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Oral squamous cell carcinoma (OSCC) is one of the most common cancer types of head and neck cancer, accounting for 95% of all cases. However, the mechanisms underlying the pathogenesis of OSCC remain unclear. Circular RNA (CircRNA) has been extensively studied in the past decades and is a promising direction for the development of OSCC therapeutic targets. In this study, we aimed to investigate the role of circMTO1 in OSCC progression. First, we validated the characterization and expression of circMTO1 in OSCC. It was found that circMTO1 was upregulated in OSCC tumor tissues and cells. Subsequently, we conducted biological experiments. It was found that circMTO1 knockdown inhibited OSCC cell proliferation, migration, and invasion. Furthermore, we conducted a series of experiments to elucidate the underlying mechanisms. A novel circMTO1/miR-320a/ATRX axis was identified. Our results suggest that circMTO1 modulates ATRX expression to accelerate OSCC progression by sponging miR-320a. Moreover, we found that circMTO1 expression in OSCC was transcriptionally regulated by Zinc Finger Protein 460 (ZNF460). Our study showed a novel ZNF460/circMTO1/miR-320a/ATRX signaling in OSCC development.

Background In this study, we determine the potential roles and uncover the regulatory mechanisms of circCCDC66 in regulating cell growth and cell metastasis of glioma. Methods qRT-PCR was used to detect the expressions of circCCDC66 in gliomas and tissues. The biological function of circCCDC66 in glioma cell lines was elucidated by functional experiments. Cell counting kit-8 and transwell were used to detect the effect of circCCDC66 on the proliferation, migration and invasion of glioma cells. Bioinformatics analysis was applied to reveal the targets of circCCDC66. Results The results showed circCCDC66 was overexpressed in glioma and acted as an oncogene. CircCCDC66 knockdown suppressed the proliferation, migration, and invasion of glioma cells. We constructed a circCCDC66 regulating miRNA network and revealed miR-320a was a potential target of circCCDC66, which was down-regulated in high-grade gliomas compared to low-grade gliomas. Bioinformatics analysis showed circCCDC66-miR-320a/b axis was involved in regulating multiple cancer-related pathways. Furthermore, we identified FOXM1 as a key target of circCCDC66, which was involved in regulating DNA damage response pathways. In mechanism study, circCCDC66 could sponge miR-320a, thereby increasing the expression of FOXM1. Conclusions CircCCDC66 could facilitate glioma cells proliferation, invasion and migration by down-regulating miR-320a and up-regulating FOXM1.

Cutaneous squamous cell carcinoma (cSCC) is a highly prevalent skin malignancy, and the effective therapy still remains a challenge. Crocin can be used for cSCC therapy. This study explored the effects of cSCC cells treatment with crocin in vitro and in vivo. The study used A431 and SCL-1 cells lines, cSCC human samples and BALB/C nude mice for investigations. Apoptosis was determined by MTT assays, while miR-320a and ATG2B expressions were validated through RT-qPCR. Interaction of miR-320a with ATG2B was examined via dual luciferase reporter assay. The autophagy and apoptosis proteins expressions were further confirmed through western blot and immunofluorescence staining assays. The results indicated a significantly upregulated miR-320a, but a down-regulated ATG2B expression in the cSCC clinical samples. Crocin significantly repressed cSCC cells growth, and induced apoptosis through autophagy. Furthermore, miR-320a expression was inhibited and ATG2B expression was increased. Dual luciferase reporter assay revealed that miR-320a regulated ATG2B expression directly. Additionally, the upregulation of ATG2B expression in cSCC cells inhibited cell proliferation and led to cell apoptosis. Crocin also reduced tumor growth and stimulated the apoptosis in vivo. In conclusion, miR-320a is upregulated and ATG2B is down-regulated in cSCC, Crocin suppresses the proliferation and induces apoptosis of cSCC cells. Further, Crocin increases autophagy while miR-320a hinders autophagy and the apoptotic effects of crocin on cSCC cells. MiR-320a binds ATG2B directly, and ATG2B expression is upregulated by crocin. Finally, Crocin triggers cSCC cells apoptosis in vivo. Crocin can target ATG2B/miR-320a and may be an effective alternative for cSCC treatment., GRAPHICAL ABSTRACT

Pathophysiological events that modulate the progression of structural changes in osteoarthritis (OA) include monocyte adhesion and infiltration, and synovial inflammation. In particular, the adhesion protein intercellular adhesion molecule type 1 (ICAM-1) promotes monocyte recruitment into the synovial tissue. Visfatin is an adipocyte hormone that promotes the release of inflammatory cytokines during OA progression. We report that visfatin enhances ICAM-1 expression in human OA synovial fibroblasts (OASFs) and facilitates the adhesion of monocytes with OASFs. AMPK and p38 inhibitors, as well as their respective siRNAs, attenuated the effects of visfatin upon ICAM-1 synthesis and monocyte adhesion. We also describe how miR-320a negatively regulates visfatin-induced promotion of ICAM-1 expression and monocyte adhesion. We detail how visfatin affects ICAM-1 expression and monocyte adhesion with OASFs by inhibiting miR-320a synthesis via the AMPK and p38 signaling pathways.

