Effects Of MIB1,lncRNA PVT1 On Ubiquitination Of BLM And Proliferation And Migration Of Prostate Cancer Cells

BLM helicase can maintain genomic stability and DNA repair,replication,recombination and transcription.Mutations in the BLM can lead to the disease Bloom syndrome,which is characterized by susceptibility to prostate cancer(PCa),breast cancer,lung cancer and other cancers.Relevant studies have shown that there are 7susceptibility genes closely related to the occurrence of PCa,and BLM ranks first.The high expression of BLM will lead to its mislocalization in the cytoplasm,thus promoting tumor growth.At the same time,the investigation and study of PCa in2020 showed that the incidence rate and mortality of PCa ranked among the top ten male malignant tumors in China,and continued to rise.PCa has gradually become one of the common tumors affecting the life and health of Chinese men.Therefore,it is particularly important to study the regulation of BLM protein expression in PCa.BLM expression is closely related to cell cycle and proliferation.The ubiquitination modification of BLM is a very important aspect.The amount of protein in BLM is closely controlled by the ubiquitin proteasome pathway.However,the specific mechanism affecting the ubiquitination of BLM is still unclear.Our work focuses on two aspects: ubiquitination and de ubiquitination of BLM.The first is the ubiquitination modification of BLM.By combing the literature,we found the function of E3 ligase MIB1.Through the intervention of cycloheximide(CHX)and the control experiment of ubiquitin proteasome inhibitor(MG132),it was found that MIB1 could significantly promote the degradation of exogenous BLM protein;MIB1 could bind to BLM by immunoprecipitation assay;Through the ubiquitination experiment,it was found that MIB1 promoted the ubiquitination modification of BLM protein;At the same time,overexpression and knockdown experiments partially proved that MIB1 could promote the degradation of BLM,which laid a foundation for the next research.Subsequently,the effects of MIB1 on the proliferation and migration of prostate cancer cells were studied.The expression of miRNA in PCa and its correlation with MIB1 expression were analyzed by bioinformatics software and database data to verify its interaction;The MIB1 si RNA knockdown and overexpression virus vectors were constructed and transfected into PCa cells to study their effects on the proliferation,migration and invasion of PCa cells.The results showed that MIB1 overexpression promoted the proliferation,migration and invasion of PCa cells,which was consistent with the function of BLM,which was inconsistent with our prediction.The analysis of the regulatory relationship between MIB1 and BLM had multiple effects.Firstly,the cancer promotion of MIB1 overexpression itself could not be ignored.Secondly,it was speculated that the change of cancer cell cycle and the wrong localization of BLM in the cytoplasm exacerbated the overexpression of MIB1 and other cancer promoting genes,but the specific mechanism was still unclear;At the same time,mir-195-5p can target and regulate the m RNA expression of MIB1 gene to inhibit the expression of MIB1 protein,thereby inhibiting the proliferation and invasion of PCa cells.Finally,in terms of de ubiquitination of BLM,we preliminarily predicted the potential interaction between PVT1 and BLM,the ubiquitination effect of PVT1 on BLM,and the proliferation and migration of prostate cancer cells.According to the relevant literature,the reports that BLM really directly acts on a certain oncogene or tumor suppressor gene are very limited,and most of them are the effects of interference or overexpression of BLM on the phenotype of cancer cells.Based on this,some studies have shown that BLM can combine with EZH2 protein to regulate the proliferation of PCa cells through BLM/EZH2/MDM2/p53 signal axis.Other studies have shown that lncRNA PVT1 can combine with EZH2 protein to regulate the proliferation and migration of cancer cells.At the same time,PVT1 can be used as a deubiquitinase scaffold to regulate the cell localization of deubiquitinase and maintain the stability of oncoprotein.Combing the literature reports,we speculate that BLM and lncRNA PVT1 may interact through EZH2 protein,there is a potential lncRNA PVT1/EZH2/ BLM/MIB1 regulation axis or lncRNA PVT1/BLM/MIB1 regulation relationship,which may be related to the de ubiquitination of BLM.The miR-27b-3p closely related to PVT1 and BLM was screened by bioinformatics software.We found a close negative correlation between the expression of PVT1 and miR-27b-3p through q RT PCR and cancer database expression profile analysis.RNA immunoprecipitation,double Luciferase Report Analysis and RNA pull-down evaluation further confirmed the direct targeting relationship between the two RNAs.RNA pull-down evaluation also revealed the targeted binding of BLM and PVT1 in PCa cells.PVT1 reduced the ubiquitination level of BLM in PCa cells and played a role in stabilizing BLM.By using CCK-8,Transwell in vitro and xenograft tumor in vivo,it was clarified that PVT1 affected the expression of its target gene BLM by adsorbing miR-27b-3p in the cytoplasm.Some functions of PVT1/miR-27b-3p/BLM/MIB1 regulatory axis in the proliferation and migration of PCa cells were elucidated.In conclusion,miR-195-5p can inhibit the proliferation and invasion of PCa cells by targeting MIB1 m RNA expression;PVT1 regulates the overexpression of its target gene BLM through the functional adsorption of miR-27b-3p by ce RNA to promote the proliferation and migration of PCa cells in vivo and in vitro.At the same time,part of the function of PVT1 is to reduce the ubiquitination of BLM and improve its stability.These findings may provide new targets and ideas for PCa therapy.