罗哌卡因通过circ_0044516/微小RNA-198通路调控肺癌A549细胞增殖、迁移和侵袭的实验研究

Ropivacaine regulates proliferation,migration and invasion of lung cancer A549 cells through the circ_0044516/microRNA-198 pathway

目的探讨罗哌卡因对肺癌A549细胞增殖、迁移和侵袭的影响及其机制。方法2019年1月至2020年4月,将体外培养A549细胞(购自中国科学院上海细胞库)分为对照组、不同剂量[25、50、100 mg/L,罗哌卡因组(Rop组)]、乱序无意义阴性序列组(si-NC组)、si-circ_0044516组、Rop+pcDNA组和Rop+pcDNA-circ_0044516组,细胞计数试剂盒(CCK-8)法检测细胞增殖,Transwell检测细胞迁移和侵袭,蛋白质印迹法(Western blot)检测细胞中细胞核增殖抗原(Ki-67)、基质金属蛋白酶(MMP)-2和MMP-9蛋白表达,实时定量反转录聚合酶链反应(RT-qPCR)法检测circ_0044516和微小RNA(miRNA,miR)-198表达。双荧光素酶报告基因实验验证circ_0044516和miR-198调控关系。两组间比较行t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果不同剂量Rop组A549细胞抑制率均高于对照组[(22.54±2.11)%、(47.01±4.28)%、(65.84±4.63)%比(0.00±0.00)%,F=670.553,P<0.05],细胞迁移数[(82.08±4.60)、(63.02±4.34)、(47.94±3.85)个比(105.64±11.21)个,F=123.952,P<0.05]、侵袭数[(70.94±5.08)、(53.63±4.11)、(31.01±3.34)个比(93.00±6.25)个,F=267.501,P<0.05]及Ki-67(0.62±0.05、0.46±0.04、0.32±0.03比0.75±0.04,F=191.409,P<0.05)、MMP-2(0.41±0.03、0.28±0.03、0.13±0.02比0.59±0.04,F=361.500,P<0.05)和MMP-9(0.67±0.05、0.52±0.03、0.36±0.03比0.82±0.06,F=177.835,P<0.05)的蛋白表达、circ_0044516表达均低于对照组(0.81±0.06、0.61±0.05、0.45±0.04比1.00±0.06,F=182.097,P<0.05),miR-198表达高于对照组(1.64±0.12、2.16±0.20、2.77±0.23比1.00±0.05,F=185.997,P<0.05),差异均有统计学意义。si-circ_0044516组细胞抑制率高于si-NC组[(51.27±4.11)%比(5.69±0.46)%,t=33.064,P<0.05],迁移数[(55.57±4.04)个比(107.65±10.84)个,t=13.506,P<0.05]、侵袭数[(43.02±4.08)个比(92.42±7.84)个,t=16.768,P<0.05]及Ki-67[0.40±0.03比0.78±0.05,t=19.551,P<0.05]、MMP-2(0.21±0.03比0.58±0.04,t=22.200,P<0.05)和MMP-9(0.42±0.04比0.86±0.06,t=18.305,P<0.05)的蛋白表达均低于si-NC组,差异均有统计学意义。circ_0044516靶向负调控miR-198。Rop+pcDNA-circ_0044516组细胞抑制率低于Rop+pcDNA组[(28.57±2.49)%比(67.48±4.78)%,t=21.658,P<0.05],迁移数[(88.57±5.31)个比(46.35±4.33)个,t=18.486,P<0.05]、侵袭数[(74.57±5.97)个比(28.26±2.19)个,t=21.848,P<0.05]及Ki-67(0.64±0.04比0.31±0.03,t=19.800,P<0.05)、MMP-2(0.46±0.04比0.12±0.02,t=22.808,P<0.05)和MMP-9(0.73±0.06比0.33±0.03,t=17.889,P<0.05)的蛋白表达均高于Rop+pcDNA组,差异均有统计学意义。结论Rop可能通过调控circ_0044516/miR-198轴抑制肺癌A549细胞增殖、迁移和侵袭。

