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MicroRNAs (miRNAs) are involved in the progression of many cancers through largely unelucidated mechanisms. The results of our present study identified a gene cluster, miR‐221/222, that is constitutively upregulated in serum exosome samples of patients with colorectal carcinoma (CRC) with liver metastasis (LM); this upregulation predicts a poor overall survival rate. Using an in vitro cell coculture model, we demonstrated that CRC exosomes harboring miR‐221/222 activate liver hepatocyte growth factor (HGF) by suppressing SPINT1 expression. Importantly, miR‐221/222 plays a key role in forming a favorable premetastatic niche (PMN) that leads to the aggressive nature of CRC, which was further shown through in vivo studies. Overall, our results show that exosomal miR‐221/222 promotes CRC progression and may serve as a novel prognostic marker and therapeutic target for CRC with LM., MiR‐221/222 is frequently upregulated in serum exosomal samples of patients with colorectal carcinoma (CRC) liver metastasis (LM). CRC exosome–delivered miR‐221/222 exerts a vital effect in promoting liver‐specific metastasis through remodeling the liver microenvironment. Tumor‐derived exosomal miR‐221/222‐3p can induce hepatic stromal cells to secrete HGF in the premetastatic niche (PMN).

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Complement factor H related protein 5 (CFHR5) may contribute to dysfunctional complement activation, thus predisposing to SLE. The expression levels of anti-dsDNA, C3 and CFHR5 in blood samples from 50 SLE patients and 50 healthy individuals were evaluated, and also their expression levels were measured in an MRL/lpr mouse model and control MRL/MPJ mice. The results showed that CFHR5 expression increased in SLE patients together with the increase of anti-dsDNA in comparison with the healthy control. Furthermore, CFHR5 expression was inversely correlated with C3, down-regulation of which was associated with worse SLE. Previous studies indicated that long noncoding RNA (lncRNA) regulates mRNA synthesis via microRNA (miRNA) inhibition. The present bioinformatics analysis revealed that the target miRNA (miR-222) was combined with both lncRNA MIAT and mRNA CFHR5. H&E staining of the kidney tissues of the MRL/lpr mice revealed that lncRNA MIAT, as a competitive inhibitor of miR-222, enhanced SLE by upregulating CFHR5 expression through the degradation of miR-222 in vivo . Thus, our study revealed for the first time the role of lncRNA MIAT in regulating CFHR5 expression in SLE in vivo and provided new insights into the role of lncRNA in regulation and complement function of SLE pathogenesis.

Type 2 diabetes mellitus (T2DM) is the most common diabetes and has numerous complications. Recent studies demonstrated that T2DM compromises bone fracture healing in which miR‐222 might be involved. Furthermore, tissue inhibitor of metalloproteinase 3 (TIMP‐3) that is the target of miR‐222 accelerates fracture healing. Therefore, we assume that miR‐222 could inhibit TIMP‐3 expression. Eight‐week‐old rats were operated femoral fracture or sham, following the injection of streptozotocin (STZ) to induce diabetes one week later in fractured rats, and then, new generated tissues were collected for measuring the expression of miR‐222 and TIMP‐3. Rat mesenchymal stem cells (MSCs) were isolated and treated with miR‐222 mimic or inhibitor to analyse osteogenic differentiation. MiR‐222 was increased in fractured rats and further induced in diabetic rats. In contrast, TIMP‐3 was reduced in fractured and further down‐regulated in diabetic rats. Luciferase report assay indicated miR‐222 directly binds and mediated TIMP‐3. Furthermore, osteogenic differentiation was suppressed by miR‐222 mimic and promoted by miR‐222 inhibitor. miR‐222 is a key regulator that is promoted in STZ‐induced diabetic rats, and it binds to TIMP3 to reduce TIMP‐3 expression and suppressed MSCs’ differentiation.

Background: Lung cancer is one of the main human health threats. Survival of lung cancer patients depends on the timely detection and diagnosis. Among the genetic irregularities that control cancer development and progression, there are microRNAs (miRNAs). This study aimed to assess the plasma level of circulating miRNA-17 and miRNA-222 as non-invasive markers in non-small-cell lung cancer (NSCLC) patients. Patients and methods: A total of 40 patients with NSCLC and 20 healthy controls who were matched in terms of age and sex with the patient group were included in this case-control study.. Estimation of miRNA-17 and miRNA-222 expression profiles in the plasma was done using quantitative real-time PCR (qRT-PCR). The relationship between both markers and their clinicopathological features were also determined. Receiver operating characteristic (ROC) curve analysis was done to evaluate the role of these microRNAs in NSCLC diagnosis and follow-up. Results: MiRNA-17 and miRNA-222 levels were significantly upregulated in NSCLC patients compared with controls (48.32±12.35 vs 1.16±0.19 and 34.53±3.1 vs 1.22±0.14) (P=0.000). Plasma miRNA-17 level was increased, and the miRNA-222 level was decreased across different stages of the disease; however, these differences d were not statistically significant (P=0.4, P=0.5, respectively). The miRNA-17 levels were higher in the lung cancer patients with metastasis , but miRNA-222 levels were lower patients without metastasis. We found no statistically significant difference in this regard(P=0.4 vs P=0.3, respectively). ROC curve analysis showed that the sensi¬tivity and specificity of miRNA-17 were 77.78% and 87.50% , and of miRNA-222 were 50% and 88.89%. Conclusion: MiRNA-17 and miRNA-222 can be considered as non-invasive biomarkers for detection of early lung carcinogenesis and metastasis in patients with NSCLC, hence providing a basis for the development of novel therapeutic approaches.

Metastasis is one of the major causes of death in colorectal cancer (CRC) patients. MiR-222 has been reported to be an oncogene in many types of cancer. However, its role in CRC cell invasion and migration as well as CRC downstream signaling pathways remains largely unknown. Our study found that miR-222 overexpression promotes the migration and invasion of CRC cell lines, and miR-222 interference results, as expected, in inhibition of migration and invasion. Bioinformatic analysis and dual luciferase reporter assay showed that mammalian STE20-like protein kinase 3 (MST3) may be the target gene of miR-222. Down-expression of MST3 in CRC cell lines enhanced their migration and invasion, but overexpression of MST3 could attenuate miR-222 overexpression in the promotion of migration and invasion in colorectal cell lines. HCT116 cell lines overexpressing miR-222 were transplanted into nude mice resulting in more lung metastases than in the control group. Further study found that MST3 may play a role in paxillin phosphorylation to reduce adhesion, or increase the invadopodia. These findings demonstrate that miR-222 modulates MST3 and therefore plays a critical role in regulating CRC cell migration and invasion. Thus, miR-222 may be a novel therapeutic target for CRC.