Osteosarcoma is a primary malignant bone tumor, which affects children, adolescents, and young adults commonly. MicroRNAs (miRNAs) have been proved to be dysregulated in different cancers, including osteosarcoma. Although miR‐320a has been implicated in many types of malignancies, little is known about the role of miR‐320a in osteosarcoma. In this study, we show that the overexpression of miR‐320a or knockdown of cytoplasmic polyadenylation element‐binding protein 1 (CPEB1) inhibited osteosarcoma cell migration and invasion. miR‐320a downregulates CPEB1 expression by directly targeting the CPEB1 3′‐UTR. Furthermore, CPEB1 reintroduction reversed the antiproliferation, antimigration, and antiinvasion roles of miR‐320a, indicating that miR‐320a might function as a tumor suppressor in osteosarcoma through CPEB1. In conclusion, our study demonstrates that miR‐320a plays a critical role in osteosarcoma progression and may provide a potential therapeutic target for osteosarcoma., Our study demonstrates that miR‐320a plays a critical role in osteosarcoma progression and may provide a potential therapeutic target for osteosarcoma.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system. It has been found that hypoxia mediates the malignant evolvement of RCC. Here, we probe the impact and potential mechanism of HECT domain E3 ubiquitin-protein ligase 2 (HECTD2) and HIF-1α on regulating RCC evolvement. RCC tissues and adjacent normal tissues were collected, and the association between the expression profiles of HECTD2 and HIF-1α and the clinicopathological features was analyzed. Additionally, we constructed HECTD2/HIF-1α overexpression and knockdown models in RCC cell lines to ascertain the impacts of HECTD2 and HIF-1α on RCC cell proliferation, apoptosis, migration, and growth in vivo. We applied bioinformatics to predict the upstream miRNA targets of HECTD2. Meanwhile, RNA immunoprecipitation (RIP), and the dual-luciferase reporter assays were employed to clarify the targeting association between HECTD2 and miR-320a. The effect of miR-320a on HECTD2-mediated RCC progression was investigated. The results suggested that both HIF-1α and HECTD2 were up-regulated in RCC (compared with adjacent non-tumor tissues), and they had positive relationship. Moreover, higher level of HECTD2 and HIF-1α is associated with poorer overall survival of RCC patients. HECTD2 overexpression heightened RCC cell proliferation and migration, and weakened cell apoptosis. On the other hand, the malignant phenotypes of RCC cells were signally impeded by HECTD2 or HIF-1α knockdown. Moreover, miR-320a targeted the 3′-untranslated region of HECTD2 and suppressed HECTD2 expression. The rescue experiments showed that miR-320a restrained HECTD2-mediated malignant progression in RCC, while up-regulation of HIF-1α hampered miR-320a expression. Collectively, HIF-1α mediated HECTD2 up-regulation and aggravated RCC progression by attenuating miR-320a.

Osteoporosis is a serious bone metabolism disease. Recent studies have shown that MYC could promote the formation of osteoclasts. Evidence has also shown that miR-320a could injure osteoblasts by inducing oxidative stress. By querying the database, we found that MYC has the potential to target and affect the expression of miR-320a. However, the effects of MYC and miR-320a on the the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclasts are unclear. In this study, we examined the relationship between MYC and miR-320a with luciferase reporter assay. To investigate the role of MYC and miR320a in osteoporosis, MYC or miR-320a expression were knocked down in RAW 264.7 cells. Meanwhile, the expression of markers of osteoclasts was detected with Western blotting. Finally, we inhibited the expression of PTEN in RAW 264.7 cells with miR-320a depletion and detected the expression of abovementioned proteins. MYC promoted the expression of miR-320a in RAW 264.7 cells by binding to the promoter of miR-320a. Inhibition of MYC and miR-320a suppressed the formation of RANKL-induced osteoclasts by inhibiting the expression of c-Fos, NFATc1, TRAP and CTSK. Moreover, the expression of c-Fos, NFATc1, TRAP and CTSK was rescued and the RANKL-induced osteoclasts was promoted after the repressing the expression of PTEN. In conclusion, MYC enhanced the formation of RANKL-induced osteoclasts by modulating the miR-320a/PTEN pathway.

