Effect of lentivirus vector-mediated RNA interference dbpA gene silencing on the biological behavior of colorectal cancer cells
10.3760/cma.j.issn.1007-631X.2019.07.017
- VernacularTitle:慢病毒载体介导的dbpA基因沉默对结直肠癌细胞生物学行为的影响
- Author:
Ruiting LIU
1
;
Yali HOU
;
Xiangtian WU
;
Guorong WANG
;
Chang LIU
;
Jirong BAI
;
Jian QIU
;
Likun YAN
;
Xiaojun LI
;
Xiaoqiang WANG
Author Information
1. 陕西省人民医院普外一科
- Keywords:
Colorectal neoplasms;
DNA binding proteins;
Apoptosis;
Cell proliferation
- From:
Chinese Journal of General Surgery
2019;34(7):613-617
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of lentivirus-mediated RNA interference (RNAi) targeting DNA binding protein A (dbpA) on the proliferation and the biological behavior of colorectal cancer cell line SW620.Methods The experiment was divided into 3 groups:KD group (siRNA-dbpA,lentivirus interference group),CON group (non-specific sequence group) and NC group (blank control group).The lentiviral vector siRNA-dbpA was constructed and verified by PCR and DNA sequencing.SW620 cells were transfected with siRNA-dbpA plasmid,nontargeting siRNA plasmid,or empty plasmid.After 48 h the transfection,the cells were examined for dbpA expression using Western blot.After 72 hrs transfection,flow cytometry was used to detect the cell apoptosis and cell cycle changes.The cell growth inhibition rate was detected by MTT (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay,and then clone formation was detected,and the ability of SW620 cells to form tumors in vivo after dbpA was silenced was studied in nude mice.Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting dbpA gene was successfully inserted into the lentiviral vector.siRNA-dbpA transfection resulted in reduced expression of dbpA in SW620 cells.After transfection,the apoptosis rate of siRNA-dbpA-transfected cells increased to 26.60％ ± 0.38％,significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid 12.54％ ± 0.25％ and 4.46％ ± 0.19％,respectively (F =28.159,P ＜0.01).The growth inhibition test indicate that the OD value of the fifth day in siRNA-dbpA group was 0.194 ±0.037,significantly lower than that in the other two groups 0.814 ±0.043 and 1.625 ±0.061,respectively(F =23.214,P ＜ 0.01).The colony formation number is 37 ± 3,64 ± 5and 175 ± 10 respectively,siRNA-dbpA is significantly higher than that in the other two groups(F =40.254,P ＜ 0.01).After the completion of nude mouse transplantation tumor model,through the detection of tumor volume,KD group (group siRNA-dbpA) tumor volume after 14 d and CON and NC group had obvious difference (F =38.256,P ＜ 0.05),and after 21d is more significant difference in tumor size (F =40.241,P ＜ 0.01),can be clearly observed after 35 d KD group (group siRNA-dbpA) growing tumors had differences with the control group (F =30.257,P ＜ 0.05).Conclusion Lentivirus-mediated RNAi targeting dbpA can effectively suppress the expression of dbpA in colorectal tumor in nude mice,it is proved that dbpA silencing has a significant inhibitory effect on the growth of living tumor cells and decrease the proliferation of the colorectal cells.
