Epinodosin Inhibits The Proliferation,Migration And Invasion Of Esophageal Squamous Cell Carcinoma By Regulating The MiR-143-3p/Bcl-2 Axis

BackgroundEsophageal cancer（Esophageal cancer,EC）is a common and highly invasive malignant digestive tract tumor,accounting for about 2%of all malignant tumors.Every year,about 400,000 people die of esophageal cancer in the world,while about150,000 people die of esophageal cancer in China every year.Esophageal cancer is mainly divided into two types:esophageal squamous cell carcinoma（Esophageal squamous cell carcinoma,ESCC）and esophageal adenocarcinoma（Esophageal Adenocarcinoma,EAC）.There is a high incidence of esophageal squamous cell carcinoma in China,especially in Linxian County of Henan Province and Cixian County of Hebei Province in Taihang Mountain area of China,which ranks first in the world.It is the first cause of death of local malignant tumor and poses great harm to people’s life and health.The medical cost of treating cancer in our country is as high as hundreds of billions of yuan every year,which has caused a heavy economic burden to the individual,patients,family and society.The search for anti-tumor compounds with safety,low toxicity and little side effects from traditional Chinese herbal medicine has been internationally recognized and has become a research hotspot in recent years.The application of Chinese herbal medicine in anti-cancer has a long history and solid foundation,and has broad medicinal resources and low cost,which can greatly reduce the economic pressure of patients.Drug targets are biological macromolecules or biological structures that can specifically combine with specific drugs and produce therapeutic effects,which play a leading role in the complex process of biological mediation,thus playing a role in the treatment of diseases.Once new drug targets are discovered,they often become a breakthrough in a series of new drug discovery.Diterpene compound Epinodosin（EP）,which is called Biao nuo duo xing in Chinese,is a monomer compound isolated from Rabdosia pubescens.Through the early screening in our laboratory,it was found that EP has a certain cytotoxicity to esophageal cancer cells,hepatocellular carcinoma cells,breast cancer cells and so on,especially has a strong inhibitory effect on the growth of esophageal cancer cells.In this study,we will further study the action target and mechanism of this compound in esophageal cancer cells,so as to provide more theoretical basis for screening anti-esophageal cancer drugs.PurposeThe purpose of this study is to elucidate the effects of Epinodosin on the proliferation,migration and invasion of esophageal squamous cell carcinoma cells in vitro and in vivo and to study its mechanism.Methods1.MTT assay was used to detect the effects of different concentrations of EP on the proliferation of esophageal squamous cell carcinoma EC9706 and KYSE105,and the 50%inhibitory concentration(IC₅₀);The effect of EP on the ability of clone formation was detected by clone formation assay;the effect of EP on migration and invasion of esophageal squamous cell carcinoma was detected by wound healing test and Transwell test.PI staining and flow cytometry were used to detect the effect of EP on the cell cycle of esophageal squamous cell carcinoma.Annexin V-FITC/PI double staining and flow cytometry were used to detect the effect of EP on apoptosis of esophageal squamous cell carcinoma cells.Western blot was used to detect the differential expression of apoptosis-related proteins and EMT pathway markers between drug-resistant group and non-drug-treated group.2.Tumor-bearing experiment in mice:Firstly,the mouse model of esophageal squamous cell carcinoma was established by inoculating EC9706 cells into mice by subcutaneous injection.When the tumor volume was larger than 200 mm³,the mice in EP group were given 100μL of EP,with the concentration of 50 mg/Kg.The control group was fed with the same volume of normal saline,and the positive group was treated with cisplatin with 100μL concentration of 1 mg/Kg intraperitoneally for 12days,.The growth of the tumor was observed and the volume of the tumor was measured every day.3.The total RNA and protein were extracted from mouse tumor tissue after grinding,and the expression differences of apoptosis-related proteins and key proteins of EMT pathway were