Construction of survivin shRNA and APC double gene vector and its expression in HT-29 cells
10.16571/j.cnki.1008-8199.2016.04.007
- VernacularTitle:Survivin shRNA 和 APC 片段双基因的载体构建及其在HT-29细胞的表达
- Author:
Xixian YUAN
;
Houyu DUAN
;
Feng CAO
;
Jingfeng DU
;
Yanli ZHU
;
Kai CHENG
;
Yantao YANG
- Publication Type:Journal Article
- Keywords:
HT-29;
Survivin shRNA;
APC efficient fragments;
Double gene;
Lentiviral vectors
- From:
Journal of Medical Postgraduates
2016;29(4):369-374
- CountryChina
- Language:Chinese
-
Abstract:
Objective Studies show that the abnormal ex-pressions of APC and survivin play important roles in the development and progression of colon cancer .Survivin shRNA and APC double gene co-expression lentiviral vector was constructed to observe whether it could be successfully expressed in HT-29 colon cancer cells and whether it could impact on colon cancer cell apoptosis . Methods We selected the best shRNA interference fragment from the construc-tion of three pairs of complementary shRNA fragments and connected it with the effective fragment of APC ( aa1020-1698 ) to construct a double gene co-expression lentiviral vector .HT-29 cells were divid-ed into experimental group , empty loading group and blank group .HT-29 cells were observed by fluorescence microscopy after infec-tion.Survivin and APC expression levels were observed by real time PCR and western blot .Apoptosis was detected by caspase-3 activi-ty assay. Results ①We successfully constructed three pairs of shRNA sequences and proved that they had no human gene homolo -gous to the rest.Real time PCR analysis showed that the best sequence was shRNA 3.②After the sequence alignment of constructed shRNA vectors, three pairs of shRNA sequences were completely the same with the first designed sequence .Green fluorescence was observed in HT-29 cells by fluorescence microscope .The survivin content in experiment group (31.71 ±1.49) was significantly de-creased compared with empty loading group (100 ±0) and blank group(95.12 ±2.15)(P<0.05).The expression level of APC mR-NA was significantly increased compared with empty loading group (0 ±0) and blank group(0.51 ±0.15)(P<0.05).③The relative ratio of apoptosis in experiment group (0.573 ±0.050) was significantly increased compared with empty loading group (0.390 ± 0.040) and blank group(0.407 ±0.030)(P<0.05). Conclusion We have successfully constructed survivin-shRNA-APC double gene co-expression lentiviral vector which can be successfully expressed in HT-29 colon cancer cells , providing references for subse-quent gene therapy by the use of the carrier .
