The Role Of DRG MZF1in The Pathogenesis Of Neuropathic Pain

Backgrounds:Voltage-gated potassium channels (Kv) in the dorsal root ganglion (DRG)govern neuronal excitability and play a key role in neuropathic pain genesis. Kv1.2, asubunit of Kv1family channels, is highly expressed in DRG. In chronic contractioninjury (CCI)-induced neuropathic pain, the level of Kv1.2mRNA and protein isdown-regulated, total Kv current reduced, and neuronal excitability increased in theDRG neurons. A recently identified long non-coding RNA, Kv1.2antisense (AS)RNA, negatively regulates Kv1.2mRNA expression. The promoter region of Kv1.2AS RNA gene contains a specific binding site (161AGTGGGGA154) of thetranscription factor myeloid zinc finger protein1(MZF1). Spinal nerve injury (SNL)increases MZF1expression and enhances the binding activity of MZF1to Kv1.2ASRNA gene in the DRG. Moreover, MZF1mRNA is co-localized with Kv1.2AS RNAin the DRG neurons. In the cultured HEK-293T cells or DRG neurons, over-expression of MZF1increases Kv1.2AS RNA expression and reduces the expressionof Kv1.2mRNA and protein, whereas knockdown of MZF1reduces Kv1.2AS RNAexpression and increases the expression of Kv1.2mRNA and protein. The evidenceindicates that MZF1alters Kv1.2expression through regulating Kv1.2AS RNA.DRG MZF1may be involved in the development and maintenance of neuropathicpain and be a potential target for preventing and treating this disorder.Objective:This study was to investigate the effects of DRG MZF1over-expression orknockdown through DRG microinjection on rat behavioral responses to mechanical,thermal and cold stimuli, the expression of Kv1.2AS RNA, MZF1mRNA andprotein, and Kv1.2mRNA and protein in the DRG, and DRG neuronal excitability inthe CCI rats or na ve rats. Methods:Experimental1: Male SD rats were randomly divided into two groups, EGFPand MZF1. Unilateral L4/5DRGs were injected with rAAV5-MZF1(2μl;4×1012GC/μl) or rAAV5-EGFP (2μl;4×1012GC/μl). Mechanical, thermal, and cold tests werecarried out prior to DRG micro-injection, and1,3,4,6, and8weeks post-micro-injection. After behavioral tests, bilateral L4/5DRGs were collected for quantitativereal-time RT-PCR to measure the levels of DRG Kv1.2AS RNA, MZF1mRNA andKv1.2mRNA and for Western blot to measure the amounts of Kv1.2protein andMZF1protein.Experimental2: Male SD rats were randomly divided into two groups, EGFPand MZF1. Unilateral L4/5DRGs were injected with a mixture of rAAV5-MZF1(1μl;4×1012GC/μl) plus rAAV-EGFP (1μl;4×1012GC/μl) or rAAV5-EGFP (2μl;4×1012GC/μl). Five weeks later, the injected DRGs were harvested and the acutelydissociated DRG neurons plated on the cover-glasses. Three hours later, whole-cellpatch clamp recording was carried out to examine neuronal excitability.Experimental3: Male SD rats were randomly divided into four groups, PBS+CCI, MZF1siRNA+Sham, MZF1siRNA+CCI, and MZF1scramble+CCI.Unilateral L4/5DRGs were injected with PBS (2μl), MZF1siRNA (2μl;10μmol/L),or MZF1scramble siRNA (2μl;10μmol/L). Seven days later, CCI model was carriedout. Mechanical, thermal, and cold tests were carried out prior to DRG micro-injection, before CCI model, and3,5, and7days post-CCI. After behavioral tests,bilateral L4/5DRGs were collected for quantitative real-time RT-PCR to measure thelevels of DRG Kv1.2AS RNA, MZF1mRNA and Kv1.2mRNA and for Westernblot to measure the amounts of Kv1.2protein and MZF1protein.Experimental4: Male SD rats were randomly divided into4groups, CCI+PBS,CCI+EGFP, CCI+MZF1siRNA, and CCI+MZF1scramble. CCI model was firstcarried out. Seven days later, Seven days later, unilateral L4/5DRGs were injectedwith PBS (2μl), MZF1siRNA (2μl;10μmol/L), or MZF1scramble siRNA (2μl;10μmol/L). Mechanical, thermal, and cold tests were carried out prior to CCI model,before DRG micro-injection, and7and10days post-injection. After behavioral tests,bilateral L4/5DRGs were collected for quantitative real-time RT-PCR to measure the levels of DRG Kv1.2AS RNA, MZF1mRNA and Kv1.2mRNA and for Westernblot to measure the amounts of Kv1.2protein and MZF1protein.Results:1. Unilateral L4/5DRG injection of rAAV5-MZF1time-dependently andsignificantly reduced paw withdrawal threshold to mechanical stimulation andpaw withdrawal latencies to thermal and stimuli on the ipsilateral (but notcontralateral) side. These reductions appeared at4weeks post-injection andlasted at least at8weeks post-injection. On the ipsilateral side, the levels ofMZF1mRNA and protein and Kv1.2AS RNA were significantly increasedand the amount of Kv1.2mRNA and protein decreased in the injected DRGs.None changes were observed on the contralateral side.2. DRG MZF1overexpression markedly increased rest membrane potential,reduced injection current threshold, and increased number of action potentialsin the medium and large DRG neurons compared to control EGFP group.3. Consistent to our previous studies, CCI increased the expression of MZF1mRNA and protein and of Kv1.2AS RNA and reduced the expression ofKv1.2mRNA and protein in the ipsilateral L4/5DRGs. DRG microinjectionof MZF1siRNA significantly and specifically blocked CCI-induced increasesin the amounts of MZF1mRNA and protein and of Kv1.2AS RNA in theipsilateral L4/5DRGs, reversed CCI-induced down-regulation of Kv1.2mRNA and protein in the ipsilateral L4/5DRGs, and attenuated CCI-inducedmechanical allodynia, thermal hyperalgesia and cold allodynia at3,5, and7days (the development) post-CCI. DRG microinjection of MZF1siRNAsignificantly reduced basal expression of MZF mRNA and protein and Kv1.2AS RNA and increased the level of Kv1.2mRNA and protein in the ipsilateralL4/5DRGs from sham rats, but did not alter basal responses to mechanical,thermal, and cold stimuli. DRG microinjection of MZF1scramble siRNA didnot affect CCI-induced changes in MZF1, Kv1.2AS RNA, and Kv1.2in theDRGs and pain hypersensitivities to mechanical, thermal, and cold stimuli. 4. Post-DRG microinjection of MZF1siRNA (but not scramble siRNA)significantly reduced CCI-induced mechanical allodynia, thermal hyperalgesiaand cold allodynia during the maintenance period of CCI-induced neuropathicpain.Conclusion:Our results suggest that DRG MZF1participates in neuropathic paindevelopment and maintenance through Kv1.2AS RNA-triggered DRG Kv1.2downregulation. Targeting DRG MZF1may have potential clinical implications inprevention and treatment of neuropathic pain.