| Objective:To investigate the effects of lncRNA NBR2 knockdown on proliferation,migration,invasion,cell cycle and apoptosis in gastric cancer cell line BGC823,and to explore the regulation of BRCA1 expression by lncRNA NBR2,in order to provide experimental basis for the clinical design of effective strategies for the treatment of gastric cancer.Methods:1.qRT-PCR and Western blot was used to detect the expressions of lncRNA NBR2 and BRCA1 in gastric epithelial cell line GES1 and gastric cancer cell lines MGC803,SGC7901 and BGC823,respectively.2.Three pPLK GFP+Puro/NBR2 shRNA interfering vectors(shRNAa,shRNAb and shRNAc)were constructed and transiently transfected into BGC823 cells respectively.qRT-PCR was performed to identify the interference efficiency.3.The effects of lncRNA NBR2 knockdown on the proliferation,migration,invasion,cell cycle and apoptosis of BGC823 cells were observed by MTT,wound-healing assay,Transwell migration and invasion assay and flow cytometry individually..4.qRT-PCR and Western blot were conducted to analyze the mRNA and protein expression levels of BRCA1 induced by lncRNA NBR2 knockdown separately.Results:1.The results of qRT-PCR showed that the expression of lncRNA NBR2 in gastric cancer cell lines MGC803,SGC7901 and BGC823 was significantly higher that in GES1 cells,and lncRNA NBR2 expression level in BGC823 cells was the highest(P < 0.05).The expression of BRCA1 was evidently upregulated in BGC823 and SGC7901 cells than that in GES1 cells(P < 0.05),but there was no significant differennce between MGC803 cells and GES1 cells(P > 0.05).2.Western blot dispalyed that the expression of BRCA1 was greatly upregulated in BGC823 and SGC7901 cells,and slightly downregulated in MGC803 cells(P < 0.05),compared with GES1 cells.3.Fluorescence microscope analysis exhibited that the transfection efficiency of three interference vector groups and empty vector group was more than 80%.qRT-PCR demonstrated that NBR2 expression in NBR2 shRNAa group and the NBR2 shRNAb group was markedly downregulated(P <0.05),but was no significant difference in the NBR2 shRNAc group(P > 0.05),compared with untransfected group and empty vector group.The NBR2 shRNAa vector was the most effective,with an interference efficiency of about 65%.4.MTT assay revealed that OD value(490nm)in the NBR2 shRNA transfection group at 48 h,72h and 96 h was notably lower than that in the untransfected group and the empty vector group(P < 0.05).Furthermore,the inhibition rate in the NBR2 shRNA transfection group enhanced in a time-dependent manner.This indicated that lncRNA NBR2 knockdown can inhibit the proliferation of gastric cancer BGC823 cells.5.The wound-healing assay illustrated that in 0 ~ 24 h and 24 ~ 48 h,the cell migration relative distance(85.867±5.254 and 56.600±4.612)in NBR2 shRNA transfection group vastly decreased than that in untransfected group(222.570±5.257 and 140.710±3.278)and emptyvector group(218.033±5.341 and 135.667±7.353)(P < 0.05).Transwell migration assay also found that the number of migration cells in the NBR2 shRNA transfection group(50.500±2.887)was much less than those in the untransfected group(194.750±6.801)and the empty vector group(191.250±5.909)(P < 0.05).All above proveed that lncRNA NBR2 knockdown can remarkably inhibit the migration ability of gastric cancer BGC823 cells.6.Transwell invasion assay described that the number of invasion cells in the NBR2 shRNA transfection group(39.500±2.380)declined sharply,compared with the untransfected group(115.750±4.573)and the empty vector group(112.250±5.315)(P < 0.05),which indicated that lncRNA NBR2 knockdown can remarkably suppressed invasion ability of gastric cancer BGC823 cells.7.Flow cytometry revealed that the ratio of G0/G1 cells in the NBR2 shRNA transfection group went up markedly,compared with the untransfection group(3.767±0.289)and the empty vector group.However,the proportion and the proliferation index of cells in S phase and G2/M phase reduced very much(P < 0.05).The above results indicated that the down-regulation of lncRNA NBR2 can suppress the proliferation and arrest BGC823 cells in the G0/G1 phase.In addition,flow cytometry also demonstrated that the apoptosis rate of NBR2 shRNA transfection group(26.240±0.325)was obviously upregulated compared with the untransfected group(3.440±0.016)and the empty vector group(6.150±0.248)(P < 0.05),which suggested the knockdown of lncRNA NBR2 can promote apoptosis of gastric cancer BGC823 cells.8.qRT-PCR and Western blot displayed that mRNA and protein expression levels of BRCA1 in NBR2 shRNA transfection group were significantly lower than those in the untransfected group and the empty vector group(P < 0.05),demonstrating that lncRNA NBR2 knockdowncan downregulate the expression of BRCA1.Conclusion:1.Knockdown of lncRNA NBR2 can inhibit proliferation,migration,invasion,and induce G0/G1 phase arrest and apoptosis in gastric cancer BGC823 cells.2.LncRNA NBR2 knockdown can downregulate the expression of BRCA1. |
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