The Role Of TMEM16A In Alveolar Liquid Clearance And Apoptosis And Regulated By PI3K/Akt Signal Pathway

Clinically, the acute phase of ALI is characterised by cyanosis, hypoxaemia, pulmonary oedema, and increasing respiratory failure over time, resulting in multiorgan failure and a high mortality rate(up to 40%). A major cause of respiratory failure in the acute phase of ALI is damage to the epithelial endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid.There is convincing evidence that the alveolar epithelium is not only a tight epithelial barrier that resists the movement of edema fluid into the alveoli, but it is also actively involved in the transport of ions and solutes, a process that is essential for edema fluid clearance and the resolution of acute lung injury. Considerable experimental evidence indicates that active Na+ transport is the dominant ion transport mechanism involved in alveolar liquid clearance. Sodium channel(ENa C) is a major determinant of the rate of AFC. Less is known about the role of specific chloride channels in regulating transepithelial fluid transport. Hower, there is now evidence that CFTR might be the chloride channel involved in AFC.transmembrane protein 16A(TMEM16A,whichwe also call anoctamin 1(ANO1))is a chloride channel that is activated by intracellular Ca2+ and Ca2+-mobilizing stimuli. TMEM16 A was observed in epithelial cells of pulmonary bronchioles, whereas expression in lung alveolar cells was less prominent. As a chloride channel, there is no evidence that TMEM16 A might be involved in AFC.The PI3K/Akt signaling pathway plays important roles in a variety of biological processes. Activated Akt migrates to both the cytosol and the nucleus, regulating multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration.Small interfering RNA(si RNA) holds a great promise for the future of genomic medicine because of its highly sequence-specific gene silencing and universality in therapeutic target. In general, gene/si RNA carriers can be divided into two main categories, i.e., viral and non-viral carriers and viral vectors are more efficient in comparison to non-viral ones. lentivirus vectors provide efficient delivery, integration and long-term expression by establishing a stable provirus in target cells.These properties led to their widespread use in research and clinical environments for in vitro and in vivo gene transfers.To futher confirm the role of TMEM16 A in AFC, we contructed lentivirus vectors of si RNA expression and confirmed the interference effect in vitro. By endotracheal lentivirus injection, TMEM16 A gene expression in lung was silenced partly. Finally, to gain a better understanding of the mechanisms, Whether PI3K/Akt pathway is in volved in the regulation of TMEM16 A gene expression, was discussed in the present study. Part 1 Construction and identification of lentiviral vector expressingsi RNA of TMEM16AObjective: To construct the mouse TMEM16A-sh RNA lentiviral vector and identify the knock-down effieieney of the si RNA in LA795 cells in in vitro.Methods:. 1 The location of TMEM16 A expression was detected in LA795 cell by streptavidin-perosidase. 2 The abundance of TMEM16 A expression was detected by Real-Time PCR compared with Hepa1-6 and NIH/3T3 cell lines. 3 In order to ensure the effect of silence, we design four small TMEM16 A si RNA sequence and construct lentivirus-sh RNA vector accordingly. 4 we get optimum MOI values by Analysis the transfection efficiency in conditions of different transfection reagents. 5 the cells were divided six groups: Control group, Negative Control group, KD1 group, KD2 group, KD3 group and KD4 group. m RNA is extracted after 72 hours and the optimum si RNA sequence was getted by Real-Time PCR. 6 We choosed the optimum si RNA sequence to verify the knockdown effects at protein level further by Western blots.Results: 1 the expression of TMEM16 A located in cell membranes in LA795. the expression of TMEM16 A is higher in LA795 cell line compared with Hepa1-6 and NIH/3T3 cell lines, so LA795 cell line is suited to be verified knockdown experiment. 