Influence Of MicroRNA Let-7b Targeting CD 147 On Invasion Of Rat Pituitary Adenomas GH3 Cells

BackgroundMicroRNA (miRNA) was found in Caenorhabditis elegans by Victor Ambros in 1993 for the first time. It existences in a variety of eukaryotic cells and viruses, approximately 21-23 nt long of endogenous and non-coding single-stranded RNA. At the same time, Gary Ruvkun laboratory identified the first miRNA target gene. From then on, one of the most important adjustment methods for protein expression was found by human. Mature miRNAs specific identification in the form of miRISC complexes and combining to the target mRNA on 3 ’UTR region to suppress the translation or degradation of the target mRNA. The important effect of MicroRNAs in regulating cell proliferation, differentiation and cell cycle had been found for a long time. Disorder regulation is closely related to the occur of diseases or tumors. As one of the earliest discovered miRNAs, Let-7b was found closely related to cell proliferation and tumor and still has been widely concerned. But the target genes of Let-7b in pituitary adenoma cells and its mechanism has not been found by people now. By adopting different bioinformatics prediction software to the target genes screening, make each outcome of different softwares into intersection. We finally found the miRNA Let-7b and CD 1473 ’UTR has complementary relationship between them, Let-7b may acts on the cell protein and its biological behavior through the expression of CD 147. CD 147 is also known as matrix metalloproteinases inducer, belongs to a member of the immunoglobulin superfamily. Participate in a variety of important cell signal transduction in cells. Have been confirmed now:high expression of CD 147 can promote matrix metalloproteinase-9 (MMP-9) expression and then promote invasive tumor growth. In the process of this research, we take the rat pituitary adenoma GH3 cells as the research object, using lentivirus transfection in oder to change the expression level of Let-7b.Then detection is closely related to the tumor invasive CD 147 and MMP-9 protein expression and the change of cell invasion ability, so as to clear the Let-7b of invasive pituitary adenomas GH3 cells growth.This experiment is divided into two parts.I:Influence of MicroRNA Let-7b targeting CD147 on the expression of CD147 and MMP-9 in rat pituitary adenomas GH3 cellsObjective To investigate target genes of microRNA Let-7b in GH3 cells of rat, and study the influence of Let-7b on the expression of CD147 and MMP-9 in GH3 cells. Methods Three different bioinformatic ways including TargetScan, PicTar and MiRanda were used to identify the target genes. The Let-7b mimic and anti-miRNA oligonuclotide (AMO) were designed and transfected into GH3 cells via lentivirus, and the GH3 cells which transfected with vehicle were used as the control group. Real-time Quantitative PCR Detecting System (Q-PCR) was used to detect the expression of Let-7b after different transfection. Westen blotting was used to detect the expression of extracellular metalloproteinase inducer (CD 147) and matrix metalloprotein-9(MMP-9). Results Let-7b directly targeting CD147 as an inhibitor, suppressed the protein expression of CD 147 and MMP-9 in GH3 cells. After transfection with Let-7b mimic, the mRNA level of Let-7b was 9.56±0.51 times higher than that in the control group (P<0.05), the protein expression of CD147 and MMP-9 were reduced. Inhibiting the Let-7b expression by transfection with AMO, the mRNA level of Let-7b was 0.31±0.01 times lower than that in the control group (P <0.05), the protein expression of CD147 and MMP-9 were increased. There were no significant differences in the expression of Let-7b, the protein expression of CD147 and MMP-9 between the control group and the natural growth group. Conclusion Let-7b targeting CD147 inhibits the experssion of CD147 and MMP-9 in rat GH3 cells, it may be used as drug target for gene treatment and clinical prognostic indices.Ⅱ:Influence of MicroRNA Let-7b on invasion of rat pituitary adenomas GH3 cellsObject To explore the influence of miRNA Let-7b on the invasion growth of rat pituitary adenoma GH3 cells. Methods Rat pituitary adenoma GH3 cells were cultured with conventional method. Transfection was done by lentivirus according to the process of instruction. Respectively transfect with Let-7b analog (Let-7b mimic), antisense oligonucleotide chains (AMO) and the virus does not contain homologous sequence of rats been used as the control group. AT 36h,48h,60h,72h after transfection, transwell experiment were used to determine in the change of the invasive tumor cells respectively. Results After transfection with Lentivirus on rat GH3 cell, between natural growth (83/vision) and blank control group (79/vision) there was no statistically significant difference (P> 0.05), Compared with the Let-7b mimic group (42/vision) and the Let-7b AMO group (125/vision) difference was statistically significant (P<0.05). Conclusion Let-7b targeting CD147 inhibits the invasive growth of GH3 cells obviously, it may be used as drug target for gene treatment and clinical prognostic indices.