CAJ | 학술논문

　　背景：转录因子 Runx2是调节成骨分化及骨发育的关键因子，过表达 Runx2可诱导间质细胞 C2C12分化为成骨细胞，但相关诱导分化分子机制并不清楚。目的：分析 miR-376家族成员在 Runx2诱导 C2C12细胞进行成骨分化过程中的作用。方法：利用可诱导表达Runx2的细胞系C2C12/Runx2Dox，在不同的时间点通过荧光定量PCR方法检测miR-376家族成员表达情况。C2C12/Runx2 Dox胞标记基因碱性磷酸酶及骨钙素表达情况，并利用碱性磷酸酶染色法分析碱性磷酸酶活性。利用在线工具miRanda, miRWalk与TargetScan共同预测miR-376b-3p潜在靶基因。利用DAVID Bioinformatics Resources数据库对得到的靶基因进行功能聚类分析。结果与结论：在 Runx2诱导 C2C12进行成骨分化过程中 miR-376b-3p 表达显著增加，其他成员表达无明显变化。转染 miR-376b-3p mimic 可上调碱性磷酸酶表达，但对骨钙素表达无影响；转染 miR-376b-3p mimic也可增加碱性磷酸酶活性。靶基因功能聚类分析结果表明 miR-376b-3p 可参与机体骨骼发育过程，表明其在成骨分化过程中的作用。研究结果表明，Runx2通过上调 miR-376b-3p 的方式对与成骨分化相关基因的表达进行调节，以促进 C2C12进行成骨细胞的早期分化。细胞转染miR-376b-3p mimic后利用荧光定量PCR检测成骨细
　　배경：전록인자 Runx2시조절성골분화급골발육적관건인자，과표체 Runx2가유도간질세포 C2C12분화위성골세포，단상관유도분화분자궤제병불청초。목적：분석 miR-376가족성원재 Runx2유도 C2C12세포진행성골분화과정중적작용。방법：이용가유도표체Runx2적세포계C2C12/Runx2Dox，재불동적시간점통과형광정량PCR방법검측miR-376가족성원표체정황。C2C12/Runx2 Dox포표기기인감성린산매급골개소표체정황，병이용감성린산매염색법분석감성린산매활성。이용재선공구miRanda, miRWalk여TargetScan공동예측miR-376b-3p잠재파기인。이용DAVID Bioinformatics Resources수거고대득도적파기인진행공능취류분석。결과여결론：재 Runx2유도 C2C12진행성골분화과정중 miR-376b-3p 표체현저증가，기타성원표체무명현변화。전염 miR-376b-3p mimic 가상조감성린산매표체，단대골개소표체무영향；전염 miR-376b-3p mimic야가증가감성린산매활성。파기인공능취류분석결과표명 miR-376b-3p 가삼여궤체골격발육과정，표명기재성골분화과정중적작용。연구결과표명，Runx2통과상조 miR-376b-3p 적방식대여성골분화상관기인적표체진행조절，이촉진 C2C12진행성골세포적조기분화。세포전염miR-376b-3p mimic후이용형광정량PCR검측성골세
BACKGROUND: The transcription factor Runx2 is the key factor that regulates osteogenic differention and bone development. It has been reported that the C2C12 mesenchymal cells can be induced to differentiate into osteoblasts by Runx2 overexpression, but the molecular mechanism of induction is stil largely unclear. OBJECTIVE: To investigate the role of the members of the miR-376 family during Runx2-induced osteogenic differentiation in C2C12 cells. METHODS: The expression of the members of the miR-376 family was detected by real-time quantitative PCR at different time points using C2C12/Runx2Dox sub-line with conditional Runx2 expression. In miR-376b-3p-transfected C2C12/Runx2Dox cells, the expression of osteoblast markers, such as alkaline phosphatase and osteocalcin, was detected by real-time quantitative PCR, and the alkaline phosphatase activity was also examined by alkaline phosphatase staining. The putative miR-376b-3p targets were commonly predicted by online tools (miRanda, miRWalk and TargetScan). The functional classification of these putative targets was performed by DAVID Bioinformatics Resources database. RESULTS AND CONCLUSION: The expression of miR-376b-3p was significantly increased during Runx2-induced osteogenic differentiation of C2C12 cells, but the expression of other members was not changed. Transfection of miR-376b-3p mimic upregulated alkaline phosphatase expression, but had no effect on osteocalcin expression. The alkaline phosphatase activity was also increased by transfection of miR-376b-3p. The functional classification of miR-376b-3p putative targets showed that miR-376b-3p is involved in the skeleton development, indicating the role of miR-376b-3p in osteoblast differentiation. Taken together, these results suggest that Runx2 promotes early osteogenic differentiation in C2C12 cells by regulating the expression of genes related to osteogenic differentiation through upregulation of miR-376b-3p.