The Effect Of TMEM16A On Proinflammatory Cytokine Secretion And Apoptosis Of Lipopolysaccharide-induced A549Cells

Acute lung injury(ALI) or acute respiratory distress syndrome (ARDS) isa severe complication that increases the mortality in critically ill patients.ALI/ARDS-associated mortality remains high at40%. The pathophysiologicalbasis of ALI consists of excessive and protracted alveolar inflammationaccompanied by alveolar epithelial injury, including epithelial cell death.Alveolar epithelial cells (AECs) are the first cells that encounter pathogenicmicroorganisms. AECs are not only the targets of inflammatory cells andmediators but also active inflammatory and responsive cells. They can beactivated by LPS, and secrete inflammatory cytokines, such as IL-8, IL-6,TNF-α, ICAM-1etc. A549cells have been extensively used to investigate theinjury-defense mechanism of AECs.TMEM16A protein is postulated as an important component of Calciumactivated chlofide channels (CaCCs) in various types of cells and tissues.CaCCs are anionic channels and play a variety of physiological roles indifferent organs and tissues. In vivo and in vitro studies have demonstratedthat TMEM16A is needed for Ca2+dependent Cl-secretion. In the airways ofpatients with cystic fibrosis, overexpression of TMEM16A could suppress theexpression and release of the proinflammatory cytokine IL-8. At present, thereare no studies focusing on TMEM16A protein in AECs and the role ofTMEM16A in ALI. In the previous studies, our research team had found thatTMEM16A was expressed in normal rats alveolar epithelial cells, andTMEM16A expression decreased in rats with LPS-induced acute lung injuryby trans-tracheal intratracheal instillations. We speculate that TMEM16Aplays a role in acute lung injury, but it is unclearly whether TMEM16Aexpress in human type Ⅱ epithelial cells or not? How does LPS regulate the expression of TMEM16A in human alveolar epithelial type Ⅱ cell? WhetherTMEM16A play a role in the proinflammatory cytokine secretion andapoptosis of LPS-induced A549? We will try to solve these problems in thepresent study. We use the human lung type Ⅱepithelial cell line A549as thecellular model，and use methods of molecular biology and cell biology to:(1)create an acult injury cell model by treating A549cell with LPS, and observethe expression of endogenous TMEM16A in A549treated with or without LPStreatment.(2) create a stable TMEM16A overexpression A549cell line.(3)study the effect of over-expression of TMEM16A on the proinflammatorycytokine secretion and apoptosis of LPS-induced A549.(4) knock downTMEM16A gene by RNA interference and study the effect of TMEM16Aknockdown on the proinflammatory cytokine secretion.Part one LPS regulates the expression of TMEM16A in human alveolarepithelial type ⅡcellObjective: Create an acult injury cell model by treating A549cell with10μg/ml LPS for24h, and observe the expression of endogenous TMEM16Ain A549treated with or without LPS treatment.Methods: TNF-α secretion of A549cells was evaluated by ELISAfollowing the manufacture’s instructions. The apoptosis of A549cells wasdetected by flow cytometry with AnnexinV-FITC/PI double staining. Theorganelle of A549cell was observed with transmissionelectron microscope.The A549cells were divided into six groups: untreated, treated with LPSfor6,12,24,36and48h. The whole cell lysates were collected respectively todetermine the endogenous TMEM16A protein expression. The TMEM16Aprotein was evaluated by Western blots.Results:1An acult injury cell model was created.(1) Results of ELISA: TNF-αlevel significantly increased at3,6,12and24h after LPS treatment.(2)Electron microscopy: compared to A549untreated with LPS, the organelles ofA549cell treated with lipopolysaccharide (10μg/ml,24h): microvilli reduced.vacuoles, the fusion of the ridge, membrane of mitochondria and even the lack of ridge appeared in the cells. Rough endoplasmic reticulum dilated gentlely,with granule fusion and degranulation.