| Background and Objective:Colorectal cancer(CRC)is a common digestive tract tumor.Although surgical resection is regarded as the standard curative treatment for CRC,the metastasis and recurrence rates after radical resection of CRC remain high.Thus,the research of molecular mechanisms involved in CRC metastasis and looking for effective new method to prevent and treat CRC metastasis were key problems in the field of oncology.Human Rho-associated coiled-coil containing protein kinase 2(Rock2)is a downstream effector of Rho.The studies have shown that Rock2 plays an important role in the tumorigenesis and progression of malignant tumors,and it is involved in the regulation of a variety of biological behavior and function of cells.However,the expression of Rock2 in CRCs and its function in the process of CRC metastasis remain unclear.The protein ?-catenin,a key mediator of the ?-catenin/TCF4 signaling pathway,plays an important role in CRC carcinogenesis and tumor metastasis.It is well known that ?-catenin is targeted to ubiquitin-proteasome-mediated degradation.In addition,we previously demonstrated that Rock2 can modify the ubiquitin degradation of MMP2 and Cdc25 A.Thus,we presumed that Rock2 might also regulate ?-catenin by affecting ?-catenin ubiquitination degradation.We first analyzed the expression of Rock2 and its clinical significance in CRCs.Next,We investigated the effect of Rock2 gene on invasion and metastasis of colon cancer cells both in vitro and in vivo.Then,the effect of Rock2 on the-catenin/TCF4 signal pathway in colon cancer cells was analyzed.Finally,we clarified the mechanism through which Rock2 regulates ?-catenin/TCF4.In this study,we investigated the mechanism of Rock2 regulates the invasion and metastasis of CRC,providing the new theoretical basis for clarifying the mechanism of CRC invasion and metastasis.Methods:1.The expression of Rock2 in 116 CRC tissue samples and the corresponding adjacent tissues were detected by qRT-PCR,immunohistochemistry(IHC)and western blotting.We also analyzed the connection between the expression of Rock2 and clinicopathological parameters of 116 CRC patients.2.The expression of Rock2 in SW480,SW620,LOVO,DLD-1 and HCT-116 cells were detected by qRT-PCR and western blotting.Four shRock2 plasmids were constructed,the best Rock2 interference plasmid was screened by qRT-PCR and western blotting.The effects of Rock2 on CRC cell invasion and metastasis was investigated by transwell assays and Wound-healing assays.The effect of down-regulation of Rock2 expression on tumor metastasis was evaluated by orthotopic transplantation model of human colorectal tumor in nude mice.3.The effect of up-or down-regulation of Rock2 on ?-catenin expression and the ?-catenin/Tcf4 transcriptional activity were detected by western blotting and luciferase reporter gene assay.Additionly,we activated the?-catenin/TCF4 pathway in Rock2 knockdown colon cancer cells or inhibited the?-catenin/TCF4 pathway in Rock2-overexpressing colon cancer cells,and then observed the expression of Rock2 and the other related downstream target genes.The cell migration and invasion abilities were also detected by transwell assays.4.The interaction between ?-catenin and ubiquitin in colon cancer cells was assessed by Co-IP and Colocalization assays.The effects of variable Rock2 on ?-catenin expression,either with or without the MG132 or CHX,were detected by western blotting.The effect of up or down-regulation of Rock2 on the level of ubiquitination of ?-catenin was assessed by by immunoprecipitation.Results:1.The qRT-PCR experiments revealed that the average fold change of Rock2 mRNA expression in tumor tissues was significantly higher than in paired nontumor tissues.The IHC results showed that the Rock2 protein was highly expressed in62.07%(72/116)of the CRC tissue samples and in only 11.21%(13/116)of the adjacent tissues.Additionally,the western blotting results showed that the Rock2 protein levels were significantly elevated in the CRC tissues,which was consistentwith the IHC results.In addition,the statistical analysis found that high Rock2 expression correlated with lymphatic invasion(P<0.001),venous invasion(P<0.001),higher T stage(P<0.001),lymph node metastasis(P<0.001),distant metastasis(P=0.011)and advanced clinical stage(P<0.001).Furthermore,we found that patients whose tumors showed high Rock2 expression had significantly shorter survival times than patients whose tumors showed low Rock2 expression,based on Kaplan–Meier analysis(P=0.027).2.We manipulated the Rock2 levels by the stable transfection of Rock2 shRNA or a Rock inhibitor(Y27632)into SW480 and LOVO cells,the results showed that the expression levels of Rock2 mRNA and protein were significantly decreased(p<0.05),and then the cell migration and invasion abilities were also decreased(p<0.05).However,the expression levels of Rock2 mRNA and protein were significantly increased after transfection with Rock2 plasmid to HCT-116 cell,and the cell migration and invasion abilities were also enhanced(p<0.05).In addition,the in vivo metastasis experiment further confirmed that the tumors formed by SW480-shRock2 cells showed decreased liver metastasis compared with the tumors formed by the control cells(p<0.05).3.Knockdown of Rock2 can significantly decrease the total and nuclear ?-catenin protein expression levels and the transcriptional activity of TCF4 in SW480 and LOVO cells.We also found that the knockdown of Rock2 decreased the other Wnt pathway signaling components,including HOXB9 and MMP-7 in SW480 and LOVO cells,whereas the upregulation of ?-catenin attenuated the loss of these protein expression levels in Rock2-knockdown SW480 and LOVO cells(p<0.05).Further transwell assay results revealed that the upregulation of ?-catenin rescued the decreased cell migration and invasion induced by the knockdown of Rock2.Additionally,We found that the upregulation of Rock2 expression significantly increased the ?-catenin expression level,the transcriptional activity of TCF4,and the expression of the HOXB9 and MMP7 proteins were also increased in HCT-116cells(p<0.05),whereas the downregulation of ?-catenin dramatically inhibited the increase in these protein expression levels in HCT-116-Rock2 cells(p<0.05).Meanwhile,transwell assay results showed that the downregulation of ?-catenindramatically decreased Rock2-induced cell migration and invasion(p<0.05).4.The co-immunoprecipitation and colocalization assays using confocal microscopy results showed that the endogenous ?-catenin and ubiquitin are directly bound in SW480 and LOVO cells,Furthermore,treatment with the proteasome inhibitor MG132 for the indicated time resulted in signi ficant accumulation of the endogenous ?-catenin protein.These results demonstrated that ?-catenin is also degraded by the biquitin-proteasome system(UPS)in CRC cells.We found that reducing or increasing Rock2 exhibited no significant impact on ?-catenin expression in SW480 and LOVO cells after treatment with MG132(Fig.4D).Moreover,the degradation dynamics assay showed that the half-life of the ectopically?-catenin was significantly increased in the Rock2-overexpressing SW480 and LOVO cells compared with the control cells fter treatment with CHX.These results suggest that Rock2 was involved in the degradation of ?-catenin.In addition,we also found that Rock2 can stabilize the expression of ?-catenin.Finally,The IP results showed that the knockdown of Rock2 could increase the levels of ?-catenin ubiquitination,whereas Rock2 overexpression significantly reduced the levels of ?-catenin ubiquitination.These results suggest that Rock2 stabilizes ?-catenin expression by regulating ?-catenin ubiquitination.Conclusion:1.Rock2 is overexpressed in CRCs,and is associated with the progression and outcome of CRC.2.Rock2 promotes the invasion and metastasis of CRC by modifying ?-catenin degradation,providing a novel mechanism for the regulation of ?-catenin expression and greater insight into our understanding of the mechanism of metastasis in human CRC. |
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