摘要
研究lncRNA LINC00958对小细胞肺癌细胞的阿帕替尼(AP)耐药性的影响及其作用机制。采用逐步增加阿帕替尼浓度法建立小细胞肺癌阿帕替尼耐药细胞株H446/AP,分别用2.5μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L的阿帕替尼处理H446、H446/AP细胞,并检测阿帕替尼对其增殖抑制率的影响。然后将H446/AP细胞分为AP+si-NC组(转染LINC00958干扰载体阴性对照+5μmol/L阿帕替尼)、AP+si-LINC00958组(转染LINC00958干扰载体+5μmol/L阿帕替尼)、AP+miR-NC组(转染miR-490-3p模拟物阴性对照+5μmol/L阿帕替尼)、AP+miR-490-3p组(转染miR-490-3p模拟物+5μmol/L阿帕替尼)、AP+si-LINC00958+anti-miR-NC组(共转染LINC00958干扰载体和miR-490-3p抑制载体阴性对照+5μmol/L阿帕替尼)、AP+si-LINC00958+anti-miR-490-3p组(共转染LINC00958干扰载体和miR-490-3p抑制载体+5μmol/L阿帕替尼)。用实时荧光定量PCR(RT-qPCR)检测LINC00958和miR-490-3p的表达水平;CCK-8检测细胞增殖抑制率;Western blot检测蛋白表达水平;细胞划痕实验检测细胞划痕愈合率;Transwell检测细胞迁移和侵袭;双荧光素酶报告实验检测LINC00958和miR-490-3p的靶向关系。结果显示,耐药和非耐药小细胞肺癌组织中LINC00958高表达,miR-490-3p低表达(P<0.05);相较于H446细胞,H446/AP细胞中LINC00958表达水平显著升高,miR-490-3p表达水平显著降低(P<0.05)。不同浓度阿帕替尼处理后,H446/AP相较于H446细胞增殖抑制率显著降低,半数抑制浓度(IC50)显著升高(P<0.05)。抑制LINC00958表达联合阿帕替尼和miR-490-3p过表达联合阿帕替尼,H446/AP细胞中CyclinD1、MMP-2、MMP-9表达水平显著降低,H446/AP细胞中p21表达水平显著升高,H446/AP细胞抑制率显著升高,划痕愈合率降低,H446/AP细胞迁移和侵袭数量显著降低(P<0.05)。LINC00958靶向调控miR-490-3p,干扰miR-490-3p表达逆转了抑制LINC00958表达对H446/AP细胞阿帕替尼耐药性的抑制作用。因此,抑制LINC00958表达联合阿帕替尼可抑制H446/AP细胞增殖、迁移和侵袭,降低小细胞肺癌对阿帕替尼的耐药性,其机制可能与miR-490-3p表达有关。
This work aimed to study the effect and mechanism of lncRNA LINC00958 on the AP(apatinib)resistance of small cell lung cancer cells.The method of gradually increasing the concentration of apatinib was used to establish a small cell lung cancer cell line H446/AP resistant to apatinib,H446 and H446/AP cells were treated with 2.5μmol/L,5μmol/L,10μmol/L,20μmol/L,40μmol/L apapitinib to detect the inhibition rate of proliferation.Then the H446/AP cells were divided into AP+si-NC group(transfected with LINC00958 interference vector negative control+5μmol/L apatinib),AP+si-LINC00958 group(transfected with LINC00958 interference vector+5μmol/L apatinib),AP+miR-NC group(transfected miR-490-3p mimic negative control+5μmol/L apatinib),AP+miR-490-3p group(transfected miR-490-3p mimetic+5μmol/L apatinib),AP+si-LINC00958+anti-miR-NC group(cotransfected with LINC00958 interference vector and miR-490-3p inhibitor vector negative control+5μmol/L apatinb),AP+si-LINC00958+anti-miR-490-3p group(cotransfected with LINC00958 interference vector and miR-490-3p suppression vector+5μmol/L apatinib).RT-qPCR(real-time quantitative PCR)was used to detect the expression of LINC00958 and miR-490-3p;CCK-8(cell counting kit 8)was used to detect the inhibition rate of cell proliferation;Western blot was used to detect protein expressions;Scratch test to detect cell scratch healing rate;Transwell was used to detect cell migration and invasion;dual luciferase reporter assay was used to detect the targeting relationship between LINC00958 and miR-490-3p.The results show that LINC00958 was highly expressed in small cell lung cancer tissues,and miR-490-3p was lowly expressed(P<0.05).Compared with H446 cells,the expression of LINC00958 in H446/AP cells was significantly increased,while the expression of miR-490-3p was significantly decreased(P<0.05).After treatment with different concentrations of apatinib,the proliferation inhibition rate of H446/AP cells was significantly decreased compared with H446 cells,while IC50(half inhibitory concentration)was significantly increased(P<0.05).For inhibition of LINC00958 expression combined with apatinib and miR-490-3p overexpression combined with apatinib,CyclinD1,MMP-2,MMP-9 expression were significantly decreased in H446/AP cells;p21 expression was significantly increased in H446/AP cells,and H446/AP cell inhibition rate was significantly increased;scratch healing rate was decreased;the migration and invasion numbers of H446/AP cells were significantly decreased(P<0.05).LINC00958 targets miR-490-3p,and interference with miR-490-3p expression reverses the inhibitory effect of inhibition of LINC00958 expression on apatatin resistance of H446/AP cells.Therefore,inhibiting the expression of LINC00958 combined with apatinib can inhibit the proliferation,migration and invasion of H446/AP cells,reducing the resistance of small cell lung cancer to apatinib.The mechanism may be related to the expression of miR-490-3p.
作者
杨帆
荣腾浩
YANG Fan;RONG Tenghao(Department of Thoracic Surgery,Chongqing People’s Hospital,Chongqing General Hospital,University of Chinese Academy of Sciences,Chongqing 401100,China;Department of Cardiothoracic Surgery,Bishan District People’s Hospital,Chongqing 402460,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2020年第11期1931-1940,共10页
Chinese Journal of Cell Biology
