| China is a major country of gastric cancer.Integrated Chinese and Western medicine treatment is a unique model of comprehensive treatment of gastric cancer in China.Clinical practice confirms that the combination of Modified Liangfu Granule(Gaoliangjiang,Xiangfu,Chuanshanlong,MLFG)and chemotherapy for advanced gastric cancer shows a higher clinical remission rate and better quality of life than chemotherapy alone.The mechanism study found that the anti-gastric cancer effect of MLFG was related to the regulation of apoptosis-related gene expression.However,its upstream regulatory mechanism has not been clarified.Based on the preliminary research,we intends to explore the regulation effect of MLFG and diosgenin(the main component of Chuanshanlong)on miR-34a/SIRT1/p53 axis,miR-34a apoptosis-related target genes and apoptosis signaling pathway,so as to further clarify the mechanism of effect of MLFG and diosgenin,and provide scientific research basis for the treatment of gastric cancer by MLFG.Objectives:To observe the inhibitory effect of MLFG and diosgenin on the function of human gastric cancer cells and to explore the underlying anti-gastric cancer mechanism of the effect of MLFG and diosgenin on the miR-34a/SIRT1/p53 pathway and the Caspase apoptosis pathway by in vitro and in vivo experiments.Methods:1.Study on the effect and mechanism of MLFG and diosgenin on gastric cancer cells①Functional experiments of MLFG and diosgenin inhibiting human gastric cancer cells:CCK-8 assay was used to detect the effect of MLFG and diosgenin on inhibiting the proliferation of gastric cancer cells.The flow cytometry was conducted to detect the regulation of cell cycle and apoptosis on gastric cancer cells by MLFG and diosgenin,and the scratch test was performed to test the effect of MLFG on inhibiting the migration of gastric cancer cells.②The effect of MLFG and diosgenin on the regulation of miR-34a:qPCR was performed to detect the expression of miR-34a in normal gastric mucosa epithelial cells GES-1 and gastric cancer cells.The gastric cancer cells HGC-27 with miR-34a overexpressing were constructed by lentiviral infection method.CCK-8 assay was used to detect the proliferation of gastric cancer HGC-2 7 cells overexpressing miR-34a and the scratch test was conducted to detect the migration of gastric cancer cells overexpressing miR-34a.The effect of MLFG and diosgenin on miR-34a gene was detected by qPCR.③Study on the mechanism of MLFG and diosgenin through anti-gastric cancer by regulating miR-34a:qPCR was used to detect the expression of miR-34a/SIRT1/p53 loop and miR-34a target genes after overexpressing miR-34a.qPCR method was conducted to detect the expression of miR-34a/SIRT1/p53 loop,miR-34a target genes and apoptosis pathway after the intervention of MLFG and diosgenin.Western blot was used to detect the activation of caspase signaling pathway by MLFG and diosgenin.2.Study on the inhibitory effect and mechanism of MLFG and diosgenin on gastric cancer xenografts in nude miceBSP method was performed to detect the methylation level of miR-34a in gastric cancer cells.By constructing a tumor-bearing nude mouse model of gastric cancer HGC-27 cells,we set a normal control group,model group,medium-dose of MLFG group,high-dose of MLFG group,diosgenin group,5-Aza-CdR group,medium-dose combined group and high-dose combined group to observe the general state of nude mice.After the administration,the length and diameter of the transplanted tumor were measured and the tumor volume,tumor weight and tumor inhibition rate were calculated;the survival time of nude mice was observed.qPCR method was used to detect the expression of miR-34a.Western blot was used to detect the expression of miR-34a/SIRT1/p53,apoptosis related proteins and the endogenous apoptosis pathway.Results:1.Study on the effect and mechanism ofMLFG and diosgenin on gastric cancer cellsMLFG inhibited the proliferation of HGC-27,MGC80-3 and AGS cells in a concentration-and time-dependent manner.MLFG could increase G1 phase of HGC-27 cells and reduce G2 phase cells.MLFG could induce apoptosis of HGC-27 and MGC80-3 cells,in which the induction of HGC-27 cell apoptosis was significant.Compared with the control group,the migration rate of HGC-27 cells in MLFG group was slower,except for the concentration of 4mg/mL in 24h.MLFG can significantly inhibit the migration function of MGC80-3 cells in a time-dependent manner,the difference was statistically significant.Each concentration of MLFG could inhibit the migration of AGS cells at 24h and 48h.Diosgenin inhibited cell proliferation in a time and concentration-dependent manner when it acted on