Objective To investigate the effect of ropivacaine(Rop)on the proliferation,migration and invasion of lung cancer A549 cells and its mechanism.Methods The study time was from January 2019 to April 2020.A549 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.A549 cells were cultured in vitro and divided into control group,different doses(25,50,100 mg/L)Rop groups,out-of-order nonsense negative sequence(si-NC)group,si-circ_0044516 group,Rop+pcDNA group and Rop+pcDNA-circ_0044516 group.The cell proliferation was detected by cell counting kit-8(CCK-8)assay,cell migration and invasion were examined by Transwell,the protein expression of proliferation cell nuclear antigen(Ki-67),matrix metalloproteinase(MMP)-2 and MMP-9 in A549 cells was detected by Western blotting,and the expression of circ_0044516 and microRNA(miRNA,miR)-198 was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The dual luciferase reporter gene experiment verified the regulatory relationship between circ_0044516 and miR-198.The two groups were compared by t test;the comparison between multiple groups was performed by one-way analysis of variance,and the pairwise comparison between groups was performed by least significant difference t(LSD-t)test.Results Compared with the control group,the inhibition rate of A549 cells in the Rop groups with different doses increased[(22.54±2.11)%,(47.01±4.28)%,(65.84±4.63)%to(0.00±0.00)%,F=670.553,P<0.05],the number of migrating cells(82.08±4.60,63.02±4.34,47.94±3.85 vs.105.64±11.21,F=123.952,P<0.05),the number of invading cells(70.94±5.08,53.63±4.11,31.01±3.34 vs.93.00±6.25,F=267.501,P<0.05)and the protein expression of Ki-67(0.62±0.05,0.46±0.04,0.32±0.03 vs.0.75±0.04,F=191.409,P<0.05),MMP-2(0.41±0.03,0.28±0.03,0.13±0.02 vs.0.59±0.04,F=361.500,P<0.05)and MMP-9(0.67±0.05,0.52±0.03,0.36±0.03 vs.0.82±0.06,F=177.835,P<0.05)decreased,and the expression of circ_0044516 decreased(0.81±0.06,0.61±0.05,0.45±0.04 vs.1.00±0.06,F=182.097,P<0.05),while the expression of miR-198 increased(1.64±0.12,2.16±0.20,2.77±0.23 vs.1.00±0.05,F=185.997,P<0.05).Compared with the si-NC group,the inhibition rate of the si-circ_0044516 group increased[(51.27±4.11)vs.(5.69±0.46),t=33.064,P<0.05],the number of migrating cells(55.57±4.04 vs.107.65±10.84,t=13.506,P<0.05),the number of invading cells(43.02±4.08 vs.92.42±7.84,t=16.768,P<0.05)and the protein expression of Ki-67(0.40±0.03 vs.0.78±0.05,t=19.551,P<0.05),MMP-2(0.21±0.03 vs.0.58±0.04,t=22.200,P<0.05)and MMP-9(0.42±0.04 vs.0.86±0.06,t=18.305,P<0.05)decreased.The circ_0044516 negatively regulated the expression of miR-198.Compared with the Rop+pcDNA group,the cell inhibition rate of the Rop+pcDNA-circ_0044516 group was reduced[(28.57±2.49)%vs.(67.48±4.78)%,t=21.658,P<0.05],the number of migrating cells(88.57±5.31 vs.46.35±4.33,t=18.486,P<0.05),the number of invading cells(74.57±5.97 vs.28.26±2.19,t=21.848,P<0.05)and the protein expression of Ki-67(0.64±0.04 vs.0.31±0.03,t=19.800,P<0.05),MMP-2(0.46±0.04 vs.0.12±0.02,t=22.808,P<0.05)and MMP-9(0.73±0.06 vs.0.33±0.03,t=17.889,P<0.05)increased.Conclusion Rop may inhibit the proliferation,migration and invasion of lung cancer A549 cells by regulating the circ_0044516/miR-198 axis.