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目的:探讨P53抑制肺腺癌A549细胞干细胞特性及其机制。方法:收集2014年3月至2015年12月解放军第85医院胸外科手术切除的30例非小细胞肺癌(non-small cell lung carcinoma,NSCLC)患者癌组织及癌旁组织,采用Real-time PCR检测NSCLC组织和癌旁组织中miR-145的表达。构建人P53基因的真核表达载体(pcDNA3.1-P53)和突变载体[pcDNA3.1-P53R273H(CGT-CAT)],设计靶向P53基因的siRNA,分别转染A549细胞;细胞分为对照转染组(Vector或NCsiRNA)和实验组(Flag-P53或P53-siRNA);Western blotting检测P53蛋白过表达和干扰效率,Real-time PCR检测OCT4和miR-145的表达,荧光双报告基因和Western blotting技术检测证实A549细胞中干细胞多能性调节基因(OCT4)为miR-145的靶标分子。结果:NSCLC组织中miR-145的表达水平明显低于癌旁组织[(2.31±0.13)vs(3.51±0.27),P<0.01];P53明显促进A549细胞中miR-145的表达[(1.84±0.14)vs(1.00±0.00),P<0.01];miR-145 mimics显著抑制A549细胞中pGL3-OCT4-3′-UTR活性(P<0.01)和OCT4蛋白表达水平(P<0.05),而转染miR-145抑制剂可进一步增加OCT4表达水平,且逆转P53对A549细胞的干细胞特性的抑制作用。结论:P53通过促进miR-145表达下调OCT4表达,进而明显抑制A549细胞的干细胞特性,该结果为肺腺癌的临床诊断和治疗提供了新途径。
Objective: To investigate the effect of P53 on inhibiting lung adenocarcinoma A549 cells and its mechanism. Methods: Tumor samples from 30 patients with non-small cell lung carcinoma (NSCLC) resected from Department of Thoracic Surgery, PLA 85 Hospital from March 2014 to December 2015 were collected. Real-time PCR The expression of miR-145 in NSCLC tissues and adjacent tissues was detected. The eukaryotic expression vector (pcDNA3.1-P53) and the mutant vector [pcDNA3.1-P53R273H (CGT-CAT)] of human P53 gene were constructed and siRNAs targeting P53 gene were designed and transfected into A549 cells respectively. Transfection group (Vector or NCsiRNA) and experimental group (Flag-P53 or P53-siRNA); Western blotting detection of P53 protein overexpression and interference efficiency, Real-time PCR detection of OCT4 and miR-145 expression, fluorescent double reporter gene and Western blotting assay confirmed that the stem cell pluripotency regulator (OCT4) in A549 cells was the target of miR-145. Results: The expression of miR-145 in NSCLC tissues was significantly lower than that in paracancer tissues [(2.31 ± 0.13) vs (3.51 ± 0.27), P <0.01]. P53 significantly promoted the expression of miR-145 in A549 cells [(1.84 ± 0.14) vs (1.00 ± 0.00), P <0.01]. MiR-145 mimics significantly inhibited the activity of pGL3-OCT4-3’-UTR and the expression of OCT4 protein in A549 cells (P <0.05) Staining miR-145 inhibitors further increases OCT4 expression and reverses the inhibitory effect of P53 on the stem cell properties of A549 cells. CONCLUSION: P53 can down-regulate the expression of OCT4 by up-regulating the expression of miR-145, thereby significantly inhibiting the stem cell characteristics of A549 cells. This result provides a new approach for the clinical diagnosis and treatment of lung adenocarcinoma.