Fuzheng Jiedu Decoction Regulates The Mechanism Of PDCD4 Inhibiting Epithelial-mesenchymal Transition In Colorectal Cancer

Background:Colorectal cancer is one of the most common gastrointestinal cancers.The number of new cases and deaths worldwide ranks third and fourth respectively among all cancers.More than 50%of patients with colorectal cancer will eventually endure recurrence and metastasis after radical resection,which is the main cause for the death of colorectal cancer patients after operation.Fuzheng Jiedu formula is an experience prescription for clinical treatment of colorectal cancer by Professor Piaobingkui,a master of Chinese medicine.Fuzheng Jiedu formula can effectively prevent invasion and metastasis of colorectal cancer.The transformation of epithelial mesenchymal transition is a common cause of tumor metastasis,and miR-21/PDCD4/AP-1 signaling pathway may be the main cause of epithelial mesenchymal transition,invasion and metastasis of colorectal cancer.We hypothesized that Fuzheng Jiedu formula can inhibit the recurrence and metastasis of colorectal cancer by acting on the miR-21/PDCD4/AP-1 signaling pathway and reversing the procedure of EMT.Objective:To clarify the possible targeting signaling pathway of Fuzheng Jiedu formula in reversing EMT formation and preventing recurrence and metastasis of colon cancer.Methods:1.Lovo subcutaneous xenograft model in nude mice was established in vivo,and mice were divided into Fuzheng Jiedu formula group and the control group.After subcutaneous inoculation of Lovo cells,mice were treated for 28 days with Fuzheng Jiedu formula or distilled water respectively.The effects of Fuzheng Jiedu formula on the weight of subcutaneous tumor and the expression of EMT related protein were observed.2.In vitro,rats were used to prepare Fuzheng-Jiedu serum and blank serum.Lovo cells were cultured with the serum containing Fuzheng Jiedu serum and blank serum respectively,CCK8 method was applied to detect the inhibion rate of the serum of Fuzheng Jiedu Decoction on Lovo cells,and observed the influence of the serum of Fuzheng Jiedu formula on the morphological and invasive and migratory ability of Lovo cells under two-dimensional culture conditions.The total protein of Lovo cells under the culture medium of Fuzheng Jiedu serum and blank serum were extracted,and Western Blotting was applied to detect the effects of Fuzheng Jiedu serum had on the expression of EMT-related proteins and possible targeting protein in Lovo cells;miR-21 overexpressing and Lovo-gfp cell lines were established by Lentivirus infection technique,and RT-qPCR was performed to investigate how Fuzheng-Jiedu serum affect target mRNA.Results:1.Fuzheng Jiedu Decoction can inhibit the proliferation of human high-metastatic colon cancer Lovo cells in vitro,high-dose drug-containing serum group inhibition rate of 35%at 72h;2.After 72 hours of blank serum culture,Lovo cells were spindle-shaped,curled and drawn,showing two forms of epithelial cells and mesenchymal cells,mainly in the form of mesenchymal cells;low dose group of Fuzheng Jiedu serum culture After 72 hours,the cell line between Lovo cells was reduced and still showed epithelial cells and interstitial cells.The Lovo cells in the middle dose group contained 72 hours of culture,and the Lovo cells grew in a plane.There were still a few cells in filamentous shape.The high dose group contained After 72 hours of drug serum incubation,Lovo cells were polygonal and fusiform,and the monolayers were tightly arranged,and most of Lovo cells recovered to epithelial cells.3.The low,middle and high dose groups of Fuzheng Jiedufang contained serum can inhibit Lovo cell migration to varying degrees,and the high dose group has the best effect.4.Transwell chamber membrane Lovo cell number:Fuzheng Jiedufang high dose group<Fuzheng Jiedufang middle dose group<Fuzhen Jiedufang low dose group<blank serum group.5.Fuzheng Jiedufang Containing serum can promote the expression of E-cadherin,PDCD4,mRNA in vitro cultured in Lovo cells,there are significant differences compared with the blank serum group,and can inhibit Lovo cell N-cadherin,MMP2,Vim,β-cantenin mRNA expression.At the same time,compared with miR-21/Lovo cells+blank serum group,Fuzheng Jiedufang drug-containing serum can reverse the down-regulation of E-cadherin and PDCD4 mRNA expression caused by overexpression of miR-21,and N-cadherin,MMP2,Vim,β-cantenin mRNA expression was up-regulated,P<0.05,both statistically significant;6.The serum containing Fuzheng Jiedu Decoction can promote the expression of E-cadherin in Lovo cell total protein.With the increase of drug-containing serum dose,the promotion effect is obviously enhanced.The content of N-cadherin in Lovo cell total protein was inhibited by Fuzheng Jiedu serum,and the inhibitory effect increased with the increase of the concentration.Fuzheng Jiedu serum could promote the expression of PDCD4 in Lovo total protein and inhibit the expression of P-cantenin and c-Jun;7.The results of the suppression of human colon carcinoma and nude mice subcutaneous transplanted tumors by Fuzheng Jiedu Decoction showed that Fuzheng Jiedu Decoction had a certain inhibitory effect on tumorigenicity of nude mice;8.Compared with the blank control group,the expression of E-cadherin and PDCD4 increased,and the expression of Vimentin and p-cantenin decreased in the tissue sections of the subcutaneously transplanted tumors of the TCM group;9.Fuzheng Jiedu Decoction can significantly up-regulate the expression of PDCD4 and E-cadherin protein in colon cancer xenografts in nude mice,decrease the expression of c-Jun,Vimentin and N-cadherin protein in tumor tissue(P<0.05),and can also decrease β-cantenin protein expression,but not significant.Conclusions:1.Fuzheng Jiedu Decoction can inhibit the proliferation of highly metastatic human colon cancer Lovo cells in vitro and in vivo.2.Fuzheng Jiedufang containing serum can effectively inhibit the invasion and metastasis of Lovo cells,which may be related to the influence of EMT process.3,Fuzhengjiedufang prescription containing serum can upregulate the expression of E-cadherin,PDCD4 protein,down-regulate the protein expression of N-cadherin,ββ-cantenin,c-Jun,was dose-dependent,high-dose group drug-containing serum effect is most significant;4.The serum containing Fuzheng Jiedufang can inhibit the expression of N-cadherin,Vimentin,MMP2,and β-cantenin mRNA in Lovo cells,and promote the expression of E-cadherin and PDCD4 mRNA.5.Fuzheng Jiedu Decoction can promote the expression of E-cadherin and PDCD4 protein in human colon cancer xenografts in nude mice and inhibit the expression of N-cadherin,Vimentin,β-cantenin and c-Jun proteins,which may affect colon cancer xenograft in nude mice of EMT.