SiRNA Mediated Inhibition Of CaV2.2e[37a] Gene Expression

N-type calcium channel is a key pharmacological target for treatment of pain, N-type calcium channel inhibitors always cause side effects.The present challenge lies in finding some ways to specifically inhibit N-type channels in the pain pathway,not only eliminating pathological pain but also maintaining normal nociception,as well as sensory and autonomic.N-type calcium channels have two isoforms,CaV2.2e[37a]and CaV2.2e[37b].All neurons express CaV2.2e[37b] but that a subset of nociceptive neurons,particularly in DRG,express CaV2.2e [37a].This study focused on specifically knockdown the expression of CaV2.2e [37a]gene by RNA interference to provide the base for exploring the N-type calcium channel isoform inhibitor.PartⅠConstruction of EGFP-e37a/e37b fusion gene plasmids and e37a cell lineObjective:To find out siRNAs specifically inhibiting CaV2.2e[37a]without inhibiting CaV2.2e[37b]gene expression Methods:①Construction of EGFP-37a/37b fusion gene plasmids as the mimic target genes.Full length cDNA fragment of EGFP was obtained from pcDNA316-EGFP,and amplified by high fidelity PCR Polymerase using sense primer with SOE- PCR.The fragment was inserted into eukaryotic reporter vector pAAV2 -neo by cutting with EcoRⅠand BglⅢ.The fusion gene vectors,named pAAV2 -neo-EGFP-37a/37b identified by restricted endonuclease digestion and DNA sequence analysis.②Establishment of stable cell line expressed EGFP-e37a fusion gene in BHK cells.The recombinant plasmids pAAV2 -neo-EGFP- e37a was transfect into BHK cells by Lipofectamine 2000.After transfection,cells were maintained with G418.pAAV2 -neo-EGFP-37a cell line was constructed with dilution.Result:①EGFP-37a/37b were characterized by PCR and digestion by EcoRⅠand BglⅡ.The length of fragments was about 829 bp.The positive clones were subjected to sequencing and the results were shown as expected.②The efficiency of recombinant plasmids pAAV2 -neo- EGFP- e37a transfection into BHK cells was about 40-50%.7 strains positive cell clones were selected by G418.Conclusions: Successfully construction of EGFP-37a/37b fusion gene plasmid as the mimic target genes by SOE-PCR.Establishment the screening platform to screen siRNA for inhibiting CaV2.2e[37a]gene expression.PartⅡDesigning and screening siRNA for inhibiting expression of CaV2.2e[37a]geneObjective:To design and construct plasmids siRNA according to CaV2.2e[37a] gene and find out siRNAs specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression.Methods:①Following siRNA design principles, four e37a siRNAs,namedⅠ,Ⅱ,Ⅲ,Ⅳ,were synthesized and clone into vector pGPU6/Neo.②Plasmids pAAV2- neo-EGFP-e37a were co-transfected into BHK cells with pGPU6 /Neo-siRNA at ratio 10:1.Plasmids pAAV2- neo- EGFP, pAAV2-neo-EGFP-e37b was used as control.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose.To assess the effect of RNAi on EGFP-37a fusion protein,microscopy and fluorescence activated cell sorting(FACS) were used.③RNAi in e37a cell line:Plasmids siRNAⅡwere transfected into e37a cell line at different dose and detected the effect of RNAi at different points of time.④Fusion protein expression was detected by western blotting analysis.Results:①Design 4 pairs of 21 bp siRNAs.The result of restricted endonuclease digestion results showed the inserted fragment was as expected.②In the 4 pairs of siRNA,siRNAⅡinduced a significant decrease of EGFP-e37a expression(84-90%),without interfering EGFP- 37b gene expression.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose,and reducing the percentage of the mean fluorescence intensity(MFI) were:10:1,86±6.1(%);5:1,82±5.9 (%);1:1,80±4.2(%);1:5 85±6.0(%);1:10,79±4.6(%).③RNAi in e37a cell line:Plasmids siRNAⅡreducing the percentage of MFI were:3.6ug, 93±4.2(%);1.8ug,95±4.9(%);0.9ug,91±3.8(%);0.45ug,86±5.4(%);0.225ug, 70±4.3(%).24h after the transfection,the efficiency of RNAi was not obvious.At the time point of 48h,the efficiency of RNAi amount to 74±4.8(%);72h,82±3.3 (%).④siRNAⅡcould decrease the expression of EGFP-e37a protein level obviously,while not decrease the expression of EGFP-e37b and EGFR Conclusions:siRNAⅡthat specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression has been found out by fluorescence microscopy and FACS.So we provide a basis tbr studying of RNAi of Cav2.2e[37a]in vitro and in vivo.PartⅢStudy of the specific siRNA inhibiting CaV2.2e[37a]gene expressionObjective:To detect the effect of siRNAⅡinhibiting CaV2.2e[37a]gene expression by western blotting.Methods:①Design the primer for sequencing four plasmids.②Plasmid siRNAⅡco-transfected into 293FT cells with plasmids Cav2.2e[37a],Cav2.2e[37b],β3,α2σ1 respectively,and detected the expression of Cav2.2e[37a]gene by western blotting.Result:①The results of DNA sequencing showed the four plasmids were as Genebank sequences.②The protein on band 6 diminished obviously which indicate that siRNAⅡdecrease the generation of CaV2.2e[37a]expression protein in 293FT cells.Conclusions: SiRNAⅡcould inhibit Cav2.2e[37a]gene expression specifically without inhibiting CaV2.2e[37b]gene expression,and providing a potential agent for studying RNAi Cav2.2e[37a]isoform in noxious stimuli transmission..