The Role Of CCAT1 In Prostate Cancer Progression And Its Regulation Mechanisms

According to the announcement of The American Cancer Society,Prostate cancer(PCa)has been the most-commonly diagnosed cancer among American men and second leading cause of cancer-related death in elderly men in 2019.Recent years,with the change of lifestyle and diet structure,the morbidity and mortality of PCa have also shown a great increase in China,which seriously threatens the health of elderly men.Since the first PCa patient received the androgen deprivation therapy(ADT)in 1941,ADT has become the front-line treatment for PCa.Even though more than 80% PCa patients initially respond well for ADT,most of them will inevitably progress to the castration-resistant prostate cancer(CRPC)within 2 years after this treatment,which accounts for the most cases of death for patients with advanced prostate cancer.Therefore,it is urgent to uncover the pathogenesis of PCa progression and to develop corresponding therapeutic strategies.Recent years,different types of RNA has been extensively studied in the progression of many kinds of tumors.Long non-coding RNAs(LncRNAs),a class of non-protein coding transcripts longer than 200 nucleotides,has been confirmed to participate in many biological processes,such as cell proliferation,differentiation,apoptosis,DNA damage response and chromosome imprinting.According to their different subcellular localization,LncRNAs maybe involved in regulating gene expression through different mechanisms.In the cytoplasm,LncRNAs can transactivate mRNA’s decay by duplexing with 3′ UTRs or by competing for miRNA binding,thereby modulating the de-repression of miRNA target gene and affecting cellular signaling cascade.In the nucleus,LncRNAs can bind to RNA or DNA to regulate chromatin modification and the cleavage of pre-mRNA,or act as scaffolds for distinct protein complexes assembly.Androgen receptor(AR),a ligand responsive protein,can mediate the effector function of androgen in PCa.As well known that even after ADT,the abnormally activated AR is essential for the PCa cells survival and the progression of CRPC.In the study of reactivation of AR signaling pathway,increasing evidences illustrate that LncRNAs can regulate the expression of AR pathway downstream genes by recruiting co-activators of specific transcription factors,thus playing crucial roles in the transition of hormone-dependent state.DDX5(p68),one of the large DEAD-box family,is an ATP-dependent RNA helicase which can regulate the cleavage and translation of RNA.As a co-activator of AR,P68 can be recruited to the AU-rich element(ARE)of PSA gene promoter region,promoting AR targeted gene expression,thus activating AR signaling pathway and stimulating CRPC progression.By combining the ISH staining results from androgen dependent prostate cancer(ADPC)tissues、CRPC tissues、metastatic lymph node samples and expression profile from prostate cancer biological database,we found that CCAT1 was significantly upregulated in metastatic lymph nodes、CRPC tissue and cell lines(PC3、Du145)relative to ADPC tissue and cell line(LNCaP),suggesting that CCAT1 may be related to the progression of CRPC.CCAT1,located on chromosome 8q24.21,was first identified as an oncogene in colorectal cancer.The abnormal expression was also found in other tumors,such as live cancer,gastric cancer,gallbladder cancer,lung cancer,ovarian cancer and esophageal squamopus cell carcinoma.However,the biologic functions of CCAT1 and molecular mechanisms underlying CCAT1’s role in prostate cancer remain unclear.Based on the above status of research,this present study identified the expression and subcellular localization of CCAT1 in different PCa cell lines by subcellular fractionation location and qRT-PCR firstly.In vitro and in vivo functional experimens were further performed to verify the effect of CCAT1 on prostate cancer cells.Finally,the molecular mechanism of CCAT1 function in PCa progression was explored by the combination of bioinformatics and molecular bioloy assays.Our findings set up the foundation for screening biomakers and searching for new therapeutic targets for PCa in the future.Part Ⅰ The differential expression of CCAT1 in prostate cancer tissuesObjective: Accompanied by the aging society procession in China in the past decade,the incidence and mortality of prostate cancer has increased in our country.In the initial stage,most tumors are ADPC and responsive to