PARENT SESSION

(14) DEVELOPMENT OF AN EFFICIENT RNA INTERFERENCE STRATEGY FOR ASSESSING THE ROLE OF hTrpC4 PROTEINS IN MYOMETRIAL CELLS.

Ulloa, Aida¹, Zhong, Miao¹, Kim, Yoon-Sun¹, Ku, Chun-Ying¹, Sanborn, Barbara¹, ¹ Colorado State University, Fort Collins, CO

ABSTRACT- During labor, increases in intracellular calcium (Ca²⁺) have been closely correlated with human myometrial contractions. Extracellular Ca²⁺ enters the cell through voltage-operated and signal-regulated Ca2+-entry (SRCE) mechanisms and is involved in actions such as stimulating the contractile apparatus and replenishing intracellular Ca²⁺ stores. In SRCE, activation of signaling receptors and/or depletion of intracellular Ca²⁺ stores stimulate Ca²⁺ uptake from the extracellular environment. This and other SRCE mechanisms contribute to the regulation of calcium homeostasis. Ion channels implicated in SRCE are the canonical transient receptor potential (TrpC) channels. The seven TrpC members (TrpC1-7) are postulated to form hetero- or homotetramers, thus allowing for the formation of a variety of channels possessing different physiological properties. Our studies show that hTrpC proteins are involved in SRCE pathways in myometrium and that human myometrium expresses hTrpC4 and hTrpC1 mRNAs in greatest relative abundance compared to other hTrpCs. In order to study the relative contributions of specific TrpCs to SRCE, we have designed an effective method for inducing RNAi (RNA interference) of specific hTrpC channels in the immortalized human myometrial cell line PHM1-41. To screen for effective RNAi sequences, short hairpin RNA (shRNA) constructs were designed to target hTrpC4 specifically. hTrpC4-shRNA-containing plasmids were placed into a miRNA (microRNA) backbone in pSHAG and tested using the psiCHECK-2 luciferase reporter system. Four hTrpC4-shRNA constructs proved effective for targeting hTrpC4-mRNA reduction (60-90%). hTrpC4-shRNA sequences were incorporated into adenoviral vectors for infecting PHM1-41 cells at >75% efficiency, monitored by co-expression of green fluorescent protein. Initial studies with the viral vectors show 85% hTrpC4 mRNA reduction in infected cells and changes in phenotype in response to agents that stimulate SRCE. In conclusion, a successful strategy for design and testing of shRNA constructs targeting hTrpC4 has been devised. This approach promises to be useful in elucidating the role of this protein and other TrpCs in SRCE in myometrium. Supported by HD38970 and T32-HD07031.

KEY WORDS: TrpC: canonical type of transient receptor potential channels, SRCE: Signal Regulated Calcium Entry, RNAi: RNA interference, shRNA: short-hairpin RNA