Background Exercise can protect myocardial infarction (MI) and downregulate cardiac Homeodomain-Interacting Protein Kinase 2 (HIPK2). However, the role of HIPK2 in MI is unclear. Methods HIPK2–/– mice and miR-222–/– rats, HIPK2 inhibitor (PKI1H) and adeno-associated virus serotype 9 (AAV9) carrying miR-222 were applied in the study. Animals were subjected to running, swimming, acute MI or post-MI remodeling. HIPK2 inhibition and P53 activator were used in neonatal rat cardiomyocytes (NRCMs) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) subjected to oxygen glucose deprivation/reperfusion (OGD/R). Serum miR-222 levels were analyzed in healthy people and MI patients that were survival or readmitted to the hospital and/or died. Findings Cardiac HIPK2 protein levels were reduced by exercise while increased in MI. In vitro, HIPK2 suppression by lentiviral vectors or inhibitor prevented apoptosis induced by OGD/R in NRCMs and hESC-CMs. HIPK2 inhibitor-treated mice and HIPK2–/– mice reduced infarct size after acute MI, and preserved cardiac function in MI remodeling. Mechanistically, protective effect against apoptosis by HIPK2 suppression was reversed by P53 activators. Furthermore, increasing levels of miR-222, targeting HIPK2, protected post-MI cardiac dysfunction, whereas cardiac dysfunction post-MI was aggravated in miR-222–/– rats. Moreover, serum miR-222 levels were significantly reduced in MI patients, as well as in MI patients that were readmitted to the hospital and/or died compared to those not. Interpretation Exercise-induced HIPK2 suppression attenuates cardiomyocytes apoptosis and protects MI by decreasing P-P53. Inhibition of HIPK2 represents a potential novel therapeutic intervention for MI. Funding This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to JJ Xiao), National Natural Science Foundation of China (82020108002, 81722008, and 81911540486 to JJ Xiao, 81400647 to MJ Xu, 81800265 to YJ Liang), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-09-E00042 to JJ Xiao), the grant from Science and Technology Commission of Shanghai Municipality (18410722200 and 17010500100 to JJ Xiao), the “Dawn” Program of Shanghai Education Commission (19SG34 to JJ Xiao), Shanghai Sailing Program (21YF1413200 to QL Zhou). JS is supported by Horizon2020 ERC-2016-COG EVICARE (725229).

Radiation-induced hair cell injury is detrimental for human health but the underlying mechanism is not clear. MicroRNAs (miRNAs) have critical roles in various types of cellular biological processes. The present study investigated the role of miR-222 in the regulation of ionizing radiation (IR)-induced cell injury in auditory cells and its underlying mechanism. Real-time PCR was performed to identify the expression profile of miR-222 in the cochlea hair cell line HEI-OC1 after IR exposure. miRNA mimics or inhibitor-mediated up- or down-regulation of indicated miRNA was applied to characterize the biological effects of miR-222 using MTT, apoptosis and DNA damage assay. Bioinformatics analyses and luciferase reporter assays were applied to identify an miRNA target gene. Our study confirmed that IR treatment significantly suppressed miR-222 levels in a dose-dependent manner. Up-regulation of miR-222 enhances cell viability and alleviated IR-induced apoptosis and DNA damage in HEI-OC1 cells. In addition, BCL-2-like protein 11 (BCL2L11) was validated as a direct target of miR-222. Overexpression of BCL2L11 abolished the protective effects of miR-222 in IR-treated HEI-OC1 cells. Moreover, miR-222 alleviated IR-induced apoptosis and DNA damage by directly targeting BCL2L11. The present study demonstrates that miR-222 exhibits protective effects against irradiation-induced cell injury by directly targeting BCL2L11 in cochlear cells.

Background Pneumonia is a serious respiratory tract infection disease in children, which threatens to the health or life of children patients. Ginsenoside Rb1 (Rb1) is a principle active ingredient extracted from the root of Panax notoginseng (Burk.) F.H.Chen with anti-inflammatory effect. Our study aimed to determine the effects and molecular mechanisms of Rb1 on lipopolysaccharide (LPS)-induced inflammatory injury of lung fibroblasts WI-38 cells. Methods Cell viability and apoptosis were evaluated by CCK-8 and flow cytometry analysis, respectively. The production of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-lβ, and IL-6 were measured by ELISA and RT-qPCR. miR-222 expression was examined by RT-qPCR. The expression levels of the nuclear factor-kappa B (NF-κB) p65 and phosphorylated p65 were detected by western blot. Results LPS stimulation induced WI-38 cell inflammatory injury by inhibiting cell viability, and inducing apoptosis and inflammatory cytokine production, while treatment with Rb1 significantly attenuated LPS-induced inflammatory injury in WI-38 cells. Additionally, Rb1 decreased LPS-induced upregulation of miR-222 and activation of the NF-κB pathway in WI-38 cells. Overexpression of miR-222 abolished the inhibitory effects of Rb1 on LPS-induced viability reduction, apoptosis, inflammatory cytokine production and activation of the NF-κB pathway. Conclusion Rb1 alleviated LPS-induced inflammatory injury in WI-38 cells via downregulating miR-222 and inactivation of the NF-κB pathway, contributing to our understanding of the effects and molecular mechanism of Rb1 in pneumonia.

MicroRNAs (miRs) are non-coding master regulators of transcriptome that could act as tumor suppressors (TSs) or oncogenes (oncomiRs). We aimed to systematically investigate the relevance of miRs as prognostic biomarkers in primary glioblastoma multiforme (GBM) treated with postoperative radio(chemo)therapy (PORT). For hypothesis generation, tumor miR expression by Agilent 8x15K human microRNA microarrays and survival data from 482 GBM patients of The Cancer Genome Atlas (TCGA cohort) were analyzed using Cox-PH models. Expression of candidate miRs with prognostic relevance (miR-221/222, miR-17-5p, miR-18a, miR-19b) was validated by qRT-PCR using Taqman technology on an independent validation cohort of GBM patients (n = 109) treated at Heidelberg University Hospital (HD cohort). In TCGA, 50 miRs showed significant association with survival. Among the top ranked prognostic miRs were members of the two miR families miR-221/222 and miR-17-92. Loss of miR-221/222 was correlated with improved prognosis in both cohorts (TCGA, HD) and was an independent prognostic marker in a multivariate analysis considering demographic characteristics (age, sex, Karnofsky performance index (KPI)), molecular markers (O-6-methylguanine-DNA methyltransferase (MGMT) methylation, IDH mutation status) and PORT as co-variables. The prognostic value of miR-17-92 family members was ambiguous and in part contradictory by direct comparison of the two cohorts, thus warranting further validation in larger prospective trials.

