miR-96通过靶向调控KRAS蛋白抑制骨肉瘤的增殖和转移

miRNA-96 inhibits proliferation and metastasis of osteosarcoma through targeting KRAS

目的 探讨miR-96在骨肉瘤组织和细胞中的表达情况及其对骨肉瘤细胞的调控作用。方法 采用RT-qPCR方法检测骨肉瘤组织和骨肉瘤细胞系MG63和U2OS中miR-96的表达;miR-96 mimics转染骨肉瘤细胞后,CCK-8法检测细胞增殖;Transwell实验和划痕实验检测细胞侵袭和转移;荧光素酶报告基因实验检测miR-96与鼠类肉瘤病毒癌基因(KRAS)的3’UTR区的靶向结合作用。Western blot检测KRAS蛋白表达变化。结果 与对照组相比,骨肉瘤组织和细胞中miR-96表达较低。体外实验中发现,miR-96的过表达能够抑制骨肉瘤细胞增殖和侵袭、转移。荧光素酶报告基因实验发现KRAS的3’UTR是miR-96的靶点。miR-96过表达能下调骨肉瘤细胞中KRAS蛋白的表达。结论 miR-96在骨肉瘤患者中表达下调,miR-96过表达能显著抑制骨肉瘤细胞的增殖和侵袭转移,该作用与靶向调控KRAS蛋白相关。

Objective To investigate the expression of miR-96 in osteosarcoma tissues and cells and its regulatory role on osteosarcoma cells. Methods The expression of miR-96 in osteosarcoma tissues and osteosarcoma cells MG63 and U2OS were detected by RT-qPCR. After transfection of osteosarcoma cells with miR-96 mimics, cell proliferation was detected by CCK-8 assay, cell invasion and metastasis were detected by transwell assay and wound healing assay. The interaction between miR-96 and 3’UTR of kirsten rat sarcoma viral oncogene(KRAS) was detected by luciferase activity. Western blot was used to detect the expression of KRAS protein. Results Compared with the control group, the expression of miR-96 was lower in osteosarcoma tissues and cells. Overexpression of miR-96 could inhibit the proliferation,invasion and metastasis of osteosarcoma cells in vitro experiments. Luciferase reporter assay revealed that the 3’UTR of KRAS was the target of miR-96. Overexpression of miR-96 could down-regulate the expression of KRAS protein in osteosarcoma cells. Conclusion miR-96 is down-regulated in patients with osteosarcoma, and overexpression of miR-96could significantly inhibit the proliferation, invasion and metastasis of osteosarcoma cells, which is related to the targeted regulation of KRAS protein.