A Study For The Role Of TRB2and Its Relative MiRNA In Adenocarcinoma Of Lung

In this research, we investigated the pathogenesis of miR-99, let-7c and TRB2gene in lung adenocarcinoma at the cellular level. We constructed the expression vector of miR-99, let-7c and the RNAi vector specific to TRB2gene, and carring out the basic research for pathogenesis of TRB2and its relative miRNA in lung adenocarcinoma, which provides theoretical basis for clinical treatment of lung adenocarcinoma.1. Research for the regulation of miRNA on TRB2(1) The Construction of pcDNA-GFP-TRB2-3’UTR vector:TRB2gene3’UTR sequence was amplied and cloned into the pcDNA-GFP vector (laboratory stored) to form pcDNA-GFP-TRB2-3’UTR vector. Our results showed that the pcDNA-GFP-TRB2-3’UTR vector was construced successfully.(2) The role of miRNA on TRB2:We co-transfected the pcDNA-GFP-TRB2-3’UTR vector and miR-99or let-7c into A549cells. NC vector was used as control. Compareing experimental group with control group48h after transfection, we found that the number of green fluorescent protein positive cells was lower in experimental group. At the same time, TRB2gene expression decreased in miRNA treatment group by western blot, which indicated miR-99and let-7c can regulate the expression of TRB2. Also the adjustive function of let-7c is much more effective.2. Inhibition of tumor cell growth by miR-99and let-7c(1) The construction of miRNA expression vector:miRNA gene (miR-99、let-7c) was amplified by PCR. Then, miRNA gene was cloned into the T vector. In addition, the aim miRNA (miR-99、let-7c) gene was cloned into pRNAi-U6.1/Neo vector by designed restriction sites, which formed the pRNAi-U6.1/Neo-miR-99or pRNAi-U6.1/Neo-let-7c vector. We treated pRNAi-U6.1/Neo-NC vector as control vector. (2) Study at the cellular level:In order to evaluate the effect of miRNA on cell proliferation and apoptosis, we chose A549cells and Hela cells as research subjects. After transfection the experimental and control vectors for48h, we detected the expression of miR-99and let-7c by real-time PCR. Further we detected cell proliferation and apoptosis by fluorescence microscopy, flow cytometry and electron microscopy. The results indicated that after transfecting these miRNA expression vectors into cells, miR-99and let-7c were expressed effectively, which can inhibit cells proliferation and promote cells apoptosis.3. The roles of TRB2and its relative factor regulated by miRNA in the pathogenesis of lung cancer(1) The Construction of pcDNA3.1-TRB2vector:We clond TRB2-cDNA gene, which obtained by PCR, into pcDNA3.1vector to form pcDNA3.1-TRB2vector.(2) The effect of miRNA on TRB2and its relative factor:TRB2gene downstream signaling factors may include:AKT, EBP-a/EBP-β and P-38by information analysis and other studies. We transfected pcDNA3.1-TRB2, RNAi-TRB2and let-7c vectors in A549cells. RT-PCR results show that siRNA specific to TRB2gene (RNAi-TRB2) and expression vector (pcDNA3.1-TRB2) can inhibit TRB2gene or enhanced TRB2gene expression, respectively. Also, miRNA-let-7c can regulate TRB2gene negatively. We detected the expression of the protein factors by western blot. The results showed that TRB2and let-7c gene can indeed lead to p-p38expression change, which confirmed P38, regulated the expression of TRB2and let-7c gene, play important role in pathogenesis of lung adenocarcinoma.In summary, miR-99and let-7c can act on TRB2gene3’UTR, which regulates TRB2expression negatively. The low expression gene TRB2and high expression gene let-7c both can make the high expression of P-P38. All of which inhibit cells proliferation and promote cells aptosis eventually.