miRNA-125b在膀胱癌中的表达和意义

目的:探讨膀胱癌组织中miR-125b的表达在膀胱癌发生发展中的作用及其临床意义。方法:采用应用茎环RT-PCR的方法检测66例膀胱尿路上皮癌组织标本的miR-125b的表达,另有16例正常膀胱黏膜组织作对照,并结合临床病理资料进行统计学分析。结果:miR-125b在膀胱癌组织中的表达显著高于非肿瘤的正常膀胱黏膜组织(p0.05),miR-125b的水平还与表达水平与膀胱癌的组织学分级、肿瘤转移、术后复发均明显相关性(P0.05)。结论:miR-125b在膀胱癌组织中表达量升高,且与组织学分级、肿瘤转移、术后复发相关,可能作为膀胱癌诊断和预后指标。     

Prognostic significance of miR-205 in endometrial cancer

Purpose

microRNAs have emerged as key regulators of gene expression, and their altered expression has been associated with tumorigenesis and tumor progression. Thus, microRNAs have potential as both cancer biomarkers and/or potential novel therapeutic targets. Although accumulating evidence suggests the role of aberrant microRNA expression in endometrial carcinogenesis, there are still limited data available about the prognostic significance of microRNAs in endometrial cancer. The goal of this study is to investigate the prognostic value of selected key microRNAs in endometrial cancer by the analysis of archival formalin-fixed paraffin-embedded tissues.

Experimental Design

Total RNAs were extracted from 48 paired normal and endometrial tumor specimens using Trizol based approach. The expression of miR-26a, let-7g, miR-21, miR-181b, miR-200c, miR-192, miR-215, miR-200c, and miR-205 were quantified by real time qRT-PCR expression analysis. Targets of the differentially expressed miRNAs were quantified using immunohistochemistry. Statistical analysis was performed by GraphPad Prism 5.0.

Results

The expression levels of miR-200c (P<0.0001) and miR-205 (P<0.0001) were significantly increased in endometrial tumors compared to normal tissues. Kaplan-Meier survival analysis revealed that high levels of miR-205 expression were associated with poor patient overall survival (hazard ratio, 0.377; Logrank test, P = 0.028). Furthermore, decreased expression of a miR-205 target PTEN was detected in endometrial cancer tissues compared to normal tissues.

Conclusion

miR-205 holds a unique potential as a prognostic biomarker in endometrial cancer.     

Roles and Mechanism of miR-199a and miR-125b in Tumor Angiogenesis

MicroRNAs (miRNAs) have been shown to be involved in different aspects of cancer biology including tumor angiogenesis. In this study, we identified that two miRNAs, miR-199a and miR-125b were downregulated in ovarian cancer tissues and cell lines. Overexpression of miR-199a and miR-125b inhibited tumor-induced angiogenesis associated with the decrease of HIF-1α and VEGF expression in ovarian cancer cells. Moreover, the levels of miR-199a and miR-125b were negatively correlated with VEGF mRNA levels in ovarian tissues. We further showed that direct targets of miR-199a and miR-125b HER2 and HER3 were functionally relevant. Forced expression of HER2 and HER3 rescued miR-199a- and miR-125b-inhibiting angiogenesis responses and Akt/p70S6K1/HIF-1α pathway. This study provides a rationale for new therapeutic approach to suppress tumor angiogenesis using miR-199a, miR-125b, or their mimics for ovarian cancer treatment in the future.     

miR-425-5p as an exosomal biomarker for metastatic prostate cancer

Prostate cancer-related deaths are mostly caused by metastasis, which indicates the importance of identifying clinical prognostic biomarkers. In this study, we evaluated the expression profile of exosomal microRNAs (miRNAs) derived from metastatic prostate cancer (mPCa) cell lines (LNCaP and PC-3). miRNA signatures in exosomes and cells were evaluated by miRNA microarray analysis. Fourteen miRNAs were identified as candidates for specific noninvasive biomarkers. The expression of five miRNAs was validated using RT-qPCR, which confirmed that miR-205-5p, miR-148a-3p, miR-125b-5p, miR-183-5p, and miR-425-5p were differentially expressed in mPCa exosomes. Bioinformatic analyses showed that miR-425-5p was associated with residual tumor, pathologic T and N stages, and TP53 status in PCa samples. Gene ontology analysis of negatively correlated and predicted targeted genes showed enrichment of genes related to bone development pathways. The LinkedOmics database indicated that the potential target HSPB8 has a significant negative correlation with miR-425-5p. In conclusion, this study identified a panel of exosomal miRNAs with potential value as prognostic biomarkers for prostate cancer.     