In this study, we investigated the role of serum exosomal miR-222 in obesity-related insulin resistance. Bioinformatics analyses showed that miR-222 levels were significantly upregulated in the white adipose tissue of obese patients with insulin resistance (GSE25402 dataset) and in serum samples from type 2 diabetes mellitus (T2DM) patients (GSE90028 dataset). Moreover, analysis of miRNA expression in adipose tissue-specific Dicer knockout mice (GitHub dataset) and diabetic model mice (GSE81976 and GSE85101 datasets), gonadal white adipose tissue (gWAT) was the main source of serum exosomal miR-222. MiR-222 levels were significantly elevated in the serum, serum exosomes and gWAT of mice fed a high-fat diet (HFD), and there was a corresponding downregulation of IRS1 and phospho-AKT levels in their liver and skeletal muscle tissues, which correlated with impaired insulin sensitivity and glucose intolerance. These effects were abrogated by surgically removing the gWAT from the HFD-fed mice. Thus, gWAT-derived serum exosomal miR-222 appears to promote insulin resistance in the liver and skeletal muscle of HFD-fed obese mice by suppressing IRS1 expression.

Sertoli cells are central and essential coordinators of spermatogenesis. Accumulating evidence has demonstrated that miRNAs participate in the regulation of Sertoli cell growth. However, the functions and the regulatory mechanisms of miRNAs in Sertoli cells of domestic animals remain largely unknown. Here we report that miR-222 overexpression repressed cell cycle progression and proliferation and promoted the apoptosis of immature porcine Sertoli cells, whereas miR-222 inhibition resulted in the opposite result. miR-222 directly targeted the 3′-UTR of the GRB10 gene and inhibited its mRNA abundance. An siRNA-induced GRB10 knockdown showed similar effects as did miR-222 overexpression on cell proliferation and apoptosis and further attenuated the role of miR-222 inhibition. Furthermore, both miR-222 overexpression and GRB10 inhibition repressed the phosphorylation of PI3K and AKT, the key elements of the PI3K/AKT signaling pathway, whereas GRB10 inhibition offsets the effects of the miR-222 knockdown. Overall, we concluded that miR-222 suppresses immature porcine Sertoli cell growth by targeting the GRB10 gene through inactivation of the PI3K/AKT signaling pathway. This study provides novel insights into the epigenetic regulation of porcine spermatogenesis by determining the fate of Sertoli cells.

Background Pulmonary arterial hypertension (PAH) is often characterized by cell proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). LncRNA cancer susceptibility candidate 2 (CASC2) has been revealed to be involved in PASMC injury in hypoxia-induced pulmonary hypertension. However, the exact molecular mechanisms whereby CASC2 regulates PASMC proliferation and migration are still incompletely understood. Methods The expression levels of CASC2, miR-222 and inhibitor of growth 5 (ING5) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. Wound healing assay was used to analyze cell migration ability. The relationship between miR-222 and CASC2 or ING5 was confirmed using bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation assay. Results CASC2 was down-regulated in hypoxia-induced PASMCs in a dose- and time-dependent manner. Functional experiments showed that CASC2 overexpression could reverse hypoxia-induced proliferation and migration of PASMCs. Bioinformatics analysis indicated that CASC2 acted as a competing endogenous RNA of miR-222, thereby regulating the expression of ING5, the downstream target of miR-222, in PASMCs. In addition, rescue assay suggested that the inhibition mediated by CASC2 of hypoxia-induced PASMC proliferation and migration could be attenuated by miR-222 inhibition or ING5 overexpression. Conclusion CASC2 attenuated hypoxia-induced PASMC proliferation and migration by regulating the miR-222/ING5 axis to prevent vascular remodeling and the development of PAH, providing a novel insight and therapeutic strategy for hypoxia-induced PAH.

Cisplatin has been widely used in the treatment of a various types of cancers including triple-negative breast cancer (TNBC) by damaging DNA and inducing apoptosis. However, its anti-cancer effects are often limited due to chemo-resistance, which is one of the main reasons causing cancer relapse and metastasis. To overcome resistance, cisplatin is often used in combination with other drugs or molecules. Our study found that the targeted inhibition of miR-221/222 in MDA-MB-231 cells promoted cisplatin-induced cell apoptosis, and increased the cell sensitivity to cisplatin in vitro. Much higher expression levels of miR-221/222 were detected in the cisplatin-resistant MDA-MB-231 cells and in cisplatin-resistant breast cancer patients. The combination chemotherapy of cisplatin with anti-miR-221/222 showed much higher efficiency in suppressing tumor growth in the mice transplanted with MDA-MB-231 cells. In addition, anti-miR-221 and anti-miR-222 showed synergetic effects on improving sensitivity to cisplatin in MDA-MB-231 cells. Suppression of SOCS1-STAT3-Bcl-2 pathway and activation of p53-Pten signaling both contribute to anti-miR-221/222-induced sensitivity to cisplatin in MDA-MB-231 cells. These findings suggest the potential of a novel approach for the combination chemotherapy of cisplatin with small non-coding RNA in treatment of human TNBC.

We analyzed five miRNA molecules (miR-221, miR-222, miR-146b, miR-21, miR-181b) in the plasma of patients with papillary thyroid cancer (PTC), nodular goiter (NG) and healthy controls (HC) and evaluated their diagnostic value for differentiation of PTC from NG and HC. Preoperative PTC plasma miRNA expression (n = 49) was compared with plasma miRNA in the HC group (n = 57) and patients with NG (n = 23). It was demonstrated that miR-221, miR-21 and miR-181b were overexpressed in preoperative PTC plasma samples compared to HC (p &lt, 0.0001, p &lt, 0.002, respectively). The upregulation in tumor tissue of these miRNAs was consistent with The Cancer Genome Atlas Thyroid Carcinoma dataset. A significant decrease in miR-21, miR-221, miR-146b and miR-181b expression was observed in the plasma of PTC patients after total thyroidectomy (p = 0.004, p = 0.001, p = 0.03, p = 0.036, respectively). The levels of miR-222 were significantly higher in the preoperative PTC compared to the NG group (p = 0.004). ROC curve (receiver operating characteristic curve) analysis revealed miR-222 as a potential marker in distinguishing PTC from NG (AUC 0.711, p = 0.004). In conclusion, circulating miR-222 profiles might be useful in discriminating PTC from NG.

Background Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts.Methods Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers.Results Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP.Conclusion Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion.

