Front Immunol. 7:389(2016) 

MicroRNA-7 Deficiency Ameliorates the Pathologies of Acute Lung Injury through Elevating KLF4

Lin Xu


摘要:

Recent evidence showed that microRNA-7 (miR-7) played an important role in the pathologies of lung-related diseases. However, the potential role of miR-7 in acute lung injury (ALI) still remains poorly understood. Here, we assessed the effect of miR-7 deficiency on the pathology of ALI. We, first, found that the expression of miR-7 was upregulated in lung tissue in murine LPS-induced ALI model. Notably, we generated miR-7 knock down mice by using miRNA-Sponge technique and found that miR-7 deficiency could ameliorate the pathologies of lung as evidenced by accelerated body weight recovery, reduced level of bronchoalveolar lavage (BAL) proinflammatory cytokines and decreased number of BAL cells in ALI mice. Moreover, the proportion and number of various immune cells in BAL, including innate immune cell F4/80+ macrophages, γδT cells, NK1.1+ T cells, and CD11c+DCs, as well as adaptive immune cell CD4+ T cells and CD8+ T cells, also significantly changed, respectively. Mechanistic evidence showed that KLF4, a target molecule of miR-7, was upregulated in lung tissues in ALI model, accompanied by altered transduction of NF-κB, AKT, and ERK pathway. These data provided a previously unknown role of miR-7 in pathology of ALI, which could ultimately aid the understanding of development of ALI and the development of new therapeutic strategies against clinical inflammatory lung diseases.We annealed and ligated, gel purified and cloned oligonucleotides for miR-7 binding sites with 5-nt spacers (one spacer: 5′-CGCG-3′) for 6 bulged sites (one bulged site: 5′-AACAAAATCGAGG- -TCTTCCA-3′), into pEGFP-C2 vector (Invitrogen) digested with HindIII and KpnI enzyme for the construction of pEGFP-C2-miR-7 sponge vector.We next established miR-7 KD (miR-7KD) FVB/N mice using pEGFP-C2-miR-7 sponge vector with the help of Cyagen Biosciences Inc. To identify miR-7KD mice, we designed the primers spanning the EGFP sites and miR-7SP sequence, forward (P1): CGCACCATCTTCTTCAAGGA, reverse (P2): TGGAAGACCTCGATTTTGTTC. The predicted PCR product was 505 bp. All animals were housed under specific pathogen-free conditions at Zunyi Medical College. And all animal experiments were performed according to the guidelines for the Care and Use of Laboratory Animals (Ministry of Health, China, 1998). The experimental procedures were approved by the ethical guidelines of Zunyi Medical College laboratory Animal Care and Use committee (permit number 2013016).To investigate the potential role of miR-7 in the development of ALI, we detected the relative expression level of miR-7 in murine 12 organs, including heart, lung, brain, kidney, and so on. Data showed that miR-7 expression had a higher level in lung tissue (Figure S1A in Supplementary Material). Expectedly, we further found that the expression of miR-7 significantly increased in pathogenic lung tissues in LPS-induced ALI mice (Figure S1B in Supplementary Material, p < 0.05). Together, these data indicated that miR-7 might be involved in the development of ALI.Accumulating evidence showed that miRNA Sponge strategy was valuable approach for the downregulation of specific miRNA expression in vivo (21, 22). Moreover, it is difficult to generate miR-7 deficiency mice using traditional strategies, including TALENs and CRISPR, because of mature miR-7 molecule is transcribed from three different genomic loci on chromosomes 9, 15 and 19, respectively (23). Then, we used miRNA Sponge (miR-SP) strategy to generate mice with miR-7 deficiency. The design and proof of principle of miR-SP technology is to construct a eukaryotic expression vector encoding an expression cassette for miR-7-SP sequence, the miR-7-SP elements included inserting tandemly arrayed miR-7 binding sites into the 3′UTR of a reporter gene encoding destabilized EGFP driven by the CMV promoter. Binding sites for miR-7 seed family were perfectly complementary in the seed region with a bulge at positions 9–12 to prevent RNA interference-type cleavage and degradation of the sponge RNA, the bulge contained six repetitive sequences complementary to miR-7 with mismatches for enhanced stability (Figure 1A). Then, transgenic mice were generated carrying one or two copies of the miR-SP cassette through putting the miR-7SP carrier to inject murine fertilized eggs, after which were transplanted into the oviduct of pseudo-pregnant mice female, and 3 weeks after birth (Figure 1B). To verify a special KD of miR-7 in FVB/N mice, we designed primers (P1 and P2) spanning the EGFP sites and miR-7SP sequence, and the predicted PCR product size was 505 bp. Then, we detected both wild-type (WT) mice and miR-7KD mice, while only miR-7KD mice could amplify 505 bp specific products (Figure 1C). Importantly, we found that the relative expression of miR-7 was obviously reduced in lung in miR-7KD mice (Figure 1D, p < 0.05). To confirm these data, we further detected the expression of miR-7 in lung tissues of miR-7KD mice using in situ hybridization and obtained similar results (Figure 1E). Consistently, the relative expression of miR-7 in other various organs and tissues in miR-7KD mice also decreased significantly (Figure S2 in Supplementary Material, p < 0.05). These results demonstrated that miR-7KD mice were successfully generated using miR-SP Technology.We acknowledged Cyagen Biosciences Inc. for technical assistance with the generation of miR-7KD mice, and Zunyi Medical College laboratory Animal Center for providing housing and breeding places.
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