Identification Of HSD17B1 As A Potential Bladder Tumor Marker And Its Participation In The Inhibitory Effect Of Curcumin On The Bladder Cancer Cells

Bladder cancer is one of the main causes of mortality among the malignancy disease that is associated with high levels of incidence.Recently,improving and developing bladder cancer therapeutics is an interesting field for scientific research.Curcumin is a polyphenol derived from turmeric that has been shown to inhibit tumor progressions such as bladder,prostate,colorectal,breast,pancreatic,brain,head,and neck cancers.However,the molecular mechanism of curcumin in the treatment of bladder cancer remains to be clarified.In this study,with the help of relevant bioinformatics databases,we aimed to find functional protein(s)involved in the inhibitory effect of curcumin on bladder cancer.BATMAN-TCM dataset was used to identify the target genes associated with the Curcumin herb.Its prediction showed that six genes were enriched in the biological process of Steroid Biosynthesis which was Curcumin’s target for cancer regulation.Among the targets of genes associated with the curcumin herb,the UALCAN database found one particular cross-gene with significant expression in bladder cancer.According to UALCAN expression results for the six genes,HSD17B1 exhibited higher mRNA expression than other relevant genes.Then,we analyzed the differences in gene expression profiles between bladder cancer and normal bladder tissues by bioinformatics datasets.We found that mRNA and protein levels of HSD17B1 were significantly associated with high expression in cancer samples than those in normal samples of patients with p=1.85E-05.However,A significant association has been found between the HSD17B1 mRNA expression levels and individual cancer stage 2,stage 3,and stage 4 compared to normal patients with(p=9.76E-03,p=9.76E-03,and p=9.76E-03)respectively.In function enrichment of HSD17B1 in patients with BLCA analysis,the biological processes of HSD17B1 and its similar genes were mainly enriched in such as metabolic process,positive regulation of the biological process,multicellular process,and so on.Moreover,to investigate which parts of HSD17B1 work in the cells,protein-protein interaction enrichment was conducted,and pathway and process enrichment analysis has been applied to each MCODE component detected by Molecular Complex Detection(MCODE)algorithm independently.We notified that three functions enriched by physical interactions were the fatty acid metabolic process,monocarboxylic acid metabolic process,and fatty acid metabolism.Furthermore,to compare different CpG sites in BLCA samples,the HSD17B1 gene’s DNA methylation levels and the CpG islands’ prognostic value were examined using the MetSurv tool.Eight methylated CpG islands,and two CpG islands cg20404150 and cg15418287 have shown an increase in levels of DNA methylation.The methylation levels of the two CpG islands cg20404150 and cg15418287 were found to be associated with the prognosis with a p-value<0.05.Elevated levels of HSD17B1 methylation in these two CpG islands,particularly cg20404150 were associated with poorer overall survival of BLCA patients when compared to individuals with lower levels of HSD17B1 CPG methylation.Subsequently,the expression levels of HSD17B1 in various tumor cells were detected by qRTPCR.Consistent with the TCGA-UALCAN results,HSD17B1 was significantly upregulated in cervical cancer cell HeLa,bladder cancer cell UM-UC3 cell lines,liver cancer cell SMMC-2271,and human breast cancer MCF-7 compared with epithelial normal kidney cell 293T.Then,different cancer cells SH-SY5Y,5637,T24,MG63,SV-HUC-1,and UM-UC3 were treated by curcumin.The cell viability assay showed that curcumin significantly inhibited cell proliferation of those cell lines in a dose-dependent manner.Next,in order to investigate whether HSD17B1 participates in the inhibitory effect of curcumin on bladder cancer cells MTT test,DAPI staining,and flow cytometry were used to evaluate the inhibitory effect of curcumin on bladder cancer UM-UC3 cells.The data showed that curcumin with IC50 10.57μM was significantly inhibited cell proliferation of bladder cancer UM-UC3 cells in a dose-dependent manner with variation statistically significant(P<0.001).In addition,the ratio of apoptotic UM-UC3 cells significantly increased after 24,48,and 72 H treatment with curcumin 5 and 10 μM respectively in a dosedependent manner.The overall apoptotic ratio of UM-UC3 cells increased from 3.63%at baseline to 5.58 and 6.26%after treatment with 5 and 10 μM Curcumin during 24H incubation.In addition,the ratio of apoptotic UM-UC-3 cells significantly increased after 24,48,and 72 H treatment with curcumin 5 and 10 μM respectively in a dose-dependent manner.The overall apoptotic ratio of UM-UC-3 cells increased from 3.63%at baseline to 5.58 and 6.26%after treatment with 5 and 10 μM Curcumin during 24H incubation.Finally,we detected the effect of curcumin on the mRNA expression of HSD17B1 and its related genes STAT2,IL1R1,PBK,KIF20A,and MYBL1 by qRT-PCR,data showed that mRNA levels of HSD17B1,STAT2,IL1R1,PBK,KIF20A,and MYBL1 were upregulated in UM-UC3 cell line while the mRNA level of RARRES3 was significantly downregulated after treated with 10 μM of curcumin for 24 H with p<0.001.In summary,using the bioinformatics databases,we identified HSD17B1 as a novel potential bladder tumor marker,which could be used to predict a better prognosis,including improved disease-specific survival and metastasis-free survival in bladder cancer.Additionally,our data showed that HSD17B1 participates in the inhibitory effect of curcumin on bladder cancer cells.However,the possible molecular mechanism needs to be further studied in the future.It is hoped to provide potential biomarkers for the diagnosis and prognosis of bladder cancer and to clarify the mode of action of curcumin.The results also provide basic data for the screening of future drug targets.