The Role Of CAPN 7 On Human Endometrial Stromal Cell

Calpains are a family of intracellular calcium-dependent cysteine proteases, functional by modulating their substrates through limited proteolytic cleavage of them. Calpains have been suggested to play important roles in many biological processes, including migration, proliferation and apoptosis. Many reports have shown that the proliferation and migration of endometrial stromal cell from women with a diagnosis of endometriosis is higher than that from normal fertile women, but the pathogenesis is still unclear.Our result suggested that calcium-activated neutral protease 7(CAPN 7) is highly expressed in eutopic endometrium from endometriosis(EMT); this implied that CAPN 7 play roles in the pathogenesis of EMT. To detect the role of CAPN 7 in the pathogenesis of EMT, we first detect whether CAPN 7 affects h ESC proliferation. When overexpressed CAPN 7 by infecting with the adenovirus-GFP-CAPN 7, the protein expression of Cyclin D1 and Cyclin D3 increased and the proliferation of h ESC increased by 1.5-fold(** P<0.01). But when CAPN 7 was knocked down, Cyclin D1 and Cyclin D3 expression markedly decreased and the proliferation reduced by 15%(* P<0.05). We found that the inhibitor of calpains: PD150606 could inhibit the effect of CAPN 7 on h ESC proliferation and significantly decreased the proliferation of eutopic endometrial stromal cell from EMT patients.Secondly, we used scratch wound healing assay and Trans Well assay to detect whether CAPN 7 affects h ESC migration and invasion. Results suggested that when CAPN 7 was overexpressed, the migration rates increased by 1.2, 1.3 and 1.7 fold after infection with the CAPN 7 adenovirus at 24, 48 and 72 hour(h), compared to the corresponding control, respectively. But following CAPN 7 knockdown in h ESC, the migration rate reduced by 10% compared to control all of time point. And Trans Well results suggest that overexpression of CAPN 7 increased the invasion of h ESC by approximately 2-fold but knocking down decreased this ability by approximately 65% compared with endometrial stromal cell exhibiting normal CAPN 7 expression.Then,real-time Quantitative PCR, Western Blotting and gelatin zymography were used to explore the mechanism of how CAPN 7 effect h ESC migration and invasion. Results suggested that CAPN 7 affect the expression and activity of matrix metalloproteinase 2(MMP-2), but little effect on matrix metalloproteinase 9(MMP-9). CAPN 7 overexpression led to increased tissue inhibitor of metalloproteinases 2(TIMP-2) expression and CAPN 7 knockdown reversed this change, but little effect on tissue inhibitor of metalloproteinases 1(TIMP-1) expression. Interestingly, we found that CAPN 7 increased the expression of MMP-2 over that expression of TIMP-2. We also found that inhibitor of MMP-2: OA-Hy decreased the effects of CAPN 7 overexpression on h ESC migration and invasion by approximately 50% and 55%, respectively. Our data collectively suggest that CAPN 7 promote h ESC migration and invasion by augmenting MMP-2 activation via an increased ratio of MMP-2 to TIMP-2. Additionally, our results suggested that PD150606 significantly decreased both the migration and invasion of eutopic endometrial stromal cells from EMT patients. Results from above suggested that CAPN 7 play important roles in the pathogenesis of EMT.In summary, the present study provides the first evidence that CAPN 7 is highly expressed in EMT. Increased expression of CAPN 7 promoted the proliferation, migration and invasion of h ESC. Delineation of the mechanisms underlying the aberrant expression of CAPN 7 in EMT may be of interest to obtain a better understanding of the pathophysiology of EMT and ultimately for the development of new diagnostic tools and targeted therapies.