The Mechanism Of Metastasis Associated Gene FMNL2 In Epithelial-mesenchymal Transition Of Colorectal Carcinoma

BACKGROUND & OBJECTIVEColorectal cancer(CRC) is one of the most common malignant tumors.During the last decade,incidence rate of colorectal cancer in our country showed an increasing trend.Metastasis is the main cause of affecting treatment and death of patients,Therefore,searching for effective and specific metastasis-related genes,and exploring their functions not only help to clarify the molecular mechanism of the metastasis of colorectal cancer,but also provide new markers for clinical diagnosis and treatment,drug screening.Epithelial mesenchymal transformation(EMT) refers to the transfer of epithelial cells to fibroblasts or mesenchymal cells morphologically and the aquirement of metastatic ability,which is a basic physiological and pathological phenomena,and plays an important role in embryonic development,organizations.In addition,EMT is also involved in a variety of pathological processes,such as wound healing,kidney fibrosis,cancer and metastasis.More and more studies have shown that EMT plays an important role in the invasion and metastasis of tumor cells,which has been a hot topic in the oncological research.In our preliminary study,we selected FMNL2 gene,a member of the family formins,as a new research target for colorectal cancer invasion and metastasis.At until now,there are only a few literatures reported about FMNL2,mainly focusing on its cloning,evolution and structural analysis.There was no other reported literature about its function especially in tumour,except for our research group.Our group firstly explored the mechanism of FMNL2 in CRC invasion and metastasis,and the results showed that FMNL2 was positively correlated with invasion and metastasis. FMNL2 promotes colon cancer cell movement and invasion through RhoA/ROCK signaling pathway.Whether is FMNL2 correlated with EMT of CRC or not? RhoA has been reported to be an important factor in the EMT of tumor.After CRC cells were silenced by FMNL2,their shape changed,from the spindle or similar to the morphology of fibroblasts to typical cobblestone-like shape.This morphological change may be caused by mesenchymal-epithelial transformation(MET).The gene chip results before and after FMNL2 silencing showed that many EMT related factors showed significantly expressed,such as paxillin,MAPK,AKT,PI3K,mainly involving in PI3K-AKT and Ras-MEK-MAPK signaling pathway.Thus,we speculated that the FMNL2 should play an important role in colorectal cancer EMT.This study focused on the relationship between FMNL2 genes and EMT of colorectal cancer and we aimed to explore the new way of FMNL2 involved in CRC metastasis increase the new insight of FMNL2 gene with unknown function,and provide the new clues for mechanism of tumor metastasis.METHODS1.Expressions of FMNL2 and EMT markers in colorectal carcinomas and their relationshipS-P immunohistochemical method was applied to detect the expression of FMNL2,EMT epithelial markers(E-cadherin,CK) and mesenchymal markers (Vimentin) in four colorectal cancer cell lines(SW620,SW480/M5,SW480,HT29) with different capacities of metastasis,138 cases of colorectal cancer and 30 cases of the corresponding normal colorectal mucosa expression.Then we analyzed their relevance. Fluorescence quantitative RT-PCR were applied to detect FMNL2,EMT epithelial markers(E-cadherin) and mesenchymal markers(Vimentin) mRNA level in above four colorectal cancer cell lines(SW620,M5,SW480,HT29).2.The effects on colorectal cancer EMT after FMNL2 silencingWe transfected the vector of FMNL2 interfere to cells;and selected obtain FMNL2 interference resistance monoclonal by G418.Then we cultured the cells and detect the stability of the interference by using fluorescence quantitative RT-PCR.The fluorescence quantitative RT-PCR and Western blot were applied to detect FMNL2, EMT epithelial markers(E-cadherin,α-catenin,γ-catenin),mesenchymal markers (Vimentin,Nβ-cateinin) and transcription factors(Snail,Slug) changes after silencing FMNL2.3.The impact of FMNL2 silencing on colorectal cancer EMT induced by TGF-β1The exogenous TGF-β1 was applied to induce EMT in colon cancer cell line (M5/mock,M5/FMNL2-) and canine kidney epithelial cell line MDCK,which was common in research in EMT.Then the morphologic was observed with inverted phase-contrast microscope.The fluorescence quantitative RT-PCR was applied to detecte the FMNL2,E-Cadherin and Vimentin mRNA level;Western blot was applied to detect the EMT epithelial marker(E-cadherin,α-catenin,γ-catenin),mesenchymal markers(Vimentin,Nβ-catenin) and