ALKBH5 Upregulates BUB1B To Promote The Proliferation Of Cholangiocarcinoma

BackgroundCholangiocarcinoma(CCA)is a serious malignant disease that develops from the epithelial cells of the bile ducts and occupies an important place in clinical practice.Cholangiocarcinoma is insidious in origin,difficult to diagnose at early stage and has poor prognosis.Cholangiocarcinoma are also challenging to treat because they are less sensitive to conventional treatment and because we cannot prevent or detect early tumor formation.Although surgery is expected to be curative,most radical resection rates are low and postoperative recurrence is likely to occur.While some patients with early-stage hilar cholangiocarcinoma can undergo neoadjuvant radiotherapy followed by liver transplantation.For patients with advanced and unresectable tumors,local and systemic radiotherapy is the most effective treatment option to achieve optimal disease control.AlkB homolog 5(ALKBH5),a major m6A RNA demethylase,is one of the nine members of the Alk B family,a suberin and 2-ketoglutarate-dependent nucleic acid oxygenase that promotes the demethylation of m6 A in RNA.ALKBH5 plays an important role in a variety of biological processes,including proliferation,migration,invasion,metastasis and tumor growth,which are critical to the treatment of cancer.However,no study has been reported on ALKBH5 in cholangiocarcinoma,so the aim of this project is to explore the role of ALKKBH5 in the development of cholangiocarcinoma.ObjectiveTo study the expression level of ALKBH5 in cholangiocarcinoma tumor tissues and paracancerous bile duct tissues and its effect on patients’ prognosis,and to investigate its role in the proliferation of cholangiocarcinoma cells and its mechanism.Methodology1.Bioinformatics analysis: To investigate the expression levels of ALKBH5 in cholangiocarcinoma cells and paraneoplastic cells and whether ALKBH5 expression correlates with the overall survival cycle of cholangiocarcinoma patients through the TCGA database.GO and KEGG function and pathway enrichment of the differentially expressed gene dataset for cholangiocarcinoma were performed through the DAVID website,and the pathways associated with ALKBH5 were analyzed using GSEA to screen for genes associated with ALKBH5.2.Tissue samples: Tumor tissues and paracancer tissues from patients with cholangiocarcinoma were collected,and the expression levels of ALKBH5 in cholangiocarcinoma tumor tissues and paracancer tissues were detected by quantitative real-time PCR(q-PCR),western blot(WB)and immunohistochemistry(IHC)assays.The 42 patients with cholangiocarcinoma were divided into two groups with high and low expression of ALKBH5 according to immunohistochemical results,and survival curve analysis was performed using the Kaplan-Meier(KM)method and log-rank test(log-rank).3.Cell function assays were performed to verify the effect of ALKBH5 on the proliferative capacity of cholangiocarcinoma: the cholangiocarcinoma cell lines QBC939,SK-CHA-1,Hu CC-T1 and HCC-9810 were cultured.The expression of ALKBH5 in bile duct cell lines was detected using Western blot(WB)to screen bile duct cancer cell lines.Hu CC-T1 cells were transiently transfected,and the protein expression of the cholangiocyte cell line Hu CC-T1 after transfection was detected by Western blot(WB).The effects of ALKBH5 on the proliferation of cholangiocarcinoma were determined by MTT proliferation assay,clone formation assay and 3D sphere formation assay.4.ALKBH5 promotes cell viability and maintains cancer stem cells in cholangiocarcinoma by regulating BUB1B: ALKBH5 expression was silenced in Hu CC-T1 cells and overexpressed in QBC939 cells,and the genes most associated with ALKBH5 were screened among 7 genes by q-PCR and WB.The effect on cell proliferation was determined by silencing BUB1 B in Hu CC-T1 cells.ALKBH5 was determined to function through BUB1 B by disruption in clone formation experiments and 3D sphere formation experiments.Also,overexpression of ALKBH5 in Hu CCT-1cells silenced BUB1 B to detect protein expression of biomarkers in tumor stem cells.Results1.By bioinformatics Analysiss,the expression levels of ALKBH5 were significantly different,with high expression in cholangiocarcinoma tumor tissues and low expression in paraneoplastic tissues,and the overall prognosis of patients with high ALKBH5 expression was poor.The increase of ALKBH5 in cholangiocarcinoma can effectively shorten the cell cycle and promote the development of cholangiocarcinoma.2.In the tissue samples,the expression of ALKBH5 in cholangiocarcinoma tumor tissues was significantly higher than that in the corresponding paraneoplastic tissues as verified by q-PCR,WB and IHC.3.Silencing the expression of ALKBH5 inhibited the proliferation of the cholangiocarcinoma cell line Hu CC-T1 cells.4.ALKBH5 promotes cell proliferation and maintains the tumor stem cell characteristics of cholangiocarcinoma by regulating BUB1B.ConclusionALKBH5 is highly expressed in cholangiocarcinoma,and the higher level of ALKBH5 expression predicts a poorer prognosis for patients.Cellular assay results indicate that ALKBH5 promotes the proliferation of bile duct cancer cells.Mechanistic studies revealed that ALKBH5 promotes cell proliferation and maintains the tumor stem cell characteristics of cholangiocarcinoma mainly by regulating BUB1 B.Our findings suggest that ALKBH5 holds promise as a potential new target for the treatment of patients with cholangiocarcinoma.