| Background and ObjectiveProstate cancer(PCa)is the most common cancer among men in the United States and Europe,which is the second and third leading cause of cancer death.It is estimated that in 2018,there will be about 160,000 new cases of prostate cancer and more than 29,000 deaths in the United States.In 2012,Europe accounted for 400,000 of the 1.1 million new cases of prostate cancer worldwide.In China,with the wide application of serum PSA and prostate biopsy,the incidence of prostate cancer in China is also increasing year by year.Androgen blocking therapy is one of the main treatments for prostate cancer at present,and most patients with prostate cancer will change to androgen-independent prostate cancer after 18-24 months of androgen blocking therapy.The function of long non-coding RNA in various biological processes and diseases has attracted wide attention.Numerous studies have revealed the importance of IncRNA in various cellular physiological processes,including transcription?intracellular transport and chromosome remodeling.In cancer cells,the expression level of certain lncRNA changes significantly,and these changes may be closely related to the process of tumorigenesis and development.It has been found that lncRNA can play an important regulatory role at the transcriptional and post-transcriptional levels,affecting the generation?proliferation?invasion and metastasis of cancer cells.Prostate cancer gene expression marker 1 is a prostate-specific gene,which is highly expressed in prostate cancer and can promote the proliferation of LNCAP cells.Its specific mechanism is not yet fully understood.PCGEM1 is considered as a potential marker of prostate cancer.The molecular mechanism of PCGEMI is still unclear.Its mechanism may be mainly from epigenetic mechanism and transcriptional level.The post-transcriptionallevel regulates gene expression.It is reported in the literature that bioinformatics software is used to predict the transcriptional level.At the transcriptional level,bioinformatics results suggest that HMG-1Y,MEF2A,HNF-3p,NF-Y,Sox-5 and Pax6 may be involved in the transcriptional level regulation of PCGEMI in prostate cancer tissues.Myocyte enhancer facter 2(MEF2)is an important transcription regulator,belonging to the MADS-box family,including MEF2A/B/C/D.MEF2 has DNA binding properties and can bind to specific DNA sequences in the promoter of muscle creatine kinase(MCK)gene.It has been found that MEF2 plays an important role in many physiological and pathological processes,such as muscle formation,skeletal development,liver fibrosis,etc.The most prominent function of MEF2 is to perform gene transcription in the process of cell differentiation of skeletal muscle,smooth muscle and myocardium.In addition,MEF2 is an important calcium-dependent regulator and plays an important role in the development and differentiation of the nervous system.The role of MEF2 in various tumors has also been found.MEF2 gene family is a hematological oncogene.In acute T-cell lymphoblastic leukemia,the expression of MEF2C is increased.The production of dazapl/MEF2 fusion protein with carcinogenic activity was found in acute B lymphoblastic leukemia.The role of MEF2 protein in the development of solid tumors has been proposed.The expression of MEF2A gene and MEF2C gene in primary hepatocellular carcinoma(HCC)cells was higher than that in normal hepatocytes.The expression of MEF2 in hepatocellular carcinoma patients suggests poor prognosis.The activity of MEF2 family proteins as tumor suppressor genes has also been determined.Studies have shown that the expression of MEF2 target gene in adipose tissue and leiomyosarcoma is lower than that in normal tissue.In this study,we will explore the molecular mechanism of PCGEMI involvement in hormone-independent prostate cancer.MiRNA is a kind of endogenous non-coding RNA with regulatory function in eukaryotic organisms.It has about 19-25 nucleotides,and it plays an important role in tumorigenesis and development.Abnormal expression of microRNAs is associated with most tumors,and can be used as oncogene or tumor suppressor gene to participate in a variety of cellular functions,including angiogenesis,proliferation,apoptosis,invasion and metastasis.Studies have shown that the expression and regulation of PCGEMI may be related to microRNAs,including microRNAs-145,microRNAs-148a,microRNAs-26a,microRNAs-26b and so on.Studies have confirmed that in androgen-dependent prostate cancer cells,the high expression of microRNA-145 can down-regulate the expression of PCGEM1,while the low expression of PCGEMI can up-regulate