SLC8A1-AS1靶向调控微小RNA-182-3p及对口腔鳞癌细胞迁移和侵袭的影响

Regulation on microRNA-182-3p of SLC8A1-AS1 and its effects on migration and invasion of oral squamous cell carcinoma cells

目的探讨长链非编码RNA溶质运载蛋白家族8成员A1反义RNA 1(SLC8A1-AS1)在口腔鳞状细胞癌(OSCC)细胞增殖、迁移和侵袭中的生物学作用及其与微小RNA-182-3p(miR-182-3p)的靶向调控关系。方法采用实时荧光定量PCR(qPCR)检测OSCC组织和细胞(CAL-27、SCC-4、SCC-9和Tca8113)的SLC8A1-AS1和miR-182-3p水平。双荧光素酶报告实验鉴定SLC8A1-AS1和miR-182-3p的相互作用。将Tca8113细胞分为4组:对照组(空白对照)、SLC8A1-AS1过表达组(转染pcDNA3.1-SLC8A1-AS1+miR-182-3p阴性对照NC)、miR-182-3p过表达组(转染SLC8A1-AS1阴性对照pcDNA3.1+miR-182-3p模拟物mimics)和共过表达组(转染pcDNA3.1-SLC8A1-AS1+miR-182-3p mimics)。利用MTT法、划痕实验和Transwell小室实验检测SLC8A1-AS1和miR-182-3p对Tca8113细胞增殖、迁移和侵袭的影响。结果与癌旁组织相比,OSCC组织的SLC8A1-AS1水平下调,而miR-182-3p水平上调(P<0.05);与正常人口腔角质上皮细胞NHOK相比,OSCC细胞的SLC8A1-AS1水平下调,而miR-182-3p水平上调(P<0.05)。双荧光素酶报告实验结果显示,miR-182-3p mimics降低了野生型SLC8A1-AS1的荧光素酶活性(P<0.05),而不影响突变型SLC8A1-AS1的荧光素酶活性(P>0.05)。与对照组相比,SLC8A1-AS1过表达组的Tca8113细胞活力降低至1.454±0.103、划痕愈合率降低至(17.416±1.645)%及穿膜细胞数量减少至(119.877±6.469)个,而miR-182-3p过表达组的Tca8113细胞活力增强至2.879±0.126、划痕愈合率升高至(73.929±7.488)%及穿膜细胞数量增多至(391.243±23.819)个,且与SLC8A1-AS1过表达组相比,共过表达组Tca8113细胞活力增强至2.228±0.109、划痕愈合率升高至(32.924±4.114)%及穿膜细胞数量增多至(234.356±38.766)个,上述差异均有统计学意义(P<0.05)。结论SLC8A1-AS1通过靶向miR-182-3p在OSCC中发挥抑癌作用,SLC8A1-AS1/miR-182-3p轴可作为OSCC治疗的新靶点。

Objective To investigate the biological role of solute carrier family 8 member A1 antisense RNA 1(SLC8A1-AS1)on proliferation,migration and invasion of oral squamous cell carcinoma(OSCC)cells and explore its targeted regulation on microRNA-182-3p(miR-182-3p).Methods Levels of SLC8A1-AS1 and miR-182-3p in OSCC tissues and cell lines(CAL-27,SCC-4,SCC-9 and Tca8113)were measured by quantitative real-time PCR(qPCR).The interaction within SLC8A1-AS1/miR-182-3p was identified using the luciferase reporter assay.Tca8113 cells were selected and allocated into four groups:control group(blank control),SLC8A1-AS1 overexpression group(cells transfected with pcDNA3.1-SLC8A1-AS1 and miR-182-3p negative control NC),miR-182-3p overexpression group(cells transfected with SLC8A1-AS1 negative control pcDNA3.1 and miR-182-3p mimics)and co-overexpression group(cells transfected with pcDNA3.1-SLC8A1-AS1 and miR-182-3p mimics).Effects of SLC8A1-AS1 and miR-182-3p on proliferation,migration and invasion of Tca8113 cells were assessed by MTT,scratch assay and Transwell chamber assay,respectively.Results Compared with the adjacent tissues,SLC8A1-AS1 level was down-regulated but miR-182-3p level was up-regulated in OSCC tissues(P<0.05).Compared with normal human oral keratino cytes NHOK,SLC8A1-AS1 level was down-regulated but miR-182-3p level was up-regulated in OSCC cells(P<0.05).Double luciferase reporter assay showed that miR-182-3p mimics decreased the luciferase activity of wild-type SLC8A1-AS1(P<0.05),but did not affect the luciferase activity of mutant SLC8A1-AS1(P>0.05).Compared with the control group,the viability of Tca8113 cells decreased to 1.454±0.103,scratch healing rate decreased to(17.416±1.645)%and number of penetrating cells decreased to 119.877±6.469 in the SLC8A1-AS1 overexpression group,while the viability of Tca8113 cells increased to 2.879±0.126,scratch healing rate increased to(73.929±7.488)%and number of penetrating cells increased to 391.243±23.819 in the miR-182-3p overexpression group(P<0.05).Compared with SLC8A1-AS1 overexpression group,viability of Tca8113 cell increased to 2.228±0.109,scratch healing rate increased to(32.924±4.114)%and number of penetrating cells increased to 234.356±38.766 in the co-overexpression group(P<0.05).Conclusion SLC8A1-AS1 acts as a tumor suppressor in OSCC by targeting miR-182-3p,and the discovery of SLC8A1-AS1/miR-182-3p axis provides new therapeutic targets for OSCC.