[1]白　杨,黄　婷,李真真,等.变异性浆细胞瘤异位1基因对高糖培养的大鼠心脏成纤维细胞的影响及其机制[J].新乡医学院学报,2021,38(11):1017-1024.[doi:10.7683/xxyxyxb.2021.11.004]
　BAI Yang,HUANG Ting,LI Zhenzhen,et al.Effect of plasma-cytoma variant translocation 1 gene on cardiac fibroblasts of rats cultured by high glucose and its mechanism[J].Journal of Xinxiang Medical University,2021,38(11):1017-1024.[doi:10.7683/xxyxyxb.2021.11.004]
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变异性浆细胞瘤异位1基因对高糖培养的大鼠心脏成纤维细胞的影响及其机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
38
期数:
2021年11
页码:
1017-1024
栏目:
基础研究
出版日期:
2021-11-05

文章信息/Info

Title:
Effect of plasma-cytoma variant translocation 1 gene on cardiac fibroblasts of rats cultured by high glucose and its mechanism
作者:
白　杨1; 黄　婷1; 李真真1; 王　琰2
（1.郑州市第七人民医院内分泌科，河南　郑州　450016；2.郑州市第七人民医院心内科，河南　郑州　450016）
Author(s):
BAI Yang1; HUANG Ting1; LI Zhenzhen1; WANG Yan2
(1.Department of Endocrinology,the 7th People′s Hospital of Zhengzhou,Zhengzhou 450016,Henan Province,China2.Department of Cardiology,the 7th People′s Hospital of Zhengzhou,Zhengzhou 450016,Henan Province,China)
关键词:
心脏成纤维细胞; 高糖; 变异性浆细胞瘤异位1基因; 微RNA-93-5p; 转化生长因子-β1
Keywords:
cardiac fibroblasthigh glucoseplasma-cytoma variant translocation 1 genemicroRNA-93-5ptransforming growth factor-β1
分类号:
R541
DOI:
10.7683/xxyxyxb.2021.11.004
文献标志码:
A
摘要:
目的　探讨变异性浆细胞瘤异位1（PVT1）基因对高糖培养的大鼠心脏成纤维细胞（CF）的影响及其作用机制。方法　取对数生长期CF并随机分为空白对照组、高糖组、PVT1 小干扰RNA（siRNA）组、PVT1 siRNA阴性对照（NC）组（PVT1 siRNA NC组）。空白对照组和高糖组细胞不进行转染，PVT1 siRNA组及PVT1 siRNA NC组细胞分别转染PVT1 siRNA及PVT1 siRNA NC试剂；转染24 h后，高糖组、PVT1 siRNA组、PVT1 siRNA NC组细胞用含25 mmol·L-1葡萄糖的培养基培养，模拟高糖培养条件，空白对照组细胞不作处理。采用天狼猩红染色法观察高糖处理24 h后4组细胞胶原纤维沉积情况。流式细胞术检测高糖处理24 h后4组细胞细胞周期分布情况。采用实时荧光定量聚合酶链反应法检测高糖处理24 h后4组细胞中PVT1、转化生长因子-β1（TGF-β1）mRNA及微RNA（miR）-93-5p的表达。采用Western blot法检测高糖处理24 h后4组细胞中TGF-β1、Smad2/3、磷酸化Smad2/3（p-Smad2/3）、Smad7、p21、α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（Col Ⅰ）表达情况。另取对数生长期CF，随机分为未转染组、PVT1 siRNA+miR-93-5p inhibitor组、PVT1 siRNA+miR-93-5p NC组、PVT1 NC+miR-93-5p inhibitor组、PVT1 NC+miR-93-5p NC组；未转染组细胞不进行转染，PVT1 siRNA+miR-93-5p inhibitor组细胞转染PVT1 siRNA和miR-93-5p inhibitor，PVT1 siRNA+miR-93-5p NC组细胞转染PVT1 siRNA和miR-93-5p NC，PVT1 NC+miR-93-5p inhibitor组细胞转染PVT1 NC和miR-93-5p inhibitor，PVT1 NC+miR-93-5p NC组细胞转染PVT1 NC和miR-93-5p NC，转染24 h后，用含25 mmol·L-1葡萄糖的培养基处理5组细胞，模拟高糖培养条件。采用Western blot法检测高糖处理24 h后5组细胞中TGF-β1、Smad7、Col Ⅰ蛋白表达表达情况。 结果　与空白对照组比较，高糖组细胞数量增多，红色胶原纤维沉积增加；与高糖组比较，PVT1 siRNA组细胞间隙变大，红色胶原纤维沉积减小；PVT1 siRNA NC组细胞红色胶原纤维沉积与高糖组相近。高糖组、PVT1 siRNA组、PVT1 siRNA NC组G0+G1期细胞比例显著低于空白对照组，S+G2+M期细胞比例显著高于空白对照组（P<0.05）；PVT1 siRNA组G0+G1期细胞比例显著高于高糖组，S+G2+M期细胞比例显著低于高糖组（P<0.05）；PVT1 siRNA NC组G0+G1期细胞比例显著低于PVT1 siRNA组，S+G2+M期细胞比例显著高于PVT1 siRNA组（P<0.05）；高糖组和PVT1 siRNA NC组G0+G1期细胞比例、S+G2+M期细胞比例比较差异无统计学意义（P>0.05）。