Extracellular vesicles (EVs) play crucial roles in intercellular communication. miRNAs derived from EVs emerge as promising diagnostic indicators and therapeutic targets in a variety of malignancies. Tremendous studies have revealed the function of miRNAs derived from EVs in tumorigenesis, metastasis and other aspects. The mechanism of action of EV-derived miRNAs, however, in ovarian cancer remains largely unknown. In this study, EVs were enriched from the ovarian cancer cell lines. EVs as a whole could promote cell proliferation, invasion and new vasculature formation. However, the down-regulated EV-derived miR-320a was demonstrated to potentially suppress tumorigenesis, metastasis and angiogenesis. Moreover, EV-derived miR-320a has been proved to directly regulate a previously unknown target, ZC3H12B. An unreported role of ZC3H12B in promoting ovarian cancer cell proliferation has been elucidated and miR-320a could mediate the expression of ZC3H12B, thereby inhibiting the downstream response. As for the practical clinic values, lower expression of EV-derived miR-320a correlates with shorter survival period, indicating that EV-derived miR-320a may also serve as a prognostic biomarker in ovarian cancer. This research provides new insight into the molecular mechanism of EV-derived miR-320a in ovarian cancer and may provide new therapeutic and prognostic strategies for ovarian cancer treatment. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-021-00437-2.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system. It has been found that hypoxia mediates the malignant evolvement of RCC. Here, we probe the impact and potential mechanism of HECT domain E3 ubiquitin-protein ligase 2 (HECTD2) and HIF-1α on regulating RCC evolvement. RCC tissues and adjacent normal tissues were collected, and the association between the expression profiles of HECTD2 and HIF-1α and the clinicopathological features was analyzed. Additionally, we constructed HECTD2/HIF-1α overexpression and knockdown models in RCC cell lines to ascertain the impacts of HECTD2 and HIF-1α on RCC cell proliferation, apoptosis, migration, and growth in vivo. We applied bioinformatics to predict the upstream miRNA targets of HECTD2. Meanwhile, RNA immunoprecipitation (RIP), and the dual-luciferase reporter assays were employed to clarify the targeting association between HECTD2 and miR-320a. The effect of miR-320a on HECTD2-mediated RCC progression was investigated. The results suggested that both HIF-1α and HECTD2 were up-regulated in RCC (compared with adjacent non-tumor tissues), and they had positive relationship. Moreover, higher level of HECTD2 and HIF-1α is associated with poorer overall survival of RCC patients. HECTD2 overexpression heightened RCC cell proliferation and migration, and weakened cell apoptosis. On the other hand, the malignant phenotypes of RCC cells were signally impeded by HECTD2 or HIF-1α knockdown. Moreover, miR-320a targeted the 3′-untranslated region of HECTD2 and suppressed HECTD2 expression. The rescue experiments showed that miR-320a restrained HECTD2-mediated malignant progression in RCC, while up-regulation of HIF-1α hampered miR-320a expression. Collectively, HIF-1α mediated HECTD2 up-regulation and aggravated RCC progression by attenuating miR-320a.

Background Curcumin has a potential therapeutic role in ovarian cancer. However, whether curcumin plays anti-cancer role in ovarian cancer by mediating the circular RNA (circRNA)/microRNA (miRNA)/mRNA network is still unclear. Methods The expression of circ-PLEKHM3, miR-320a, and suppressor of morphogenesis in genitalia 1 (SMG1) was detected via qRT-PCR. Cell viability, colony-formation ability and apoptosis were analyzed via cell counting kit-8 assay, colony formation analysis, and flow cytometry. Protein expression was measured using western blot. The in vivo experiments were performed using a xenograft model. Target association was evaluated via dual-luciferase reporter analysis and RIP assay. Results Curcumin suppressed ovarian cancer cell proliferation and promoted apoptosis. Circ-PLEKHM3 was downregulated in ovarian cancer, and its expression could be promoted by curcumin treatment. Circ-PLEKHM3 overexpression exacerbated the effect of curcumin on ovarian cancer cell proliferation and apoptosis, as well as anti-tumor effect. MiR-320a was targeted by circ-PLEKHM3. The inhibition effect of circ-PLEKHM3 overexpression on cell proliferation and the enhancing effect on cell apoptosis could be reversed by miR-320a mimic. SMG1 was targeted by miR-320a, and its knockdown also reversed the regulation of miR-320a inhibitor on the proliferation and apoptosis of ovarian cancer cells. In addition, circ-PLEKHM3 could upregulate SMG1 expression via sponging miR-320a. Conclusion Curcumin restrained proliferation and facilitated apoptosis in ovarian cancer by regulating the circ-PLEKHM3/miR-320a/SMG1 axis.

Dysregulated expression profiles of microRNAs (miRNAs) have been observed in several types of cancer, including non-small cell lung cancer (NSCLC); however, the diagnostic and prognostic potential of circulating miRNAs in NSCLC remains largely undefined. Here we found that circulating miR-320a was significantly down-regulated (~5.87-fold;p< 0.0001) in NSCLC patients (n= 80) compared to matched control plasma samples from healthy subjects (n= 80). Kaplan-Meier survival analysis revealed that NSCLC patients with lower levels of circulating miR-320a had overall poorer prognosis and survival rates compared to patients with higher levels (p< 0.0001). Moreover, the diagnostic and prognostic potential of miR-320a correlated with clinicopathological characteristics such as tumor size, tumor node metastasis (TNM) stage, and lymph node metastasis. Functionally, depletion of miR-320a in human A549 lung adenocarcinoma cells induced their metastatic potential and reduced apoptosis, which was reversed by exogenous re-expression of miR-320a mimics, indicating that miR-320a has a tumor-suppressive role in NSCLC. These results were further supported by high levels of epithelial-mesenchymal transition (EMT) marker proteins (e.g., Beta-catenin, MMP9, and E-cadherin) in lung cancer cells and tissuesviaimmunoblot and immunohistochemistry experiments. Moreover, through bioinformatics and dual-luciferase reporter assays, we demonstrated thatAKT3was a direct target of miR-320a. In addition, AKT3-associated PI3K/AKT/mTOR protein-signaling pathways were elevated with down-regulated miR-320a levels in NSCLC. These composite data indicate that circulating miR-320a may function as a tumor-suppressor miRNA with potential as a prognostic marker for NSCLC patients.