To identify molecular markers that can accurately predict aggressive tumor behavior at the time of surgery, a propensity-matching score analysis of archived specimens yielded two similar datasets of DTC patients (with and without RAI). Bioinformatically selected microRNAs were quantified by qRT-PCR. The risk score was generated using Cox regression and assessed using ROC, C-statistic, and Brier-score. A predictive Bayesian nomogram was established. External validation was performed, and causal network analysis was generated. Within the eight-year follow-up period, progression was reported in 51.5% of cases, of these, 48.6% had the T1a/b stage. Analysis showed upregulation of miR-221-3p and miR-222-3p and downregulation of miR-204-5p in 68 paired cancer tissues (p &lt, 0.001). These three miRNAs were not differentially expressed in RAI and non-RAI groups. The ATA risk score showed poor discriminative ability (AUC = 0.518, p = 0.80). In contrast, the microRNA-based risk score showed high accuracy in predicting tumor progression in the whole cohorts (median = 1.87 vs. 0.39, AUC = 0.944) and RAI group (2.23 vs. 0.37, AUC = 0.979) at the cutoff &gt, 0.86 (92.6% accuracy, 88.6% sensitivity, 97% specificity) in the whole cohorts (C-statistics = 0.943/Brier = 0.083) and RAI subgroup (C-statistic = 0.978/Brier = 0.049). The high-score group had a three-fold increased progression risk (hazard ratio = 2.71, 95%CI = 1.86–3.96, p &lt, 0.001) and shorter survival times (17.3 vs. 70.79 months, p &lt, 0.001). Our prognostic microRNA signature and nomogram showed excellent predictive accuracy for progression-free survival in DTC.

Background/Aims: Aberrant vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of pulmonary artery hypertension (PAH). Dysregulated microRNAs (miRNAs, miRs) have been implicated in the progression of PAH. miR-222 has a pro-proliferation effect on VSMCs while it has an anti-proliferation effect on vascular endothelial cells (ECs). As the biological function of a single miRNA could be cell-type specific, the role of miR-222 in pulmonary artery smooth muscle cell (PASMC) proliferation is not clear and deserves to be explored. Methods: PASMCs were transfected with miR-222 mimic or inhibitor and PASMC proliferation was determined by Western blot for PCNA, Ki-67 and EdU staining, and cell number counting. The target genes of miR-222 including P27 and TIMP3 were determined by luciferase assay and Western blot. In addition, the functional rescue experiments were performed based on miR-222 inhibitor and siRNAs to target genes. Results: miR-222 mimic promoted PASMC proliferation while miR-222 inhibitor decreased that. TIMP3 was identified to be a direct target gene of miR-222 based on luciferase assay. Meanwhile, P27 and TIMP3 were up-regulated by miR-222 inhibitor and down-regulated by miR-222 mimic. Moreover, P27 siRNA and TIMP3 siRNA could both attenuate the anti-proliferation effect of miR-222 inhibitor in PASMCs, supporting that P27 and TIMP3 are at least partially responsible for the regulatory effect of miR-222 in PASMCs. Conclusion: miR-222 promotes PASMC proliferation at least partially through targeting P27 and TIMP3.

Background: miR-222 is one of the most consistently overexpressed miRNAs in papillary thyroid carcinoma (PTC). Previous studies demonstrated that miR-222 overexpression conferred high-risk features in PTC patients, suggesting its value in risk-stratification. However, studies in term of miR-222's utility on stratifying PTCs are lacking. Methods: One hundred patients including 10 with multinodular goiter and 90 with PTC were enrolled. Formalin-fixed paraffin-embedded samples were exploited for miR-222 quantitative reverse transcriptase- polymerase chain reaction (RT-PCR) analysis. Correlations between miR-222 expression and different clinicopathological features, Tumor-node-metastasis (TNM) staging and ATA risk level were analyzed. Results: miR-222 expression of the PTC group was significantly higher than that of the goiter group (P

Resistance to doxorubicin (DOX) is the most common clinical problem in breast cancer therapy, and the underlying molecular mechanism remains to be investigated. MicroRNAs (miRNAs) exhibit important regulatory functions in various malignant tumors including breast cancer. The aim of the present study was to find the relationship between miR-222 and DOX resistance. We found that miR-222 was highly expressed in patients’ serum and DOX-resistant cell line MCF-7-R and that miR-222 could promote proliferation and migration of breast cancer cells. Our results also showed that inhibition of miR-222 in MCF-7-R significantly increased Bcl-2 interacting mediator (Bim) expression both in mRNA and protein levels by using quantitative real-time PCR (qRT-PCR) and Western blot. MTT and flow cytometry suggested that lower expressed miR-222 enhanced apoptosis and decreased IC50 of MCF-7-R cells. Conversely, in MCF-7 cells transfected with miR-222 mimics, up-regulation of miR-222 was associated with decreased Bim level accompanied by less apoptosis and higher IC50. Moreover, miR-222 inhibitors reversed DOX resistance via miR-222-Bim-caspase pathway. Collectively, these data first elucidated that miR-222 could function as an oncogene and was able to reduce the sensitivity of breast cancer cells to DOX through miR-222-Bim-caspase pathway, which provided a potential target to increase DOX sensitivity in clinical breast cancer treatment.

It has been demonstrated that miR‑222 is upregulated in human intervertebral disc (IVD) degeneration tissues; however, the underlying mechanisms remain unclear. In this study, we aimed to elucidate the mechanisms of action of miR‑222 in IVD tissues. Nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) to simulate IVD degeneration. The expression level of miR‑222 was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) in cells and tissues. Cell apoptosis was analyzed by flow cytometry. Additionally, western blot analysis was used to determine the levels of Toll‑like receptor 4 (TLR4), Iκβ‑alpha (IκBα) and p65. Interleukin (IL)‑1β, tumor necrosis factor‑α (TNF‑α) and IL‑6 protein expression levels were determined by enzyme‑linked immunosorbent assay (ELISA). The target gene of miR‑222 was determined by TargetScan7.2 and dual luciferase reporter gene analysis. Western blot analysis and RT‑qPCR were used to determine the mRNA and protein levels of tissue inhibitor of metalloproteinase 3 (TIMP3). The mRNA expression level of miR‑222 was found to be increased in IVD tissues and in LPS‑stimulated cells, and its expression was positively associated with the clinical MRI grade. In vitro, apoptosis was promoted/inhibited by miR‑222 mimics/inhibitors. Transfection with miR‑222 mimics/inhibitors significantly increased/decreased the production of TNF‑α, IL‑1β and IL‑6 and suppressed/enhanced collagen II and aggrecan expression. The protein levels of TLR4, p‑IκΒα and p‑p65 were upregulated/downregulated by transfection with the mimics/inhibitors. In addition, it was demonstrated that TIMP3 was a direct target gene of miR‑222, and was negatively regulated by miR‑222 in NP cells. The silencing of TIMP3 reversed the inhibitory effects of miR‑222 inhibitor on cell apoptosis, which was induced by LPS. Thus, on the whole, the findings of this study demonstrate that miR‑222 functions as a promoter of IVD development, partly via the regulation of TIMP3.

