Inhibition Of Brd4 Specific SiRNA On Prostate Cancer Cells By Cationic Nano Carrier PGEA-CHO

Background: The incidence rate of prostate cancer(PCa)has been increasing in China in recent years.Because of the relatively hidden incidence of prostate cancer,many patients find it late,which has increased the difficulty of treatment.At present,the treatment of prostate cancer mainly includes surgery,radiotherapy,chemotherapy and hormone therapy,which easily lead sequelae and relapse.With the development of gene research,gene therapy has become a hot topic.It has become a common means of cancer treatment to reduce the expression of cancer-related genes through gene interference.At present,the most commonly used interference method is to use small interfering RNA(siRNA)entering into the cell which plays the role of gene interference.Brd4(bromodomain containing protein 4)is a member of the BET family.It has been reported that Brd4 is related to the development of a variety of tumors.It participates in the transcription of MYC,BCL2 and other genes,so as to regulate cell proliferation and apoptosis.In order to deliver Brd4 specific siRNA(siBrd4)to tumor cells,we need ideal gene vectors.Cationic polymer gene carrier,has become a research hotspot because of its good biocompatibility,low toxicity,structural diversity,relatively high transfection efficiency,easy synthesis and low cost.Nowadays,there are two ways to evaluate the transfection effect of cationic polymer: high transfection efficiency(including the binding efficiency of cationic polymer and gene and the release efficiency in cell)and low cytotoxicity.From these two points of view,a new cationic polymer PGEA-CHO was used as siBrd4 carrier to complete the treatment of prostate cancer,and the related mechanism was preliminarily explored.Objective: A new cationic nanoparticle PGEA-CHO was used to transport Brd4 specific siRNA.PEI,a common cationic polymer transfection reagent,was used as a control to study and evaluate the transfection efficiency and cytotoxicity of PGEA-CHO compound containing cholesterol end group.In vitro experiments showed that PGEA-CHO could effectively inhibit the growth of prostate cancer cells.And the relevant mechanisms were preliminarily explored in this research.Methods: PGEA-CHO was synthesized by chemical method.Agarose gel and transfection fluorescence test were conducted to detect the ability of PGEA-CHO to bind siRNA under different N/P ratios,which can determine the best transfection ratio of N/P.CCK-8 method was used to detect the toxic effects of PGEA-CHO at different concentrations on PC-3 and RM-1 cells.Flow cytometry was used to detect the effect of sibrd4 carried by PGEA-CHO on prostate cancer cell apoptosis.CCK-8 and clone formation were used to detect the effect of siBrd4 carried by PGEA-CHO on the growth and reproduction of prostate cancer cells.Western blot and real-time quantitative qPCR were used to detect the expression of Brd4 and c-MYC protein and m RNA.Results: PGEA-CHO was synthesized successfully,and the expression of FAM in the cells was observed by in vitro transfection experiment.It can be confirmed that PGEA-CHO can be used as an effective gene carrier,carrying siBrd4 to transfect PC-3 cells and RM-1 cells.CCK-8 showed that the cytotoxicity of PGEA-CHO was significantly lower than PEI.qPCR and Western blot showed that the expression of Brd4 m RNA and protein was significantly lower in the control group and PEI transfected group.CCK8 results showed that the proliferation inhibition rate of PGEA-CHO-siBrd4 group was significantly higher than PEI-Brd4;flow cytometry results showed that the apoptosis rate of PGEA-CHO-siBrd4 group was significantly higher than other groups with statistical difference.In addition,qPCR and Western blot showed that the expression level of c-MYC in PGEA-CHO-siBrd4 cells was significantly down regulated.Conclusion: Compared with PEI,PGEA-CHO is safer,and its transfection efficiency is more efficient and stable.Also siBrd4 transfected with PGEA-CHO is more effective in the treatment of prostate cancer cell lines PC-3 and RM-1.In addition,the therapeutic effect of pgea-cho-sibrd4 on prostate cancer cells may be due to the down-regulation of c-MYC expression,and then regulates apoptosis,growth and other related genes.These results indicate that PGEA-CHO can be used as a safe and effective siRNA vector for gene targeted therapy of tumor.