Mechanisms Of MiRNAs Regulating Key Members Of TGF-β Signaling Canonical Pathway In Non-Small-Cell Lung Cancer

[Background and Objective]lung cancer is the most common cancer type worldwide, and non-small-cell lung cancer(NSCLC) accounts for 85% of all cases of lung cancer. Genetic or epigenetic factors may play a essential role on the tumorigenesis and cancer development. The transforming growth factor-beta(TGF-β) signaling pathway regulates a series of activities ranging from embryonic development to homeostasis, and to tumor genesis and development when itself happens some disorders. The aberrant expression of members of TGF-β signaling is a main cause of the pathway functional disorders. TGFβR1 and SMAD4, two key components of TGF-β canonical pathway, are observed frequently down-regulated in NSCLC. However, mutations of this two genes were rarely identified in primary NSCLC. This encourages us to elucidate an alternately epigenetic mechanism such as microRNAs(miRNAs) regulation, by which the expression of TGFβR1 and SMAD4 are down-regulated in NSCLC. MiRNAs are small noncoding RNAs which regulate gene expression by targeting m RNAs and affect the the stability of the mRNAs and post-transcriptional protein translation. Most human protein-coding genes are predicted to be potentially regulated by miRNAs. In silico analyses suggest that TGFβR1 and SMAD4 may be potential targets of some miRNAs. In this study, we sought to elucidate the miRNA regulating machanisms by which the aberrant expression pattern of TGFβR1 and SMAD4 in NSCLC, to illustrate the important role of miRNAs on fine turning of TGF-β signaling and the occurrence and development of NSCLC, further, to supply new sight for the diagnosis and treatment of lung cancer.[Methods]1) With the use of real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR) and Western blot, we detected the expression levels of genes and miRNAs in cells and tissues. 2) Interactions between miRNAs and 3’-untranslated region(3’-UTR) of gene transcripts were detected using dual-luciferase report gene plasmid technology. 3) In order to obtain NSCLC cells in which miR-142-3p was consistently over-expressed or silenced, two types of lentiviral plasmids systems were constructed. 4) The cell growth ability was tested using cell number counting method or CCK-8 cell counting assay. 5) The migration and invasion of cells were observed with the use of transwell assay. 6) Using MassARRAY assay, we determined the quantified methylated frequencies of miR-205 promoter in NSCLCs.[Results]Part1 miR-142-3p represses TGF-β-induced growth inhibition through repression of TGFβR1 in NSCLCCompared with HBE cells, the expression level of miR-142-3p and TGFβR1 was significantly higher and lower in most of NSCLC cells, respectively. Moreover, NSCLC tissues had markedly elevated levels of miR-142-3p and decreased level of TGFβR1, respectively, compared with paired noncancerous lung. Interestingly, the ratio of miR-142-3p level(T/N) was reversely correlated with that of TGFβR1 mRNA level(T/N) in 49 paired tissues, suggesting that miR-142-3p played an important role in reducing TGFβR1 expression in NSCLCs.we predicted in silico that TGFβR1 was a direct target of miR-142-3p. Overexpression of miR-142-3p significantly down-regulated TGFβR1 mRNA and protein level in A549 cells, while silencing of miR-142-3p remarkably increased TGFβR1 mRNA and protein level. Experimental results indicates that miR-142-3p significantly repressed the luciferase activities in cells transfected with the TGFβR1 3’-UTR wild type.miR-142-3p was stably over-expressed in A549 cells and consistently down-regulated in monoclonal cells using a lentiviral expression system, respectively. TGF-β1-induced phosphorylation of SMAD3(pSMAD3), which is an indispensable downstream transcriptional regulator in canonical TGF-β/Smads signaling, was inhibited in A549 cells over-expressing miR-142-3p but activated in cells under-expressing miR-142-3p. These findings illustrate that increased miR-142-3p blocks activity of TGF-β signaling pathway through reducing TGFβR1 expression in NSCLC cells. Moreover, in the present study, our findings indicated that miR-142-3p impaired TGF-β-mediated NSCLC cell growth arrest via repression of TGFβR1 and in turn inhibited the expression of p21, which is a key regulator of cell cycle. On the other hand, miR-142-3p repressed TGF-β-induced NSCLC cell EMT progress and abilities of migration and invasion.Part2 miR-205 regulates proliferation NSCLC cells by modulating expression of SMAD4Compared with paired noncancerous lung tissues, the expression level of miR-205 and SMAD4 was significantly elevated and decreased in NSCLC tissues, respectively. The ratio of miR-205 level(T/N) was reversely correlated with that of SMAD4 mRNA level(T/N) in 52 paired tissues, suggesting that miR-205 played an important role in reducing SMAD4 expression in NSCLCs.In silico prediction indicates that SMAD4 is a potential direct target of miR-205. Over-expression of mi R-205 significantly down-regulated SMAD4 mRNA and protein level in NSCLC cells. Experimental results indicates that miR-205 significantly repressed the luciferase activities in cells transfected with the SMAD4 3’-UTR wild type.Our data showed that over-expression of mi R-205 markedly enhanced the proliferation of A549 cells. The si RNA-based knockdown of SMAD4 led to a similar effect on promoting tumor cell growth.We also determined the methylation status of miR-205 promoter region and the results showed us that among the 12 tested CpG sites, only one site(-77 CpG site) methylated less frequently in NSCLC tissues, compared with the paired noncancerous lung tissues. These results demonstrate that the hypomethylation of promoter may be one of the possible mechanisms of downregulation of miR-205 in NSCLC.