高糖组、PVT1 siRNA组、PVT1 siRNA NC组细胞中PVT1、TGF-β1 mRNA相对表达量均显著高于空白对照组，miR-93-5p相对表达量显著低于空白对照组（P<0.05）；PVT1 siRNA组细胞中PVT1、TGF-β1 mRNA相对表达量显著低于高糖组，miR-93-5p相对表达量显著高于高糖组（P<0.05）；PVT1 siRNA NC组细胞中PVT1、TGF-β1 mRNA相对表达量均显著高于PVT1 siRNA组，miR-93-5p相对表达量显著低于PVT1 siRNA组（P<0.05）；高糖组和PVT1 siRNA NC组细胞中PVT1、TGF-β1 mRNA及miR-93-5p相对表达量比较差异无统计学意义（P>0.05）。高糖组、PVT1 siRNA组、PVT1 siRNA NC组细胞中α-SMA、Col Ⅰ、TGF-β1蛋白相对表达量及（p-Smad2/3）/（Smad2/3）显著高于空白对照组，p21、Smad7蛋白相对表达量显著低于空白对照组（P<0.05）；PVT1 siRNA组细胞中α-SMA、Col Ⅰ、TGF-β1蛋白相对表达量及（p-Smad2/3）/（Smad2/3）显著低于高糖组，p21、Smad7蛋白相对表达量显著高于高糖组（P<0.05）；PVT1 siRNA NC组细胞中α-SMA、Col Ⅰ、TGF-β1蛋白相对表达量及（p-Smad2/3）/(Smad2/3）显著高于PVT1 siRNA组，p21、Smad7蛋白相对表达量显著低于PVT1 siRNA组（P<0.05）；高糖组和PVT1 siRNA NC组细胞中α-SMA、Col Ⅰ、p21、TGF-β1、Smad7蛋白相对表达量及(p-Smad2/3)/(Smad2/3)比较差异无统计学意义（P>0.05）。PVT1 siRNA +miR-93-5p NC组细胞中TGF-β1、Col Ⅰ蛋白相对表达量显著低于未转染组，Smad7蛋白相对表达量显著高于未转染组（P<0.05）；PVT1 NC+miR-93-5p inhibitor组细胞中TGF-β1、Col Ⅰ蛋白相对表达量显著高于未转染组，Smad7蛋白相对表达量显著低于未转染组；未转染组细胞中TGF-β1、Smad7、Col Ⅰ蛋白相对表达量与PVT1 NC+miR-93-5p NC组、PVT1 siRNA+miR-93-5p inhibitor组比较差异无统计学意义（P>0.05）。PVT1 NC+miR-93-5p inhibitor组、PVT1 siRNA+miR-93-5p inhibitor组、PVT1 NC+miR-93-5p NC组细胞中TGF-β1、ColⅠ蛋白相对表达量显著高于PVT1 siRNA +miR-93-5p NC组，Smad7蛋白相对表达量显著低于PVT1 siRNA +miR-93-5p NC组（P<0.05）；PVT1 siRNA+miR-93-5p inhibitor组、PVT1 NC+miR-93-5p NC组细胞中TGF-β1、ColⅠ蛋白相对表达量显著低于PVT1 NC+miR-93-5p inhibitor组，Smad7蛋白相对表达量显著高于PVT1 NC+miR-93-5p inhibitor组（P<0.05）；PVT1 siRNA+miR-93-5p inhibitor组与PVT1 NC+miR-93-5pNC组细胞中TGF-β1、Smad7、Col Ⅰ蛋白相对表达量比较差异无统计学意义（P>0.05）。结论　PVT1基因沉默可促进miR-93-5p/Smad7通路活化，抑制TGF-β1/Smad通路激活，进而抑制高糖培养的CF纤维化进程。
Abstract:
Objective　To explore the effect of plasma-cytoma variant translocation 1 (PVT1) gene on rat cardiac fibroblast (CF) cultured by high glucose and its mechanism.Methods　The CFs in logarithmic growth phase were taken and randomly divided into blank control group,high glucose group,PVT1 small interfering RNA(siRNA) group and PVT1 siRNA negative control(NC) group (PVT1 siRNA NC group).The cells in the blank control group and high glucose group were not transfected,the cells in the PVT1 siRNA group and PVT1 siRNA NC group were transfected with PVT1 siRNA and NC reagents,respectivelyafter transfection for 24 h,the cells in the high glucose group,PVT1 siRNA group and NC group were cultured with a medium containing 25 mmol·L-1 glucose to simulate high-glucose culture conditions,the cells in the blank control group were not treated.After transfection for 24 h,cells in the five groups were treated with a medium containing 25 mmol·L-1 glucose to simulate high-glucose culture conditions.The collagenous fiber deposition of cells treated with high-glucose for 24 h in the 4 groups was observed by the sirius scarlet staining method.The cell cycle distribution of cells in the four groups after 24 h of high glucose treatment was detected by flow cytometry.The expressions of PVT1,transforming growth factor-β1 (TGF-β1) mRNA and microRNA(miRNA)-93-5p of cells in the four groups after 24 