| Objective:Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 (TRPV6) is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of TRPV6 in chickens. In this study, we evaluated the effects of TRPV6 gene interference on the expression of calcium transport genes to investigate the mechanism underlying the regulation of TRPV6. Methods:According to the known Genebank TRPV6 mRNA sequences and siRNA design principles, U.S. Ambion’s online design software was used for conducting genome-wide scanning and analysis. Three possible RNA interference points were filtered, three optimal siRNAs were selected and one negative control were designed. And then corresponding shRNAs were designed and linked to the linearized RNAi-Ready pSIREN-RetroQ-ZsGreen expression vector, The positive colones were selected by transformation, screening, identification. To probe the effects of the TRPV6 gene RNA interference plasmids on the expression of TRPV6 gene, we extracted the plasmids of high purity without endotoxin and transfected them into chicken osteoblasts. The expression of TRPV6 was evaluated by Real-time PCR and Western-blot. The plasmids which could effectively inhibit the expression of TRPV6 were transfected into chicken osteoblasts. The expression of calcium transport genes, OPG and RANKL was evaluated by Real-time PCR and Western-blot. Results:The digestion and sequencing results showed that we successfully constructed chicken TRPV6 RNA interference plasmids. In the experiment of inhibiting effect of chicken TRPV6 RNA interference plasmids on the expression of TRPV6 gens chicken osteoblasts, the Real-time PCR results showed that the mRNA expression level of TRPV6 was reduced by 45.7% with pSIREN-TRPV6-3 (P<0.01) and was reduced by 27.8% with pSIREN-TRPV6-2 (P < 0.05) 48 h after transfection compared with the untreated group. In contrast, pSIREN-TRPV6-1 had no significant interference effect on TRPV6 expression; TheWestern-blot results showed that the protein expression level of TRPV6 was decreased significantly with pSIREN-TRPV6-3 (P<0.01) compared with the untreated group. Transfecting the pSIREN-TRPV6-3 plasmids into chicken osteoblasts, The Real-time PCR results showed that the expression of calbindin-D28K was reduced by 27.9% (P<0.01), while the expression of PMCAlb, NCX1, OPG and RANKL did not change. The Western-blot results showed that the protein expression level of calbindin-D28K decreased significantly (P<0.01), while the expression of RANKL did not change. Conclusion:we successfully constructed chicken TRPV6 RNA interference plasmids. TRPV6 only regulates the expression of calbindin-D28K during Ca2+ transport, while there was no effect on the expression of NCX1, PMCA1b, OPG and RANKL by silencing TRPV6. |
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