lncRNA RHPN1-AS1靶向miR-485-5p调控骨肉瘤细胞增殖、凋亡、迁移、侵袭及其分子机制

目的:探讨lncRNA RHPN1反义RNA1（RHPN1-AS1）靶向miR-485-5p对骨肉瘤细胞增殖、凋亡、迁移、侵袭的影响和机制。方法:实时荧光定量PCR（qRT-PCR）检测20例骨肉瘤组织与其对应的癌旁组织、人正常成骨细胞hFOB1.19以及3种骨肉瘤细胞（U-2OS、SAOS-2、HOS）中RHPN1-AS1和miR-485-5p的表达水平。利用脂质体转染法将RHPN1-AS1小干扰RNA（si-RHPN1-AS1）、小干扰RNA阴性对照（si-NC）、miR-485-5p模拟物（miR-485-5p mimics）、miRNA阴性对照（miR-NC）分别转染U-2OS细胞，四甲基偶氮唑蓝（MTT）法检测细胞活力，流式细胞术检测细胞凋亡，Transwell实验检测细胞迁移和侵袭能力，蛋白质印记（Western blot）检测细胞周期蛋白D1（Cyclin D1）、p21、p27、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）和基质金属蛋白酶14（MMP-14）蛋白的表达。双荧光素酶报告基因实验和qRT-PCR验证RHPN1-AS1和miR-485-5p的靶向调控关系。结果:与癌旁组织比较，骨肉瘤组织中RHPN1-AS1的表达水平显著升高，miR-485-5p的表达水平显著降低（P＜0.05）；与hFOB1.19细胞比较，3种骨肉瘤细胞中RHPN1-AS1的表达水平显著升高，miR-485-5p的表达水平显著降低（P＜0.05）。与si-NC组比较，si-RHPN1-AS1组U-2OS细胞的活力显著降低，迁移和侵袭能力显著降低，细胞凋亡率显著升高，Cyclin D1、Bcl-2、MMP-2、MMP-9和MMP-14蛋白的表达水平显著降低，p21、p27和Bax蛋白的表达水平显著升高（P＜0.05）；与miR-NC组比较，miR-485-5p组U-2OS细胞的活力显著降低，迁移和侵袭能力显著降低，细胞凋亡率显著升高，Cyclin D1、Bcl-2、MMP-2和MMP-9蛋白的表达水平显著降低，p21和Bax蛋白的表达水平显著升高（P＜0.05）。RHPN1-AS1靶向负性调控miR-485-5p表达。干扰miR-485-5p表达逆转了抑制lncRNA RHPN1-AS1表达对骨肉瘤U-2OS细胞增殖、凋亡、迁移和侵袭的影响。结论:抑制RHPN1-AS1通过上调miR-485-5p抑制骨肉瘤细胞增殖、迁移和侵袭，诱导细胞凋亡。

lncRNA RHPN1-AS1 regulates the proliferation,apoptosis,migration and invasion of osteosarcoma cells by targeting miR-485-5p and its molecular mechanism

Objective:To investigate the effect of long non-coding RNA RHPN1-AS1 on proliferation,apoptosis,migration and invasion of osteosarcoma cells by targeting miR-485-5p and its mechanism.Methods:Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of RHPN1-AS1 and miR-485-5p in 20 cases of osteosarcoma and its corresponding paracancerous tissues,human normal osteoblast hFOB1.19 and 3 kinds of osteosarcoma cells (U-2OS,SAOS-2,HOS).RHPN1-AS1 small interfering RNA (si-RHPN1-AS1),small interfering RNA negative control (si-NC),miR-485-5p mimics (miR-485-5p mimics),miRNA negative control (miR-NC) were transfected into U-2OS cells by lipofection,respectively.Cell viability was detected by MTT assay.The apoptosis was detected by flow cytometry.Transwell assay was used to detect cell migration and invasion,and the expression of Cyclin D1,p21,p27,B-cell lymphoma/leukemia-2 (Bcl-2),Bcl-2 related X protein (Bax),matrix metalloproteinase 2 (MMP-2),matrix metalloproteinase 9 (MMP-9) and matrix metalloproteinase 14 (MMP-14) proteins was detected by Western blot.The dual luciferase reporter gene assay and qRT-PCR were used to verify the targeted and regulatory relationship between RHPN1-AS1 and miR-485-5p.Results:Compared with adjacent tissues,the expression level of RHPN1-AS1 was significantly increased in osteosarcoma tissues,while the expression level of miR-485-5p was significantly decreased (P＜0.05).Compared with hFOB1.19 cells,the expression level of RHPN1-AS1 in three osteosarcoma cells was significantly increased,while the expression level of miR-485-5p was significantly decreased (P＜0.05).Compared with the si-NC group,the cell viability of U-2OS cells in the si-RHPN1-AS1 group was significantly decreased,and the migration and invasion ability were significantly decreased,and the apoptosis rate was significantly increased,and the expression levels of Cyclin D1,Bcl-2,MMP-2,MMP-9 and MMP-14 proteins were significantly decreased,and the expression levels of p21,p27 and Bax proteins were significantly increased (P＜0.05).Compared with miR-NC group,the cell viability of U-2OS cells in miR-485-5p group was significantly decreased,and the migration and invasion ability were significantly decreased,and the apoptosis rate was significantly increased,and the expression levels of Cyclin D1,Bcl-2,MMP-2 and MMP-9 proteins were significantly reduced,and the expression levels of p21 and Bax proteins were significantly increased (P＜0.05).RHPN1-AS1 targeted and negatively regulated the expression of miR-485-5p.The interference of miR-485-5p reversed the effect of si-RHPN1-AS1 on proliferation,apoptosis,migration and invasion of osteosarcoma U-2OS cells.Conclusion:Inhibition of RHPN1-AS1 inhibits the proliferation,migration and invasion and induces apoptosis of osteosarcoma cells by up-regulating miR-485-5p.