ARRB2 Promotes Colorectal Cancer Cell Growth Through Triggering WTAP

BackgroundsColorectal cancer(CRC)is the third cause of cancer-related deaths among and women that annually is killing more than 700 000 patients in the world.Colorectal cancer patients with metastases have an extremely poor prognosis,and the 5-year overall survival(OS)rate of CRC is about 65%.Thus,exploring new targets for colorectal cancer treatment is important.There are two β-Arrestin proteins,namely β-Arrestin1(ARRB1)and β-Arrestin2 ARRB2),which are expressed ubiquitously.They act downstream of G-protein coupled receptors(GPCRs),are well known negative regulators of GPCR signaling.β-Arrestins regulate every physiological process in fundamental cellular functions,including growth and migration.For example,Likewise,ARRB2 promotes myeloid leukemia initiation and progression via regulating Wnt signaling,and it also induces tumor cell proliferation and metastasis.Several studies have been reported that cancers express high levels of ARRB2,which was suggested to regulate the cancer cell proliferation and invasion.However,hepatocellular carcinoma tumor with downregulation of ARRB2 expression promotes invasion.Masannat study showed that ARRB2 expression is increased in CRC tumor compared to normal tissue and that high levels of ARRB2 correlate with worse patient survival.And found ARRB2 promotes tumor growth via regulates c-Src activity,Cyclin A expression.However,the precise role of ARRB2 in migration and proliferation of CRC cells is unknown.In the present study,we report that ARRB2 is highly expressed in colorectal cancer tissue and colorectal cancer cells compare with normal tissue and normal cell,which promotes cell proliferation and migration.Furthermore,we found that inhibition of ARRB2 by shRNA attenuates the progression of CRC mediated by downregulating WTAP expression.These results suggest that colorectal cancer cells regulating WTAP expression to promote colorectal cell proliferation and migration.Targeting ARRB2 may represent a promising approach for selectively causing cell proliferation and migration in colorectal cancer cells.Contents and methods1.To identify the ARRB2 expression in colorectal cancer tissue and colorectal cancer cells.(1)Based on the TCGA database,using the Kaplan-Meier survival analysis determined the expression of ARRB2 in colorectal cancer tissues.(2)Immunohistochemistry(IHC)staining showed the expression of ARRB2 in the in specimen of normal colonic mucosal,ulcerative colitis and colorectal cancer tissue.(3)Western Blotting and IHC staining showed the expression of ARRB2 in colorectal cancer cell.2.To identify the deficiency of ARRB2 influenced the Progression of CRC.(1)ARRB2 WT and KO mice treated with AOM/DSS to determine the role of ARRB2 in the Progression of colorectal cancer.(2)Appling the colonoscopic imaging monitored the progression of CRC,colonoscopic imaging was obtained after AOM/DSS treatment at week 4 and 18.(3)Histological morphology of hematoxylin-eosin stained colon tissue sections of representative ARRB2 WT and KO colon sections after AOM/DSS treatment at week 4 and 18.(4)Western blotting showed the expression of PCNA,p-STAT3 and MMP2 in ARRB2 WT not in KO AOM/DSS treated group colon tissues.3.To identify the Suppression of ARRB2 inhibit colorectal cancer cell proliferation,cell cycle,migration and invasion in vitro.(1)Establishing ARRB2 stable knock down HCT116 and RKO cells using two shRNA.(2)The in vitro proliferation function of ARRB2 was measured by the CCK8 assay in HCT116and RKO.(3)The colony formation assays revealed the influence of influence with the inhibition of ARRB2 by shRNA.(4)Flow cytometry analyzed the influence in ARRB2 stable knocked down cells.(5)Transwell migration assay showed that ARRB2 knockdown influenced colorectal cancer HCT116 and RKO cells migration and invasion in vitro.(6)Scratch Test showing a reduced number of invasion and migration formed by HCT116 and RKO ARRB2 knockdown cells compared to control.(7)Western blotting assay showed that knockdown ARRB2 influenced the expression of PCNA,MMP2 and MMP9.4.To identify the Suppression between shRNA and shRNA-resistant ARRB2,and revealed the differences in cell proliferation,cell cycle,migration and invasion.(1)CCK8 assay measured the in vitro proliferation function of ARRB2 in control,shRNA2 and shRNA-resistant HCT116 cells.shRNA-resistant ARRB2 inhibits ARRB2 knockdown-induced cell growth defect.(2)Flow CytoMetry showed the representative image in control,shRNA2 and shRNA-resistant HCT116 cells.(3)Representative images of harvested tumors from nude mice subcutaneously injected with control,shRNA2 and shRNA-resistant HCT116 cells,then measured the difference in volume and weight.5.To identify the genes expression Profile and Key Pathways in CRC by Blocking of ARRB2,and analysed ARRB2 influences colorectal cancer cell proliferation via interacting with WTAP.(1)To identify ARRB2 downstream effectors by RNA sequencing(RNA-seq)using ARRB2 stable knockdown(KD)HCT116 colorectal cancer cell under TNF-αtreated.(2)Establishing ARRB2 stable knocked down HCT116 cells using shRNA and shRNA-resistant ARRB2,and revealed the differences in cell proliferation,cell cycle and the expression of WTAP.(3)Applying the Chip assay showed that ARRB2 binding to WTAP DNA.(4)Applying immunohistochemistry(IHC)staining assay determined the expression of ARRB2 and WTAP in colon cancer specimens and showed the correlation between ARB2 and WTAP by Pearson correlation coefficient analysis.Results1.Based on the TCGA database,the expression of ARRB2 in the colorectal cancer tissue is higher than in specimen of normal colonic mucosal.Higher levels of ARRB2are significantly correlated with reduced overall survival compared to low ARRB2 levels.2.Tumor rate and the number of macroscopic neoplasms were increased in ARRB2 WT not KO group when treated with AOM/DSS,and western blotting showed that PCNA,p-STAT3 and MMP2 in increase in ARRB2 WT not in KO AOM/DSS treated group colon tissues.3.The inhibition of ARRB2 by shRNA in HCT116 and RKO cells decreased cancer cell proliferation and reduced the colony formation,then induced robust apoptosis.ARRB2 knockdown suppressed colorectal cancer HCT116 and RKO cells migration and invasion in vitro.Western blotting assay showed that knockdown ARRB2 reducated the expression of PCNA,MMP2 and MMP9.4.Compared with shRNA-2,shRNA-resistant has the ability of cell proliferation,migration and invasion restored and approached the level of CTL group.5.RNA sequencing showed that the WTAP gene is the top one in the down-regulated gene in TNF-α treated HCT116 cells with ARRB2 knocked down.The staining level for ARRB2 and WTAP were decreased in in xenograft specimens when knocking down ARRB2 in shRNA group not in shRNA-resistant group.KEGG analysis confirmed that the ability of proliferation,migration and invasion of colon cancer was ultimately reduced by affecting wtap transcription and Wnt pathway expression after the knockdown of ARRB2.The ChIP assay showed that ARRB2 binding to WTAP DNA,and Pearson correlation coefficient analysis showed that WTAP are correlation with ARRB2 upregulated in colorectal cancer.ConclusionsIn this study,we found that ARRB2 is commonly upregulated in colorectal cancer and is important for tumorigenesis.Knockdown ARRB2 inhibit colorectal cancer cell proliferation and migration.ARRB2-mediated WTAP expression increases,resulting in promotion of cell proliferation and migration.Thus,ARRB2 may be a therapeutic strategy to suppress tumor growth and metastasis.