沉默lncRNA SLC16A1-AS1对脑胶质瘤细胞恶性生物学行为的影响
Effects of silencing lncRNA SLC16A1-AS1 on malignant biological behaviors of brain glioma cells
龙银波 1李贺扬 1金治宾1
作者信息
- 1. 061000 河北,沧州市中心医院神经外科
- 折叠
摘要
目的 探讨沉默长链非编码RNA(lncRNA)SLC16A1-AS1对脑胶质瘤细胞恶性生物学行为的影响.方法 PCR检测2021年5月至2023年1月手术切除的62例脑胶质瘤组织lncRNA SLC16A1-AS1、微小RNA(miR)-584-5p以细胞外蛋白调节激酶1(MAPK1)mRNA,免疫印迹法检测MAPK1蛋白表达;以瘤旁组织(距离肿瘤边缘>2 cm)作为正常脑组织.从胶质瘤组织中分离、培养胶质瘤细胞,进行CD133、Neatin免疫荧光染色鉴定;转染不同质粒沉默lncRNA SLC16A1-AS1、上调或下调miR-584-5p表达;应用CCK-8法、划痕实验、Transwell实验和流式细胞术检测细胞增殖、迁移、侵袭和凋亡情况;免疫印迹法检测细胞MAPK1、细胞周期素D1(CyclinD1)、基质金属蛋白酶(MMP)-2、caspase-3蛋白表达;双荧光素酶报告基因实验验证lncRNA SLC16A1-AS1、miR-584-5p和MAPK1的靶向关系.结果 胶质瘤组织lncRNA SLC16A1-AS1、MAPK1呈高表达(P<0.05),miR-584-5p呈低表达(P<0.05).免疫荧光染色显示,分离培养的细胞CD133、Nestin均呈阳性表达.沉默lncRNA SLC16A1-AS1表达,明显抑制体外培养的胶质瘤细胞增殖、迁移、侵袭(P<0.05),促进细胞凋亡(P<0.05),明显下调细胞CyclinD1、MMP-2、MMP-9蛋白表达(P<0.05),明显上调miR-584-5p、caspase-3表达.抑制miR-584-5p明显逆转沉默lncRNA SLC16A1-AS1对体外培养的价胶质瘤细胞的作用.双荧光素酶报告基因实验证实lncRNA SLC16A1-AS1与miR-584-5p/MAPK1存在靶向调节关系.结论 胶质瘤组织lncRNA SLC16A1-AS1呈高表达,沉默lncRNA SLC16A1-AS1可以上调miR-584-5p表达,抑制MAPK1表达,进而抑制胶质瘤细胞的增殖、迁移、侵袭,促进细胞凋亡.
Abstract
Objective To investigate the effects of silencing long non-coding RNA(lncRNA)SLC16A1-AS1 on the malignant biological behaviors of glioma cells.Methods LncRNA SLC16A1-AS1,miR-584-5p and MAPK1 mRNA were detected by PCR in glioma tissues obtained from 62 patients who underwent surgery from May 2021 to January 2023,and MAPK1 protein expression was detected by Western blotting.Para-tumor tissues(>2 cm from the edge of the tumor)were used as control.Glioma cells were isolated and cultured from the glioma tissues,and CD133 and Neatin were identified by immunofluorescence staining.Different plasmids were transfected to silence lncRNA SLC16A1-AS1,up-regulate or down-regulate the expression of miR-584-5p.CCK-8 method,scratch experiment,Transwell experiment and flow cytometry were used to detect the proliferation,migration,invasion and apoptosis of the cultured glioma cells.The protein expressions of MAPK1,CyclinD1,MMP-2 and caspase-3 were detected by Western blotting.The dual luciferase reporter gene assay was used to verify the targeting relationship between lncRNA SLC16A1-AS1,miR-584-5p and MAPK1.Results LncRNA SLC16A1-AS1 and MAPK1 were significantly highly expressed in glioma tissues(P<0.05),and miR-584-5p was significantly lowly expressed(P<0.05).The immunofluorescence staining showed that the isolated and cultured cells were positive for CD133 and Nestin.Silencing of lncRNA SLC16A1-AS1 significantly inhibited the proliferation,migration and invasion of the glioma cells in vitro(P<0.05),promoted cell apoptosis(P<0.05),significantly down-regulated the protein expressions of CyclinD1,MMP-2 and MMP-9(P<0.05),and significantly up-regulated the expressions of miR-584-5p and caspase-3.Inhibition of miR-584-5p significantly reversed the effects of lncRNA SLC16A1-AS1 silencing on the glioma cells in vitro.Dual luciferase reporter gene assay confirmed that lncRNA SLC16A1-AS1 was targeted to regulate miR-584-5p/MAPK1.Conclusions LncRNA SLC16A1-AS1 is highly expressed in glioma tissues.Silencing lncRNA SLC16A1-AS1 can up-regulate the expression of miR-584-5p and inhibit the expression of MAPK1,thereby inhibiting the proliferation,migration and invasion of glioma cells and promoting cell apoptosis.
关键词
脑胶质瘤/长链非编码RNA/SLC16A1-AS1/微小RNA-584-5p/细胞外蛋白调节激酶1/细胞增殖/细胞凋亡Key words
Brain glioma/Long non-coding RNA SLC16A1-AS1/miRNA-584-5p/MAPK1/Malignant biological behaviors引用本文复制引用
基金项目
河北省2021年度医学科学研究课题计划(20210971)
出版年
2024
NETL
NSTL
万方数据