| Background and objectiveProstate cancer is one of the most common malignant tumors in men and one of the major causes of cancer death.Prostate cancer accounts for 1 to 2%of men with both screening and non-screening[1].The worldwide incidence of prostate cancer varies as much as 25 times and Asia is the region with the lowest incidence of prostate cancer[2].However,the prevalence of prostate cancer in China has risen markedly as lifestyles change,improvemnts of health awareness,healthcare services,data reporting systems,and especially,the wide spread adoption of PSA screening.Data from Center for Disease Control?CDC?show that prostate cancer is one of the most rapidly growing cancers in China between 2001 and 2011[3].In our country,PCa was mostly diagosed too late,and the tumor had metastasized locally or remotely and could not be cured.After the endocrine therapy,the patients became castration resistant prostate cancer?CRPC?with poor prognosis[4].Prostate cancer has become one of the major cancer"killers"of men in our country.In recent years,great progresses have been made in the research and clinical treatment of prostate cancer.Biological studies have found that the vast majority of castration-resistant prostate cancers are still driven by androgen[5].Clinical treatment of androgen receptor signaling pathway drugs such as enzalutamide[6]and Abiraterone acetate[7]and immunotherapy Sipuleucel-T[8]improved the prognosis of castration-resistant prostate cancer,prolonging the patient's survival.However,due to the complicated mehcamism of prostate cancer,these treatments still only prolong the survival time of patients for several months.Thus,Biological and clinical studies of prostate cancer are still in urgent need of breakthrough[1].Long noncoding RNAs?lncRNAs?are a class of noncoding RNAs>200bp in length[9].The number is far greater than the protein coding gene[10].LncRNAs have higher tissue expression specificity than protein-coding genes[11-13].LncRNAs are involved in a variety of networks that regulate gene expression and function[14].It affects cell proliferation,metastasis,renewal,survival and apoptosis through the transcriptional and post-transcriptional regulatory mechanisms leading to tumorigenesis and progression[15-25].Almost all these well studied lncRNAs are on the autosomes and our knowledge about the sex-specific lncRNAs is rare.The incidence of many sex-nonspecific cancers is higher in men than that in women,but no well-documented causes exist currently[26,27].Prostate cancer is a male-specific tumor,and Y chromosome is a male-specific chromosome.But the link between the development of prostate cancer and Y chromosome abnormalities remained unclear.A few of researches showed that abnormalities of Y chromosome might cause prostate cancer[28,29].The deletion of Y chromosome fragments and the abnormal expression of specific genes involved in the occurrence and development of prostate cancer[28,30].These alterations of Y chromosome may also influnce the genes of lncRNAs.But literature about Y chromosomal located lncRNAs is scarce.Therefore,this Ph.D.research project focused on prostate cancer-associated long non-coding RNAs located on the Y chromosome.We analyzed the roles of these lncRNAs on the development and progression of prostate cancer using CRISPR/Cas9and other technologies,and thsese results might provide us new treatments for prostate cancer.Based on data of transcriptome of 65 pairs of cancer and paracancerous tissues,we found lncRNAs locating on the Y chromosome,which were differentially expressed between the cancer and paracancerous tissues.TTTY15 was the most differentially expressed lncRNA,and further experiments found that it had the ability to promote tumor growth,migration and invasion.Part ?:Screening of Long Non-coding Prostate Cancer-Associated RNAs on Y chromosome and Identification of Clinical Significance of TTTY15Objective:To find prostate cancer associated long non-coding RNA on the Y chromosome and provide a new target molecule for the diagnosis and treatment of prostate cancer.Methods:?1?The transcriptome sequencing data of 65 pairs of prostate cancer tissues and adjacent cancer tissue samples obtained from the previous study were used to further analyze the lncRNAs located on the Y chromosome in order to find out differentially expressed lncRNAs between the cancer and paracancerous tissues?2?We collected 145 pairs of cancer and paracancerous tissues from patients undergoing radical prostatectomy in our hospital and sent them to