贫氘水抑制肺癌细胞增殖的miRNAs差异表达谱分析及功能研究

Some reports suggest that Deuterium depleted water(DDW) treatment, when integrated into other forms of conventional cancer therapy, can significantly decreased the growth rate of various tumor cells and also block the metastasis and recurrence in vitro and in vivo, however the underlying mechanisms remain unclear. In our previous investigation, we found that DDW with 50 ppm significantly inhibited the ability of cell proliferation cloned formation and migration. Furthermore,we preliminarily evaluated the miRNA expression profiles of A549 cell lines with or without DDW(50ppm) treatment using the Affymetrix microRNA array and we also revealed a number of potential tumor suppressor miRNAs and lots of novel miRNAs. In this study, we will address the decrease of D concerntration(5~100ppm) can intervene in the signal transduction pathways thus inducing apoptosis in lung cancer cell lines originated from different tissues(A549/H1650/DMS53). Further, we will investigate the expression pattern of potential tumor suppressor miRNAs(such as miRNA-34a、miR-106a、miR-211、miR-17~92) through potential targeting PTEN or P53. and identifying that down-regulation of ATP-binding cassette C1 protein expression in lung cancer cells after DDW(50ppm) treatment is mediated by microRNA-1291.

体内外研究表明贫氘水作为多种癌症的辅助治疗剂可抑制癌组织生长、转移与复发，但分子机制有待阐明。课题组希望在已经开展（50~100ppm）贫氘水对多种肿瘤细胞生长有明确抑制作用和初步差异miRNAs表达谱分析的前期工作基础上，降低氘浓度对肺癌不同组织类型细胞株（肺腺癌细胞株A549、肺泡细胞癌细胞株H1650及小细胞肺癌细胞株DMS53）抑制增殖作用，进一步分析随着贫氘水（D/H）比值变化而抑制肺癌细胞增殖的过程中，miRNAs表达谱差异表达规律；候选显著差异表达miRNA-34a、miR-106a、miR-211、miR-17~92调控抑癌靶基因P53、PTEN表达的分子机制；明确miR-1291是否能通过靶击ATP酶，调控多药耐药蛋白MRP1/ABCC1的表达，增加肺癌细胞化疗的药敏性。

本项目拟探讨贫氘水抗肺癌作用的相关分子机制，研究发现随着培养基中氘含量下降，肺癌A549和A549/DDP细胞增殖显著被抑制(P＜0.05)，克隆生成和游走侵袭能力显著下降(P＜0.01)，但对前成骨细胞和16HBE细胞生长无明显抑制作用；流式细胞仪检测显示，贫氘水能诱导肺癌A549细胞生长周期阻滞：S期减少，G1期显著增加（P＜0.05），而对正常细胞生长周期无明显抑制；ATP酶试剂盒检测结果显示贫氘水能显著抑制A549细胞Na+ K+-ATP酶活力(P＜0.01)、Ca++ Mg++-ATP酶活力(P＜0.01)和T-ATP酶活力(P＜0.001)；western-blot 检测结果显示贫氘水能引起PTEN、P-PTEN、Non-p-PTEN和NQO1蛋白表达水平显著上调，MRP1、VEGF、ATP、PDK1、P-PDK1和PCNA蛋白表达水平显著下调；免疫荧光结果发现贫氘水能降低ATP、VEGF、MMP9和PCNA的表达，升高NQO1和Nrf2的表达。Affymetrix microRNA芯片比较贫氘水（50ppm）与普通水（150ppm）培养肺癌细胞A549后，miRNA表达谱差异结果显示差异倍数在两倍以上的miRNA有182个，其中96个高表达，86个低表达；QPCR验证贫氘水对肺癌A549细胞的miRNA差异表达谱中miR-1291、miR-143、miR-15b和let-7b的表达，结果显示miR-1291升高16.89倍，miR-143升高2.29倍，miR-15b升高6.69倍和let-7b升高13.88倍；QPCR检测贫氘水对肺癌A549/DDP细胞的miR-1291、miR-143、miR-15b和let-7b表达，结果显示miR-143升高3.05倍，miR-1291升高3.43倍，miR-15b和let-7b的表达变化不明显；进一步功能分析结果显示miR-1291、miR-143、miR-15b和let-7b调控的靶基因与肿瘤增殖、凋亡、转移和多药耐药等相关；通过对33例非癌组织与100例肺癌患者组织切片的免疫组化分析，PTEN、ATP蛋白的表达水平与肺癌患者的T分期、N分期、TNM分期和预后相关。