Long Non-coding RNA PlncRNA-1 Functions As A Competing Endogenous RNA Of Androgen Receptor To Promote Prostate Cancer Progression

Based on the comprehensive cancer statistic in 2015, the incidence of prostate cancer was 26% of all new cancer cases and the mortality rate was 9% of the total cancer related deaths。And it ranks second as the leading causes of cancer mortality.The initiation of PCa is a complex and dynamic biological process that involves multiple genomic and epigenomic alterations that occur over an extended time period of approximately 10 years. The human genome encodes approximately 20,000 protein-coding genes, a number similar to that observed in many other species. As first reported in 2007, most human DNA is transcribed into non-protein coding regulatory molecules, referred to as noncoding RNAs, that play critical roles in multiple biological processes. The discovery of numerous long non-coding RNA(lncRNA) transcripts in species ranging from yeast to mammals has dramatically altered our understanding of cell biology, particularly with respect to the pathophysiology of diseases, such as cancer. Early evidence suggested that lncRNAs play essential roles in tumorigenesis and that lncRNA-mediated biological processes are at the core of cancer progression.Several lncRNAs(i.e.PCA3, PCAT-1, PCAT-29, PCGEM1, PRNCR1 and CTBP1-AS) are reported to be involved in PCa. As all stages of PCa are critically dependent on androgen receptor(AR) signaling for growth and survival, androgen-regulated lncRNAs are of fundamental significance to PCa.In a previous study using RNA-seq data from 14 pairs of PCa tissues and patient-matched normal tissues, we found that the lncRNA referred to as PlncRNA-1 plays an important role in PCa pathogenesis. PlncRNA-1, also referred to as CBR3-AS1, comprises five exons(Gene ID: BC038671). Using knockdown experiments, we found that PlncRNA-1 expression was directly correlated with cell proliferation and the expression of AR in LNCaP cells. We also found that the Plnc RNA-1 transcript levels were significantly associated with the AR mRNA levels in human PCa tissue samples. These findings indicated that PlncRNA-1 and ARs are associated with a common regulatory mechanism in PCa; however, this mechanism has yet to be characterized.Several recent reports proposed a model in which lncRNA functions as a competing endogenous RNA(ceRNA) to protect critical target genes from miRNA-mediated suppression. ceRNAs generally recognize miRNA-response elements in the transcripts of several important genes and prevent the mRNA transcripts of these genes from miRNA-mediated degradation. The results of the current study suggest that PlncRNA-1 functions as a ceRNA to regulate the expression of ARs and AR-regulated miRNAs in PCa, as demonstrated by bioinformatics analysis and other experimental approaches. Furthermore, PlncRNA-1 is a direct transcriptional target of ARs. Finally, we demonstrated that PlncRNA-1 negatively regulated miR-34c-5p and miR-297-3p, which may partially account for the PlncRNA-1-mediated cell proliferation observed in PCa tissues. Of considerable interest, miR-34c-5p and miR-297-3p inhibit cell growth and simultaneously repress PlncRNA-1 expression, thereby forming a reciprocal inhibitory feedback loop that might contribute to a competitive endogenous RNA network in PCa. Part Ⅰ Identification and Characterization of the Long Noncoding RNA PlncRNA-1Methods:(1) PlncRNA-1 Gene annotations using UCSC and H3K27 ac ChIP data from ENCODE project are shown.(2)Comparison of PlncRNA-1 isoform expressions in prostate cancer cell lines(RWPE-1, LNCaP, 22RV1, and DU145) by northern blot and qPCR. Comparison of PlncRNA-1 expression in various cell lines(A498, 7860, EJ, LNCaP, PC3 and 293T).(3)Fluorescence in situ hybridization of PlncRNA-1 in prostate cancer cell lines(22RV1 and LNCaP). Nuclear and cytoplasmic distributions of various non-coding and protein coding transcripts in LNCaP and 22RV1 cells(4)Using RT-PCR and Western blot to verify the relationship between AR and PlncRNA-1(5) AR antibody mediated RNA-IP analysis in LNCaP cells.Results:(1) PlncRNA-1 is located on chr2(q22,12) and is one of the three alternatively spliced isoforms transcribed from the Cbr3-as1 locus.