Objective: This study was set out to explore the expression and related mechanism of XIST and miR-200a-3p in retinoblastoma (Rb).
Patients and Methods: Fifty-four children with Rb who came to our hospital for surgery from January 2018 to September 2019 were collected. In addition, Rb cells and human retinal epithelial cells were purchased. XIST-siRNA (si-XIST), XIST-shRNA (sh-XIST), empty vector plasmid (siRNA-NC), miR-200a-3p-mimics and miR − 200a-3p-inhibition were transfected into Y79 cells. The expression of XIST and miR-200a-3p in the samples were determined by qRT-PCR. β-catenin, cyclin B1, cyclin D1, Bax, Caspase-3, N-cadherin, vimentin, Snail, E-Cadherin and ZO-1 protein levels were measured by WB. MTT, Transwell and flow cytometry were utilized to detect cell proliferation, invasion, and apoptosis, respectively.
Results: XIST was highly expressed while miR-200a-3p was lowly expressed in patients’ tissues, and the AUC of both was over 0.8. XIST and miR-200a-3p was related to differentiation degree in Rb patients. Y79 cells were selected for transfection. Compared with the siRNA-NC group, XIST was significantly reduced in the siRNA-XIST group, and it was significantly increased in the shRNA-XIST group (P< 0.01). The proliferation capacity of siRNA-XIST group was decreased, while that of shRNA-XIST group was up-regulated. The apoptosis rate of siRNA-XIST group was significantly up-regulated, while that of shRNA-XIST group was decreased (P< 0.001). The invasive capacity of siRNA-XIST group was decreased, while that of shRNA-XIST group was up-regulated (P< 0.001). Silencing XIST and over-expressed miR-200a-3p could inhibit cell epithelial–mesenchymal transition (EMT), proliferation, invasion, and promote apoptosis. WB detection showed that Carboplatin + LncRNA XIST intervention group could more significantly inhibit β-catenin, cyclin B1, cyclin D1, N-cadherin, vimentin, Snail protein, and promote the up-regulation of Bax, Caspase-3, E-Cadherin and ZO-1 expression.
Conclusion: Inhibition of XIST expression can up-regulate miR-200a-3p-mediated PI3K-Akt/MAPK-ERK signaling pathway and affect cell EMT, proliferation, invasion, and apoptosis, which is expected to be a potential therapeutic target for Rb.

Keywords: lncRNA-XIST, miR-200a-3p, NRP1, epithelial–mesenchymal transition, retinoblastoma, biological mechanism


中文翻译：

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卡铂通过 IncRNA XIST/miR-200a-3p/NRP1 Axis 抑制视网膜母细胞瘤的进展。

目的：本研究旨在探讨XIST和miR-200a-3p在视网膜母细胞瘤（Rb）中的表达及相关机制。
患者和方法：收集2018年1月至2019年9月来我院手术的Rb患儿54例。此外，还购买了 Rb 细胞和人视网膜上皮细胞。将 XIST-siRNA (si-XIST)、XIST-shRNA (sh-XIST)、空载体质粒 (siRNA-NC)、miR-200a-3p-mimics 和 miR-200a-3p-抑制转染到 Y79 细胞中。通过qRT-PCR测定样品中XIST和miR-200a-3p的表达。通过WB测量β-连环蛋白、细胞周期蛋白B1、细胞周期蛋白D1、Bax、Caspase-3、N-钙粘蛋白、波形蛋白、Snail、E-钙粘蛋白和ZO-1蛋白水平。MTT、Transwell和流式细胞仪分别用于检测细胞增殖、侵袭和凋亡。
结果：XIST在患者组织中高表达，而miR-200a-3p低表达，两者的AUC均大于0.8。XIST和miR-200a-3p与Rb患者的分化程度有关。选择 Y79 细胞进行转染。与siRNA-NC组相比，siRNA-XIST组XIST显着降低，shRNA-XIST组显着升高（P<0.01）。siRNA-XIST组增殖能力下降，shRNA-XIST组增殖能力上调。siRNA-XIST组细胞凋亡率明显上调，而shRNA-XIST组细胞凋亡率降低（P<0.001）。siRNA-XIST组侵袭能力降低，shRNA-XIST组侵袭能力上调（P<0.001）。沉默 XIST 和过表达的 miR-200a-3p 可以抑制细胞上皮-间质转化 (EMT)、增殖、侵袭并促进细胞凋亡。WB检测显示卡铂+LncRNA XIST干预组能更显着抑制β-catenin、cyclin B1、cyclin D1、N-cadherin、vimentin、Snail蛋白，促进Bax、Caspase-3、E-Cadherin和ZO-1 表达。
结论：抑制XIST表达可上调miR-200a-3p介导的PI3K-Akt/MAPK-ERK信号通路，影响细胞EMT、增殖、侵袭和凋亡，有望成为Rb的潜在治疗靶点。

关键词： lncRNA-XIST，miR-200a-3p，NRP1，上皮-间质转化，视网膜母细胞瘤，生物学机制