Study On Biological Functions Of Gstπ Gene In Cervix Cancer

Objective:To study the effects of overexpression of exogenous GSTπgene on cell growth,migration,invasion and radiosensitivity in human cervix cancer cell line HeLa,and to preliminarily investigate the underlying mechanisms.Methods:(1) A full-length sequence of human Gstπgene was obtained by a polymerase chain reaction using the primers based on to the GSTπsequence in the GeneBank, and inserted into a recombinant eukaryotic expression plasmid by molecular cloning, and identified by restriction analysis and DNA sequencing. (2) The HeLa cells with a high expression of GSTπwere obtained by stable transfection of plasmids by using lipofectamine and G418 resistant screening. The mRNA and protein expression of GSTπwere detected by RT-PCR assay and Western Blot assay. (3) Cell counting was used to evaluate the effects of GSTπon HeLa cell growth. (4) In vitro scratch assay and Boyden chamber assay were used to identify the functions of GSTπin cell migration and invasion. (5) Colony formation assay was used to detect the influence of GSTπgene on HeLa cells'radiosensitivity ofχray. (6) Flow cytometry assay was performed to explore the impacts of GSTπon cell cycle prograssion. And the protein expression of cell cycle regulating factor was detected by Western Blot assay.Results:(1) The recombinant eukaryotic expression plasmid of GSTπgene (pcDNA3/GSTπ) was successfully constructed. (2) A HeLa cell line stably expressing high level GSTπwas obtained,as demonstrated by Western blot and RT-PCR assay. (3) A significant inhibition of cell growth was observed in GSTπoverexpressing HeLa cells compared to untransfected HeLa parent cells and HeLa/Neo cells transfected with control"empty"pcDNA3 vector. (4) Increased expression of GSTπsignificantly inhibited the migration and invasion of HeLa cells. (5) HeLa/GSTπcells were more resistant to radiotherapy compared to HeLa/Neo cells; (6) Analyzed by flow cytometry assay, GSTπdelayed cell cycle progression by arresting cells at G0/G1 phase. And the expression level of cyclin D1 and PTEN was increased in HeLa/GSTπcells compared to HeLa/Neo cells as determined by Western blot assay.Conclusion: These results demonstrated that the increased expression of Gstπgene inhibited HeLa cell growth through decreasing the protein level of G0/G1 phase controller-cyclin D1. GSTπsuppressed migration and invasion of HeLa cells, and the mechanisms may be related to its ability to increase the expression of metastasis suppressor PTEN. GSTπalso reduced cell sensitivity to radiotherapy, and the mechanisms may be related to its ability to increase the expression of G2/M phase controller-cyclin B1. These findings will provide a basis and evidence for further investigation of biological activities of GSTπin cervical cancer.