The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer

The incidence of oral squamous cell carcinoma (OSCC) is rising rapidly in developed countries, posing a growing challenge due to the poor management of this type of malignancy at present. In this study, we profiled tumor suppressive microRNAs (miRNAs) that are silenced by DNA hypermethylation in OSCC using a function-based screening approach. This approach employed a cell proliferation assay for 327 synthetic miRNAs in two OSCC cell lines. Among the 110 miRNAs identified in this set that exhibited inhibitory properties, we compared DNA methylation and expression status in a wider panel of OSCC cell lines and primary tumor tissues, resulting in the identification of miR-218 and miR-585 as functionally significant miRNA genes that are frequently silenced in OSCC by DNA hypermethylation. Ectopic expression of miR-218 and miR-585 in OSCC cells lacking endogenous expression reduced cell growth in part through caspase-mediated apoptosis. Notably, miR-218 reduced levels of the rapamycin-insensitive component of mTOR, Rictor, in a manner associated with a suppression of Akt S473 phosphorylation. Together our findings define miR-585 as a tumor suppressive function that is often epigenetically silenced in OSCC, and they identify Rictor as a novel target of miR-218, suggesting that activation of the mTOR-Akt signaling pathway induced by Rictor contributes centrally to oral carcinogenesis.     

CpG island methylation status of miRNAs in esophageal squamous cell carcinoma

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5' regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148-59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2'-deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253-66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.     

Down-regulation of miR-517a and miR-517c promotes proliferation of hepatocellular carcinoma cells via targeting Pyk2

Growing evidence indicates that some tumor suppressive miRNAs are subject to epigenetic modifications during carcinogenesis. Here, we found that a large miRNA cluster of C19MC was upregulated in HCC cells after combined treatment with DNA methylation inhibitor and histone deacetylase inhibitor. MiR-517a and miR-517c were strikingly different from the remaining 41 miRNAs in C19MC. Ectopic expression of MiR-517a and miR-517c inhibited cell proliferation by blocking G2/M transition, whereas down-regulation of miR-517a and miR-517c facilitated cell growth. We further showed Pyk2 is a target of miR-517a and miR-517c and both the miRNAs are downregulated in HCC samples. These data collectively suggest that down-regulation of both miR-517a and miR-517c contribute to HCC development through regulating Pyk2.     

MicroRNA expression signature of oral squamous cell carcinoma: functional role of microRNA-26a/b in the modulation of novel cancer pathways

Background:

MicroRNAs (miRNAs) have been shown to play major roles in carcinogenesis in a variety of cancers. The aim of this study was to determine the miRNA expression signature of oral squamous cell carcinoma (OSCC) and to investigate the functional roles of miR-26a and miR-26b in OSCC cells.

Methods:

An OSCC miRNA signature was constructed by PCR-based array methods. Functional studies of differentially expressed miRNAs were performed to investigate cell proliferation, migration, and invasion in OSCC cells. In silico database and genome-wide gene expression analyses were performed to identify molecular targets and pathways mediated by miR-26a/b.

Results:

miR-26a and miR-26b were significantly downregulated in OSCC. Restoration of both miR-26a and miR-26b in cancer cell lines revealed that these miRNAs significantly inhibited cancer cell migration and invasion. Our data demonstrated that the novel transmembrane TMEM184B gene was a direct target of miR-26a/b regulation. Silencing of TMEM184B inhibited cancer cell migration and invasion, and regulated the actin cytoskeleton-pathway related genes.

Conclusions:

Loss of tumour-suppressive miR-26a/b enhanced cancer cell migration and invasion in OSCC through direct regulation of TMEM184B. Our data describing pathways regulated by tumour-suppressive miR-26a/b provide new insights into the potential mechanisms of OSCC oncogenesis and metastasis.     

