate chondrocytes. Further, perturbation of miR-365 expression also had a significant effect on the expression of COL2A and ACAN and on matrix degeneration. Overexpression of HDAC4 abolished miR-365 rescued end plate chondrocyte degeneration during ICMT application. Furthermore, we found that the wnt/β-catenin signal pathway was related to HDAC4 and promoted end plate chondrocyte degeneration. Overall, our results suggest that miR-365 is a mechanosensitive miRNA that regulates human chondrocyte degeneration by directly targeting HDAC4. We propose that therapeutic regulation of miR-365 may be an efficient anabolic strategy for inhibiting end plate chondrocyte degeneration. • miR-365 is a mechanosensitive miRNA that regulates human chondrocyte degeneration by directly targeting HDAC4. • HDAC4 expression induced endplate chondrocyte degeneration by activating the Wnt/β-catenin signaling pathway. • Regulating miR-365 or HDAC4 expression can relieve endplate chondrocyte degeneration induced by ICMT. [ABSTRACT FROM AUTHOR]

The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4 , in turn, enhancing the chondrogenesis of BMSCs. [ABSTRACT FROM AUTHOR]