The Effect Of SiRNA-Mediated Silence UPAR And Cathepsin B On The Proliferation, Invasion, And Apoptosis Of Hepatocellular Carcinoma Cells

Objective:To investigate the effect on the proliferation、invasion、and apoptosis of hepatocellular carcinoma cells after alone or simultaneously the uPAR and cathepsin B gene expression.Methods:transfected human hepatocellular carcinoma cells SMMC-7721 with the fluorescent-labeled transfection control siRNA duplex to find out the most suitable concentration of siRNA, and conducted the follow trials with this comcentration. Three siRNA was been respectively designed and compounded to silence uPAR or cathepsin B, respectively. And one negative control siRNA was designed and compounded, too. Transfected human hepatocellular carcinoma cells SMMC-7721, Q-PCR and WB was used to detected which siRNA piece possessed the best silence effect toward uPAR or Cathepsin B. These two filtered siRNA pieces were used in the followed tests. In the siRNA-mediated alone or simultaneously silence of uPAR and cathepsin B tests, Q-PCR and WB was used to detect the mRNA level and protein level of uPAR and Cathepsin B, respectively. MTT trial was used to detect the cell proliferation. Matrigel invasion assay was applied to detect cells invasive ability, flow cytometry was used to detect the cells apoptosis.Results1. Transfected human hepatocellular carcinoma cells SMMC-7721 with different proportions of the transfection reagents and siRNA concentration according to Roch instruction, transfection efficiency can reach 80% and the cell in tip-top condition in the low concentration of siRNA.2. Transfection was conducted with the low concentration, Q-PCR was used to detect the gene level of uPAR and Cathepsin B after transfection 48 hours, respectively. The result shown that, statistical significance (P<0.05) was found between the mRNA level of uPAR and Cathepsin B in three siRNA groups and no-treatment control (NTC) after transfection 48 hour. These results show that si-uPAR-C could silence the most of mRNA of uPAR, and si-CTSB-B could silence the most of mRNA of Cathepsin B.3. After transfection 72 hours, WB was used to detect the protein of uPAR and Cathepsin B in different groups. The result shown that, statistical significance (P<0.05) was found between the protein level of uPAR and Cathepsin B in three siRNA groups and NTC group. In a word, si-uPAR-C and si-CTSB-B achieve the best silence effect, and used them in the simultaneously silence trials.4. Five groups (NTC、LC、si-uPAR、si-CTSB、si-UC) were set up in the simultaneously silence trials. Q-PCR was used to detect the gene level of uPAR and Cathepsin B after simultaneously silence human hepatocellular carcinoma cells SMMC-7721. The result shown that, compared with NTC group, uPAR gene was significant inhibited in si-uPAR and si-UC group, CTSB gene was significant inhibited in si-CTSB and si-UC group. and the simultaneously silence group achieve the better silence effect than the alone silence group (P<0.05).5. WB was used to detect the protein level of uPAR and Cathepsin B after simultaneously silence human hepatocellular carcinoma cells SMMC-7721. The result shown that, compared with NTC group, uPAR protein was significant inhibited in si-uPAR and si-UC group, CTSB protein was significant inhibited in si-CTSB and si-UC group, and the simultaneously silence group achieve the better silence effect than the alone silence group (P<0.05)6. MTT assay was used to evaluate the cells proliferation after simultaneously silence uPAR and Cathepsin B at different times (0 h,24 h,48 h, 72 h). Cells was significantly decrease in the si-CTSB group、si-UC group when compared with NTC group after transfection 24h (P<0.05), cells in si-uPAR group was significantly decrease when compare with NTC group after transfection 48h (P<0.05), and the simultaneously silence group achieve the better silence effect than si-CTSB group (P<0.05). No statistical difference between NTC group and LC group (P>0.05)7. The result of cell invasion assays show that, simultaneously (or alone) silence uPAR and Cathepsin B induced a significantly decrease in the invasion of SMMC-7721 cells according to the NTC groups (P<0.05), and the invasive cells in the simultaneously silence group were least than any other groups(P<0.05).8. Detect apoptotic cell with flow cytometry:after transfection 48h, the apoptosis rate of cells in the alone or simultaneously silence group were significantly higher than the NTC group (P<0.05), and the simultaneously silence group higher than the alone silence group (P<0.05)Conclusion:siRNA is an effective way to silence gene of uPAR and Cathepsin B in the hepatocellular carcinoma cells SMMC-7721. Simultaneously or alone silence uPAR and Cathepsin B can effective inhibit HCC cells SMMC-7721 proliferation, invasion and induce cells apoptosis, ferthmore, the simultaneously silence more effective.