The Effect Of Jianpi Huayu Formula On EMT And Migration And Invasion Of Hepatocarcinoma Cells Mediated By MiR-23a-3p Through Exosomes

ObjectiveHepatic carcinoma is one of the top ten malignant tumors,which has become the third cancer slayer in China.The hepatic carcinoma is difficult to be detected in the early stage with an increased incidence,alone with several symptoms of emaciation,abdominal pain and even abdominal distension,which indicates it may have reached an advanced stage.The hepatic carcinoma shows a higher degree of malignancy,which could be metastasized to the liver and lung easily and relapse,especially after surgery.The Jianpi Huayu formula is an important prescription for treating liver cancer in traditional Chinese medicine.It has been demonstrated previously in vitro that Jianpi Huayu formula could inhibit the migration,invasion and epithelial-mesenchymal transition（EMT）of hepatocellular carcinoma cell line MHCC-97H through exosomes.In addition,the results showed that the exosome miR-23a-3p was related to EMT of hepatic carcinoma,and the expression of the exosome miR-23a-3p was inhibited by Jianpi Huayu formula,but the mechanism of Jianpi Huayu formula is still unclear.Therefore,the purpose of this study is proposed as follows:1.Take the regulation of Exosome miR-23a-3p on target gene translation as the breakthrough point,the effects of miR-23a-3p on EMT,invasion and metastasis of HCC cells were explored by promoting or inhibiting the expression of miR-23a-3p in exosomes of HCC cells.2.To further study the antagonistic effect and the mechanism of Jianpi Huayu formula on the regulation of miR-23a-3p in exosomes on Smads pathway to promote EMT,invasion and metastasis.Methods1.Liver cancer cells were infected by slow virus with miR-23a-3p genes of overexpress or inhibition,and then miR-23a-3p level in the infected cells was detected by qPCR experiments,the changes of the function of liver cancer cells proliferation and migration were detected via CCK-8 and transwell detection tests,the m RNA and protein expression level of Smads pathways and EMT related gene（E-cadherin,Vimentin,Smad3,Snail）were verified by western blot method.2.The subcutaneous xenograft models of hepatoma cells in nude mice were established,which were randomly divided into 3 groups:control group,high concentration group（2g crude drug/ml）and low concentration group（1g crude drug/ml）of Jianpi Huayu formula.Within 48-72 hours of model establishment,the control group was given 0.2ml/d of sterilized normal saline,and the high and low concentration of Jianpi Huayu formula group were given with 0.2ml/d of traditional Chinese medicine,respectively.Then,record the body weight,observe the weight changes and adverse reactions.At the 5th week after intragastric administration,the peripheral blood of nude mice was collected to detect exosomes.Subsequently,The nude mice were killed,tumor tissues were collected,tumor weight was recorded,and the long and short diameters of tumor lesions were calculated.Serum exosomes were obtained by overspeed centrifugation method.And then transmission electron microscopy（TEM）and nanoparticle tracking analysis（NTA）were used to identify the obtained exosomes.And the expression level of exosomes proteins CD63,TSG101,HSP70 and Alix were detected by Western blot,and the expression level of miR-23a-3p in serum exosomes was verified by qPCR.The expression of Smads pathway and EMT-related genes and proteins（Smad3/p-Smad3,Smad7,Snail,ZEB1,E-cadherin/N-cadherin/vimentin）were detected by qPCR/immunofluorescence/Western blot.Results1.The qPCR results of liver cancer cells showed that,the gene expression abundance of hsa-miR-23a-3p in the normal cells with slow virus infection of hsa-miR-23a-3p miRNA-up（OE）group is that of normal cells with negative control virus infected cells（NC-OE）group of 6.110 times after infected by the overexpression or inhibition of miR-23a-3p genes slow virus.Besides,the relative expression level of miR-23a-3p in OE group was increased when compared with NC-OE group（P<0.05）.While the gene expression abundance of hsa-miR-23a-3p in the normal cells with slow virus infection of hsa-miR-23a-3p miRNA-down（KD）group is that of normal cells with negative control virus infected cells（NC-KD）group of 0.467 times,and the relative expression level of miR-23a-3p in KD group was decreased when compared with NC-KD group（P<0.05）.Importantly,The results of transwell tests showed that the cells’transwell transfer rate in the KD group was decreased when contrast with the NC-KD group.On the contrary,the transwell transfer rate of OE group was increased when compared with the NC-OE group.The results of western blot indicated that,the protein expression level of E-cadherin in the KD group was up-regulated than that of NC-KD group（P<0.05）,while the expression level of Vimentin,Smad3,Snail protein were down-regulated（P<0.05）.Conversely,the protein expression level of E-cadherin in the OE group was down-regulated when compared with the NC-OE group（P<0.05）,while the expression of Vimentin,Smad3 and Snails protein were up-regulated（P<0.05）.2.The results of tumor size of the three groups intuitively showed that the control group was larger than the other two groups,and the tumor volume and weight of the control group were also larger than the other two groups（P<0.05）.the concentration of exosomes which was detected by the NTA technology was distributed in the range of7.5×10¹¹-11×10¹¹particles/m L,and the results of NTA were consistent with TEM,the diameter of exosomes detected by them was about 80 nm.In the qPCR experiments of exosomes extracted from serum and tumor tissues,the relative expression levels of miR-23a-3p treated with Jianpi Huayu formula of different concentrations were lower than the control group（P<0.05）;when comparing with the control group,the m RNA expression levels of E-cadherin and Smad7 protein in tumor tissues were increased with Jianpi Huayu formula of different concentrations（P<0.05）;while the miRNA expression levels of N-cadherin,Vimentin,Zeb1,Snail and Smad3 proteins were decreased（P<0.05）.The results of western blot showed that the protein expression levels of E-cadherin and Smad7 treated with Jianpi Huayu formula were increased when contrast with the control group（P<0.05）.ConclusionJianpi Huayu formula can inhibit the proliferation,invasion and migration of hepatocellular carcinoma cells.The mechanism of Jianpi Huayu formula to inhibit the expression of miR-23a-3p in exocrine bodies,may be to regulate Smads pathway and inhibit epithelial-mesenchymal transformation of hepatocellular carcinoma cells.