简介:AbstractBackground:Long non-coding RNA (lncRNA) actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) functions as a competing endogenous RNA to regulate target genes expression by sponging microRNAs (miRs) to play cancer-promoting roles in cancer stem cells. However, the regulatory mechanism of AFAP1-AS1 in cervical cancer (CC) stem cells is unknown. The present study aimed to provide a new therapeutic target for the clinical treatment of CC.Methods:Hyaluronic acid receptor cluster of differentiation 44 variant exon 6 (CD44v6)(+) CC cells were isolated by flow cytometry (FCM). Small interfering RNAs of AFAP1-AS1 (siAFAP1-AS1) were transfected into the (CD44v6)(+) cells. The levels of AFAP1-AS1 were measured by quantitative real-time PCR (qRT-PCR). Sphere formation assay, cell cycle analysis, and Western blotting were used to detect the effect of siAFAP1-AS1. RNA pull-down and luciferase reporter assay were used to verify the relationship between miR-27b-3p and AFAP1-AS1 or vascular endothelial growth factor (VEGF)-C.Results:CD44v6(+) CC cells had remarkable stemness and a high level of AFAP1-AS1. However, AFAP1-AS1 knockdown with siAFAP1-AS1 suppressed the cell cycle transition of G(1)/S phase and inhibited self-renewal of CD44v6(+) CC cells, the levels of the stemness markers octamer-binding transcription factor 4 (OCT4), osteopontin (OPN), and cluster of differentiation 133 (CD133), and the epithelial-mesenchymal transition (EMT)-related proteins Twist1, matrix metalloprotease (MMP)-9, and VEGF-C. In the mechanism study, miR-27b-3p/VEGF-C signaling was demonstrated to be a key downstream of AFAP1-AS1 in the CD44v6(+) CC cells.Conclusions:LncRNA AFAP1-AS1 knockdown inhibits the CC cell stemness by upregulating miR-27b-3p to suppress VEGF-C.
简介:摘要目的研究长非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(AFAP1-AS1)对甲状腺癌细胞增殖和侵袭的影响,并探讨其机制。方法采用实时荧光定量PCR(qRT-PCR)检测正常甲状腺和甲状腺癌细胞株中AFAP1-AS1的表达。将甲状腺癌细胞株WRO分成3组,AFAP1-AS1沉默表达组(AFAP1-AS1-siRNA组)、阴性对照组(NT-siRNA组)及空白对照组(Blank组),AFAP1-AS1-siRNA组和NT-siRNA组采用Lipofectamine™ 3000分别转染AFAP1-AS1-siRNA、NT-siRNA,Blank组加入PBST对照。CCK-8检测3组细胞增殖能力,Transwell实验检测侵袭能力,Western blotting检测Rho A、Cyclin D1和MMP-9蛋白的表达情况。结果正常甲状腺细胞株FRTL-5、甲状腺癌细胞株SW579、CAL-62、FRO、WRO的AFAP1-AS1相对表达量分别为1.03±0.04、2.95±0.17、5.31±0.35、7.26±0.49、9.67±0.53,5组间比较差异具有统计学意义(F=16.932,P=0.027);AFAP1-AS1在WRO细胞中表达量最高,因此,选择WRO细胞进行后续实验。AFAP1-AS1-siRNA组、NT-siRNA组、Blank组3组细胞AFAP1-AS1的相对表达量分别为0.23±0.02、1.02±0.04、1.03±0.05,差异具有统计学意义(F=13.590,P=0.006),与NT-siRNA组相比,AFAP1-AS1-siRNA组AFAP1-AS1表达量显著降低(P<0.001)。3组细胞转染后3、4 d的A450值分别为0.68±0.06、1.17±0.09、1.22±0.09,0.96±0.08、1.69±0.11、1.72±0.12,差异均具有统计学意义(F=7.318,P=0.016;F=10.351,P=0.004),第3、4 d AFAP1-AS1-siRNA组、NT-siRNA组两组比较,差异均有统计学意义(P=0.043;P=0.013)。3组侵袭细胞数分别为(72.8±5.7)个、(145.6±8.9)个、(148.4±7.3)个,差异具有统计学意义(F=37.273,P=0.034),AFAP1-AS1-siRNA组侵袭细胞数少于NT-siRNA组(P=0.021)。3组细胞Rho A蛋白表达量分别为0.34±0.03、1.02±0.04、1.04±0.03,差异具有统计学意义(F=9.667,P=0.013),AFAP1-AS1-siRNA组Rho A蛋白表达量显著低于NT-siRNA组(P=0.018);3组Cyclin D1蛋白表达量分别为0.52±0.04、1.03±0.02、1.05±0.04,差异具有统计学意义(F=15.464,P=0.010),AFAP1-AS1-siRNA组Cyclin D1蛋白表达量显著低于NT-siRNA组(P=0.023);3组MMP-9蛋白表达量分别为0.42±0.04、1.05±0.03、1.02±0.04,差异有统计学意义(F=10.328,P=0.032),AFAP1-AS1-siRNA组MMP-9蛋白表达量显著低于NT-siRNA组(P=0.035)。结论AFAP1-AS1沉默可抑制甲状腺癌细胞增殖、侵袭,其机制可能与下调Rho A、Cyclin D1、MMP-9蛋白表达相关。
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