Construction Of Genetic Diagnosis Model For Early Hepatocellular Carcinoma And The Role Of WNT5A In The Oncogenesis Of Hepatocellular Carcinoma

ObjectiveDelayed diagnosis is the main cause leading to poor prognosis of liver cancer.The regularity and mechanism of transcriptomic changes in hepatocellular carcinoma(HCC)are unclear.A systematic and in-depth study of the transcriptome will have important significance for the early diagnosis and precise treatment of HCC.Materials and Methods1.Utilizing transcriptome data of 826 early HCC from multiple international platform database(GEO,TCGA,GTEx,ICGC),gene expression characteristics were screened by using elastic net and RobustRankAggreg(RRA),and diagnosis prediction model for early HCC(DP.eHCC)was constructed by logistic regression analysis.Survival risk prediction model for early HCC(SP.eHCC)was established by selecting 256 cases of early HCC patients with survival information and using univariate Cox regression and lasso Cox regression analysis.2.CIBERSORT and LM22 were used to estimate the type of immune cell infiltration and the corresponding immune cell fraction in early hepatocellular carcinomas and non-cancerous tissues to analyze the tumor immune microenvironment(TIME)and clinical significance.Weighted co-expression genes modules were constructed by WGCNA.The potential functional pathways of co-expressed genes in those modules were analyzed by GSEA.3.WNT5A expression was detected in Ncoa5 and Tip30 knockout female mice.WNT5A knockout plasmids were constructed by CRISPR-Cas9 technique.Monoclonal WNT5A gene knockout cell lines were established in vitro in liver cancer cell line PP5 cells.By using cell function experiments,we observed the effect of WNT5A gene knockout on cell growth and proliferation,migration and invasion ability of liver cancer cells.Results1.Nine genes(AFM,AKR1C3,CYP1A2,CYP2E1,GPC3,HAMP,HBB,MT1G and SPINK1)were screened for construction of the DP.eHCC model.With a diagnostic threshold value of 0.0324,the AUC of DP.eHCC model for the diagnosis of early HCC was 0.956(95%CI:0.941-0.972;P<0.001).The results showed that DP.eHCC model significantly improved prediction performance for early HCC over its 9 genes alone.2.Based on nine genes(UBLCP1,CCDC42,AQP5,KCTD8,LARS,SMS,TNNT3,RUVBL1 and YIF1B),survival risk prediction model for early HCC(SP.eHCC)was constructed.The median 1.755 was selected as the SP.eHCC risk score threshold.The results showed that early HCC patients with low scores obtained longer mean overall survival times than patients with high scores(95.705±5.642 months vs.55.901±3.763 months,P<0.0001).3.A total of 12 cell subtypes were obtained in the tumor immune microenvironment(TIME)by CIBERSORT algorithm.In addition,9 cell subtypes of TIME infiltrating cells were hierarchically clustered by Consensus Cluster Plus,and 4 clusters of TIME(A-D)were obtained.The average overall survival time in patients of TIME ClusterB was significantly longer than that in TIME ClusterD.4.Hub genes and their molecular relations were visualized and screened in the PPI network among three modules(turquoise module,blue module and yellow module showed by WGCNA.The results suggested that the occurrence and development of early HCC are closely related to the signal pathways of cell proliferation and immune response.5.Down-regulation of Wnt5a was observed in Ncoa5+/-and Tip30+/-mice.And as the key member Wnt/β-catenin pathway,the activation of Ccndl was also demonstrated in Ncoa5+/-and Tip30+/-mice.Monoclonal liver cancer cell line PP5 with WNT5A gene knockout targeting two sites of the WNT5A coding region(ENSE00001032709)was successfully established.Results showed that WNT5A coding segment gene knockout can increase the proliferation,migration and invasion of liver cancer cells by CCK8,Transwell experiment and so on.And,deletion of the WNT5A gene can improve the transcription and expression of key genes CCNB1 and TCF(HNF4A)involved in Wnt/β-catenin pathway.These results confirmed that the tumor suppressor role of WNT5A involved in Wnt/β-catenin and P53 signaling pathway during the oncogenesis of HCC.Conclusions1.The DP.eHCC model was constructed and confirmed to be an effective model for early diagnosis of hepatocellular carcinoma,with a sensitivity of 90.91%and a specificity of 92.97%.2.The SP.eHCC model was constructed and validated to be an independent risk predictor of HCC.3.By applying CRISPR-Cas9 gene knockout technology,we demonstrated that WNT5A gene could inhibit the oncogenesis of hepatocellular carcinoma in vitro and in vivo.