| Prostate cancer is one of the most common tumors in men,with the second highest incidence rate of male malignant tumors and the fifth highest mortality rate in the world.Prostate cancer accounts for 27%of all new cancer cases and 11%of cancer-related mortality,according to the American Cancer Society.Previous studies have shown that the androgen receptor is one of the key factors driving prostate cancer progression.Therefore,androgen deprivation therapy that blocks androgen synthesis or inhibits its interaction with the androgen receptor remains the mainstay of treatment for locally advanced high-risk prostate cancer as well as for metastatic prostate cancer.However,resistance to this treatment regimen is unavoidable,because androgen receptors can continue to drive tumor growth in the absence of androgens in circulating fluids,resulting in 10-50%of prostate cancer patients in the progression to castration-resistant prostate cancer continued within three years of diagnosis,and nearly all prostate cancer-related adverse events occurred at this stage.With the aging of China’s population and the trend of westernization of living and eating habits,the incidence and mortality of prostate cancer have been increasing in the past decade in China,and the medical burden of prostate cancer patients has also gradually increased.Therefore,exploring molecular markers and therapeutic targets for the diagnosis of prostate cancer in the early stage and strengthening the research on the pathogenesis of prostate cancer are particularly important for improving the survival and prognosis of patients and reducing the medical burden.With the continuous improvement of gene sequencing technology,studies have found that in the human genome,only about 2%of genes can encode proteins,and most of the other genes are transcribed into non-coding RNAs.Long noncoding RNAs(lncRNAs)are a class of noncoding RNAs longer than 200 nucleotides.In recent years,many functional lncRNAs have been discovered,and are involved in regulation of gene transcription,translation modification,epigenetic modification,regulation of cell proliferation,apoptosis,and cell differentiation.In the regulation of epigenetic modification by lncRNAs,lncRNAs located in the cytoplasm usually bind to miRNAs by competing with endogenous RNAs,regulate the degradation and translation of transcription products,and then participate in the malignant tumor progression.In previous experiments,we screened the differentially expressed lncRNAs,miRNAs and mRNAs and validated them in prostate cancer cell lines through the prostate cancer tissue transcriptome data and clinical information in the TCGA-PRAD and GSE70770 databases,and found that lncRNA SNHG5(SNHG5)was highly expressed in prostate cancer cell lines.SNHG5 is highly expressed in prostate cancer and is associated with poor prognosis,suggesting that SNHG5 may be closely related to prostate cancer progression.SNHG5 is located on human chromosome 6q14.36 and is first reported to be abnormally expressed in gastric cancer,and is involved in the regulation of malignant progression of breast cancer,renal clear cell carcinoma and hepatocellular carcinoma,but the role of SNHG5 in prostate cancer remains unclear.Thus,this study firstly detected the expression of SNHG5 in prostate cancer tissues and cell lines,and analyzed its relationship with poor prognosis.Then,the subcellular localization of SNHG5 was clarified in the nucleocytoplasmic separation experiment.The effects of SNHG5 on the biological functions of prostate cancer cells were explored through in vivo and in vitro biological experiments,and the target molecules interacting with SNHG5 were predicted with the help of online databases,and verified in the dual-luciferase reporter gene experiment.Finally,combined with bioinformatics analysis and recovery experiments,the mechanism of SNHG5 in the malignant progression of prostate cancer was further explored,which provided novel ideas for finding diagnostic and prognostic biomarkers and therapeutic targets in prostate cancer.Part Ⅰ:SNHG5 expression and clinical significances in prostate cancer Objective:Abnormally expressed lncRNAs in prostate cancer were screened in TCGA and GEO databases and validated in prostate cancer tissues and cell lines to identify their correlation with prognosis.Methods:(1)Screening of differentially expressed genes in prostate cancer by TCGA-PRAD and GSE70770 data,and constructing