Wang Ya-Peng, Huang Yan, Hou Tao, Lu Min
Department of Oncology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Transl Cancer Res. 2020 Mar;9(3):1427-1438. doi: 10.21037/tcr.2020.01.26.
Long non-coding RNAs (lncRNAs) have been proved to be involved in the occurrence and progression of various tumors including pancreatic cancer (PC). Growing evidence shows that lncRNA X inactive-specific transcript (XIST) functions as an oncogene in multiple tumorigenesis. However, the underlying mechanism of lncRNA XIST in the progression of PC remains elusive.
Expression levels of XIST and miR-185-5p both in PC tissues or PC cells were determined using real-time quantitative PCR (qRT-PCR). Gain and loss-of-function of XIST or miR-185-5p was performed for further exploration. Moreover, colony formation assay was performed to assess cell proliferation. Flow cytometry analysis was performed to measure cell cycle and apoptosis. Dual-luciferase reporter assay was conducted to verify the correlation between XIST, miR-185-5p and CCND2, respectively. Additionally, western blot analysis was conducted to determine the expression pattern of apoptosis-related proteins and cell cycle-associated proteins.
Herein, we found that XIST expression was up-regulated while miR-185-5p was down-regulated both in PC tissues and cell lines, compared with that of controls. Moreover, there was a negative correlation between XIST and miR-185-5p. Following that, functional experiments displayed that knockdown of XIST or overexpression of miR-185-5p inhibited cell proliferation, induced cell cycle arrest and promoted apoptosis in PC cells. Furthermore, mechanistic experiments displayed that XIST could negatively regulate miR-185-5p via direct binding. In addition, CCND2 was shown to be a downstream target of miR-185-5p. Importantly, overexpression or knockdown of XIST significantly increased or decreased the expression of CCND2, while these effects were reversed by miR-185-5p.
Taken together, our study demonstrated that lncRNA XIST functions as an oncogene and exerts its regulation via miR-185-5p/CCND2 axis, promoting proliferation and inhibiting apoptosis in PC.
长链非编码RNA（lncRNAs）已被证明参与包括胰腺癌（PC）在内的各种肿瘤的发生和发展。越来越多的证据表明，lncRNA X失活特异性转录本（XIST）在多种肿瘤发生过程中发挥癌基因的作用。然而，lncRNA XIST在PC进展中的潜在机制仍不清楚。
采用实时定量PCR（qRT-PCR）检测PC组织或PC细胞中XIST和miR-185-5p的表达水平。对XIST或miR-185-5p进行功能获得和功能缺失实验以进一步探索。此外，进行集落形成试验以评估细胞增殖。进行流式细胞术分析以测量细胞周期和凋亡。进行双荧光素酶报告基因试验以分别验证XIST、miR-185-5p和细胞周期蛋白D2（CCND2）之间的相关性。此外，进行蛋白质免疫印迹分析以确定凋亡相关蛋白和细胞周期相关蛋白的表达模式。
在此，我们发现与对照相比，PC组织和细胞系中XIST表达上调而miR-185-5p表达下调。此外，XIST与miR-185-5p之间存在负相关。随后，功能实验表明，敲低XIST或过表达miR-185-5p可抑制PC细胞的增殖，诱导细胞周期停滞并促进细胞凋亡。此外，机制实验表明，XIST可通过直接结合负向调节miR-185-5p。此外，CCND2被证明是miR-185-5p的下游靶点。重要的是，过表达或敲低XIST显著增加或降低CCND2的表达，而这些作用被miR-185-5p逆转。
综上所述，我们的研究表明lncRNA XIST作为癌基因发挥作用，并通过miR-185-5p/CCND2轴发挥其调节作用，促进PC细胞增殖并抑制细胞凋亡。
