To construct retroviral vector carrying human interleukin-3 c0mplementary DNA(HuIL-3 cDNA) under control of human a-fetoprotein gene enhancer core sequence and human SV4O pro-moter. Methods and Results: HuIL-3 cDNA was inserted into polylinker site of retroviral vector pMNSMto construct retr0viral vector pMNS-IL-3, in which the transcription of HuIL-3 cDNA was drived by SV40early region promoter. Human Q-fetoprotein gene enhancer core sequence was released from plasmidpGEM. 7Zf-AFPe and inserted into the polylinker site of pMNSM. Then human interleukin-3 cDNA wasinserted int0 p0lylinker site to construct retroviral vector pMNSA-IL-3, in which HuIL-3 cDNA transcrip-tion was drived by SV40 early region promoter and enhanced by human a-fetoprotein enhancer core se-quence- Conclusion: The vectors are of significance for hepatoma-specific gene therapy.

Objective To investigate the effect and mechanism of miR-141-3p on pulmonary fibrosis in rats with acute respiratory distress syndrome(ARDS).Methods Rats were divided into the control group,the model group,the agomir negative control group and the miR-141-3p agomir group according to random number table,with 10 rats in each group.In addition to the control group,the ARDS rat model was established by lipopolysaccharide(LPS)infusion.Rat alveolar typeⅡepithelial cells RLE-6TN cells were divided into the NC group,the LPS group,the miR-NC group,the miR-141-3p mimics group,the miR-141-3p mimics+pcDNA group and the miR-141-3p mimics+NRF2 and Kelch-like ring associated protein 1(Keap1)group.LPS cell model was established in all groups except the NC group.The mRNA expression levels of miR-141-3p and Keap1 in lung tissue and cells were detected by qPCR.Western blot assay was used to analyze lung tissue and cell epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),microtubule associated protein light chain 3B(LC3B),autophagy associated gene Beclin-1,α-smooth muscle actin(α-SMA),type I collagen(Col-Ⅰ),Keap1 and nuclear factors E2 related factor 2(NRF2)and heme oxygenase 1(HO-1).HE staining and Masson staining were used to observe pathological changes of lung tissue and to estimate the area of lung tissue injury and pulmonary fibrosis.Hydroxyproline(Hyp)in lung tissue was detected by the kit.Levels of inflammatory factor interleukin-1β,tumor necrosis factor(TNF-α)and oxidative stress index malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.Dual luciferase reporting experiment was used to verify the targeting relationship between miR-141-3p and Keap1.Results The expression of miR-141-3p was down-regulated and the expression of Keap1 was up-regulated in lung tissue and cells(P＜0.05).Overexpression of miR-141-3p can reduce the degree of pathological damage and fibrosis of lung tissue in rats,Hyp content,and up-regulate expression levels of SOD,E-cadherin,LC3B,Beclin-1,NRF2 and HO-1 in lung tissue and cells,and down-regulate the expression levels of IL-1β,TNF-α,MDA,N-cadherin,α-SMA,Col-I and Keap1(P＜0.05).Overexpression of Keap1 was able to reverse the improvement effect of overexpression of miR-141-3p on alveolar epithelial cell damage in ARDS rats(P＜0.05).Double Luciferase reporter gene experiment confirmed that miR-141-3p and Keap1 may have a targeted regulatory relationship.Conclusion Overexpression of miR-141-3p may activate the Keap1-NRF2/ARE signaling pathway,activate autophagy,inhibit inflammatory response,oxidative stress,and EMT progression,and improve pulmonary fibrosis in ARDS rats.

