摘要
目的:探讨长链非编码RNA LINC00478对口腔鳞癌细胞顺铂耐药性的影响及其对微小RNA-214(miR-214)的靶向调控作用.方法:采用qRT-PCR法检测口腔鳞癌组织、癌旁组织中LINC00478、miR-214的表达量;体外培养口腔鳞癌细胞CAL-27,建立顺铂耐药性细胞CAL-27/DDP,分别将pcDNA、pcDNA-LINC00478、pcDNA-LINC00478与miR-NC、pcDNA-LINC00478与miR-214 mimics转染至CAL-27/DDP细胞;采用qRT-PCR法检测CAL-27细胞与CAL-27/DDP细胞中LINC00478、miR-214的表达量;MTT法与克隆形成实验检测细胞增殖能力;流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测LINC00478、miR-214的靶向关系;Western blot法检测Ki67、Pro-caspase3、Cleaved-caspase3蛋白表达量.结果:与癌旁组织比较,口腔鳞癌组织LINC00478的表达水平显著降低(P<0.05),miR-214的表达水平显著升高(P<0.05);与CAL-27组比较,CAL-27/DDP组LINC00478的表达水平显著降低(P<0.05),miR-214的表达水平显著升高(P<0.05);与转染pcDNA比较,CAL-27/DDP细胞中转染pcDNA-LINC00478可提高增殖抑制率、凋亡率及Cleaved-caspase3蛋白水平(P<0.05),减少克隆形成数(P<0.05),降低Ki67、Pro-caspase3蛋白水平(P<0.05);双荧光素酶报告实验证实LINC00478能够靶向调控miR-214的表达及活性;与共转染pcDNA-LINC00478与miR-NC比较,共转染pcDNA-LINC00478与miR-214 mimics可降低增殖抑制率、凋亡率及Cleaved-caspase3蛋白水平(P<0.05),增加克隆形成数(P<0.05),提高Ki67、Pro-caspase3蛋白水平(P<0.05).结论:LINC00478通过靶向抑制miR-214的表达而抑制口腔鳞癌细胞增殖及诱导细胞凋亡,从而降低细胞顺铂耐药性.
Objective:To explore the effect of LncRNA LINC00478 on cisplatin resistance of oral squamous cell carcinoma and its targeted regulation on miR-214.Methods:The expression levels of LINC00478 and miR-214 in oral squamous cell carcinoma and adjacent tissues were detected by qRT-PCR.Oral squamous carcinoma cells CAL-27 were cultured in vitro to establish cisplatin-resistant cells CAL-27/DDP,and pcDNA,pcDNA-LINC00478,pcDNA-LINC00478 and miR-NC,pcDNA-LINC00478 and miR-214 mimics were transfected into CAL-27/DDP cells.The expression of LINC00478 and miR-214 in CAL-27 cells and CAL-27/DDP cells were detected by qRT-PCR method.MTT method and clone formation experiment were used to detect cell proliferation ability.Flow cytometry was used to detect the apoptosis rate.The dual luciferase report experiment detected the targeting relationship between LINC00478 and miR-214.Western blot was used to detect the protein expression of Ki67,Pro-caspase3 and Cleared-caspase3.Results:Compared with the adjacent tissues,the expression level of LINC00478 in oral squamous cell carcinoma was significantly decreased(P<0.05),and the expression level of miR-214 was significantly increased(P<0.05).Compared with the CAL-27 group,the expression level of LINC00478 in the CAL-27/DDP group was significantly reduced(P<0.05),and the expression level of miR-214 was significantly increased(P<0.05).Compared with transfection of pcDNA,transfection of pcDNA-LINC00478 in CAL-27/DDP cells could increase the proliferation inhibition rate,apoptosis rate,and the protein level of Cleaved-caspase3(P<0.05),reduce the number of clone formation(P<0.05),reduce the protein levels of Ki67 and Pro-caspase3(P<0.05).The dual luciferase report experiment confirmed that LINC00478 could target and regulate the expression and activity of miR-214.Compared with co-transfection of pcDNA-LINC00478 and miR-NC,co-transfection of pcDNA-LINC00478 and miR-214 mimics can reduce the proliferation inhibition rate,apoptosis rate and the level of Cleared-caspase3 protein(P<0.05),and increase the number of clone formation(P<0.05),increase the protein levels of Ki67 and Pro-caspase3(P<0.05).Conclusion:LINC00478 inhibits the proliferation of oral squamous carcinoma cells and induces apoptosis by targeting the expression of miR-214 to reduce cell cisplatin resistance.
作者
李冠睿
李建武
颜家渝
LI Guanrui;LI Jianwu;YAN Jiayu(Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu 610072, China;Linyi Traditional Chinese Medical Hospital, Linyi 276003, China;Guang'an Hospital Affiliated to Chengdu University of Traditional Chinese Medicine/Guang'an Hospital of Traditional Chinese Medicine, Guang'an 638600, China)
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2021年第1期19-25,70,共8页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
四川省科技计划项目(18ZDYF0878)。
