miR-193a-3p通过...制口腔鳞状细胞癌增殖与侵袭_何思思_.pdf

532 CHINESE JOURNAL OF ANATOMY Vol.45 No.6 2022 解剖学杂志 2022 年第 45 卷第 6 期doi：10.3969/j.issn.1001-1633.2022.06.008论 著miR-193a-3p通过富亮氨酸2糖蛋白1/鞘氨醇激酶1信号通路 抑制口腔鳞状细胞癌增殖与侵袭何思思 李国良 黄鹏程（郴州市第一人民医院南院，1 健康管理中心，2 口腔科，郴州 423000）摘要 目的：目的：探究miR-193a-3p通过介导富亮氨酸2糖蛋白1（LRG-1）/鞘氨醇激酶1（SPHK1）信号通路对口腔鳞状细胞癌生物作用机制作用。方法：方法：选取本院2018年1月 2020年1月20例已确诊口腔鳞癌患者的癌组织与癌旁组织标本进行研究，分为口腔鳞癌组与癌旁组。HN4细胞分为3组，鳞癌组（鳞癌细胞常规培养）、转染NC组（转染miR-193a-3p-mimics-NC）、转染组（转染miR-193a-3p-mimics）。RT-PCR检测miR-193a-3p水平。CCK8检测增殖能力。Transwell小室检测侵袭能力。ELISA检测各组p53、caspase-3、Bcl-2、survivin水平。免疫印迹检测SPHK1、LRG-1水平。结果：结果：口腔鳞癌组织中miR-193a-3p水平低于癌旁组织。鳞癌组miR-193a-3p水平与转染NC组较为接近；转染组miR-193a-3p水平高于鳞癌组、转染NC组。转染组在0 h时的OD值均较为接近；鳞癌组、转染NC组在不同时间点的OD值均较为接近；转染组在24、48、72 h时的OD值低于鳞癌组、转染NC组。鳞癌组癌细胞侵袭数量与转染NC组侵袭数量较为接近；转染组癌细胞侵袭数量较鳞癌组、转染NC组明显降低。鳞癌组与转染NC组SPHK1、LRG-1、caspase-3、p53、survivin和Bcl-2水平相比无差异；与鳞癌组相比，转染组、敲除LRG-1组、转染SPHK1组LRG-1、SPHK1、survivin、Bcl-2水平降低，caspase-3、p53水平升高，转染组、敲除LRG-1组和转染SPHK1组上述各指标水平对比无差异.结论：结论：miR-193a-3p或通过降低SPHK1和LRG-1表达以抑制口腔鳞癌细胞的增殖及侵袭。关键词 miR-193a-3p；富亮氨酸2糖蛋白1；鞘氨醇激酶1；口腔鳞状细胞癌；增殖；侵袭miR-193a-3p inhibits proliferation and invasion of oral squamous cell carcinoma by mediating leucine-rich alpha-2-glycoprotein-1/sphingosine kinase 1 signaling pathwayHe Sisi，Li Guoliang，Huang Pengcheng（1.Health Management Center，2.Department of Stomatology，South Hospital of Chenzhou First Peoples Hospital，Chenzhou 423000，China）Abstract Objective：To explore the biological mechanism of miR-193A-3p in oral squamous cell carcinoma by mediating leucine rich alpha-2-glycoprotein-1/sphingosine kinase 1（LRG-1/SPHK1）signaling pathway.Methods：Twenty patients with confirmed oral squamous cell carcinoma in our hospital（from January 2018 to January 2020）were selected for the study of cancer tissue and paracancer tissue samples，which were divided into oral squamous cell carcinoma group and paracancer group.HN4 cells were divided into three groups：a squamous cell carcinoma group（conventional culture of squamous cell carcinoma），an NC group（transfection with miR-193a-3P-NC），and a transfection group（transfection with miR-193a-3p-mimics）.miR-193a-3p levels were detected by RT-PCR.CCK8 was used to detect proliferation.The transwell chamber assesses invasive ability.The levels of p53，caspase-3，Bcl-2 and survivin were detected by ELISA.The levels of SPHK1 and LRG-1 were detected by Western blotting.Results：miR-193a-3p level in oral squamous cell carcinoma was lower than that in paracancer tissues.The level of miR-193A-3p in squamous cell carcinoma group