The Biological Effects Of Bpes Related Gene Foxl2 On Hela And Siha Cells

Foxl2 is a single-exon gene encoding a forkhead transcription factor, whose germline mutations are responsible for the blepharophimosis ptosis epicanthus inversus syndrome(BPES; MIM 110100) [1]. This genetic disorder is characterized by malformation of the external eye with blepharophimosis, ptosis and a variable degree of epicanthus inversus. BPES is divided into two types: Type I with POF and Type II with normal fertility [1,2]. Other members of the large family of winged helix/forkhead transcription factors are involved in different developmental and metabolic processes and some of them are responsible for genetic, developmental diseases. In mice, Foxl2 m RNA is expressed in embryonic eyelids and in embryonic and adult ovary [1,3], and the murine Foxl2 gene is essential for differentiation of granulosa cells and maintenance of ovarian structure and function. Furthermore, two independent Foxl2-/- knock-out mice models have been generated and have confirmed the key role of Foxl2 in ovarian determination and granulose cell physiology [8, 9].Foxl2 is expressed in ovarian granulosa cells [4]. A recent study revealed that a markedly reduced expression of Foxl2 in a series of juvenile ovarian granulose cell tumors(OGCTs) [5]. Moreover, Shah et al discovered a recurring somatic mutation 402C>G in the gene Foxl2 in 2009 [5]. This mutation was confirmed to be present in 97% adult ovarian granulose cell tumors(OGCTs) and specific to this tumor type [6-10]. However, inactivation of the Foxl2 locus by somatic hypermethylation was found in colorectal cancer [11]. Most interestingly, this mutation was not found in any other sex-cord stromal tumors, nor in any unrelated ovarian or breast tumors [12, 13]. In addition, Wegman P found that Foxl2 was expressed in breast cancer and correlated with aromatase as well as with clinical outcome [14].To the best of our knowledge, Foxl2 has not been previously studied in cervical cancer and we therefore aimed to investigate its expression and the effect on cervical cancer cells.1、THE EXPRESSION OF BPES RELATED GENE FOXL2 IN CERVICAL CARCINOMAObjective To detect the expression level of BPES related gene Foxl2 in cervical carcinoma and cervical cancer cell lines Hela and Siha.Methods We collected cervical cancer tissues of 37 patients, and they were divided into two groups according to adenocarcinoma and squamous cell carcinoma. In addition, cervical intraepithelial neoplasia(CIN) tissues of 8 patients were also collected and divided into low-grade squamous intraepithelial lesions(LSIL) group and high-grade squamous intraepithelial lesions(HSIL) group. Moreover, cervicitis tissues of 10 cases were taken as control. We measured the expression of Foxl2 in the control group, CIN and cervical cancer by immunohistochemistry. Then we cultured cervical cancer cell lines Hela and Siha, and the total RNA was extracted from Hela and Siha cells with Trizol reagent. The complementary DNA(c DNA) was generated with oligo(d T) 18 primers by using Revert Aid? First Strand c DNA Synthesis Kit. Furthermore, we investigated the m RNA level of Foxl2 in Hela and Siha cells in order to confirm whether these two cell lines could transcribe Foxl2.Results We determined the expression of Foxl2 in tissue by immunohistochemistry, and the results showed that the expression of Foxl2 in cervical squamous cell carcinoma(CSC) was higher than CIN group and the control group(cervicitis). Moreover, the expression level of Foxl2 in cervical squamous carcinoma was significantly higher than cervical adenocarcinoma(CAC). Foxl2 was not espressed in cervical adenocarcinoma, CIN and cervicitis, and there was no significant difference among them. We extracted the total RNA from Hela and Siha cells and obtained c DNA template after reverse transcription. The RT-PCR result demonstrated that Hela and Siha cells could transcribe the Foxl2 m RNA.Conclusion The expression of Foxl2 in cervical squamous carcinoma was significantly higher than it in CIN and cervicitis. Hela and Siha cells could transcribe the m RNA of Foxl2, and the expression level of Foxl2 in Siha cells was markedly higher than Hela cells. Foxl2 was expressed at a low level in Hela cells and at a high level in Hela cells. Therefore