DNA Methylation-mediated Silencing Of MicroRNA-145 Is A Potential Prognostic Marker In Patients With Lung Adenocarcinoma

Section I Molecular mechanism of miR-145 down-regulating expression by DNA methylation in lung adenocarcinomaBackground With approximately 1.5 million new cases diagnosed every year, lung cancer is the most prevalent cause of cancer deaths worldwide, and lung adenocarcinoma (LAC) accounts for almost 40% of lung cancer deaths. Previously, we have demonstrated that miR-145 is down-regulated in LAC tissues and mir-145 suppresses the proliferation of lung cancer-initiating cells(LCICs) derived from A549 in vitro by targeting POU5F1 mRNA, our following study further validated that miR-145 could also modulate the EMT properties of LCICs by targeting Oct4. Downstream mechanisms of miR-145 altering cellular processes are partially confirmed, but the molecular mechanisms of down-regulated miR-145 in lung cancer cells remain largely unknown. By Bioinformatics method, we found several CpG sites upstream of miR-145 coding gene. We hypothesized that aberrant hyper-methylation of the CpG sites silences the expression of mir-145 in LAC.Methods Data were downloaded from the TCGA portal and processed partly with the MethHC (http://MethHC.mbc.nctu.edu.tw). The discriminating methylat ion levels of the three CpG sites (cg08537847, cg22941668, cg11671363) in LAC and non-tumor tissues were analyzed by us on the basis of the data (TCGA_LUAD_hMethyl450) from Cancer Browser (https://genome-cancer.ucsc. edu/). Expression of miR-145 was tested in LAC cells after treated with the d e-methylating agent,5-aza-2’-deoxycytidine (5-Aza-CdR). Genomic DNA was extra cted from 22 resected LAC specimens and their adjacent normal lung tissues using the genomic DNA rapid extraction kit (TIANGEN BIOTECH CO, LTD. Beiji ng, China). Then, we analyzed the methylation level of CpG sites at the prom oter region of miR-145 by using the Sequenom MassARRAY platform and semi-quantitative RT-PCR.Results MethHC (http://MethHC.mbc.nctu.edu.tw) revealed that miR-145 is co mmonly down-regulated in human LAC accompanied by increased DNA methyl ation of its 300-upstream region. Data downloaded from Cancer Browser(https: //genome-cancer.ucsc.edu/)showed that all of the three CpG sites are hyper-methylated in LAC tissues.5-Aza-CdR treatment of LAC cell lines (A549, H1975 and Spc-A-1) led to elevated miR-145 expression. A significant negative correlation between the miR-145 expression and methylation levels was observed in lung adenocarcinoma tissues.Conclusion Expression of miR-145 is associated with methylation level of CpG sites upstream of miR-145. Methylation of the promoter region might contribute to down-regulated expression of miR-145 in lung adenocarcinoma.Section Ⅱ mir-145 is a potential prognostic marker for LACBackground MiRNAs act as tumor suppressors or oncogenes in a wide variety of biological processes, such as cell differentiation, proliferation, and apoptosis. A growing number of studies have shown that aberrant miRNA expressions contribute to tumorigenesis and cancer progression. In view of its pivotal role in tumor development and progression, we also explored the clinical utility of mir-145 as a prognostic marker in lung adenocarcinoma.Methods We applied a tissue microarray (TMA) with follow-up data containing 92 pairs of tumor and paired non-tumor tissues to evaluate the expression of mir-145 by in situ hybridization.The associations between clinical characteristics and mir-145 expressions were analyzed by Student’s t-test or one-way ANOVA. Survival curves were plotted using the Kaplan-Meier method, and differences between survival curves were tested using the log-rank test. Cox’s proportional hazards model was used to identify the factors that have significantly independent influence on survival. Results Down-regulated mir-145, to some degree, implies a trend of lymph node metastasis, pleural invasion, and advanced TNM staging, however, no statistical significance was observed to support that. The univariate and multivariate analysis further revealed that mir-145 expression level was an independent risk factor for both OS and DFS in LAC patients.ConclusionThese results showed that the expression of miR-145 was an independent prognostic factor for LAC.