Background This study incorporates fundamental research referring to considerable amounts of gene-sequencing data and bioinformatics tools to analyze the pathological mechanisms of diffuse large B-cell lymphoma (DLBCL). Methods A lncRNA-miRNA-mRNA ceRNA network of DLBCL was constructed through database analysis combining GTEx and TCGA. qPCR was used to detect the expression of LINC00963 and miR-320a in DLBCL cell lines. After LINC00963 or miR-320a overexpression in vitro, western blot was performed to assess the protein levels of UPR sensors (GRP78, p-IRE1, IRE1, active ATF6, ATF4 and XBP1), along with apoptosis markers (Bcl-2, Bax, caspase 3) and autophagy indicators (Beclin1, LC3II, LC3I and p62). Additionally, the expression of LC3 was analyzed through immunofluorescence (IF) assay. Results Following LINC00963 overexpression in vitro, SUDHL4 cell line showed a marked increase in the level of UPR-related GRP78, p-IRE1 and spliced XBP-1/XBP-1(s), apoptosis-related Bax and cleaved caspase 3, as well as autophagy-related Beclin1 and LC3II, whereas miR-320a mimic greatly diminished the effects of LINC00963 overexpression. Moreover, LINC00963 targeted miR-320a while miR-320a bound to the 3’UTR of XBP1. It was also found that LINC00963 overexpression resulted in significantly delayed tumor growth in a xenograft model of DLBCL. Conclusion Mechanistically, LINC00963/miR-320a regulated XBP1-apoptosis pathway and autophagy, implying the therapeutic potential of this pathway for selective targeting. The data presented here illustrated the mechanism of LINC00963/miR-320a/XBP1 in DLBCL for the first time.

Background Previous evidence have shown that long non-coding RNA (lncRNA) TMPO antisense RNA 1 (TMPO-AS1) is involved in the aggressiveness of several cancers. Nevertheless, the precise functions of TMOP-AS1 in hepatocellular carcinoma (HCC) are still unresolved. Materials and Methods The expressions of TMPO-AS1 and miR-320a were detected in HCC tissues and cells by qRT-RCR. The cell growth, migration and invasion were detected by colony formation, wound healing assay and Transwell assay, respectively. The targeting relation between miR-320a and TMPO-AS1 was predicted by bioinformatics analysis and identified by luciferase reporter gene as well as FISH assay. The expression of SERPINE1 MRNA Binding Protein 1 (SERBP1) was detected by Western blot. The growth of HCC cell was analyzed using transplanted tumor model. Results Currently, we revealed that TMPO-AS1 was overexpressed in clinical HCC samples and a panel of HCC cell lines. Clinically, a higher level of TMPO-AS1 was connected to the advanced stage of HCC and worse prognosis of patients. Depletion of TMPO-AS1 repressed HCC cell viability, migration ability and invasiveness. Nevertheless, upregulation of TMPO-AS1 caused opposite results. Further studies revealed that lncRNA TMPO-AS1 was largely located in the cytoplasm of HCC cell and sponge miR-320a, resulting in increasing the level of SERBP1 in HCC cell. Finally, TMPO-AS1 silencing suppressed tumor growth of HCC cell in vivo. Conclusion Collectively, our results suggested that TMPO-AS1 was a promoting factor for the aggressive behaviors of HCC cell.

AFAP1‐AS1 is a long non‐coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1‐AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1‐AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp‐2 cells. Indeed, we found not only that AFAP1‐AS1 was up‐regulated in laryngeal carcinoma specimens and cells, but also that stemness‐associated genes were overexpressed. Silencing of AFAP1‐AS1 promoted HEp‐2 cell chemoresistance under cisplatin treatment. Expression of AFAP1‐AS1 was increased in drug‐resistant Hep‐2 cells. We then probed the mechanism of AFAP1‐AS1 activity and determined that miR‐320a was a potential molecular target of AFAP1‐AS1. Luciferase reporter and qRT‐PCR assays of AFAP1‐AS1 and miR‐320a levels in human specimens and cell cultures indicated that AFAP1‐AS1 negatively regulates miR‐320a. To discover the molecular mechanism of miR‐320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3′‐untranslated regions (3′‐UTR) of RBPJ into a luciferase reporter, we determined that miR‐320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1‐AS1 increases RBPJ expression by negatively regulating miR‐320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1‐AS1 silencing. Taken together, these results suggest that AFAP1‐AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR‐320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.