transcription factors(Snail,Slug) expression.4.Colorectal cancer EMT-related signaling pathway involved by FMNL2The fluorescent quantitative PCR was applied to detect a number of target genes before and after FMNL2 silencing on the chip,in order to test the reliability of this gene chip.The Western Blot was applied to detect the level of EMT-related signaling molecules,including PI3K,AKT,MAPK,MEK,P-PI3K,P-AKT,P-MAPK and P-MEK,in stable-interference FMNL2 cell lines.RESULTS1.Expression of FMNL2 and EMT marker in colorectal carcinomas and their relationship The mesenchymal markers(Vimentin) and FMNL2 expression in high metastasis capacity SW620 and SW480/M5 colon cancer cell line is higher than the low capacity metastatic cell lines SW480 and HT29;But the expression of epithelial markers(E-cadherin,CK) showed an opposite trend to the mesenchymal markers.The expression of FMNL2 and Vimentin in the colorectal cancer was significantly higher than in normal mucosa.Their expression in the metastatic lymph node is higher than in colorectal cancer(P＜0.05),But the epithelial markers (E-cadherin,CK) expression in normal mucosa higher than in either colorectal cancer or metastatic ones.There was a negative correlation between expression of FMNL2 and epithelial marker(E-Cadherin and CK)(r=-0.377;P=0.000;r=-0.327,P= 0.000);While a positive correlation between expression of FMNL2 and mesenchymal marker Vimentin(r = 0.503,P = 0.000).These results suggest that the FMNL2 associated with colorectal cancer EMT may be through promote colorectal cancer cells transfer to EMT.2.The effects on colorectal cancer EMT after FMNL2 silencingThe interference efficiency of FMNL2 was above 75%.After FMNL2 silencing, the colorectal cancer cell morphologically changed,from spindle-shaped or similar fibroblasts morphology to the typical round or oval shape.The expression of epithelial markers E-cadherin,α-catenin,γ-catenin increased;the mesenchymal markers Vimentin and Nβ-catenin expression and transcription factors Snail and Slug expression have reduced.These results suggest that exogenous FMNL2 occurred in colorectal cancer cells can promote EMT.3.The impact of FMNL2 silencing on colorectal cancer EMT induced by TGF-β1TGF-β1 was applied to stimulate colon cancer cell line(M5/mock,M5/FMNL2-) and canine kidney epithelial MDCK cell line for about 48 hours,three cell lines morphological can changed obviously.The MDCK morphologically changed from round or oval to a spindle and fibroblast-like mesenchymal cells,which is a typical EMT changes;The M5/mock cells typically showed spindle or fibroblast similar cells and the M5/FMNL2- cells mostly round or oval under normal condition.After TGF-β1 stimulation,the M5/mock cells change into mesenchymal-like cells which some M5/FMNL2- cells also change into mesenchymal-like cells,but the also have some mesenchymal-like cells.The M5/FMNL2- cells changed into spindle-like stromal cells significantly reduced compare to M5/mock cells.By fluorescence quantitative PCR,we found that the expression of epithelial marker E-cadherin was significantly decreased,while the mesenchymal marker Vimentin and FMNL2 significantly increased.However,the M5/mock group cells EMT epithelial marker E-cadherin and the mesenchymal marker Vimentin changes of mRNA expression relative significantly compared to M5/FMNL2- cells.Western blot results showed a concordant result on protein level with mRNA levels.Expression ofα-Catenin andγ-Catenin were decreased,while nuclearβ-Catenin was decreased.The expression of transcription factors Snail and Slug were increased.The same result that the M5/mock group cells EMT epithelial marker,the mesenchymal marker and transcription factor changes of protein expression relative significantly compared to M5/FMNL2- cells.These results suggest that FMNL2 occurred in colorectal cancer cells can promote EMT.4.Colorectal cancer EMT-related signaling pathway involved by FMNL2The reliability of the result on chip have verified by the Fluorescence quantitative PCR.The EMT-related signaling molecules PI3K,AKT,MAPK,MEK, P-PI3K,P-AKT,P-MAPK,P-MEK protein decreased when FMNL2 was silenced. These results suggest that FMNL2 caused EMT mainly through PI3K-AKT and Ras-MEK-MAPK signaling pathway.CONCLUSION1.Metastasis-related gene FMNL2 associated with colorectal cancer EMT.2.Metastasis-related gene FMNL2 may promote the colorectal cancer EMT through PI3K-AKT and Ras-Raf-MAPK pathways.INNOVATION1.We put forward a new idea that FMNL2 take part in colorectal cancer metastasis through EMT for the first time. 2.Our study provided a new target for the mechanism of colorectal cancer metastasis.