摘要
目的 探讨长链非编码RNA生长停滞特异性转录本5(LncRNA GAS5)调节微小RNA-182-5p(miR-182-5p)/叉头盒蛋白F2(FOXF2)对口腔鳞状细胞癌(OSCC)细胞增殖、迁移、侵袭及凋亡的影响.方法 收集45例确诊为OSCC的癌组织以及癌旁组织,体外培养人口腔上皮细胞系HOEC及人OSCC细胞系CAL-27、HSC-3、SCC-25,qRT-PCR 检测组织、细胞中 LncRNA GAS5、miR-182-5p、FOXF2 的表达;择细胞株 CAL-27 分为 ctrl 组、pcDNA 组、pcDNA-GAS5 组、pcDNA-GAS5+miR-NC 组及 pcDNA-GAS5+miR-182-5p mimics 组,采用 CCK-8 试剂盒检测细胞增殖活性,Transwell实验检测细胞迁移和侵袭能力,流式细胞术检测细胞凋亡水平,Western Blot检测癌细胞中FOXF2、bax、Bcl-2、cleaved caspase-3及E-cadherin蛋白表达,双萤光素酶报告实验验证miR-182-5p与LncRNA GAS5、FOXF2的关系.结果 与癌旁组织相比,OSCC组织中LncRNA GAS5、FOXF2 mRNA表达降低,miR-182-5p 表达升高(P＜0.05);CAL-27、HSC-3、SCC-25 细胞与 HOEC 相比,LncRNA GAS5、miR-182-5p、FOXF2表达趋势与OSCC组织内一致,且在CAL-27表达最明显,选择其作为后续实验的细胞株;与ctrl组、pcD-NA 组比较,pcDNA-GAS5 组CAL-27细胞中GAS5、FOXF2 mRNA、细胞凋亡率及BAX、E-cadherin蛋白表达升高(P＜0.05),miR-182-5p表达、细胞增殖、迁移和侵袭数及Bcl-2蛋白表达降低(P＜0.05);上调miR-182-5p可减弱过表达LncRNA GAS5对CAL-27细胞增殖、迁移和侵袭的抑制作用,也可抑制癌细胞凋亡;LncRNA GAS5靶向负调控miR-182-5p表达,miR-182-5p靶向负调控FOXF2表达.结论 上调GAS5可能通过抑制miR-182-5p来增加FOXF2表达,进而抑制CAL-27细胞增殖、迁移及侵袭,促进CAL-27细胞的凋亡.
Abstract
Objective To investigate the impact of long non-coding RNA growth arrest specific transcript 5(LncRNA GAS5)on proliferation,migration,invasion and apoptosis of oral squamous cell carcinoma(OSCC)cells by regulating microRNA-182-5p(miR-182-5p)/fork head box F2(FOXF2).Methods OSCC tissues and adjacent tissues were collected from 45 patients diagnosed as OSCC.Human oral epithelial cell line HOEC,human OSCC cell lines CAL-27,HSC-3,and SCC-25 were cultured in vitro.qRT-PCR was applied to detect the expressions of LncRNA GAS5,miR-182-5p and FOXF2 in tissues and cells.CAL-27 cell line was selected and divided into the following groups:ctrl group,pcDNA group,pcDNA-GAS5 group,pcDNA-GAS5+miR-NC group,and pcDNA-GAS5+miR-182-5p mimics group.CCK-8 kit was applied to detect cell proliferation activity.Transwell assay was applied to detect cell migration and invasion capacities.Flow cytometry was applied to detect the level of cellular apoptosis.Western blot was applied to detect the protein expressions of FOXF2,bax,Bcl-2,cleaved caspase-3,and E-cadherin in cancer cells.Dual Luciferase reporter assay was applied to verify the association of miR-182-5p with LncRNA GAS5 and FOXF2.Results When compared to adjacent cancer tissues,the expressions of LncRNA GAS5 and FOXF2 mRNA in OSCC tissues were decreased,while miR-182-5p expression was increased(P＜0.05).When compared to HOEC,the expression trends of LncRNA GAS5,miR-182-5p,and FOXF2 in CAL-27,HSC-3,and SCC-25 cells were consistent with those in OSCC tissues,and their expressions were pronounced in CAL-27 cell line.Therefore,CAL-27 was selected as the cell line for subsequent experiments.When compared to ctrl and pcDNA groups,pcDNA-GAS5 group showed the increases in GAS5,FOXF2 mRNA,apoptosis rate,bax,and E-cadherin protein expressions in CAL-27 cells(P＜0.05),while miR-182-5p expression,cell proliferation,migrated cell numbers,invaded cell numbers and Bcl-2 protein expression were decreased(P＜0.05).Upregulating miR-182-5p weakened the inhibitory effect of LncRNA GAS5 overexpression on CAL-27 cell proliferation,migration and invasion,and inhibited cancer cellular apoptosis.LncRNA GAS5 negatively regulated miR-182-5p expression,while miR-182-5p negatively regulated the FOXF2 expression.Conclusion Upregulating GAS5 might increase FOXF2 expression by inhibiting miR-182-5p,thereby inhibiting CAL-27 cell proliferation,migration and invasion,and promoting CAL-27 cell apoptosis.
基金项目
陕西省自然科学基础研究计划项目(2024JC-YBQN-0769)
NETL
NSTL
万方数据