ADT.But after the sensitive period of about 14-30 months,most of tumors will develop into CRPC.Once tumors become resistant to ADT,the median survival time will be less than 20 months due to the lack of effective drugs and tumor resistant to radiotherapy and chemotherapy.With the completion of Human Genome Project Sequencing,numbers of non-coding RNAs,considered to be “noisy” transcripts,have been confirmed to be invovled in the regulation of cell proliferation,apoptosis,differentiation,metabolism,development of individuals and cancer progression.LncRNA,a relatively long non-coding RNA moleclue,can form the complex RNA secondary structure,thus binding to proteins to regulate gene function and participating in the tumor progression.In this part,CCAT1 expression in different PCa stage samples and different type of cell lines was detected by ISH and IF staining.By using subcellular fractionation location and qRT-PCR,CCAT1 subcellular localization was identified.The correlation between CCAT1 and prostate cancer clinical onset and prognosis was evaluated by re-analyzing profile from prostate cancer biological database.Methods: Tumor samples of ADPC patients(n=8)and CRPC patients(n=4)were collected after radical prostatectomy(RP).CCAT1 expression in different stage samples were analyzed by RNA ISH.The expression and subcellular localization were detected by FISH,subcellular fractionation location and qRT-PCR.LncRNA expression data obtained from TCGA and MSKCC database were re-analyzed to study the relationship between CCAT1 expression and tumor biochemical recurrence.Results: The expression of CCAT1 in CRPC tissues、cell lines(PC3 and Du145)and metastatic lymph node was signifcantly up-regulated compared with ADPC tissues and cell line(LNCaP)(P < 0.05).Subcellular fractionation location assay and FISH staining suggested CCAT1 located differently in different cell lines.The cytoplasm location for CCAT1 mainly existed in Du145 cell line and in LNCaP cell line,CCAT1 location could be observed both in cytoplasm and nucleus.By analyzing TCGA database,CCAT1 expression was proven to get upregulated with the malignant progression of prostate cancer(P < 0.05)and in different distant metastatic sites,its expression was also significantly higher(P<0.001)than the primary lesion of prostate cancer.Combined with the MSKCC prostate cancer database,Kaplan-Meier analysis using the log-rank test showed that among those patients who underwent RP with a follow-up longer than 12 months,the biochemical relapse-free survival in those patients with low levels of CCAT1(CCAT1 expression for the first quarter of about 32 patients weas defined as low level)was significantly longer than that in patients with high levels of CCAT1(CCAT1 expression for the rest 83 patients was defined as high level).Similarly,based on the tissue microarray results of another 150 patients,we found that for patients with high levels of CCAT1(for the first third of about 41 patients,CCAT1 expression was defined as low and for the rest of 109 patients,CCAT1 expression was defined as high),the overall survival in months was much shorter compared with patients with low levels of CCAT1.Conclusion: The above results suggested that CCAT1 expression in CRPC tissues and cell lines was significantly higher than that of ADPC tissues and cell.Results from TCGA and MSKCC databases showed that upregulation of CCAT1 representsed a poor prognostic factor for PCa patients.The different subcellular localization in different type of cells illustrated that CCAT1 may participate in the malignant progression of PCa through different mechanisms.Part Ⅱ Functional analyses of CCAT1 in prostate cancerObjective: The malignant progression of PCa is a continuous,multi-factor and multistep invovled dynamic process.Many regulatory molecules,such as co-activator,chromatin-modifying enzyme and AR-cleavage variant,can interact with each other to promote the androgen dependent state transition in cancer cells.In part ? study,results of RNA ISH,qRT-PCR suggested that CCAT1 expression in CRPC tissues and cell lines was significantly higher than that of ADPC tissues and cell.Subcellular fractionation location assay and IF staining suggested CCAT1 located differently in different cell lines.By analysing the LncRNA microarray results in TCGA and MSKCC databases,we found that CCAT1 is related to the progression and prognosis of PCa.These above results confirmed that CCAT1 may take part