2 optimum MOI values is 20 in the condition of 5ug/ml Eni.S+polybrene. 3 TMEM16 A m RNA relative quantity is decreased in KD1,KD2,KD3,KD4 groups compared that of Negative Control group and the effect of knockdown of KD1 group is the best. 4. the effect of knockdown of KD1 group in protein level is about 81.35% by Western blots in vitro.Conclusions: Lentivial Vector expressing TMEM16A-sh RNA for mouse has been constructed sueeessfully and it can knockdown TMEM16 A protein expression of LA795 effectively.Part 2 Effect of Lenti-TMEM16A-sh RNA on AFC and apoptosis innormal and acute lung injury miceObjective: to determine the role of TMEM16 A in the regulation of AFC and apoptosis in mice under both physiological and pathophysiological conditions such as acute lung injury(ALI) by Lenti-TMEM16A-sh RNA endotracheal injectionMethods: 1 Concentrated Lenti-Neg-sh RNA was delivered into male wild-type C57BL/6 mice. 14 days after lentiviral supernatant delivery, the lung tissue was harvested to be observed the pathological variation. At the same time, bronchoalveolar lavage(BAL) was performed to detected the epithelial permeability which is impacted by Lenti-Neg-sh RNA. The reporter gene(GFP) and blue(Hoechst) fluorescence were analyzed by immunofluorescence and confocal microscopy.The protein was drawed to be verified the knock-out effect by Western-blot. 2 Male wild-type C57BL/6 mice were divided three groups :cotrol group, Lenti-Neg-sh RNA group and Lenti-TMEM16A-sh RNA group, then AFC were measured in live mice. The expression of α-ENa C and CFTR related with AFC and the expression of Bcl-2 and Bax related with apoptosis were detected by Western-blot. 3 ALI mouse models were established by intraperitoneal injection of LPS. At 24 hs after injection, the location expression of TMEM16 A and ZO-1 was observed by immunofluorescence and confocal microscopy both in control and ALI group. 4 Because of the failure of TMEM16 A knock-out in ALI mice, TMEM16 A inhibitor was emploied. Male wild-type C57BL/6 mice were divided four groups: control group, inhibitor-TMEM16 A group, ALI group and ALI+ inhibitor-TMEM16 A group, then AFC were measured in live mice. The expression of α-ENa C and CFTR related with AFC is detected both in control and ALI group by Western-blot.Results: 1 The verification of the silent effect of transfection of the slow virus into lung tissue:(1)The protein concentrations was no significant difference between control group and Lenti-Neg-sh RNA group in BALF( control group: 0.685±0.0823 g/L VS Lenti-NC group: 0.708±0.0826 g/L, P >0.05). Therefore, intratracheal instillation of lentiviral vectors did not increase epithelial permeability of lung.The structure of lung tissue is nomal with light microscope and confocal microscopy showed that transduced(GFP+) cells mainly localized at airway epithelias of the lung. The expression was also present in alveolar epithelial cells in scattered lung regions.(2) Western blot analysis revealed that the level of TMEM16 A protein in lenti-sh RNA group was reduced about 50% compared with that of Lenti-Neg-sh RNA group(P>0.05). TMEM16 A sh RNA efficiently knock down the protein in lung tissue in normal mice. 2 Even though there are 50% drop in the TMEM16 A expression after sh RNA transduction in lung tissue of untreated mice, we did not detect statistical significance in AFC among the groups(P>0.05). With TMEM16 A expression reduction in lung tissue, we did not detect the change of expression of α-ENa C and CFTR. Conversely, the expression of Bcl-2 was decreased and Bax was increased(P<0.05). 3 Tight junction protein ZO-1 was continuous in control group and disrupt in ALI group. Moreover, Twenty-four hours after intraperitoneal injection of LPS, the increased TMEM16 A expression in lung after LPS treatment was much more prominent in alveolar epithelial region than in bigger airway epithelial cells. 