| Objective: In this study,we investigated the effect of β-arrestin1 on epithelial-mesenchymal transition and invasion and metastasis of colorectal cancer through GSK-3β/β-catenin signaling pathway.In order to provide experimental evidence for targeting therapy of tanshinone ⅡA against metastasis of colorectal cancer,we investigate the regulation mechanism of inhibitory effect of tanshinone ⅡA on EMT and lung metastasis in colorectal cancer.Methods:(1)The relationship between the expression of β-arrestin1 and the prognosis of colorectal cancer was analyzed using GSE41258 database.(2)Bioinformatics was used to analyze the target and pathway of β-arrestin1,and the biological network of“β-arrestin1-target” was constructed.(3)The protein expression of β-arrestin1 in primary and metastatic lesions of colorectal cancer was analyzed by immunohistochemistry,and the correlation between the expression of β-arrestin1 and clinicopathological features of colorectal cancer was discussed.(4)To construct recombinant lentiviral vectors with interference and overexpression of β-arrestin1 gene,and to infect and screen stable interference and overexpression of β-arrestin1 gene in colorectal cancer cell lines.(5)We used transwell,cell scratch,western blot and immunofluorescence to investigate the effects of β-arrestin1 on epithelial-mesenchymal transformation and invasion and metastasis of colorectal cancer cells.The effects of β-arrestin1 on Wnt/beta-catenin signaling pathway and the expression of downstream gene were detected by western blot,the location and expression of β-catenin in colorectal cancer cells were detected by immunofluorescence.Luciferase-labeled lentiviruses were used to construct sh RNA/β-arrestin1/Luc and over/βarrestin1/Luc HCT-116 cells for animal experiment.Lung metastasis model of colorectal cancer in nude mice was established by tail vein injection.We observed the effects of β-arrestin1 on lung metastasis and survival time of tumor-bearing mice.The expressions of E-cadherin,N-cadherin,Vimentin and Snail in lung metastases were detected by immunohistochemistry and western blot.(6)After the intervention of tanshinone ⅡA on HCT-116 and Lo Vo cells,we used transwell,scratch and western blot to study the effects of tanshinone ⅡA on EMT,invasion and metastasis through β-arrestin1/GSK-3β/β-catenin signaling pathway.Luciferase-labeled HCT-116 cells were used to establish lung metastasis model of colorectal cancer in nude mice.The effects on lung metastasis and survival time of tumor-bearing mice were observed by intervention with tanshinone ⅡA.The expressions of E-cadherin,N-cadherin,Vimentin and Snail,GSK-3β/β-catenin signaling pathway,c-Myc and Cyclin D1,MMP-2 and MMP-9 were detected by immunohistochemistry and Western blot.Results:(1)The m RNA expression of β-arrestin1 was closely related to metastasis of colorectal cancer.In the GSE41258 database,the expression of β-arrestin1 in patients with lung metastasis of colorectal cancer was significantly higher than patients with primary colorectal cancer.It was also found that β-arrestin1 was significantly higher in lung metastasis specimens than primary colorectal cancer in our hospital.It was suggested that the high expression of β-arrestin1 was positively correlated with invasion and metastasis of colorectal cancer.Two hundred colorectal cancer patients with survival data were screened from GSE41258 database.The data showed that the high expression of β-arrestin1 was negatively correlated with survival.We used pathway common,KEGG,GO and dbemt databases to analyze the pathway and regulation direction of β-arrestin1.The results showed that β-arrestin1 interacted with Wnt signaling pathway,and was related to the proteins of Snail,E-cadherin,Vimentin,MMP2,MMP9.(2)β-arrestin1 could promote epithelial-mesenchymal transformation,invasion and metastasis in HCT-116 and Lo Vo cells,inhibit the expression of E-cadherin,promote the expression of N-cadherin,Snail and Vimentin,it also could up-regulate the protein expression of MMP2 and MMP9,and increase the abilitiy of invasion and metastasis.(3)β-arrestin1 could promote epithelial-mesenchymal transformation and lung metastasis of colorectal cancer in nude mice,and affect the survival of tumor-bearing mice.The mechanism of β-arrestin1 regulating EMT and metastasis through GSK-3β/β-catenin signaling pathway,which could down-regulate the expression of GSK-3β,promote the accumulation of β-catenin in the cytoplasm,to a certain extent,β-catenin enter the nucleus,and activate the expression downstream genes of β-catenin,including c-Myc,Cyclin D1.(4)Tanshinone ⅡA could significantly inhibit CRC EMT,invasion and metastasis,and reduced the rate of lung metastases were respectively 8.73%,19.09%,34.63% in low,mid,and high-dose groups.The effect of Tanshinone ⅡA regulating CRC EMT and metastasis through inhibiting the GSK-3 β/β-catenin signaling pathway mediated byβ-arrestin1,up-regulate the expression of E-cadherin and down-regulate the expression of N-cadherin,Snail and Vimentin.Tanshinone ⅡA also could inhibit the ability of cell invasion and migration via decreasing the expression of MMP2,MMP9 which could reduce the degradation of extracellular matrix.Conclusion:(1)Differential expression of β-arrestin1 was found in surgical specimens of patients with primary colorectal cancer and lung metastasis of CRC.The expression of β-arrestin1 was closely related to the epithelial-mesenchymal transformation,invasion and metastasis of colorectal cancer.Further survival analysis showed that high expression of β-arrestin1 was suggested poor prognosis,andβ-arrestin1 could be a potential predictor of metastasis and prognosis of colorectal cancer.(2)β-arrestin1 could promote colorectal cancer cells EMT,invasion and metastasis through activating GSK-3β/β-catenin signaling pathway,repressing the expression of GSK-3β and inhibiting the degradation of β-catenin.When β-catenin accumulated to a certain extent,it entered the nucleus,promoted the expression of c-Myc and Cyclin D1,increased the expression of E-cadherin,reduced the expression of N-cadherin,Snail and Vimentin.(3)Tanshinone ⅡA regulated the expression of Snai/E-cadherin through β-arrestin1/GSK-3β/β-catenin signaling pathway,thus inhibiting the epithelial-mesenchymal transformation of colorectal cancer.This may be one of the mechanisms of tanshinone ⅡA inhibiting invasion and metastasis of colorectal cancer. |
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