Design,preparation and screening of siRNA of proliferating cell nuclear antigen
- VernacularTitle:增殖细胞核抗原siRNA序列的设计、合成与筛选
- Author:
Jianwei JIANG
;
Jintao SHI
;
Zhihui WU
;
Huilan ZENG
;
Yuxia YAN
;
Tao CHEN
;
Wenting DING
- Publication Type:Journal Article
- Keywords:
Proliferating cell nuclear antigen;
siRNA;
Colorectal neoplasms;
Liver neoplasms;
Stomach neoplasms
- From:
Chinese Journal of Pathophysiology
1986;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To design,prepare and screen out functional small interfering RNA(siRNA) for specifically silencing proliferating cell nuclear antigen(PCNA) gene expression and effectively inhibiting cell proliferation on human hepatoma cell line SMMC-7721,human gastric carcinoma cell line SGC-7901 and human colorectal carcinoma cell line Caco2.METHODS: PCNA siRNA was designed based on previous studies about design guidelines and synthesized in vitro by T7 Mega short script reaction kit according to the manufacturer's instructions.After purification,the integrities of siRNA were identified through 19% denaturing polyacrylamide gel electrophoresis,and the concentrations of the generated siRNA were measured by testing the absorbance at 260 nm using a spectrophotometer.Four synthesized double-strand siRNA were transfected into three types of carcinoma cell lines by LipofectamineTM2000 reagent,respectively.WST-8 assay was employed to examine the proliferative inhibitions of these three cell lines.The subsequent alterations on PCNA mRNA and protein levels were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and immunocytochemistry,respectively.RESULTS: 3 sequences,No.2,No.3 and No.4 PCNA siRNA showed effective inhibitions on tumor cells among the 4 candidate siRNA,and a single dose of 50 nmol/L of No.2,No.4 PCNA siRNA showed the most effective inhibitory rates as more than 62% at 48 h after transfection.50 nmol/L of No.2 and No.4 PCNA siRNA transfection caused 72% decrease of PCNA mRNA level and almost completely loss of the protein in human Caco2 cells.CONCLUSION: No.2 and No.4 PCNA siRNA have been screened out in this study,which show the capability to effectively down-regulated PCNA expression and significantly inhibit the proliferation of carcinoma cells.The optimal concentration is 50 nmol/L and satisfactory effects are achieved 48 h after transfection in vitro.
