Department of Cardiac Surgery, Second Hospital of Hebei Medical University, Shi Jiazhuang, China.
Eur Rev Med Pharmacol Sci. 2019 Oct;23(19):8540-8550. doi: 10.26355/eurrev_201910_19168.
Atherosclerosis is one of the most important risk factors for coronary heart disease (CHD), and growing evidence has shown that long non-coding RNAs (lncRNAs) can serve as prospective markers for atherosclerosis. In this study, we mainly focused on the potential roles of linc00961 in CHD patients.
qRT-PCR was used to detect the expressions of linc00961 and miR-367 in CHD patients and ApoE-/-mice, and the correlations were analyzed. Then, HA-VAMC was respectively treated with 5 inflammatory factors and hypoxia conditions to explore the factors that affect linc00961 levels. Furthermore, the linc00961 overexpression lentivirus (LV-linc00961) and linc00961 downregulation lentivirus (LV-sh linc00961) were purchased and transfected into human vascular smooth muscle cells (VSMCs). CCK8 assay was carried out to measure the cell proliferation of VSMC, and the levels of Cyclin D1, Bcl-2, Bax, and cleaved caspase-3 were detected by RT-PCR and Western blot. Moreover, the Luciferase assay was performed to explore the binding site of linc00961 and miR-367. Finally, the miR-367 inhibitor was transfected into LV-sh linc00961 VSMCs to confirm the linc00961 functions via miR-367.
We found that linc00961 was significantly decreased in patients with CHD and ApoE-/-mice. Additionally, linc00961 was reduced in VSMCs at the conditions of hypoxia and C-reactive protein (CRP). Most importantly, the overexpression of linc00961 significantly inhibited the VSMCs proliferation, repressed the levels of Cyclin D1 and Bcl-2, and increased the levels of Bax and cleaved caspase-3. However, the downregulation of linc00961 promoted VSMCs proliferation, increased the levels of Cyclin D1 and Bcl-2, and repressed the levels of Bax and cleaved caspase-3. We also found that miR-367 was downregulated following the upregulation of linc00961, while it was upregulated following the downregulation of linc00961. The Luciferase gene reporter assay indicated that linc00961 could directly bind with miR-367 in VSMCs. Finally, we found that linc00961 could inhibit proliferation and promote apoptosis of VSMCs via binding with miR-367.
According to the results, our study revealed that linc00961 was significantly decreased in patients with CHD and ApoE-/-mice. Furthermore, our findings firstly uncovered that linc00961 was reduced by hypoxia and CRP in VSMCs. The downregulation of linc00961 contributed to promote proliferation and inhibit apoptosis of VSMCs by sponging miR-367 in CHD patients, which might provide a potential target for treating atherosclerosis.
动脉粥样硬化是冠心病（CHD）最重要的危险因素之一，越来越多的证据表明，长链非编码 RNA（lncRNA）可以作为动脉粥样硬化的前瞻性标志物。在这项研究中，我们主要关注 linc00961 在 CHD 患者中的潜在作用。
采用 qRT-PCR 检测 CHD 患者和 ApoE-/-小鼠中 linc00961 和 miR-367 的表达，并分析其相关性。然后，HA-VAMC 分别用 5 种炎症因子和低氧条件处理，以探讨影响 linc00961 水平的因素。此外，购买 linc00961 过表达慢病毒（LV-linc00961）和 linc00961 下调慢病毒（LV-sh linc00961）并转染入人血管平滑肌细胞（VSMCs）。采用 CCK8 法检测 VSMC 的增殖，采用 RT-PCR 和 Western blot 检测细胞周期蛋白 D1、Bcl-2、Bax 和 cleaved caspase-3 的水平。此外，采用荧光素酶报告基因实验探讨 linc00961 与 miR-367 的结合位点。最后，将 miR-367 抑制剂转染至 LV-sh linc00961 VSMCs，通过 miR-367 验证 linc00961 的功能。
我们发现，CHD 患者和 ApoE-/-小鼠中 linc00961 的表达明显降低。此外，在缺氧和 C-反应蛋白（CRP）条件下，VSMCs 中的 linc00961 减少。最重要的是，linc00961 的过表达显著抑制了 VSMC 的增殖，降低了细胞周期蛋白 D1 和 Bcl-2 的水平，增加了 Bax 和 cleaved caspase-3 的水平。然而，下调 linc00961 促进了 VSMC 的增殖，增加了细胞周期蛋白 D1 和 Bcl-2 的水平，降低了 Bax 和 cleaved caspase-3 的水平。我们还发现，linc00961 的上调导致 miR-367 下调，而 linc00961 的下调导致 miR-367 上调。荧光素酶基因报告实验表明，linc00961 可在 VSMCs 中直接与 miR-367 结合。最后，我们发现 linc00961 可通过与 miR-367 结合抑制 VSMC 的增殖并促进其凋亡。
根据研究结果，我们发现 CHD 患者和 ApoE-/-小鼠中 linc00961 的表达明显降低。此外，我们的研究首次揭示了 linc00961 在 VSMCs 中受低氧和 CRP 下调。下调 linc00961 通过海绵吸附 miR-367 促进 CHD 患者 VSMC 的增殖和抑制凋亡，可能为治疗动脉粥样硬化提供了一个潜在的靶点。