the expression of microRNA-145.PCGEM1 can promote the proliferation of androgen-dependent prostate cancer cells by down-regulating the expression of microRNA-145,which is considered as a potential target for the treatment of prostate cancer.It has been found that microRNA-148a is closely related to tumors.In esophageal,gastric,hepatic,colorectal,pancreatic,non-small cell lung,breast and urogenital cancers and other cancers,down-regulation of microRNA-148a can be detected.However,the up-regulation of microRNA-148a can also be seen in glioma and osteosarcoma.The expression of MiR-148a is related to the clinical classification,curative effect and prognosis of tumors.It has been confirmed that the expression level of microRNA148a in normal prostate cells and hormone-sensitive prostate cancer cells is significantly higher than that in castrate-resistant prostate cancer cells,suggesting that microRNA-148a is not only involved in the occurrence of prostate cancer,but also in the transformation of hormone-sensitive prostate cancer to castrate-resistant prostate cancer.However,it is still unclear whether PCGEMI and microRNA-148a have regulatory relationship and what kind of regulatory relationship exists.Objective:There is no effective treatment for castration-resistant prostate cancer,which is an important cause of death of prostate cancer.There is an urgent need to find new treatment methods.The biological role of PCGEM1 in prostate cancer and its molecular mechanism are still unclear.In this study,the expression of PCGEMI in prostate cancer tissues and cell lines and its effect on the proliferation of prostate cancer cells were detected to determine the important role of PCGEMI in the occurrence and development of prostate cancer.By further exploring the molecular mechanism of PCGEM1 involvement in castration-resistant prostate cancer,we hope to provide new tumor markers for prostate cancer and provide new therapeutic targets for prostate cancer.Methods:To investigate the biological role of PCGEM1 in prostate cancer and its molecular mechanism,this study was divided into three parts:1.To detect the expression of PCGEMI in prostate cancer,we collected prostate cancer tissues and their corresponding non-cancer tissues,cultured normal prostate epithelial cell lines(PrEC),hormone-sensitive prostate cancer cell lines(LNCAP)and castrate-resistant prostate cancer cells(PC3 and DU145).The expression of PCGEMI was detected by real-time quantitative PCR in prostate cancer tissues,adjacent adjacent tissues and different prostate cancer cell lines.2.In order to study the biological role of PCGEM1 in prostate cancer cells,by up-regulating and down-regulating the expression of PCGEM1 in prostate cancer PC3 cells,MTT assay and plate cell cloning assay were used to observed the effect of PCGEM1 on the proliferation of prostate cancer cells.3.To study the mechanism of PCGEM1 on the proliferation of prostate cancer cells,we first confirmed whether MEF2A was involved in the transcriptional regulation of PCGEM1 in prostate cancer tissues,cultured PC3 cells,and ascertained the effect of MEF2A on the expression of PCGEM1 by up-regulating and down-regulating the level of MEF2A.In order to verify the existence of MEF2A binding site on PCGEM1 promoter,the concentration of MEF2A was evaluated by chromatin immunoprecipitation assay(CHIP).To clarify that MEF2A can regulate the expression of PCGEM1 by directly interacting with promoter and driving gene transcription,luciferase analysis was used to determine the effect of MEF2A on the transcription efficiency of PCGEM1 promoter.In order to further analyze the role of PCGEM1 in regulating the expression of microRNA-148a,the effects of PCGEM1 on the expression of microRNA-148a were analyzed by silencing or overexpressing PCGEM1.Finally,the effects of PCGEM1 and miR-148a on PC3 cell apoptosis were evaluated by flow cytometry.Results:1.PCGEM1 overexpressed in prostate tumor tissues and cancer cells.To determine the difference of PCGEM1 expression in prostate cancer tissues and adjacent non-cancerous tissues,we studied the expression of PCGEM1 in prostate cancer and adjacent non-cancerous tissues by reverse transcription polymerase chain reaction(RT-PCR).The expression of PCGEM1 in prostate cancer tissue was significantly higher than that in adjacent adjacent tissues.In addition,we examined the expression of PCGEM1 in normal prostate epithelial cell line(PrEC),hormone-sensitive prostate cancer cell line LNCaP and hormone-resistant prostate cancer cell line PC3 and DU145.The expression of PCGEM1 in PC-3 and DU145 cells was