【作者】 张晓文；

【导师】 陈明；

【作者基本信息】 东南大学 ， 泌尿外科， 2017， 博士

【摘要】 前列腺癌的发病率呈爆发性增长,但其具体的病因尚且不能完全清楚。在发病的早期,几乎都为激素依赖性前列腺癌(ADPC),经过手术去势或者药物去势,病情都会得到有效控制。但颇为遗憾的是,几乎所有患者最终都将逐渐发展为去势抵抗性前列腺癌(CRPC)。去势抵抗性前列腺癌的临床处理棘手,在一系列的化疗放疗等全身治疗后,最终出现转移,并导致患者死亡。目前研究热点主要集中在如何抑制患者从激素依赖性前列腺癌转化为去势抵抗性前列腺癌,哪些分子学和生物学因素参与其中,如何寻找到调控这个变化的药物和方法。miRNAs是目前基础研究热点,主要通过转录后水平调控靶基因的表达,在细胞的多种生命活动,如增殖、分化、生长、凋亡、迁移、侵袭等生物学过程中发挥重要作用。miRNA的基因发生缺失、突变等改变及合成过程异常,将导致其表达水平失调,可能扰乱机体细胞增殖、分化及凋亡等调控过程,参与肿瘤的发生与发展过程,在肿瘤中发挥癌基因或抑癌基因的作用。目前研究发现多种miRNAs在前列腺癌的发生发展发挥重要作用。通过体外及体内实验研究miR-195对激素非依赖性前列腺癌细胞株生物学行为的影响,并鉴定miR-195影响的下游基因,并研究其下游基因高迁移率族蛋白A1(HMGA1)的生物学功能。充实去势抵抗性前列腺癌发生发展的理论基础,为新药的研发提供实验依据。第一部分临床相关性研究miR-195在激素依赖性前列腺癌与去势抵抗性前列腺癌中表达的差异性研究目的:通过体外及体内实验研究miR-195对激素非依赖性前列腺癌细胞株DU145及PC3细胞生物学行为的影响,鉴定miR-195影响的下游基因,并研究其下游基因高迁移率族蛋白A1(HMGA1)的生物学功能。研究方法:通过miRNAs芯片检测发现在临床获得的早期ADPC与晚期CRPC组织中有差异性表达的miRNA,并进一步通过qRT-PCR实验检测验证。对GSE21034数据库中获得的关于前列腺癌miRNA表达的基因芯片数据进行二次分析,比较miR-195在不同分化程度前列腺癌中的表达差异,并分析miR-195表达与患者预后之间的关系。研究结果:microRNAs芯片检测提示一些mi-RNA在ADPC与CRPC组织中表达有异常,同时通过qRT-PCR实验验证miR-195在CRPC较ADPC中显著下降(p<0.05=。对GSE21034数据库资料的二次分析表明,miR-195在前列腺癌中的表达与前列腺患者的Gleson评分和无瘤生存率均成正相关。研究结论:在CRPC中miR-195的表达较ADPC中显著下降,提示其可能是前列腺癌潜在的重要的抑癌基因。对GSE21034数据库资料的二次分析提示,miR-195可以作为前列腺癌的预后预测因子。第二部分细胞功能性研究miR-195对前列腺癌细胞周期、凋亡、增殖、迁移、侵袭及体内成瘤的影响研究目的:第一部分研究提示在CRPC中miR-195的表达较ADPC中显著下降,提示其可能是前列腺癌潜在的重要的抑癌基因。我们希望通过相关功能实验进一步验证miR-195在前列腺癌中的具体作用。探讨miR-195对去势抵抗性前列腺癌细胞DU145与PC3的细胞周期变化、细胞凋亡比例和细胞增殖能力的影响,以及裸鼠活体皮下成瘤能力的影响。研究方法:在体外功能实验中,DU145及PC3细胞株体外转染miR-195,分别通过四甲基偶氮唑蓝(MTT)法及细胞集落形成实验法检测细胞增殖的能力;通过流式细胞术检测细胞周期改变及凋亡率。在裸鼠成瘤实验中,检验转染miR-195的PC3细胞裸鼠皮下肿瘤情况。研究结果:MTT实验表明在激素抵抗性前列腺癌细胞DU145和PC3中,转染miR-195后细胞增殖率显著低于阴性对照(NC)组。在体外克隆形成实验中,miR-195抑制前列腺癌细胞形成集落的数目和大小。流式细胞术检测中,miR-195组细胞阻滞在G0/G1期比率增加,凋亡率显著高于NC组。在裸鼠成瘤实验中,转染miR-195的PC3细胞裸鼠皮下肿瘤的大小明显小于对照组。研究结论:在激素非依赖性前列腺癌细胞株中,miR-195可以调控前列腺癌细胞的增殖、周期和凋亡。第三部分分子机制研究miR-195直接作用于HMGA1促进前列腺癌细胞凋亡研究目的:通过生物信息学分析,预测出构架转录因子HMGA1可能是miR-195在前列腺癌中的潜在靶基因。我们试图通过相关实验进一步验证HMGA1在前列腺癌中的作用,以及进一步实验验证miR-195能够与HMGA1的3’-UTR直接结合抑制HMGA1的表达。研究方法:GSEA进行富集分析,初步观察miR-195调控靶基因。通过RNA sequence检测转染miR-195后DU145及PC3细胞中表达变化的基因,并将这些差异基因通过GSEA进行富集分析。通过靶基因预测软件miRanda、TargetScan等预测miR-195的潜在靶基因HMGA1,荧光素酶报告基因检测miR-195与潜在靶基因3’UTR的结合能力,使用Western Blotting检测潜在靶基因HMGA1表达变化。同样,我们对GSE35988数据库资料进行二次分析,比较HMGA1在不同类型前列腺组织中的表达,分析HMGA1表达与前列腺癌生存率等预后相关性分析。将低表达及高表达HMGA1患者之间差异表达的基因通过GSEA进行富集分析,寻找HMGA1可能调控的基因通路。然后通过小干扰RNA(small interferingRNA,si-RNA)抑制HMGA1的表达。分别通过MTT实验及体外克隆形成实验检测DU145及PC3细胞增殖能力的改变;同时通过流式细胞技术检测其对细胞周期及细胞凋亡的影响。研究结果:GSEA进行富集分析,提示miR-195可能主要通过调控周期相关基因影响细胞功能。预测软件预测HMGA1可能是miR-195的靶基因;荧光素酶报告基因结果表明,miR-195能够明显抑制野生型报告基因质粒的荧光值。Western Blot检测DU145和PC3细胞中HMGA1的表达水平,与转染NC的细胞株相比转染miR-195后HMGA1表达明显下降。对GSE35988数据进行二次分析表明,HMGA1在去势抵抗性前列腺癌组织中表达升高,HMGA1高表达组的患者无病生存时间和总生存时间较HMGA1低表达组短。GSEA富集分析表明HMGA1调控的基因与细胞周期有关。体外转染si-HMGA1,MTT实验表明在Du145和PC3细胞中,si-HMGA1组能够抑制前列腺癌细胞的增殖能力。流式细胞术检测发现si-HMGA1组细胞阻滞在G0/G1期比率增加,表明凋亡率明显高于对照组。裸鼠成瘤实验中,肿瘤免疫组化病理提示转染miR-195组的HMGA1表达明显下降。研究结论:HMGA1是miR-195的直接靶基因,HMGA1在前列腺癌中起促癌作用。更多还原

【Abstract】 The incidence of prostate cancer was explosive growth,but the specific cause is still not completely clear.In the early stages of the disease,almost all of the hormone-dependent prostate cancer(ADPC),after surgery castration or drug castration,the condition will be effectively controlled.Unfortunately,almost all patients will eventually develop into castration-resistant prostate cancer(CRPC).The clinical treatment of castration-resistant prostate cancer is tricky,after a series of chemotherapy radiotherapy and other systemic treatment,the final occurrence of metastasis,and lead to death.The current research focus is focused on how to prevent patients from hormone-dependent prostate cancer into castration-resistant prostate cancer,which molecular and biological factors involved,how to find the regulation of this change in drugs and methods.MiRNAs are the basic research hotspots at present.They