Construction and identification of the pshRNA-CACNA1G-SH-SY5Ycells targeted to silence Cav3.1 mRNA expression

摘要

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.