目的探讨microRNA-78(miR-78)抑制心肌部分梗死模型大鼠心肌纤维化的作用机制。方法采用结扎左前降支建立大鼠心肌梗死模型,分析miR-78在模型大鼠心肌梗死部位的表达,采用miR-78前体慢病毒载体过表达来分析miR-78在大鼠心肌梗死过程中...目的探讨microRNA-78(miR-78)抑制心肌部分梗死模型大鼠心肌纤维化的作用机制。方法采用结扎左前降支建立大鼠心肌梗死模型,分析miR-78在模型大鼠心肌梗死部位的表达,采用miR-78前体慢病毒载体过表达来分析miR-78在大鼠心肌梗死过程中的作用。评价心肌功能,并测定纤维化相关因子Ⅰ胶原(COL1)和ACTA2表达水平。从新生的大鼠中分离出心肌纤维化细胞,进行荧光素酶试验,转染野生型或突变型GATA43’UTR质粒和miR-78,研究GATA4和miR-78表达水平之间的关系。并通过转染GATA4特异性小干扰RNA验证COL1和ACTA2表达水平的变化。结果与假手术组相比,手术组I区和B区的miR-78表达水平均显著降低(P<0.05),术后2周的表达水平降低最明显,术后4周表达水平略有升高。实时荧光定量聚合酶链式反应(RT-PCR)结果显示,premiR-78组的miR-78水平明显高于转染对照组(P<0.05);转染对照组梗死面积约为50%,但miR-78过表达后,梗死面积明显减少至约30%。假手术组左室射血分数(LVEF)约为60%,术后降至约20%;左室缩短率(LVFS)由手术前的30%左右减少到手术后的15%左右,miR-78表达水平明显恢复。心肌梗死诱导后COL1 m RNA和ACTA2 m RNA表达水平均明显升高(P<0.05),而miR-78过表达时两者均受到抑制(P<0.05)。当靶序列发生突变(mut-GATA43’UTR)时,miR-78的过表达对荧光素酶活性影响不显著(P>0.05);Si-GATA4组与对照组相比,GATA4 m RNA显著下调表达水平(P<0.05);当GATA4被抑制时,COL1和ACTA2的蛋白水平均被抑制。结论miR-78可抑制心肌部分梗死模型大鼠心肌纤维化,作用机制可能为下调GATA4表达,导致COL1和ACTA2的表达下调。展开更多
BACKGROUND Diabetic retinopathy(DR)is one of the major eye diseases contributing to blindness worldwide.Endoplasmic reticulum(ER)stress in retinal cells is a key factor leading to retinal inflammation and vascular lea...BACKGROUND Diabetic retinopathy(DR)is one of the major eye diseases contributing to blindness worldwide.Endoplasmic reticulum(ER)stress in retinal cells is a key factor leading to retinal inflammation and vascular leakage in DR,but its mechanism is still unclear.AIM To investigate the potential mechanism of LEF1 and related RNAs in DR.METHODS ARPE-19 cells were exposed to high levels of glucose for 24 hours to simulate a diabetic environment.Intraperitoneally injected streptozotocin was used to induce the rat model of DR.The expression levels of genes and related proteins were measured by RT-qPCR and Western blotting;lnc-MGC and miR-495-3p were detected by fluorescent in situ hybridization;CCK-8 and TUNEL assays were used to detect cell viability and apoptosis;enzyme-linked immunosorbent assay was used to detect inflammatory factors;dual-luciferase gene assays were used to verify the targeting relationship;and the retina was observed by HE staining.RESULTS LEF1 and lnc-MGC have binding sites,and lnc-MGC can regulate the miR-495-3p/GRP78 molecular axis.In high glucose-treated cells,inflammation was aggravated,the intracellular reactive oxygen species concentration was increased,cell viability was reduced,apoptosis was increased,the ER response was intensified,and ferroptosis was increased.As an ER molecular chaperone,GRP78 regulates the ER and ferroptosis under the targeting of miR-495-3p,whereas inhibiting LEF1 can further downregulate the expression of lnc-MGC,increase the level of miR-495-3p,and sequentially regulate the level of GRP78 to alleviate the occurrence and development of DR.Animal experiments indicated that the knockdown of LEF1 can affect the lnc-MGC/miR-495-3p/GRP78 signaling axis to restrain the progression of DR.CONCLUSION LEF1 knockdown can regulate the miR-495-3p/GRP78 molecular axis through lnc-MGC,which affects ER stress and restrains the progression of DR and ferroptosis in retinal pigment epithelial cells.展开更多