sequencing company for RNA sequencing to validate the expression of TTTY15.?3?The expression of TTTY15 was determined by qPCR.?4?Expression level validation of TTTY15 using public TCGA data.?5?We applied the RNA ISH to explored the expression of TTTY15 in both prostate cancer and normal tissues?formalin-fixed,paraffin-embedded samples?.?6?Expression assessment of TTTY15 in prostate cancer cell lines.Res?lts:?1?In-depth analysis of the transcriptome sequencing data of 65 pairs of prostate cancer and paracancerous tissues obtained from the previous study revealed some differentially expressed lncRNAs located on the Y chromosome,and TTTY15 was the most significantly differentially expressed.?2?After the hospital ethics committee aproved,another patch of prostate cancer and adjacent normal tissues were collected and saved.Then,the transcriptome was sequenced and the expression of TTTY15 was analyzed preliminarily.The results were consistent with the results of previous sequencing data,and TTTY15 expression in prostate cancer is up-regulated.?3?The expression of TTTY15 in35 pairs of prostate cancer and paracancer was determined using qPCR.As with the sequencing data,the expression of TTTY15 in cancer tissue was up-regulated.?4?Further analysis of public TCGA data confirmed that TTTY15 was highly expressed in cancer tissues.?5?Semi-quantitative analysis of the expression of TTTY15 using RNA ISH in 70prostate cancers and 16 prostate normal tissues shown that the expression of TTTY15 was higher in canerous tissues.?6?TTTY15 expression in prostate cancer cell lines?LNCaP,C42,DU145?was significantly higher than that in normal epithelium RWPE-1,which is evaluated by PCR.Conclusion:Focusing on the lncRNA on the Y chromosome,and through further analysis of the sequencing results of the previous transcriptome,we found that the most significantlly differentially expressed lncRNA was TTTY15.Data from another large numbers of samples assessed by a variety of methods showed that the expression of TTTY15 in prostate cancer is highly expressed.These discovery and validation data suggested that TTTY15 might play a role in the development of prostate cancer.Part ?:construction of lncRNA TTTY15 knockout model usingCRISPR/Cas9,and evaluating the effects of TTTY15 on the phenotypes of prostate cancer cellsObjective:To construct a TTTY15 knockout cell model and evaluate the role of TTTY15 on the phenotypes of prostate cancer cellsMethods:?1?Small interference RNAs which could effectively knock-down TTTY15were screened.?2?Overexpression adenovirus was produced.?3?Selected prostate cancer cell line were used for test the influences after TTTY15 up-regulated and down-regulated.EdU incorporation assay?analyzed by fluorescence microscopy or flow cytometry?,clonegenic assay and cck-8 test were used to detect cell proliferation;Annexin-V/PI double staining flow cytometry to detect apoptosis;Transwell assay was employed to assess the cell migration and invasion ability.?4?Two novel CRISPR/Cas9-mediated lncRNA knockout methods were designed.Then TTTY15 knockout cell model was constructed,and related phenotypes were studied.?5?The tumor-bearing experiments of TTTY15knockout and rescue cells were carried out by using the mouse subcutaneous tumor-bearing model.Res?lts:?1?Two siRNAs with significant interference efficiency were successfully obtained.Knock-down efficiency of both siRNAs were over 70%.?2?Adenovirus with high TTTY15 overexpression efficiency was successfully obtained.?3?Based on the distribution of TTTY15 in prostate cancer cell lines,the main research on cell level was performed on LNCaP and DU145.In vitro functional assays showed that the proliferation,invasion and migration were significantly increased after TTTY15 was overexpressed,while the proliferation and invasion and migration ability were down-regulated after TTTY15 was down-regulated.The change of TTTY15 had no significant effect on the apoptosis of prostate cancer cells.?4?In view of the fact that lncRNA can not be knocked out by CRISPR/Cas9-induced mutation simply,we creatively knock-out TTTY15 using two different methods and obtain knock-out cell lines A6,TKO2 and TKO8 respectively.In vitro phenotypic experiments found that proliferation and invasion and migration of prostate cancer cell decreased after TTTY15 was knocked-out.But if TTTY15 rescued later,proliferation and invasion and migration significantly increased.?5?Using the mouse tumor-bearing model,we found that the tumorigenic ability of TTTY15 knock-out cells decreased,while