Effects Of Serine-arginine Protein Kinase 1 On The Phenotype Of Stemness And Epithelial-Mesenchymal Transition In HepG2 Cells

BackgroundHepatocellular cancer(HCC)is the major of primary liver cancer.The main reason for the poor prognosis of HCC patients is the high recurrence rate,and the existence of cancer stem cells(CSCs)is considered to be the root cause of HCC recurrence.Excessive activation of Epithelial mesenchymal transformation(EMT)promotes tumor heterogeneity and chemoresistance.Serine-arginine protein kinase 1(SRPK1)is a splicing factor of pre-mRNA,which regulated the progression of EMT in gastric cancer and promoted the accumulation of CSCs phenotype in non-small cell lung cancer meanwhile.Previously,we found that high expression of SRPK1 is associated with HCC development,but its mechanism has not been fully elucidated.In this study,we further analyzed the effect of regulating SRPK1 expression on tumor stemness and EMT in HepG2 cell line.The conclusion may provide an experimental basis for HCC therapy targeting SRPK1.Method1.The relationship between SRPK1 and the disease progression and clinical data of HCC patients.LIHC(liver hepatocellular carcinoma)in TCGA(The Cancer Genome Atlas)GTEx(Genotype-Tissue Expression)and THPA(The human preotein atlas,https://www.proteinatlas.org/)hepatocellular carcinoma were visited.The expression levels of SRPK1 in different tumors were analyzed,and the Wilcoxon rank sum test was used to compare the expression differences of SRPK1 between liver cancer and adjacent tissue samples in the TCGA database.The relationship between clinical data such as patient survival time and SRPK1 expression levels in 50 paired liver cancer and its adjacent tissue samples was also analyzed.The expression of SRPK1 in liver cancer tissues and adjacent tissues was analyzed by comprehensive evaluation of the proportion and staining intensity of antibody-stained cells in the THPA database.The TIMER database was used to analyze the correlation between SRPK1 expression and infiltrating immune cells in liver cancer tissue,including CD4+T cells,and the relationship between the infiltration level of various immune cells and the prognosis of liver cancer patients.2.The effect of up-regulation or down-regulation of SRPK1 expression on the biological characteristics of tumor stemness in liver cancer cells.:HepG2 cells with over expression and suppressed expression of SRPK1 protein were constructed.Stem cell sphere formation test was used to detect the self-renewal ability of liver cancer stem cells;Side population(SP)cells with Hoechst33342 staining were analyzed by flow cytometry;Real-time PCR was used to compare the mRNA levels of tumor stemness markers Nanog,Oct4,CD133 and Bmi1.3.The effect of regulating SRPK1 expression on EMT in HepG2 cells:Western Blot was used to detect the protein levels of EMT molecular markers E-cadherin and Vimentin in HepG2 cells by regulating the expression of SRPK1.Immunofluorescence was used to futher verify the distribution of EMT molecular markers E-cadherin and Vimentin in HepG2 cells.4.The effect of SRPK1 on Wnt/β-catenin pathway activation in HepG2 cells:The correlation between SRPK1 and Wnt/β-catenin pathway activation was predicted by Gene set Enrichment analysis(GSEA).The difference of β-catenin expression in the nucleoplasm of HepG2 cells with different SRPK1 expression level was compared by nucleoplasmic protein separation and Western Blot.Real-time PCR was used to compare the expression differences of downstream genes such as Twist,MYC,MMP 9,CD44,CCND 1 in Wnt/β-catenin pathway in HepG2 cells.Gene correlation analysis in GEPIA2 was used to observe the correlation between SRPKl and Twist,MYC,MMP9,CD44 and CCND 1 in LIHC tissue.Results:1.TCGA data analysis showed that the expression of SRPK1 mRNA in tumor tissues of LICH patients was significantly higher than that of normal controls,and it increased with the increase of disease stage and pathological grade.The overall survival time of patients with high expression of SRPK1 was lower than that of patients with low expression,there were no significant differences in race,gender,age,and weight.The SRPK1 expression was significantly up-regulated in liver cancer tissues of 50 paired samples.The results of the HPA database analysis showed that the expression of SRPK1 was significantly up-regulated in liver cancer.The expression of SRPK1 is related to B cells,CD8+T cells,CD4+T cells.The infiltration levels of macrophages,neutrophils,and dendritic cells were positively correlated with SRPK1 expression level,and HCC patients with high levels of the above cells infiltration had a worse prognosis.2.Compared with the control group,HepG2 cells with high expression of SRPK1 had increased spheroidization ability,and the mRNA expression of stemness genes Nanog,Oct4,CD 133 and Bmil also increased.SRPK1 expression was positively correlated with the expression of Nanog,Oct4,CD133 and Bmil in LICH tissue,respectively.3.The results of Western Blot and immunofluorescence showed that the expression of epithelial marker E-cadherin decreased and the expression of mesenchymal marker Vimentin increased in HepG2 cells with high SRPK1 expression.4.GSEA analysis found that the overexpression of SRPK1 in LICH was positively correlated with the activation of the Wnt/β-catenin pathway.The expression of the βcatenin protein in the nucleus of HepG2 cells with high SRPK1 expression was increased,and the mRNA levels of Twist,MYC,MMP 9,CD44 and CCND 1 were increased.Conclusions:High expression of SRPK1 is associated with poor prognosis in HCC and SRPK1 may promote the progression of EMT and the acquisition of tumor stemness phenotype by activating the Wnt/β-catenin pathway in HepG2 cells.