a ceRNA regulatory network based on molecular interactions predicted by online database websites(starBase,miRcode,miRDB,TargetScan);(2)Using Kaplan-Meier survival analysis to screen out lncRNA and miRNA related to survival prognosis;(3)Validating the expression level of SNHG5 in prostate cancer tissues and cells by RT-qPCR;(4)Analyzing the secondary molecules structure of SNHG5 and its correlation with immune cell infiltration with the help of online database RNAfold and bioinformatics software;(5)Clarifying the subcellular localization of SNHG5 in prostate cancer cell lines by nucleocytoplasmic separation experiments.Results:(1)Screening of differentially expressed genes in prostate cancer based on TCGA-PRAD and GSE70770 databases,including 203 lncRNAs,74 miRNAs and 1492 mRNAs,the molecular correlations predicted by online databases(starBase,miRcode,miRDB,TargetScan),and constructing a ceRNA regulatory network;(2)9 lncRNAs and 11 miRNAs related to survival prognosis were screened by Kaplan-Meier survival analysis;(3)RT-qPCR analysis showed that SNHG5 was highly expressed in prostate cancer tissues and cells;(4)The secondary molecular structure of SNHG5 was obtained in RNAfold,and the ssGSEA method was used to analyze that the expression of SNHG5 was positively correlated with the infiltration levels of CD8 T cells,plasmacytoid dendritic cells and cytotoxic cells,and was negatively correlated with the infiltration levels of active central T cells,dendritic cells and eosinophils;(5)The results of nucleocytoplasmic separation experiments showed that SNHG5 was mainly distributed in the cytoplasm of prostate cancer cells.Conclusion:According to TCGA-PRAD and GSE70770 analysis,it was found that SNHG5 was highly expressed in prostate cancer tissues and cells,mainly localized in the cytoplasm,and correlated with poor prognosis and immune cell infiltration.Part Ⅱ:Effects of SNHG5 on the biological behavior of prostate cancerObjective:To explore the effects of SNHG5 on the biological behavior of prostate cancer cells in vivo and in vitro experiments.Methods:(1)Plasmid interfering siRNA,lentiviral sh-RNA and lentiviral oe-RNA were transfected in prostate cancer cell lines,respectively,and their transfection efficiency was detected by RT-qPCR;(2)CCK8 assay and plate cloning assay were used to detect the changes of prostate cancer cell proliferation activity and clone formation ability after down-regulating or up-regulating the expression of SNHG5 respectively;(3)Transwell assay detected the changes in the migration and invasion abilities of prostate cancer cells after down-regulating or up-regulating the expression of SNHG5;(4)The effects of down-regulation of SNHG5 on the growth and proliferation of prostate cancer xenografts in vivo were detected by subcutaneous tumor-bearing experiments and immunohistochemical experiments in nude mice.Results:(1)Transfection of plasmid siRNA and lentiviral sh-RNA in prostate cancer cell lines could significantly down-regulate SNHG5 expression,and transfection of lentiviral oe-RNA could significantly up-regulate SNHG5 expression;(2)CCK8 experiments and plate cloning experiments showed that SNHG5 could significantly enhance the proliferative activity and clone formation ability of prostate cancer cells;(3)Transwell experiments results showed that SNHG5 could significantly enhance the migration and invasion ability of prostate cancer cells;(4)The results of subcutaneous tumor-bearing experiments and immunohistochemical experiments in nude mice showed that down-regulation of SNHG5 could significantly inhibit the proliferative activity and growth of prostate cancer xenografts in vivo.Conclusion:In prostate cancer cells,SNHG5 can significantly promote the ability of tumor cell proliferation,migration and invasion;in vivo nude mice subcutaneous tumor-bearing experiments and immunohistochemical staining experiments,SNHG5 can significantly promote the growth and proliferation activity of prostate cancer cells.Part Ⅲ:SNHG5 promotes malignant progression of prostate cancer through miR-23a-3pObjective:To explore the potential target molecules and mechanisms of SNHG5 in prostate cancer.RT-qPCR verified the expression of miR-23a-3p in prostate cancer cell lines,and the expression changes of miR-23a-3p after up-regulation and down-regulation of SNHG5;Methods:(1)Combining online databases(starBase,lncbase and lnCAR)and transcriptome sequencing data to predict the target