Screening of optimal siRNA interference sequence of CIT gene and its inhibition expression in hepatoma SK-Hep-1
10.3760/cma.j.issn.1007-3418.2019.04.008
- VernacularTitle: CIT siRNA最佳干扰序列的筛选及其在SK-Hep-1肝癌细胞中对CIT表达的抑制
- Author:
Yue HU
1
;
Qi LIU
1
;
Xiaofang ZHOU
1
;
Lili HUANG
2
;
Hang SUN
1
Author Information
1. Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education and Department of Infectious Diseases, Chongqing Medical University, Chongqing 400016, China
2. Department of Nephrology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
- Publication Type:Journal Article
- Keywords:
siRNA;
CIT;
SK-Hep-1 human hepatoma cells
- From:
Chinese Journal of Hepatology
2019;27(4):281-285
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct and screen optimal siRNA interference sequence of CIT gene and to detect its interference efficiency as well as proliferation effect in human hepatoma cell line SK-Hep-1.
Methods:Three siRNA target spots were designed and synthesized according to the CIT gene sequence. SK-Hep-1 HCC cells were transfected by liposome transfection. The knockdown efficiency of the target CIT gene was detected by real-time PCR and Western blot. Expressional change of CIT in SK-Hep-1 cells after 48 hours of siRNA interference were observed by immunohistochemistry and confocal microscopy. The proliferation of SK-Hep-1 cells after 48 hours of siRNA interference was detected by EdU cell proliferation assay. A t-test was used to compare the mean of two samples, and one-way ANOVA was used to compare the mean of multiple samples.
Results:Western blot results showed that the three interference sequences were targeted at different target spots. The expression level of CIT protein in KD-1,-2, and-3 groups were decreased (P < 0.01) than control, while the protein expression level of KD1 group was the lowest. Real-time PCR results showed that compared with the control group, the expression level of CIT mRNA in KD-1, -2, and -3 groups decreased (P < 0.01), while that in KD1 group was the lowest. Laser confocal microscopy also confirmed that the morphological expression of CIT attenuated significantly after transfection with siRNA. The results of EdU proliferation assay showed that siRNA transfected with CIT significantly attenuated the proliferation of SK-Hep-1 hepatoma cells (P < 0.05).
Conclusion:The successful construction and screening of siRNA fragments can effectively inhibit the expression and proliferation of CIT gene in hepatoma SK-Hep-1.