Association of Circulating Serum miR-34a and miR-122 with Dyslipidemia among Patients with Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of diseases from simple steatosis to non-alcoholic steatohepatitis, with approximately 20% risk of progressing to fibrosis and cirrhosis. The aim of this study was to compare the relative expression levels of circulating miR-21, miR-34a, miR-122, miR-125b and miR-375 between healthy controls and NAFLD patients, and to assess the feasibility of microRNAs as potential biomarkers for NAFLD. A cross-sectional study was conducted to evaluate circulating serum miRNAs as potential diagnostic markers for NAFLD. Twenty-eight clinically diagnosed and histologically-confirmed NAFLD patients, as well as 36 healthy controls were enrolled in this study. The relative expression of serum microRNAs were calculated using the comparative cycle threshold with spiked-in C. elegans miR-39 as exogenous internal control. Serum levels of miR-34a and miR-122 were significantly higher in NAFLD patients than in healthy controls (P = <0.0001). Positive correlations were observed between serum miR-34a with very low density lipoprotein cholesterol (VLDL-C) and triglyceride levels. However, the expression levels of miR-34a and miR-122 did not correlate with the histological features of NAFLD. Interestingly, receiver operating characteristic (ROC) curve analysis revealed that miR-34a and miR-122 are potential markers for discriminating NAFLD patients from healthy controls with an area under the curve (AUC) values of 0.781 and 0.858, respectively. Serum levels of miR-34a and miR-122 were found to be significantly higher among NAFLD patients, and were positively correlated with VLDL-C and triglyceride levels. Thus, circulating miR-34a and miR-122 can be used as potential biomarkers for discriminating NAFLD patients from healthy controls. Larger cohorts are required to validate the utility of miR-34a and miR-122 in monitoring liver injury.     

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.     

Interferon-β Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.     

Analyses in human urothelial cells identify methylation of miR-152, miR-200b and miR-10a genes as candidate bladder cancer biomarkers

Urinary miRNAs are discussed as potential biomarkers for bladder cancer. The majority of miRNAs, however, are downregulated, making it difficult to utilize reduced miRNA signals as reliable diagnostic tools. Because the downregulation of miRNAs is frequently associated with hypermethylation of the respective regulative sequences, we studied whether DNA hypermethylation might serve as an improved diagnostic tool compared to measuring downregulated miRNAs. miRNA expression arrays and individual qPCR were used to identify and confirm miRNAs that were downregulated in malignant urothelial cells (RT4, 5637 and J82) when compared to primary, non-malignant urothelial cells (HUEPC). DNA methylation was determined by customized PCR-arrays subsequent to methylation-sensitive DNA-restriction and by mass spectrometry. miRNA expression and DNA methylation were determined in untreated cells and in cultures treated with the demethylating agent 5-Aza-2′-deoxycytidine. miR-200b, miR-152 and miR-10a displayed differential expression and methylation among untreated cancer cell lines. In addition, reduced miRNA expression of miR-200b, miR-152, and miR-10a was associated with increased DNA methylation in malignant cells versus HUEPC. Finally, the demethylation approach revealed a causal relationship between both parameters for miR-152 in 5637 and also suggests a causal connection of both parameters for miR-200b in J82 and miR-10a in 5637. In conclusion, our studies in multiple bladder cancer cell lines and primary non-malignant urothelial cells suggest that hypermethylation of miR-152, miR-10a and miR-200b regulative DNA sequences might serve as epigenetic bladder cancer biomarkers.     

Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth

MicroRNAs (miRNAs) play important roles in cancer development. By cloning and sequencing of a HPV16(+) CaSki cell small RNA library, we isolated 174 miRNAs (including the novel miR-193c) which could be grouped into 46 different miRNA species, with miR-21, miR-24, miR-27a, and miR-205 being most abundant. We chose for further study 10 miRNAs according to their cloning frequency and associated their levels in 10 cervical cancer- or cervical intraepithelial neoplasia-derived cell lines. No correlation was observed between their expression with the presence or absence of an integrated or episomal HPV genome. All cell lines examined contained no detectable miR-143 and miR-145. HPV-infected cell lines expressed a different set of miRNAs when grown in organotypic raft cultured as compared to monolayer cell culture, including expression of miR-143 and miR-145. This suggests a correlation between miRNA expression and tissue differentiation. Using miRNA array analyses for age-matched normal cervix and cervical cancer tissues, in combination with northern blot verification, we identified significantly deregulated miRNAs in cervical cancer tissues, with miR-126, miR-143, and miR-145 downregulation and miR-15b, miR-16, miR-146a, and miR-155 upregulation. Functional studies showed that both miR-143 and miR-145 are suppressive to cell growth. When introduced into cell lines, miR-146a was found to promote cell proliferation. Collectively, our data indicate that downregulation of miR-143 and miR-145 and upregulation of miR-146a play a role in cervical carcinogenesis.     

KDM4B promotes gastric cancer metastasis by regulating miR-125b-mediated activation of Wnt signaling

Emerging evidence has demonstrated that the aberrant expression of histone-modifying enzymes such as histone demethylases contributes to gastric carcinogenesis and progression. The role of KDM4B in cancer progression has been gradually revealed. However, the underlying mechanisms regulating gastric cancer metastasis of KDM4B remain unclear. In the present study we determined KDM4B expression in gastric cancer and its biologic function in vitro and in vivo. We found that KDM4B expression was significantly increased in most gastric cancer tissues compared with the adjacent normal tissues. Upregulated expression of KDM4B in human gastric cancer was correlated with poor prognosis. In vitro, KDM4B overexpression in AGS cells promoted cell invasion, whereas knockdown of KDM4B inhibited cell invasion. Furthermore, KDM4B overexpression also promoted tumor metastasis in vivo. Mechanistically, KDM4B upregulated miR-125b expression and activated Wnt signaling pathway. More important, miR-125b partially mediated KDM4B-induced activation of Wnt signaling. Finally, we demonstrated that KDM4B promoted gastric cancer cell invasion in vitro and cancer metastasis in vivo, at least in part, by upregulating miR-125b expression. These data provided novel insights on the role of KDM4B-driven gastric cancer metastasis and indicated that KDM4B may be served as a potential target for gastric cancer.     

Combined analysis of HPV DNA and p16INK4a expression to predict prognosis in ASCUS and LSIL pap smears

Human papillomavirus (HPV) is known to play an important etiological role in the genesis of cervical cancer, but only a very small proportion of infected women develop invasive cervical cancer. The purpose of cervical cancer prevention is early diagnosis of its precursors. The molecular detection of HPV DNA as a diagnostic test to cervical carcinogenesis gave a low positive predictive value as compared to the use of biomarkers. p16INK4a has been proposed as putative surrogate biomarkers that would allow identification of dysplastic cervical epithelia. Serial consecutive cervical smears were test for high-risk HPV stained with immunocytochemistry for p16INK4a and followed-up for 36 months. The aim of the study was to evaluate the immunohistochemical expression of pl6INK4a as a marker of progression risk in low-grade dysplastic lesions of the cervix uteri. In the present series, significant pl6 overexpression was observed in the group that progressed from low to high-grade squamous intraepithelial lesion when compared with the group that did not progress. In conclusion, overexpression of p16INK4a acts as potential biomarkers for cervical cancer progression from premalignant lesions.     

miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP,CK2-α, CCNJ,and MEGF9

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3’-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40–56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.