Inhibition of the expression and function of TRPV4 by RNA interference in dorsal root ganglion.
BACKGROUND
Transient receptor potential vanilloid 4 (TRPV4) calcium channel is an essential mediator of spinal nociceptive transmission in dorsal root ganglion (DRG), but its mechanism in pain transmission is poorly understood. The present study intended to evaluate the effect of small interfering RNA (siRNA) on the expression and function of TRPV4 in DRG neurons and its possible usage in pain relief treatment.


METHODS
Two pairs of siRNA targeting TRPV4 (siRNAa and siRNAb) were designed and synthesized. A mismatched double-strand RNA was chosen as negative control. siRNAs were transfected into primary culture DRG neurons with cationic lipid-based CodeBreaker reagent. Real-time reverse transcription polymerase chain reaction and Western blot analysis were performed to assess the change of TRPV4 messenger RNA (mRNA) and protein expression in DRG. Calcium imaging and ELISA of substance P were carried out to evaluate the function change of TRPV4. The relationship between TRPV4 and PKC was also observed by immunocytochemistry.


RESULTS
TRPV4 mRNA levels in DRG of mismatch and control groups were significantly higher (3.88- and 3.75-fold) than normal. However, siRNAa and siRNAb increased only 1.26- and 1.87-fold, respectively. TRPV4 protein expression was also decreased significantly, as indicated by Western blot. In addition, the function of TRPV4 as ion channel was evaluated by calcium imaging and P substance release before and after siRNA treatment. Inhibition of TRPV4 activity was observed after transfection with both siRNA sequences. Protein kinase C expression, which was thought to be related with TRPV4 function, was inhibited as well.


CONCLUSIONS
TRPV4 is a key component in hypotonic signal transduction, and designed siRNA inhibits mRNA level, protein expression and function of TRPV4 in DRG neurons. Therefore, synthesized siRNA is suggested to be a potential treatment for TRPV4-related pathological conditions, specifically pain.
Liu
Ting-Ting
TT
Department of Physical Medicine & Rehabilitation, Qilu Hospital, Medical School of Shandong University, Jinan, China.
Bi
Hong-Sheng
HS
Lv
Shan-Yun
SY
Wang
Xing-Rong
XR
Yue
Shou-Wei
SW
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005727:Ganglia, Spinal / Q000201:enzymology / Q000378:metabolism*; D007150:Immunohistochemistry; D009474:Neurons / Q000201:enzymology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013373:Substance P / Q000378:metabolism; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D050916:TRPV Cation Channels; D013373:Substance P; D012313:RNA; D011493:Protein Kinase C; D002118:Calcium