play an important role in the biological processes such as proliferation,differentiation,growth,apoptosis,migration and invasion of cells through transcriptional regulation of target gene expression.MiRNA gene deletion,mutation and other changes in the synthesis process abnormalities,will lead to its level of expression disorders,may disrupt the cell proliferation,differentiation and apoptosis and other regulatory processes involved in the development and progression of the tumor,in cancer play an oncogene or The role of tumor suppressor gene.The current study found that a variety of miRNAs in the occurrence and development of prostate cancer play an important role.The effect of miR-195 on the biological behavior of hormone-independent prostate cancer cell lines was studied by in vitro and in vivo experiments.The downstream genes affected by miR-195 were identified and the genes of the high mobility group protein A1(HMGA1)Learning function.To enrich the theoretical basis of castration resistance of prostate cancer can provide experimental basis for new drugs.PART I Study of clinical relevanceDifferences in the expression of miR-195 in hormone-dependent prostate cancer and castration resistant prostate cancerOBJECTIVE:To study the effect of miR-195 on the biological behavior of hormone-independent prostate cancer cell lines DU145 and PC3 in vitro and in vivo.To investigate the downstream genes affected by miR-195 and to study the effect of miR-A1(HMGA1)biological function.METHODS:miRNAs were detected by miRNAs chip detection and found to be differentially expressed in the early ADPC and late CRPC tissues,and were further verified by qRT-PCR.The expression of miR-195 in the prostate cancer was analyzed by comparing the miR-195 expression in the differentiated differentiation of prostate cancer.The relationship between the expression of miR-195 and the prognosis of the patients was analyzed by using the gene chip data obtained from the GSE21034 database on the miRNA expression of prostate cancer.The results of microRNAs showed that some mi-RNAs were abnormal in ADPC and CRPC tissues,and qRT-PCR showed that miR-195 was significantly lower in CRPC than ADPC(p<0.05).In the data of GSE21034 database,the results of miR-195 expression in prostate cancer were positively correlated with Gleson score and tumor-free survival in prostate patients.CONCLUSIONS:The expression of miR-195 in CRPC is significantly lower than that in ADPC,suggesting that it may be a potentially important tumor suppressor gene for prostate cancer.The secondary analysis of GSE21034 database data suggests that miR-195 can be used as a prognostic factor for prostate cancer.PART Ⅱ Functional analysisThe influence of miR-195 on cell cycle,apoptosis,proliferation,migration,invasion and in vivo tumorigenesis of prostate cancer were observed.OBJECTIVE:The first part of the study suggests that the expression of miR-195 in CRPC is significantly lower than that in ADPC,suggesting that it may be a potentially important tumor suppressor gene for prostate cancer.We hope to further verify the specific role of miR-195 in prostate cancer through related functional experiments.To investigate the effect of miR-195 on cell cycle,cell apoptosis and cell proliferation of DU 145 cells and PC3 in ovariectomized prostate cancer cells and the effect of subcutaneous tumorigenesis in nude mice.Methods:In vitro experiments,DU 145 and PC3 cell lines were transfected into miR-195 in vitro.The ability of cell proliferation was detected by MTT assay and cell colony formation assay.The cell cycle and apoptotic rate were measured.In subcutaneous tumor of nude mice,the subcutaneous tumor of PC3 cells transfected with miR-195 was examined.RESULTS:MTT assay showed that cell proliferation rates were significantly