Construction, Transfection And Expression Of Letivirus ShRNA-LKB1

Objective:To construct a specific short hairpin RNA(shRNA) expressing vectors against human liver kinase B1(LKB1) or serine/threonine protein kinase11(STK11) gene and study its effect of silencing LKB1 gene expression after transfecting the hepatocellular carcinoma Huh7 cells.Methods:Three siRNA(siRNA1、siRNA2、siRNA3) sequences targeting the LKB1 gene and one negative sequence which was used as negative control group were designed and synthesized, and the corresponding four pairs of complementary single strand DNA of shRNA were formed. The synthetic shRNA sequence was inserted into the empty hU6-MCS-CMV-EGFP shRNA vector. Then the recombinant lentivirus vector was transfected into 293 T cell line after being detected by DNA sequencing. Virus yielded by 293 T cell was transfected into human hepatocellular carcinoma Huh7 cells.Cellular morphology and transfection efficiency were observed under fluorescence microscope. The inhibitory effect of LKB1 shRNA construct on LKB1 mRNA and protein expression was examined with real-time fluorescence quantitative-polymerase chain reaction(RFQ- PCR) and Western blotting, respectively.Results:The shRNA LKB1 lentivirus vectors were successfully constructed, and it can effectively inhibit the expression of LKB1 mRNA. We decided the shRNA LKB1-2(KD2) was the effective target, according to the cellular morphology and transfection efficiency.Conlusion:We successfully get the shRNA sequences which can silence the LKB1 geneexpression, and construct the lentivirus. After being transfected, the expression of LKB1 mRNA and protein were obviously inhibited. Our expriments provided experimental basis for the later research.