(2)V3(PlncRNA-1) is highly expressed in PCa cells compared with normal cells. With respect to the urinary tumor cell lines, PlncRNA-1 was strongly expressed in prostate cancer cell lines.(3) PlncRNA-1 was mainly expressed in the cytoplasm of both LNCaP and 22RV1 cells.(4) Knockdown of PlncRNA-1 significantly decreased AR mRNA level and protein level. Overexpression of PlncRNA-1 significantly increased AR mRNA level and protein level in LNCaP and 22RV1 cells.(5)PlncRNA-1 was not significantly enriched in RNAs coimmunoprecipitated with AR.Conclusion:PlncRNA-1 is highly expressed and mainly located in the cytoplasm of PCa cells might interact with cytoplasmic components to exert its effects in PCa.PlncRNA-1 was not significantly enriched in RNAs coimmunoprecipitated with AR. Part Ⅱ Androgen receptor directly regulates PlncRNA-1 expressionMethods:(1)PlncRNA-1 expression was measured upon stimulation with DHT(stimulated with 10nM) in a time-dependent fashion in LNCaP cells. LNCaP cells was stimulated with 0, 0.01, 1, 10, 100 nM of androgen for 48 h and a dose-dependent PlncRNA-1 induction was indeed observed.(2) We used siRNA knockdown of AR and tested PlncRNA-1 expression in LNCaP cells.(3) Sub-cloned the PlncRNA-1 promoter into a luciferase reporter. Testing the activities of reporter on the condition of the presence of androgen and AR antagonist MDV3100.(4) Using PROMO transcription factor binding prediction algorithm, tested predicted AR responsive element(ARE) in PlncRNA-1 promoter. We performed chromatin immunoprecipitation(Ch IP) assays for AR followed by qPCR testing the enrichment of PlncRNA-1 promoter.Results:(1) PlncRNA-1 expression was promoted upon stimulation with DHT in a time-dependent and a dose-dependent fasion(2) SiRNA knockdown of AR and showed significantly decreased PlncRNA-1 in LNCaP cells(3) Androgen significantly induced the activities of reporter. On the other hand, AR antagonist MDV3100 significantly inhibited the activities.(4) Using PROMO transcription factor binding prediction algorithm, we found three predicted AR responsive element(ARE) in between 1,500 bp upstream PlncRNA-1 transcription starting site. CHIP confirmed it.Conclusion:Androgen receptor directly regulates PlncRNA-1 expression. Part Ⅲ PlncRNA-1 regulates AR through competing endogenous RNA mechanismMethods:(1) we used Miranda algorithm and identify miRNAs shared by PlncRNA-1.(2)Expression profiling of miRNAs after PlncRNA-1 siRNA treatment. Chose miRNA which target PlncRNA-1.(3)AR and PlncRNA-1 mRNA levels were assayed after cotransfection with the indicated miRNAs.Using luciferase reporters assay, RT-PCR to confirm these miRNA can target both PlncRNA-1 and AR.(4)The relative luciferase activities of luciferase reporters containing wild-type or mutant PlncRNA-1 transcripts were assayed 48 hrs after cotransfection with the indicated mi RNAs(5) RIP analysis test whether PlncRNA-1 and miRNAs bind to AGO2.(6)Assessing the binding ability of miRNAs to PlncRNA-1 and mutated PlncRNA-1.Results:(1)We used Miranda algorithm and identify 13 miRNAs shared by PlncRNA-1 and AR.(2)Identified miR-34c-5p and miR-297-3p are shared by PlncRNA-1 and AR through bioinformatics tool.(3)Luciferase reporters assay, RT-PCR confirmed that these miRNA can target both PlncRNA-1 and AR.(4)PlncRNA-1 –mut without MREs for miR-34c-5p and miR-297-3p abolished their effect on PlncRNA-1.(5) RIP analysis suggested PlncRNA-1 directly bind to these mi RNAs.