The Role Of Human TIMP-2 Protein In The Migration And Invasion Of Hepatocellular Carcinoma

Objective: To obtain the human TIMP-2 protein by prokaryotic expression, and explores the role of TIMP-2 in the migration and invasion of HCC. Methods:(1) Total RNA was extracted from human hepatoma cell line(SMMC-7721), and the TIMP-2 c DNA was obtained with RT-PCR. Then, the amplified TIMP-2 coding region by PCR technology was cloned into p MD18-T; after identification and sequencing, the TIMP-2 DNA fragment was inserted into the prokaryotic expression vector of p GEX-4T-1, to construct prokaryotic expression vector p GEX-TIMP-2, which was then transformed into Origami(DE3) for expression. After induced by IPTG, the expression product was purified with affinity chromatography and identified by Western blot.(2) Analysis the role of TIMP-2 protein in the in the migration and invasion of HCC with RNAi technique and adding extraneous protein. Detection of m RNA and protein expression levels of endogenous TIMP-2 in hepatocellular carcinoma by real-time quantitative PCR and western blot after transfection with si RNA-TIMP-2. Results:(1) TIMP-2 gene coding region was cloned from SMMC-7721 cell and the recombinant plasmid of p GEX-TIMP-2 was successfully constructed and expressed fusion protein GST-TIMP-2 in Origami(DE3) when induced by IPTG, then purified protein was obtained.(2) Compared with control group, adding exogenous TIMP-2 suppressed the migration and invasion of HCCs(P<0.05), on the contrary, the expression of TIMP-2 decreased and enhanced the migration and invasion of HCCs after knock-down of TIMP-2(P<0.05); The conditional medium of hepatic stellate cells can promote the migration and invasion of HCC cells, which can be inhibited by exogenous TIMP-2 protein. Conclusion: The p GEX-TIMP-2 recombinant vector was successfully constructed and obtained the recombinant protein; The level of TIMP-2 protein was closely related to the migration and invasion of HCCs in the microenvironment of hepatocellular carcinoma.