| BackgroundsColorectal cancer(CRC)is one of the most common epithelial malignant gastrointestinal tumour.The incidence of CRC sustainable high and the mortality rate is on the rise.Recent years,studies shown that the activation of epithelial-mesenchymal transition(EMT)played important roles in the invasion and metastasis of cancer cells.EMT characterized by down-regulation of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers such as Vimentin,which epithelial cells acquired mesenchymal properties,resulting in generating cell metastases.Down-regulation of E-cadherin and the up-regulation of Vimentin were the specific hallmarks of activating EMT in colon cancer cells.Sphingosine kinase 1(SphK1)is an essential rate-limiting enzyme for the maintenance of sphingolipid balance in cells and plays an important role in the development and progression of variousmalignancies including colon cancer.In previous study,we found that the expression of SphK1 protein was associated with the expression of FAK and p-FAK proteins in colon cancer tissues and SphK1 maybe promoted the metastasis of colon cancer by regulating FAK/p-FAK axis.FAK is a non-receptor tyrosine kinase and known as focal adhesions.The signal transduction mediated by FAK promoted the adhesion of tumor cells to the extracellular matrix,which influenced cell adhesion,movement and migration.Besides,FAK played a key role in the EMT of cancer cells.However,it has not been reported whether SphK1 promotes the migration and EMT through FAK/p-FAK axis in colorectal cancer.This study intends to explore by vivo cell experiments and clinicopathological studies to illuminate whether SphK1 promotes the migration and EMT through FAK/p-FAK axis in colorectal cancer.ObjectivesPart I.To reveal that FAK inhibitor(PF-562271)suppress EMT and metastasis of colon cancer cells and SphK1 inhibitor(SKI-II)suppress EMT and metastasis through inhibiting FAK/p-FAK.Part II.To reveal that FAK knockdown suppressed the EMT and migration of RKO cells.SphK1 knockdown also suppressed the EMT and migration of RKO cells through inhibiting FAK/p-FAK.Part III.To reveal that over-expression of SphK1 strengthened the EMT and migration of HT29 cells through promoting FAK/p-FAK.Part IV.To reveal that FAK inhibitor(PF-562271)suppress EMT andmetastasis of SphK1-overexpressing HT29 cells,compared to without being treated by PF-562271.Part V.To reveal that the expression of SphK1,FAK,p-FAK,E-cadherin and Vimentin is related to the metastasis of cancer cells in colorectal cancer patients and SphK1 maybe used as a prognostic indicator on postoperative survival time of colorectal cancer patients.MethodsCell transfection was used to establish the stable cell lines model with over-expression of SphK1,down-regulation of SphK1 and down-regulation of FAK.MTT assay was used to detect the drug toxicity to cells.Transwell and wound healing assay were used to detect the ability of cell migration.Real-time reverse transcription-polymerase chain reaction(RT-PCR)analysis was used to detect the expression of mRNA.Western Blotting was used to detect the expression of protein.Scanning electron microscope was used to observe the microvilli and pseudopodia of the cells.Immunohistochemistry staining was used to detect the expression of protein in normal colonic mucosa tissues and colorectal cancer tissues.Part I.To detect the expression of EMT markers such as E-cadherin and vimentin,and cell migration with and without inhibiting the expression of FAK/p-FAK by FAK inhibitor(PF-562271)in RKO and HT29 cells.To detect the expression of FAK,p-FAK,E-cadherin and vimentin,and cell migration with and without inhibiting the expression of SphK1 by SphK1 inhibitor(SKI-II)in RKO and HT29 cells.Part II.Construction of stably transfected FAK down-regulation of RKO cell line to detect the changes on the expression of p-FAK,E-cadherin and vimentin,the microvilli and pseudopodia of cells,cell migration.Construction of stably transfected SphK1 down-regulation of RKO cell line to detect the changes on the expression of FAK,p-FAK,E-cadherin and vimentin,the microvilli and pseudopodia of cells,cell migration.Part III.Construction of stably transfected SphK1 over-expression of HT29 cell line to detect the changes on the expression of FAK,p-FAK,E-cadherin and vimentin,the microvilli and pseudopodia of cells,cell migration.Part IV.To detect the expression of FAK,p-FAK,E-cadherin and vimentin,the microvilli and pseudopodia of cells,cell migration with or without inhibiting the expression of FAK by FAK inhibitor(PF-562271)in SphK1 over-expression of HT29 cell.Part V.To detect the expression and the clinical significancer of SphK1,FAK,p-FAK,E-cadherin and Vimentin in 50 cases tissues of normal intestinal mucosa,63 