FHL2 Regulate KGN Cell Proliferation By Interacting With TGF-α/EGFR Singnaling Pathway

FHL2( Four and a half LIM domains protein 2)gene is one of the most studied FHLgene family members. FHL2 not only can combine with different transcription factor to regulate related gene expression, but also palys an important role in the cell signal transduction pathway. Studies have shown that FHL2 expressed in granulosa cell tumor cell, and TGF-α can regulate human ovarian granular cells proliferation by PI3K/AKT and MAPK signaling pathways. But the mechanism that FHL2 regulates KGN cell, the interactions of FHL2 with TGF-α, and the mutual function to granulosa cells remains poorly understood. This study used KGN as cellular model, researched these problems. The results will be an in-depth understanding of the mechanism and regulation of follicular development, as well as provide new research ideas to signal transduction pathways of follicles. The main results were as follows:1. FHL2 regulates the cell proliferation, apoptosis and the TGF-α expression of KGN cellAfter knockdown FHL2, we detected the cell proliferation, apoptosis and the expression of TGF-α in KGN cell using CCk-8 kit, flow cytometry and RT-PCR.1) Cell proliferation assays using the CCK-8 assay, revealed that knockdown the FHL2 can reduce the cell activity(1.00±0.01 vs 0.32±0.05, p<0.001). Cell counting test found that the ratio of the cell number of FHL2 konckdown group was significantly lower than the control group(1.00 ± 0.01 vs 0.44 ± 0.10, p<0.05);2) Analysis of the cell cycle, the result showed that a few cells in the experimental group were in S-phase(13.17±0.13 vs 8.90±0.04, p<0.001), suggesting that FHL2 can arrest cell cycle.3) Compared with control group, treatment of KGN cells with FHL2 siRNA to knockdown FHL2 significantly reduced the expression of both TGF-α and EGFR(1.03±0.10 vs 0.19±0.01, p<0.001; 1.08±0.12 vs 0.70±0.30, p<0.05).2. TGF-α stimulates KGN cells proliferationTo determine the effect of TGF-α on KGN cells proliferation, we used cell counting test. The results showed that the number of TGF-α(100 ng/m L, 72 h) treated group was significantly higher than control group(92.67 ± 11.55 vs 149.00 ± 8.89, p<0.001).3. TGF-α regulates the FHL2, EGFR and TGF-α expression in KGN cellsKGN cells which were cultured with TGF-αincreased the expression of FHL2(1.00±0.08 vs 2.16 ±0.29, p<0.001), EGFR(1.02±0.22 vs 2.01 ±0.13, p<0.01)and TGF-α( 1.03±0.10 vs 2.08±0.03, p<0.01). When cultured cells with TGF-α after FHL2 RNAi, RT-PCR results showed that TGF-α had marginal impact on the expression of FHL2, EGFR and TGF-α.4 TGF-α regulates the expression of FHL2 via MAPK pathwayWhen cultured KGN cells with TGF-α after treatment with the signaling pathway inhibitor U0126, AG1478, LY294002 for 3 h. Following are the experimental results:1) Compared with the control group, the expression of FHL2(2.16 ±0.29 vs 0.79 ±0.07, p<0.001)、EGFR(1.84 ±0.20 vs 0.68 ±0.04, p<0.001)and TGF-α(1.46 ±0.32 vs 0.52 ±0.25, p<0.01)were reduced when KGN cells were treated with MEK inhibitor and TGF-α.2) Compared with the control group, there were no significant difference of FHL2 expression(2.16 ± 0.29 vs 1.79 ±0.11, p> 0.05), meanwhile the expression of EGFR(1.84 ±0.20 vs 0.90 ±0.27, p<0.01)and TGF-α(1.46 ±0.32 vs 0.28 ±0.01, p<0.01)were reduced when KGN cells were treated with PI3 K inhibitor followed by TGF-α.3) Compared with the control group, the expression of FHL2(2.16 ±0.29 vs 1.26 ±0.02, p<0.05)、EGFR(1.84 ±0.20 vs 1.32 ±0.11, p<0.01)and TGF-α(1.46 ±0.32 vs 0.28 ±0.24, p<0.01)were reduced when KGN cells were treated with EGFR inhibitor inhibitor and TGF-α.These results suggestesd that TGF-α can regulate the expression of FHL2 through MAPK signaling pathway via binding to its receptor EGFR.5. TGF-α/EGFR/FHL2 autocrine loop regulates the proliferation of KGN cellsIn the present study, we used KGN cell as cellular model to investigate the interaction between TGF-α and FHL2 as well as its function on KGN cell. The results indicated that FHL2 can regulate the expression of TGF-α and EGFR, meanwhile TGF-α plays an critical role in KGN cell proliferation. TGF-α can regulate FHL2 expression through activting MAPK signal pathway with binding with EGFR. TGF-α/ EGFR / FHL2 formed autocrine loop, involving in the growth of KGN cells.