⑶Compared to A549untreated withLPS, the early apotosis rate and late apoptosis/necrosis rate of A549celltreated with LPS(10μg/ml,24h) increased significantly.2The endogenous TMEM16A protein was expressed in the alveolarepithelial A549cell lines. There were no significant differences in theTMEM16A protein levels at0,3,6and12h after LPS treatment, however,TMEM16A protein significantly increased at24and36h and decreasedmarkedly at48h after LPS treatment.Conclusions: LPS could successfully induce acute injury in A549cells:LPS activated AECs-II and induce proinflammatory cytokine secretion andapoptosis.We provided the first evidence that AEC-II (A549) cells expressedendogenous TMEM16A protein, and TMEM16A protein significantlyup-regulated at24and36h and decreased at48h after LPS treatment,indicating TMEM16A participates in the process of acute Alveolar epithelialcells injury.Part two TMEM16A overexpression inhibits LPS-induced proinflamma-tory cytokine secretion and apoptosis of A549Cell LineObjective:⑴Create stable TMEM16A overexpression A549Cell Line.⑵Study the effects of overexpression of TMEM16A on proinflammatorycytokine secretion and apoptosis of A549Cell Line treated with LPS.Methods: TMEM16A gene was transfected into A549cell withLipofectamine2000, and then cell clones were picked up in the presence ofG418to create stable TMEM16A expression A549cell line. TMEM16AmRNA and protein expressions were measured by real-time quantitative PCRand western blots, respectively. Proinflammatory cytokine TNF-α、IL-8levelswere determined by ELISA following the manufacturer’s instructions. Theapoptosis of cells was detected by flow cytometry with AnnexinV-FITC/PIdouble staining.Results:1TMEM16A gene was transfected into A549cell with Lipofectamine 2000, and then cell clones were picked up in the presence of G418. To verifywhether TMEM16A gene was over expressed by pCMV6-TMEM16A plasmidDNA transfection，we detected TMEM16A mRNA and protein expression inA549cells transfected with the pCMV6-TMEM16A plasmid DNA，emptypCMV6plasmid（negative control）and non-transfected A549. The resultsshowed that TMEM16A mRNA and protein expressions were significantlyhigher in TMEM16A stable overexpression A549cells than that in thenegative control group and non-transfected A549group. TMEM16Aoverexpression cell line of A549was established.2The levels of TNF-α and IL-8at multiple time points after LPSexposure were obviously higher than that under normal conditions in threegroups, respectively. Under normal conditions, there were no differenceamong the levels of TNF-α and IL-8of three groups, respectively. Afterlipopolysaccharide treatment, the levels of TNF-α and IL-8secreted byTMEM16A overexpression A549cells at multiple time points were obviouslylower than the levels of TNF-α and IL-8secreted by cells transfected with theempty vector and non-transfected cells. In brief, LPS induced A549, negativecontrol A549and stable TMEM16A overexpression A549to secrete TNF-αand IL-8. Under normal conditions, TMEM16A overexpression alone did notaffect the secretion of TNF-α and IL-8, however, TMEM16A overexpressionreduced LPS-induced TNF-α and IL-8secretion.3Early apoptosis rate and late apoptosis/necrosis rate of cells in threegroups after LPS exposure were obviously higher than that under normalconditions, respectively. Under normal conditions, early apoptosis rate andlate apoptosis/necrosis rate of cells in three groups were not different,respectively. After lipopolysaccharide treatment, the early apoptosis rate inTMEM16A overexpression A549cells were obviously lower than that in cellstransfected with the empty vector and non-transfected cells. In brief, LPStreatment increased the apoptosis rate of A549, negative control A549andstable TMEM16A overexpression A549. Under normal conditions,TMEM16A overexpression alone did not affect the apoptosis of cells, however, TMEM16A overexpression reduced LPS-induced early apoptosis rate of cells.Conclusions: TMEM16A overexpression cell line of A549wasestablished. TMEM16A overexpression attenuates the proinflammatorycytokine secretion of A549treated with LPS and can suspressed LPS-inducedapoptosis of A549cell.Part three The effect of TMEM16A knockdown on proinflammatorycytokine secretion from LPS-induced A549Objetive:⑴Observe the influence of lentiviral vector mediated RNA interferenceon expression of human TMEM16A gene in human Pulmonaryadenoeareinoma cell line A549, and creat stable TMEM16A knockdown A549cell.