the tumorigenic ability of TTTY15 rescued cells recovered to some extent.Conclusion:The change of TTTY15 expression could affect the proliferation,migration and invasion of cells,but had no significant effect on apoptosis.Part ?:TTTY15 played it's role as an endogenous Competitive RNAObjective:To clarify the specific molecular mechanism of TTTY15 in promoting proliferation,invasion and migration of prostate cancerMethods:?1?series analysis methods including literature mining,TCGA transcriptome data analysis,transcription factor?TF?binding motif analysis using JASPAR and experiments such as TF interference and overexpression experiments as well as ChIP-PCR experiments were performed to find transcription factors regulating expression of TTTY15.?2?Real time quantitive PCR after nuclear and cytoplasmic RNA extraction and RNA ISH clearly demostated the expression and localization of TTTY15 in cells.?3?By means of network analysis tools,the possible target miRNAs and target genes of TTTY15were obtained.The dual luciferase reporter assay,qPCR and sequencing were used to assess the binding of miRNAs and the target genes.?4?Expression of some genes targeted by associated microRNAs was determined in cells using western blot after expression of TTTY15 changed.The role of associated microRNA in this regulatory process was evaluated by mutational experiments.Res?lts:?1?After a series of literature mining,public data analysis and experimental verification,we found that FOXA1 could bind to TTTY15 promoter and regulate the expression of TTTTY15.?2?Real time quantitative PCR after nuclear and cytoplasmic RNA extraction,and RNA ISH assay proved that TTTY15 distributed in both the nucleus and the cytoplasm,and RNA in the cytoplasm accounted for about two-thirds.?3?TTTY15could bind to the menbers of let-7 famiy which is predicted by miRanda,MIRDB,RNA22and other tools.The results of dual luciferase reporter assay showed that the expression of hsa-let-7a,hsa-let-7c,hsa-let-7f,and miR98 Reduced relative luciferase activity.Through the prediction of miRNA targeting genes,valuating the changes of related genes after knockout by qPCR and sequencing,we found that the expression of CDK6 and FN1decreased after TTTY15 down-regulation.?4?Western blot results showed that expression of CDK6 and FN1 was down-regulated,and it could increase after TTTY15overexpression or rescue in knockout cells.But if the binding site of let-7 mutated in TTTY15,this effect could disapear.Therefore,let-7 might be an important intermediate in the TTTY15 mediated regulation of CDK6 and FN1.CDK6 and FN1 as oncogenes,play a role in promoting tumors.Conclusion:The TTTY15 sequence contains binding sites for multiple let7 family members.It could compete with other important mRNA molecules to bind let7 family members and thus played a regulatory role in other molecules.Through sequencing and qPCR results analysis,we found that CDK6 and FN1 are important regulatory targets of TTTY15.TTTY15 inhibits the expression of CDK6 and FN1 protein via blocking the effect of let7 on CDK6 and FN1.ConclusionsBased on previous transcriptome sequencing data of large samples,this study focuses on prostate cancer associated lncRNAs that located on the Y chromosome.By analyzing the differential expression of lncRNAs in prostate cancer associated with the Y chromosome,TTTY15 was found to be most significantly elevated in prostate cancer.Further transcriptome sequencing of large samples,qPCR validation and public database data validation confirmed that TTTY15 indeed significantly increased in prostate cancer,suggesting that TTTY15 may play a role in prostate cancer.Subsequent in vitro tumor phenotype experiments showed that TTTY15 could enhance the proliferation,invasion and migration of prostate cells,but the effect on apoptosis is not significant.Further mechanistic studies revealed that the expression of TTTY15 was not affected by androgens but was regulated by the transcription factor FOXA1.FOXA1 promotes TTTY15expression by binding to the TTTY15 promoter.Meanwhile TTTY15,as a predominantly cytoplasmic lncRNA,bounds to multiple members of the let7 family and promoted the expression of CDK6 and FN1 proteins.The results of this study provided new evidence to reveal the role of Y chromosome in the development of prostate cancer and shed light on the mechanism of the development of prostate cancer.Moreover,this study shown that TTTY15 might be a potential therapeutic target in prostate cancer. |
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