significantly higher than that in PrEC and LNCaP cells,and the difference was statistically significant.2.Effect of PCGEM1 on Proliferation of Prostate Cancer Cell PC3.To determine the effect of PCGEM1 expression on PC3 cell proliferation,we knocked down the expression of PCGEM1 in PC3 cells by PCGEM1 siRNA(Fig.2A).Then we determined the effect of PCGEM1 knockdown on PC3 cell proliferation by MTT assay.We found that when PCGEM1 expression was knocked down,PC3 cell proliferation was significantly inhibited(Fig.2B).To further determine the effect of PCGEM1 on the proliferation of prostate cancer,pcDNA3.1-PCGEM1 was overexpressed in PC3 cells(Fig.2C).When the expression of PCGEM1 was up-regulated,the proliferation of PC3 cells increased significantly(Fig.2D).3.Effect of PCGEM1 on cloning of prostate cancer cells.To determine the effect of PCGEM1 expression on the cloning of PC3 cells,we knocked down the expression of PCGEM1 in PC3 cells by PCGEM1 siRNA.Then,the PC3 cells were planted in six-well plate for 2 weeks.Then we observed the number of clones formed.We found that when the expression of PCGEM1 was knocked down,the number of clones decreased significantly(Figure 3A).When PCGEM1 was overexpressed in PC3 cells,the number of clones increased significantly(Fig.3B).4.MEF2A up-regulates the expression of PCGEM1.PC3 cells were transfected with pcDNA3.1-MEF2A and si-MEF2A to detect the expression of MEF2A.PC3 cells were transfected with pcDNA3.1 as control group.After transfection of pcDNA3.1-MEF2A,the expression levels of MEF2A gene and protein were significantly higher than those of the control.When the expression of MEF2A increased,the expression of PCGEM1 also increased.After transfection of MEF2A siRNA,the expression of MEF2A decreased,and the expression of PCGEM1 also decreased significantly.These results suggest that MEF2A can regulate the expression of PCGEM1 in prostate cancer cells.5.MEF2A regulates the transcriptional activity of PCGEM1.MEF2A can regulate the expression of PCGEM1.As a transcription factor,we speculated that MEF2A can regulate the promoter binding to PCGEM1,thus promoting the expression of PCGEM1.To verify this hypothesis,pcDNA3.1-MEF2A and pcDNA3.1 were transfected into PC3 cells.It was observed that MEF2A increased the activity of PCGEM1 promoter by 2.0-2.5 times.On the contrary,after transfection of si-MEF2A into PC3,its activity decreased significantly.In order to verify the binding site of PCGEM1,the chromatin immunoprecipitation technique was used to analyze its enrichment.After transfection of pcDNA3.1-MEF2A,the enriched promoter fragment of PCGEM1 was twice as high as that of the control.In addition,when siRNA knocked down MEF2A expression,PCGEM1 enrichment was significantly reduced.These results suggest that MEF2A can activate the expression of PCGEM1 by targeting its promoter.6.PCGEM1 has a negative regulatory effect on microRNA-148a.In order to elucidate the molecular mechanism of PCGEM1 regulating the proliferation of prostate cancer cells,we further explored the regulatory effect of PCGEM1 on microRNA-148a.Based on the analysis of software,microRNA-148a contains a binding site with the 5'UTR end of PCGEM1.To confirm the regulatory effect of PCGEM1 on microRNA-148a,the expression of PCGEM1 was silenced in PC3 cells.When the expression of PCGEM1 was knocked down,the expression of microRNA-148a increased significantly.On the contrary,when PCGEM1 was overexpressed,the expression of microRNA-148a decreased significantly.These results suggest that there is a negative regulatory relationship between PCGEM1 and microRNA-148a.In addition,we studied the effects of microRNA-148a and PCGEM1 on PC3 cell apoptosis by flow cytometry.The results showed that the apoptotic rate in si-PCGEM1 group was significantly higher than that in control group.At the same time,compared with only si-PCGEM1 treatment,inhibition of the expression of microRNA-148a,cell apoptosis decreased to a certain extent.These results suggest that knockdown of PCGEM1 can promote apoptosis of PC3 cells by up-regulating the expression of microRNA-148a.Conclusion1.PCGEM1 was overexpressed in prostate cancer tissues and cancer cells.2.PCGEM1 can promote proliferation of prostate cancer cells3.MEF2A can activate the expression of IncRNA PCGEM1 by targeting its promoter.There is a positive regulatory relationship between MEF2A and PCGEM14.There is a negative regulatory relationship between PCGEM1 and miR-148a,knockdown of PCGEM1 can promote apoptosis of PC3 cells by up-regulating the expression of microRNA-148a. |
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