h of high glucose treatment were detected by real-time fluorescent quantitative polymerase chain reaction method.The expressions of TGF-β1,Smad2/3,phosphorylation (p-Smad2/3),Smad7,p21,α-smooth muscle actin (α-SMA),type Ⅰcollagenous (Col Ⅰ) of cells in the four groups after 24 h of high glucose treatment were detected by Western blot.In addition,the CFs in the logarithmic growth phase were taken and randomly divided into untransfected group,PVT1 siRNA+miR-93-5p inhibitor group,NC group,PVT1 NC+miR-93-5p inhibitor group,NC group.The cells in the untransfected group were not transfected,the cells in the PVT1 siRNA+miR-93-5p inhibitor group were transfected with PVT1 siRNA and miR-93-5p inhibitor,the cells in the NC group were transfected with PVT1 siRNA and miR-93-5p NC,the cells in the PVT1 NC+miR-93-5p inhibitor group were transfected with PVT1 NC and miR-93-5p inhibitor,the cells in the NC group were transfected with PVT1 NC and miR-93-5p NC.The expressions of TGF-β1,Smad7,Col Ⅰ protein of cells in the five groups after 24 h of high glucose treatment were detected by Western blot.Results　Compared with the blank control group,the number of cells in the high glucose group was increased,and the deposition of red collagenous fibers was increasedcompared with the high glucose group,the intercellular spaces of cells in the PVT1 siRNA group became larger and the deposition of red collagenous fibers were decreasedthe deposition of red collagenous fibers of cells in the PVT1 siRNA NC group was similar to the high glucose group.The proportion of cells in the G0+G1 phase in the high glucose group,PVT1 siRNA group and PVT1 siRNA NC group was significantly lower than that in the blank control group,and the proportion of cells in the S+G2+M phase was significantly higher than that in the blank control group (P<0.05)the proportion of cells in the G0+G1 phase in the PVT1 siRNA group was significantly higher than that in the high glucose group,and the proportion of cells in the S+G2+M phase was significantly lower than that in the high glucose group (P<0.05)the proportion of cells in the G0+G1 phase in the PVT1 siRNA NC group was significantly lower than that in the PVT1 siRNA group,and the proportion of cells in the S+G2+M phase was significantly higher than that in the PVT1 siRNA group (P<0.05).The relative expression levels of PVT1 and TGF-β1 mRNA in the high glucose group,PVT1 siRNA group and PVT1 siRNA NC group were significantly higher than those in the blank control group,and the relative expression level of miR-93-5p was significantly lower than that in the blank control group (P<0.05)the relative expression levels of PVT1 and TGF-β1 mRNA in the PVT1 siRNA group were significantly lower than those in the high glucose group,and the relative expression level of miR-93-5p was significantly higher than