[Screening of differently expressed miRNAs and the mechanism of miR-3907 in meibomian gland carcinoma].

Objective: To screen the differently expressed microRNAs (miRNAs) and to explore the effect and mechanism of microRNA-3907 (miR-3907) in meibomian gland carcinoma (MGC). Methods: 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 t test, two-factor repeated measure analysis of variance. Results: 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 (P=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 (F=3.70, 2.65; both P<0.01), and the migration rate at 24 h was significantly higher (54.6%±3.4% vs. 34.2%±0.6%; t=8.34, P<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 (F=3.10, 2.17, 3.09; all P<0.05), and the migration rate at 24 h was significantly lower (40.8%±2.8% vs. 69.7%±2.7%; t=10.42, P<0.01). Compared with the negative control group, the THBS1 knock-down group promoted cell proliferation at 24 h, 48 h and 72 h (F=3.84, 3.79, 2.24; all P<0.05), and the migration rate at 24 h was significantly increased (82.5%±1.9% vs. 37.6%±5.1%; t=11.74, P<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 (F=3.97, 3.31; both P<0.05), and the migration healing rate at 24 h was significantly increased (56.9%±2.2% vs. 41.9%±4.3%; t=3.53, P<0.05). Conclusions: 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.目的： 筛选睑板腺癌（MGC）差异表达微小RNA（miRNA）并探究微小RNA-3907（miR-3907）对MGC增殖和迁移的影响及作用机制。 方法： 实验研究。收集2011年7月至2019年1月于天津医科大学眼科医院经组织病理学确诊的MGC患者的癌组织样本及癌旁组织样本。应用miRNA芯片对其中5份MGC与癌旁组织差异表达的miRNA进行筛选。选取MGC中表达显著上调的miR-3907为观察对象，通过生物数据库和双荧光素酶实验预测并鉴定miR-3907靶基因。采用免疫组织化学染色法测定18份MGC组织及6份癌旁组织样本中靶基因的蛋白表达水平。在培养的MGC原代细胞中分别进行miR-3907过表达、miR-3907敲减、靶基因敲减、miR-3907敲减+靶基因敲减转染并分别设组，采用荧光定量PCR和Western印迹法检测转染后各基因mRNA和蛋白的表达水平，采用细胞计数试剂盒（CCK-8）和划痕实验评估转染后MGC细胞的增殖和迁移能力，并进行比较。统计学方法主要为Fisher确切概率检验、独立样本t检验、双因素重复测量方差分析。 结果： 与癌旁组织样本相比，MGC样本共筛选出表达上调的miRNA 22个，表达下调的miRNA 5个，其中miR-3907表达显著上调。生信数据库与双荧光素酶报告显示血小板凝血蛋白酶1（THBS1）为miR-3907的下游靶基因。癌旁组织中THBS1蛋白阳性表达率（6/6）显著高于MGC组织（5/18），差异有统计学意义（P=0.003）。与对照组比较，miR-3907过表达组48、72 h细胞增殖能力显著增强（F=3.70、2.65）；24 h后细胞迁移率显著增高（54.6%±3.4%与34.2%±0.6%比较；t=8.34）；miR-3907敲减组24、48、72 h细胞增殖能力降低（F=3.10、2.17、3.09）、24 h细胞迁移率显著降低（40.8%±2.8%与69.7%±2.7%比较；t=10.42）；THBS1敲减组24、48和72 h细胞增殖能力增强（F=3.84、3.79、2.24）、24 h后细胞迁移率显著增加（82.5%±1.9%与37.6%±5.1%比较；t=11.74）；miR-3907敲减+THBS1敲减组24、48 h细胞增殖能力增强（F=3.97、3.31）、24 h细胞迁移率显著增高（56.9%±2.2%与41.9%±4.3%比较；t=3.53）；差异均有统计学意义（均P<0.05）。 结论： MGC与癌旁组织之间存在差异表达miRNA，其可能与MGC发生和发展有关；其中差异显著的miR-3907可以促进MGC增殖和迁移，并主要通过抑制THBS1表达发挥作用。.