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shRNA沉默CCAT2对非小细胞肺癌耐顺铂细胞A549的影响

Effects of shRNA on Cisplatin-resistant Non-small Cell Lung Cancer Cell A549 via Silencing CCAT2

    摘要
  • 摘要:
      目的  研究短发夹RNA（short hairpin RNA，shRNA）沉默结肠癌相关转录因子2（colon cancer associated transcript 2, CCAT2）对非小细胞肺癌耐顺铂（cisplatin，DDP）细胞株（A549/DDP）的增殖、侵袭和凋亡以及体内肿瘤形成的影响。
      方法  shRNA-CCAT2（sh-CCAT2）或shRNA-阴性对照（shRNA-NC）转染A549/DDP细胞（并设未经处理的A549/DDP细胞为对照组），通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测3组A549/DDP细胞CCAT2 mRNA表达；通过MTT实验检测3组A549/DDP细胞经过不同质量浓度（0～8 mg/L）DDP处理后的增殖能力变化，并依此选定2 mg/L DDP进行后续实验。采用克隆形成实验、流式细胞法、Transwell实验检测2 mg/L DDP处理对各组细胞（并设未经处理的A549/DDP细胞为对照组）增殖、凋亡、侵袭的影响，用Western blot检测各组细胞增殖标记蛋白〔Ki67蛋白、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）〕、凋亡标记蛋白（Caspase-3、Caspase-9）、侵袭标记蛋白〔血管内皮生长因子（vascular endothelial growth factor，VEGF）、基质金属蛋白酶-14（matrix metalloproteinase-14，MMP-14）〕蛋白的表达。裸鼠皮下注射A549/DDP细胞、转染shRNA-NC和转染sh-CCAT2的 A549/DDP细胞，每3 d腹腔注射DDP 2 mg/kg，连续给药7次，另设对照组（皮下注射A549/DDP细胞，腹腔注射等量生理盐水）。检测每5 d肿瘤体积，共30 d。30 d后处死取肿瘤组织，RT-PCR检测CCAT2 mRNA表达，TUNEL染色检测肿瘤细胞凋亡。
      结果  与对照组和shRNA-NC转染组相比，sh-CCAT2转染A549/DDP细胞后，细胞CCAT2 mRNA表达降低（P<0.01），2～8 mg/L DDP作用后细胞增殖抑制更明显（P<0.01）。与对照组相比，DDP处理的3组细胞克隆形成减少，增殖标记蛋白Ki67和PCNA表达减少（P<0.01）；细胞凋亡率增加，凋亡标记蛋白Caspase-3和Caspase-9表达增多（P<0.01）；细胞侵袭数目减少，侵袭标记蛋白VEGF和MMP-14表达降低（P<0.01）；DDP处理的3组细胞中，sh-CCAT 2转染的细胞上述表现最为明显（P<0.01）。体内实验显示，与对照组相比，3个DDP组肿瘤体积减小，30 d时差异有统计学意义（P<0.01）；肿瘤组织CCAT2 mRNA表达减少（P<0.01），细胞凋亡增加（P<0.01）。3个DDP组中，接种sh-CCAT2转染的 A549/DDP细胞组上述表现最为明显（P<0.01）。
      结论  sh-CCAT2可以抑制A549/DDP细胞的增殖、诱导细胞凋亡和降低细胞的侵袭能力，从而抑制A549/ DDP细胞生长。

     

    Abstract:
      Objective  To investigate the effects of short hairpin RNA (shRNA) on the proliferation, invasion, apoptosis and tumor formation of non-small cell lung cancer cisplatin-resistant cell line (A549/DDP) via silencing of colon cancer associated transcript 2 (CCAT2).
      Methods  TA549/DDP cells were transfected with shRNA-CCAT2 (sh-CCAT2) or shRNA-negative control (shRNA-NC), and untransfected A549/DDP cells were used as the control group. CCAT2 mRNA expression in three groups of A549/DDP cells was detected by quantitative real-time PCR (qRT-PCR). The proliferation of three groups of A549/DDP cells treated with different mass concentrations of DDP (0-8 mg/L) was detected by MTT. According to the proliferation experiment results, 2 mg/L was selected as DDP concentration for subsequent experiments. The effects of 2 mg/L DDP treatment on the proliferation, apoptosis, and invasion ability of each group of cells (with untreated A549/DDP cells as the control group) were tested by clone formation experiments, flow cytometry analysis and Transwell experiments. The expression levels of cell proliferation marker proteins (Ki67, PCNA), apoptosis marker proteins (Caspase-3, Caspase-9) and invasion marker proteins (VEGF, MMP-14) were detected by Western blot. Nude mice were injected subcutaneously with A549/DDP cells, A549/DDP cells transfected with shRNA-NC or A549/DDP cells transfected with sh-CCAT2. DDP was intraperitoneally injected at the concentration of 2 mg per kilogram of mice body weight totally for 7 times with an interval of 3 d. A control group was injected subcutaneously with A549/DDP cells, and an equal volume of normal saline instead of DDP was injected intraperitoneally. The tumor volume was detected every 5 d for a total of 30 d. Mice were sacrificed and tumor tissues were taken out 30 d later. CCAT2 mRNA expression level in tumor tissues was detected by RT-PCR, and tumor cell apoptosis was detected by TUNEL staining.
      Results  Compared with the control group and the shRNA-NC transfection group, the expression level of CCAT2 mRNA was decreased in sh-CCAT2 transfected A549/DDP cells (P<0.01). The decrease degree of cell proliferation was more pronounced after treating with 2 to 8 mg/L of DDP (P<0.01). Compared with the control group, in the three groups that treated with DDP, the formation of clones and the expression of proliferation marker proteins Ki67 and PCNA were reduce (P<0.01), while the rate of apoptosis and the expression of apoptosis marker proteins Caspase-3 and Caspase-9 were increased (P<0.01). Also, the number of invasion cell and the expression of invasion marker proteins VEGF and MMP-14 were reduced in the three groups that treated with DDP (P<0.01). Among the three groups of DDP-treated cells, the changes in sh-CCAT2 transfected cells was the most obvious (P<0.01). Compared with the control group, the tumor volume of the three DDP treatment groups was smaller and the differences were statistically significant at 30 d (P<0.01). The expression of CCAT2 mRNA was decreased in tumor tissues (P<0.01), while apoptosis increased (P<0.01). Among the three DDP treatment groups, the A549/DDP cell group transfected with sh-CCAT2 showed the most notable changes (P<0.01).
      Conclusion   sh-CCAT2 can inhibit the proliferation of A549/DDP cells, induce apoptosis and reduce the cell invasion ability, thereby inhibiting the growth of A549/DDP cells.

     

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