黄芪多糖小檗碱配伍调控lncRNAs改善胰岛素抵抗的作用机制研究

Astragalus polysaccharides and Berberine (APBBR) are known to improve insulin resistance (IR), but the molecular mechanism of lncRNAs underlying this effect remains unclear. Our previous study showed that IRS1 expression was increased significantly after APBBR intervention in an IR INS1 model induced by palmitic acid (PA), suggesting that lncRNA MALAT1 and PVT1 may act with miR-144 and miR-33, and that overexpression of miR-144 or miR-33 in beta cells could significantly inhibit the expression of IRS1 and PDK1 respectively. These findings suggest that APBBR may participate in IR via the above-mention pathway. In the present study, we plan to detect differential expression of lncRNAs by lncRNA microarray in an INS1 model induced by high concentrations of PA, verify the probable miRNA targeting IR by dual luciferase reporter assay, interfere with the expression of the MALAT1 and PVT1 by siRNA, and finally measure the level of IRS1 and PDK1, the phosphorylation level of AKT, the intracellular calcium concentration, proliferation, apoptosis and active oxygen by using qRT-PCR, Western Blot, flow cytometry and confocal laser scanning microscopy, in an attempt to clarify the molecular mechanism of lncRNA regulation by APBBR in improving IR.

本课题组前期研究观察到：黄芪多糖和小檗碱配伍（APBBR)能显著升高经软脂酸诱导产生胰岛素抵抗（IR)的INS1细胞IRS1的表达，显示其有改善IR作用；过表达miR-144和miR-33能分别抑制INS1细胞IRS1及PDK1的表达；生物信息学提示：长链非编码RNA：lncRNA MALAT1和PVT1可能与miR-144和miR-33发生作用。我们分析APBBR很可能通过上述途径改善IR。本课题拟在软脂酸诱导产生IR的INS1细胞模型，应用lncRNA芯片技术，检测APBBR干预前后lncRNAs的差异表达，双荧光素酶检测APBBR对miRNAs水平的影响；应用siRNA干扰INS1细胞MALAT1和PVT1后，RT-PCR等方法检测IRS1、PDK1等表达、AKT磷酸化水平、胞内钙离子浓度、细胞增殖与凋亡及活性氧簇等变化；以期探明APBBR在lncRNA水平改善IR的作用与机制。

本课题研究了黄芪多糖小檗碱（APBBR）对胰岛素抵抗HepG2细胞（IR- HepG2）的表型影响及其对lncRNA的调控作用。研究表明：IR-HepG2细胞造模最佳时间为胰岛素干预48h，APBBR可以改善IR-HepG2细胞胰岛素抵抗效应，最佳给药浓度为10mg。APBBR可以降低细胞内H2O2含量和细胞内钙瞬变，对IR-HepG2细胞凋亡无影响。本次研究共注释了66980个lncRNA；其中12962个lncRNA为已知的lncRNA，32433个为新鉴定的lncRNA。从RNA-seq结果中，共检测到521个lncRNA：与正常对照组比较，模型组中有351个lncRNA有显著性差异表达（47个上调，304个下调）；与模型组比较，APBBR组中有170个lncRNAs 有显著性差异表达（100上调和70下调）。APBBR可以逆转IR模型中70个下调基因中的其中两个基因（CTD-2600O9.2，MIR4435-2HG）和IR模型中100个上调lncRNA的其中5个lncRNA（RP5-1057I20.5，CTD-3014M21.1，HOXB-AS3，LAMA5-AS1，CTD-2517M22.14）。用qPCR检测筛选了差异表达的lncRNA，其中lncRNA LAMA5-AS1在模型组中高表达，在APBBR组中表达量显著下降，与芯片结果相符。采用2-NBDG检测葡萄糖摄取量，发现LAMA5-AS1可以促进胰岛素抵抗，APBBR可以通过下调LAMA5-AS1改善胰岛素抵抗。lncRNA LAMA5-AS1可上调chemeri，FABP-4，fetuin-A和myostatin蛋白表达，下调AMPK， p38MAPK，GLUT2， PPARa 和 PPARγ的蛋白表达，而APBBR可以下调lncRNA LAMA5-AS1，上调AMPK，p38MAPK，GLUT2，PPARa 和 PPARγ的表达。该研究首次提供了HepG2细胞胰岛素抵抗lncRNAs的表达谱，为lncRNA调控IR的机制提供了研究基础，也为APBBR治疗IR的lncRNA调控机制奠定了基础。