Construction Of CD147-shRNA Vector And Its Influence On Biological Behavior Of Hepatocellular Carcinoma Cell Lines SMMC-7721 And HepG2

BACKGROUNDS Primary hepatocellular carcinoma(HCC) is a common malignant tumor with poor prognosis; the treatment to HCC is various,but the overall curative effect is bad.The occurrence and development of HCC is a multi-gene and multi-step process, with the development of gene technology,the research of gene treatment to HCC become a hotspot worldwide.RNA interference(RNAi) means degradation of homologous mRNA induced by endogenous or exogenous short double-stranded RNA(dsRNA) molecule,its effector molecule is 21-23 nucleotide(nt) dsRNA called small interfering RNA(siRNA).The gene knockout effect caused by RNAi technology can inhibit the expression of oncogene ,tumor suppression gene and gene mutatin et al.The short hairpin RNA(shRNA) expressed by vector transfected into target cells can be recognized and splitted into siRNA.Use the plasmid vector system which can steadily express shRNA can inhibit gene expression permanently.CD147,also named extracellular matrix metalloproteinase inducer(EMMPRIN),is a member of immunoglobulin superfamily,highly expressed in HCC and other malignant tumors.It can stimulate fibroblast and endothelial cell to secrete matrix metalloproteinases(MMPs),destroy the basal membrane and promote the infiltration and metastasis of malignant tumor.This study is to establish the CD147-shRNA vector and transfect the hepatoma cell lines SMMC-7721 and HepG2,detect the inhibition effect of CD147 expression,and observe the growth,proliferation,mobility of hepatoma cells and the expression of MMP-9 and AFP;establish the nude mice model transplanted with hepatoma cells subcutaneously to observe the growth of transplanted tumor.Thus to provide a new effective target for the treatment of HCC.METHODS1 Construction and identification of CD147-shRNA and its inhibition effect on CD147 expression of hepatoma cells.According to the CD147 mRNA sequence in Genebank,use the siRNA design software of Promega to find the right target sequence,BLAST to confirm no homologous with known human gene.3 pair of oligonucleotide strand was synthesized.The sequence is 21nt sense sequence,6nt loop and 21nt anti-sense sequence and terminal signal,cloned the sequence to pBS/U6 vector.Rename the recombinant plasmid pBS/U6/CD147-shRNA1,pBS/U6/CD147-shRNA2,pBS/U6/CD147-shRNA3,and restriction enzyme digestion and sequencing to confirm the correctness.Transfect the plasmid into hepatoma cells SMMC-7721 and HepG2 to detect the expression of CD147 by Westernblot and immunofluorescence.2 Influence of CD147-shRNA to proliferation and mobility and expression of MMP-9 and AFP of hepatoma cells.The growth curve of ransfected cells SMMC-7721,SMMC-7721/pBS/U6-control,SMMC-7721/pBS/U6/CD147-shRNA3 and HepG2,HepG2/ pBS/U6-control,HepG2/pBS/U6/CD147-shRNA3 was detected by count number metheod;the cells proliferation was detected by MTT,and calculate the inhibition rate.The cells mobility was detected by wound healing experiment. Detect MMP-9 and AFP expression level by Westernblot.3 Effect of CD147-shRNA vector on nude mice model transplanted with hepatoma cells subcutaneously.24 BALB/c-nu/nu mice was randomly divided into 3 groups,8 each group;group A was inoculated with SMMC-7721,and B group with SMMC-7721/pBS/U6-control and C group SMMC-7721/ pBS/U6/CD147-shRNA3. The nude mice model was established with 5×106/0.2ml cells inoculated subcutaneously in the armpit.After the tumor grown,measure the length and width and calculate the volume of tumor.Execute the nude mice on the 40th day and cut the tumor and weigh,calculate the inhibition rate.Detect the expression of CD147 of the tumor by immunohistochemistry.RESULTS1 CD147-shRNAs recombinant plasmid is completely correct comfirmed by restriction enzyme digestion and sequencing.All the plasmids have gene silencing effect and the pBS/U6/CD147- shRNA3 is most significant.It can inhibit the endogenous CD147 expression about 70%-80%.The fluorescence intensity of hepatoma cells transfected by pBS/U6/CD147- shRNA3 is much lower.2 The growth of hepatoma cells transfected by pBS/U6/CD147-shRNA3 is slower.The absorbance of SMMC-7721 cells transfected by 0.3μg and 1μg pBS/U6/ CD147-shRNA3 is much lower in MTT experiment,and there is a dose-dependent relationship,the maximum inhibition rate is 32.2%.A similar result can be seen in HepG2 cells,the maximum inhibition rate is 25.4%.The wound healing capatility of SMMC-7721 and HepG2 cells transfected by pBS/U6/CD147-shRNA3 is weaker than pBS/U6-control group.The precursor and activated MMP-9 is siginificantly decreased after RNAi. And the AFP expression of hepatoma cells is siginificantly inhibited after the pBS/U6/CD147- shRNA3 was transfected compared with pBS/U6-control group,the inhibition rate is about 80%-90%.3 The tumor mass can be seen on 8th-10th day after inoculation on all nude mice,the mean diameter is 2-4mm.The tumor volume of group A and group B is about same otherwise group C is siginificantly smaller.All the nude mice were executed on 40th day and the tumor were weight,the tumor weight of group C is much lighter than group A and B,the inhibition rate is 64.8%.The CD147 expression is strong positive in group A and B,but weak positive in group C.CONCLUSIONS1 Constructed CD147-shRNA recombinant plasmid successfully and transfected into hepatoma cells SMMC-7721 and HepG2;the CD147 protein expression is siginificantly inhibited detected by Westernblot and immunofluorescence.Screen the optimal plasmid pBS/U6/CD147- shRNA3.2 The growth,proliferation and mobility of SMMC-7721 and HepG2 cells is siginificantly inhibited by pBS/U6/CD147- shRNA3;the MMP-9 and AFP expression level of hepatoma cells is siginificantly decreased,thus reduce the invasion and metastasis ability of hepatoma cells.3 Establish the nude mice model transplanted hepatoma cells subcutaneously successfully. pBS/U6/CD147-shRNA3 can siginificantly inhibit the tumor growth in vivo.The CD147 expression of transplanted tumor is much weaker.4 This study indicate that CD147 is an important molecule in the development of HCC,the growth of hepatoma cells can be inhibited by silencing CD147 gene,and provide evidence for choosing CD147 as a new target to treat HCC.