molecules of SNHG5;(2)Applying RT-qPCR to verify the expression of miR-23a-3p in prostate cancer cell lines,and the expression changes of miR-23a-3p after up-regulation and down-regulation of SNHG5;(3)RT-qPCR verified the expression changes of miR-23a-3p in cell lines after transfection of miR-23a-3p inhibitor and miR-23a-3p mimic respectively;(4)Combining database prediction and dual luciferase reporter gene detection experiments to detect whether there is a binding site between SNHG5 and miR-23a-3p;(5)Using CCK8 experiments and plate cloning experiments to detect whether inhibiting the expression of miR-23a-3p can restore the promoting effect of SNHG5 on prostate cancer cell proliferation and colony formation ability;(6)Using transwell assay to detect whether inhibiting the expression of miR-23a-3p can slow down the promoting effect of SNHG5 on prostate cancer cell migration and invasion ability.Results:(1)Through the prediction results of online databases(starBase,lncbase and lnCAR)and transcriptome sequencing data,miR-23a-3p may be the target molecule of SNHG5;(2)RT-qPCR validation results showed that the expression of miR-23a-3p decreased in prostate cancer cell lines,and SNHG5 was regulated miR-23a-3p expression;(3)RT-qPCR results showed that after transfection of miR-23a-3p inhibitor and miR-23a-3p mimic respectively in prostate cancer cell lines,the expression of miR-23a-3p was correspondingly decreased or increased;(4)Combining online database prediction and dual luciferase reporter gene detection experiments,it was showed that there was a direct binding site between SNHG5 and miR-23a-3p;(5)CCK8 experiments and plate cloning experiments showed that miR-23a-3p could slow down the promoting effect of SNHG5 on prostate cancer cell proliferation and colony formation ability;(6)Transwell experiments showed that miR-23a-3p could slow down the promoting effect of SNHG5 on prostate cancer cell migration and invasion ability.Conclusion:SNHG5 can directly bind to miR-23a-3p,and promotes the proliferation,migration,and invasion of prostate cancer cells by inhibiting the expression of miR-23a-3p for participating in the malignant progression of tumors.Part Ⅳ:SNHG5 promotes prostate cancer malignant progression through the miR-23a-3p/GPRC5B signaling axisObjective:To screen the target molecules of miR-23a-3p and further explore the mechanisms of SNHG5 in prostate cancer.Methods:(1)The target molecules of miR-23a-3p were predicted by combining online database and transcriptome sequencing data;(2)The expression of GPRC5B in prostate cancer cell lines was detected by RT-qPCR,and the expression changes of GPRC5B were detected after down-regulating SNHG5 and interfering with miR-23a-3p;(3)CCK8 assay and plate clone assay were used to detect the changes of prostate cancer cell proliferation activity and clone formation ability after down-regulating GPRC5B expression;(4)Combining database prediction and dual luciferase reporter gene detection experiments to detect whether there is a binding site between miR-23a-3p and GPRC5B;(5)sh-SNHG5 and miR-23a-inhibitor were co-transfected in prostate cancer cell lines,and the expression changes of GPRC5B were detected by RT-qPCR and Western blot.Results:(1)Combining the online database prediction results and transcriptome sequencing data,it was showed that GPRC5B may be the target molecule of miR-23a-3p;(2)RT-qPCR results showed that GPRC5B was highly expressed in prostate cancer tissues and tumor cell lines.After down-regulation of SNHG5,the expression of GPRC5B was decreased in prostate cancer cell lines.And the expression of GPRC5B was increased in prostate cancer cell lines after interfering with miR-23a-3p;(3)The results of CCK8 assay and plate cloning assay showed that GPRC5B could enhance the proliferative activity and clone formation ability of prostate cancer cells;(4)Combining database prediction and dual luciferase reporter gene detection experiments,it was showed that there was a direct binding site between miR-23a-3p and GPRC5B;(4)RT-qPCR and Western blot results showed that miR-23a-3p could attenuate the promoting effect of SNHG5 on the expression of GPRC5B.Conclusion:GPRC5B can directly bind to miR-23a-3p,and SNHG5 regulates the expression of GPRC5B by inhibiting miR-23a-3p to promote the proliferation,migration and invasion ability of prostate cancer cells for participating in the malignant progression of tumors. |
PDF Full Text Request