Study On The Correlation Between Matrine Inhibiting The Formation Of Vascularized Mimicry In Colon Cancer Cells And Its Targeted Regulation Of Claudin-9

Objective:Colon cancer is the third most common new cancer in the world,with the second highest mortality.Unrestricted tumor growth,metastasis and lack of effective treatment are the root causes of high mortality of colon cancer.Studies have shown that vasculogenic mimicry（VM）,as a new way of blood supply for malignant tumors,plays an important role in the rapid growth and escape of tumors.Matrine is the main active component of traditional Chinese medicine Sophora Flavescens,which has a wide range of antitumor effects,including colon cancer;However,whether it can participate in tumor inhibition by regulating VM formation has not been reported.The purpose of this study is to clarify the pharmacological activity and potential mechanism of Matrine in inhibiting the proliferation,invasion and VM formation of colon cancer cells,so as to provide a new strategy for the clinical treatment of colon cancer.Methods:Three dimensional culture technology of matrix gel was used to evaluate the ability of different colon cancer cells to form VM,and CT26 cells with strong VM formation ability were selected for subsequent research.CT26 cells were treated with different concentrations of Matrine for 24 and 48 hours respectively.The inhibitory effect of Matrine on the proliferation of colon cancer cells was studied by CCK-8 method;At the same time,the effect of Matrine on VM formation was observed.Flow cytometry was used to analyze the apoptosis after Matrine treatment;The effects of Matrine on cell migration and invasion were analyzed by cell scratch test,transwell migration and invasion test;The effect of Matrine on the cytoskeleton formation of colon cancer cells was measured by stress fiber formation experiment;RT-qPCR and Western Blot were used to detect the expression changes of VM key factors E-cadherin,N-cadherin,MMP2 and MMP9 after Matrine treatment,which were verified in Human Colon Cancer Cell SW480.The differentially expressed mRNA of colon cancer cells treated with Matrine was screened by high-throughput sequencing,and the go and KEGG pathway were enriched and analyzed;RT-qPCR was used to verify the mRNA levels of the enriched cytochrome P450 genes Aldh31a1,Gstp2,cell adhesion molecules（CAMs）pathway related genes Jam3,Claudin-9,Claudin-18 and MAPK signaling pathway related genes Efna4,Efnal,Mapkapk5,Angpt2,Hspa2.Further,the expression changes of key genes ERK and JNK of MAPK pathway after Matrine treatment were detected by Western Blot.Finally,we designed the gene of interest claudin-9 siRNA,transfected CT26 cells with Lipofectamine 3000,detected the effects of silencing claudin-9 on cell activity,VM formation,apoptosis,invasion and migration,and detected the expression changes of VM key factors and MAPK Pathway key genes by RT-qPCR and Western Blot.Results:（1）Matrine inhibited the proliferation and VM formation of colon cancer cells in a time and concentration dependent manner,significantly promoted cell apoptosis,inhibited cell migration,invasion and the formation of stress fibers;The expression levels of N-cadherin,MMP2,MMP9 mRNA and protein were significantly inhibited,while the expression level of E-cadherin was up-regulated;Moreover,the mRNA level of VM key factor in Human Colon Cancer Cell SW480 treated with Matrine was consistent with that in CT26 cells.（2）High throughput sequencing showed that 163 genes were up-regulated and 333 genes were down-regulated after Matrine treatment（log2foldchange>1,P<0.05）.Go analysis of differentially expressed genes enriched most in fluid transport,extracellular matrix,receptor regulatory activity and so on;KEGG pathway analysis showed that the top ranked were cytochrome P450 exogenous substance metabolism,cell adhesion molecules（CAMs）and MAPK signaling pathways,and confirmed that the mRNA expression levels of some genes enriched in these pathways were consistent with the sequencing results;It was clear that Matrine treatment significantly inhibited the expression of key genes pERK and p-JNK of MAPK pathway in CT26 cells.（3）Cell adhesion molecules（CAMs）are closely related to the formation of VM,so claudin-9 gene with significant difference in this pathway was selected for follow-up study.Silencing claudin-9 expression significantly inhibited the proliferation and VM formation of colon cancer cells,promoted cell apoptosis,significantly inhibited cell migration,invasion and the formation of stress fibers;Significantly down regulated the expression levels of N-cadherin,MMP2 and MMP9,and up regulated the expression of Ecadherin;Significantly inhibited the expression of p-ERK and p-JNK in MAPK pathway;The intervention of Matrine will further enhance the above trend.It is preliminarily clear that Matrine may regulate the expression of key proteins of MAPK pathway through claudin-9,so as to inhibit the proliferation,invasion and migration of colon cancer cells and the formation of VM.Conclusion:（1）Matrine treatment significantly inhibited the proliferation and VM formation of colon cancer cells,significantly promoted cell apoptosis,inhibited cell invasion and migration and stress fiber formation in a time and concentration dependent manner.（2）The mRNA differential expression profile of colon cancer cells treated with Matrine was established.The analysis of go and KEGG pathway showed that the differentially expressed genes were involved in regulating a variety of biological processes and involved many important signal pathways,such as fluid transport,extracellular matrix,receptor regulatory activity,cytochrome P450 metabolism of exogenous substances,cell adhesion molecules（CAMs）and MAPK signal pathway.It was further clarified that Matrine treatment significantly inhibited MAPK signal pathway.（3）It is clear that Matrine regulates MAPK signal pathway through claudin-9,inhibits the proliferation,VM formation,invasion and migration of colon cancer cells,and exerts its pharmacological activity of inhibiting colon cancer.