| Colon cancer is the third most commonly diagnosed cancer among both men and women in China,only next to lung cancer and gastric cancer.The mortality of colon cancer ranks the fifth.Its pathogenesis mainly includes gene mutation,epigenetic changes,inflammatory damage,intestinal barrier dysfunction and so on.Metastasis accounts for the main reason for poor prognosis of colon cancer.Tumor metastasis is a multi-step process in which tumor cells move from the primary site to other tissues,but many of the mechanisms involved in metastasis remain unclear.In the past few decades,many studies showed that the endothelial-to-mesenchymal transition(EMT)is one of the most important factors leading to tumor invasion and metastasis,and plays an important role in the early stage of disease progression.EMT refers to the transformation of epithelial phenotype into mesenchymal phenotype.The hallmarks of EMT include the loss of expression or function of E-cadherin and reduced abundance of tight junction proteins,such as zona occludens 1(ZO-1)and occluding,and cytokeratins,as well as concomitant increase in abundance of mesenchymal markers,such as vimentin,fibronectin,fibroblast specific protein 1(FSP-1),a-smooth muscle actin(a-SMA),and N-cadherin;in addition,the morphology of cells also changes to fibroblast-like cells;cell to cell junction is weakened;the cell polarization changes,which will cause decreased intercellular association and junction and become more invasive.The process of EMT is very complex and is influenced by many signaling pathways.Some regulators have promotion or inhibition roles on EMT,such as transforming growth factor-beta(TGF-?),tumor necrosis factor-alpha(TNF-a),interleukin-6(IL-6),macrophage migration inhibitory factor(MIF)and so on.In colon cancer,it was found that TGF-?1 and TNF-a synergistically promote the EMT of colon epithelial cells,but it still needs further studies to clarify the precise regulatory mechanisms and find new targets to interfere with the occurrence and development of colon cancer.In addition to transcriptional regulation,many studies have suggested that cell motility may also participates in tumor progression through the effects on EMT.Recent studies have found that cytoskeleton regulatory proteins may also have regulatory roles on EMT.For example,the high expression of profibrin 2(PFN2)as a cytoskeleton regulatory protein in head and neck squamous epithelial cell carcinoma is associated with poor prognosis of the disease;in highly invasive non-small cell lung cancer,targeting PFN2 to inhibit EMT may be an effective intervention for the cancer progression,but the specific molecular mechanism has not been elucidated meanwhile very little study on colon cancer.Based on the theory mentioned above,the aim of this study:is to analyze the regulatory effect of PFN2 on the EMT of colon cancer,therefore,to investigate its impact on the metastasis of colon cancer and the within cell molecular mechanism.Thus,to offer some theoretical and experimental proofs.Main research methods:The expression of PFN2 was checked by immunohistochemistry,Westernblot and Realtime-PCR in normal colon tissues as well as colon cancer tissues,combined with the clinical manifestation and the prognosis,thus to evaluate the relationship between the expression level of tumorigenic phenotypes.Then,we tested the PFN2 expression in different human colon cancer cell lines by Westernblot,and chose the high PFN2 expression cell-HCT116(low metastatic ability)and low PFN2 expression cell-SW620(high metastatic ability)as experimental colon cancer cell representants.The wound healing and Tranwell assay were performed to verify the correlation between PFN2 and cell metastasis ability,also tested the expression of EMT related molecules by Westernblot in HCT116 and SW620 cells.After that,PFN2 was overexpressed in SW620 cells by transfection with plasmid.The metastatic ability of PFN2 overexpressed SW620 cells were tested by wound healing and Tranwell assay,meanwhile,in vivo liver and lung cancer metastasis nude mice model was established to strengthen the result.The phosphorylation of myosin light chain(MLC)was checked by Westernblot in PFN2 overexpressed SW620 cells and the changing of EMT of such cells was observed as well,thus to analyze the molecular mechanism of how PFN2 regulates the EMT in colon cancer cells.Results:In our study,we found that PFN2 expression was negatively associated with the degree of tumor metastasis.Low PFN2 expression in colon cancer cells was related with enhanced epithelial-mesenchymal transition(EMT)and,in turn,may increase migratory capabilities.Overexpression of PFN2 in colon cancer cell lines with a low level of endogenous PFN2 inhibited the EMT process,as well as the associated migration;in addition,myosin light chain(MLC)phosphorylation was upregulated.Inhibition of MLC phosphorylation attenuated the inhibition of EMT and cell migratory abilities induced by PFN2 overexpression in colon cancer cell lines,the results suggested that PFN2 may suppress colon cancer EMT and the subsequent metastasis by regulating cytoskeletal reorganization.Conclusion:from a new perspective of cytoskeleton regulation,these results demonstrated that PFN2 may serve a suppressive role in the metastasis of colon cancer by inhibition of EMT,which provides a theoretical basis for finding new prognostic indicators and intervention targets of colon cancer. |
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