Detection Serum MiRNAs Expression Profiles Of Prostate Cancer And Preliminary Study On Regulating Function Of MiR-101 Targeting To EZH2

Prostate cancer(PCa)is one of the most common malignant tumours in urinary system.Radical prostatectomy,hormonal therapy,radiotherapy and chemotherapy are considered as major therapeutic approaches for prostate cancer.Patients with recurrence or metastasis after radical prostatectomy and patients who lost opportunity of operation usually choose hormonal therapy that is effective in the initial period.But almost all prostate cancer would progressed into androgen-independent and hormone-refractory prostate cancer,and hormonal therapy would become ineffective.Therefore,revealing the internal molecular mechanism in the progression of prostate cancer has been a research hotspot.MicroRNA(microRNA,miRNA,miR)is a short non-coding RNA of 21～25 nucleotides in length that usually function as endogenous repressors of target genes mainly by affecting both the stability and translation of mRNA at post-transcriptional level.In the past few years,miRNA profiling of various human cancers has revealed many miRNAs that function as tumor suppressors.MiRNA-101 is one of the down-regulated miRNAs in a diverse of malignancies relative to their non-tumor counterparts,whitch is suggested that miRNA-101 is possibly a potential target for therapeutic interventions in aggressive tumors.In the present study,we detected and compared the differential expression profiling of miRNA in serums of benign prostatic hyperplasia patients and prostate cancer patients in different clinical stages,and confirmed the correlations between those miRNAs and prostate cancer.Furthermore,we further analysed whether miR-101 in serum of prostate cancer was beneficial to diagnosis and evaluation of malignant degree.Lastly,we investigated the regulative effect and mechanism of miR-101 targeting to EZH2 in prostate cancer by transfecting synthetic miR-101 mimic into human prostate cancer PC-3 cellsThis study consists of two parts.Part I The expression profile of miRNA in serum of prostate cancer patientsObjective:To investigate the differential expression profiling of miRNA in serums of benign prostatic hyperplasia patients and prostate cancer patients in different clinical stages by using miRNA microarray and confirm the correlations between those miRNAs and prostate cancer.Methods:Expressions of miRNA in serum of ADPC、AIPC and BPH patients were detected by using microRNA microarray.All the targeted gene regulated by these miRNA were acquired in microRNA database for target gene Pathway analysis and GO analysis.Results:9 serum samples(3 cases of ADPC,3 cases of AIPC and 3 BPH)were carried out miRNA microarray analysis and screened 31 differentially expressed miRNA in three groups.8 aberrant miRNAs were detected in ADPC in comparison to BPH.Furthermore,7 upregulated miRNAs including miR-181a-2,miR-29a,miR-423-5p,miR-424,miR-1184,miR-671-3p,miR-548b-3p and 1 downregulated miRNA comprising of let-7b(P<0.001).9 upregulated miRNAs(miR-181a-2,miR-3664,miR-519e,miR-144,miR-603,miR-187,miR-491-3p,miR-BART20-5p,miR-3139)were detected in AIPC in comparison to BPH,and 6 downregulated miRNAs included miRPlus-A1073,miR-542-3p,miR-660,miR-134,miR-15b,miR-H4-3p(P<0.001).Particularly,miR-181 a-2 was simultaneously aberrantly expressed in ADPC and AIPC.In addition,4 upregulated miRNAs(miR-BART16,miR-3142,miR-491-3p,miR-214)were detected in AIPC in comparison to ADPC,and 4 downregulated miRNAs included miR-369-3p,miR-371-3p,miR-495,miR-671-3p(P<0.001).Ultimately,all the targeted gene regulated by these aberrant miRNAs were acquired in miRNA database for target gene Pathway analysis and GO analysis.Conclusions:MiRNA might be involving in prostatic tumorgenesis and prostate cancer androgen-independent conversion.The differentially expressed miRNA in serum might have diognosis potential and be seen as theraputic targets in human prostate cancer.Interesting,miR-181a-2 might anticipate in the progression of prostate cancer.Differential expression of miR-101 in serum of prostate cancer had not been detected,which confirmed the relationship of miRNA expression between serum and tissue was controversial.Part II The expressions of miR-101 and EZH2 in prostate cancer and the effect of miR-101on biological