| ObjectiveThis study explored the expression of RASSF1 A in normal gastric mucosa epithelial cells and different gastric cancer cell lines,analyzed the correlation between the expression of RASSF1 A and survival prognosis in gastric cancer.The effect of RASSF1 A on cisplatin toxicity in gastric cancer cells was verified in vitro experiment,and intend to reveal the mechanism of RASSF1 A mediated gastric cancer EMT by regulating JNK pathway.Methods1.Bioinformatics analysis of RASSF1 A gene differentially expressed Gastric cancer and normal gastric tissues,as well as RASSF1 A related to prognosis.Gastric cancer cell lines GES-1,MGC-803,SGC-7901,HGC-27 and AGS as the research objects were classified to five groups.Western Blot was used to detect the protein expression level of RASSF1 A in different gastric epithelial cells;2.Lentivirus harboring NC and RASSF1 A plasmids was transfected into MGC-803 and SGC-7901 cell lines.RASSF1 A protein expression was verified the transfection efficiency by Western blot.CCK8 experiment was used to detect the effects of RASSF1 A on the cytotoxicity of cisplatin in gastric cancer;3.The gastric cancer cell lines MGC-803 and SGC-7901 were classified to4 groups.DMSO group as the control group,0.5 umol/l,1.0 umol/l and 2.0umol/l were the experimental group.MGC-803 and SGC-7901 cell lines were treated to the corresponding concentration anisomycin for 1 hour,2 hours and 4hours.The protein expression level of JNK,P-JNK,EMT markers,transcription factor Snail and other indicators were detected by Western Blot.As dose regimens for following cell experiments;4.MGC-803 and SGC-7901 cell lines were treated to anisomycin after transfected with NC and RASSF1 A lentivirus respectively,and classify the experiment to four groups: DMSO + NC,Anisomycin + NC,DMSO +RASSF1A,Anisomicin + RASSF1 A,The protein expression level of JNK,P-JNK,EMT markers,RASSF1 A and other indicators were detected by Western Blot.Then explore the mechanism of RASSF1 A mediated gastric cancer EMT by regulating JNK pathway.Results1.GES-1,RASSF1 A protein expression was lower in MGC-803,HGC-27 and AGS than GES-1(P < 0.0001).Comparing to the patients with low expression of RASSF1 A,the patients with high expression of RASSF1 A had longer total survival time(OS),the difference was statistically significant(P <0.0001).2.The RASSF1 A promotes the cytotoxicity of cisplatin to gastric cancerThe result of CCK8 assay showed that RASSF1 A enhanced cisplatin’s inhibitory effect of proliferation in MGC-803 gastric cancer cell line(IC50 of NC group was 1.932 ug/ml,IC50 of RASSF1 A group was 1.215 ug/ml);in SGC-7901 gastric cancer cell line,RASSF1 A group was more sensitive to cisplatin than NC group(IC50 of NC group was 3.040 ug/ml,the IC50 of RASSF1 A group was 1.737ug/ml).3.Anisomycin stimulated MGC-803 and SGC-7901 gastric cancer cell lines that were successfully transfected with NC and RASSF1 A.Western Blot showed that P-JNK and Vimentin protein in RASSF1 A + Anisomycin group were significantly higher than those in RASSF1 A + DMSO group,and E-cadherin protein were significantly lower(P < 0.05).Conclusion1.Gastric cancer patients with high expression of RASSF1 A have longer OS.2.RASSF1 A promotes the drug sensitivity of cisplatin in gastric cancer.3.RASSF1 A inhibits gastric cancer EMT by down-regulating JNK pathway phosphorylation. |
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