【作者】 王凯；

【导师】 许斌；

【作者基本信息】 东南大学 ， 外科学， 2022， 硕士

【摘要】 背景:脂肪与肥胖相关蛋白(fat mass and obesity-associated protein,FTO)和alk B同源物5(alk B homologue 5,ALKBH5)作为m~6A最重要的两种去甲基化酶,已被发现在多种癌症中发挥重要作用。然而,它们在前列腺癌(prostatic cancer,PCa)的发生和进展中的作用仍然知之甚少。在这里,我们的目的是研究FTO和ALKBH5在前列腺癌中的功能和临床相关性。方法:RT-qPCR和western blot检测基因表达。通过慢病毒转染在前列腺癌细胞系(DU145,LNCaP)中构建FTO和ALKBH5的沉默和过表达细胞系。在体外层面:CCK-8实验及细胞克隆形成实验检测细胞增殖能力;Transwell迁移实验和细胞划痕愈合实验检测细胞迁移能力;用β-半乳糖苷酶染色试剂盒检测细胞衰老情况。在体内层面:裸鼠皮下成瘤实验评价细胞在体内的生长情况。采用Dot blot和Epic Qick m~6A RNA甲基化定量试剂盒测定细胞总RNA的m~6A水平。通过RNA-seq研究其中的分子机制。结果:通过ONCOMINE和TCGA数据库我们发现,在前列腺癌中FTO和ALKBH5下调表达,并且均与患者更差的预后显著相关。在体外过表达FTO后明显抑制前列腺癌细胞DU145的增殖能力,克隆形成能力,迁移能力;抑制LNCa P细胞的增殖和克隆形成能力。裸鼠皮下瘤实验表明过表达FTO后DU145细胞的体内成瘤能力明显下降。Dot blot结果表明过表达FTO后DU145细胞中总RNA的m~6A水平明显下降。在体外过表达ALKBH5后明显抑制前列腺癌细胞DU145的增殖能力,克隆形成能力,迁移能力;抑制LNCa P细胞的增殖和克隆形成能力。Epic Qick m~6A RNA甲基化定量实验表明过表达ALKBH5后LNCa P细胞中总RNA的m~6A水平明显上升。结论:本研究确定m~6A去甲基化酶FTO和ALKBH5在前列腺癌中主要发挥抑癌作用,主要抑制前列腺癌的增殖及迁移,有可能作为前列腺癌治疗的潜在靶点。更多还原

【Abstract】 Background:Fat mass and obesity-associated protein(FTO)and alk B homologue 5(ALKBH5),as the two most important demethylases of m~6A,have been found to play important roles in a variety of cancers.However,their roles in the occurrence and progression of prostate cancer are still poorly understood.Here,we aim to study the functional and clinical relevance of FTO and ALKBH5 in prostate cancer.Methods:The gene expression of prostate cancer DU145,LNCa P cell lines were detected by RT-q PCR and Western blot.Gene silencing and over expression of cell lines were constructed by lentiviral transfection.Cell proliferation was detected by CCK-8 assay and cell clonal formation assay in vitro.Transwell migration assay and scratch healing assay were used to detect cell migration.Cell senescence was detected byβ-galactosidase staining kit.Valuation of cell growth in vivo by subcutaneous tumor-formation assay in nude mice.And m~6A levels of total RNA were measured by Dot blot and Epic Qick m~6A RNA methylation quantitative kit.RNA-seq was used to investigate the underlying molecular mechanism.Results:Through ONCOMINE and TCGA databases,we found that FTO and ALKBH5 were down-regulated in PCa,and both of them were significantly correlated with worse prognosis of patients.Over-expression of FTO in DU145 significantly inhibited the proliferation,clonogenesis and migration in vitro;and decreased tumor forming ability in subcutaneous mouse model in vivo.Dot blot results showed that m~6A level of total RNA in DU145 cells was significantly/greatly decreased after over-expression of FTO.Similar results were found in LNCa P,FTO overexpression decreased cell proliferation and clonaogenesis in vitro.Increased ALKBH5expression in vitro significantly/enormously suppressed the proliferation,clonal formation and migration of DU145.Quantitative experiments of Epic Qick m~6A RNA methylation showed that the m~6A level of total RNA in LNCa P cells increased significantly after overexpression of ALKBH5.Conclusion:In this study,it was determined that m~6A demethylase FTO and ALKBH5 play a major role in inhibiting the proliferation and migration of prostate cancer,and may be used as a potential target for prostate cancer treatment.更多还原