Background: A wealth of evidence has shown that microRNAs (miRNAs) can modulate specific genes, increasing our knowledge on the fine-tuning regulation of protein expression. miR-221 and miR-222 have been frequently identified as deregulated across different cancer types; however, their prognostic significance in cancer remains controversial. In view of these considerations, we performed an updated systematic review and meta-analysis of published data investigating the effects of miR-221/222 on overall survival (OS) and other secondary outcomes among cancer patients. A systematic search of PubMed, Web of Knowledge, and Cochrane Library databases was performed. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were used to assess the strength of association. Results: Fifty studies, analyzing 6086 patients, were included in the systematic review. Twenty-five studies for miR-221 and 17 studies for miR-222 which assessed OS were included in the meta-analysis. High expression of miR-221 and miR-222 significantly predicted poor OS (HR: 1.48, 95% CI: 1.14−1.93, p = 0.003 and HR: 1.90, 95% CI: 1.43−2.54, p < 0.001, respectively). Subgroup analysis revealed that the finding on miR-221 was not as robust as the one on miR-222. Furthermore, high miR-222 expression was also associated with worse progression-free survival and disease-free survival pooled with recurrence-free survival. Conclusions: The meta-analysis demonstrated that high expression of miR-222 is associated with poor prognosis in cancer patients, whereas the significance of miR-221 remains unclear. More work is required to fully elucidate the role of miR-221 and miR-222 in cancer prognosis, particularly in view of the limitations of existing results, including the significant heterogeneity and limited number of studies for some cancers.

We analyzed miR-146b, miR-21, miR-221, miR-21, and miR-181b in formalin fixed paraffin-embedded papillary thyroid carcinoma (PTC) tissue samples of 312 individuals and evaluated their expression relationship with clinicopathological parameters. A higher expression of miR-21 was related to unifocal lesions (p &lt, 0.011) and autoimmune thyroiditis (0.007). miR-221, miR-222 expression was higher in the PTC tissue samples with extrathyroidal extension (p = 0.049, 0.003, respectively). In a group of PTC patients with pT1a and pT1b sized tumors, the expression of miR-146b, miR-21, miR-221, and miR-222 in PTC tissue samples was lower than in patients with pT2, pT3, and pT4 (p = 0.032, 0.0044, 0.003, 0.001, 0.001, respectively). Patients with lymph node metastases had higher expression of miR-21, -221, -222, and -181b (p &lt, 0.05). A high expression of miR-146b, miR-21, miR-221 panel was associated with decreased overall survival (OS) (Log rank p = 0.019). Univariate analysis revealed that presence of metastatic lymph nodes and high expression of miR-146b, miR-21, and miR-221 panels were associated with increased hazard of shorter OS. After multivariate analysis, only sex (male) and age (≥55 years) emerged as independent prognostic factors associated with shorter OS (HR 0.28 (95% CI 0.09–0.86) and HR 0.05 (95% CI 0.01–0.22), respectively). In conclusion, 5 analyzed miRs expression have significant relations to clinicopathologic parameters so further investigations of these molecules are expedient while searching for prognostic PTC biomarkers.

Summary MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-β. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

Background: MicroRNAs play a crucial role in the regulation of pathological cardiac remodeling and heart failure. Previously, we found that overexpression of miR-221 induces heart failure in mice. The miR-222 and miR-221 share the same gene cluster, however, the role of miR-222 in the regulation of cardiac function remained ill-defined. Methods and Results: Transgenic mice with cardiac-specific expression of miR-222 (Tg-miR-222) mice were generated. The Tg-miR-222 mice developed significantly enlarged hearts at 4 weeks of age. Transthoracic echocardiograph data indicated that the hearts of Tg-miR-222 mice exhibited an increased left ventricular end-diastolic internal diameter and decreased fractional shortening. We observed that the LC3-II in Tg-miR-222 mice was decreased accompanied with the upregulation of p62, indicating the autophagy inhibition in the hearts of Tg-miR-222 mice. The mTOR pathway, a negative regulator of autophagy, was activated in the hearts of Tg-miR-222 mice. The expression of p27 was downregulated by miR-222 overexpression. Conclusion: Our data indicate that miR-222 overexpression induces heart failure in mice. The downregulation of p27 and the activation of mTOR pathway may be involved in miR-222-induced heart failure and autophagy inhibition. Thus, targeting miR-222 expression may be a therapeutic strategy against pathological cardiac remodeling.

High levels of arsenic in drinking water, soil, and air are associated with the higher incidences of several kinds of cancers worldwide, but the mechanism is yet to be fully discovered. Recently, a number of evidences show that dysregulation of microRNAs (miRNAs) induces carcinogenesis. In this study, we found miR-222 was upregulated in arsenic-transformed human lung epithelial BEAS-2B cells (As-T cells). Anti-miR-222 inhibitor treatment decreased cell proliferation, migration, tube formation, and induced apoptosis. In addition, anti-miR-222 inhibitor expression decreased tumor growth in vivo. We also found that inhibition of miR-222 induced the expression of its direct targets ARID1A and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and activated apoptosis of As-T cells in part through ARID1A downregulation. These results indicate that miR-222 plays an important role in arsenic-induced tumor growth.

Relationship between the expression of miR-222 and uterine cancer was investigated to explore its prognostic value. A total of 66 patients with uterine cancer diagnosed by pathological examination in Dongying People's Hospital were enrolled from March 2014 to October 2016. Uterine cancer and adjacent tissues were collected, and the expression of miR-222 in the tissues was detected by stem-loop RT-PCR. The relationship between miR-222 expression and various clinicopathological features of uterine cancer was analyzed. All the patients were followed up to record the survival conditions. The results revealed that stem-loop RT-PCR method could specifically amplify miR-222. The expression of miR-222 in uterine cancer tissues was significantly upregulated compared with that in adjacent tissues (p0.05). There was a significant negative correlation between the expression of miR-222 and the survival of patients with uterine cancer. In conclusion, the expression of miR-222 in uterine cancer tissues was significantly upregulated in uterine cancer and negatively correlated with prognosis. miR-222 may play a pivotal role in the development and progression of uterine cancer. It is expected that miR-222 will be an indicator and target for the treatment and prognosis of uterine cancer.