Genome-wide pharmacologic unmasking identifies tumor suppressive microRNAs in multiple myeloma

Epigenetic alterations have emerged as an important cause of microRNA (miRNA) deregulation. In Multiple Myeloma (MM), a few tumor suppressive miRNAs silenced by DNA hypermethylation have been reported, but so far there are few systemic investigations on epigenetically silenced miRNAs. We conducted genome-wide screening for tumor suppressive miRNAs epigenetically silenced in MM. Four Human MM Cell lines were treated with demethylating agent 5′azacytidine (5′aza). Consistently upregulated miRNAs include miR-155, miR-198, miR-135a*, miR-200c, miR-125a-3p, miR-188-5p, miR-483-5p, miR-663, and miR-630. Methylation array analysis revealed increased methylation at or near miRNA-associated CpG islands in MM patients. Ectopic restoration of miR-155, miR-198, miR-135a*, miR-200c, miR-663 and miR-483-5p significantly repressed MM cell proliferation, migration and colony formation. Furthermore, we derived a 33-gene signature from predicted miRNA target genes that were also upregulated in MM patients and associated with patient survival in three independent myeloma datasets. In summary, we have revealed important, epigenetically silenced tumor suppressive miRNAs by pharmacologic reversal of epigenetic silencing.     

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.     

miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer

The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer.     

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study

Background:

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV).

Methods:

Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature.

Results:

One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%.

Conclusion:

MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.     

Down-regulation of miR-203 induced by Helicobacter pylori infection promotes the proliferation and invasion of gastric cancer by targeting CASK

Several microRNAs (miRNA) have been implicated in H. pylori related gastric cancer (GC). However, the molecular mechanism of miRNAs in GC has not been fully understood. In this study, we reported that miR-203 is significantly down-regulated in H. pylori positive tissues and cells and in tumor tissues with important functional consequences. Ectopic expression of miR-203 dramatically suppressed cell proliferation and invasion. We found that miR-203 strongly reduced the expression of CASK oncogene in GC cells. Similar to the restoring miR-203 expression, CASK down-regulation inhibited cell growth and invasion, whereas CASK over-expression rescued the suppressive effect of miR-203. These results can also be found in nude mice. In clinical specimens, CASK was over-expressed in tumors and H. pylori positive tissues and its mRNA levels were inversely correlated with miR-203 expression. Taken together, our results indicated that miR-203 functions as a growth-suppressive miRNA in H. pylori related GC, and that its suppressive effects are mediated mainly by repressing CASK expression.     

MicroRNA-34b functions as a potential tumor suppressor in endometrial serous adenocarcinoma

Endometrial serous adenocarcinoma (ESC) is aggressive and carries a poor prognosis. p53 is frequently mutated in ESC. microRNAs (miRNAs) are a direct p53 target and have been implicated in cancer cell behavior. In this study, we compared miRNA expression levels in ESC with the levels in endometrial endometrioid adenocarcinoma (EEC) and normal endometria. Six miRNAs were identified as having aberrant down-regulation specific to ESC with miR-34b being most pronounced. miR-34b was found to have promoter hypermethylation, which when reversed, restored miR-34b expression in the cell lines treated with 5-aza-2' deoxycytidine (DAC). Ectopic expression of miR-34b in turn inhibited cell growth, migration and most notably invasion. Our findings suggest a relationship among p53 mutation, miR-34b promoter methylation and tumor cell behavior. These effects are likely mediated by the downstream target of miR-34b, the proto-oncogene mesenchymal-epithelial transition factor (MET), a known prognostic factor in endometrial carcinomas. The expression of MET was reduced following the restoration of miR-34b in cell lines. In summary, our data suggest that miR-34b plays a role in the molecular pathogenesis of endometrial cancer.     

