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

HE Liang, DU Ze-dong, WANG Yang, MENG Rong-qin, ZHAO Wen-wu

Abstract

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.

 

Keywords: CCAT2, Non-small cell lung cancer, Cisplatin

 

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