in the transformation from ADPC to CRPC to some extent.However,there is no systematic functional study on the role of CCAT1 in PCa until now.Therefore,it is worthy to explore whether CCAT1 participate in the transition of androgen dependent state of cell.In this part,we intend to explore the effects of CCAT1 on cell proliferation,apoptosis,cell cycle progression in vitro and subcutaneous tumorigenicity in vivo.The cellular functional pathways effected by CCAT1 were initially summarized at the same time.Methods: After construction of abnormal CCAT1 expression cell lines(knockdown in CRPC cell lines and overexpress in ADPC cell line),proliferation of PCa cells was measured by CCK8 proliferation assay and colony formation assay.Cell cycle and apoptosis were detected by flow cytometry.GSEA and GO annotation were performed after knockdown CCAT1 in PC3 cell by lentiviral transfection.The genetics change of related pathway was detected by Western Blot.In vivo assay,we tested effect of CCAT1 on subcutaneous tumorigenesis in nude mice.Ki-67,Caspase-3 of implanted tumor were tested by IHC to further verify CCAT1 effect on cell proliferation and apoptosis.Results: After construction of CCAT1 overexpression and knockdown cell lines,CCK8 and colony formation assay showed upreglated expression of CCAT1 in LNCaP cell could promote cell proliferation(P < 0.05),while downregulation of CCAT1 would suppress cell proliferation in PC3 and Du145 cell(P < 0.05).Flow cytometry showed that CCAT1 overexpression in LNCaP could promote cell cycle progression and decrease percentage of aopotosis cell(P < 0.05),on the contrary,CCAT1 downregulation in PC3、Du145 cell would inhibit cell cycle progression and increase percentage of aopotosis cell(P < 0.05).Western Blot results showed that the expression level of cyclin dependent kinase inhibitor-p21 protein and tumor suppressor gene-p53 protein both get decreased in CCAT1 overexpression LNCaP cell,however,these two protein expression level get increased in CCAT1 knockdown PC3、Du145 cell lines.From in vitro assays,we can draw the conclusion that CCAT1 act as an oncogene in PCa.In vivo assay,silencing CCAT1 by lentiviral transfection could obviously suppressed tumor growth as measured by tumor size and weight.Hematoxylin and eosin(H&E)and IHC staining of Ki67,activated caspase-3 in the endpoint tumors revealed that significantly reduced Ki67-positive cells and increased caspase 3-positive cells in CCAT1 knockdown PC3 tumors.GSEA and GO annotation analysis showed that DNA repair process and p53 apoptosis pathway were closely related to decrease expression of CCAT1 in PC3 cell.Conclusion: CCAT1 can promote PCa cells proliferation、cell cycle progression and inhibit cell apoptosis.In vivo experiment demonstrated that reduced CCAT1 expression inhibited prostate tumor regeneration and growth by suppression of proliferation and promotion of apoptosis.Altogether,the above experiments further confirmed that CCAT1 serves as an oncogenic factor in tumorigenesis of prostate cancer.Part Ⅲ CCAT1 binds to miR-28-5p to reverse the tumor suppressive effectObjective: In the second part,in vitro and in vivo assays showed that CCAT1 acted as an oncogene by promoting PCa cells proliferation,cell cycle progression and reducing cell apoptosis.However,the mechanism by which CCAT1 is involved in the progression of PCa is not clear yet.LncRNAs can participate in the development of tumor through different mechanisms according to their different subcellular localizations.In the cytoplasm,LncRNA can regulate gene expression by binding to the 3’ UTRs duplex of mRNA or by competing for miRNA binding to transactivate mRNA’s decay,thus modulating the de-repression of miRNA target gene.While in the nucleus,LncRNAs can affect mRNA function in different machanisms.Therefore,the molecular mechanism of CCAT1 oncogenic role need to be further investigated.Methods: By in vitro reverse transcription,we constructed the biotin-labeled CCAT1 sense and CCAT1 antisense single stranded RNA.After got folded to form the secondary structure,biotin-labeled CCAT1 sense and CCAT1 antisense were incubated with PCa cell lysates,followed by RNA-seq analysis.According to the different subcellular localization of CCAT1,we verified the relationship between CCAT1 and the targeted miRNA(miR-28-5p)in Du145 cell by dual luciferase reporter assay and qRT-PCR.The interaction between them were further confirmed by a series of functional