睑板腺癌差异表达miRNA筛选及miR-3907功能机制研究

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

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。

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.