miR-486-3p靶向DDR1对口腔鳞状细胞癌的抑制作用及槟榔致口腔鳞状细胞癌的可能机制

Inhibition of miR-486-3p targeted DDR1 on oral squamous cell carcinoma and possible mechanism of oral squamous cell carcinoma induced by betel-nut

目的探讨miR-486-3p靶向盘蛋白结构域受体1(DDR1)对口腔鳞状细胞癌(OSCC)的抑制作用,并基于miR-486-3p和DDR1分析槟榔致OSCC的可能机制。方法(1)收集20例原发性OSCC患者肿瘤组织及癌旁组织以检测miR-486-3p的表达水平。(2)取人口腔表皮癌(OEC)-M1细胞,分为对照1组(常规培养细胞,不给予任何特殊处理)、miR-486-3p mimic组(转染miR-486-3p mimic)、miR-486-3p inhibitor组(转染miR-486-3p inhibitor),以及对照2组(常规培养细胞,不给予任何特殊处理)、OE-DDR1组(转染过表达DDR1的慢病毒质粒)、sh-DDR1组(转染低表达DDR1的慢病毒质粒)。检测各组细胞增殖能力,以及活化型Caspase-3(cleaved Caspase-3)、Caspase-3蛋白表达水平。检测对照1组、miR-486-3p mimic组、miR-486-3p inhibitor组DDR1 mRNA的表达水平。(3)取HEK293细胞,分别转染miR-486-3p mimic和野生型或突变型DDR13端非翻译区。通过TargetScan数据库及双荧光素酶实验分析miR-486-3p与DDR1的结合情况。(4)取OKF4/TERT-1细胞,分为无槟榔碱组(不给予槟榔碱处理)和槟榔碱组(用槟榔碱连续刺激5 d),检测两组细胞miR-486-3p、DDR1 mRNA及蛋白表达水平。结果(1)与癌旁组织相比,OSCC组织中miR-486-3p表达水平降低(P<0.05)。(2)与对照1组/对照2组相比,miR-486-3p mimic组/sh-DDR1组OEC-M1细胞增殖能力降低,cleaved Caspase-3表达水平升高,而miR-486-3p inhibitor组/OE-DDR1组OEC-M1细胞增殖能力增高,cleaved Caspase-3表达水平降低(均P<0.05)。与对照1组相比,miR-486-3p mimic组OEC-M1细胞的DDR1 mRNA表达水平降低,miR-486-3p inhibitor组DDR1 mRNA表达水平升高(均P<0.05)。(3)miR-486-3p与野生型DDR1存在结合位点。(4)与无槟榔碱组相比,槟榔碱组OKF4/TERT-1细胞中miR-486-3p表达水平降低,DDR1 mRNA和蛋白表达水平升高(均P<0.05)。结论miR-486-3p通过靶向下调DDR1的表达,抑制OSCC细胞增殖,促进OSCC细胞凋亡,进而发挥抑癌作用。槟榔可能通过调控miR-486-3p和DDR1的表达从而诱发OSCC。

Objective To investigate the inhibition of miR-486-3p targeted discoidin domain receptor-1(DDR1)on oral squamous cell carcinoma(OSCC),and to analyze the possible mechanism of OSCC induced by betel-nut based on miR-486-3p and DDR1.Methods(1)Cancerous and paracancerous tissues from 20 patients with primary OSCC were collected to detect the expression of miR-486-3p.(2)The human oral epidermal carcinoma(OEC)-M1 cells were obtained,and they were assigned to control 1 group(with routine cell culture but without any special treatment),miR-486-3p mimic group(transfected with miR-486-3p mimic)or miR-486-3p inhibitor group(transfected with miR-486-3p inhibitor),as well as control 2 group(with routine cell culture but without any special treatment),OE-DDR1 group(transfected with lentivirus plasmid of over-expressed DDR1)or sh-DDR1 group(transfected with lentivirus plasmid of low-expressed DDR1).The cell proliferation ability,and protein expressions of cleaved Caspase-3,and Caspase-3 were detected in various groups.The DDR1 mRNA expression in the control 1 group,miR-486-3p mimic group,and miR-486-3p inhibitor group was detected.(3)HEK293 cells were obtained to transfect with miR-486-3p mimic,and DDR13′untranslated region in wild type or mutant type,respectively.The binding condition of miR-486-3p with DDR1 was analyzed by the TargetScan database and dual-luciferase assay.(4)OKF4/TERT-1 cells were acquired to assign to the non-arecoline group(no treatment of arecoline)or the arecoline group(continuously stimulated with arecoline for 5 days).The mRNA and protein expressions of miR-486-3p and DDR1 were detected in the two groups.Results(1)Compared with paracancerous tissues,miR-486-3p expression in OSCC tissues was decreased(P<0.05).(2)Compared with the control 1 group/control 2 group,the miR-486-3p mimic group/sh-DDR1 group exhibited a decreased ability of cell proliferation,an elevation of cleaved Caspase-3 expression,whereas the miR-486-3p inhibitor group/OE-DDR1 group interpreted an increased ability of OEC-M1 cell proliferation,and a decline of cleaved Caspase-3 expression(all P<0.05).Compared with the control 1 group,the miR-486-3p mimic group yielded a decline of DDR1 mRNA expression of OEC-M1 cells,and the miR-486-3p inhibitor group implied an elevation of DDR1 mRNA expression(all P<0.05).(3)There was binding locus of miR-486-3p with DDR1 in wild type.(4)Compared with the non-arecoline group,the arecoline group depicted a decline of miR-486-3p expression in OKF4/TERT-1 cells,and elevations of DDR1 mRNA and protein expressions(all P<0.05).Conclusion miR-486-3p inhibits the proliferation of OSCC cells,promotes the apoptosis of OSCC cells,and further exerts an effect of cancer inhibition by targetedly down-regulating the expression of DDR1.Betel-nut may induce OSCC by regulating the expressions of miR-486-3p and DDR1.