that in the high glucose group (P<0.05)the relative expression levels of PVT1 and TGF-β1 mRNA in the PVT1 siRNA NC group were significantly higher than those in the PVT1 siRNA group,and the relative expression level of miR-93-5p was significantly lower than that in the PVT1 siRNA group (P<0.05).The relative expression levels of α-SMA,Col Ⅰ,TGF-β1 protein and (p-Smad2/3)/(Smad2/3) in the high glucose group,PVT1 siRNA group and PVT1 siRNA NC group were significantly higher than those in the blank control group,while the relative expression levels of p21 and Smad7 proteins were significantly lower than those in the blank control group (P<0.05)the relative expression levels of α-SMA,Col Ⅰ,TGF-β1 protein and (p-Smad2/3)/(Smad2/3) in the PVT1 siRNA group were significantly lower than those in the high glucose group,and the relative expression levels of p21 and Smad7 protein were significantly higher than those in the high glucose group (P<0.05)the relative expression levels of α-SMA,Col Ⅰ,TGF-β1 protein and (p-Smad2/3)/(Smad2/3) in the PVT1 siRNA NC group were significantly higher than those in the PVT1 siRNA group,and the relative expression levels of p21 and Smad7 protein were significantly lower than those in the PVT1 siRNA group (P<0.05).The relative expression levels of TGF-β1 and Col Ⅰ protein in the PVT1 siRNA+miR-93-5p NC group were significantly lower than those in the untransfected group,and the relative expression level of Smad7 protein was significantly higher than that in the untransfected group (P<0.05)the relative expression levels of TGF-β1 and Col Ⅰ protein in the PVT1 NC+miR-93-5p inhibitor group were significantly higher than those in the untransfected group,and the relative expression level of Smad7 protein was significantly lower than that in the untransfected group(P<0.05).The relative expression levels of TGF-β1 and Col Ⅰ protein in the PVT1 NC+miR-93-5p inhibitor group,PVT1 siRNA+miR-93-5p inhibitor group,PVT1 NC+miR-93-5p NC group were significantly higher than those in the PVT1 siRNA+miR-93-5p NC group,and the relative expression level of Smad7 protein was significantly lower than that in the PVT1 siRNA+miR-93-5p NC group (P<0.05)the relative expression levels of TGF-β1 and ColⅠ protein in PVT1 NC+miR-93-5p inhibitor group,PVT1 siRNA+miR-93-5p inhibitor group,PVT1 NC+miR-93-5p NC group were significantly higher than those in the PVT1 siRNA+miR-93-5p NC group,and the relative expression level of Smad7 protein was significantly lower than that in the PVT1 siRNA+miR-93-5p NC group (P<0.05).Conclusion　The PVT1 gene silencing can promote the activation of miR-93-5p/Smad7 pathway and inhibit the activation of TGF-β1/Smad pathway,thereby inhibiting the fibrosis process of CF cultured by high glucose.

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更新日期/Last Update: 2021-11-05