was similar to that in NC group.The level of miR-193a-3p in the transfection group was higher than that in the squamous cell carcinoma group and the transfection NC group.OD values of transfection group at 0 h were similar.OD values of squamous cell carcinoma group and transfection NC group were similar at different time points.OD values of the transfection group at 24，48 and 72 h were lower than those of the squamous cell carcinoma group and the transfection NC group.The number of invasive cells in squamous cell carcinoma group was close to that in NC transfection group.Compared with squamous cell carcinoma group and 第 1 作者 E-mail：收稿日期：2021-04-06；修回日期：2022-09-26NC group，the number of invasive cancer cells in the transfection group was significantly lower.There were 533 no differences in SPHK1，LRG-1，caspase-3，p53，survivin and Bcl-2 levels between the squamous cell carcinoma group and the NC transfection group.Compared to squamous cell caecinoma group，the levels of LRG-1，SPHK1，survivin and Bcl-2 were decreased in the transfection group，lRG-1 knockout group and SPHK1 transfection group，while the levels of caspase-3 and p53 were increased.Conclusion：miR-193a-3p may inhibit the proliferation and invasion of oral squamous cell carcinoma cells by decreasing the expression of SPHK1 and LRG-1.Key words miR-193a-3p；leucine rich alpha-2-glycoprotein-1；sphingosine kinase 1；sphingosine kinase 1；oral squamous cell carcinoma；proliferation；invasion口腔鳞状细胞癌占头颈部鳞状细胞癌的50%左右1。其中，舌癌在临床上最为常见，约占50%2。口腔鳞癌的转移能力较强，可通过淋巴等浸润周围组织，还可造成口腔功能异常，影响患者生活质量、危害患者生命安全3-5。有关研究表明，microRNAs和蛋白因子对癌细胞癌具有一定的治疗作用，microRNAs具有调控基因水平的能力，机体内有近半数的基因受microRNAs调控，且其还贯穿肿瘤产生及发展的全过程，并可影响癌细胞的生物学行为。miR-193a-3p是microRNAs的一种，为抑癌基因，且已有研究证实其在食管癌等多种肿瘤中为低表达6-7。富亮氨酸2糖蛋白1（LRG-1）属于富亮氨酸重复蛋白（LRR）类，有关研究表明，LRG-1在舌癌等多种癌症中均呈高表达，且其对癌细胞的生物学行为具有一定调控作用8。鞘氨醇激酶1（SPHK1）具有维持鞘脂代谢平衡的作用，对肿瘤的生物学行为也具有一定的影响9。因此本研究拟转染miR-193a-3p模拟物通过LRG-1/SPHK1信号通路介导对口腔鳞状细胞癌的作用，并探讨机制。1 材料和方法1.1 一般资料1.1 一般资料人口腔鳞癌细胞HN4来自上海泽叶生物公司，于实验室保存。选取本院2018年1月 2020年1月20例口腔鳞癌患者的癌组织（已确诊）与癌旁组织标本进行研究，分为口腔鳞癌组与癌旁组，每组10例，男女各半，年龄（49.375.22）岁，所有病例术前均未进行放疗、化疗以及其他辅助治疗。患者本人同意参加本次实验。1.2 实验试剂1.2 实验试剂胰蛋白酶（河南瑞仁公司）；链霉素（武汉罗森杰公司）；CCK8溶液（青岛达斐公司）；酶标仪（山东霍尔德公司）；基质胶（杭州仟诺公司）；酶联免疫试剂盒（上海劲马公司）；青霉素（湖南泰仁公司）。1.3 细胞的分离与培养1.3 细胞的分离与培养将HN4细胞用含1105 U/L青霉素、0.1 g/L链霉素、5 mol/L PTX 培养液常规培养，细胞融合 至80%90%时胰蛋白酶消化，离心1 250 r/min，共5 min，重悬，传代，取第4代细胞进行实验。在6孔板中接种即将要转染的细胞，待细胞长至65%融合时，采用LipofectamineTM 2000转染50 nmol/L相应的microRNA mimics以及阴性对照miR-193a-3p-NC，继续培养3 d。实验分为3组，鳞癌组（口腔鳞癌细胞）、转染NC组、转染组（转染miR-193a-3p）。1.4 PT-PCR检测miR-193a-3p水平1.4 PT-PCR检