The Mechanism Of BLM Helicase In Coordination With HDGF In Prostate Cancer Cells

Prostate cancer（PCa）has been the leading cause of cancer death among male worldwide.The incidence of PCa in China is also reported to be increasing year by year.In addition,the characteristic of histomorphology and molecular tumor showcase substantial discrepancies between patients and within specific tumors,which pose an immense diagnostic challenge for PCa.As a guardian of the genome,BLM is mainly involved in DNA replication,repair and telomere maintenance.It has also been reported that BLM upregulation may be a hallmark of cancer susceptibility,tumor formation,and carcinogenic activation.Previous studies of our group indicated that the proliferation ability of PCa PC3 cells was blunted significantly upon BLM knockdown.Similar to BLM,HDGF is also a nucleolar protein,which is highly expressed in a variety of malignant tumors.The PWWP domain of HDGF is principally participated in protein-protein,protein-RNA,protein-DNA interactions,so as to complete the regulation of biological functions including chromosome remodeling,gene transcription,cell growth and differentiation.Of note,HDGF is a vital oncogene,which is associated with the proliferation,invasion and metastasis ability of liver cancer,gastric cancer,prostate cancer and non-small cell lung cancer.On the flipside,studies have shown that HDGF is linked to the survival prognosis of PCa.Thus far,the molecular mechanism of BLM and HDGF was rarely reported in PCa.Therefore,the mechanism of BLM helicase and HDGF in prostate cancer cells was studied,and the results are as follows:1.BLM and HDGF have interaction in PC3 cellsWestern blotting suggested that BLM was discrepantly expressed in normal prostate cells and PCa cell lines,and the highest expression level was found in PC3cells.Therefore,using BLM as the decoy protein in PC3 cells,541 proteins interacting with BLM were found by Co-IP combined with mass spectrometry.TCGA database analysis exhibited that BLM and HDGF were highly expressed in PCa.Prostate adenocarcinoma（PRAD）samples in TCGA database were divided into 4groups according to the optimal expression of BLM and HDGF m RNA.The results showcased that BLM and HDGF m RNA levels were highly expressed and negatively correlated with survival time.Bioinformatics analysis indicated a significant positive correlation between the expression of BLM and HDGF in PCa(P^-Value=4.4e-16).Immunofluorescence assay determined that BLM and HDGF were primarily localized in the nucleus in PC3.Finally,Co-IP assay was conducted to demonstrate the direct interaction between BLM and HDGF via IP antibody of BLM and HDGF as decoy proteins.2.The elevated expression of BLM and HDGF in PCaThe expression of BLM and HDGF in normal prostate tissue,hyperplasia prostate tissue and PCa tissue were detected by immunohistochemistry.Compared with normal prostate tissues,both BLM and HDGF were highly expressed in PCa tissues（P<0.001）,and there was a significant positive correlation between BLM and HDGF in PCa（P=0.0009,R²=0.207）.Combined with clinicopathological data,the positive rate of BLM and HDGF expression in PCa tissues were analyzed.The results implied that the expression levels of BLM and HDGF in PCa tissues were remarkably correlated with Gleason score（P=0.000,P=0.016）and Gleason grade（P=0.001,P=0.009）.However,there was no significant correlation with age,lymph node metastasis and distant metastasis.3.Both konckdown of BLM and HDGF expression can conspicuously inhibit the proliferation,migration and invasion abilities of PC3 cellsEdU,CCK8,clonogenesis assay,scratch assay and Transwell invasion assay were employed to delve the influences of BLM and HDGF knockdown on PC3 cells.The results evinced that PC3 proliferation,migration and invasion ability were more impaired when both BLM and HDGF knockdown compared with that BLM or HDGF knockdown solely.As a consequence,BLM and HDGF have synergistic effects on the regulation of PC3 cell biology.In parallel,the Western blotting results indicated that BLM depletion failed to influence HDGF expression.Likewise,BLM expression was unaffected by HDGF silencing.4.Influences of BLM and HDGF knockdown or overexpression on proliferation,migration and invasion of PC3 cellsThe expression levels of BLM,HDGF,Ki67 and PCNA were quantified upon BLM and HDGF knockdown or overexpression.BLM expression was impervious upon HDGF knockdown or overexpression.Equally,HDGF expression was insusceptible to BLM knockdown or overexpression.Ki67 and PCNA expression levels were positively correlated with BLM and HDGF,indicating that BLM and HDGF could contribute to the proliferation of PC3 cells.EdU,CCK8,clonogenesis,cell scratch and Transwell invasion assay were employed to pinpoint the influence of BLM and HDGF knockdown or overexpression on biology of PC3 cells.The results displayed that HDGF overexpression partially abolished the promotion effect on cell invasion,clonogenicity,migration and proliferation elicited by BLM defciency in PC3.Conversely,silencing HDGF attenuated PC3 invasion,clonogenicity,migration and proliferation.Nonetheless,the inhibition was alleviated following BLM overexpression.Cell function recovery experiments certified the synergistic effect of BLM and HDGF on the regulation of PC3 cells.5.HDGF activates MAPK/ERK pathway by regulating downstream genes KRAS and Rho A in PC3ChIP-seq assay was conducted via ChIP grade HDGF antibody,and the data were analyzed by GO functional annotation and KEGG pathway enrichment.We found that HDGF could bind to the promoter of KRAS and Rho A,and the above results were confirmed by double luciferase assay.Meanwhile,HDGF could transcriptionally activate KRAS gene and transcriptionally inhibit Rho A gene.Western blotting results manifested that KRAS,p MEK and p ERK expression levels were positively correlated with HDGF in MAPK/ERK pathway,while Rho A,ROCK1 and ROCK2 expression levels were negatively correlated with HDGF in Rho-Rac pathway.Accordingly,the interaction between BLM and HDGF activated the MAPK/ERK pathway,and thus modulating the proliferation,migration and invasion of PC3.The growth trend of tumor in nude mice was basically consistent with that of BLM and HDGF in vitro.To recapitulate,high expression of BLM and HDGF may be employed as potential biomarkers candidates for poor prognosis in PCa patients.The interplay of BLM and HDGF was involved in the regulation of proliferation,migration and invasion of PCa cells.HDGF can transcriptionally activate KRAS while transcriptionally repress Rho A.The regulation of KRAS and Rho A by HDGF has signal crosstalk.MAPK/ERK is the predominant downstream signaling pathway of HDGF.Interaction between BLM and HDGF can facilitate the growth of prostate cancer.BLM and HDGF are expected to be utilized as diagnostic markers or potential therapeutic targets for PCa.