Construction and identification of the pshRNA‑CACNA1G‑SH‑SY5Ycells targeted to silence Cav3.1 mRNA expression
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- Published online on: May 29, 2013 https://doi.org/10.3892/br.2013.117
- Pages: 669-673
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Abstract
T‑type calcium channels are a class of low voltage‑dependent calcium channels that may be activated following minor depolarizations of the cell membrane. Cav3.1 is the dominant subtype of the T‑type calcium channel in SH‑SY5Y cells. T‑type channels play a key role in the regulation of the intracellular calcium concentration, which is involved in the neurotoxic effect of local anesthetics. However, there is a lack of specific inhibitors of T‑type calcium channels. The existing T‑type calcium channel inhibitors exhibit poor specificity and may block the high voltage‑dependent calcium channels, such as the L- and N‑type channels. Furthermore, there is no selectivity to the subtype of the T‑type calcium channel. Therefore, the development of a specific T‑type calcium channel inhibitor may contribute to the elucidation of the functions and characteristics of T‑type calcium channels. the aim of this study was to silence the Cav3.1 mRNA expression in SH‑SY5Y cells via the RNA interference (RNAi) method in order to construct pshRNA‑CACNA1G‑SH‑SY5Y cells and assess Cav3.1 mRNA and protein expression by western blot analysis and reverse transcription-polymerase chain reaction (RT‑PCR) to identify the constructed cell line. The results demonstrated that Cav3.1 mRNA and protein expression were significantly reduced following transfection with the SH‑SY5Y cells by the supernatant liquors. The results also demonstrated that the pshRNA‑CACNA1G‑SH‑SY5Y cells were successfully constructed. These findings may contribute to the elucidation of the functions of Cav3.1 in SH‑SY5Y cells.