Objective:To discuss the effect of inhibiting microRNA(miR)-193a-5p expression on pulmonary fibrosis in the rats with acute respiratory distress syndrome(ARDS),and to clarify the related mechanism.Methods:A total of 60 male SD rats were divided into sham operation group,model group,miR-193a-5p antagonist group(Antagomir group),and negative control group(Antagomir-NC group),and there were 15 rats in each group.The ARDS animal model was induced by administering 10 mg·kg-1 lipopolysaccharide(LPS)via tracheal instillation,while the rats in sham operation group received an equal volume of saline.After successful modeling,the rats in Antagomir group and Antagomir-NC group were treated with miR-193a-5p Antagomir or Antagomir-NC via tail vein injection.The arterial partial pressure of oxygen(PaO2)and oxygenation index(OI)of the rats in various groups were measured;HE staining and Masson staining were used to observe the pathology and collagen fiber deposition in lung tissue of the rats;kit was used to detect the level of hydroxyproline(Hyp)in lung tissue of the rats in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the levels of inflammatory factors tumor necrosis factor α(TNF-α),interleukin(IL)-1β,and IL-6 in bronchoalveolar lavage fluid(BALF)of the rats in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-193a-5p in lung tissue of the rats in various groups;Western blotting method was used to detect the expression levels of β-catenin,Snail family transcriptional repressor 1(Snail1),and α-smooth muscle actin(α-SMA)proteins in lung tissue of the rats in various groups.Results:Compared with sham operation group,the PaO2 and OI of the rats in model group were significantly decreased(P<0.05);compared with model group,the PaO2 and OI of the rats in Antagomir group were significantly increased(P<0.05).The HE staining results showed that the lung tissue structure of the rats in sham operation group was normal,and there were no obvious inflammatory changes;compared with sham operation group,mild abnormalities in lung tissue structure,alveolar atrophy,and collapse were observed in the rats in model group and Antagomir-NC group,with a large number of lymphocytes and a small number of neutrophils infiltrating in the alveolar cavities,and widened alveolar spaces;compared with model group,the rats in Antagomir group showed a significant reduction in lymphocytes and neutrophil infiltration in the alveolar cavities and there were no obvious hyperplasia.The Masson staining results showed no obvious blue collagen fiber deposition in lung tissue of the rats in sham operation group;compared with sham operation group,significant blue collagen fiber deposition was observed in lung tissue of the rats in model group and Antagomir-NC group,with severe damage of the alveolar structure,indicating obvious pulmonary fibrosis;compared with model group,the deposition of blue-stained collagen fibers in lung tissue of the rats in Antagomir group was significantly reduced.Compared with sham operation group,the level of Hyp in lung tissue of the rats in model group was significantly increased(P<0.05);compared with model group,the level of Hyp of the rats in Antagomir group was significantly decreased(P<0.05).The ELISA results showed that compared with sham operation group,the levels of TNF-α,IL-1β,and IL-6 in BALF of the rats in model group were significantly increased(P<0.05);compared with model group,the levels of TNF-α,IL-1β,and IL-6 of the rats in Antagomir group were significantly decreased(P<0.05).The RT-qPCR results showed that compared with sham operation group,the expression level of miR-193a-5p in lung tissue of the rats in model group was significantly increased(P<0.05);compared with model group,the expression level of miR-193a-5p of the rats in Antagomir group was significantly decreased(P<0.05).The Western blotting results showed that compared with sham operation group,the expression levels of β-catenin,Snail1,and α-SMA proteins in lung tissue of the rats in model group were significantly increased(P<0.05);compared with model group,the expression levels of β-catenin,Snail1,and α-SMA proteins in lung tissue of the rats in Antagomir group were significantly decreased(P<0.05).Conclusion:Inhibition of miR-193a-5p expression can improve the lung function and alleviate the pulmonary fibrosis in the ARDS rats by reducing the inflammatory responses and downregulating the expressions of β-catenin,Snail1,and α-SMA proteins.

Objective : To investigate the effect of miR-141-3p on LPS induced A549 cell injury by targeting high mobility group protein 1 (HMGB1) ． Methods : A549 cells derived from type Ⅱ alveolar epithelial cells were taken as the study object，miR-141-3p mimics，mimics NC，HMGB1 gene overexpression plasmid (pcDNA3. 1-HMGB1) and empty Vector were transfected into A549 cells respectively or co-transfected，then 10 μg / ml LPS was used for 24 h．Cell proliferation activity was detected by cell counting kit-8 ( CCK-8) ．The activity of lactate dehydrogenase ( LDH) in the supernatant of cell culture was detected by colorimetry．The apoptosis level of each group was detec- ted by flow cytometry．The levels of interleukin (IL) -1 β , IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (Elisa) ．Dual luciferase reporter gene assay verified the targeted regulatory relationship between miR-141-3p and HMGB1 . Results : After treatment with LPS ，the proliferative activity of A549 cells and the expression level of miR-141-3p decreased ( P ＜0. 05 ) ，the apoptosis rate increased ( P < 0. 05) ，the levels of IL-1 β , IL-6，TNF-α and the activity of LDH in supernatant increased (P＜0. 05) ．Overex- pression of miR-141-3p increased the proliferation activity of A549 cells treated with LPS (P ＜0. 05 ) ，and de- creased the apoptosis rate and the levels of IL-1 β , IL-6，TNF-α in cells and LDH activity in supernatant (P < 0. 05) ．However，overexpression of HMGB1 gene could reverse the ameliorative effect of miR-141-3p on LPS-in- duced A549 cell injury．Dual luciferase reporter gene experiment confirmed that HMGB1 was the downstream target gene of miR-141-3p． Conclusion miR-141-3p can inhibit LPS-induced apoptosis，reduce the expression level of inflammatory factors，and improve the damage of A549 cells，which may be related to the targeted regulation of HMGB1 expression.