The Mechanism Research Of LASP1 And ECHS1 On Promoting The Progression In Colorectal Cancer

Background and ObjectiveColorectal cancer is one of the most common digestive tract malignant tumors in China,and its diagnosis and treatment have attracted wide attention.It has been widely reported that a variety of molecules were involved in tumor invasion and metastasis.So far,little was known about the exact mechanism of colorectal cancer metastasis.The previous research of our group had expounded the importance of LASP1 in colorectal cancer.Using the proteomic research strategy of two-dimensional fluorescence differential gel electrophoresis combined with mass spectrometry,50 proteins related to LASP1 action were obtained.ECH was one of the proteins positively related to LASP1 action,and ECHS1 was its short chain.Some studies had shown that ECHS1 was involved in the occurrence and development of tumors,and this enzyme could be helpful in the diagnosis,treatment and prognosis of some tumors,but it was not very clear about the role and mechanism of ECHS1 in colorectal cancer,as well as the interaction and mechanism between LASP1 and ECHS1,and how they played molecular biological functions in colorectal cancer.We tried to further explain the relationship between them.It may provide a new target for screening,treatment and prognosis of colorectal cancer.Method1.the result of Western blot and q-PCR were displayed in colorectal cancer cell lines.Western blot and q-PCR analysis found that ECHS1 was a differentially expressed gene between colorectal cancer and normal intestinal mucosa.2.LASP1 positively regulated the expression of ECHS 1 in a protein level.2.1 We transferred LASP1 cDNA to RKO cell,ECHS1 mRNA or protein expression were measured by qPCR and Western Bloting.The results of qPCR indicated that ECHS1 mRNA expression were not influenced by LASP1(P>0.05)and Western Bloting revealed that LASP1 regulated the expression of ECHS 1.2.2 We used siRNA interference in sw480 cell line,in order to interfere the expression of LASP1.ECHS 1 mRNA or protein expression were measured by qPCR and Western Bloting.The results of qPCR indicated that ECHS1 mRNA expression were not influenced by LASP1(P>0.05)and Western Bloting revealed that LASP1 regulated the expression of ECHS1.2.3 Immunoflurescence found that LASP1 and ECHS1 were related and colocalized in protein level.3.The impact of silencing ECHS1 transiently on the capacity of proliferation,invasion in CRC cells.3.1 validation the efficiency of ECHS 1 which was transiently silencing siRNA were transfected in SW480 cell,Western blot test results showed that the level of ECHS1 protein was successfully knocked down than the paired SW480NC cell.3.2 The effect of silencing ECHS1 transiently on the capability of proliferation in CRC cancer cell.The results of CCK8 showed that interferencing ECHS1 transiently in SW480 cell resulted in statistically significantly decreasing the ability of growth compared with control(P<0.05).3.3 The effect of silencing ECHS1 transient on the capability of migration in CRC cancer cells.The results of transwell showed that ECHS1 interference transiently in SW480 cells resulted in statistically significantly decreasing the number of migrated cells compared with control(P<0.05).This result indicated that ECHS 1 silencing in vitro could inhibit the migration capability of CRC cells.4.LASP1 mediated the invasive phenotype of colorectal carcinoma through ECHS1.4.1 The verify of ECHS 1 transient silencing.Western blot results showed that:the expression levels of ECHS 1 protein drop after transfect interfering fragments,it means that the design and synthesis of siRNA can interfere significantly knock down of ECHS 1.4.2 In order to detect the changes of invasive phenotype induced by ECHS1’s protein levels in cells mediated by LASP1,we performed rescue assays.Firstly,we established several groups:NC,LASP1 and LASP1+si-ECHS1.We found that overexpression of LASP1 and ECHS1 knockdown meanwhile could significantly weaken the migration capacity mediated by LASP1(P<0.05).5.Immunohistochemistry(IHC)was used to detect the expression of LASP1 and ECHS1 in colorectal cancer samples and normal intestinal mucosa.The relationship between ECHS1 and clinicopathological parameters were analyzed.RESULTS1.Analysis the protein and mRNA expression of ECHS1 gene,we found that ECHS1 was a differentially expressed gene between colorectal cancerand normal colorectal cells.2.The results of qPCR indicated that ECHS 1 mRNA expression were not influenced by LASP1(P>0.05)and Western Bloting revealed that LASP1 positive correlated with the expression of ECHS 1.Immunoflurescence found that L ASP1 and ECHS1 were related colocalized in protein level.3.The effects of ECHS1 transient silencing on proliferation and invasion in colorectal cancer cell.The results of CCK8 showed that interferencing ECHS1 transiently in SW480 resulted in statistically significantly decreasing the ability of growth compared with control(P<0.05).Transwell assay showed that transient silencing of ECHS 1 in vitro can suppress the ability of invasion of colorectal cancer cells.4.LASP1 mediated the invasive phenotype of colorectal carcinoma through ECHS14.1 The verify of ECHS 1 transient silencing.Western blot results showed that:the expression levels of ECHS 1 protein droped after transfect interfering fragments,it means that the design and synthesis of siRNA can interfere significantly knock down of ECHS 1.4.2 In order to detect the changes of invasive phenotype induced by ECHS1’s protein levels in cells mediated by LASP1,we performed rescue assays.Firstly,we established several groups:BLANK,LASP1 and LASP1+si-ECHS1.We found that overexpression of LASP1 and ECHS1 knockdown meanwhile could significantly weaken the migration capacity mediated by LASP1(P<0.05).5.IHC experiments shows that ECHS1 was mainly localized in the cytoplasm of normal colorectal tissue;There were no significant differences of ECHS 1 expression in different age groups,different genders and different differentiation(P>0.05),but there were significant differences of ECHS 1 expression in different TNM stage.The overexpression rate of ECHS 1 in cytoplasm of lymph node metastasisstage was higher than those without lymph node metastasis.The overexpression rate of ECHS 1 protein expressed in cytoplasm in colorectal cancer tissue was higher than the overexpression rate of ECHS 1 protein expressed in cytoplasm in normal colorectal tissue.Conclusion1.The expression of ECHS1 protein and mRNA in colorectal cancer cells was higher than that in normal intestinal mucosal cells.2.Colorectal cancer metastasis-related protein LASP1 positively regulated the expression of ECHS1,and LASP1 and ECHS1 were colocated.3.In vitro experiments showed that interfering with ECHS1 significantly inhibited the proliferation and migration of colorectal cancer cells.4.LASP1 mediates the invasive phenotype of colorectal cancer cells through ECHS1.5.Compared with normal intestinal mucosa,the expression of ECHS 1 in colorectal cancer was significantly up-regulated.The high expression of ECHS 1 was related to the lymphatic metastasis of colorectal cancer.