cases tissues of colorectal cancer without metastasis and 51 cases tissues of colorectal cancer with metastasis.To regular telephone follow-up of the above patients with colorectal cancer.ResultsPart I.Suppression of FAK by FAK inhibitor(PF-562271)decreased the expression of p-FAK and Vimentin,while the expression of E-cadherin wasincreased in HT29 and RKO cells.The ability of cell migration of HT29 and RKO cells was weaken.Suppression of SphK1 by SphK1 inhibitor(SKI-II)decreased the expression of FAK,p-FAK and Vimentin in HT29 and RKO cells,while the expression of E-cadherin was increased.The ability of cell migration of HT29 and RKO cells was also weaken.The results indicated that suppression of FAK by FAK inhibitor(PF-562271)inhibited EMT through inhibiting FAK/p-FAK,resulting in weakening cell migration in colon cancer.Suppression of SphK1 by SphK1 inhibitor(SKI-II)inhibited EMT through inhibiting FAK phosphorylation to regulate FAK/p-FAK axis,resulting in weakening cell migration in colon cancer.Part II.The expression of FAK,p-FAK and Vimentin was decreased in stably transfected FAK down-regulation of RKO cells,while the expression of E-cadherin was increased.With stably transfected FAK down-regulation,the microvilli and pseudopodia of RKO cells were decreased and the ability of cell migration was weaken.The expression of p-FAK,Vimentin and SphK1 was decreased,FAK was no significant change while E-cadherin was increased in RKO cells with stably transfected SphK1 down-regulation.Moreover,with stably transfected SphK1 down-regulation,the microvilli and pseudopodia of RKO cells were decreased and the ability of cell migration was weaken.The results indicated that FAK knockdown inhibited EMT through inhibiting FAK/p-FAK,resulting in weakening cell migration in colon cancer.And SphK1 knockdown inhibited EMT through inhibiting FAK phosphorylation to regulateFAK/p-FAK axis,resulting in weakening cell migration in colon cancer.Part III.The expression of SphK1,p-FAK and Vimentin was increased,FAK was no significant change while E-cadherin was decreased in HT29 cells with stably transfected SphK1 over-expression.Moreover,with stably transfected SphK1 over-expression,the microvilli and pseudopodia of HT29 cells were increased and the ability of cell migration was strengthen.The results indicated that over-expression of SphK1 promoted EMT through increasing FAK phosphorylation to regulate FAK/p-FAK axis,resulting in strengthening cell migration in colon cancer.Part IV.Compared to without being treated by PF-562271,the expression of FAK,p-FAK and Vimentin was decreased,while E-cadherin was increased with suppressing the expression of FAK by PF-562271 in stably transfected SphK1 over-expression of HT29 cells.Compared to without being treated by PF-562271,the microvilli and pseudopodia of HT29 cells were decreased and the ability of cell migration was weaken with suppressing the expression of FAK by PF-562271 in stably transfected SphK1 over-expression of HT29 cells.The results indicated that the EMT and metastatic potency induced by over-expression of SphK1 were suppressed by FAK inhibitor in HT29 cells.Part V.The expression density and positive rate of SphK1,FAK,p-FAK and Vimentin in colorectal cancer tissues were higher than those of normal colonic mucosa tissues,while the expression of E-cadherin was slighter than that of normal colonic mucosa tissues.Moreover,there was a significant difference of the proteins expression between non-metastatic cancer tissues and metastatic cancer tissues.The positive expression rates of SphK1,FAK,p-FAK and Vimentin were higher in colorectal cancer tissues with the later stages(stage III and stage IV),lymph nodes metastasis and distant metastasis than those of colorectal cancer tissues with early stages(stage I and stage II),non-lymph nodes metastasis and non-distant metastasis,while the positive expression rate of E-cadherin was slighter.The prognosis of colorectal cancer patients with SphK1-positive was poorer than that of colorectal cancer patients with SphK1-negative.The results indicated that the expression of SphK1,FAK,p-FAK,E-cadherin and Vimentin were associated with metastasis in colorectal cancer.And SphK1 could be used as a prognostic indicator on postoperative survival time of colorectal cancer patients.Conclusions(1)The expression of SphK1 increased in advanced colorectal cancer patients and was associated with lymph node and distant metastasis.SphK1 could be used as an important molecule to predict metastasis and prognosis of colorectal cancer.(2)SphK1 promoted EMT through increasing FAK phosphorylation to regulate FAK/p-FAK axis,resulting in strengthening cell migration in colon cancer. |
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