lower in patients with hormone-resistant prostate cancer cells DU145 and PC3 than in negative control group(NC).In vitro clonal formation experiments,miR-195 inhibited the number and size of prostate cancer cell colonies.In the flow cytometry,the percentage of cells in the miR-195 group was increased at G0/G1 phase and the apoptotic rate was significantly higher than that of the NC group.In nude mice,the size of subcutaneous tumors of PC3 cells transfected with miR-195 was significantly smaller than that of the control group.CONCLUSIONS:In hormone-independent prostate cancer cell lines,miR-195 regulates the proliferation,cycle and apoptosis of prostate cancer cells.PART Ⅲ Molecular mechanism researchMiR-195 directly acts on HMGA1 to promote apoptosis of prostate cancer cellsOBJECTIVE:To predict the potential target gene of miR-195 in prostate cancer by bioinformatics analysis,suggesting that the structural transcription factor HMGA1 may be a potential target gene for miR-195 in prostate cancer.We attempted to further validate the role of HMGA1 in prostate cancer by experiments,and further experiments confirmed that miR-195 was able to inhibit the expression of HMGA1 directly with HMGA1 3’-UTR.Methods:GSEA was enriched and analyzed,and the target gene of miR-195 was observed.The expression of DU145 and PC3 cells transfected with miR-195 was detected by RNA sequence,and these differential genes were analyzed by GSEA.The potential target gene HMGA1 and the luciferase reporter gene of miR-195 were predicted by miRanda and TargetScan,and the binding ability of miR-195 to the potential target gene 3’UTR was detected.The expression of HMGA1 was detected by Western Blotting.Similarly,we analyzed the GSE35988 database data,compared the expression of HMGA1 in different types of prostate tissue,and analyzed the correlation between the expression of HMGA1 and the survival rate of prostate cancer.The differentially expressed genes between low expression and high expression of HMGA1 were analyzed by GSEA to find the gene pathway that HMGA1 might control.The expression of HMGA1 was then inhibited by small interfering RNA(siRNA).The proliferation of DU145 and PC3 cells was detected by MTT assay and in vitro cloning assay.The effects of cell cycle and apoptosis were detected by flow cytometry.The results of the study:GSEA enrichment analysis,suggesting that miR-195 may be mainly through the regulation of cycle-related genes affect cell function.Predictive software predicts that HMGA1 may be the target gene of miR-195.The results of luciferase reporter gene show that miR-195 can significantly inhibit the fluorescence of wild-type reporter gene.Western blot was used to detect the expression of HMGA1 in DU145 and PC3 cells.The expression of HMGA1 was significantly decreased after transfection with miR-195 compared with the transfected cells.The secondary analysis of GSE35988 data showed that the expression of HMGA1 in castration resistant prostate cancer was higher than that in HMGA1 group.The survival time and overall survival time of HMGA1 overexpression group were shorter than that of HMGA1 group.GSEA enrichment analysis showed that HMGA1 regulated genes were associated with cell cycle.In vitro transfection of si-HMGA1,MTT assay showed that si-HMGA1 could inhibit the proliferation of prostate cancer cells in Du145 and PC3 cells.Flow cytometry showed that the percentage of cells in the Si-HMGA1 group was increased at GO/G1 phase,indicating that the apoptotic rate was significantly higher than that of the control group.In nude mice,the expression of HMGA1 in miR-195 group was significantly decreased by immunohistochemical staining.CONCLUSIONS:HMGA1 is a direct target gene for miR-195,and HMGA1 plays a role in cancer progression in prostate cancer.更多还原