N-myc下游调节基因对胰腺癌细胞增殖、侵袭及迁移的影响及其机制研究

The role of NDRG1 in the proliferation, invasion and migration of pancreatic cancer cells and its mechanisms

摘要:

目的 探究N-myc下游调节基因（NDRG1）对胰腺癌细胞增殖、侵袭及迁移的影响，并分析可能的机制。方法 体外培养人胰腺癌细胞系MZ-3262细胞，并分为空白对照组（细胞不做特殊处理）、pcDNA-NC组（细胞转染pcDNA-NC）、pcDNA-NDRG1组（细胞转染pcDNA-NDRG1）、通路抑制剂组［转染pcDNA-NDRG1后加入含终浓度为1 mmol/L 磷脂肌醇3-激酶（PI3K）通路激活剂bpv的培养液］；采用实时荧光定量聚合酶链反应检测NDRG1 mRNA的表达；CCK-8法检测细胞存活情况；划痕实验、Transwell实验分别检测细胞迁移、侵袭能力；Western blotting检测NDRG1、增殖及侵袭迁移相关蛋白［细胞增殖相关核抗原（PCNA）、E-钙黏附蛋白（E-Cadherin）、N-钙黏附蛋白（N-Cadherin）、波形蛋白（Vimentin）］及蛋白激酶B/转录激活因子3（Akt/STAT3）通路蛋白［PI3K、p-PI3K、Akt、p-Akt、STAT3］的表达。结果 与空白对照组、pcDNA-NC组比较，pcDNA-NDRG1组、通路激活剂组MZ-3262细胞NDRG1 mRNA和蛋白、E-Cadherin蛋白相对表达量升高（P <0.05），细胞存活率、侵袭细胞数、划痕愈合率、PCNA、N-Cadherin、Vimentin蛋白及p-PI3K/PI3K、p-Akt/Akt、p-STAT3/STAT3相对表达量降低（P <0.05）；但通路激活剂组MZ-3262细胞NDRG1 mRNA和蛋白、E-Cadherin蛋白相对表达量低于pcDNA-NDRG1组（P <0.05），细胞存活率、侵袭细胞数、划痕愈合率、PCNA、N-Cadherin、Vimentin蛋白及p-PI3K/PI3K、p-Akt/Akt、p-STAT3/STAT3相对表达量高于pcDNA-NDRG1组（P <0.05）。结论 过表达NDRG1表达可能通过抑制PI3K/Akt/STAT3通路活化，抑制胰腺癌MZ-3262细胞增殖、侵袭及迁移。

Abstract:

Objective To investigate the role of N-Myc downstream-regulated gene 1 (NDRG1) in the proliferation, invasion and migration of pancreatic cancer (PC) cells, and to analyze the potential mechanisms.Methods The human PC MZ-3262 cells were cultured in vitro and divided into control group (no special treatment), pcDNA-NC group (transfected with pcDNA-NC), pcDNA-NDRG1 group (transfected with pcDNA-NDRG1) and pathway activation group (transfected with pcDNA-NDRG1 and cultured in the medium containing 1 mmol/L of phosphatidylinositol 3-kinase (PI3K) pathway activator bpv). The mRNA expression level of NDRG1 was measured via quantitative real-time polymerase chain reaction (qRT-PCR). The cell survival was detected via CCK-8, and the migration and invasion abilities of the cells were detected via scratch assay and transwell assay. The protein levels of NDRG1, molecules associated with proliferation, invasion and migration including proliferating cell nuclear antigen (PCNA), E-cadherin, N-cadherin and vimentin, and those associated with Akt/STAT3 pathway including PI3K, p-PI3K, Akt, p-Akt, and STAT3, were detected via Western blotting.Results The mRNA expression of NDRG1 and protein expressions of NDRG1 and E-Cadherin in MZ-3262 cells were higher in the pcDNA-NDRG1 and pathway activation group than those in the control group and pcDNA-NC group (P < 0.05). The cell survival rate, the number of invasive cells, the rate of gap closure, the protein expressions of PCNA, N-cadherin, and vimentin, and the relative expressions of P-PI3K/PI3K, p-Akt/Akt, and p-STAT3/STAT3 were lower in the pcDNA-NDRG1 and the pathway activation group than those in the control group and pcDNA-NC group (P < 0.05). However, mRNA expression of NDRG1 and protein expressions of NDRG1 and E-cadherin in MZ-3262 cells were lower in the pathway activation group compared with those in the pcDNA-NDRG1 group (P < 0.05). Besides, the cell survival rate, the number of invasive cells, the rate of gap closure, the protein expressions of PCNA, N-cadherin, and vimentin, and the relative expressions of P-PI3K/PI3K, p-Akt/Akt, and p-STAT3/STAT3 were higher in the pathway activation group than those in the pcDNA-NDRG1 group (P < 0.05).Conclusions Overexpression of NDRG1 may inhibit the proliferation, invasion and migration of MZ-3262 cells by attenuating the activation of PI3K/Akt/STAT3 pathway.