Zhang C L, Liu X, Jiang M X, Zhu L M, Lin T T, He Y J
Tianjin Medical University Eye Hospital, Eye Institute and School of Optometry, Tianjin Branch of National Clinical Research Center for Ocular Disease, Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin 300384, China.
Zhonghua Yan Ke Za Zhi. 2022 Mar 11;58(3):205-212. doi: 10.3760/cma.j.cn112142-20210630-00313.
To screen the differently expressed microRNAs (miRNAs) and to explore the effect and mechanism of microRNA-3907 (miR-3907) in meibomian gland carcinoma (MGC). Experimental research. MGC tissues and para-carcinoma tissues of patients diagnosed with MGC by histopathology were collected from July 2011 to January 2019 in Tianjin Medical University Eye Hospital. The miRNA microarray analysis of MGC and para-carcinoma tissue samples from 5 patients was performed. miR-3907 with a significant up-regulation was selected as a research object. Bioinformatics predicted and dual-luciferase gene reporter assay verified miR-3907 target genes. The protein expression levels of target genes in 18 MGC tissues and 6 para-carcinoma tissue samples were determined by immunohistochemical staining. miR-3907 over-expression, miR-3907 knock-down, target gene knock-down and miR-3907 knock-down with target gene knock-down were respectively performed in MGC cell. The mRNA and protein expressions were validated by real-time PCR and Western blotting after transfection. The cell proliferation and migration ability was detected by cell counting kit-8 and scratch experiment after transfection. The main statistical methods were Fisher's exact test, independent sample test, two-factor repeated measure analysis of variance. There were 22 differently up-regulated miRNAs and 5 differently down-regulated miRNAs in MGC tissues, of which miR-3907 was significantly up-regulated. Thrombospondin-1 (THBS1) was a target gene of miR-3907 according to bioinformatics and dual-luciferase gene reporter assay. The positive expression rate of THBS1 protein in para-carcinoma tissues (6/6) was significantly higher than that in MGC tissues (5/18), and the difference was statistically significant (=0.003). Compared with the negative control group, the proliferation ability of the miR-3907 over-expression group was increased at 48 h and 72 h (=3.70, 2.65; both <0.01), and the migration rate at 24 h was significantly higher (54.6%±3.4% . 34.2%±0.6%; =8.34, <0.01). Compared with the negative control group, the proliferation ability of the miR-3907 knock-down group was decreased at 24 h, 48 h and 72 h (=3.10, 2.17, 3.09; all <0.05), and the migration rate at 24 h was significantly lower (40.8%±2.8% . 69.7%±2.7%; =10.42, <0.01). Compared with the negative control group, the THBS1 knock-down group promoted cell proliferation at 24 h, 48 h and 72 h (=3.84, 3.79, 2.24; all <0.05), and the migration rate at 24 h was significantly increased (82.5%±1.9% . 37.6%±5.1%; =11.74, <0.01). Compared with the control group, the miR-3907 knock-down with THBS1 knock-down group increased proliferation at 24 h and 48 h (=3.97, 3.31; both <0.05), and the migration healing rate at 24 h was significantly increased (56.9%±2.2% . 41.9%±4.3%; =3.53, <0.05). There are differently expressed miRNAs between MGC and para-carcinoma tissues, which may be related to the occurrence and development of MGC. miR-3907 in MGC tissues has a significant difference from that in para-carcinoma tissues. Moreover, miR-3907 can play a role in promoting proliferation and migration of MGC by inhibiting the expression of THBS1.
筛选差异表达的微小RNA（miRNA），并探讨微小RNA - 3907（miR - 3907）在睑板腺癌（MGC）中的作用及机制。实验研究。收集2011年7月至2019年1月在天津医科大学眼科医院经组织病理学诊断为MGC患者的MGC组织及癌旁组织。对5例患者的MGC及癌旁组织样本进行miRNA芯片分析。选择上调显著的miR - 3907作为研究对象。通过生物信息学预测并经双荧光素酶基因报告基因检测验证miR - 3907的靶基因。采用免疫组织化学染色法检测18例MGC组织和6例癌旁组织样本中靶基因的蛋白表达水平。在MGC细胞中分别进行miR - 3907过表达、miR - 3907敲低、靶基因敲低以及miR - 3907敲低联合靶基因敲低。转染后通过实时荧光定量PCR和蛋白质免疫印迹法验证mRNA和蛋白表达。转染后通过细胞计数试剂盒 - 8和划痕实验检测细胞增殖和迁移能力。主要统计方法为Fisher确切概率法、独立样本t检验、两因素重复测量方差分析。MGC组织中有22个上调差异显著的miRNA和5个下调差异显著的miRNA，其中miR - 3907上调显著。根据生物信息学和双荧光素酶基因报告基因检测，血小板反应蛋白1（THBS1）是miR - 3907的靶基因。THBS1蛋白在癌旁组织中的阳性表达率（6/6）显著高于MGC组织（5/18），差异有统计学意义（P = 0.003）。与阴性对照组相比，miR - 3907过表达组在48 h和72 h时增殖能力增强（P = 3.70，2.65；均<0.01），24 h时迁移率显著升高（54.6%±3.4%对34.2%±0.6%；P = 8.34，<0.01）。与阴性对照组相比，miR - 3907敲低组在24 h、48 h和72 h时增殖能力降低（P = 3.10，2.17，3.09；均<0.05），24 h时迁移率显著降低（40.8%±2.8%对69.7%±2.%; P = 10.42，<0.01）。与阴性对照组相比，THBS1敲低组在24 h、48 h和72 h时促进细胞增殖（P = 3.84，3.79，2.24；均<0.05），24 h时迁移率显著升高（82.5%±1.9%对37.6%±5.1%；P = 11.74，<0.01）。与对照组相比，miR - 3907敲低联合THBS1敲低组在24 h和48 h时增殖增加（P = 3.97，3.31；均<0.05），24 h时迁移愈合率显著升高（56.9%±2.2%对41.9%±4.3%；P = 3.53，<0.05）。MGC与癌旁组织之间存在差异表达的miRNA，这可能与MGC的发生发展有关。MGC组织中的miR - 3907与癌旁组织中的miR - 3907有显著差异。此外，miR - 3907可通过抑制THBS1的表达促进MGC的增殖和迁移。