文摘目的探讨microRNA-78(miR-78)抑制心肌部分梗死模型大鼠心肌纤维化的作用机制。方法采用结扎左前降支建立大鼠心肌梗死模型,分析miR-78在模型大鼠心肌梗死部位的表达,采用miR-78前体慢病毒载体过表达来分析miR-78在大鼠心肌梗死过程中的作用。评价心肌功能,并测定纤维化相关因子Ⅰ胶原(COL1)和ACTA2表达水平。从新生的大鼠中分离出心肌纤维化细胞,进行荧光素酶试验,转染野生型或突变型GATA43’UTR质粒和miR-78,研究GATA4和miR-78表达水平之间的关系。并通过转染GATA4特异性小干扰RNA验证COL1和ACTA2表达水平的变化。结果与假手术组相比,手术组I区和B区的miR-78表达水平均显著降低(P<0.05),术后2周的表达水平降低最明显,术后4周表达水平略有升高。实时荧光定量聚合酶链式反应(RT-PCR)结果显示,premiR-78组的miR-78水平明显高于转染对照组(P<0.05);转染对照组梗死面积约为50%,但miR-78过表达后,梗死面积明显减少至约30%。假手术组左室射血分数(LVEF)约为60%,术后降至约20%;左室缩短率(LVFS)由手术前的30%左右减少到手术后的15%左右,miR-78表达水平明显恢复。心肌梗死诱导后COL1 m RNA和ACTA2 m RNA表达水平均明显升高(P<0.05),而miR-78过表达时两者均受到抑制(P<0.05)。当靶序列发生突变(mut-GATA43’UTR)时,miR-78的过表达对荧光素酶活性影响不显著(P>0.05);Si-GATA4组与对照组相比,GATA4 m RNA显著下调表达水平(P<0.05);当GATA4被抑制时,COL1和ACTA2的蛋白水平均被抑制。结论miR-78可抑制心肌部分梗死模型大鼠心肌纤维化,作用机制可能为下调GATA4表达,导致COL1和ACTA2的表达下调。
基金Supported by Science and Technology Program of Yunnan Provincial Department of Science and Technology-Basic Research Program,No.202301BA070001-025.
文摘BACKGROUND Diabetic retinopathy(DR)is one of the major eye diseases contributing to blindness worldwide.Endoplasmic reticulum(ER)stress in retinal cells is a key factor leading to retinal inflammation and vascular leakage in DR,but its mechanism is still unclear.AIM To investigate the potential mechanism of LEF1 and related RNAs in DR.METHODS ARPE-19 cells were exposed to high levels of glucose for 24 hours to simulate a diabetic environment.Intraperitoneally injected streptozotocin was used to induce the rat model of DR.The expression levels of genes and related proteins were measured by RT-qPCR and Western blotting;lnc-MGC and miR-495-3p were detected by fluorescent in situ hybridization;CCK-8 and TUNEL assays were used to detect cell viability and apoptosis;enzyme-linked immunosorbent assay was used to detect inflammatory factors;dual-luciferase gene assays were used to verify the targeting relationship;and the retina was observed by HE staining.RESULTS LEF1 and lnc-MGC have binding sites,and lnc-MGC can regulate the miR-495-3p/GRP78 molecular axis.In high glucose-treated cells,inflammation was aggravated,the intracellular reactive oxygen species concentration was increased,cell viability was reduced,apoptosis was increased,the ER response was intensified,and ferroptosis was increased.As an ER molecular chaperone,GRP78 regulates the ER and ferroptosis under the targeting of miR-495-3p,whereas inhibiting LEF1 can further downregulate the expression of lnc-MGC,increase the level of miR-495-3p,and sequentially regulate the level of GRP78 to alleviate the occurrence and development of DR.Animal experiments indicated that the knockdown of LEF1 can affect the lnc-MGC/miR-495-3p/GRP78 signaling axis to restrain the progression of DR.CONCLUSION LEF1 knockdown can regulate the miR-495-3p/GRP78 molecular axis through lnc-MGC,which affects ER stress and restrains the progression of DR and ferroptosis in retinal pigment epithelial cells.
