Calcification is a major cause of clinical failure of bioprosthetic and other tissue cardiac valve substitutes.The mechanism of calcification of cardiac valve substitutes is quite complex and has a variety of determinants,involving reaction of calcium-containing extracellular fluid with membrane-associated phosphorus to yield calcium phosphate mineral deposits.The most promising anticalcification strategies have included binding of calcification inhibitors to glutaraldehyde fixed tissue,removal or modification of calcifiable components,modification of glutaraldehyde fixation and use of tissue cross linking agents other than glutaraldehyde.This article reviews current progress toward the research of calcification and anticalcification strategies that are under investigation either in advanced animal models or in clinical trials.

Objective:To investigate the effect of siRNA targeting inhibition of α-enolase (ENO1) combined with paclitaxel on the proliferation, invasion and apoptosis of hepatocellular carcinoma SK-HEP-1 cell and its mechanism.Methods:siRNA-ENO1 (siRNA-ENO1 group) and siRNA-negative control (siRNA-NC group) were transfected into SK-HEP-1 cells in vitro, the untransfected SK-HEP-1 cells were used as the control group, and the transfection effect was detected by real-time fluorescent quantitative polymerase chain reaction and western blotting. After SK-HEP-1 cells were treated with 0, 2.5, 5, 10, 20 and 40 μg/L paclitaxel for 48 hours, the cell survival rate was measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) method and the semi inhibitory concentration of paclitaxel was calculated. SK-HEP-1 cells transfected with siRNA-ENO1 or siRNA-NC were treated with 10 μg/L paclitaxel as paclitaxel+ siRNA-ENO1 group and paclitaxel+ siRNA-NC group. The proliferation, clonogenesis, invasion and apoptosis of siRNA-NC group, siRNA-ENO1 group, paclitaxel+ siRNA-ENO1 group and paclitaxel+ siRNA-NC group were detected by MTT, clonogenesis, Transwell chamber and flow cytometry respectively. The expression levels of the phosphorylation of phosphatidylinositol-3-kinase (p-PI3K), p-protein kinase B (Akt) and proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 9 (MMP-9) and B lymphocytoma-2 gene (Bcl-2) were detected by western blotting. Results:Compared with the control group (1.00±0.00 and 0.69±0.04, respectively), the expression levels of ENO1 mRNA and protein (0.25±0.03 and 0.23±0.02, respectively) in siRNA-ENO1 group decreased significantly ( P<0.05), but there were no significant differences in the expression levels of ENO1 mRNA and protein in siRNA-NC group ( P>0.05). Compared without treatment group [(100.00±0.00)%, P<0.05], the survival rates of SK-HEP-1 cells treated with 2.5, 5, 10, 20 and 40 μg/L paclitaxel [(88.65±6.46)%, (72.36±6.08)%, (60.48±4.23)%, (38.52±3.56)% and (20.75±2.32)%, respectively] decreased significantly ( P<0.05), and the semi inhibitory concentration of paclitaxel was 13.26 μg/L. The cell survival rate and clone formation rate of siRNA-ENO1 group [(68.86±5.12)% and (18.12±2.25)%, respectively] were lower than those of siRNA-NC group [(100.00±0.00)% and (29.65±3.06)%, respectively, P<0.05]. The cell survival rate and clone formation rate of the paclitaxel+ siRNA-ENO1 group [(43.28±2.64)% and (8.72±0.52)%, respectively] were significantly different from those of the paclitaxel+ siRNA-NC group [(61.75±5.06)% and (13.48±2.16)%, respectively, P<0.05] and siRNA-ENO1 groups [(68.86±5.12)% and (18.12±2.25)%, respectively, P<0.05]. Cell invasion number in paclitaxel+ siRNA-ENO1 group (23.64±2.12) was lower than that in siRNA-ENO1 group and paclitaxel+ siRNA-NC group (42.16±2.75 and 37.35±2.42, respectively, P<0.05). The apoptosis rates of paclitaxel+ siRNA-NC group and siRNA-ENO1 group [(17.49±1.35)% and (15.29±1.50)%, respectively] were higher than that of siRNA-NC group [(7.21±0.70)%, P<0.05]. The apoptosis rate in the paclitaxel+ siRNA-ENO1 group [(24.59±2.40)%] was higher than those in the paclitaxel+ siRNA-NC group and siRNA-ENO1 group [(17.49±1.35)% and (15.29±1.50)%, respectively, P<0.05]. The expression levels of ENO1, PI3K/Akt signaling pathway related proteins including p-PI3K and p-Akt and the expression levels of PCNA, MMP-9 and Bcl-2 in siRNA-ENO1 group and paclitaxel+ siRNA-NC group were lower than those in siRNA-NC group ( P<0.05). The expression levels of ENO1, p-PI3K, p-Akt, PCNA, MMP-9 and Bcl-2 in paclitaxel+ siRNA-ENO1 group were lower than those in siRNA-ENO1 group or paclitaxel+ siRNA-NC group ( P<0.05). Conclusion:siRNA targeting inhibition of ENO1 expression can enhance the inhibitory effect of paclitaxel on proliferation, invasion and apoptosis of SK-HEP-1 cells, and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway.