Ovarian carcinoma is the most lethal of the gynecologic malignancies worldwide. Increasing evidence suggests dysfunction of microRNAs (miRNAs) plays an important role in human cancers. The function of miR-222 was detected in ovarian carcinoma to verify the regulation of phosphatase and tensin homolog (PTEN) by miR-222. miR-222 expression in ovarian carcinoma tissues and cell lines were examined using RT-qPCR. Transwell assay was used to detect miR-222 effects on ovarian carcinoma cell migration and invasion. Western blot analysis and luciferase assays were performed to validate PTEN as miR-222 targets. miR-222 expression was upregulated in ovarian carcinoma tissues and three cell lines (A2780, SKOV-3 and OVCAR-3). Ectopic overexpression of miR-222 in ovarian carcinoma cells was sufficient to promote invasion and migration. PTEN acted as a direct target of miR-222. Overexpression of PTEN inhibited human ovarian carcinoma cell migration and invasion. In summary, our findings suggest that miR-222 plays an important role in promoting ovarian carcinoma cell invasion and migration and miR-222/PTEN may be a novel therapeutic target of miRNA-mediated promotion of cell invasion and migration in ovarian carcinoma.

Background: Our present study of the microRNA (miRNA) expression signature in castration-resistant prostate cancer (CRPC) revealed that the clustered miRNAs microRNA-221 (miR-221) and microRNA-222 (miR-222) are significantly downregulated in cancer tissues. The aim of this study was to investigate the functional roles of miR-221 and miR-222 in prostate cancer (PCa) cells. Methods: A CRPC miRNA signature was constructed by PCR-based array methods. Functional studies of differentially expressed miRNAs were analysed using PCa cells. The association between miRNA expression and overall survival was estimated by the Kaplan–Meier method. In silico database and genome-wide gene expression analyses were performed to identify molecular targets regulated by the miR-221/222 cluster. Results: miR-221 and miR-222 were significantly downregulated in PCa and CRPC specimens. Kaplan–Meier survival curves showed that low expression of miR-222 predicted a short duration of progression to CRPC. Restoration of miR-221 or miR-222 in cancer cells revealed that both miRNAs significantly inhibited cancer cell migration and invasion. Ecm29 was directly regulated by the miR-221/222 cluster in PCa cells. Conclusions: Loss of the tumour-suppressive miR-221/222 cluster enhanced migration and invasion in PCa cells. Our data describing targets regulated by the tumour-suppressive miR-221/222 cluster provide insights into the mechanisms of PCa and CRPC progression.

miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associated with metastasis and poor prognosis in breast cancer, including the triple-negative subtype (TNBC). Modification of components of uPAS and involved miRNAs may contribute to targeted therapy for breast cancer patients. miR-221-/-222-overexpressing or miR-221-depleted cells were employed for qRT-PCR and Western blots to show associations of uPAR with miR-221/-222. To substantiate direct targeting of miR-221/-222 within 3' UTR of the uPAR isoform 2, in silico analysesand in vitro assays were conducted. Significant associations between miR-221 and uPAR isoform 2 expressions were observed at the mRNA and protein levels in breast cancer cells representing TNBC. For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222. Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC. These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222. By targeting uPAR isoforms and/or miRNA-221/-222, the diagnosis and therapy of breast cancer, in particular in TNBC, could be significantly improved.

Human immunodeficiency virus type-1 (HIV-1) infection of monocyte/macrophages is modulated by the levels of entry receptors cluster of differentiation 4 (CD4) and C-C chemokine receptor type 5 (CCR5), as well as by host antiviral restriction factors, which mediate several post-entry blocks. We recently identified two microRNAs, miR-221 and miR-222, which limit HIV-1 entry during infection of monocyte-derived macrophages (MDMs) by down-regulating CD4 expression. Interestingly, CD4 is also down-regulated during the differentiation of monocytes into macrophages. In this study, we compared microRNA expression profiles in primary monocytes and macrophages by RNAseq and found that miR-221/miR-222 are enhanced in macrophages. We took advantage of the monocytic THP-1 cell line that, once differentiated, is poorly susceptible to HIV-1. Accordingly, we found that CD4 levels are very low in THP-1 differentiated cells and that this down-regulation of the virus receptor is the result of miR-221/miR-222 up-regulation during differentiation. We thus established a THP-1 cell line stably expressing a modified CD4 (THP-1-CD4R) that is not modulated by miR-221/miR-222. We show that in contrast to parental THP-1, this line is productively infected by HIV-1 following differentiation, sustaining efficient HIV-1 CD4-dependent replication and spread. This new THP-1-CD4R cell line represents a useful tool for the study of HIV-1-macrophage interactions particularly in contexts where spreading of viral infection is necessary.

Background: Selective serotonin reuptake inhibitor (SSRI) antidepressant drugs are the first-line of treatment for major depressive disorder (MDD) but are effective in

Objective Today, many single nucleotide polymorphisms in microRNA genes are known to alter the microRNA expression levels or processing causing susceptibility of several human diseases. The present study aimed to investigate the association of microRNA-146a (rs2910164) and microRNA-222 (rs2858060) polymorphisms with susceptibility to polycystic ovary syndrome (PCOS) in an Iranian population. Materials and methods This case–control study was performed on 205 patients with PCOS and 205 normal women as the control group. After DNA extraction, Tetra-amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) was used to detect the polymorphisms. The association between genotypes and the risk of PCOS was examined by odds ratios (OR) and 95% of confidence intervals (CIs). Results Our results showed that there are significant differences in CG genotype frequencies between case and control groups regarding miR-146a rs2910164 polymorphism (OR = 2.03, CI = 1.3–3, P = 0.001). In a dominant model for the C allele, CC + CG genotypes were associated with PCOS risk (OR = 2, 95% CI = 1.3–2.9, P = 0.001) and the C allele increased the risk of PCOS (OR = 1.6, 95% CI = 1.1–2.1, P = 0.004). Furthermore, a positive association was observed between miR-222 CG genotype and the risk of PCOS (OR = 2.2, 95% CI = 1.1–4.1, P = 0.02). These results were evident after adjustment for age and body mass index. Conclusion The present results suggest that the miR-146a rs2910164 and miR-222 rs2858060 polymorphisms are associated with an increased risk of PCOS. Therefore, both polymorphisms could play an important role as a genetic risk factor for development of PCOS in the Iranian population.

microRNAs have been shown to play critical roles in regulating the chemosensitivity of cancer cells. As a member of the oncogenic miRNAs (oncomiRs), miR-222 has been reported to drive the oncogenesis of many types of malignancies. However, little is known concerning the specific role of miR-222 in human oral squamous cell carcinoma (OSCC). The present study explored the role and mechanism of miR-222 in increasing the expression of p53 up-regulated modulator of apoptosis (PUMA) and enhancing the sensitivity of OSCC to cisplatin (CDDP). Results showed that antisense (As)-miR-222 inhibits the expression of miR-222. In contrast, PUMA was dramaticallyup-regulated. IC50 values were significantly decreased in cells treated with As-miR-222 combined with CDDP, to a greater extent than in cells treated with CDDP alone. Furthermore, As-miR-222 enhanced apoptosis and inhibited the invasiveness of UM1 cells. Analysis of the above data suggested that, in UM1 cells, there might be a regulatory loop between miR-222 and PUMA, and that miR-222 inhibition increased the chemosensitivity to CDDP. These findings demonstrated that down-regulation of miR-222 could enhance the chemosensitivity of human OSCC cells to CDDP, and that the combination of As-miR-222 and CDDP could be an effective therapeutic strategy by boosting the expression of PUMA for controlling the growth of OSCC.

