Effects Of Antisense Oligodeoxynuleotides Targeted Mcl-1 On The Biological Function Of Hepatocellular Carcinoma HepG2 Cells.

Objective:To screen out the strongest member of Bcl-2 family ASODNs,which inhibit the proliferation and induce apoptosis of digestive tract carcinoma cell lines(HepG2 LOVO and MGC-803 cells).To transfect the Mcl-1 ASODN into hepatocellular carcinoma HepG2 cells and test the effect of the ASODN on several biological fuctions(inhibit proliferation,induce apoptosis,sensitize HepG2 cells to chemotherapeutic drugs and gene expression) of HepG2 cells,to provide a new target.MethodsPartⅠScreen out the strongest one of Bcl-2 family member ASODNs inhibiting the growth of hepatocellular carcinoma,gastric cancer and colon cancer.1.Screen out the strongest one inhibiting the proliferation of hepatocellular carcinoma,gastric cancer and colon cancer cell lines by WST-8 test.2.Screen out the strongest one inducing apoptosis of hepatocellular carcinoma and colon cancer cell lines by flow cytometry.PartⅡThe effect of mcl-1 ASODN on the biological function of hepatocellular carcinoma cells1.Test the effect of Mcl-1 ASODN on inhibiting proliferation of hepatocellular carcinoma cells by WST-8 test.2.Test the effect of Mcl-1 ASODN on Mcl-1 mRNA of hepatocellular carcinoma cells by realtime PCR.3.Observe the morphology of apoptosis of hepatocellular carcinoma cells by Hoechst 33258 staining4.Test the effect of Mcl-1 ASODN on cell cycle of hepatocellular carcinoma cells by flow cytometry.5.Test the effect of Mcl-1 ASODN on apoptosis of hepatocellular carinoma cells by flow cytometry.6.Test the effect of Mcl-1 ASODN on sensitizing hepatocellular carcinoma cells to chemotherapeutic drugs(cisplatin and epirubicin) by WST-8 test.ResultsPartⅠScreen out the strongest one of Bcl-2 family member ASODNs inhibiting the growth of hepatocellular carcinoma,gastric cancer and colon cancer.1.Among all of Bcl-2 family member ASODNs,Bcl-xl ASODN and Mcl-1 ASODN are the strongest members which inhibit the proliferation of hepatocellular carciona,colon cancer and gastric cancer cell lines.2.Among all of Bcl-2 family member ASODNs,Bcl-xl ASODN and Mcl-1 ASODN are the strongest members which induce apopotosis of hepatocellular carciona and colon cancerPartⅡThe effect of Mcl-1 ASODN on the biological function of hepatocellular carcinoma cells1.After Mcl-1 ASODN was transfected into hepatocellular carcinoma cells 48h, it can inhibit the expression of mcl-1 mRNA effectively,the low effective concentration is 0.25μmol/L,when the concentration is higher,the inhibitory rates is more higher,it can reach up to 80%.2.After Mcl-1 ASODN was transfected into hepatocellular carcinoma cells 48h, compared with random Oligodeoxynuleotides control(RODN) group,the difference is of statistic significance,when the concentration is higher,the inhibitory rate is higher,when the concentration is 0.25μmol/L,the inhibitory rate is 29.66%,when the concentration is 1.0μmol/L,the inhibitory rate is 52.36%.3.After Mcl-1 ASODN was transfected into hepatocellular carcinoma cells 48h, apoptotic morphological alterations,such as chromatin condensation and apoptotic bodies were also observed by staining with Hoechst33258 under the fluorescence microscope.4.Compared with control group,the cells in s phase increase obviously,resulted in cell cycle arrest at s phase5.Compared with control group,the apoptotic rates of Mcl-1 ASODN group increases,it can reach up to 13.6%,the difference is of statistical significance; but the apoptotic rates of RODN group is only 2.8%,the difference is of no statistical significance.6.The results of sensitizing hepatocellular carcinoma cells to chemotherapeutic drugs(1) The IC50 of single cisplatin is 27.5μg/mL,after combined with Mcl-1 ASODN,the IC50 is 7.26μg/mL,it is 3.79 times of single cisplatin,and their combined effect is of synergism.(2) The IC50 of single epirubicin is 1.54μg/mL,after combined with Mcl-1 ASODN,the IC50 is 0.48μg/mL,the IC50 is 3.21 times of single epirubicin, and their combined effect is additionalConclusion1.In HepG2,MGC-803 and LOVO cells,they all display that Bcl-xl ASODN and Mcl-1 ASODN are the strongest members which inhibit the proliferation and induce apoptosis,Bcl-2 ASODN is weaker(except in HepG2 cells),Bcl-w and A1 ASODN is the weakest one.2.Mcl-1 ASODN can inhibit the expression the mcl-1 mRNA effectively,inhibit the proliferation of hepatocellular carcinoma cells,resulted in cell cycle arrest at s phase,and induce apoptosis.3.Mcl-1 ASODN can sensitize hepatocellular carcinoma cells to cisplatin and epirubicin...