| Objective:Built on our previous research,we set out to test the role of miR-539 in bacterial infection in the lung.We transfected miR-539 mimic into mouse alveolar macrophages(MH-S cells)to examine whether upregulated miR-539impact TNF-αand IL-1βexpression in MH-S cells after Pseudomonas aeruginosa infection.We next transfected miR-539 mimic into mouse lungs and studied how up-regulated miR-539 affects P.aeruginosa pneumonia in mice.Methods:(1)MH-S cells were studied and P.aeruginosa laboratory strain PA14 stimulated MH-S cells,and the changes in the relative expression levels of miR-539 in MH-S cells were detected by RT-q PCR.(2)Transfection of miR-539mimic(539-m)with FAM marker,cell flowmetry to detect cell transfection efficiency followed by RT-q PCR to detect the expression level of miR-539 using Negative Control mimic(NC-m)as control.(3)MH-S cells cultured in vitro were randomly divided into 4 groups,NC-m(-),539-m(-)group(control)and NC-m(+),539-m(+)group(bacterial infection),respectively.The bacterial infection group was infected with the transfected cells for 1 h after transfection with miRNA mimic for 24 h at MOI(multiplicity of infection)=20.Then the medium containing bacteria was discarded,and 100 ng/m L polymyxin B was added and the culture was continued for 24 h.The cells were collected and RNA and protein were extracted separately.We next detected the m RNA expression levels of miR-539,TNF-α,IL-1βin each group by RT-q PCR,and detected the protein expression changes of miR-539,TNF-α,IL-1βin each group by Western blot.(4)The model of P.aeruginosa pneumonia in C57BL/6 mice was set up by instilling50μl of bacterial solution containing 5 x 10~6 bacteria into the airway.The general condition,weight change,lung histopathology and CFU(colony forming units)count of the mice were also observed.(5)After tail vein injection of miR-539mimic 50μg/mouse for 24 h,lung tissues were removed and RT-q PCR was performed to detect the effect of miR-539 mimic transfection and to establish a mouse lung transfection model.(6)C57BL/6 mice transfected with miR-539mimic(539-m)or Negative Control mimic(NC-m)were airway perfused with P.aeruginosa 5x10~6 CFU/mouse for 24 h.The left lung was formalin-fixed and subjected to HE staining was performed to observe morphological changes in the lung tissue.The right lung was homogenized after CFU counting and RNA extraction to detect the expression of miR-539,TNF-αm RNA and IL-1βm RNA.Results:(1)Compared to normal MH-S cells,the relative expression of miR-539 was increased in MH-S cells after PA14 stimulation,suggesting that miR-539 is associated with the development of inflammation induced by P.aeruginosa.(2)Compared to untransfected MH-S cells,transfection efficiency was 93.2%by cell flowmetry after transfection of miR-539 mimic labeled with FAM marker.RT-q PCR analyses showed that the relative expression of miR-539in cells transfected with miR-539 mimic was significantly increased by approximately 10-fold compared to negatively transfected cells.(3)MH-S cells transfected with miR-539 mimic were infected with PA14 and the relative expression of miR-539 was reduced in the 539-m group compared to the uninfected 539-m group.m RNA expression of TNF-αand IL-1βincreased significantly in 539-m-transfected cells vs.NC-m-transfected cells after P.aeruginosa infection.We also detected TNF-α,IL-1βprotein expression using Western blot,which was also increased in the 539-m group compared to the NC-m group after P.aeruginosa infection.The results of both transcript and protein levels suggest that miR-539 promotes the expression of TNF-αand IL-1βin MH-S cells in response to P.aeruginosa infection indicating that miR-539 may have a regulatory role in mouse alveolar macrophages in response to P.aeruginosa-induced inflammation.Meanwhile miR-539 inhibitors can block the elevation of these cytokines.(4)Mice infected with PA14 lost more weight compared to mice in the PBS group,with the proportion of the original weight loss being 2%versus12%,respectively.Further,the weight of lung tissue increased by about 1%of body weight in the PA14 group of mice and by about 0.6%in the PBS group.CFU counts showed that P.aeruginosa was not detected in the PBS group and approximately 10~6 CFU/mg of lung tissue in the PA14 group of mice.Lung histopathological sections showed that the lung tissue of PBS-treated mice was structurally intact,with no exudate in the alveolar lumen,no inflammatory cell infiltration,and no thickening of the alveolar wall.In mice with pneumonia,the alveolar structure was disorganized,the alveolar wall was edematous and thickened,some of the alveolar cavities were fused,and a large number of scattered inflammatory cell infiltrates were seen in the tissues.(5)By tail vein injection of miRNA mimic,the relative expression of miR-539 in cells within lung tissue was increased in the 539-m group compared to the NC-m group.Based on transfected mice,the role of miR-539 in P.aeruginosa pneumonia was observed,and the results showed that the relative expression of miR-539 was upregulated in the 539-m group after PA14 infection compared to the NC-m(-)group.Compared with the NC-m(+)group,both TNF-αm RNA and IL-1βm RNA were significantly increased in the lung tissue of mice in the 539-m(+)group.Lung histopathological sections showed scattered hemorrhages,alveolar wall edema and inflammatory cell infiltration in the NC-m(+)group,and more severe damage in the 539-m(+)group,with massive structural destruction of alveoli,alveolar collapse or alveolar wall edema,fusion,diffuse inflammatory cell infiltration and hemorrhage.(6)The macrophages within the lung tissue showed increased expression of miR-539 after P.aeruginosa infection compared to the negative control group.Meanwhile,miR-539 mimic promoted m RNA expression of TNF-αand IL-1βwhen infected with P.aeruginosa compared to NC-m(-)group.(7)Lung homogenate colony counts suggested that NC-m(+)mice had approximately 10~6 CFU/mg of lung tissue colonies,while miR-539mice had more bacteria in their lung tissue at approximately 10~7 CFU/mg.(8).Mouse survival curves showed that miR-539 increased mortality in mice with median survival times of 69 h(95%CI:0.4459,3.948)versus 52 h(95%CI:0.2533,2.242)in the NC-m(+)and 539-m(+)groups,respectively,with an observation time of 120 h(P=0.0305).Conclusion:Our preliminary research demonstrate:1.The miR-539transfection rate was efficient.2.miR-539 promotes the expression of TNF-αand IL-1βin P.aeruginosa-infected alveolar macrophages and was involved in regulating the P.aeruginosa-induced inflammatory response.3.miR-539stimulates inflammatory response and increases mortality in mice in P.aeruginosa pneumonia. |
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