Effect on invasion ability of cervical cancer cells after silence heparanase gene expression in Hela cells
10.3760/cma.j.issn.0529-567x.2013.07.013
- VernacularTitle:RNAi技术沉默子宫颈癌HeLa细胞中HPA基因的表达对细胞侵袭力的影响
- Author:
Qiongying Lü
;
Wei ZHANG
;
Jing CHENG
;
Wenting ZHANG
;
Yajuan ZHONG
- Publication Type:Journal Article
- Keywords:
Uterine cervical neoplasms;
Hela cells;
Glucuronidase;
RNA interference;
Neoplasm invasiveness
- From:
Chinese Journal of Obstetrics and Gynecology
2013;48(7):532-537
- CountryChina
- Language:Chinese
-
Abstract:
Objective Design and synthesize short hairpin RNA (shRNA) expression vector of RNA for specific silencing of heparanase (HPA) gene,screened plasmid which silence effects is the best.Observe the function of ceil invasion after inhibiting the expression of HPA in cervical carcinoma cell lines (HeLa).Methods The genomic sequence of HPA gene was retrieved from GenBank database.Designed four pairs of specific oligonucleotide sequences and a negative control according to the shRNA design principles.They were inserted into the vector pYr-1.1,vectors,and transfected into HeLa cells via lipofectamine.Reverse transcription (RT)-PCR and immunofluorescence were employed to detect the expression of HPA gene in the transfected cells at the mRNA and protein levels,respectively.The plasmid were screened and transfected into HeLa cells,then transwell small room stromal invasion experiment were employed to observe the cervical carcinoma cell invasion.Results RT-PCR results of transfected HeLa cells shown that the mRNA amplification multiples were 0.54 ±0.05 in the HPA-592 group,0.89 ±0.18 in HPA-995 group,0.82 ±0.22 in the HPA-1351 group,0.91 ±0.47 in HPA-1658 group.While,they were 1.31 ±0.72 and 1.09 ±0.16 in negative control and blank control group,respectively.Green fluorescence was visible in the cytoplasm,which indicated that the HPA protein was expressed in the cytoplasm,of them the weakest green fluorescence in the HPA-592 group.The relative numbers of invasive cells among the HeLa cells were as follows:182 ±6 in the blank control group,258 ± 17 in the negative control group,and 44 ± 4 in the HPA-592-specific interference group(P ＜ 0.01).Conclusion Successfully screened shRNA vector targeting human HPA,efficiently inhibit expression of HPA gene when transfected into HeLa cells,and significantly reduced the invasion capacity of cervical carcinoma cells.
