长链非编码RNA SPATA31D5P吸附微小RNA-320a促进乳腺癌细胞的增殖、迁移和侵袭

doi:10.16098/j.issn.0529-1356.2021.06.014

摘要/Abstract

摘要：

目的乳腺癌（BC）中存在多种长链非编码RNA（lncRNA）的异常表达，并且与生存预后密切相关。本研究旨在探讨lncRNA SPATA31D5P在乳腺癌中的表达并通过吸附miR-320a影响乳腺癌细胞增殖、侵袭和迁移能力。方法收集乳腺癌组织及癌旁组织各30例，采用Real-time PCR检测SPATA31D5P在乳腺癌组织及乳腺癌细胞系中的表达水平。选择乳腺癌MDA-MB-231细胞转染针对SPATA31D5P siRNA干扰载体，采用CCK-8法、5-乙炔基-2’脱氧尿嘧啶核苷（EdU）法、Transwell小室实验和细胞划痕实验测定细胞增殖、侵袭和迁移能力，流式细胞术检测细胞周期和凋亡的改变。采用生物信息学方法筛选可与SPATA31D5P互补结合的miRNAs, Realtime PCR及双荧光素酶报告实验验证SPATA31D5P对miR-320a的调控作用。结果乳腺癌组织中SPATA31D5P水平显著高于邻近正常乳腺组织，各乳腺癌细胞系中SPATA31D5P表达都高于正常乳腺上皮细胞MCF10 A。siRNA干扰组的SPATA31D5P水平为 0.288±0.052，低于空白对照组的1.114±0.096和阴性对照（NC）组的1.079±0.128 (P＜0.01)。与NC组和空白对照组相比，干扰组MDA-MB-231细胞的增殖活力明显下降并出现G1期阻滞，凋亡率明显增加(P＜0.01)；干扰组的划痕愈合率和穿膜细胞数目分别为（14.36 ± 1.75）%和（26 ± 1.52）个，低于NC组的（52.25 ± 1.87）% 和（67.33 ± 2.91）个 (P＜0. 01)。双荧光素酶实验证实，SPATA31D5P能够直接调控miR-320a的表达及荧光素酶活性。结论 SPATA31D5P在乳腺癌中高表达，干扰其表达能有效抑制MDA-MB-231细胞增殖、迁移和侵袭，其作用机制可能与靶向调控miR-320a有关。

关键词: 乳腺癌, 长链非编码RNA, SPATA31D5P, 微小RNA-320a, 侵袭, 实时定量聚合酶链反应, 人

Abstract:

Objective Long non-coding RNA(lncRNA) are aberrantly expressed in breast cancer(BC) and strongly associated with its survival prognosis. The aim of this study is to investigate the expression and effect of lncRNA SPATA31D5P on the invasion and migration capacity of breast cancer cells through adsorption of miR-320a. Methods Totally 30 cases of BC tissues and paraneoplastic tissues were collected, and the expression levels of SPATA31D5P in BC tissues and BC cell lines were detected by Real-time PCR. MDA-MB-231 cells were transfected with SPATA31D5P siRNA interference vector, and cell proliferation, invasion and migration capacity were determined using the cell counting kit-8 assay (CCK-8), 5-ethynyl-2- deoxyuridine(EdU), Transwell and wound-healing assay respectively. And cell cycle and apoptosis were detected by flow cytometry. Bioinformatics approachs were used to screen for miRNAs that could bind complementarily to SPATA31D5P, and the regulatory effect of SPATA31D5P on miR-320a was detected by Real-time PCR and dual luciferase reporter assay. Results SPATA31D5P levels were significantly higher in BC tissues than in adjacent normal breast tissues, and SPATA31D5P expression was higher in each BC cell line than in normal breast epithelial cells MCF10 A. The level of SPATA31D5P in the interference group was 0.288±0.052, which was lower than that of the blank control group 1.114±0.096 and negative control(NC) group 1.079±0.128 (P<0.01). The proliferation activity of MDA-MB-231 cells in the interfered group was significantly reduced and apoptotic rate was obviously increased compared to the NC and control groups (P<0.01);the G1 phase block was observed in the interfered group; the scratch healing rate and number of perforated cells in the interference group were (14.36±1.75)% and (26±1.52), which were lower than (52.25±1.87)% and (67.33±2.91) of the NC group (P<0.01). Dual luciferase experiments confirmed that SPATA31D5P could directly regulate miR-320a expression and luciferase activity. Conclusion SPATA31D5P is highly expressed in BC, interfering with SPATA31D5P expression effectively inhibits the proliferation, migration and invasion of MDA-MB-231 cells, and the mechanism may be related to the targeted regulation of miR-320a.

Key words: Breast cancer, Long non-coding RNA, SPATA31D5P, MicroRNA-320a, Invasion, Real-time PCR, Human

中图分类号:

R737.9

危敏余海浪王涵多郜蕊雷洁. 长链非编码RNA SPATA31D5P吸附微小RNA-320a促进乳腺癌细胞的增殖、迁移和侵袭[J]. 解剖学报, 2021, 52(6): 925-932.

WEI Min YU Hai-lang WANG Han-duo GAO Rui LEI Jie. Long non-coding RNA SPATA31D5P promoting proliferation, migration and invasion of breast cancer cells through adsorption of microRNA-320a[J]. Acta Anatomica Sinica, 2021, 52(6): 925-932.

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