Regulation Of Long Non-coding RNA MALAT1 In Children With Mycoplasma Pneumoniae Pneumonia

ObjectiveOur aim was to determine whether the long non-coding RNA(lnc RNA)metastasisassociated lung adenocarcinoma transcript 1(MALAT1)is involved in Mycoplasma pneumoniae pneumonia(MPP),and its possible mechanism.MethodsBronchoalveolar lavage fluid(BALF)of 50 hospitalized children diagnosed with MPP in Children's Hospital affiliated to Nanjing Medical University and 30 hospitalized children with tracheal foreign body(FB)were collected.The total RNA in BALF was extracted,the expression MALAT1 was detected by q RT-PCR,and the concentrations of inflammatory factors IL-8 and TNF-? in BALF were detected by ELISA.Human airway epithelial cells A549 and beas-2b were cultured,and the expression of MALAT1 in the cells was down-regulated by RNA interference(RNAi).The cells were infected with Mycoplasma pneumoniae(MP)strain M129,and was divided into negative control group,MALAT1 knockdown group,MP infection group,and MP infection +MALAT1 knockdown group.The expression levels of MALAT1,IL-8 and TNF-? in supernatant were detected.The expression of pp65 protein in the nucleus was detected by western blot.The transcriptional activity of nuclear factor-?B(NF-?B)in cells was detected by double luciferase reporter gene.Mouse MALAT1 knockdown model and mouse MP pulmonary infection model were established,and the lung tissues,peripheral blood and BALF of the mice were collected,and the lung histopathology was observed by HE staining.The expression of MALAT1 in lung tissues was detected by q RT-PCR.Oxidation-antioxidant indexes(MDA,SOD)and pulmonary vascular permeability damage indexes(lung homogenate protein concentration,lung homogenate protein concentration/total serum protein concentration)were detected.The levels of IL-8 and TNF-? in lung homogenate and BALF were determined by ELISA.The expression of pp65 in lung tissues was detected by western-blot.ResultsThe expression levels of MALAT1,IL-8 and TNF-? in BALF of children in the MPP group were significantly increased compared with those in the tracheal foreign body group(all P <0.01).Correlation analysis showed that MALAT1 expression in BALF of children in MPP group was positively correlated with IL-8 and TNF-?(P <0.001).Following incubation with MP strain,the expression of MALAT1 in the human airway epithelial cells A549 and BEAS-2B was increased significantly compared to that in the control group(P <0.01),and the concentrations of IL-8 and TNF-? in the supernatant were increased compared with that in the control group(P <0.01).The increased expression of IL-8 and TNF-? caused by MP infection could be suppressed by MALAT1 knockdown(P <0.01).Following incubation with MP strain,the expression of pp65 in the nucleus of the human airway epithelial cells A549 and BEAS-2B was increased than that in the control group,and this change was reversed after MALAT1 was knocked down.Following incubation with MP strain,detected by luciferase reporter gene,NF-?B activation of the human airway epithelial cells A549 and BEAS-2B was higher than that in the control group(P<0.01),and MALAT1 knockdown inhibited the activation of NF-?B in airway epithelial cells caused by MP infection(P<0.05).After infected with MP strain,lung histopathology of BALB/c mice showed obvious infiltration of inflammatory cells.The expression of MALAT1,the concentration of lung homogenate protein,the ratio of lung homogenate/ serum total protein and the MDA concentration increased(P<0.05),while SOD concentration decreased(P<0.05),compared with the uninfected group.Knockdown of MALAT1 reduced the histopathological damage caused by MP infection in BALB/c mice,and the increase range of MDA,lung homogenate protein concentration,lung homogenate protein concentration/total serum protein concentration and reduction range of SOD were all lower than those in the negative transfection group,with statistically significant differences(P<0.05).After infected with MP strain,the concentrations of IL-8 and TNF-? in the lung homogenate and BALF of BALB/c mice were significantly increased compared with those in the uninfected group(P<0.01),while MALAT1 knockdown supressed the elevated level of IL-8 and TNF-? caused by MP infection(P<0.05).After infected with MP strain,the expression of pp65 in the lung tissues of BALB/c mice was increased compared with that of the control group,while the expression of pp65 in MP-infected mice was decreased after MALAT1 knockdown.ConclusionMALAT1 is highly expressed in BALF of children with MPP and is related to the level of inflammatory factors.MALAT1 is involved in the regulation of MP induced epithelial cell secretion by mediating NF-?B.Knocking down MALAT1 in mice can effectively improve the pulmonary inflammatory damage caused by MP infection in mice.