Background Vascular homeostasis abnormalities may involve in doxorubicin induced cardiotoxicity. Methods Enhanced cardiac miR-320a expression, reduced cardiac microvessel density and impaired cardiac function were observed in mice treated by anthracycline doxorubicin. To further explore the role of miR-320a in doxorubicin induced cardiotoxicity, microRNA mimics/inhibitor in vitro and rAAV administration in vivo were employed in mice. Results Knockdown of miR-320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR-320a enhanced apoptosis in vitro, and aggravated vessel abnormalities in heart and subsequent cardiac dysfunction in mice. Furthermore, Western blot assays showed that VEGF-A was a potential target of miR-320a, which was verified by anti-Ago2 co-immunoprecipitation. Moreover, as same as miR-320a, siRNA against VEGF-A reinforced doxorubicin induced endothelial cells injury. Finally, the negative effects of miR-320a on vascular homeostasis and cardiac function were alleviated by VEGF-A re-expression in doxorubicin treated mice. Conclusion Our observations demonstrate that miR-320a play important roles in doxorubicin induced cardiotoxicity via vessel homeostasis in heart and thus, inhibition of miR-320a may be applied to the treatment of cardiac dysfunction induced by anthracycline.

// Jian-Yong Sun 1, * , Zheng-Wei Zhao 1, * , Wei-Miao Li 1, 2, * , Guang Yang 1 , Peng-Yu Jing 1 , Pei Li 1 , Hai-Zhou Dang 1 , Zhao Chen 1 , Yong-An Zhou 1 and Xiao-Fei Li 1 1 Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China 2 Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China * These authors have contributed equally to this work Correspondence to: Xiao-Fei Li, email: lixfchest@163.com Yong-An Zhou, email: chuyan22@fmmu.edu.cn Keywords: MALAT1, HUVECs, proliferation, miR-320a, FOXM1 Received: March 20, 2017 Accepted: May 21, 2017 Published: June 16, 2017 ABSTRACT Regulation of cancer angiogenesis could be a useful strategy in cancer therapy. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA), and can induce cancer cell proliferation, while lncRNAs, generally are able to act as microRNA (miRNA) sponges. The latter is a type of competitive endogenous RNA (ceRNA) that regulates expression of the targeting miRNAs and protein-coding genes. This study investigated the proliferative role of MALAT1 in human umbilical vein endothelial cells (HUVECs) and the underlying molecular events. The data showed that knockdown of MALAT1 expression using MALAT1 siRNA inhibited HUVEC proliferation and also significantly decreased levels of FOXM1 mRNA and protein in vitro , while knockdown of FOXM1 expression reduced HUVEC proliferation. Annotation of HUVEC microarray data revealed that seven miRNAs, including miR-320a, were upregulated after knockdown of MALAT1 expression in HUVECs. MALAT1 was shown to reciprocally interact with miR-320a, i.e., expression of one negatively regulated levels of the other, whereas knockdown of MALAT1 expression promoted miR-320a levels. Furthermore, miR-320a could directly target and inhibit FOXM1 expression in HUVECs. Knockdown of MALAT1 expression enhanced miR-320a expression but reduced FOXM1 expression resulting in downregulation of HUVEC proliferation. However, such an effect was inhibited by miR-320a depletion. In conclusion, this study demonstrates that miR-320a plays an important role in mediating the effects of MALAT1 on HUVEC proliferation by suppression of FOXM1 expression. Thus, targeting of this gene pathway could be a novel strategy in cancer therapy.

// Sun Yong 1 , Yu Yabin 1 , Zhou Bing 1 , Zhu Chuanrong 1 , Gu Dianhua 1 , Zhang Jianhuai 1 , Yuan Weidong 1 , Wang Shuming 1 and Liu Ling 1 1 Department of Hepatobiliary and Pancreatic Surgery, Huai’an First People’s Hospital, Nanjing Medical University, Huai'an, Jiangsu 223300, People’s Republic of China Correspondence to: Liu Ling, email: liulingdoc_2002@aliyun.com Keywords: PDAC, DGCR5, miR-320a, lncRNA Received: March 13, 2017 Accepted: April 17, 2017 Published: June 06, 2017 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies. Long non-coding microRNAs (lncRNAs) are a newly discovered type of regulatory molecule with both diagnostic and prognostic value, but the role of lncRNA in PDAC has not been well investigated until now. Here, we present evidence that shows that the lncRNA DGCR5 is significantly reduced in PDAC tissues as well as in PDAC cell lines and that the downregulation of DGCR5 predicts poor prognosis. Ectopic expression of DGCR5 inhibits the proliferation and migration, and promotes 5-FU resistances of PDAC cells. Further experiments demonstrated that DGCR5 and miR-320a regulate each other in a reciprocal manner and that DGCR5 reverses the inhibition of PDCD4 by miR-320a, which is involved in the regulation of the PDAC cell phenotype and response to 5-FU. Our findings provide novel information about the functions of lncRNAs in PDAC, some of which might be beneficial to the precise diagnosis, prognosis and individualized therapy of patients with PDAC in the future.