recovery experiments.Results: After construction of the biotin-labeled CCAT1 sense and CCAT1 antisense,these two single stranded RNAs(already folded to form the second structure)were used in vitro RNA pulldown assay and the following RNA-seq analysis showed that miR-28-5p was the most sensitive RNA to bind to CCAT1 sense compared with the antisense(P < 0.05).The dual luciferase reporter assay demonstrated that miR-28-5p significantly reduced luciferase activity(wide type),while a mutation of the miR-28-5p binding site in CCAT1 abrogated the inhibitory effects.At the same time,qRT-PCR showed low expression of CCAT1 could increase miR-28-5p expression level in cells,while miR-28-5p mimics and miR-28-5p inhibitor could respectively downregulate the expression of CCAT1 and upregulate the expression of CCAT1.Functional experiments,CCK8 and colony formation assays,revealed that miR-28-5p acted as a tumor suppressor gene in PCa.Further functional recovery assays indaicated that the inhibitory action of CCAT1 downregulation on proliferation and colony formation could be partially reversed by the miR-28-5p inhibitor.The above experiments further confirmed the direct interaction between CCAT1 and miR-28-5p.Conclusion: CCAT1 could directly bind to miR-28-5p,thus reversing the tumor suppressive effect and exerting the oncogene role in PCa cell proliferation.Part Ⅳ Mechanism of CCAT1 recruiting AR co-activator DDX5 in PCa progressionObjective: In part Ⅲ,by constructing CCAT1 sense、CCAT antisense and the following RNA pulldown assay and RNA-seq analysis,it was confirmed that CCAT1 can directly bind to the tumor suppressor gene miR-28-5p to promote PCa mailgnant progression.As mentioned above,in addition to the role of sponge absorption in cytoplasm,LncRNA can bridge different moleclues to form the nuclear protein complex in the nucleus,regulating gene expression.In part ?,CCAT1 showed different subcellular localizatin in different PCa cell lines,indicating that except for the role of competing endogenous RNA in cytoplasm,CCAT1 may promote CRPC progression by acting as the scaffold to recruit regulatory molecules in the nucleus.Therefore,the oncogenic role of CCAT1 in the nucleus and its mechanism are worthy to be studied.Methods: We constructed the biotin-labeled CCAT1 sense and CCAT1 antisense single stranded RNA in vitro.After forming the secondary structure,biotin-labeled CCAT1 sense and CCAT1 antisense were incubated with PCa cell nuclear lysates,followed by qualitative detection and analysis of differentially enriched proteins.Differential proteins enriched by CCAT1 sense and CCAT1 antisense were verified by Wester Blot、protein electrophoresis gel sliver staining and RIP assay.Whether there is a corresponding increase or decrease in different PSA fragments recruited by P68 and AR after upregulated or downregulated CCAT1 was explored by Ch IP and qRT-PCR assay.The change of PSA、hNKX3.1(downstream gene of AR pathway)and UBE2C(ARregulated castration resistant gene)expression in cells with abnormal expression of CCAT1 was confirmed by Wester Blot and qRT-PCR.Whether the tumor-promoting effects of CCAT1 will be abolished or reduced dramatically after knockdown P68 in PCa cells was also studied by CCK8、colony formation and apoptosis assay.Results: RNA pulldown assay,combined with the result of qualitative detection of differentially enriched proteins,revealed that CCAT1 sense transcript was more sensitive to interacting with p68 compared with the antisense transcript.Wester Blot、protein sliver staining and RIP assay also confirmed p68 interacting with CCAT1 sense and CCAT1 was dramatically enriched in the p68-immunoprecipitation compared to control Ig G.Ch IP and qRT-PCR assay demonstrated that overexpression of CCAT1 increased the enrichment levels of PSA fragments、PSA、hNKX3.1、UBE2C gene expression(P<0.05),while knockdown CCAT1 decreased the enrichment levels of PSA fragments、 PSA、 hNKX3.1、UBE2C gene expression(P < 0.05).In addition,CCK8,cell colony formation and apoptosis assays showed that downregulation of p68 in CCAT1 overexpression cell could dramatically inhibit cell proliferation(P < 0.05)and increased cell apoptosis(P<0.05).Conclusion: All the results indicated that CCAT1 could recruit p68 and AR to localize on different segments of AR targeted gene-PSA and increase the expression of PSA、hNKX3.1、UBE2C,thereby activating AR pathway abnormally and promoting PCa progression and transformation of CRPC.