behavior of PC-3 cells by targeting EZH2Objective:To analyze the regulative effect and mechanism of miR-101 targeting to EZH2 in prostate cancer,then detect the expression of miR-101 and EZH2 in prostate cancer cell lines and normal prostate epithelial cell line,and investigate the effect on the biological features of human prostate cancer PC-3 cells transfected by synthetic miR-101 mimic.Methods:The expression of miR-101 and EZH2 mRNA in n prostate cancer PC-3 cell line,LNCaP cell line and normal prostate epithelial RWPE-1 cell line were detected by Real-time PCR.The expression level of EZH2 protein in these cell lines were detected by Western-Blot.PC-3 cells were divided into three groups:untransfected blank control group B(B group);transfected negative control group NC(NC group);transfected experimental group with miR-101 mimics(miR-101 group).MiR-101 mimic was transfected into PC-3 cell and expression changes of EZH2mRNA and protein were measured with Real-time qPCR and Western blot respectively.MTT assays were used to measure the proliferation of the PC-3 cells;flow cytometry was used to analyze the cell cycle distribution;Annexin V/PI test was used to measure cell apoptosis;the wound scrape assays were used to measure the cell motility;transwell invasion assays were used to analyze the invasiveness in vitro.And all data were analyzed statistically.Results:(1)The expression levels of miR-101 in PC-3 cell line and LNCaP cell line were lower than the RWPE-1 cell line,the expression levels of miR-101 had a statistically significant difference in different prostate cell lines(p<0.05).(2)The expression levels of EZH2 mRNA in PC-3 cell line and LNCaP cell line were higher than the RWPE-1 cell line,and the expression levels of EZH2 protein in PC-3 cell line and LNCaP cell line were higher than the RWPE-1 cell line;The expression levels of EZH2 mRNA and protein had a statistically significant difference in different prostate cell lines(p<0.05,and p<0.05).It prompts that the expressions of miR-101 and EZH2 are negative correlated direction(p<0.05).(3)After miR-101 mimic was transfected into prostate cancer PC-3 cell,the expression of miR-101 was up-regulated(p<0.05),and the expressions of EZH2 mRNA and protein were down-regulated significantly(p<0.05).(4)The growth rate of miR-101 group is lower significantly than two control groups at 48h,72h and 96h after transfection(p<0.01),while there was no difference between control groups(p>0.05).(5)Flow cytometry analysis showed there was G0/G1 phase arrest in miR-101group than two control groups(p<0.05),while there is no difference in cell cycle distribution of the two control groups(p>0.05).(6)Cell apoptosis ratios of miR-101 group,B group and NC group at 48h after transfection was 9.84 ± 0.83%,2.53 ± 0.36%and 2.71±0.42%respectively.The apoptosis rate of miR-101 group was higher than two control groups(p<0.01).(7)Scarification test demonstrated that cell migrate of miR-101 group was more slowly than two control groups(p<0.01).The difference between B group and NC group was not obvious(p>0.05).(8)Transwell test showed that the number of invaded PC-3 cells of miR-101 group that went through the Matrigel gel were significantly less than two control groups(p<0.01),There is no difference between B group and NC group(p>0.05).Conclusions:(1)The expression of miR-101 was lower,while the expression of EZH2 was higher in prostate cancer cell lines than that in normal prostate cell lines.It prompts the abnormal expression of miR-101 and EZH2 involve in the occurrence and development of prostate cancer.EZH2 may be one of target genes of miR-101 which plays a negative regulation role.(2)The expression levels of miR-101 are lower in AIPC cell than in ADPC cell,however,EZH2 are higher.It suggests that miR-101 and EZH2 may take part in the transformation from ADPC to AIPC.(3)The downregulated expression of miR-101 in prostate cancer may cause the loss of inhibition to EZH2 expression,the EZH2 abnormal overexpression may play important role in the carcinogenic mechanism of prostate.The upregulation of miR-101 can lead to the lower expression of EZH2 in PC3 cells.(4)The upregulation of miR-101 may inhibit proliferation and reinforce apoptotis,and may decrease the ability of migration and invasion in the human prostate cancer PC3 cell.