【摘要】 该研究探讨长链非编码RNA(LncRNA)胰岛素样生长因子家族成员2反义RNA 1(IGFL2-AS1)调节miR-138-5p/叉头蛋白4(FOXP4)轴对肺癌细胞恶性生物学行为的影响。该研究收集沧州市人民医院2023年6月至2024年12月手术切除的肺癌组织及癌旁组织标本各55例。该研究培养人正常上皮肺细胞(BEAS-2B)和肺癌细胞系(A549、NCI-H1975、PC-9),将A549细胞随机分为Control组、sh-NC组、sh-IGFL2-AS1组、sh-IGFL2-AS1+anti-NC组、sh-IGFL2-AS1+anti-miR-138-5p组。该研究采用qRT-PCR检测细胞中LncRNA IGFL2-AS1、miR-138-5p、FOXP4 mRNA表达水平,双荧光素酶实验检测LncRNA IGFL2-AS1与miR-138-5p、miR-138-5p与FOXP4之间关系,CCK8和克隆形成实验测定细胞增殖水平, Transwell分析细胞迁移和侵袭能力,流式细胞仪检测各组细胞凋亡情况, Western blot检测E-cadherin、Vimentin、Bcl-2、Bax、FOXP4蛋白表达情况。将裸鼠分为对照组(皮下注射10~6个sh-NC组细胞)和实验组(皮下注射10~6个sh-IGFL2-AS1组细胞),荷瘤裸鼠实验验证敲除LncRNA IGFL2-AS1对肺癌肿瘤生长及LncRNA IGFL2-AS1、miR-138-5p、FOXP4 mRNA表达的影响。qRT-PCR结果显示,相比癌旁组织,肺癌组织中LncRNA IGFL2-AS1及FOXP4 mRNA表达水平升高, miR-138-5p表达水平下降(P<0.05)。相比正常上皮肺细胞,肺癌细胞系(A549、NCI-H1975、PC-9)中LncRNA IGFL2-AS1及FOXP4 mRNA相对表达水平升高、miR-138-5p相对表达水平降低,且A549细胞中LncRNA IGFL2-AS1、miR-138-5p、FOXP4 mRNA表达变化最为明显(P<0.05),因此选用该细胞开展进一步研究。双荧光素酶实验结果显示,转染WTIGFL2-AS1、WT-FOXP4后,相比mimic-NC组, miR-138-3p mimic组荧光素酶活性下降(P<0.05)。相比sh-NC、Control组, sh-IGFL2-AS1组存活率、迁移数、克隆数、侵袭数、FOXP4 mRNA水平以及Vimentin、Bcl-2、FOXP4蛋白水平下降,细胞凋亡率、miR-138-5p、E-cadherin和Bax水平上升(P<0.05);相比sh-IGFL2-AS1组、sh-IGFL2-AS1+anti-NC组, sh-IGFL2-AS1+anti-miR-138-5p组存活率、迁移数、克隆数、侵袭数、FOXP4 mRNA水平以及Vimentin、Bcl-2、FOXP4蛋白水平上升,细胞凋亡率、miR-138-5p、E-cadherin和Bax水平下降(P<0.05)。实验组肿瘤质量和体积及LncRNA IGFL2-AS1、FOXP4 mRNA表达水平均低于对照组, miR-138-5p表达水平高于对照组(P<0.05)。总之,干扰LncRNA IGFL2-AS1可能通过影响mi R-138-5p/FOXP4轴,进而诱导肺癌细胞凋亡,并阻滞肺癌细胞迁移、增殖和侵袭,最终逆转其恶性表型。更多还原

【Abstract】 This research was to investigate the effect of LncRNA(long non coding RNA) IGFL2-AS1(insulin-like growth factor family member 2 antisense RNA 1) on the malignant biological behavior of lung cancer cells by regulating the miR-138-5p/FOXP4(forkhead protein 4) axis. This research adopted a total of 55 lung cancer tissues and paracancer tissue specimens collected from June 2023 to December 2024 in Cangzhou People’s Hospital. This research adopted human normal epithelial lung cells(BEAS-2B) and lung cancer cell lines(A549, NCI-H1975, PC-9). A549 cells were assigned into Control group, sh-NC group, sh-IGFL2-AS1 group, sh-IGFL2-AS1+anti-NC group, and sh-IGFL2-AS1+anti-miR-138-5p group randomly. qRT-PCR was applied to detect the expression levels of LncRNA IGFL2-AS1, miR-138-5p, and FOXP4 mRNA in cells. The relationship between LncRNA IGFL2-AS1 and miR-138-5p, between miR-138-5p and FOXP4 were detected by dual luciferase assay. Cell proliferation in each group was detected by CCK8 and clone formation assays. Transwell was applied to detect cell migration and invasion in each group. Flow cytometry was applied to detect apoptosis in each group. Western blot was applied to detect the expression levels of E-cadherin, Vimentin, Bcl-2, Bax, and FOXP4 proteins in each group. Nude mice were divided into control group(subcutaneous injection of 10~6 sh-NC group cells) and experimental group(subcutaneous injection of 10~6 sh-IGFL2-AS1 group cells). The effect of LncRNA IGFL2-AS1 deletion on the growth of lung cancer and the expression of LncRNA IGFL2-AS1, miR-138-5p and FOXP4 mRNA were verified in nude mice. This research concluded that qRT-PCR results showed that compared with the paracancer tissue, the expression of LncRNA IGFL2-AS1 and FOXP4 mRNA in lung cancer tissues were increased, while the expression of miR-138-5p was decreased(P<0.05). Compared with normal epithelial lung cells, the relative expression levels of LncRNA IGFL2-AS1 and FOXP4 mRNA in lung cancer cell lines(A549, NCI-H1975, PC-9) increased, while the relative expression level of miR-138-5p decreased. The expression levels of LncRNA IGFL2-AS1, miR-138-5p, and FOXP4 mRNA in A549 cells showed the most great differences(P<0.05), therefore A549 cells were selected for subsequent experiments. Dual luciferase assay showed that after transfection with WT-IGFL2-AS1 and WT-FOXP4, compared with the mimic-NC group, the luciferase activity of the miR-138-3p mimic group decreased(P<0.05). Compared with the sh-NC and Control groups, the survival rate, clone number, migration number, invasion number, FOXP4 mRNA level, and the protein expression levels of Vimentin, Bcl-2, FOXP4 of A549 cells in the sh-IGFL2-AS1 group decreased, while the apoptosis rate, miR-138-5p, the levels of E-cadherin, and Bax protein increased(P<0.05). Compared with the sh-IGFL2-AS1 group and sh-IGFL2-AS1+anti-NC group, the survival rate, clone number, migration number, invasion number, FOXP4 mRNA level, and Vimentin, Bcl-2, FOXP4 protein expression levels of A549 cells in the sh-IGFL2-AS1+anti miR-138-5p group increased, while the apoptosis rate, miR-138-5p, E-cadherin, and Bax protein expression levels decreased(P<0.05). The tumor mass and volume, LncRNA IGFL2-AS1 and FOXP4 mRNA expressions in the experimental group were lower than those of the control group, and miR-138-5p expression was higher than that in control group(P<0.05). In summary, interfering with LncRNA IGFL2-AS1 may affect the miR-138-5p/FOXP4 axis, thereby inducing apoptosis of lung cancer cells, blocking the migration, proliferation and invasion of lung cancer cells, and ultimately reversing their malignant phenotype.更多还原