关键词: RNA, 长链非编码, RNA, 小分子干扰, 胰岛素样生长因子Ⅱ, 间充质基质细胞, 肌细胞, 心脏, 细胞分化

Abstract: Objective To investigate the effect of long non-coding RNA insulin-like growth factor 2 antisense transcript (lncRNA IGF2-AS) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cells (BMSCs) by regulating insulin-like growth factor 2 (IGF2). Methods BMSCs were isolated and cultured from SD rats. The morphology of primary and third-generation cells was observed under inverted microscope. Flow cytometry was used to detect the expressions of CD29, CD90 and CD45 on the surface of BMSCs. BMSCs with good growth in the third generation were divided into the control group, the empty vector group (transfected with pLVX-IRES-Zs Green1 vector), the lncRNA IGF2- AS group (transfected with pLVX-IRES-Zs Green1-IGF2-AS), the 5-azacytosine group (8 μmol/L 5-AZA treatment), the si-NC group, the si-IGF2-AS group, the si-IGF2 group, lncRNA IGF2-AS+si-NC group and the lncRNA IGF2-AS+siIGF2 group. Quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of IGF2- AS. MTT method was used to detect cell viability. Western blot assay was used to detect the IGF2, gap junction protein 43 (Cx43), cardiac troponin T (cTnT) and cardiac troponin I (cTnI) protein expression. RNA pull-down and RNA immunoprecipitation (RIP) were used to detect the IGF2-AS and IGF2 protein binding. Results The primary cells were in suspension at the beginning of the culture flask, most of them were round, and they began to grow adherently after 48 h of culture. The third-generation cells were long spindle-shaped, irregularly arranged, closely connected between adjacent cells, and fibroblast-like. The third-generation BMSCs showed high expression of CD29 (98.21%), CD90 (92.54%), and low expression of CD45 (3.67%). The overexpression of IGF2-AS or 5-AZA could promote protein expressions of Cx43, cTnT and cTnI in BMSCs, and reduce cell viability, and the corresponding indicators were significantly lower in the 5-AZA group than those in the lncRNA IGF2-AS group (P＜0.05). Silencing IGF2-AS or inhibiting the expression of IGF2 could reduce the protein expressions of Cx43, cTnT, and cTnI in BMSCs, and reduce cell viability (P＜0.05). lncRNA IGF2-AS could target up-regulate the expression of IGF2 protein (P＜0.05). Silencing IGF2 reversed the promotion effect of overexpression of IGF2-AS on cardiomyocyte-like differentiation of BMSCs. Conclusion Overexpression of IGF2-AS promotes cardiomyocyte-like differentiation of BMSCs by up-regulating IGF2.

Key words: RNA, long noncoding, RNA, small interfering, insulin-like growth factor Ⅱ, mesenchymal stromal cells, myocytes, cardiac, cell differentiation