LncRNA MCM3AP-AS1/miR-335对骨肉瘤细胞增殖及凋亡的影响

Effects of LncRNA MCM3AP-AS1/miR-335 on the proliferation and apoptosis of osteosarcoma cells

目的:探讨长链非编码RNA MCM3AP反义RNA 1(LncRNA MCM3AP-AS1)对骨肉瘤细胞增殖、凋亡的影响及其对miR-335的调控作用。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)法检测骨肉瘤组织、瘤旁组织中MCM3AP-AS1、miR-335的表达量;体外培养人骨肉瘤细胞U2OS,分别将si-NC、si-MCM3AP-AS1、si-MCM3AP-AS1与NC-inhibitor、si-MCM3AP-AS1与miR-335 inhibitor转染至U2OS细胞;采用qRT-PCR法检测U2OS细胞中MCM3AP-AS1、miR-335的表达量;采用MTT实验与平板克隆形成实验检测细胞增殖能力;采用流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测MCM3AP-AS1、miR-335的靶向关系;蛋白质印迹法检测B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白表达量。结果:与瘤旁组织比较,骨肉瘤组织中MCM3AP-AS1的表达水平升高(P<0.05),miR-335的表达水平降低(P<0.05);转染si-MCM3AP-AS1可明显降低光密度值及Bcl-2蛋白水平、减少克隆形成数、提高凋亡率及Bax蛋白水平(P<0.05);双荧光素酶报告实验证实MCM3AP-AS1可靶向结合miR-335;转染miR-335 inhibitor可明显逆转干扰MCM3AP-AS1对U2OS细胞增殖及凋亡的作用。结论:MCM3AP-AS1可通过负向调控miR-335的表达调节骨肉瘤细胞增殖及凋亡。

Objective:To explore the effect of long non-coding RNA MCM3 AP antisense RNA1(LncRNA MCM3 AP-AS1)on the proliferation and apoptosis of osteosarcoma cells and its regulatory effect on miR-335.Methods:The qRT-PCR method was used to detect the expression of MCM3 AP-AS1 and miR-335 in osteosarcoma tissue and adjacent tissues.Human osteosarcoma cells U2 OS were cultured in vitro,and si-NC,si-MCM3 AP-AS1,si-MCM3 AP-AS1 and NC-inhibitor,si-MCM3 AP-AS1 and miR-335 inhibitor were respectively transfected into U2 OS cells.The qRT-PCR method was used to detect the expression of MCM3 AP-AS1 and miR-335 in U2 OS cells.MTT experiment and plate clone formation experiment were used to detect cell proliferation ability.Flow cytometry was used to detect the apoptosis rate.The dual luciferase reporter experiment was used to detect the targeting relationship of MCM3 AP-AS1 and miR-335.Western blot method was used to detect the protein expression of Bax and Bcl-2.Results:Compared with adjacent tissues,the expression level of MCM3 AP-AS1 in osteosarcoma tissue was significantly increased(P<0.05),and the expression level of miR-335 was significantly decreased(P<0.05).Transfection of si-MCM3 AP-AS1 could significantly reduce the OD value and the protein level of Bcl-2,reduce the number of clones,increase the apoptosis rate and the protein level of Bax(P<0.05).The dual luciferase report experiment confirmed that MCM3 AP-AS1 could target miR-335.Transfection of miR-335 inhibitor could significantly reverse the effect of interference MCM3 AP-AS1 on the proliferation and apoptosis of U2 OS cells.Conclusion:MCM3 AP-AS1 can regulate the proliferation and apoptosis of osteosarcoma cells by negatively regulating the expression of miR-335.