Association between microRNA-527 and glypican-3 in hepatocellular carcinoma

The present study aimed to identify the specific microRNAs (miRNAs/miRs) and their corresponding target genes involved in hepatocellular carcinomas (HCCs). Microarray analysis was performed to examine the miRNA expression profiles of four paired HCC and corresponding non-cancerous (N) liver tissues using 985 miRNA probes. The Human miRNA Target database was used to identify the target genes of differentially expressed miRNAs between the HCC and N tissues. The protein expression levels of target genes in the HCC tissues and cell lines were evaluated using western blotting. miRNA-mediated suppression of target gene expression was evaluated by transiently transfecting the miRNA into the HCC cell lines. Of the 985 miRNAs evaluated, four miRNAs were differentially expressed (three upregulated and one downregulated miRNAs). Of these four miRNAs, miRNA-527 was highly downregulated in the HCC tissues. Glypican-3 (GPC-3) was predicted as a target gene of miRNA-527. Western blotting revealed that GPC-3 protein is highly expressed in the HCC tissues and HCC cell lines compared with N and normal cell lines. Transfection with miR-527 resulted in suppression of GPC-3 protein expression in the Cos7 cells. Furthermore, transfection with miR-527 also inhibited the intrinsic expression of GPC-3 in the Huh-7 cell line. This indicated that miR-527 in the HCC tissues may be an important novel miRNA that targets the GPC-3 gene expression. GPC-3, whose expression is regulated by miR-527, may be involved in the development and progression of HCC.     

Association of miR-193b Down-regulation and miR-196a up-Regulation with Clinicopathological Features and Prognosis in Gastric Cancer

Dysregulated expression of microRNAs (miRNAs) has been shown to be closely associated with tumordevelopment, progression, and carcinogenesis. However, their clinical implications for gastric cancer remainelusive. To investigate the hypothesis that genome-wide alternations of miRNAs differentiate gastric cancer tissuesfrom those matched adjacent non-tumor tissues (ANTTs), miRNA arrays were employed to examine miRNAexpression profiles for the 5-pair discovery stage, and the quantitative real-time polymerase chain reaction (qRTPCR)was applied to validate candidate miRNAs for 48-pair validation stage. Furthermore, the relationshipbetween altered miRNA and clinicopathological features and prognosis of gastric cancer was explored. Amonga total of 1,146 miRNAs analyzed, 16 miRNAs were found to be significantly different expressed in tissues fromgastric cancer compared to ANTTs (p<0.05). qRT-PCR further confirmed the variation in expression of miR-193band miR-196a in the validation stage. Down-expression of miR-193b was significantly correlated with Laurentype, differentiation, UICC stage, invasion, and metastasis of gastric cancer (p<0.05), while over-expression ofmiR-196a was significantly associated with poor differentiation (p=0.022). Moreover, binary logistic regressionanalysis demonstrated that the UICC stage was a significant risk factor for down-expression of miR-193b (adjustedOR=8.69; 95%CI=1.06-56.91; p=0.043). Additionally, Kaplan-Meier survival curves indicated that patientswith a high fold-change of down-regulated miR-193b had a significantly shorter survival time (n=19; mediansurvival=29 months) compared to patients with a low fold-change of down-regulated miR-193b (n=29; mediansurvival=54 months) (p=0.001). Overall survival time of patients with a low fold-change of up-regulated miR-196a (n=27; median survival=52 months) was significantly longer than that of patients with a high fold-changeof up-regulated miR-196a (n=21; median survival=46 months) (p=0.003). Hence, miR-193b and miR-196a maybe applied as novel and promising prognostic markers in gastric cancer.     

RETRACTED ARTICLE: miR-203 suppression in gastric carcinoma promotes Slug-mediated cancer metastasis

MicroRNAs (miRNAs) play critical roles in tumorigenesis and cancer metastasis. Recently, miR-203 was reported as a tumor suppressor microRNA silenced in different malignancies including hepatocellular carcinoma, prostate cancer, oral cancer, breast cancer, and hematopoietic malignancy, whereas its role in the carcinogenesis of gastric carcinoma (GC) has not been evaluated. Here, we analyzed the levels of miR-203 and Slug in the GC specimen and studied their correlation. We analyzed the binding of miR-203 to the 3′-UTR of Slug messenger RNA (mRNA) and its effects on Slug translation by bioinformatics analysis and by luciferase-reporter assay, respectively. We modified miR-203 levels in GC cells and studied their effects on the cell invasiveness in transwell cell migration assay. We found that in GC, miR-203 levels were significantly decreased and Slug levels were significantly increased. miR-203 and Slug inversely correlated in patients’ specimen. Bioinformatic analysis predicted that miR-203 may target the 3′-UTR of Slug mRNA to inhibit its translation, which was confirmed by luciferase-reporter assay. Overexpression of miR-203 inhibited Slug and cell invasiveness, while depletion of miR-203 increased Slug and cell invasiveness. These data suggest that miR-203 suppression in GC promotes Slug-mediated cancer metastasis.     

