Department of Urology, Qujing No. 1 Hospital, Qujing, China.
Eur Rev Med Pharmacol Sci. 2020 Sep;24(17):8747-8755. doi: 10.26355/eurrev_202009_22813.
It was the aim of this study to explore the role and mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 in the progression of bladder cancer (BCa) by in vitro experiments.
AFAP1-AS1 levels in 40 pairs of clinical BCa tissue samples and normal ones collected from BCa patients were determined, and paired sample t-test was applied to compare the differences between groups. The prognosis data of patients with BCa were collected, and survival analysis and t-test were performed to specify the interplay between AFAP1-AS1 and the prognosis of BCa patients. Subsequently, AFAP1-AS1 expression level in BCa and normal cells were further confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), and transwell assays were performed to figure out the influence of this lncRNA on the proliferation ability and invasiveness of BCa cells. Meanwhile, the interaction between AFAP1-AS1 and its sense mRNA was analyzed. We used co-transfection technology to simultaneously transfect si-AFAP1-AS1 and pcDNA3.1-AFAP1 or their corresponding negative controls into BCa cells, and cell proliferation and invasion ability in different subgroups were determined to explore the underlying mechanism through which AFAP1-AS1 plays a role in BCa progression.
No matter in BCa tissues or in cell samples, compared to the corresponding normal controls, AFAP1-AS1 was found highly expressed; at the same time, in invasive bladder cancer tissues, the expression level of AFAP1-AS1 was also higher than that in non-invasive tissues. Meanwhile, survival analysis revealed that patients with BCa with high expression of AFAP1-AS1 owned a shorter overall survival rate than those with low expression, indicating a negative interplay between AFAP1-AS1 expression and patients' prognosis. In addition, in BCa cell lines, according to the results of CCK-8, EDU, and transwell assays, the proliferative capacity, as well as the invasive ability of BCa cells, were found weakened after downregulation of AFAP1-AS1. Meanwhile, a negative interplay was discovered between AFAP1-AS1 and its sense mRNA. Finally, the results of cell reversal experiment using co-transfection technique revealed that overexpression of AFAP1 can reverse the inhibitory impact of lncRNAAFAP1-AS1 on the malignant ability of BCa cells.
AFAP1-AS1 may enhance the proliferation ability as well as the invasiveness of BCa cells so as to aggravate the degree of BCa malignancy.
本研究旨在通过体外实验探讨长链非编码 RNA（lncRNA）AFAP1-AS1 在膀胱癌（BCa）进展中的作用和机制。
检测 40 对膀胱癌患者临床组织样本和正常组织中 AFAP1-AS1 水平，采用配对样本 t 检验比较组间差异。收集膀胱癌患者的预后资料，进行生存分析和 t 检验，明确 AFAP1-AS1 与膀胱癌患者预后的相互关系。随后，通过实时定量聚合酶链反应（qRT-PCR）进一步证实膀胱癌和正常细胞中 AFAP1-AS1 的表达水平，并进行细胞计数试剂盒-8（CCK-8）、5-乙炔基-2'-脱氧尿苷（EdU）和 Transwell 检测，以了解该 lncRNA 对膀胱癌细胞增殖能力和侵袭性的影响。同时，分析 AFAP1-AS1 与其有义 mRNA 之间的相互作用。我们采用共转染技术，将 si-AFAP1-AS1 和 pcDNA3.1-AFAP1 或其相应的阴性对照同时转染入膀胱癌细胞，通过不同亚组细胞增殖和侵袭能力的检测，探讨 AFAP1-AS1 在 BCa 进展中发挥作用的潜在机制。
无论是在膀胱癌组织还是细胞样本中，与相应的正常对照组相比，AFAP1-AS1 均呈高表达；同时，在侵袭性膀胱癌组织中，AFAP1-AS1 的表达水平也高于非侵袭性组织。同时，生存分析显示，AFAP1-AS1 高表达的膀胱癌患者总生存率低于低表达患者，表明 AFAP1-AS1 表达与患者预后呈负相关。此外，在膀胱癌细胞系中，根据 CCK-8、EDU 和 Transwell 检测结果，下调 AFAP1-AS1 后，膀胱癌细胞的增殖能力和侵袭能力均减弱。同时，发现 AFAP1-AS1 与其有义 mRNA 之间存在负相互作用。最后，采用共转染技术的细胞逆转实验结果表明，过表达 AFAP1 可以逆转 lncRNA AFAP1-AS1 对膀胱癌细胞恶性能力的抑制作用。
AFAP1-AS1 可能通过增强膀胱癌细胞的增殖能力和侵袭能力，从而加重膀胱癌的恶性程度。
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