Autophagy-related Gene WD Repeat Domain 45B Promoted Proliferation And Migration In Hepatocellular Carcinoma

Objective Hepatocellular carcinoma（HCC）is a highly invasive and malignant tumor with high morbidity and mortality.It has been found that autophagy plays role both as a tumor promoter and inhibitor in HCC carcinogenesis.However,the mechanism is unknown.This study aims to examine for the key autophagy-related proteins in HCC,and then to explore their functions and mechanism,in order to shed light on novel clinical diagnosis and treatment targets of HCC.Methods RNA-Seq transcription data along with clinical information of HCC patients were obtained from public databases TCGA,ICGC and UCSC Xena.Autophagyrelated genes were downloaded from the HADb database.The autophagy-related gene WDR45 B was identified by rank-sum test and Lasso-Cox regression analysis,which was differentially overexpressed in tumor tissues.Kaplan-Meier survival curves were plotted to compare the prognosis of HCC patients with high and low WDR45 B expression levels.Gene set enrichment analysis（GSEA）was used to analyze potential pathway mechanisms.Immunohistochemical（IHC）was performed on paraffin-embedded tissues originated from 58 HCC patients and 3 patients with hepatic blunt trauma from our pathology archives from 2020 to 2021.RNA and protein of human normal liver cell line LO2,human HCC cell line Hep G2 and Huh-7 were extracted,and QRT-PCR and Western blot test were performed to validate the differential expression of WDR45 B.WDR45B-knockdown HCC cell lines were constructed by lentivirus infection.CCK8 and wound-healing assay were applied to identify the effects of WDR45 B inhibition on the proliferation and migration of HCC in vitro.The expression levels of autophagy marker proteins LC3 and p62/SQSTM1,Akt and m TOR phosphorylation levels of Akt/m TOR pathway in WDR45 Bknockdown HCC cell lines were detected to explore the specific mechanism of WDR45 B affecting the occurrence and development of HCC.Results Firstly,bioinformatics analysis showed that autophagy-related gene WDR45 B is up-regulated in HCC.Patients with high WDR45 B expression levels tend to have worse prognosis and advanced clinical stage.Genomic analysis showed that methylation level of WDR45 B was related to the prognosis of the HCC patients.Gene set enrichment analysis revealed that high expression of WDR45 B influenced the Akt/m TOR signaling pathway.Next,WDR45 B overexpression was validated in HCC tissue by using QRT-PCR,western blot and IHC.Autophagy marker LC3-II/LC3-I was found to be down-regulated,and p62/SQSTM1 was up-regulated in the Hep G2 cell lines after knockdown of WDR45 B.There was no significant difference in the expression of Akt and m TOR of the Akt/m TOR signaling pathway,and the phosphorylation levels of Akt and m TOR are significantly increased.The effects of WDR45 B knockdown on autophagy and Akt/m TOR signaling pathways could be reversed by the autophagy inducer rapamycin,validating the inhibition of autophagy after the knockdown of WDR45 B.Lastly,we found that proliferation and migration of HCC was inhibited after the knockdown of WDR45 B through the CCK8 assay and wound-healing assay.Conclusion Autophagy-related gene WDR45 B is differentially expressed in HCC,and associated with poor prognosis and advanced clinical stage.Knockdown of WDR45 B can activate the Akt/m TOR signaling pathway by up-regulating the phosphorylation levels of Akt and m TOR leading to the inhibition of autophagy,and promoting proliferation and migration of HCC.WDR45 B may become a new biomarker for HCC prognosis assessment and potential target for molecular therapy.