| Objective:Hepatocellular carcinoma(HCC)is one of the most common primary hepatic carcinomas and has become the leading cause of cancer-related death in many regions and countries.Although the technology is becoming more and more mature and the means for the treatment of HCC are increasing,the "three HIGH"(high morbidity,high mortality and high recurrence rate)is still a major problem troubling clinicians and endangering patients.Therefore,it is urgent to find markers that can early predict the incidence,metastasis,recurrence and prognosis of HCC.The purpose of this study is to explore and verify the role and mechanism of CLEC3 B in HCC through cell.To analyze the relationship between the expression of CLEC3 B in HCC and related clinical indexes,hoping to provide predictive signatures for early diagnosis of recurrence and metastasis and potential treatment targets of HCC.Methods:We analyzed the expression of CLEC3 B in HCC and GSEA analysis by bioinformatics.The clinical samples of HCC were collected,and the expression of CLEC3 B in HCC and adjacent normal tissues was verified by IHC and RT-q PCR.CLEC3 B was overexpressed in HCC cell lines HUH-7 and PLC/PRF/5 by lentivirus.Wound healing assay and Transwell experiment were used to explore the effects of CLEC3 B on migration and invasion of HCC cells.Flow-cytometry was used to detect the effects of CLEC3 B on apoptosis of HCC cells.Finally,Western Blotting was used to explore the effects of CLEC3 B on PI3K/AKT signal pathway and apoptosis signal pathway.Results:(1)The expression of CLEC3 B was analyzed by TCGA,and it was found that the expression of CLEC3 B was low in a variety of malignant tumors.In HCC,compared with the adjacent tissues,the expression of CLEC3 B was down-regulated in HCC.CLEC3 B can be used as one of the molecular indexes for predicting HCC,AUC=0.7152(95% CI:0.6585?0.7720,p < 0.0001),and the prediction ability is above medium level.(2)The total RNA of clinical samples was extracted to construct c DNA library,and real-time quantitative PCR showed that the expression of CLEC3 B was down-regulated in HCC(p < 0.0001),which was consistent with the result of TCGA database.The results of IHC showed that the expression of CLEC3 B was higher in normal tissues,on the contrary,it was decreased in HCC tissues.Based on the survival analysis of RT-q PCR and IHC score,it was found that the OS of the group expressed lower CLEC3 B was significantly poorer than the higher group.(3)Through GSEA analysis of TCGA database,it was found that the activity of cancerrelated pathway,m TOR signal pathway,apoptosis pathway and autophagy in low expression group was lower than that in high expression group of CLEC3 B.(4)Through scratch test and Transwell test,it was found that the metastatic and invasive ability of HCC cells decreased when HUH-7 cells and PLC/PRF/5 cells expressed CLEC3 B.(5)Flow cytometry showed that the apoptosis level of HCC cells increased after overexpression of CLEC3 B in HUH-7 cells and PLC/PRF/5 cells,and colony formation assay showed that the ability of clone formation was significantly decreased after overexpression of CLEC3 B.(6)Through Western blotting,CLEC3 B can regulate the apoptosis level of HCC cells by regulating PI3K-AKT signal pathway.Conclusion:The low-expression of CLEC3 B weakened the ability of CLEC3 B to regulate PI3K/AKT signal pathway in HCC,resulting in increased the expression of bcl-2(antiapoptotic protein)and decreased the expression of bax(apoptotic protein),thereby inhibiting apoptosis and promoting proliferation of HCC.Objective: Hepatocellular carcinoma(HCC)is the most common malignant tumor of the liver,with high morbidity and mortality.Autophagy has been proved to play an important role in the occurrence and development of liver cancer.Our aim is to study the expression characteristics of autophagy-related genes in hepatocellular carcinoma and to find out the polygene expression characteristics that predict the prognosis of patients with hepatocellular carcinoma.Considering the complexity of gene transcription and translation,we hope to construct a ceRNA network related to liver cancer autophagy to provide a direction for follow-up research to better and more comprehensively understand the status and role of autophagy in the occurrence and development of liver cancer.Methods: Differentially expressed autophagy-related genes(DE-ATGs)and differentially expressed mi RNAs and lnc RNAs in HCC and normal tissues were screened from TCGA.With the cutoff value of | log Fc | ? 1.5 and FDR< 0.05.The differentially expressed genes related to autophagy(DE-ATGs)were screened and analyzed by GO and KEGG.Univariate and multivariate Cox regression analysis was performed to determine the best genes related to prognosis,survival analysis was performed and verified in clinical specimens and ICGC database.Then a line chart was established to verify the clinical features,including DE-ATGs,gender,staging and TNM staging.Finally,we use all the DEATGs and differentially expressed mi RNAs and lnc RNAs to depict a ceRNA network associated with autophagy genes.Results:(1)27 DE-ATGs were screened from TCGA.According to GO and KEGG analysis,they were all related to autophagy or cancer.(2)Cox regression analysis confirmed that BIRC5,HSPB8 and SQSTM1 were closely related to prognosis,and a risk score model was established and evaluated.K-M curve showed that the overall survival status of high-risk patients was significantly poor.ROC curve was used to evaluate the predictive ability of the model,and AUC=0.749,was verified in clinical specimens and ICGC database.(3)The clinical features such as DE-ATGs,sex,staging and TNM work together to construct a line map,c-index 0.736,which can improve the predictive specificity of autophagy-related risk signals.(4)The ceRNA network related to autophagy genes was constructed by using all differentially expressed DE-ATGs and differentially expressed mi RNAs and lnc RNAs,which laid a foundation for further study of post-transcriptional modification and regulation of autophagy-related genes.Conclusion:In this study,we observed that an autophagy marker composed of three genes can be used as a biomarker and therapeutic target for predicting prognosis,constructed a diagram containing this marker and clinicopathological features,and described the ceRNA network of DE-ATGs in hepatocellular carcinoma. |
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