⑵Study the effects of TMEM16A knockdown on proinflammatorycytokine secretion of A549Cell Line treated with LPS.Methods: We commissioned Genechem Co. Ltd to design，screen andcompound four RNA interference sequeneces targeting TMEM16A gene and ascramble interference sequenece（as RNAi negative control），and then thesequences were separately cloned into the CGV115-GFP vector and constructlentiviral RNAi vectors: TMEM16A-RNAi-LV1#, TMEM16A-RNAi-LV2#,TMEM16A-RNAi-LV3#, TMEM16A-RNAi-LV4#and scramble-RNAi-LV.These lentiviral RNAi vectors were then transfected into competent cells DH5α, then positive clones of recombinant gene were screened by PCR andidentified by sequencing. The titer of lentivirus was determined after293Tcells were contransfected with TMEM16A-RNAi-LV、pHelper1.0、pHelper2.0.The recombinant lentiviruses were transfected into A549cells. The expressionof GFP co-contransfected were detected by fluorescence microscopy todetermine whether the recombinant vectors were efficiently transfected intoA549. After TMEM16A protein expression was examined by western blots，we screened that TMEM16A-RNAi-LV3#recombinant lentivirus knockdownTMEM16A more efficiently, so we selected TMEM16A-RNAi-LV3#recombinant lentivirus to proceed the following experiment, and named it as TMEM16A-siRNA-LV TMEM16A mRNA and protein expression wereexamined by real-time quantitative PCR and western blots, respectively, andthe results were compared with those of the non-transfected and Scr-RNAi-LVtransfered A549cells. Proinflammatory cytokine TNF-α、IL-8secretion ofA549cells, scr-RNAi-LV transfered A549cell and TMEM16A-RNAi-LVwere evaluated by ELASA following the manufacturer’s instructions.Results:1Fluorescence microscopy results showed: More than80%of A549cellstransfected with TMEM16A-RNAi-LV1#, TMEM16A-RNAi-LV2#, TMEM16A-RNAi-LV3#and Scr-siRNA-LV expressed GFP at72h post transfection,but cells transfected with TMEM16A-RNAi-LV4#didn’t express GFP.2TMEM16A Protein expression was examined by western blots，and wescreened that TMEM16A-RNAi-LV3#recombinant lentiviruses knockdownTMEM16A more efficiently, so we selected TMEM16A-RNAi-LV3#recombinant lentiviruses to proceed the following experiment, and named it asTMEM16A-siRNA-LV.3TMEM16A expression in A549cells transfected with the TMEM16A-siRNA-LV was significantly inhibited at both mRNA and protein levelscompared with the non-tansfected and Scr-SiRNA-LV transfered A549cells.TMEM16A stable knockdown cell line of A549was established.4The levels of TNF-α and IL-8at multiple time points after LPSexposure were obviously higher than that under normal conditions in threegroups, respectively. Under normal conditions, there were no differenceamong the levels of TNF-α and IL-8of three groups, respectively. Afterlipopolysaccharide treatment, the levels of TNF-α and IL-8secreted byTMEM16A knockdown A549cells at multiple time points were obviouslyhigher than the levels of TNF-α and IL-8secreted by cells transfected withScr-SiRNA-lentivirus and non-transfected cells. In brief, LPS induced A549,RNC and TMEM16A stable knockdown A549to secrete TNF-α and IL-8.Under normal conditions, TMEM16A knockdown alone did not affect thesecretion of TNF-α and IL-8, however, TMEM16A knockdown increased LPS-induced TNF-α and IL-8secretion.Conclusion: TMEM16A knockdown A549cell line was created by RNAinterference with the lentivirus vector system. TMEM16A knockdownincreased the secretion of TNF-α and IL-8from LPS-induced A549.