Effect And Molecular Mechanism Of Protein 4.1N On EMT Of Colon Cancer

BackgroundColon cancer is one of the most common malignant tumors in the world.It originates from tumors with extremely high malignancy of colonic epithelial cells.According to cancer data analysis,colon cancer has the highest incidence and mortality in the forefront of various malignant tumors.Its invasion and metastasis are affecting the prognosis of patients seriously,and they cause high mortality either.Tumor metastasis is a multi-step dynamic process which tumor cells migrate out of the blood vessels and then spread to distant organs.Epithelial-mesenchymal transition(EMT)is an important step in tumor metastasis and could affect colon cancer cells metastatic ability seriously.Therefore,studying the molecular mechanism of EMT in colon cancer cells may provide new directions and ideas for treating colon cancer.A large number of studies have proved that the EMT process of tumor cells is related to the remodeling of cytoskeleton closely,and it also involves the coordinated regulation of various signaling pathways,especially the EMT transcription factors interact with miRNAs,regulating the process of tumor EMT.Researching the molecular mechanism of tumor metastasis has become one of the hotspots.As a key molecule of membrane skeletal protein,protein 4.1 plays an important role in the regulation of tumor EMT,and related to tumorigenesis,invasion and metastasis closely.Protein 4.1N is a member of the protein 4.1 family and associated with a variety of tumor metastases.Some researchers overexpressed protein 4.1N in lung cancer cells,and the results indicate that the expression of various EMT-related marker proteins in lung cancer cells has changed.In conclusion,protein 4.1N is related to the process of colon cancer development and metastasis,and may involve in the regulation of colon cancer EMT.Therefore,protein 4.1N affects the process of tumor metastasis,but the mechanism has not been revealed,it still remains to be explored.So,to explore the molecular mechanism of how protein 4.1N regulates EMT of colon csancer is significant for understanding the metastasis of colon cancer.Objective The regulation and biological significance of protein 4.1N in colon cancer EMT were preliminarily elucidated by studying the regulatory relationship between protein 4.1N and various important molecules in colon cancer EMT.Methods 1.Indicate the expression of protein 4.1N in colon cancer cells HT29 and RKO.Construct the RKO cells overexpress protein 4.1N and detect the expression of protein 4.1N.2.Detect the effects of protein 4.1N on EMT marker(E-cadherin,Vimentin)、EMT related transcription factors(ZEB1/2,Snail1,Slug,Twist)and EMT effect protein matrix metalloproteinases(MMPs)in colon cancer cells.3.Detect the effects of protein 4.1N on the proliferation 、migration and invasion ability of RKO cells.4.MiRNA-Seq was used to detect the different expression of miRNA associated with EMT in colon cancer cell lines with different protein 4.1N expression and verify the expression of candidate miRNA,miR-200 s.5.Detect the effect of protein 4.1N on the Raf-MEK-ERK pathway,and affirm the interaction between protein 4.1N and RhoA initially.ERK inhibitor was used and then detected miR-200 family、EMT marker and EMT related transcription factors expression.6.Use miR-200 s inhibitor and then detect the expression of the expression of EMT marker and transcription factors.On the other hand,use miR-200 s mimics and detect the expression of protein 4.1N.7.The subcutaneous graft tumor and lung metastasis model were established on balb/c nude mice,count the tumor size 、growth curve and number of pulmonary surface metastases.HE staining and immunehistochemical staining were used to analyze the EMT marker,to further clarify the role of 4.1N in the invasion and metastasis of colon cancer.Results 1.In human colon cancer cell line HT29,4.1N expression is normal,but in RKO cells,4.1N expression was lack.Constructed the eukaryotic expression vector pEGFP-C3-4.1N and transfected EPB41L1 gene to RKO cells successfully and RKO cells were expressed 4.1N after transfected EPB41L1 gene.2.After expressed protein 4.1N exogenously,the epithelial marker E-cadherin was increased,and the mesenchymal marker waveform protein(Vimentin),EMT effect protein matrix metalloproteinases MMP-2 and MMP-9 appeared reduced.3.Protein 4.1N has inhibitory effect on the proliferation,migration and invasion of RKO cells in vitro.4.After expressed protein 4.1N exogenously,miR-200b、c in RKO cells increase obviously,ZEB expression was reduced significantly.After co-transfection of 4.1N and miR-200 family inhibitor,the expression variation of EMT marker and effect protein was inhibited.After transfection of miR-200 family mimics,there was no significant effect on 4.1N expression.5.After expressed protein 4.1N exogenously,the phosphorylation level of protein in Raf-MEK-ERK signaling pathway was inhibited,miR-200b/c expression increased obviously,and 4.1N was interacting with RhoA protein.6.The subcutaneous graft tumor and lung metastasis model were established in balb/c nude mice to verify that protein 4.1N had some inhibitory effect on subcutaneous graft tumor and lung metastasis of colon cancer.ConclusionProtein 4.1N inhibits the proliferation、migration and invasion in RKO cells,and inhibits the EMT pathway of RKO cells by inhibiting the Raf-MEK-ERK signaling pathway.It interacts with RhoA and then affects the negative feedback regulation loop of miR-200 and transcription factor ZEB.Thus,inhibits the metastasis of tumor cells.The results of animal experiments further confirmed that protein 4.1N could inhibit the growth of subcutaneous transplanted tumor of colon cancer,and also had some inhibitory effect on lung metastasis in mice lung metastasis model significantly.