Objective: To determine if changes in levels of serum microRNAs (miRNAs) were seen preceding the diagnosis of AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Design: Serum miRNA levels were compared in 3 subject groups from the Multicenter AIDS Cohort Study: HIV-negative men (n = 43), HIV-positive men who did not develop NHL (n = 45), and HIV-positive men before AIDS-NHL diagnosis (n = 62, median time before diagnosis, 8.8 months). Methods: A total of 175 serum-enriched miRNAs were initially screened to identify differentially expressed miRNAs among these groups and the results validated by quantitative polymerase chain reaction. Receiver-operating characteristic analysis was then performed to assess biomarker utility. Results: Higher levels of miR-21 and miR-122, and a lower level of miR-223, were able to discriminate HIV-infected from the HIV-uninfected groups, suggesting that these miRNAs are biomarkers for HIV infection but are not AIDS-NHL specific. Among the HIV-infected groups, a higher level of miR-222 was able to discriminate diffuse large B-cell lymphoma (DLBCL) and primary central nervous system lymphoma (PCNSL) subjects from HIV-infected subjects who did not develop NHL, with area under the receiver-operating characteristic curve of 0.777 and 0.792, respectively. At miR-222 cutoff values of 0.105 for DLBCL and 0.109 for PCNSL, the sensitivity and specificity were 75% and 77%, and 80% and 82%, respectively. Conclusions: Altered serum levels of miR-21, miR-122, and miR-223 are seen in HIV-infected individuals. Higher serum level of miR-222 has clear potential as a serum biomarker for earlier detection of DLBCL and PCNSL among HIV-infected individuals. Copyright © 2014 by Lippincott Williams & Wilkins.

The miR-221/222 cluster has been demonstrated to function as oncomiR in human cancers. miR-221/222 promotes epithelial-to-mesenchymal transition (EMT) and confers tamoxifen resistance in breast cancer. However, the effects and mechanisms by which miR-221/222 regulates breast cancer aggressiveness remain unclear. Here we detected a much higher expression of miR-221/222 in highly invasive basal-like breast cancer (BLBC) cells than that in non-invasive luminal cells. A microRNA dataset from breast cancer patients indicated an elevated expression of miR-221/222 in BLBC subtype. S-phase entry of the cell cycle was associated with the induction of miR-221/222 expression. miRNA inhibitors specially targeting miR-221 or miR-222 both significantly suppressed cellular migration, invasion and G1/S transition of the cell cycle in BLBC cell types. Proteomic analysis demonstrated the down-regulation of two tumor suppressor genes, suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibit 1B (CDKN1B), by miR-221/222. This is the first report to reveal miR-221/222 regulation of G1/S transition of the cell cycle. These findings demonstrate that miR-221/222 contribute to the aggressiveness in control of BLBC.

// Prashant K. Singh 1 , Leah Preus 2 , Qiang Hu 3 , Li Yan 3 , Mark D. Long 1 , Carl D. Morrison 4 , Mary Nesline 2 , Candace S. Johnson 1 , Shahriar Koochekpour 5 , Manish Kohli 6 , Song Liu 3 , Donald L. Trump 7 , Lara E Sucheston-Campbell 2,* and Moray J. Campbell 1,* 1 Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 2 Department of Cancer Prevention & Control, Roswell Park Cancer Institute, Buffalo, NY 3 Department of Biostatistics & Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY 4 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 5 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 6 Department of Medical Oncology, Mayo Clinic, Rochester, MN 7 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY * The authors share position as senior and corresponding authors Correspondence: Moray J. Campbell, email: // Lara E Sucheston-Campbell, email: // Keywords : Prostate cancer, microRNA, biochemical progression, miR-103, miR-125b, miR-222, cancer epigenetics Received : January 13, 2014 Accepted : February 13, 2014 Published : February 13, 2014 Abstract We aimed to identify microRNA (miRNA) expression patterns in the serum of prostate cancer (CaP) patients that predict the risk of early treatment failure following radical prostatectomy (RP). Microarray and Q-RT-PCR analyses identified 43 miRNAs as differentiating disease stages within 14 prostate cell lines and reflectedpublically available patient data. 34 of these miRNA were detectable in the serum of CaP patients. Association with time to biochemical progression was examined in a cohort of CaP patients following RP. A greater than two-fold increase in hazard of biochemical progression associated with altered expression of miR-103, miR-125b and miR-222 ( p

Background: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. Methods: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT–PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. Results: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. Conclusion: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.

BACKGROUND: Gastrointestinal stromal tumours (GIST) are characterised by high expression of KIT and ETV1, which cooperate in GIST oncogenesis. Our aim was to identify microRNAs that are deregulated in GIST, have a role in GIST pathogenesis, and could potentially be used as therapeutic tool.METHODS: Differentially expressed microRNAs between primary GIST (n=50) and gastrointestinal leiomyosarcomas (GI-LMS, n=10) were determined using microarrays. Selected microRNA mimics were transfected into GIST-882 and GIST-T1 cell lines to study the effects of microRNA overexpression on GIST cells. Luciferase reporter assays were used to establish regulation of target genes by selected microRNAs.RESULTS: MiR-17-92 and miR-221/222 cluster members were significantly (PCONCLUSION: MicroRNAs that may have an essential role in GIST pathogenesis were identified, in particular miR-17/20a/222 that target KIT and ETV1. Delivering these microRNAs therapeutically could hold great potential for GIST management, especially in imatinib-resistant disease.

Epithelial ovarian cancer (EOC) is the leading cause of female reproductive system cancer mortality in females. The majority of cases of ovarian carcinomas are not identified until a late stage. Identifying the molecular changes that occur during the development and progression of ovarian cancer is an urgent requirement. MicroRNAs (miRNAs) have been identified as gene expression regulators that induce mRNA degradation or translation blockade through pairing to the 3′ untranslated region (3-‘UTR) of the target mRNAs. In the present study, miR-222 was observed to be frequently upregulated in ovarian cancer. miR-222 upregulation induced an enhancement of ovarian cancer cell proliferation potential, possibly by downregulating its target, P27Kip1. A bioinformatic analysis showed that the 3′-UTR of the P27Kip1 mRNA contained a highly-conserved putative miR-222 binding site. Luciferase reporter assays demonstrated that P27Kip1 was a direct target of miR-222. Consistently, there was an inverse correlation between the P27Kip1 and miR-222 expression levels in the ovarian cancer cell lines and tissues. Overall, the present results suggest that miR-222 upregulation in human ovarian cancer may promote ovarian cancer cell proliferation during ovarian carcinogenesis.