HGC-27,MGC80-3 and AGS cells and the difference was statistically significant.There was no significant change in the cycle of gastric cancer cells after the intervention of diosgenin.Diosgenin could induce apoptosis of HGC-27 and MGC80-3 cells,of which the induction of HGC-27 cell apoptosis was more significant.The relative expression of miR-34a in HGC-27,MGC80-3 and AGS cells was significantly lower than that in normal cells.Compared with the negative control group,after transfection with miR-34a,the proliferation and migration ability of HGC-27 cells was significantly inhibited.After the intervention of MLFG and diosgenin,the expression level of miR-34a in HGC-27 and AGS cells increased.Over-expression of miR-34a could significantly up-regulate p53,Bax and Caspase-3 expression levels,and down-regulate SIRT1,Bcl-2 and Survivin mRNA expression levels.The intervention of MLFG and diosgenin increased p53,Bax and Caspase-3 mRNA expression level,reduced the mRNA expression levels of SIRT1,Bcl-2 and Survivin and the difference was statistically significant.Z-VAD-FMK could decrease HGC-27 cell apoptosis induced by MLFG and diosgenin.MLFG can reduce the level of Caspase-9 and Caspase-3 and increase the expression level of cleaved Caspase-3.2.Study on the inhibitory effect and mechanism of MLFG and diosgenin on gastric cancer xenografts in nude miceGastric cancer cell HGC-27 showed a higher methylation level at each site of miR-34a.HGC-27 cells were selected to construct a gastric cancer tumor-bearing mouse model.After drug intervention,it was observed that the nude mice in medium dose of MLFG group,high-dose of MLFG group,diosgenin group and medium-dose MLFG combined group,high-dose MLFG combined group were in good condition.The nude mice in 5-Aza-CdR group were in a worse condition.In the 5-Aza-CdR group,the body weight of nude mice gradually decreased after the 8th day of the experimental administration.On the 11 th day,the body weight of 5-Aza-CdR group was lower than that of the high-dose MLFG group and the difference was statistically significant.On the 15th day,the weight was lower than that of the model group,the MLFG high-dose group,the diosgenin group and the high-dose MLFG combined group.On the 18th day,the body weight of 5-Aza-CdR group was lower than the other groups and the difference was statistically significant.The tumor inhibition rate in the 5-Aza-CdR group was 32.08%.Compared with the model group,the difference was not statistically significant.The tumor inhibition rate in the medium-dose MLFG combined group was 35.00%,and that in the high-dose MLFG combined group was 37.18%.Compared with the model group,the difference was statistically significant.The average survival time of the model group was 46.33±0.50 d.There were no deaths in nude mice of medium dose of MLFG group,high-dose of MLFG group,diosgenin group and medium-dose MLFG combined group,high-dose MLFG combined group,with an average survival time of 57.00±0.00 d,showing the trend of prolonging the survival time of nude mice.However,the average survival time of the 5-Aza-CdR group was 40.33±7.37 d,which was shorter than the model group.Except for the middle-dose of MLFG group,the other groups could increase the expression of miR-34a in gastric cancer,and the combination group showed the most significant effect.The medium-dose of MLFG,high-dose of MLFG,diosgenin and high-dose MLFG combined groups could significantly increase the expression of p53 protein.Each group of drug intervention significantly inhibited the expression of SIRT1 protein,and reduced the expression of Bcl-2 and Survivin,and increased the expression of Bax,Cleaved Caspase-3,Cleaved Caspase-9 and Caspase-3 substrate Cleaved PARP protein.Conclusions:1.Abnormal expression of miR-34a is closely related to gastric cancer.2.MLFG can inhibit the proliferation and migration of gastric cancer cells,regulate the cell cycle,and induce apoptosis.Its component diosgenin can inhibit the proliferation of gastric cancer cells and induce apoptosis of gastric cancer cells.The mechanism may be related to the regulation of miR-34a/SIRT1/p53 axis,miR-34a apoptosis-related target genes and activation of Caspase apoptosis pathways.3.MLFG combined with 5-Aza-CdR can significantly inhibit the growth of transplanted tumors in gastric cancer nude mice,and can improve the general state of nude mice,with a tendency to maintain body weight and prolong their survival time.The mechanism may be related to the regulation of miR-34a/SIRT1/p53 axis,miR-34a apoptosis-related target genes and Caspase apoptosis signaling pathways. |
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