目的探讨miR-497对糖尿病小鼠角膜上皮愈合的抑制作用及其可能机制。
方法取40只健康清洁级野生型C57BL/J6小鼠，采用随机数字表法平均分为空白对照组和模型对照组，另取CRISPR/Cas9介导的miR-497敲除小鼠和miR-497过表达小鼠各20只，分别作为miR-497敲除组和miR-497过表达组。对模型对照组、miR-497敲除组、miR-497过表达组小鼠连续腹腔注射链脲佐菌素(STZ)构建糖尿病模型，空白对照组小鼠注射等量枸橼酸钠缓冲液，正常饲养8周。糖尿病模型建立成功后通过刮除角膜中央直径2 mm上皮，进一步构建角膜上皮损伤模型。采用角膜荧光素钠染色观察角膜上皮损伤后0、12、24和36 h各组小鼠角膜上皮缺损面积。采用Western blot法检测各组小鼠角膜组织中wnt3a、β-catenin蛋白表达；采用实时荧光定量PCR法检测各组小鼠角膜组织miR-497以及细胞增生相关基因CyclinD1、c-Myc、Ki-67 mRNA水平表达。采用双荧光素酶报告基因实验检测miR-497与wnt3a的靶向性关系。体外培养人角膜上皮细胞(HCEC)，通过Lipo8000分别转染miR-497 mimics、miR-497 mimics阴性对照、miR-497 inhibitor、miR-497 inhibitor阴性对照，作为miR-497 mimics组、mimics阴性对照组、miR-497 inhibitor组、inhibitor阴性对照组，并在含25%葡萄糖的高糖培养基中培养；另取2个组HCEC分别置于含5%及25%葡萄糖的培养基进行培养，作为正常对照组及高糖组。采用CCK8检测各组细胞增生活力。
结果注射STZ后8周，各糖尿病模型组小鼠血糖浓度明显高于空白对照组，体质量明显低于空白对照组，差异均有统计学意义(均P<0.05)。模型对照组损伤角膜上皮后12、24和36 h角膜上皮缺损面积百分比明显高于相应时间点空白对照组和miR-497敲除组，低于miR-497过表达组，差异均有统计学意义(均P<0.05)。模型对照组角膜组织中wnt3a、β-catenin蛋白相对表达量明显低于空白对照组和miR-497敲除组，高于miR-497过表达组，差异均有统计学意义(均P<0.05)。模型对照组CyclinD1、c-Myc和Ki-67 mRNA相对表达量均低于miR-497敲除组，高于miR-497过表达组，差异均有统计学意义(均P<0.05)。模型对照组、miR-497敲除组和miR-497过表达组miR-497相对表达量分别为1.00±0.02、0.63±0.06和1.48±0.03，总体比较差异均有统计学意义(F＝19.62，P<0.01)。野生型Wnt3a转染细胞中miR-497-5p mimics组荧光素酶活性低于miR-497-5p阴性对照组和空载体组，差异均有统计学意义(均P<0.05)；突变型wnt3a转染细胞中，各组荧光素酶活性比较差异无统计学意义(F＝0.73，P＝0.59)。高糖组细胞增生A值为0.59±0.03，明显低于正常对照组的0.59±0.03和miR-497 inhibitor组的0.88±0.08，明显高于miR-497 mimics组的0.48±0.11，差异均有统计学意义(均P<0.05)。
结论沉默miR-497表达可能通过靶向激活wnt/β-catenin通路促进糖尿病小鼠角膜上皮缺损修复。
ObjectiveTo investigate the inhibitory effect of miR-497 on the corneal epithelial healing in diabetic mice and its possible mechanism.
MethodsForty healthy clean-grade wild-type C57BL/J6 mice were randomly divided into a blank control group and a model control group, with 20 mice in each group.Another 20 CRISPR/Cas9-mediated miR-497 knockout mice and miR-497 overexpression mice were taken as miR-497 knockout and miR-497 overexpression groups, respectively.The diabetes model was constructed by continuous intraperitoneal injection of streptozotocin (STZ) to the mice in model control, miR-497 knockout and miR-497 overexpression groups, and the mice in blank control group were injected with an equal amount of citrate buffer, followed by 8-week normal feeding.After the establishment of diabetes model, the corneal epithelial injury model was further constructed by scraping off part of the corneal epithelium with a central diameter of 2 mm.The corneal epithelial defect area of mice in 0, 12, 24 and 36 hours after corneal epithelial injury was observed by corneal fluorescein sodium staining.The expression of Wnt3a and β-catenin proteins in mice corneal tissues was detected by Western blot.The expression of miR-497 as well as the mRNA expression levels of cell proliferation-associated factor genes CyclinD1, c-Myc, and Ki-67 mRNA was detected by real-time quantitative fluorescence PCR.The targeting relationship between miR-497 and wnt3a was detected by a dual luciferase reporter gene assay.Human corneal epithelial cells (HCEC) were cultured in vitro and transfected with miR-497 mimics, miR-497 mimics negative control, miR-497 inhibitor, and miR-497 inhibitor negative control by Lipo8000 as miR-497 mimics group, mimics negative control group, miR-497 inhibitor group, andmiR-497 inhibitor negative control group, respectively, all of which were cultured in high glucose medium containing 25% glucose.Another two groups of HCEC were taken and cultured in medium containing 5% and 25% glucose as control and high glucose groups, respectively.The cell proliferation viability was determined by CCK8 method.The use and care of animals complied ith the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (2019K-K010).
ResultsEight weeks after STZ injection, the blood glucose of mice was significantly higher and the weight was significantly lower in each diabetic model group than those of blank control group (all at P<0.05). At 12, 24 and 36 hours after the corneal epithelial injury, the percentages of corneal epithelial defect area observed by slit-lamp microscopy in model control group were significantly higher than those in blank control group and miR-497 knockout group and lower than those in miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expressions of wnt3a and β-catenin proteins in the corneal tissues of model control group were significantly lower than those of blank control group and miR-497 knockout group, but higher than those of miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expressions of CyclinD1, c-Myc and Ki-67 mRNA in model control group were lower than those in miR-497 knockout group, but higher than those in miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expression of miR-497 in model control group, miR-497 knockout group and miR-497 overexpression group was 1.00±0.02, 0.63±0.06 and 1.48±0.03, respectively, with a statistically significant difference (F=19.62, P<0.01). The luciferase activity of miR-497-5p mimics group in wild-type wnt3a transfected cells was lower than that of miR-497-5p negative control group and empty vector group, and the differences were statistically significant (all at P<0.05). In the mutant wnt3a transfected cells, there was no significant difference in the luciferase activity among various groups (F=0.73, P=0.59). The cell proliferation A value of high glucose group was 0.59±0.03, which was significantly lower than 0.59±0.03 of normal control group and 0.88±0.08 of miR-497 inhibitor group, but significantly higher than 0.48±0.11 of miR-497 mimics group (all at P<0.05).
ConclusionsThe silencing of miR-497 may promote the repair of diabetic corneal epithelial defects by targeting wnt/β-catenin pathway.
黄钰清,杨燕宁,王杨,等. miR-497对糖尿病小鼠角膜上皮损伤修复的抑制作用及其靶向wnt3a调控机制[J]. 中华实验眼科杂志,2023,41(09)：856-863.
DOI:10.3760/cma.j.cn115989-20220108-00005版权归中华医学会所有。
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黄钰清：参与选题设计、收集数据、分析和解释数据、论文撰写及修改；杨燕宁：参与资料的分析和解释、对文章知识性内容作批评性审阅；王杨、潘玉苗、程思敏：参与实验设计、论文修改
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