Background miR-222 has been reported to be overexpressed in colorectal cancer and it influences cancer cell proliferation, drug resistance and metastasis. However, the underlying molecular mechanism of miR-222 in colorectal cancer cell invasion and migration has not been thoroughly elucidated to date. Methods The cell cycle distribution and apoptosis were assessed by flow cytometry. Cell migration and invasion were analyzed by Transwell assays. The possible target gene of miR-222 was searched and identified by bioinformatics, dual luciferase reporter assay and western blot analysis. The siRNA method was used to confirm the function of the target gene. Results Overexpression of miR-222 effectively promotes migration and invasion of colorectal cancer (CRC) cells in vitro. Bioinformatics and the dual luciferase reporter assay revealed that miR-222 specifically targeted the 3′-UTR of melanoma inhibitory activity member 3 (MIA3), down-regulating its expression at the protein level. Inhibition of MIA3 by siRNA enhanced the migration and invasion of CRC cell lines. Conclusions Our study showed that miR-222 enhances the migration and invasion in CRC cells, primarily by down-regulation of MIA3. Electronic supplementary material The online version of this article (doi:10.1186/s12935-017-0447-1) contains supplementary material, which is available to authorized users.

Background : Micro-RNAs (miRNAs) are expressed in a tissue-specific manner and are known to demonstrate differential expression even among the various subtypes of a given tumor. This differential expression has been harnessed successfully in the development of diagnostic assays for various malignant tumors. These assays have been found to be relevant and of value as additional diagnostic tools even among thyroid tumors, particularly with regard to thyroid carcinomas of follicular morphology. Materials and Methods : A limited set of miRNA have been assessed as part of this study in an effort to use minimal number of miRNA markers (miR-187, miR-221, miR-222, and miR-224) to differentiate the benign from the malignant thyroid tumors using miRNA derived from paraffin embedded material. Results : While miR-221 and miR-222 were found to provide good accuracy as individual markers (86% and 84%), a combination of the two provided slightly better accuracy (91%). Both miR-221 and 222 were able to significantly differentiate malignant tumors from the benign samples (P< 0.001) individually and as a combination of markers. However, inclusion of miR-187 and miR-224 in the panel did not provide any additional benefit. Conclusion : While a combination of miR-221 and 222 when used in a diagnostic panel could provide fairly good accuracy additional markers may need to be investigated to augment their diagnostic utility.

The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR‑221 and miR‑222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR‑221 and anti-miR‑222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222.

Purpose The microRNA-221/222 (miR-221/222) gene cluster has been reported to be associated with the promotion of epithelial-mesenchymal transition (EMT), downregulation of estrogen receptor-α, and tamoxifen resistance in breast cancer. We studied the expression of miR-222 in human breast cancer samples to analyze its relationship with clinicopathologic features of the tumor, including estrogen receptor status, expression of EMT markers, and clinical outcomes. Methods Quantitative real-time polymerase chain reaction was performed to detect the expression of miR-222 in 197 invasive breast cancers. Expression of EMT markers (vimentin, smooth muscle actin, osteonectin, N-cadherin, and E-cadherin) was evaluated using immunohistochemistry. Results High miR-222 levels were associated with high T stage, high histologic grade, high Ki-67 proliferation index, and HER2 gene amplification. Its expression was significantly higher in the luminal B and human epidermal growth factor receptor 2-positive (HER2+) subtypes than in the luminal A and triple-negative subtypes. In the hormone receptor-positive subgroup, there was a significant negative correlation between miR-222 and estrogen receptor expression, and miR-222 expression was associated with EMT marker expression. In the group as a whole, high miR-222 expression was not associated with clinical outcome. However, subgroup analyses by hormone receptor status revealed that high miR-222 expression was a poor prognostic factor in the hormone receptor-positive subgroup, but not in the hormone receptor-negative subgroup. Conclusion This study showed that miR-222 is associated with down-regulation of the estrogen receptor, EMT, and tumor progression in hormone receptor-positive breast cancer, indicating that miR-222 might be associated with endocrine therapy resistance and poor clinical outcome in hormone receptor-positive breast cancer.

Background Even though increasing evidences on miRNA involvement in human pathological responses, the distinct roles and related mechanisms of miRNAs in the pathology of osteoarthritis (OA) are not yet fully understood. Method RNA levels or protein levels of Apoptotic genes, HDACs, MMP-13, and miRNAs in human chondrocytes isolated from normal biopsy sample and OA cartilages were analyzed by real-time PCR or western blotting. Exogenous modulation of miR-222 level was performed using delivery of its specific precursor or specific inhibitor and target validation assay was applied to identify its potent target. In vivo study using DMM mice model was performed and assessed the degree of cartilage degradation. Results According to miRNA profiling, miR-222 was significantly down-regulated in OA chondrocytes. Over-expression of miR-222 significantly suppressed apoptotic death by down-regulating HDAC-4 and MMP-13 level. Moreover, 3′-UTR reporter assays showed that HDAC-4 is a direct target of miR-222. The treatment of chondrocytes with the HDAC inhibitor, trichostatin A (TSA), suppressed MMP-13 protein level and apoptosis, whereas the over-expression of HDAC-4 displayed opposite effects. The introduction of miR-222 into the cartilage of medial meniscus destabilized mice significantly reduced cartilage destruction and MMP-13 level. Conclusion Taken together, our data suggest that miR-222 may be involved in cartilage destruction by targeting HDAC-4 and regulating MMP-13 level., Highlights • MiR-222 controls OA pathogenesis by targeting HDAC-4. • HADC-4 regulated by miR-222 modulates MMP-13 expression during cartilage destruction. • Our study indicates the possibility that miR-222 could act as a protective factor against OA.

Little is known about the potential role of microRNAs (miRNAs) in the carcinogenesis of gastric cancer induced by Helicobacter pylori (H. pylori). Here, we showed that microRNA-222 (miR-222) was up-regulated in H. pylori-infected gastric mucosa and gastric cancer. Ectopic expression of miR-222 promoted cell proliferation and colony formation in vitro. Mechanistically, we identified RECK as a novel target of miR-222, and also confirmed their relationship by the inverse correlation of mRNA expression ex vivo. Furthermore, we found that RNA interference silencing of RECK can mimic the oncogenic effects of miR-222. Collectively, H. pylori may function as an initiator in the process of carcinogenesis by up-regulating miR-222, which further participates in the progression of cancer by promoting proliferation and inhibiting RECK.

BackgroundMiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity.Methodology/principal findingsHere we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression.Conclusions/significanceThese findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.