Silencing ARK5 Reduces Metastasis And Aggressiveness Of Colorectal Cancer By Regulating EMT

Objective: Colorectal cancer is a common malignant tumor of the digestive tract.Previous studies have found that protein kinase-related kinase 5(ARK5)can promote the metastasis of various cancers and enhance the aggressiveness of cancer.Recent evidence supports epithelial-mesenchymal transition(EMT)as the only way for tumor cell metastasis.In this study,we will explore whether ARK5 affects the metastasis and invasion of colorectal cancer by regulating the EMT.Method:1.A total of 134 patients with colorectal cancer who underwent radical surgery and no preoperative chemoradiotherapy were selected randomly from January 2013 to January 2014.Colorectal tumor tissues and adjacent normal tissues were obtained from 134 patients.ARK5 and epithelial-mesenchymal transition(EMT)related markers were detected and scored by immunohistochemistry.The scores were statistically analyzed with the patient's clinical stage,OS and other related data.2.Design RNA interference(RNAi)sequences and negative control(NC)sequences for ARK5 gene and TWIST gene,and complete the LV-ARK5-RNAi recombinant lentiviral vector and LV-TWIST-RNAi recombinant slow construction of viral vectors and negative recombinant lentiviral vectors.3.Infect SW480 and HT29 cells with a negative recombinant lentiviral vector carrying fluorescence(GFP),observe with a fluorescent inverted microscope and select the most effective infection conditions.4.Culture two human colon cancer cells,SW480 and HT29,for further experiments.5.Taking SW480 as an example,it is divided into six groups: I is SW480;II is infected with SW480 + ARK5 shRNA virus;III is SW480 + TWIST shRNA infection;IV is SW480 + TGF-?1 enhanced group;V SW480 + TGF-?1 + ARK5 shRNA infection group;VI was infected with SW480 + NC.HT29 cells were divided into the same 6 groups.6.Western blot technology was used to test ARK5,E-cadherin,Vimentin and ?-actin in 12 groups of cells.7.Use the wound healing assay to reflect the migration ability of the two groups of 12 cells.8.Trans-well chamber model was used to reflect the invasion ability of two groups of 12 groups of cells.Results:1.Clinical statistics: The expression levels of ARK5 and Vimentin in colorectal cancer tissues are significantly higher than those in adjacent normal intestinal mucosa tissues(p=0.002 and p=0.0144,respectively).In contrast,E-cadherin showed lower expression levels in colorectal cancer tissues(p<0.001).The expression level of ARK5 in colorectal cancer tissues was positively correlated with the expression of Vimentin(p=0.0144),but negatively correlated with the expression of E-cadherin(p <0.001).Clinicopathological analysis of 134 colorectal cancer patients showed that the expression levels of ARK5,E-cadherin,and Vimentin were correlated with T stage,pTNM stage,and lymph node metastasis,(p<0.05).The immunohistochemical score of ARK5 in patients with lymph node metastasis was significantly higher than that in patients without metastasis(p<0.001).ARK5 positive,Vimentin positive but Ecadherin negative in colorectal cancer patients were associated with shorter overall survival,(p <0.05).2.Pre-experimental results of lentiviral infection: LV-ARK5-RNAi recombinant lentivirus transfects SW480 cells with an optimal MOI of 20 and the infection system is a 1640 low-sugar medium containing 10% fetal bovine serum(FBS)+ HiTransG A(40ul),infection time is 24h;LV-ARK5-RNAi recombinant lentivirus transfects HT29 cells with the best MOI of 20,and the infection system is 10% FBS-containing DMEM low-sugar medium + HiTransG P(40ul),the infection time is 24 hours;the optimal reinfection index MOI of SW480 cells transfected with LV-TWIST-RNAi recombinant lentivirus is 60,and the infection system is 1640 low-sugar medium containing 10% FBS + HiTransG P(40ul),infection time the optimal reinfection index MOI for HT29 cells transfected with LV-TWIST-RNAi recombinant lentivirus was 60.The infection system was DMEM low-sugar medium containing 10% FBS + HiTransG A(40ul),and the infection time was 24 h.3.Western Blot: In both SW480 and HT29 cells,E-cadherin was up-regulated when ARK5 was blocked down by lentivirus,and Vimentin was down-regulated.In the same two cells,after the EMT pathway was blocked by TWIST,E-cadherin was up-regulated and Vimentin was down-regulated;and ARK5 protein expression did not change.Finally,after the EMT pathway was enhanced with TGF-?1,and then infected with ARK5 lentivirus to prevent expression,ARK5 and Vimentin was down-regulated,Ecadherin was up-regulated.The statistical difference analysis showed that the difference was statistically significant(P <0.05).4.Wound healing assay: In SW480 and HT29 strains.The wound healing speed of ARK5 lentivirus was significantly lower than that of parental cell group and negative virus control group.Similarly,the group of cells with reduced wound healing speed was blocked by the TWIST lentivirus,and the group of cells blocked by the ARK5 lentivirus after the EMT pathway was enhanced by TGF-?1.After enhancing the EMT pathway by TGF-?1,the healing speed increased significantly,significantly faster than the parental cells and the negative virus control group.The difference in the results of scratch area analysis was statistically significant(P <0.05).5.Cell migration and invasion assays: In SW480 and HT29 strains.After blocking by ARK5 lentivirus,the number of cells fixed in the compartment was significantly smaller than that of the parental cell group and the negative virus control group.The group of cells that blocked the EMT pathway by TWIST lentivirus,and the group of cells that blocked ARK5 expression by ARK5 lentivirus after enhancing the EMT pathway by TGF-?1,all showed that the number of cells fixed in the upper compartment was less than that of parent cells and Negative virus control group.After the EMT pathway was enhanced by TGF-?1,the number of fixed cells in the upper compartment was significantly higher than that of parental cells and negative virus control group.The difference in cell counts in the upper compartment was statistically significant(P <0.05).Conclusions:The expression of ARK5 is closely related to the clinical stage of the tumor,lymph node metastasis,and the survival time of patients.The ability of ARK5 to invade and metastasize tumor cells in colorectal cancer can be regulated by epithelialmesenchymal transition(EMT).In cell experiments that simply blocked ARK5 and blocked ARK5 again after enhancing the EMT pathway with TGF-?1,it can be seen that after ARK5 is blocked,ARK5 is down-regulated,E-cadherin is up-regulated,and Vimentin is down-regulated;the healing of the scratch area is slowed down and the invasion ability decline.However,E-cadherin up-regulation and Vimentin downregulation also occurred after the TWIST lentivirus was used to block the EMT pathway.The healing of the scratch area was slowed and the invasion capacity was reduced,but the expression of ARK5 protein was not affected.It can be seen that ARK5 may regulate the EMT pathway upstream.