miR-146b-3p通过靶向结合LCN1抑制肺鳞癌生物学行为

miR-146b-3p inhibiting the biological behavior of lung squamous cell carcinoma through targeted binding to LCN1

目的探讨miR-146b-3p通过靶向结合脂质运载蛋白1(LCN1)抑制肺鳞癌生物学行为的效果。方法构建稳定过表达miR-146b-3p和稳定敲低LCN1的NCI-H2170和NCI-H226细胞系,CCK-8法和克隆实验检测细胞增殖能力,流式细胞仪检测细胞周期和凋亡情况。生物信息学分析miR-146b-3p和LCN1与肺鳞癌临床病理特征的关系,并预测两者之间的靶向关系。双荧光素酶报告实验验证miR-146b-3p和LCN1的靶向关系。RT-PCR检测miR-146b-3p在肺鳞癌细胞中的表达。裸鼠成瘤实验检测肿瘤体质量、体积和肺鳞癌组织中miR-146b-3p的表达。Western blot检测细胞蛋白表达。结果miR-146b-3p在肺鳞癌组织中低表达,肺鳞癌细胞miR-146b-3p过表达后,细胞增殖能力降低,克隆数减少,细胞周期受到阻滞,细胞凋亡率增加,裸鼠移植瘤生长受到抑制,BCL2、CCNB1和CDK2蛋白减少,BAX蛋白增加,差异均有统计学意义(P<0.05)。LCN1在肺鳞癌组织中高表达,敲低LCN1表达可抑制肺鳞癌细胞增殖,影响细胞周期,促进细胞凋亡,差异均有统计学意义(P<0.05)。生物信息学分析显示,LCN1是miR-146b-3p的靶基因;双荧光素酶报告实验显示miR-146b-3p mimic与LCN1-WT共转染使荧光素酶活性降低(P<0.05)。肺鳞癌细胞共转染miR-146b-3p和LCN1后,可以逆转miR-146b-3p抑制细胞增殖和克隆、促凋亡的作用。结论miR-146b-3p可能是通过下调LCN1表达抑制肺鳞癌细胞增殖,影响细胞周期,促进细胞凋亡。

Objective To explore the effect of miR-146b-3p in inhibiting the biological behavior of lung squamous cell carcinoma by targeted binding to lipocalin-1(LCN1).Methods NCI-H2170 and NCI-H226 cell lines with stable overexpression of miR-146b-3p and stable knockdown of LCN1 were constructed.The cell proliferation was detected by CCK-8 assay and clone formation assay.The cell cycle and apoptosis were detected by flow cytometry.Bioinformatics analysis was used to analyze the relationships of miR-146b-3p and LCN1 with clinicopathological features of lung squamous cell carcinoma,and predict the targeted relationship of miR-146b-3p and LCN1.The targeted relationship between miR-146b-3p and LCN1 was verified by dual-luciferase reporter assay.The expression of miR-146b-3p in lung squamous cell carcinoma cells was detected by RT-PCR.The mass and volume of tumors and expression of miR-146b-3p in lung squamous cell carcinoma tissues were detected by the tumor formation assay in nude mice.The expression of different proteins were detected by Western blot.Results The expression of miR-146b-3p was down-regulated in lung squamous cell carcinoma tissues.After overexpression of miR-146b-3p in lung squamous cell carcinoma cells,the cell proliferation ability decreased,the number of clones reduced,the cell cycle was blocked,the apoptosis rate increased,the growths of transplanted tumor in nude mice was inhibited,the protein expression of BCL2,CCNB1 and CDK2 decreased,and the protein expression of BAX increased,with statistically significant differences(P<0.05).The expression of LCN1 was up-regulated in lung squamous cell carcinoma tissues,knockdown LCN1 could inhibit the proliferation of lung squamous cell carcinoma cells,affect cells cycle and promote cell apoptosis,with statistically significant differences(P<0.05).Bioinformatics analysis showed that LCN1 was the targeted gene of miR-146b-3p.Dual-luciferase reporter assay showed that the luciferase activity was decreased after the co-transfection of miR-146b-3p mimic and LCN1-WT(P<0.05).After co-transfection of miR-146b-3p and LCN1,lung squamous cell carcinoma cells could reverse the effect of miR-146b-3p in inhibiting cell proliferation,cloning,and pro-apoptosis.Conclusion miR-146b-3p can inhibit the proliferation of lung squamous cell carcinoma cells,affect cells cycle and promote apoptosis by down-regulating the expression of LCN1.