The Effects Of RNA Interference In GIRK Gene Expression In Human Esophageal Smooth Muscle Cells

Background and objective :Disoerder of the esophageal function frequently results in an impotence of esophageal smooth muscles contraction [1], impermanency relaxation of lower esophageal sphinctor[2], inefficacy of the esophagus elimination [3; 4] that consequently leads to the gastroesophageal reflux diseases, such as reflux esophagitis, Barrett esophagus and esophageal carcinoma[5; 6; 7]. Contraction of the esophageal muscles is dominated and regulated by Ach which is abele to active M2AChR and IKACh. GIRK channels, or acetylcholine-sensitive potassium channels (IKACh), were constructed by GIRK family including GIRK1, GIRK2, GIRK3 and GIRK4 subunits [8; 9; 10]. GIRK subunits are expressed in human various tissues. Thereinto, GIRK1, GIRK2 and GIRK3 subunits are expressed in various regions of the CNS, such as the olfactory bulb, cerebral cortex, amygdala, hippocampus, thalamus, cerebellum, substantia nigra, ventral tegmental area, locus coeruleus, some nuclei of brainstem and spinal cord [11; 12; 13; 14], indicating their possible involvement in various CNS functions such as cognition, memory, emotions and motor coordination. In contrast, GIRK4 subunits are expressed in only a few regions of the brain [12; 14; 15]. Neuronal GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK2 subunits in most brain regions [13; 16] or homomultimers composed of GIRK2 subunits in the substantia nigra[17]. GIRK1 subunits do not form functional homomeric channels [18; 19]. In the heart, atrial GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK4 subunits [18]. GIRK4 is the functional subunit of IKACh and ablation of GIRK4 should result in the functional elimination of IKACh. GIRK1 could not form a functional ion channel without GIRK4. Humman esophageal IKACh was activated by various receptors coupled to G-proteins of pertussis toxin-sensitive class. Among these receptors, muscarinic 2 acetylcholine receptor was the most important one. However, it was still unclear whether GIRK channels exist in esophageal SMCs.RNA interference (RNAi) by short double stranded RNA represents an efficient and frequently used way to study gene function. RNAi utilizes sequence-specific double-stranded small interfering RNA to silence gene expression in mammalian cells. RNAi in human esophageal smooth muscles (SMCs) would increase the value for applications, however the related experiments were seldom performed in human SMCs. To investigate the expression of GIRK gene and its subunits in esophageal smooth muscles, a series of experiments was designed in this study.Methods GIRK gene expression and was detected in human esophageal SMCs with the methods of reverse transcription polymerase chain reaction (RT-PCR) and western blotting. The human esophageal SMCs were cultured for experiment models. The plasmid was constructed and then rearranged into adenovirus to achive vector Ad-GIRK4-shRNA.The human esophageal smooth muscles were transfected by Ad-GIRK4-shRNA and the expression of GIRK4,M2AChR was detected. The density of IKACh was observed using electrophysiological recording system.Results (1) The mRNA and protein expression of GIRK 2, 3, 4 subunits except for GIRK 1 were detected in human esophageal either longitudinal muscle or circular muscle cells.(2) A high effective RNAi sequence (GGCTGAGCAGAATGAAGAA) for GIRK4 was sccessfully achieved. (3) The plasmid pGCsi-GIRK4-shRNA-U6 with expression of GIRK4-shRNA was constructed successfully. (4)The adenoviral vector (Ad-GIRK4-shRNA) to express GIRK4-shRNA was constructed successfully. (5)The efficiency of knockdown in GIRK4 expression was different between the exogenous systems in 48.9% and endogenous in 94.3%. (6) RNA interference to GIRK4 expression was demonstrated both in time-dependent and dose-dependent model. Comparing the efficiency of knockdown in experimental and control group, the valiue of ratio from two groups was 15.1%, 35.2% and 51.1% in MOI 5, 20, 50 respectively in protein level. It was 49.1%, 85.3%, 1.7% at 24th, 48 th, 96 th hours in MOI 50 respectively in mRNA level. (7) Adenovirus was a sutiable vector for transferring shRNA into the human esophageal SMCs with more than 95% of efficiency. (8) Expression of M2AChR in sh-GIRK4 group was 0.609±0.027 which was more 19.6% than 0.509±0.028 in the control in mRNA level (p<0.05). It was 0.531±0.022 in sh-GIRK4 group which was more 18.6% than 0.448±0.023 in the control in protein level (p<0.05). (9)The current density of IKACh in Ad-GIRK4-shRNA group was–0.93±0.28pA/pF which was 53% less tha–1.95±0.51pA/pF in the control (p<0.01).Conclusion: Expression of GIRK 2-4 subunits was found in human esophageal SMCs.That indicated there would be functional GIRK channels in the SMCs.The expression of GIRK4 in human esophageal SMCs was not only decreased by adenovirus-delivered short hairpin RNA targeting GIRK4, but also the expression of M2AChRand the amplitude of IKACh were affected. GIRK4 might play an important role in the pathway(Ach-M2AChR-G protein- IKACh) and IKACh channel.