[Effect and mechanism of siRNA targeting α-enolase gene combined with paclitaxel on proliferation, invasion and apoptosis of hepatocellular carcinoma cell].

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.

Ribosomal protein RPS15A augments proliferation of colorectal cancer RKO cells via regulation of BIRC3, p38 MAPK and Chk1.

OBJECTIVE: Ribosomal protein S15A (RPS15A) has been implicated in tumorigenesis, but its role in colorectal cancer (CRC) is not fully studied. The objective of this study was to investigate the role of RPS15A in CRC carcinogenesis. PATIENTS AND METHODS: RBSP15A expression was detected in 120 colorectal adenocarcinoma biopsies by immunohistological staining, and we examined the association of RSP15A expression with clinicopathological outcomes. We generated RPS15A stable knockdown CRC cell lines using shRNAs and assessed cell proliferation by MTT assays, clonogenicity by colony formation assays, and apoptosis and cell cycle arrest by flow cytometric analyses. A mouse tumor xenograft model was used to confirm the influence of RPS15A expression on CRC in vivo. RESULTS: RPS15A expression was predictive for poor disease-free survival. Knockdown of RPS15A expression significantly inhibited cell proliferation and colony formation and augmented apoptosis in both the RKO and SW620 CRC cell lines. Moreover, RPS15A knockdown arrested RKO cells at the G2/M phase and SW620 cells at the G0/G1 phase. KEGG pathway analysis of 785 genes differentially expressed between wild-type and shRPS15A RKO cells showed enrichment for the pathway in cancer and MAPK signaling pathway KEGG terms. RPS15A knockdown induced apoptosis via regulation of BIRC3, p38 MAPK, and Chk1. Consistently, RPS15A knockdown significantly impaired the growth of subcutaneous CRC xenografts in nude mice. CONCLUSIONS: These results indicate that RPS15A is a novel, potentially oncogenic gene involved in colorectal carcinogenesis. RPS15A knockdown may be an attractive strategy for treating CRC with gene therapy.