Effects On Autophagy Of Mouse Forestomach Carcinoma Cells Induced With Anti-cancer Drugs By Silencing HMGB1Gene

Gastric cancer is one of the most common gastrointestinal tumors. Chemotherapy is still one of major means for treatment of gastric carcinoma. Clinical application of chemotherapy drugs have induced MDR which causes the failure of chemotherapy. The detailed mechanism of MDR is needed to be further investigated. It has been reported that HMGB1activated autophagy and increased the development of drug resistance in colorectal cancer, leukemia and myeloma. However, it is still unclear that the role of HMGB1in drug-resistance of gastric cancer. The aim of this study is to reveal the effects on autophagy of mouse forestomach carcinoma cells(MCF) induced with anti-cancer drugs by silencing HMGB1gene.Part1. Constructing lentivirus of ShRNA targeting on HMGB1gene: Four specific single-strand DNA sequences which targeted on mouse HMGB1mRNA were designed and synthesized. After annealing, the double-strand DNAs were cloned to pSilencer1.0vector. The open reading frame of ShRNA on pSilencer1.0vector were digested with restriction enzymes and cloned to lentivirus vector pNL. The recombinants were screened by PCR, restriction enzyme digestion and DAN sequencing.Part2. Screening the anti-tumor drugs which induced autophagy on MCF cells:50%inhibitory concentration of Gefitinib, Doxorubicin, Geldanamycin and mitomycin were tested which inhibited the proliferation of MFC with MTT. The expression of LC3-Ⅱwas analysed by western blot with the concentration of IC50of Gef, DOX, GA and MMC in order to screening the anti-tumor drug which induce autophagy effectively. The results showed that the autophagy was effectively induced by Gef, DOX and GA respectively with dose-time-dependent maner.Part3. Impact on autophagy induced with chemotherapy drugs by down regulating HMGB1: After transient transfection of pNL-ShHMGB1vectors to MFC cells, the expression of HMGB1mRNA and protein were analysed by real-time RT-PCR and western blot. The results showed that all four targeting sequences could lower the expression of HMGB1mRNA respectively. One of these targeting vectors showed the greatest efectiveness on HMGB1expression. Then the lentivirus particles of pNL-ShHMGB1were produced by cotransfection into239FT with pHelper and pVSVG. The functional titer of unconcentrated viral supernatant was2×107TU/ml.Impact on autophagy induced with DOX was checked in order to down regulating HMGB1by transient transfecting with pSilencer-ShHMGB1instead of lentivirus particles due to lower infection on MCF cells with unknown reasons. Expression of HMGB1and LC3-Ⅱwere analysised by western blot. The results showed that HMGB1was downregulated by transfecting with pSilencer-ShHMGB1after36hours, expression of LC3-Ⅱ was greatly decreased in cells with low expression of HMGB1than that in normal cells.Conclusion: The autophagy was inhibited in MFC cells induced with DOX by silencing HMGB1gene. This may indicate that autophagy could serve as a new target for chemertherapy in drug resistance of gastric cancer.