【作者】 王军；

【导师】 章小平； 梁华庚；

【作者基本信息】 华中科技大学 ， 泌尿外科， 2022， 硕士

【摘要】 目的:在肾透明细胞癌(ccRCC)的进展中,脂质的异常积累是其中一个重要的影响因素。然而,其背后的机制仍不清楚。随着医学的不断发展,有新的证据表明,长链非编码RNAs(lncRNAs)也参与了脂质代谢的调节。本研究发现lncRNA COL18A1-AS1在ccRCC中处于一个低表达的状态,并且COL18A1-AS1表达量越高,预后越好。本研究旨在阐明lncRNA COL18A1-AS1如何通过影响ccRCC的脂质代谢从而影响ccRCC的发生进展的。方法:通过qRT-PCR、Western blot、IHC等实验方法来检测组肿瘤组织和癌旁组织标本中COL18A1-AS1的表达水平,并分析其表达水平与患者临床分期、分级、各种临床特征以及总体生存率等之间的相关性。利用生物信息学分析COL18A1-AS1在肿瘤中低表达的原因并证实lncRNA与miRNA的结合,MSP实验和BSP实验进一步验证COL18A1-AS1在肿瘤组织中低表达的原因,RIP实验、双荧光素酶报告基因和荧光原位杂交实验验证了COL18A1-AS1和miR-1268的结合和共定位。CCK-8、Transwell、克隆形成、划痕实验、油红染色等实验验证COL18A1-AS1、miR-1286、KLF12在ccRCC中的增殖、迁移、侵袭能力及对脂质代谢的影响。小鼠体内实验验证了恢复COL18A1-AS1表观遗传作用能够抑制肿瘤细胞生长。结果:COL18A1-AS1在ccRCC中由于DNA的高甲基化而被下调,间接导致KLF12的表达水平降低。恢复COL18A1-AS1的表达可以竞争性地结合miR-1286进而增加Krüppel-like factor 12(KLF12)的表达。生物信息学分析、Western blotting和原位免疫荧光显示COL18A1-AS1/KLF12正向调节解偶联蛋白1(UCP1)介导的脂质褐变,从而促进肿瘤细胞“瘦身”并抑制肿瘤的进展。当肿瘤细胞发生"瘦身"时,脂滴变成了小块,脂质被消耗而不产生ATP能量,从而抑制ccRCC增殖生长,达到抑制肿瘤生长的效果。结论:COL18A1-AS1表达的减少是由其启动子内CpG岛的DNA甲基化引起的。恢复被表观遗传学沉默的COL18A1-AS1可以抑制肿瘤的增殖和转移,促进体外和体内的脂质褐变和消耗。综上所述,本研究对COL18A1-AS1-miR-1286/KLF12轴的研究结果揭示了ccRCC中脂质异常积累的一个潜在机制,并为ccRCC患者的治疗提供了一个新的方向。更多还原

【Abstract】 Purpose: In the progression of clear cell renal cell carcinoma(ccRCC),abnormal accumulation of lipids is one of the important influencing factors.However,the mechanism behind it remains unclear.With the development of medical science,there is new evidence that long non-coding RNAs(lncRNAs)are also involved in the regulation of lipid metabolism.In this study,we found that lncRNA COL18A1-AS1 is in a low expression state in ccRCC,and the higher the COL18A1-AS1 expression indicates the better prognosis.The aim of this study was to elucidate how lncRNA COL18A1-AS1 affects the progression of ccRCC by influencing lipid metabolism in ccRCC.Methods: The expression levels of COL18A1-AS1 in tissue specimens were detected by qRT-PCR,Western blot,and IHC,and correlations between them and patients’ clinical staging,grading,and overall survival were analyzed.Bioinformatics analysis of the reason of low expression of COL18A1-AS1 in tumors and the binding of lncRNA and miRNA.MSP assay and BSP assay verified the reason of low expression of COL18A1-AS1 in tumor tissues,RIP assay,luciferase reporter gene assay and FISH verified the binding and colocalization of COL18A1-AS1 and miR-1268.CCK-8,Transwell,clone formation and scratch assay,oil red staining were performed to verify the proliferation,migration and invasion ability of COL18A1-AS1,miRNA and KLF12 in ccRCC and the effect on lipid metabolism.In vivo experiments verified that restoring the epigenetic effect of COL18A1-AS1 could suppress tumor cell growth.Result: COL18A1-AS1 is downregulated in ccRCC due to DNA hypermethylation,resulting in low expression of KLF12.COL18A1-AS1 competitively binds miR-1286 to increase the expression of Krüppel-like factor 12(KLF12).Bioinformatics analysis,Western blotting and immunofluorescence showed that COL18A1-AS1/KLF12 positively regulated uncoupling protein 1(UCP1)-mediated lipid browning,thereby promoting tumor cell "thinning" and inhibiting tumor progression.When tumor cells undergo "thinning",lipid droplets become small pieces and lipids are consumed without producing ATP energy.Thus,the proliferation of ccRCC was inhibited.Conclusion: The reduction in COL18A1-AS1 expression is caused by DNA methylation of CpG islands within its promoter.Restoration of epigenetically silenced COL18A1-AS1 inhibits tumor proliferation and metastasis and promotes lipid browning and depletion in vitro and in vivo.Taken together,our findings on the COL18A1-AS1-miR-1286/KLF12 axis reveal a potential mechanism for aberrant lipid accumulation in ccRCC and may be a promising therapeutic target for ccRCC patients.更多还原