Methylation-associated silencing of MicroRNA-335 contributes tumor cell invasion and migration by interacting with RASA1 in gastric cancer

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes, previous studies have shown that miR-335 play an important role in suppressing metastasis and migration in human cancer including gastric cancer (GC). However, the mechanisms which result in aberrant expression of miR-335 in GC are still unknown. Recent studies have shown that the silencing of some miRNAs is associated with DNA hypermethylation. In this study, we find the promoter of miR-335 we embedded in CpG island by accessing to bioinformatics data and the low expression of miR-335 in 5 gastric cell lines can be restored by 5-aza-2’-deoxycytidine (5-Aza-dC) treatment. So we postulated that the miR-335 genes undergo epigenetic inactivation in GC. Subsequently, in GC cells and tissues, we performed quantitative real-time PCR (RTQ-PCR) to assess the expression of miR-335, and methylation-specific PCR (MSP) and bisulfite sequence-PCR (BSP) to evaluate the DNA methylation status in the CpG islands upstream of MiR-335. The result showed that the expression of miR-335 was significantly reduce in gastric cancer cell lines and tumor tissues compared to matched normal gastric tissues, and cell lines, and which is inverse correlation with DNA hypermethylation of miR-335 both in GC cells lines and tissues, but not in normal tissues. In addition, we found that the lower miR-335 expression induced by abnormal methylation may be mainly involved in gastric cell invasion and metastasis in GC tissues. No statistical significance was found about miR-335 expression and methylation level between healthy individuals with and without H. pylori (HP) infection. Finally, we carry out miRNA transfection, RTQ-PCR and western blot assay to find the RAS p21 protein activator (GTPase activating protein) 1 (RASA1) may be the possible target genes which lead to the gastric cell invasion and metastasis, furthermore, the re-expression of endogenous miR-335 by 5-Aza-dC treatment can exert effects similar to exogenous miRNAs transfection. Taken together, our results suggest that miR-335 may be silenced by promoter hypermethylation and play important roles in gastric cell invasion and metastasis through its target genes, such as RASA1. Its methylation level might be a predictive epigenetic marker of GC and remodeling on the expression by demethylation can provided a potential therapeutic strategy.     

miR-27a在猿猴病毒小T抗原诱导支气管上皮细胞恶性转化中的作用

目的:检测猿猴病毒40(simian virus 40,SV40)小T抗原(small T antigen,ST)诱导人支气管上皮细胞(human bronchialepithelial cell,HBE)恶性转化中miRNAs的表达谱,寻找与细胞转化相关的miRNAs。方法:选择HBE、HBER和HBERST细胞株,提取总RNA,利用miRNA芯片和实时荧光定量PCR技术检测和验证永生化HBE、HBER和HBERST细胞中差异表达的miRNAs。通过细胞生长曲线检测、细胞周期分析、细胞克隆形成试验等确证与SV40 ST诱导HBE细胞恶性转化相关的miRNAs。结果:在HBE、HBER和HBERST细胞856个miRNA的表达谱中筛选出6个与SV40 ST诱导细胞转化相关的miRNA,2个表达上调(miR-20a和miR-27a).4个表达下调(let-7d,let-7f,miR-1246和miR-3746)。抑制miR-27a能减缓HBERST细胞的增殖速度(P0.01),延长细胞在G0～G1期的停留时间(P0.01)和降低HBERST细胞在软琼脂上形成克隆的数目(P0.01)。结论:miR-27a的异常表达参与了SV40ST诱导的HBE细胞恶性转化。