4 AFC was decreased in mice with LPS-induced lung injury compared with that in the control group(P<0.05). However, there was no statistical significance in AFC between inhibitor-TMEM16 A group and control group in untreated mice(P>0.05). On the contrary, administration of inhibitor-TMEM16 A significantly decreased AFC in ALI group(P<0.05). The expression of α-ENa C was decreased in ALI group compared that in control group. On the contrary, the expression of CFTR was increased in ALI group compared that in control group(P<0.05).Conclusions: In the present study, intratracheal instillation of lentiviral vectors did not increase epithelial permeability of lung and damage lung histopathology, so the lentiviral vector itself is safe. It showed lentivirus transduced successfully in the airway epithelias of the lung. Hower transfection rate was low in alveolar epithelial cells. TMEM16 A is not a primary ion channel to regulate AFC in lung at physiological condition. During acute lung injury, however, increased TMEM16 A is involved in the regulation of AFC. TMEM16 A maybe has anti-apoptotic effect. With TMEM16 A expression reduction in lung tissue, the expression of Bcl-2 was decreased and Bax was increased. ZO-1 was disrupt in ALI group and we postulate that the damage of cell-cell junction also contributes to the decreased AFC in ALI mice. Part 3 TMEM16 A protein expression of injuried cell by LPS regulated by PI3K/Akt signaling pathwayObjective: To explore whether PI3K/Akt signal pathway regulating TMEM16 A expression in the process of cell injury induced by LPS.Methods: 1 LA795 cells were stimulated with LPS for 0, 6, 12, 18, 24, 48 hrs and then harvested. The expression of TMEM16 A, t-Akt and p-Akt was detected by western-blot. The cells were divided four groups: PS, LPS+5μmol/L LY294002, LPS+20μmol/L LY294002 and LPS+ 40μmol/L LY294002. After 24 hours, the expression of TMEM16 A,t-Akt and p-Akt was detected given various concentrations of LY294002. 2 The cells were divided four groups: control group, control+40μmol/L LY294002 group, LPS and LPS group +40umol/L LY294002 group. After 24 hours, the expression of TMEM16 A,t-Akt and p-Akt was detected. 3 The expression and distribution of TMEM16 and α-tubulin were observed by immunofluorescence and confocal microscopy in different groups.Results: 1 There were no significant differences in the TMEM16 A protein levels at 0 and 6 hour after LPS treatment, however, TMEM16 A protein significantly increased at 12,18,24, and 48 h and peaked at 24 hour. p-Akt protein gradually increased and peaked at 18 hour with the extension of LPS stimulation(P<0.05). However, the expression of t-akt did not change at anypoint-in-time(P>0.05). 2 P-Akt protein gradually decreased and with LY294002 concentration ascent. The expression of p-Akt significantly decreased when LY294002 concentration reached 40umol/L(P<0.05). There were no significant differences in t-akt protein among groups(P>0.05). TMEM16 A protein gradually increased and with LY294002 concentration ascent. The expression of TMEM16 A significantly increased when LY294002 concentration reached 40umol/L(P<0.05). 3 There were no significant differences in TMEM16 A and p-Akt proteins between control and control+40μmol/L LY294002 group. However, The expression of TMEM16 A significantly increased given 40umol/L LY294002 in LPS group(P<0.05). 4 TMEM16 A is located in cytomembrane which declared it is a transmembrane protein. α-tubulin, a cytoskeletal protein, was used to mark the microtubule structure of the cells. The intensity of red fluorescence(TMEM16A) was increased in LPS+40umol/L LY294002 group. It was consistent with the results of Western blot.Conclusions: The expression of TMEM16 A varied dynamically after LPS stimulation that stated TMEM16 A protein participates in the process of cell injury. The expression of p-Akt increased in the form of time-dependent after LPS stimulation. A specific blocker(LY294002)of PI3K/Akt pathway suppressed the expression of p-Akt in the form of concentration-dependent and improved the expression of TMEM16 A. TMEM16 A protein expression of injuried cell induced by LPS is regulated by PI3K/Akt signaling pathway.