CircFAT1调节miR-296-3p/MAPRE1轴对鼻咽癌细胞增殖、凋亡和放疗敏感性的影响

Impacts of CircFAT1 on the proliferation, apoptosis and radiosensitivity of nasopharyngeal carcinoma cells by regulating miR-296-3p/MAPRE1 axis

目的研究环状RNA脂肪非典型钙黏蛋白1(CircFAT1)调节miR-296-3p/微管关联蛋白RP/EB家族成员1(MAPRE1)轴对鼻咽癌细胞增殖、凋亡和放疗敏感性的影响。方法以实时荧光定量PCR和免疫印迹法检测鼻咽上皮细胞NP69、人鼻咽癌细胞株CNE2及其放射线抵抗细胞CNE2-RR中CircFAT1、miR-296-3p、MAPRE1表达。将体外培养的CNE2细胞随机分为对照组、CircFAT1敲低组、阴性对照组、CircFAT1敲低+miR-296-3p inhibitor组,分组转染后以实时荧光定量PCR和免疫印迹法检测各组CNE2细胞CircFAT1、miR-296-3p、MAPRE1表达;以CCK-8法、5-乙炔基-2'-脱氧尿苷(Edu)染色与流式细胞实验检测各组CNE2细胞增殖、凋亡。将体外培养的CNE2-RR细胞随机分为对照组、放射组、放射+阴性对照组、放射+CircFAT1敲低组、放射+CircFAT1敲低+miR-296-3p inhibitor组,分组转染后以实时荧光定量PCR和免疫印迹法检测各组CNE2-RR细胞CircFAT1、miR-296-3p、MAPRE1表达;以CCK-8法、Edu染色与流式细胞实验检测各组CNE2-RR细胞增殖、凋亡。以双荧光素酶报告基因实验检测CNE2-RR中CircFAT1对miR-296-3p的靶向调节与miR-296-3p对MAPRE1的靶向调节。结果与NP69细胞相比,CNE2、CNE2-RR细胞CircFAT1表达、MAPRE1 mRNA与蛋白表达升高(P<0.05),miR-296-3p表达降低(P<0.05);与CNE2细胞相比,CNE2-RR细胞CircFAT1表达、MAPRE1 mRNA与蛋白表达升高(P<0.05),miR-296-3p表达降低(P<0.05)。与对照组相比,CircFAT1敲低组CNE2细胞CircFAT1表达、MAPRE1 mRNA与蛋白表达、存活率、增殖率降低(P<0.05),miR-296-3p表达、凋亡率升高(P<0.05);阴性对照组CNE2细胞各指标无明显变化(P>0.05),miR-296-3p inhibitor可逆转敲低CircFAT1对CNE2细胞各指标的影响。与对照组、放射组分别相比,放射+CircFAT1敲低组CNE2-RR细胞CircFAT1表达、MAPRE1 mRNA与蛋白表达、存活率、增殖率降低(P<0.05),miR-296-3p表达、凋亡率升高(P<0.05);放射组、放射+阴性对照组CNE2-RR细胞各指标无明显变化(P>0.05),miR-296-3p inhibitor可逆转敲低CircFAT1对放射处理下CNE2-RR细胞各指标的影响。CircFAT1可靶向下调CNE2-RR细胞miR-296-3p表达,而miR-296-3p可靶向下调CNE2-RR细胞MAPRE1表达。结论敲低CircFAT1可通过上调miR-296-3p而降低MAPRE1表达,从而抑制鼻咽癌细胞增殖,促进其凋亡并增强其放射敏感性。

Objective To explore the impacts of circular RNA fat atypical cadherin 1(CircFAT1)on the proliferation,apoptosis and radiosensitivity of nasopharyngeal carcinoma cells by regulating miR-296-3p/microtubule-associated protein RP/EB family member 1(MAPRE1)axis.Methods The expressions of CircFAT1,miR-296-3p and MAPRE1 in NP69,CNE2 and CNE2-RR cells were detected with real-time fluorescent quantitative PCR and Western blotting.CNE2 cells cultured in vitro were randomly divided into the control group,CircFAT1 knockdown group,negative control group,and CircFAT1 knockdown+miR-296-3p inhibitor group.After transfection,the expressions of CircFAT1,miR-296-3p and MAPRE1 were detected with real-time fluorescent quantitative PCR and Western blotting.The proliferation and apoptosis of CNE2 cells in each group were detected with CCK-8 assay,5-Ethynyl-2’-deoxyuridine_(Edu)staining and flow cytometry.CNE2-RR cells cultured in vitro were randomly divided into the control group,radiation group,radiation+negative control group,radiation+CircFAT1 knockdown group,and radiation+CircFAT1 knockdown+miR-296-3p inhibitor group.After transfection,the expressions of CircFAT1,miR-296-3p and MAPRE1 were detected with real-time fluorescent quantitative PCR and Western boltting;the proliferation and apoptosis of CNE2-RR cells in each group were detected with CCK-8 assay,Edu staining and flow cytometry.The targeting regulation of CircFAT1 on miR-296-3p and miR-296-3p on MAPRE1 were detected with double luciferase reporter gene assay.Results Compared with NP69 cells,CNE2 and CNE2-RR cells had increased expression of CircFAT1 and mRNA and protein expressions of MAPRE1(P<0.05),but decreased expression of miR-296-3p(P<0.05).Compared with CNE2 cells,CNE2-RR cells had increased mRNA and protein expressions of CircFAT1 and MAPRE1(P<0.05),but decreased expression of miR-296-3p(P<0.05).Compared with the control group,the CircFAT1 knockdown group had decreased mRNA and protein expressions of CircFAT1 and MAPRE1,and reduced survival rate and proliferation rate(P<0.05),but increased expression of miR-296-3p and apoptosis rate(P<0.05).There were no significant changes in the indexes of the negative control group(P>0.05);miR-296-3p inhibitor reversed the effects of CircFAT1 knockdown on the indexes.Compared with the control group and radiation group,the radiation+CircFAT1 knockdown group had decreased mRNA and protein expressions of CircFAT1 and MAPRE1,and reduced survival rate and proliferation rate(P<0.05),but increased expression of miR-296-3p and apoptosis rate(P<0.05).There were no significant changes in the cell indexes of the radiation group and radiation+negative control group(P>0.05);miR-296-3p inhibitor reversed the effects of CircFAT1 knockdown on the indicators of CNE2-RR cells under radiation treatment.CircFAT1 targeted and downregulated the expression of miR-296-3p,and miR-296-3p targeted and downregulated the expression of MAPRE1 in CNE2-RR cells.Conclusion Knockdown of CircFAT1 can reduce the expression of MAPRE1 by upregulating miR-296-3p,thereby inhibiting the proliferation,promoting the apoptosis,and enhancing the radiosensitivity of nasopharyngeal carcinoma cells.