Fei Xie,1,* Yuncang Yuan,1,* Luyang Xie,2,* Pengzhan Ran,1 Xudong Xiang,3 Qionglin Huang,1 Guoxiang Qi,1 Xiaopeng Guo,1 Chunjie Xiao,1 Shangyong Zheng1 1School of Medicine, Yunnan University, Kunming, Yunnan, 2Department of Stomatology, Shanghai Tenth People’s Hospital, Shanghai, 3Department of Gastrointestinal Surgery, The Third Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, People’s Republic of China *These authors contributed equally to this work Background: Downregulated expression levels of microRNA-320a (miR-320a) were found in primary breast cancers and colorectal cancer. Previous findings indicated that miRNA-320a may involve in the cancer development. In this study, we explored the roles of miR-320a by targeting c-Myc in the tumor growth of hepatocellular carcinoma (HCC). Methods: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-320a in 50 HCC tissues and four HCC cells. Luciferase reporter assay was conducted to confirm the direct downstream target of miR-320a in HEK-293 cells. The effect of miR-320a on endogenous c-Myc expression was investigated by transfecting miR-320a mimics into HepG2 and QGY-7703 cell lines. The c-Myc and miR-320a expressions were analyzed by immunohistochemistry (IHC) and qRT-PCR in the same HCC tissues. Furthermore, the biological functional correlation of miR-320a with c-Myc was determined by studying the effect of miR-320a mimics or c-Myc small interfering RNA (siRNA) on HCC cell proliferation and invasion. Results: The expression of miR-320a was downregulated in 50 HCC tissues and 4 HCC cells. Luciferase assay revealed that c-Myc is a direct target of miR-320a. IHC and Western blot analysis showed that the c-Myc expression was inhibited by miR-320a in HCC tissues and cell lines. Upregulation of miR-320a suppressed the HCC cell proliferation and invasion capacity induced by inhibiting c-Myc, and the results were consistent with the effects of c-Myc siRNA on tumor suppression. These results revealed that miRNA-320a inhibits tumor proliferation and invasion by targeting c-Myc in HCC cells.Conclusion: Our results showed that miR-320a functions as a tumor suppressor in HCC. By targeting c-Myc directly, miR-320a inhibits the HCC cell growth. Our studies provide evidence of miR-320a as a potentially target for HCC treatment. Keywords: miR-320a, hepatocellular carcinoma, c-Myc, tumor suppressors&nbsp

// Nan Xie 1, 3, * , Cheng Wang 2, 3, * , Zehang Zhuang 2, 3 , Jinson Hou 2, 3 , Xiqiang Liu 2, 3 , Yue Wu 2, 3 , Haichao Liu 2, 3 , Hongzhang Huang 2, 3 1 Department of Oral Pathology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China 2 Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China 3 Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China * These authors have contributed equally to this work Correspondence to: Hongzhang Huang, email: drhuang52@163.com Keywords: tongue squamous cell carcinoma, tumor budding, miR-320a, Suz12, invasion and metastasis Received: June 06, 2016 Accepted: August 15, 2016 Published: August 25, 2016 ABSTRACT We aimed to determine the specific miRNA profile of tumor budding cells and investigate the potential role of miR-320a in invasion and metastasis of tongue squamous cell carcinoma (TSCC). We collected tumor budding cells and paired central tumor samples from five TSCC specimens with laser capture microdissection and examined the specimens using a miRNA microarray. The specific miRNA signature of tumor budding cells was identified. We found that miR-320a was dramatically decreased in tumor budding cells. Knockdown of miR-320a significantly enhanced migration and invasion of TSCC cell lines. Suz12 was shown to be a direct target of miR-320a. Similar results were also observed in nude mouse models. Multivariate analysis indicated that miR-320a was an independent prognostic factor. Kaplan–Meier analysis demonstrated that decreased miR-320a and high intensity of tumor budding were correlated with poor survival rate, especially in the subgroup with high-intensity tumor budding and low expression of miR-320a. We concluded that decreased expression of miR-320a could promote invasion and metastasis of tumor budding cells by targeting Suz12 in TSCC. A combination of tumor budding and miR-320a may serve as an index to identify an aggressive sub-population of TSCC cells with high metastatic potential.

// Yangyang Wang 1, * , Jiping Zeng 2, * , Jianyong Pan 3 , Xue Geng 1 , Lupeng Li 2 , Jing Wu 1 , Ping Song 2 , Ying Wang 2 , Jilan Liu 2 , Lixiang Wang 1 1 Department of Pharmacology, Shandong University School of Medicine, Jinan 250012, P.R.China 2 Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, Jinan 250012, P.R. China 3 Department of Hepatobiliary Surgery, Qilu Hospital of Shandong University, Jinan 250012, P.R. China * These authors have contributed equally to this work Correspondence to: Lixiang Wang, email: wanglx@sdu.edu.cn Keywords: miR-320a, FoxM1, proliferation, gastric cancer, P27 KIP1 Received: December 06, 2015 Accepted: March 18, 2016 Published: April 11, 2016 ABSTRACT MicroRNAs (miRNAs) regulate tumorigenesis by inhibiting gene expression. In this study, we showed that miR-320a expression is decreased in human gastric cancer tissues and correlates inversely with expression of FoxM1, a key cell cycle regulator involved in gastric carcinoma. By contrast, the expression of P27 KIP1 , a downstream effector of FoxM1, correlates positively with miR-320a levels. Luciferase assays indicate that miR-320a suppresses FoxM1 expression, and in vitro recovery tests using FoxM1 siRNA indicate miR-320a inhibits gastric cancer cell proliferation by suppressing activity in the FoxM1-P27 KIP1 axis. In vivo , nude mice injected with BGC-823 gastric cancer cells expressing a miR-320a inhibitor exhibit faster tumor growth than mice injected with control cells. Analysis of FoxM1 and P27 KIP1 expression in tumor tissues indicates that miR-320a suppression increases the tumor growth by enhancing FoxM1-P27 KIP1 signaling. These results thus reveal the crucial role played by miR-320a in limiting gastric carcinoma by directly targeting FoxM1- P27 KIP1 axis.

