STAT1 gene lentiviral mediated by STAT1 expression and gene silencing in MHCC97L cells in hepatocellular carcinoma
|Beijing University of Traditional Chinese Medicine
|Traditional Chinese Medicine
|STAT1Gene Gene Overexpression Gene silencing MHCC97L cell
Objective:To explore the changes of lentiviral vector-mediated STAT1gene’s volume of expression and its molecular mechanisms after STAT gene’s overexpression and its s ilence,thereby,to ascertain whether the STAT1gene is the target one to affect the liver cancer cell’s biological effects.Methods:①Synthesizing total STAT1gene according to the human STAT1gene’s mR NA sequence to construct over-expression vector and transform into competent DH5a c ells to be verified by sequencing.Transfect293T cells by liposome-mediating and pack those to generate lentivirus.And transfect the lentivirus vector into human hepatoma M HCC97L cells.Detect the expression of STAT1of MHCC97L original cell lines and M HCC97-statl stably transfected cell lines to determine the effectiveness of over-expresse d STAT1.②Design three pairs of shRNA interference sequence and one pair of control sequence,Construct three shRNA interfering lentivirus vectors and transform into comp etent DH5a cells to be verified by sequencing.Transfect293T cells by liposome-mediati ng and pack those to generate lentivirus.Then transfect the lentivirus vector into human hepatoma MHCC97L cells.Detect the expression of STAT1of MHCC97L original cell lines,MHCC97-statl stably transfected cell lines and MHCC97-shSTAT1interfering cell lines to clear the feasibility of STAT1gene interference.Results:The results of STAT1gene overexpression:①STATl gene synthesis:Synthesize total STAT1gene (CDS region sequence) according to the human STAT1gene’s mRN A sequence and verify the gene sequence successfully.②Packing overexpression lentivir us particle:Transfect293T cells by liposome-mediating successfully.Lentivirus infects M HCC97L cells.Observe the cells under visible light microscope after24hours that most of the cells emits fluorescence which seems successful infection.③Detecting the expre ssion of STAT1gene by PCR:Gene expression of STAT1stably transfected cell lines MHCC97-statl was increased by10,938times than original MHCC97L cells after48h ours by infection.It is a statistically significant difference (P<0.01) that lentiviral STAT1gene overexpression in human hepatoma cells MHCC97L infection rate is more than90%.④Overexpression of STAT1gene detected by Western blot:The protein bands of S TAT1gene stably transfected cell lines MHCC97-statl was significantly thicker than th e original MHCC97L cells’.The results of Interfering STAT1gene silence:①Constructio n and identification of lentiviral vector interference:Three pairs of shRNA interference sequence and one pair of control sequence are ligated into pLKO-1-EGFP-puro and ver ified the gene sequence successfully.②Packing interference lentiviral particles:Transfect293T cells by liposome-mediating successfully.Lentivirus infects MHCC97L-STAT1cells. Observe the cells under visible light microscope after24hours that most of the cells e mits fluorescence which seems successful infection.③Detecting the expression of STAT1gene by PCR:The interference effect of pLKO-1-statl-shRNA-land pLKO-1-statl-shRNA-2is better than original MHCC97L cells after48hours by infection.The interference efficiency is81.4%and72.1%for each. It is a statistically significant difference (P<0.01) and indicated that interference lentiviral STAT1gene in human hepatoma MHCC97L cell interference is better.④Silence of STAT1gene detected by Western blot:The protein bands of STAT1gene stably transfected cell lines MHCC97-statl was significantly thicker than the original MHCC97L cells lines’and overexpression of STAT1stably transfected.Conclusion:The technology of STAT1gene lentivirus-mediated overexpression is successfully constructed in hepatoma MHCC97L cell and achieves gene overexpression; Interference lentiviral gene silencing of STAT1is successfully constructed in hepatoma MHCC97L cell and confirmes that expression levels of the purpose of gene are significantly reduced.It is to pave the way for further research that STAT1gene is as the targeted one to treat liver cancer and its overexpression and silencing affect the changes of Shenqifuzheng injection inhibitory effect.It is provided a reliable theoretical basis for the vitro studies of Shenqifuzheng injection subsequently.