Research Article

Segment-specific targeting via RNA interference mediates down-regulation of OPN expression in hepatocellular carcinoma cells

Published: November 19, 2015
Genet. Mol. Res. 14 (4) : 14440-14447 DOI: 10.4238/2015.November.18.6

Abstract

Osteopontin (OPN) plays an important role in the metastasis and recurrence of tumors after resection of hepatocellular carcinoma (HCC). In this study, the down-regulation effect on OPN expression in HCC cells of RNA interference (RNAi) molecules designed to target different segments of OPN was investigated to identify a more effective site for OPN knockdown. Specific small interfering RNAs (siRNAs A, B, and C) of OPN were synthesized and transfected into an HCC cell line (HEP-G2; representing the OPNi-A, OPNi-B, and OPNi-C groups). Fluorescent quantitative polymerase chain reaction and immunohistochemical methods were used to detect the mRNA and protein expression of OPN before and after RNAi. Results showed that after transfection, the fluorescence intensity of the OPNi-A group was greater than those of the OPNi-B and OPNi-C groups. After 48 h of transfection, the ΔCT values of OPN mRNA expression in the OPNi-A-C groups increased from 8.31 ± 1.58, 8.78 ± 1.49, and 8.25 ± 1.51 to 12.14 ± 1.43, 10.22 ± 1.97, and 10.48 ± 1.88, respectively (P OPN gene had different down-regulatory effects on OPN expression. Synthesis of targeted siRNA aimed at specific OPN segments might have important significance for dealing with the invasiveness and metastasis of HCC cells.

Osteopontin (OPN) plays an important role in the metastasis and recurrence of tumors after resection of hepatocellular carcinoma (HCC). In this study, the down-regulation effect on OPN expression in HCC cells of RNA interference (RNAi) molecules designed to target different segments of OPN was investigated to identify a more effective site for OPN knockdown. Specific small interfering RNAs (siRNAs A, B, and C) of OPN were synthesized and transfected into an HCC cell line (HEP-G2; representing the OPNi-A, OPNi-B, and OPNi-C groups). Fluorescent quantitative polymerase chain reaction and immunohistochemical methods were used to detect the mRNA and protein expression of OPN before and after RNAi. Results showed that after transfection, the fluorescence intensity of the OPNi-A group was greater than those of the OPNi-B and OPNi-C groups. After 48 h of transfection, the ΔCT values of OPN mRNA expression in the OPNi-A-C groups increased from 8.31 ± 1.58, 8.78 ± 1.49, and 8.25 ± 1.51 to 12.14 ± 1.43, 10.22 ± 1.97, and 10.48 ± 1.88, respectively (P < 0.05), and the OPN protein levels (immunohistochemistry scores) decreased from 6.44 ± 1.67, 5.43 ± 2.05, and 5.45 ± 2.52 to 2.84 ± 1.52, 4.43 ± 1.65, and 3.95 ± 1.43 points, respectively. These results indicated that RNAi based on different segments of the OPN gene had different down-regulatory effects on OPN expression. Synthesis of targeted siRNA aimed at specific OPN segments might have important significance for dealing with the invasiveness and metastasis of HCC cells.