| ObjectiveThrough vivo and vitro experiments,to clarify whether Jianpi-Huayu formula can promote the apoptosis of hepatocellular cacrinoma cells and affect the development of hepatocellular cacrinoma cells by regulating miRNA-602/RASSF1A pathway.MethodsThe experiment was conducted by two parts.In vivo,We culture the human hepatocellular cacrinoma cell line SMMC-7721,injected it into the liver of nude mice to establish the orthotopic hepatocellular cacrinoma model.7 days later,after the development of tumor,nude mice was divided into the control group,Jianpi-Huayu formula group and continuously treated for 21 days.During the experiment,We observed the general situation of the nude mice regularly,and record the tumor size on the body surface.The tumor size and weight were measured and evaluated.All mice were anesthetized and decapitated after administration.The tumor tissues of nude mice were saved and the tumor morphology in each group was observed under microscope by H.E staining.The expression level of miR-602 and RASSF1A mRNA and protein were detected by qRT-PCR and Western bolt.Western bolt was used to detect the protein expression level of tumor cell apoptosis-related markers,such as Caspase 8,Casepase 9,BAX,Bcl-2.In vitro experiment,human hepatocellular cacrinoma cell line SMMC-7721 was cultured,the effect of Jianpi-Huayu formula on cell proliferation was observed by MTT,and the dosage was determined.The experiment was divided into control group,Jianpi-Huayu low dose group,Jianpi-Huayu medium dose group and Jianpi-Huayu high dose group.The morphological changes of cells in each group were observed under microscope,DAPI staining and flow cytometry were used for detecting the apoptosis of hepatocellular cacrinoma cell.The mRNA and protein levels of miR-602 and RASSF1A were detected by qRT-PCR and Western bolt.Western bolt was used to detect the protein expression level of tumor cell apoptosis-related markers,Caspase 8,Casepase 9,BAX,Bcl-2.ResultsI.In vivo experiment(1)Daily observation found that after the establishment of orthotopic hepatocellular cacrinoma model,some of the nude mice will appear tumor on the body surface.At the later stage of the experiment,the tumor volume of the nude mice in the Jianpi-Huayu formula group was significantly lower than that in the control group on the 12th,15th,18th and 21st days(P<0.05,P<0.05,P<0.05,P<0.01).We found that in the later period of the experiment,the tumor of the nude mice in each group increased,the tumor of nude mice in the Jianpi-Huayu formula group was significantly smaller than that in the control group on the 12th,15th,18th and 21st days(P<0.05,P<0.05,P<0.05,P<0.01).At the same time,we found that compared with the spleen invigorating and blood stasis removing group,the nude mice in the control group showed cachexia in the later period of the experiment,which was characterized by weakness,severe emaciation,dragging and eating.But there was no such change in Jianpi-Huayu group.In terms of body weight,the body weight of nude mice in Jianpi-Huayu formula group was also significantly higher than that of the control group on the 21st day(P<0.001).In addition,the weight of tumor tissue in the Jianpi-Huayu formula group was significantly lower than that in the control group(P<0.001).(2)H.E staining showed that the tumor from each group was confirmed to be hepatocellular cacrinoma,with inflammatory infiltration;Compared with the control group,the cancer nodules in Jianpi-Huayu formula group was less and tumor tissues with less inflammatory infiltration,and the tumor invasion to the hepatic-lobule structure was weaker.(3)qRT-PCR was used to detect the expression of miR-602 in tumor tissues of each group,and the expression of miR-602 in control group was higher than that in Jianpi-Huayu group(P<0.01).The expression level of RASSF1A mRNA and protein in the control group was lower than that in the Jianpi-Huayu formula group(P<0.001,P<0.001,respectively).(4)Westren bolt detected the protein expression of apoptosis related makers such as Caspase 8,Casepase 9,BAX and Bcl-2 in liver cancer tissues of each group,compared with Jianpi-Huayu group,the protein expression of casepase8,casepase 9 and Bax in control group was up-regulated(P<0.001,P<0.05,P<0.001,respectively),while Bcl-2(P<0.001)The protein expression level was down regulated.II.In vitro experiment(1)The result of MTT showed that Jianpi-Huayu formula has the effect of inhibiting the proliferation of tumor cells,and was dose-dependent(P<0.001).The corresponding concentrations of IC75,IC50 and IC25 inhibition rates of Jianpi-Huayu formula for SMMC-7721 cells were 5mg/ml,2.5mg/ml and 1.25mg/ml,respectively.Therefore 5mg/ml,2.5mg/ml and 1.25mg/ml were used as high dose,medium dose and low dose of Jianpi-Huayu formula.(2)DAPI staining showed that the number of apoptosis of tuomor cells in the Jianpi-Huayu group was more than that in the control group.The results of flow cytometry showed that the apoptotic rate of hepatocellular cacrinoma cells in Jianpi-Huayu low,meddle and high dose groups was higher than that in the control group(P<0.05,P<0.01,P<0.001,respectively).We also observed that after treated with Jianpi-Huayu formula 48 hours,compared with the control group,the morphology tumor cells became irregular and the cell membrane was disintegrated in Jianpi-Huayu group,which was dose-dependent.(3)qRT-PCR was used to detect the expression of miR-602 in the cells of each group,and the expression of miR-602 in the control group was higher than that in the low,meddle and high dose groups of Jianpi-Huayu formula(P<0.001,P<0.001,P<0.001,respectively).Although the expression of RASSF1A mRNA in the high,middle and low dose group of Jianpi-Huayu formula was higher than that in the control group,the expression of RASSF1A mRNA in the high dose group of Jianpi-Huayu formula was statistically significant(P<0.001),but there was no statistical significance in the low and middle dose group of Jianpi-Huayu formula.The protein expression level of RASSF1A also showed the same trend,that is,the difference between the high dose group and the control group was statistically significant(P<0.05),but the difference between the middle dose group and the low dose group was not statistically significant.(4)Using westren bolt to detect the protein expression level of apoptosis related makers such as Caspase 8,Casepase 9,BAX,and Bcl-2 in each group of hepatocarcinoma cells.Compared with the control group,the protein expression level of Casepase8(P<0.01,P<0.001,P<0.01,respectively),Casepase9(P<0.001,P<0.001,P<0.001,respectively),BAX(P<0.001,P<0.05,P<0.001,respectively)in the low,medium and high dose group of Jianpi-Huayu formula was up-regulated,while the protein expression level of Bcl-2 was down regulated(P<0.01,P<0.001,P<0.05,respectively),but the change of the protein expression level was not dose-dependent.ConclusionJianpi-Huayu formula can down regulate the expression of miR-602 and up regulate the expression of RASSF1A,so as to promote the apoptosis of hepatocellular carcinoma cells and achieve the purpose of inhibiting the development of hepatocellular carcinoma. |
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