Biological effect of down-regulating of MTRR gene on cisplatin-resistant ovarian cancer SKOV3 cells in vitro and in vivo studies
10.3760/cma.j.issn.0529-567x.2016.02.009
- VernacularTitle:下调MTRR基因表达对顺铂耐药的卵巢上皮性癌SKOV3细胞体内外生物学特性的影响
- Author:
Jia CHEN
;
Qi WANG
;
Wei ZHANG
;
Li LI
- Publication Type:Journal Article
- Keywords:
Ovarian neoplasms;
Cell line,tumor;
Ferredoxin-NADP reductase;
Cisplatin;
Cell cycle
- From:
Chinese Journal of Obstetrics and Gynecology
2016;(2):126-134
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the biological effects of down-regulatingof methionine synthase reductase (MTRR) gene on cisplatin resistant ovarian cancer SKOV3/DDP cell in vitro and in vivo. Methods (1) Establishing the cell line of MTRR down-regulated. Four short hairpin RNA (shRNA) for MTRR gene (U6-GFP-Neo-homo-1106, U6-GFP-Neo-homo-1931, U6-GFP-Neo-homo-419, U6-GFP-Neo-homo-1460) were designed respectively. Western blot was used to detect the interference efficiency and selected the most efficient shRNA. The MTRR 1106 was selected as the best silencing effect of interference fragment and then therecombinant plasmid vector pSicoR-1106 was constructed and transfected into SKOV3/DDP cells.The stably transfected cells was obtained by screening of flow cytometry(FCM).Fluorescence quantitative reverse transcription (RT)-PCR and western blot were used to detect the expression of MTRR mRNA and protein. (2) Study in vitro: recombinant plasmid expression vector pSicoR-1106, pSicoR-NC and packaging plasmid were respectively transfected into 293T cell. SKOV3/DDP cells were transfected by viral supernatant. The experiment was divided into three groups, namely SKOV3/DDP-MTRRi (down-regulated MTRR group), SKOV3/DDP-NC (negative control group), and the SKOV3/DDP (blank control group). The cell growth curves and half maximal inhibitory concentration (IC50) of cisplatin were made by methyl thiazolyl tetrazolium (MTT) method. Three groups cells were treated with different concentration of cisplatin (0, 1, 2 and 4 μg/ml). The clonogenicity efficiency was observed by clony formation test. The cell cycles were measured by FCM . (3) Study in vivo: three groups cells were subcutaneously inoculated into the nude mice to develop a tumor model. Mice were injected intraperitoneally with cisplatin at 2.5 mg/kg (once every 2 days, in 21 rounds), then the tumor growth was observed. The expression of MTRR and proliferation-related Ki-67 antigen by immunohistochemistry in xenograft tumors were measured. Results (1) Results showed that U6-GFP-Neo-homo-1106 was the best shRNA with interference effect to MTRR. The recombinant plasmid pSicoR-1106 was constructed and transfected into SKOV3/DDP. The MTRR mRNA and protein were down-regulated after transfected. This result showed that MTRR down-regulated SKOV3/DDP cell line was constructed successfully. (2) The cell growth curves showed that the growth of SKOV3/DDP-MTRRi cells were significantly decreased compared with that in the SKOV3/DDP-NC cells and SKOV3/DDP cells (P<0.05). The IC50 of SKOV3/DDP-MTRRi, SKOV3/DDP-NC and SKOV3/DDP were 4.01, 7.90, and 8.91 μg/ml, respectively. The IC50 of SKOV3/DDP-MTRRi was significantly lower than that in control cell groups (P<0.05).Clony formation tests showed that the clony numbers of varied concentration of cisplatin of SKOV3/DDP-MTRRi were significantly less than those of SKOV3/DDP-NC cells and SKOV3/DDP cells (P<0.05). FCM showed that when the cisplatin concentration rose to 4 μg/ml, the G0/G1 phase cell ratio in SKOV3/DDP-MTRRi cells group was (72.8±5.0)%, which was significantly higher than those in the SKOV3/DDP-NC cells group and SKOV3/DDP cells group [(64.4±2.5)%and (64.3±3.0)%], respectively (all P<0.05).(3) Six weeks after nude mice intraperitoneal injection with cisplatin, the tumor volume of SKOV3/DDP-MTRRi, SKOV3/DDP-NC and SKOV3/DDP were respectively (97 ± 32), (168 ± 45), and (173 ± 32) mm3, the tumor weight were (0.36±0.17), (1.08±0.17), and (1.11±0.20) g, in which tumor volume and weight of SKOV3/DDP-MTRRi were significantly less than those of SKOV3/DDP-NC cells and SKOV3/DDP cells (all P<0.05). In three groups tumor tissue, positive rates of MTRR were respectively 2/8, 5/8, and 7/8, the positive rates of Ki-67 were respectively1/8, 6/8, and 7/8, in which SKOV3/DDP-MTRRi was significantly lower those SKOV3/DDP-NC cells and SKOV3/DDP cells (all P<0.05). Conclusion The growth and cisplatin resistance of ovarian cancer cells could be decreased by down-expressing of MTRR gene in vitro and in vivo.