The altered expression of miRNAs in response to stresses contributes to cancer pathogenesis. However, little is known regarding the mechanism by which cellular stresses drive alterations in miRNA expression. Here, we found that serum starvation enhanced mitophagy by downregulating the mitophagy-associated protein voltage-dependent anion channel 1 (VDAC1) and by inducing the expression of miR-320a and the transcription factor cAMP responsive element binding protein 1(CREB1). Furthermore, we cloned the promoter of miR-320a and identified the core promoter of miR-320a in the upstream -16 to -130 region of pre-miR-320a. Moreover, CREB1 was found to bind to the promoter of miR-320a to activate its expression and to induce mitophagy during serum starvation. Collectively, our results reveal a new mechanism underlying serum starvation-induced mitophagy in which serum starvation induces CREB1 expression, in turn activating miR-320a expression, which then down-regulates VDAC1 expression to facilitate mitophagy. These findings may provide new insights into cancer cell survival in response to environmental stresses.

Chaperone-mediated autophagy (CMA) is involved in wild-type α-synuclein degradation in Parkinson’s disease (PD), and LAMP2A and Hsc 70 have recently been indicated to be deregulated by microRNAs. To recognize the regularory role of miR-320a in CMA and the possible role in α-synuclein degradation, in the present study, we examined the targeting and regulating role of miR-320 in Hsc 70 expression. We first constructed an α-synuclein-overexpressed human neuroblastoma cell line, SH-SY5Y-Syn(+), stably over-expressing wild-type α-synuclein and sensitive to an autophagy inhibitor, which exerted no effect on the expression of LAMP2A and Hsc 70. Then we evaluated the influence on the CMA by miR-320a in the SH-SY5Y-Syn(+) cells. It was shown that miR-320a mimics transfection of specifically targeted Hsc 70 and reduced its expression at both mRNA and protein levels, however, the other key CMA molecule, LAMP2A was not regulated by miR-320a. Further, the reduced Hsc 70 attenuated the α-synuclein degradation in the SH-SY5Y-Syn(+) cells, and induced a significantly high level of α-synuclein accumulation. In conclusion, we demonstrate that miR-320a specifically targeted the 3' UTR of Hsc 70, decreased Hsc 70 expression at both protein and mRNA levels in α-synuclein-over-expressed SH-SY5Y cells, and resulted in significant α-synuclein intracellular accumulation. These results imply that miR-320a might be implicated in the α-synuclein aggravation in PD.

Daphne genkwa, a Chinese medicinal herb, is used frequently in Southeast Asian countries to treat diseases, the flavonoid hydroxygenkwanin (HGK) is extracted from its flower buds. The bioactivity of HGK, particularly as an anti-liver cancer agent, has not been explored. In this study, human hepatocellular carcinoma (HCC) cell lines and an animal xenograft model were employed to investigate both the activity of HGK against liver cancer and its cellular signaling mechanisms. HCC cells treated with HGK were subjected to cell function assays. Whole transcriptome sequencing was used to identify genes whose expression was influenced by HGK, and the flavonoid&rsquo, s cancer suppression mechanisms were further investigated through gain- and loss-of-function assays. Finally, in vitro findings were tested in a mouse xenograft model. The data showed that HGK induced the expression of the microRNA miR-320a, which in turn inhibited the expression of the transcription factor &lsquo, forkhead box protein M1&rsquo, (FOXM1) and downstream FOXM1-regulated proteins related to epithelial&ndash, mesenchymal transition, thereby leading to the suppression of liver cancer cell growth and invasion. Significant inhibition of tumor growth was also observed in HGK-treated mice. Hence, the present study demonstrated the activity of HGK against liver cancer and validated its potential use as a therapeutic agent.