we would like to over-expressed Foxl2 in Hela cells and silenced its expression in Siha cells, in order to study its effect in cervical cancer by subsequent experiments.2、THE BIOLOGICAL EFFECTS OF BPES RELATED GENE FOXL2 ON CERVICAL CANCER CELL LINESObjective According to the results obtained in the first part of the experiments, we constructed the models of these two cell lines. In brief, we over-expressed Foxl2 in Hela cells and silenced it in Siha cells. Subsequently, we investigated the effects of Foxl2 on proliferation, apoptosis, adhesion and invasion of Hela and Siha cells, and analyzed its roles in cervical cancer.Methods We transfected plasmids into Hela cells in order to over-expressed Foxl2 in Hela cells. Meanwhile, we transfected sh RNA into Siha cells in order to silence its expression. Furthermore, we verified the transfection sufficiency and identify whether the transfection was successful by western blotting. After that Foxl2 was over-expressed or silenced, Brdu assay was utilized to detect the proliferation of Hela and Siha cells; FITC-Annexin V/PI assay was used to measure the apoptosis of Hela and Siha cells; the adhesion kit was utilized to determine the adhesion of Hela and Siha cells. The transwell invasion assay was exploitedto measure the effect of Foxl2 on the invasiveness of cervical cancer cells. Flow cytometry(FCM) was exploited to detect the expression of Ki67, proliferating cell nuclear antigen(PCNA) and Fas L in Foxl2-overexoressed Hela cells and Foxl2-silenced Siha cells. Results The western blotting results showed that the expression of Foxl2 dramatically increased in Hela cells transfected with plasmids when compared to the control Hela cells(P<0.01). Moreover, the expression of Foxl2 markedly decreased in Siha cells ttransfected with sh RNA when compared to the control Siha cells(P<0.01). And it indicated that the transfection models were successfully constructed. The results of Brdu assay and Annexin V/PI illustrated that the proliferation of Foxl2-overexpressed Hela cells was significantly inhibited in contrast with Hela cells(P<0.01). The proliferation of Foxl2-silenced Siha cells dramatically increased in contrast with Siha cells, and its apoptosis obviously declined(P<0.001). Moerover, silencing Foxl2 enhanced the adhesion of Siha cells, but over-expressiing Foxl2 had no effect on Hela cells adhesion. Meanwhile, the transwell results indicated that the invasiveness of Foxl2-overexpressd Hela cells notably decreased in contrast to Hela cells(P<0.05); on the other hand, Foxl2 silencing dramatically promoted the invasiveness of Siha cells(P<0.05).The flow cytometry(FCM) results indicated that the expression of Ki67 in Foxl2-overexpressed Hela cells was significantly lower than Hela cells(P<0.001), and the expression of Fas L in Foxl2-overexpressed Hela cells was markedly higher than Hela cells(P<0.01). Compared with Siha cells, the expression of Ki67 in Foxl2-silenced Siha cells significantly increased(P<0.001), and the expression of Fas L in Foxl2-silenced Siha cells obviously diminished(P<0.01). However, there was no significant difference of PCNA among these groups regardless of Foxl2 over-expression or silence.Conclusion The results indicated that Foxl2 suppressed the proliferation of Hela and Siha cells, and it promoted their apoptosis. Foxl2 inhibited the invasiveness of Hela and Siha cells. Moreover, Foxl2 facilitated the adhesion of Siha cells, but it had no effect on adhesion of Hela cells. In addition, Foxl2 down-regulated Ki67 expression in Hela and Siha cells in order to inhibit the proliferation of cervical cancer cells. At the same time, Foxl2 up-regulated the expression of Fas L in Hela and Siha cells in order to facilitate the apoptosis of these cells.In summary, our study demonstrated that Foxl2 was expressed in cervical squamous cancer. On the one hand, Foxl2 suppressed the proliferation and promoted apoptosis of cervical cancer cells mainly through decreasing Ki67 expression and increasing Fas L expression; on the other hand, Foxl2 restrained the invasiveness of cervical cancer cells. Hence, Foxl2 might be a novel tumor suppressor, at least in cervical cancer. It may be valuable for estimating the metastasis and recurrence o f cervical cancer, and it needs further investigation.