老年性白内障患者circKMT2E表达变化及其对晶状体上皮细胞凋亡的影响

目的　探讨circKMT2E在老年性白内障（ARC）患者中的表达变化及其对晶状体上皮细胞凋亡的影响。方法　取10例在我院接受白内障手术的ARC患者晶状体前囊膜，另选取4例健康捐献者透明晶状体前囊膜作为对照。使用Trizol试剂从人晶状体前囊膜组织中提取总RNA。依次使用cutadapt软件、DCC软件(v0.4.4)、STAR软件 (v2.51 b)、CircBase数据库和circ2Trait数据库对所测得的环状RNA（circRNA）进行注释,筛选circRNA差异表达。将体外培养的SRA01-04细胞随机分组，其中，空白对照组:用正常培养液培养，不作特殊处理；miR-control组:转染miR-control无序序列；miR-34a组:转染miR-34a-5p mimics序列；miR-34a+control组:共转染miR-34a-5p mimics序列和pcDNA3.1-control空载体；miR-34a+HMGB1组:共转染miR-34a-5p mimics序列和pcDNA3.1-HMGB1质粒。采用实时荧光定量PCR与双荧光素酶报告基因检测基因表达情况。 通过流式细胞仪检测细胞凋亡率，Western blot检测蛋白表达。结果　经生物信息学分析显示，circKMT2E在ARC患者晶状体前囊膜组织中表达上调最为显著。双荧光素酶报告基因检测结果显示，转染miR-34a-5p后circKMT2E-WT报告基因的荧光素酶活性由1.000±0.023降低至0.502±0.057（P<0.05）；而circKMT2E-MUT报告基因的荧光素酶活性由1.005±0.018降低至0.989±0.053（P>0.05）。miR-34a组SRA01-04细胞凋亡率（23.59%±3.47%）较miR-control组（3.82%±0.41%）增高（P<0.05），而miR-34a-HMGB1组SRA01-04细胞凋亡率（8.97%±1.20%）较miR-34a-control组（24.59%±4.33 %）降低（P<0.05）。miR-34a组SRA01-04细胞Cyt-C蛋白、Bax蛋白相对表达量较miR-control组均升高，同时Bcl-2蛋白相对表达量则降低（均为P<0.05）；miR-34a-HMGB1组SRA01-04细胞Cyt-C蛋白和Bax蛋白相对表达量较miR-34a-control组均降低，同时Bcl-2蛋白相对表达量较miR-34a-control组增加（均为P<0.05）。结论　circKMT2E可通过海绵吸附miR-34a-5p减弱其对下游靶点HMGB1的抑制作用，进而有效地阻止SRA01-04细胞凋亡，在一定程度起到延缓ARC发展的作用。

Change in the expression level of circKMT2E in patients with age-related cataract and its effect on the apoptosis of lens epithelial cells

Objective To investigate the change in the expression level of circKMT2E in patients with age-related cataract(ARC)and its effect on the apoptosis of lens epithelial cells.Methods Anterior lens capsules were derived from 10 ARC patients who received cataract surgery in our hospital,and those from 4 healthy donors were used as controls.Total RNA was extracted from the anterior lens capsules using Trizol reagent.Then the cutadapt software,DCC software(V0.4.4),STAR software(V2.51 b),CircBase database,and circ2Trait database were sequentially used to annotate the detected circular RNAs(circRNAs)and screen out differences in the expression of circRNAs.Human lens epithelial SRA01-04 cells cultured in vitro were divided into the blank control group(cultured with normal medium and without any treatment),miR-control group(transfected with miR-control),miR-34a group(transfected with miR-34a-5p mimics),miR-34a+control group(co-transfected with miR-34a-5p mimics and pcDNA3.1-control vector),and miR-34a+HMGB1 group(co-transfected with miR-34a-5p mimics and pcDNA3.1-HMGB1 plasmid).Real-time quantitative polymerase chain reaction and dual-luciferase reporter gene assay were used to measure gene expression.Cell apoptosis rates were measured by flow cytometry,and protein expression levels were detected by Western blot.Results According to bioinformatics analysis,the expression of circKMT2E was most significantly elevated in the anterior lens capsule of ARC patients.Dual-luciferase reporter gene assay results showed that the luciferase activity of the circKMT2E-WT reporter gene decreased from 1.000±0.023 to 0.502±0.057 after miR-34a-5p transfection(P<0.05),while that of the circKMT2E-MUT reporter gene decreased from 1.005±0.018 to 0.989±0.053(P>0.05).The apoptosis rate of SRA01-04 cells in the miR-34a group(23.59%±3.47%)was significantly higher than that in the miR-control group(3.82%±0.41%)(P<0.05),while the apoptosis rate of SRA01-04 cells in the miR-34a+HMGB1 group(8.97%±1.20%)was significantly lower than that in the miR-34a-control group(24.59%±4.33%)(P<0.05).The relative expression levels of cytochrome C(Cyt-C)and Bax proteins in SRA01-04 cells in the miR-34a group were significantly higher than those in the miR-control group,while the relative expression level of Bcl-2 protein was significantly lower(all P<0.05).The relative expression levels of Cyt-C and Bax proteins in SRA01-04 cells in the miR-34a-HMGB1 group were lower than those in the miR-34a-control group,while the relative expression level of Bcl-2 protein was higher(all P<0.05).Conclusion circKMT2E can alleviate the inhibitory effect of miR-34a-5p on its downstream target HMGB1 via sponge adsorption,and in turn effectively prevent the apoptosis of SRA01-04 cells,to some extent delaying the development of ARC.