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Abstract：
BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective.
METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRT‒PCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male Sprague‒Dawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRT‒PCR, Western blot analysis, and immunohistochemical staining.
RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3\'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups.
CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.
METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRT‒PCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male Sprague‒Dawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRT‒PCR, Western blot analysis, and immunohistochemical staining.
RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3\'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups.
CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.
摘要：
背景：勃起功能障碍（ED）是一种常见的男性性功能障碍，随着发病率的增加，目前的治疗往往是无效的。
方法：用血管内皮生长因子(VEGFA)治疗骨髓间充质干细胞(BM-MSCs),并通过Transwell测定法测定它们的细胞迁移速率。vonWillebrand因子(vWF)VE-cadherin的表达，通过qRT-PCR和Westernblot分析确定内皮型一氧化氮合酶（eNOS）内皮标志物。通过用si-MALAT1转染VEGFA诱导的BM-MSC并过表达CDC42和PAK1，探索了MALAT1诱导的BM-MC通过CDC42/PAK1/桩蛋白途径向EC分化。通过蛋白质免疫沉淀检查了VEGFA处理和非VEGFA处理的BM-MSC中CDC42，PAK1和桩蛋白之间的结合能力。MiR-206在VEGFA诱导的BM-MSC中过表达，MALAT1、miR-206和CDC42的结合位点使用荧光素酶测定进行鉴定。将60只雄性SD大鼠分为6组（n=10/组）。通过APO实验证明了DMED建模，并通过测量血糖水平进行了评估。通过测量海绵体内压（ICP）和平均动脉压（MAP）评估勃起功能。通过qRT-PCR分析阴茎勃起组织，蛋白质印迹分析,和免疫组织化学染色。
结果：VEGFA处理条件下的MALAT1通过调节CDC42/PAK1/桩蛋白轴来调节BM-MSCs向ECs的分化。体外实验表明，干扰CDC42和MALAT1的表达抑制了BM-MSCs向EC的分化。CDC42与PAK1结合,PAK1与桩蛋白结合。此外,VEGFA组中的CDC42具有更大的与PAK1结合的能力,而VEGFA组中的PAK1具有更大的与桩蛋白结合的能力。miR-206在VEGFA诱导的BM-MSC中的过表达表明MALAT1与CDC423'-UTR竞争结合miR-206，进而参与BM-MSC向EC的分化。与DMED模型组相比，3个BM-MSCs治疗组的ICP/MAP比值显著增高.
结论：MALAT1通过调节miR-206/CDC42/PAK1/桩蛋白轴来促进BM-MSC分化为ECs，从而改善ED。本发现揭示了MALAT1在修复BM-MSCs勃起功能中的重要作用，并为BM-MSC介导的DMED修复提供了新的机制见解。
方法：用血管内皮生长因子(VEGFA)治疗骨髓间充质干细胞(BM-MSCs),并通过Transwell测定法测定它们的细胞迁移速率。vonWillebrand因子(vWF)VE-cadherin的表达，通过qRT-PCR和Westernblot分析确定内皮型一氧化氮合酶（eNOS）内皮标志物。通过用si-MALAT1转染VEGFA诱导的BM-MSC并过表达CDC42和PAK1，探索了MALAT1诱导的BM-MC通过CDC42/PAK1/桩蛋白途径向EC分化。通过蛋白质免疫沉淀检查了VEGFA处理和非VEGFA处理的BM-MSC中CDC42，PAK1和桩蛋白之间的结合能力。MiR-206在VEGFA诱导的BM-MSC中过表达，MALAT1、miR-206和CDC42的结合位点使用荧光素酶测定进行鉴定。将60只雄性SD大鼠分为6组（n=10/组）。通过APO实验证明了DMED建模，并通过测量血糖水平进行了评估。通过测量海绵体内压（ICP）和平均动脉压（MAP）评估勃起功能。通过qRT-PCR分析阴茎勃起组织，蛋白质印迹分析,和免疫组织化学染色。
结果：VEGFA处理条件下的MALAT1通过调节CDC42/PAK1/桩蛋白轴来调节BM-MSCs向ECs的分化。体外实验表明，干扰CDC42和MALAT1的表达抑制了BM-MSCs向EC的分化。CDC42与PAK1结合,PAK1与桩蛋白结合。此外,VEGFA组中的CDC42具有更大的与PAK1结合的能力,而VEGFA组中的PAK1具有更大的与桩蛋白结合的能力。miR-206在VEGFA诱导的BM-MSC中的过表达表明MALAT1与CDC423'-UTR竞争结合miR-206，进而参与BM-MSC向EC的分化。与DMED模型组相比，3个BM-MSCs治疗组的ICP/MAP比值显著增高.
结论：MALAT1通过调节miR-206/CDC42/PAK1/桩蛋白轴来促进BM-MSC分化为ECs，从而改善ED。本发现揭示了MALAT1在修复BM-MSCs勃起功能中的重要作用，并为BM-MSC介导的DMED修复提供了新的机制见解。
