ABSTRACT

Long non-coding RNA LOC285194 (LOC285194) has reported to regulate vascular smooth muscle cells (VSMCs) proliferation and apoptosis in vitro and in vivo. Here we aimed to determine the role of LOC285194 in the proliferation, migration and apoptosis of VSMCs and its underlying mechanisms. A7r5 cells were transfected with Lv-LOC285194 or control Lv-NC for 24–72 h, or small interfering RNA targeting S100A4 (S100A4 siRNA) for 24–48 h, or co-transfected with Lv-LOC285194 and PUMA siRNA for 72 h, or treated with miR-211 inhibitor or co-transfected with Lv-LOC285194 and miR-211 mimics for 72 h. A7r5 cells were also treated with transforming growth factor – β(TGF-β) (5 ng/ml) after Lv-LOC285194 transfection for 24 h. The relationship between LOC285194 and TGF-β was confirmed using luciferase reporter assay. Cell proliferation and cell apoptosis were analyzed by Cell Counting Kit-8 (CCK-8) assay, ELISA and TUNEL staining. LOC285194 and miR-211 expression were detected by qPCR assay. S100A4, pro-apoptotic and anti-apoptotic protein were detected by Western blot assay. LOC285194 inhibited cell proliferation, invasion and migration and promoted cell apoptosis accompanied by upregulation of PUMA and downregulation of miR-211 and S100A4. Targeting PUMA reversed the effect of LOC285194 on cell apoptosis and proliferation. miR-211 mimic inhibited LOC285194-induced PUMA upregulation and decreased LOC285194-induced cell apoptosis. TGF-β (5 ng/ml) treatment reversed S100A4 siRNA or LOC285194-induced S100A4 expression. Luciferase reporter assay showed that TGF-β was the target of LOC285194. LOC285194 inhibits proliferation and promoted apoptosis in vascular smooth muscle cells via targeting miR-211/PUMA signal; In addition, LOC285194 decreased cell invasion and migration by targeting TGF-β1/S100A4 signal.

摘要

长的非编码RNA LOC285194（LOC285194）报告，以调节血管平滑肌细胞（VSMC）的增殖和细胞凋亡在体外和体内。在这里，我们旨在确定LOC285194在VSMCs增殖，迁移和凋亡中的作用及其潜在机制。将A7r5细胞用Lv-LOC285194或对照Lv-NC转染24-72小时，或靶向S100A4的小干扰RNA（S100A4 siRNA）转染24-48 h，或与Lv-LOC285194和PUMA siRNA共转染72小时，或用miR-211抑制剂处理或与Lv-LOC285194和miR-211模拟物共转染72小时。Lv-LOC285194转染24小时后，还用转化生长因子–β（TGF-β）（5 ng / ml）处理A7r5细胞。使用荧光素酶报告基因分析证实了LOC285194和TGF-β之间的关系。细胞增殖和细胞凋亡通过细胞计数试剂盒8（CCK-8）测定，ELISA和TUNEL染色进行分析。通过qPCR测定法检测LOC285194和miR-211表达。S100A4，通过蛋白质印迹法检测促凋亡和抗凋亡蛋白。LOC285194抑制细胞增殖，侵袭和迁移，并促进细胞凋亡，并伴随着PUMA的上调和miR-211和S100A4的下调。靶向PUMA逆转了LOC285194对细胞凋亡和增殖的作用。miR-211模拟物抑制LOC285194诱导的PUMA上调并降低LOC285194诱导的细胞凋亡。TGF-β（5 ng / ml）处理可逆转S100A4 siRNA或LOC285194诱导的S100A4表达。萤光素酶报告基因检测表明，TGF-β是LOC285194的靶标。LOC285194通过靶向miR-211 / PUMA信号抑制血管平滑肌细胞的增殖并促进其凋亡。此外，LOC285194通过靶向TGF-β1/ S100A4信号减少了细胞侵袭和迁移。