Department of Endocrinology, Caoxian People's Hospital, East Qinghe Road, South Fumin Avenue, Caoxian Development Zone, Heze City 274400, Shandong Province, China.
Department of Medical, First People's Hospital of Jinan City, No. 132, Daminghu Road, Lixia District, Jinan City 250011, Shandong Province, China.
Biosci Rep. 2020 May 29;40(5). doi: 10.1042/BSR20194427.
The present study aimed to investigate the regulatory role of long non-coding RNA plasmacytoma variant translocation 1 (PVT1) on high glucose (HG)-induced mouse mesangial cells (MMCs).
PVT1 expression in diabetic nephropathy (DN) mice and HG-induced MMCs was detected by qRT-PCR. EdU and Colony formation, Annexin V-PI staining, Muse cell cycle, Scratch, and Transwell assays were performed to detect the cell proliferation, apoptosis, cell cycle, migration, and invasion, respectively. The contents of fibrosis factors in cell-culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was performed to detect the expression of factors involved in apoptosis, cell cycle, migration and invasion, fibrosis, and PI3K/Akt/mTOR pathway. The targeting relation between miR-93-5p and PVT1 was predicted by StarBase3.0 (an online software for analyzing the targeting relationship) and identified by Dual-luciferase reporter (DLR) assay.
PVT1 was overexpressed in DN kidney tissues and HG-induced MMCs. HG-induced MMCs exhibited significantly increased EdU-positive cells, cell colonies, S and G2/M phase cells, migration and invasion ability, and contents of fibrosis factors, as well as significantly decreased apoptosis rate compared with NG-induced MMCs. HG significantly up-regulated Bcl-2, CyclinD1, CDK4, N-cadherin, vimentin, Col. IV, FN, TGF-β1 and PAI-1, and down-regulated Bax, cleaved caspase-3, cleaved PARP, and E-cadherin in MMCs. Silencing of PVT1 eliminated the effects of HG in MMCs and blocked PI3K/Akt/mTOR pathway. MiR-93-5p was a target of PVT1, which eliminated the effects of PVT1 on HG-induced MMCs.
PVT1 silencing inhibited the proliferation, migration, invasion and fibrosis, promoted the apoptosis, and blocked PI3K/Akt/mTOR pathway in HG-induced MMCs via up-regulating miR-93-5p.
本研究旨在探讨长链非编码 RNA 浆细胞瘤变异易位 1（PVT1）对高糖（HG）诱导的小鼠系膜细胞（MMCs）的调控作用。
采用 qRT-PCR 检测糖尿病肾病（DN）小鼠和 HG 诱导的 MMCs 中 PVT1 的表达。采用 EdU 和集落形成、Annexin V-PI 染色、Muse 细胞周期、划痕和 Transwell 实验分别检测细胞增殖、凋亡、细胞周期、迁移和侵袭，细胞培养上清液中纤维化因子的含量采用酶联免疫吸附试验（ELISA）检测。Western blot 检测凋亡、细胞周期、迁移和侵袭、纤维化及 PI3K/Akt/mTOR 通路相关因子的表达。采用 StarBase3.0（分析靶向关系的在线软件）预测 miR-93-5p 与 PVT1 的靶向关系，并通过双荧光素酶报告（DLR）实验进行验证。
DN 肾脏组织和 HG 诱导的 MMCs 中 PVT1 表达上调。与 NG 诱导的 MMCs 相比，HG 诱导的 MMCs 中 EdU 阳性细胞、细胞集落、S 和 G2/M 期细胞、迁移和侵袭能力以及纤维化因子含量显著增加，凋亡率显著降低。HG 显著上调 MMCs 中 Bcl-2、CyclinD1、CDK4、N-cadherin、vimentin、Col.IV、FN、TGF-β1 和 PAI-1 的表达，下调 Bax、cleaved caspase-3、cleaved PARP 和 E-cadherin 的表达。沉默 PVT1 消除了 HG 对 MMCs 的作用，并阻断了 PI3K/Akt/mTOR 通路。miR-93-5p 是 PVT1 的靶标，消除了 PVT1 对 HG 诱导的 MMCs 的作用。
沉默 PVT1 通过上调 miR-93-5p 抑制 HG 诱导的 MMCs 的增殖、迁移、侵袭和纤维化，促进凋亡，并阻断 PI3K/Akt/mTOR 通路。
