| PART I THE STUDY ON THE EXPRESSION OF MATRIX METALLOPROTEINASE AND MICRO RIBONUCLEIC ACIDObjective:To explore the interaction of different levels of matrix metalloproteinase (MMP) and micro ribonucleic acid (miRNA) in kaposiform haemangioendothelioma (KHE).Methods:A total of 29 resected specimens from KHE patients were collected for this study. All specimens had been histologically and clinically diagnosed at the Department of Surgical Oncology of Children’s Hospital of Chongqing Medical University from 2004 to 2013. MMP 3, MMP 7, MMP 9, MMP 13 and miR99a were examined through Immunohistochemistry, Western blot and RT-qPCR. Bivariate correlations between different levels of MMP and miRNA were calculated by Spearman’s rank correlation coefficients.Results:MMP7, MMP9 and MMP 13 without MMP3 were detected in KHE through Immunohistochemistry, Western blot and RT-qPCR. Strong positive correlation was detected between miR99a and MMP7 (γ2= 0.76; P< 0.001), and between miR99a and MMP13 (γ2= 0.81; P< 0.001), but not between miR99a and MMP9 (γ2= 0.0052; P=0.96).Conclusions:MMPs and miRNAs are associated with KHE tumorigenesis. Significant correlation of miR99a, with MMP7 and MMP13, but not with MMP9 was found.PART Ⅱ THE STUDY ON MIR99A MODULATING MMP7 AND MMP13 TO REGULATE INVASIVENESS OF]Objective:To further understand the molecular bases of MMP7 and MMP13 modulated by miR99a to regulate invasiveness of KHE.Methods:SLK Cells, a human KHE cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum. SLK was transfected with a miR99a overexpressing plasmid, or short hair interferon RNA for miR99a, Cells were analyzed 48 hours after transfection by RT-qPCR. MMP7, MMP9 and MMP13 were examined in SLK cells transfected with plasmids that overexpress miR99a (SLK-miR99a) and SLK cells transfected with plasmids that overexpress short hairpin miRNA for miR99a (SLK-ShmiR99a) through RT-qPCR, Western blot and ELISA. And their changes in cell invasiveness were observed by a transwell matrix penetration assay. LY294002, a specific inhibitor to the key component of PI3k pathway-Akt, and PD98059, a specific inhibitor to the key component of ERK/MAPK pathway-ERK1/2, were added into miR99a-knockdown SLK cells, then MMP7 and MMP13 were detected in miR99a-knockdown SLK cells with RT-qPCR, Western blot and ELISA, and their changes in cell invasiveness were observed by a transwell matrix penetration assay.Results:The miR99a levels were confirmed to be overexpressed in SLK-miR99a cells than SLK-N by RT-qPCR (p<0.05), and obviously inhibited in SLK-ShmiR99a cells than SLK-N (p<0.05). MMP7 and MMP13 decreased by overexpression of miR99a in SLK cells, while MMP7 and MMP13 increased in miR99a-knockdown SLK cells were examined, by RT-qPCR (p<0.05), by Western Blot (p<0.05), by secreted proteins in the conditioned media by ELISA (p<0.05), but no difference was found in MMP9 of these three SLK cells(p>0.05). SLK-miR-99a cell invasiveness was weaker than SLK-N cell (p<0.05), while SLK-ShmiR-99a cell invasiveness was stronger than SLK-N cell in the transwell matrix penetration assay (p<0.05). MMP7, expressed in SLK-ShmiR-99a+LY294002 cells was obviously less than SLK-ShmiR-99a cells (p<0.05), but MMP13 had no difference, while MMP 13 expressed in SLK-ShmiR-99a+PD98059 cells was obvious than SLK-ShmiR-99a cells (p<0.05), but MMP7 had no difference, which were all conformed by RT-qPCR, Western Blot and ELISA. The cell invasivenee of SLK-ShmiR-99a+LY294002 and SLK-ShmiR-99a+PD98059 were all stronger than SLK-N (p<0.05), while, no difference was found between SLK-ShmiR-99a+LY294002 and SLK-ShmiR-99a+PD98059 through the transwell matrix penetration assay (p>0.05)Conclusions:SLK cell invasiveness was seemed to be regulated by miR99a through MMP7 and MMP13, while MMP9 seems to be regulated in a miR99a-independent manner. Inhibition of PI3k/Akt signaling pathway significantly abolished the effect of miR99a-knockdown on MMP7, but not MMP 13 activation, while inhibition of ERK/MAPK signaling pathway significantly abolished the effect of miR99a-knockdown on MMP 13, but not MMP7 activation. Thus, miR99a, MMP7 and MMP 13 appear to be promising therapeutic targets for preventing the metastasis of KHE. |
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