Follistatin-like 1 is important for ovarian and endometrial carcinogenesis: a differential expression and functional analysis

Abstract

Background and Objectives: Endometrial (EmCA) and ovarian carcinoma (OvCA) is the most common and the most lethal gynecologic malignancies worldwide respectively. In this study, we aimed at identifying differentially expressed genes that were probably important in their carcinogenesis and at characterizing roles of these genes by in vitro functional assays.

Materials and Methods: For gene identification, mRNA was extracted from frozen clinical samples of 37 ovarian and 40 endometrial carcinoma and their corresponding non-tumor tissues and then amplified using 40 pairs of random arbitrary primers. The PCR products were purified and sequenced. Genes were identified by blasting these sequences against the validated sequences available on NCBI webpage. Quantitative real-time RT-PCR was performed to confirm the expression of these differentially expressed genes in these 87 clinical samples, five ovarian and three endometrial cancer cell lines as well as three immortal normal ovarian and one normal endometrial cell lines. Western blotting was also performed. Full length cDNA of identified gene was cloned into a mammalian expression vector pcDNA3.1-V5-His, and transiently transfected into OvCA420 cell line with re-expression confirmed by western blotting. Cell proliferation and its rate was assessed by MTT assay and cell count. Apoptosis was assessed by TUNEL and flow cytometry. Cell migration was assessed using transwell migration chamber.

Results: We demonstrated significant reduced expression of follistatin-like 1 in 75% (30/40) and 91.9% (34/37) of endometrial and ovarian tumor samples respectively. All the ovarian and endometrial cell lines also showed reduced mRNA and protein expressions when compared with normal cell lines. Functional assays showed that OvCA420 cell line with increased follistatin-like 1 expression restored by transfection exhibited significantly reduced cell proliferation (P = 0.009) and prolonged doubling time (3.5 fold) in transfected cells when compared with cells with mock transfection. The proportion of cells in sub-G1 phase increased from 1.52% to 11.82% after transfection. Increased apoptotic activities and marked reduction of migrated cells was demonstrated in transfected cells. Conclusion: Follistatin-like 1 was frequently down-regulated in ovarian and endometrial carcinomas and probably exhibit its effect on carcinogenesis via reducing cell proliferation and migration whilst promoting apoptosis.