Mechanisms Of Mutiple MicroRNAs Regulating Aberrant Expression Of TGFβRI Gene In Non-small Cell Lung Cancer

Background and Objective: Lung cancer, includes nearly85%non-small celllung cancer (NSCLC), is the most common cancer worldwide. Aberrant expression ofTGFβRI can serve a role in affecting the transmission of Transforming Growth Factor β(TGF-β) directly as well as the growth and progression of lung cancer.Previously wereported that the haplotype of TGFβRI gene was associated with NSCLC,however, it isstill elusive that the epigenetic mechanism underlied the role of TGFβRI in NSCLCcarcinogenesis. MicroRNAs regulate gene expression in the form of binding to the3’-untranslated region (3’UTR) of specific target genes. Besides,studies have detectedthat microRNAs expression are changed in the occurrence and development ofcancer.Silico analysis demonstrated that mutiple microRNAs are able to potentiallytargeted the TGFβRI3’UTR. This study aimed to explore the epigenetic mechanisms ofabnormal expression in NSCLC regulated by microRNAs,to provide conclusiveexperimental evidence for the early diagnosis and treatment of NSCLC.Methods:(1) To research the relative level of TGFβR1mRNA and protein inNSCLC, quantitative real-time PCR and Western Blot were initially performed in fiveNSCLC cells and HBE and49paired NSCLC tissue specimens.(2) Firstly,predicted thepotential microRNAs targets of TGFβRI3’UTR by bioinformatics software. Afterthat,A549cells were transfected with microRNAs mimics, inhibitor mimics and theircorresponding negative controls,respectively. Screened the related microRNAs whichcould affect TGFβRI gene expression by detecting the relative expression of TGFβR1mRNA and protein in transfected cells using quantitative Real-Time PCR and WesternBlot. Finally, performed dual-luciferase assay to verify whether TGFβRI is a directtarget gene of the selcted microRNAs in NSCLC.(3) Detected the relative expression of the target microRNAs in HBE and five NSCLC cell lines and49paired NSCLC tissuesamples by using quantitative real-time PCR.Meanwhile, performed regression analysisof the target microRNAs and TGFβR1expression to explore their correlation.(4) Toconstruct A549cells, selected through a Lenti-X expression system, in which thetarget microRNAs can stably overexpress or the target microRNAs are sustainlydown-regulated. Identified the A549cell lines stably transfected using qRT-PCR andWestern Blot to obtain A549cell lines with the highest transfection efficiency. Ultimatly,examination of pSMAD3expression in A549cell lines stably transfected by WesternBlot. Before examination, these cells were grown in RPMI1640medium which don’thave FBS for24h and next treated with or without TGF-β1(2ng/ml) for another6h.Results:(1)a. Both the levels of TGFβR1mRNA and protein were obviouslydecreased in H1299, A549,95C, and LTEP-a-2cells when compared with HBE. b. TheTGFβR1mRNA levels were significantly lower in49paired NSCLC tissues whencompared with the corresponding non-tumor tissues (p<0.05).c. NSCLC tissuesdemonstrated a distinct reduction in TGFβR1protein expression than in thecorresponding non-tumor tissues.In a word, the level of TGFβR1is decreased inNSCLC cells and tissues.(2) Bioinformatics software analysis showed that multiplemicroRNAs are able to potentially target the3’UTR of TGFβR1mRNA. It wasvalidated that only miR-142-3p decreased TGFβR1expression by directly targeting theseed sequence of TGFβR13’UTR in NSCLC cells through microRNAs mimicstransient transfection and Dual Luciferase Reporter Assay experiment.(3) Apparentlyhigher expression of miR-142-3p was detected in H1299, A549,95C, and LTEP-a-2celllines when compaired to that in HBE. Meanwhile, the expression level of miR-142-3pwas found to be increased in49paired NSCLC tissue specimens compared with thepaired noncancerous lung tissues. In conclusion, the level of miR-142-3p is elevated inNSCLC cells and tissues. Furthermore, regression analysis revealed that the ratio ofmiR-142-3p and TGFβR1mRNA level was negative correlated in49paired tissues.(4)Successful construction of a stable cell line which could make miR-142-3p expressionincreased and cell clones which inhibited miR-142-3p expression. The stable cells were cultured starvaion for24h and next treated with or without TGF-β1(2ng/ml) forsupernumerary6h. Western Blot results showed that TGFβR1mRNA expression wasreduced in cells overexpressing miR-142-3p, in contrast, TGFβR1mRNA was increasedin cells underexpressing miR-142-3p.Conclusion: miR-142-3p expression is increased in NSCLC and miR-142-3p candecreases the level of TGFβR1by directly binding to the TGFβR13’UTR seedsequences in NSCLC cells.Besides, the ratio of miR-142-3p and TGFβR1mRNAlevel was negatively correlated in NSCLC.Our findings demonstrating that miR-142-3pmay be an oncogene and play a critical role in decreasing TGFβR1expression inNSCLC. More importantly,miR-142-3p can reduce TGF-β-induced phosphorylation ofSMAD3by decreasing TGFβR1expression in NSCLC which would inhibit TGF-βcanonical signaling pathway. The study of down-regulation of TGFβR1that mediated bymiR-142-3p may increase our comprehending of the mechanisms of NSCLCcarcinogenesis and provide a theoretical basis for the diagnosis and treatment ofNSCLC.