Diagnosis of Arrhythmogenic CardioMyopathy (ACM) is challenging and often late after disease onset. No circulating biomarkers are available to date. Given their involvement in several cardiovascular diseases, plasma microRNAs warranted investigation as potential non-invasive diagnostic tools in ACM. We sought to identify circulating microRNAs differentially expressed in ACM with respect to Healthy Controls (HC) and Idiopathic Ventricular Tachycardia patients (IVT), often in differential diagnosis. ACM and HC subjects were screened for plasmatic expression of 377 microRNAs and validation was performed in 36 ACM, 53 HC, 21 IVT. Variable importance in data partition was estimated through Random Forest analysis and accuracy by Receiver Operating Curves. Plasmatic miR-320a showed 0.53 ± 0.04 fold expression difference in ACM vs. HC (p

Advanced prostate cancer (PCa) metastasizes to bone and lymph nodes, and currently available treatments cannot prevent the progression and metastasis of the disease. Therefore, an improved understanding of the molecular mechanisms of the progression and metastasis of advanced PCa using current genomic approaches is needed. Our miRNA expression signature in castration-resistant prostate cancer (CRPC) revealed that microRNA-320a (miR‑320a) was significantly reduced in cancer tissues, suggesting that miR‑320a may be a promising anticancer miRNA. The aim of this study was to investigate the functional roles of miR‑320a in naive PCa and CRPC cells and to identify miR‑320a-regulated genes involved in PCa metastasis. The expression levels of miR‑320a were significantly reduced in naive PCa, CRPC specimens, and PCa cell lines. Restoration of mature miR‑320a in PCa cell lines showed that miR‑320a significantly inhibited cancer cell migration and invasion. Moreover, we found that lysosomal-associated membrane protein 1 (LAMP1) was a direct target of miR‑320a in PCa cells. Silencing of LAMP1 using siRNA significantly inhibited cell proliferation, migration, and invasion in PCa cells. Overexpression of LAMP1 was observed in PCa and CRPC clinical specimens. Moreover, downstream pathways were identified using si-LAMP1-transfected cells. The discovery of tumor-suppressive miR‑320a-mediated pathways may provide important insights into the potential mechanisms of PCa metastasis.

// Juan Yu 1, * , Ji-Gang Wang 1, 2, * , Lei Zhang 1 , Hai-Ping Yang 3 , Lei Wang 1 , Di Ding 1 , Qi Chen 1 , Wen-Lin Yang 1 , Ke-Han Ren 1 , Dan-Mei Zhou 1 , Qiang Zou 4 , Yi-Ting Jin 4 , Xiu-Ping Liu 1, 5 1 Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China 2 Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao 266003, China 3 Department of Pathology, People’s Hospital, Linzi District, Zibo 255400, China 4 Department of Breast Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China 5 Department of Pathology, The Fifth People’s Hospital, Fudan University, Shanghai 200240, China * These authors contributed equally to this work and are co-first authors Correspondence to: Xiu-Ping Liu, email: xpliu1228@fudan.edu.cn Yi-Ting Jin, email: clara_raky@aliyun.com Keywords: miR-320a, MTDH, metastasis, breast cancer Received: September 05, 2015 Accepted: May 01, 2016 Published: May 24, 2016 ABSTRACT Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro , while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3′-untranslated region luciferase of metadherin ( MTDH ), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo . Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.

Mink enteritis virus (MEV) is one of the most important pathogens in the mink industry. Recent studies have shed light into the role of microRNAs (miRNAs), small noncoding RNAs of length ranging from 18–23 nucleotides (nt), as critical modulators in the host-pathogen interaction networks. We previously showed that miRNA miR-181b can inhibit MEV replication by repression of viral non-structural protein 1 expression. Here, we report that two other miRNAs (miR-320a and miR-140) inhibit MEV entry into feline kidney (F81) cells by downregulating its receptor, transferrin receptor (TfR), by targeting the 3′ untranslated region (UTR) of TfR mRNA, while being themselves upregulated.

In the present study, we investigated the roles and molecular mechanisms of miR-320a in human nasopharyngeal carcinoma (NPC). miR-320a expression was strongly reduced in NPC tissues and cell lines. Overexpression of miR-320a significantly suppressed NPC cell growth, migration, invasion and tumor growth in a xenograft mouse model. A luciferase reporter assay revealed that miR-320a could directly bind to the 3′ UTR of BMI-1. Overexpression of BMI-1 rescued miR-320a-mediated biological function. BMI-1 expression was found to be up-regulated and inversely correlated with miR-320a expression in NPC. Collectively, our data indicate that miR-320a plays a tumor suppressor role in the development and progression of NPC and may be a novel therapeutic target against NPC.

Breast cancer is one of the most common malignancies worldwide and is the second leading cause of cancer-related mortality among females. miRNAs are a class of small noncoding RNAs that are aberrantly expressed in human cancers. Due to their small size and stability, miRNAs have the potential to be efficacious clinical targets. MicroRNA-320a (miR-320a) has been shown to be dysregulated in multiple malignancies. In the present study, the expression levels of miR-320a were investigated in 15 paraffin-embedded in situ breast carcinoma and 130 invasive breast cancer tissues, and the prognostic value for breast cancer patients was assessed. Chromogenic in situ hybridization revealed that 60/130 (46%) invasive breast cancer tissues exhibited high expression levels of miR-320a (staining index score of ≥4). Furthermore, miR-320a staining was found to significantly correlate with tumor size (P=0.046), clinical stage (P