LINC02381靶向miR-4500/CCNE2对肺腺癌细胞生物学行为的影响

Effects of LINC02381 silencing on the miR-4500 and CCNE2 expression and biological behavior of lung adenocarcinoma cells

    摘要
  • 摘要:
    目的 探讨LINC02381是否可调控miR-4500/CCNE2轴以影响肺腺癌细胞生物学行为。
    方法 收集2022年6月至2024年5月于复旦大学附属华山医院治疗41例肺腺癌患者的癌组织和癌旁组织，RT-PCR检测肺腺癌组织和细胞中LINC02381、miR-4500和CCNE2基因的表达水平；将肺腺癌细胞Calu-3分为对照组、si-NC组、si-LINC02381组、si-LINC02381+inhibitor NC组、si-LINC02381+miR-4500 inhibitor组、si-LINC02381+oe-NC组，si-LINC02381+oe-CCNE2组；qRT-PCR检测各组细胞中LINC02381、miR-4500和CCNE2基因表达水平；CCK-8法检测细胞增殖；细胞划痕实验检测细胞迁移；Transwell检测细胞侵袭；流式细胞术检测细胞凋亡率；Western blot检测细胞中E-cadherin、N-cadherin、vimentin、cleaved caspase-3、PCNA、MMP-2、CCNE2蛋白表达水平；双荧光素酶报告实验验证miR-4500与LINC02381和CCNE2的靶向关系。
    结果 肺腺癌组织和细胞中LINC02381和CCNE2 基因表达水平升高，miR-4500表达水平降低；与对照组和si-NC组相比，si-LINC02381组Calu-3细胞中LINC02381和CCNE2基因表达水平、OD450（24 h、48 h）值、划痕愈合率和细胞侵袭个数、N-cadherin、vimentin、PCNA、MMP-2、CCNE2蛋白表达水平降低，miR-4500表达、细胞凋亡率、E-cadherin和cleaved caspase-3蛋白表达水平升高（P<0.05）；降低miR-4500表达或提高CCNE2表达均可减弱沉默LINC02381表达对Calu-3细胞生物学行为的抑制作用（P<0.05）。
    结论 沉默LINC02381表达可提高miR-4500表达，抑制CCNE2表达来抑制肺腺癌细胞增殖和迁移，并促进细胞凋亡。

     

    Abstract:
    Objective To investigate whether LINC02381 impacts the biological behavior of lung adenocarcinoma cells by regulating the miR-4500/CCNE2 axis.
    Methods Cancer and paracancerous tissues were collected from 41 patients with lung adenocarcinoma treated at Huashan Hospital Affiliated to Fudan University, from June 2022 to May 2024. RT-PCR was used to detect the expression levels of LINC02381, miR-4500, and CCNE2 in lung adenocarcinoma tissues and cells. Calu-3 lung adenocarcinoma cells were divided into the control, si-NC, si-LINC02381, si-LINC02381+inhibitor NC, si-LINC02381+miR-4500 inhibitor, si-LINC02381+oe-NC, and si-LINC02381+oe-CCNE2 groups. qRT-PCR was used to determine the expression levels of LINC02381, miR-4500, and CCNE2 in each group of cells. The CCK-8 assay was used to detect cell proliferation. A monolayer scratch assay was performed to detect cell migration. The Transwell assay was used to detect cell invasion. Flow cytometry was performed to determine the apoptosis rate. Western blot was performed to detect E-cadherin, N-cadherin, vimentin, cleaved caspase-3, PCNA, MMP-2, and CCNE2 protein levels in the cells. The dual-luciferase reporter assay was used to verify the relationship among miR-4500, LINC02381, and CCNE2.
    Results  LINC02381 and CCNE2 expression was increased, whereas miR-4500 expression was decreased in lung adenocarcinoma tissues and cells. Compared with those in the control and si-NC groups, LINC02381 and CCNE2 expression, the OD450 (24 and 48 h) values, scratch healing rate, number of invading cells, N-cadherin, vimentin, PCNA, MMP-2, and CCNE2 protein levels in Calu-3 cells in the si-LINC02381 group were reduced, whereas miR-4500 expression levels, apoptosis rate, E-cadherin, and cleaved caspase-3 protein levels were increased (P< 0.05). Reducing miR-4500 expression or increasing CCNE2 expression weakened the inhibitory effects of LINC02381 silencing on the biological behavior of Calu-3 cells (P< 0.05).
    Conclusions  LINC02381 silencing can result in increased miR-4500 expression, inhibition of CCNE2 expression, suppression of lung adenocarcinoma cell proliferation and migration, and promotion of apoptosis.

     

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