Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells

Abstract

Objective

The aim of this study was to construct THY1 eukaryotic expression plasmid and study its effects on ovarian cancer SKOV3 cells.

Methods

The gene fragment coding for THY1 was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1 (+) to construct the recombinant plasmid pcDNA3.1(+)-THY1, which was transfected into SKOV3 cells. The experimental cells were classified into three groups: SKOV3-THY1, SKOV3-Null and SKOV3. The expression of gene was measured using RT-PCR and Western blotting. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. Both SKOV3-THY1 and SKOV3-null cells were inoculated subcutaneously into nude mice to determine in vivo tumorigenicity.

Results

The gene fragment of THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1 (+) and verified by PCR, restriction endonucleases digestion and DNA sequencing and the plasmid of pcDNA3.1(+)-THY1 (THY1 gene overexpression) has been stably transfected into SKOV3 cells. The analysis of flow cytometry indicated that the pcDNA3.1(+)-THY1 transfected cells in G1 phase were significantly elevated, but in S phase were decreased. The growth of transfected cells was suppressed, and more apoptosis cells were identified in pcDNA3.1(+)-THY1 transfectants compared with vector vehicle transfectants. The tumor suppressing activity of THY1 in SKOV3 cells was associated with inhibition of SKOV3 cellular proliferation, in vivo tumorigenesis in nude mice.

Conclusion

THY1 transfection can inhibit the growth of SKOV-3 cells in vitro and in vivo. THY1 gene may play an important role in generation and development of ovarian cancers.