detected by Western blot,and the expression of Ki-67 and Bcl-2 were detected by immunohistochemistry.4.Transcriptional sequencing was used to screen the differentially expressed mi RNA s,between drug resistant cells and control cells,and a variety of bioinformatics methods were used to predict the downstream target genes of differentially expressed mi RNA s,as well as the biological pathways and signal pathways of candidate target genes.5.Esophageal squamous cell carcinoma cells were treated with gradient concentration of EP for 24 hours,and total RNA and protein were extracted.q PCR and Western blot experiments were used to verify the differentially expressed mi RNA s and corresponding target gene m RNA and protein expression differences,and double luciferase reporter gene experiment was used to verify their targeting relationship.6.Transfection of mi R-143-3p mimic/NC and mimic into EC9706,KYSE105cells were transfected with mi R-143-3p inhibitor/NC and inhibitor.After 48 hours of culture,the cells were collected,total RNA and protein were extracted,and the changes of mi R-143-3p and downstream target gene expression were verified by q PCR and Western blot;the expression of mi R-143-3p in mouse tumor tissue was detected by q PCR;the effect of mi R-143-3p on the proliferation of esophageal squamous cell carcinoma cells was detected by CCK-8 assay;and the effect of mi R-143-3p on the migration and invasion of esophageal squamous cell carcinoma cells was detected by Transwell experiment.Flow cytometry was used to detect the effect of mi R-143-3p on apoptosis of esophageal squamous cell carcinoma cells,and dual luciferase reporter gene assay was used to verify the targeting relationship between mi R-143-3p and Bcl-2;Western blot was used to detect the effect of mi R-143-3p on apoptosis-related proteins and EMT pathway markers.7.The effect of EP and mi R-143-3p on the expression of MAPK signal pathway proteins were detected by Western blot;KYSE105 cells were transfected with mi R-143-3p inhibitor/NC and inhibitor,and then treated with EP,the expression of MAPK signal pathway proteins were detected by Western blot.Results1.EP significantly inhibited the proliferation and clone formation ability of esophageal squamous cell carcinoma cells,inhibited the wound healing rate and the ability of migration and invasion,induced the apoptosis of esophageal squamous cell carcinoma cells,blocked the cell cycle of esophageal squamous cell carcinoma cells in G2/M phase,promoted the expression of apoptotic proteins such as p53,Bax and Bim,inhibited the expression of Bcl-2,and inhibited the activation of EMT pathway.2.The results of tumor-bearing mice showed that the tumor volumes of normal saline group were significantly larger than that of EP treatment group and positive control group,but there was no significant difference between EP treatment group and positive control group.EP promoted the expression of p53,Bax,Bim in esophageal squamous cell carcinoma cells of mice,inhibited the expression of Bcl-2,and the activation of EMT pathway of EC9706 cells in mice.3.The results of transcriptional group showed that EP caused abnormal expression of 174 kinds of mi RNA s,of which 112 kinds of mi RNA s expression decreased and 62 kinds of mi RNA s expression increased compared with the control group;pathway enrichment results showed that the candidate target genes of differentially expressed mi RNA s were involved in cancer-related pathways such as PI3K/Akt,MAPK and glucose metabolism pathway;EP up-regulated the expression of mi R-143-3p in esophageal squamous cell carcinoma cells;the downstream target gene of mi R-143-3p was Bcl-2.4.Over expression of mi R-143-3p significantly inhibited the proliferation,migration and invasion of EC9706 cells,induced apoptosis,promoted the expression of p53,Bax,Bim and other proteins,decreased the expression of Bcl-2 and inhibited the activation of EMT pathway.The result of reducing the expression of mi R-143-3p in KYSE105 cells was the opposite.5.EP inhibited the activation of MAPKs signal pathway by regulating mi R-143-3p.ConclusionsEP inhibited the proliferation,migration and invasion of esophageal squamous cell carcinoma cells in vivo and in vitro by regulating the mi R-143-3p/Bcl-2 axis;While EP inhibits the activation of MAPKs signal pathway by regulating mi R-143-3p.