Objective:To investigate the effect of siRNA targeting inhibition of α-enolase (ENO1) combined with paclitaxel on the proliferation, invasion and apoptosis of hepatocellular carcinoma SK-HEP-1 cell and its mechanism.Methods:siRNA-ENO1 (siRNA-ENO1 group) and siRNA-negative control (siRNA-NC group) were transfected into SK-HEP-1 cells in vitro, the untransfected SK-HEP-1 cells were used as the control group, and the transfection effect was detected by real-time fluorescent quantitative polymerase chain reaction and western blotting. After SK-HEP-1 cells were treated with 0, 2.5, 5, 10, 20 and 40 μg/L paclitaxel for 48 hours, the cell survival rate was measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) method and the semi inhibitory concentration of paclitaxel was calculated. SK-HEP-1 cells transfected with siRNA-ENO1 or siRNA-NC were treated with 10 μg/L paclitaxel as paclitaxel+ siRNA-ENO1 group and paclitaxel+ siRNA-NC group. The proliferation, clonogenesis, invasion and apoptosis of siRNA-NC group, siRNA-ENO1 group, paclitaxel+ siRNA-ENO1 group and paclitaxel+ siRNA-NC group were detected by MTT, clonogenesis, Transwell chamber and flow cytometry respectively. The expression levels of the phosphorylation of phosphatidylinositol-3-kinase (p-PI3K), p-protein kinase B (Akt) and proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 9 (MMP-9) and B lymphocytoma-2 gene (Bcl-2) were detected by western blotting. Results:Compared with the control group (1.00±0.00 and 0.69±0.04, respectively), the expression levels of ENO1 mRNA and protein (0.25±0.03 and 0.23±0.02, respectively) in siRNA-ENO1 group decreased significantly ( P<0.05), but there were no significant differences in the expression levels of ENO1 mRNA and protein in siRNA-NC group ( P>0.05). Compared without treatment group [(100.00±0.00)%, P<0.05], the survival rates of SK-HEP-1 cells treated with 2.5, 5, 10, 20 and 40 μg/L paclitaxel [(88.65±6.46)%, (72.36±6.08)%, (60.48±4.23)%, (38.52±3.56)% and (20.75±2.32)%, respectively] decreased significantly ( P<0.05), and the semi inhibitory concentration of paclitaxel was 13.26 μg/L. The cell survival rate and clone formation rate of siRNA-ENO1 group [(68.86±5.12)% and (18.12±2.25)%, respectively] were lower than those of siRNA-NC group [(100.00±0.00)% and (29.65±3.06)%, respectively, P<0.05]. The cell survival rate and clone formation rate of the paclitaxel+ siRNA-ENO1 group [(43.28±2.64)% and (8.72±0.52)%, respectively] were significantly different from those of the paclitaxel+ siRNA-NC group [(61.75±5.06)% and (13.48±2.16)%, respectively, P<0.05] and siRNA-ENO1 groups [(68.86±5.12)% and (18.12±2.25)%, respectively, P<0.05]. Cell invasion number in paclitaxel+ siRNA-ENO1 group (23.64±2.12) was lower than that in siRNA-ENO1 group and paclitaxel+ siRNA-NC group (42.16±2.75 and 37.35±2.42, respectively, P<0.05). The apoptosis rates of paclitaxel+ siRNA-NC group and siRNA-ENO1 group [(17.49±1.35)% and (15.29±1.50)%, respectively] were higher than that of siRNA-NC group [(7.21±0.70)%, P<0.05]. The apoptosis rate in the paclitaxel+ siRNA-ENO1 group [(24.59±2.40)%] was higher than those in the paclitaxel+ siRNA-NC group and siRNA-ENO1 group [(17.49±1.35)% and (15.29±1.50)%, respectively, P<0.05]. The expression levels of ENO1, PI3K/Akt signaling pathway related proteins including p-PI3K and p-Akt and the expression levels of PCNA, MMP-9 and Bcl-2 in siRNA-ENO1 group and paclitaxel+ siRNA-NC group were lower than those in siRNA-NC group ( P<0.05). The expression levels of ENO1, p-PI3K, p-Akt, PCNA, MMP-9 and Bcl-2 in paclitaxel+ siRNA-ENO1 group were lower than those in siRNA-ENO1 group or paclitaxel+ siRNA-NC group ( P<0.05). Conclusion:siRNA targeting inhibition of ENO1 expression can enhance the inhibitory effect of paclitaxel on proliferation, invasion and apoptosis of SK-HEP-1 cells, and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway.