Effects Of Wnt2 On Proliferation,Migration And Invasion Of CRC Cells Via Regulation Of Aerobic Glycolysis And Its Association With Clinical Prognosis

Colorectal cancer(CRC)is one of the most common gastrointestinal malignancy in the world.It is a long-term hazard to human health.According to the White Paper on CRC Epidemiology,Prevention and Screening of CRC in China,the incidence of CRC in China ranks third in the incidence of malignant tumors.However,the mechanisms involved in the occurrence and progression of CRC remain unclear.Therefore,we focus on genes that influence the progression of CRC to provide potential clinical markers for early diagnosis of CRC.In this article,through TCGA database analysis,we found Wnt2 gene,which is on the human chromosome 7q31,expressed in the tumor tissue of patients with CRC,and through the GSEA pathway enrichment analysis,the enrichment of Wnt2 gene is found in Amino Suger and Nucleotide Suger,Metabolism pathway.This phenomenon prompted Wnt2 might affected the progress of CRC by the aerobic glycolysis of tumor cells,and could provide help for early diagnosis of CRC.Subsequently,we verified the expression of Wnt2 in seven existing wild-type CRC cell lines in our research group,and constructed Caco2 overexpression and HCT8 knockdown stable transgenic lines.At the same time,m RNA expression levels of the symbolic molecules of Amino Suger and Nucleotide Suger Metabolism pathways were determined in the stable transgenic cells,and it was found that the expression of HK2 was correlated with the expression of Wnt2.AG-490,an inhibitor of STAT3 pathway,was used to inhibit the phosphorylation of STAT3 to explore the mechanism of Wnt2’s influence on the expression of HK2.The results showed that the m RNA and protein levels of Wnt2 in Caco2 cells were significantly increased after stable overexpression of Wnt2 gene.After stable knockdown of Wnt2 gene,the m RNA and protein levels of Wnt2 in HCT-8 cells decreased significantly.After the addition of 2-deoxy-D-glucose(2-DG),the level of HK2 in the stable transgenic Caco2 strains overexpressing Wnt2 gene was significantly reduced.Inhibition of STAT3 pathway in Caco-2 cells overexpressing Wnt2 gene reduced the expression of HK2.In addition,the effects of Wnt2 on aerobic glycolysis of CRC cells were investigated by measuring glucose uptake and lactate production capacity of Wnt2 stable transgenic strains.Meanwhile,the effects of Wnt2 on the proliferation capacity of CRC cells were investigated by CCK-8,Ed U staining and clone formation tests.The effect of Wnt2 on the migration and invasion of CRC cells was investigated by wound healing and Transwell assay.In Caco2 cells with stable overexpression of Wnt2,the ability of aerobic glycolysis,proliferation,migration and invasion was detected after 2-DG was used to inhibit the expression of HK2.Overexpression of Wnt2 gene enhanced the aerobic glycolysis ability of Caco2 cells,knockdown of Wnt2 gene decreased the aerobic glycolysis of HCT8 cells,and inhibition of HK2 decreased the glycolysis of stable transgenic Caco2 strains with overexpression of Wnt2 gene.CCK-8,Ed U staining and clone formation test results showed that overexpression of Wnt2 gene enhanced the proliferation ability of Caco2 cells,knockdown of Wnt2 gene reduced the proliferation of HCT-8 cells,and inhibition of HK2 reduced the proliferation of Caco2 with stable overexpression of Wnt2 gene.Wound healing and Transwell test results showed that overexpression of Wnt2 gene enhanced the migration and invasion ability of Caco2 cells,knockdown of Wnt2 gene reduced the migration and invasion of HCT8 cells,and inhibition of HK2 reduced the migration and invasion ability of Caco2 with stable overexpression of Wnt2 gene.Finally,the expression of Wnt2 and HK2 in cancer tissues and adjacent normal tissues of 211 CRC patients was measured by immunohistochemical(IHC)analysis to explore the correlation between Wnt2 and HK2 expression in tissues.Cox univariate and multivariate regression models were established,and Kaplan-Meier method was used to plot survival curves to explore the relationship between Wnt2 and HK2 and clinicopathological parameters,also the prognostic value of Wnt2 and HK2 in CRC patients.Correlation analysis between IHC results and clinicopathological data showed that Wnt2 was highly expressed in the tumor tissues of CRC patients,and the expression of Wnt2 was correlated with T stage(P<0.001),nerve invasion(P=0.035),and expression of HK2(P<0.001).Cox univariate regression analysis showed that gender(male and female,P=0.032),tumor location(colon and rectum,P=0.036),T stage(T3/4,T1/2,P<0.001),N staging(N1/2,N0,P=0.016),TNM staging(III/IV,I/II,P=0.038),nerve invasion(yes,no,P=0.021),vascular invasion(yes,no,P=0.008),WNT2 expression(high,low,P=0.038)and HK2 expression(high,low,P=0.002)were indicators that affected overall survival in CRC patients.Multivariate Cox regression analysis for these indicators showed that patient gender(male,female,P=0.013),T staging(T3/4,T1/2,P=0.001),Wnt2 expression(high,low,P=0.049),and HK2 expression(high,low,P=0.001)were associated with prognosis in CRC patients.Kaplan-Meier survival analysis showed that patients with high Wnt2 expression and high HK2 expression had worse overall survival.In this study,we found that Wnt2 may affect aerobic glycolysis and tumor cell proliferation,migration and invasion by regulating HK2 in human CRC.