(6)Competitive experiments confirm that PlncRNA-1 protected AR by competitive binding miRNA.miR-34 and miR-297-3p were significantly enriched in the products that co-precipitated wild-type PlncRNA-1 compared with mutated PlncRNA-1Conclusions : MiR-34c-5p and MiR-297-3p target both PlncRNA-1 and AR. PlncRNA-1 can play as a competing endogenous RNA(ceRNA) of AR and bind to these miRNAs to protect AR, resulting the incresase of AR. Part Ⅳ PlncRNA-1 regulates PCa apoptosis,migration and proliferationMethods:(1)we use CCK-8 to test the effect of PlncRNA-1 on cell viability. And we use Annexin V-FITC Apoptosis Detection Kit to test the effect of PlncRNA-1 on cell apoptosis. We use corning transwell to test the effect on cell migration(2) we use CCK-8 to test the effect of miR-297-3p/miR-34c-5p on cell viability. And we use Annexin V-FITC Apoptosis Detection Kit to test the effect of mi R-297-3p/miR-34c-5p on cell apoptosis.(3) We transfected prostate cancer cells with Lv-PlncRNA-1 and LV-siPlncRNA-1 to study the effect in vivo.(4) We tested PlncRNA-1 and its related gene expression in xenografts in nude mouse model。Results:(1) We observed significant impact of PlncRNA-1 on cell viability, cell apoptotic and migration in PCa cells compared to control.PlncRNA-1 promote viability and migration of prostate cancer and prevent apoptosis.(2) miR-297-3p/miR-34c-5p show opposite effect on prostate cancer cells from PlncRNA-1(3)The tumor volume was increased in overexpressed LV-PlncRNA-1 compared to the LV-NC and decreased in LV-siPlncRNA-1 in vivo.(4) We also evaluated the expression of PlncRNA-1, AR, miR-34c-5p and miR-297-3p in the xenograft-derived tumor tissues using qPCR, and the results were consistent with those observed in the PCa cell in vitro experimentsConclusions:PlncRNA-1 promotes prostate tumor growth in vivo via miR-34c-5p and miR-297-3pPart Ⅴ PlncRNA-1 and its related genes expression in the diagnosis of Prostate cancerMethods:(1)Testing PlncRNA-1,miRNAs in 16 pairs of PCa tissues. Using RT-PCR and ISH method.Testing the correlation between PlncRNA-1 and its related genes expression in 48 PCa tissues(2)Testing PlncRNA-1 in plasma samples from 72 consecutively enrolled patients who underwent prostate biopsy. Thirty-seven samples were obtained from PCa patients(positive prostate biopsy), and 35 samples were obtained from non-PCs patients(negative prostate biopsy)Results:(1) In the same 16 pairs of tissues demonstrated that miR-34c-5p and miR-297-3p were down-regulated in PCa tissues while PlncRNA-1 were up-regulated.(2)PlncRNA-1 transcript levels were significantly correlated with the mRNA levels of AR and the expression of the anti-apoptotic protein Bcl-xL in human PCa samples. And were inversely correlated with miR-34c-5p and mi R-297-3p.(3)The PlncRNA-1 expression levels were elevated in the samples derivrd from PCa patients compared with those derived from the non-PCa patients.Conclusions : PlncRNA-1 expression levels were inversely correlated with miR-34c-5p and miR-297-3p expression levels in PCa specimens. PlncRNA-1 has the potential to serve as biomarker to distinguish between patients with positive prostate biopsies from patients with negative prostate biopsies. SummaryThis paper mainly focused on the function and the mechanism of long noncoding RNA in the development of prostate cancer. By constructing PlncRNA-1 overexpression and silenced cell lines, we tested its tumor promoting function in vivo and in vitro. In the research of mechanism, through a variety of strategies, we systematically confirmed the major form of PlncRNA-1 and PlncRNA-1 was mainly located in the cytoplasm of cell. It is stable in cytoplasm and may play a regulatory role. Prediction of advanced bioinformatics and dual luciferase reporter can verify that PlncRNA-1 can interact directly with mi R-34c-5p and mi R-297-3p.And the target gene prediction and related research confirmed that the two miRNA can target AR which play tumor suppressor role in the development of prostate cancer. At last, we confirmed the correlation between PlncRNA-1 and two miRNA in Prostate cancer tissues. Previously found on the basis of AR-regulated PlncRNA-1, we found miR-34c-5p and miR-297-3p inhibit cell growth and simultaneously repress PlncRNA-1 expression, thereby forming a reciprocal inhibitory feedback loop that might contribute to a competitive endogenous RNA network in prostate cancer. The mechanism is important for understanding the progression of prostate cancer associated with Androgen receptor.