Mir-17-5p Alleviates Mitochondrial Dysfunction Resulted From Chronic Intermittent Hypoxia In Genioglossus Muscle Satellite Cells Of Rats

AimsObstructive sleep apnea hypopnea syndrome(OSAHS) is a breathing disordered diseases characterized by the repeated partial or complete upper airway obstruction of breathduring sleep. Upper airway obstruction depends mainly on two factors: the anatomy of the upper airway and the expansion of upper airway dilator muscle. Genioglossus is one of the most important upper airway dilators, and it can lead to upper airway trapping or even apnea. The basis maintaining genioglossus function normal is the balance of mitochondrial energy metabolism. Some study shows:The way of muscle functions changes from genioglossus metabolic to glycolytic which results in lower energy transformation as the muscle fiber type of OSAHS patients changes, and the increasing byproducts lactic acid makes muscle tissue much more easier to fatigue, aggravating the degree of airway obstruction. However, this process of mitochondrial adaptation to hypoxia is very complex, and its exact mechanism is still unknown. Some researchers have found that, miRNAs can regulate the mitochondrial function precisely at the post-transcription level. With hypoxia regulated miRNAs emergence, researchers began to notice these miRNAs could regulate the cellular energy metabolism in hypoxic environment and achieve the recovery of mitochondrial function. Therefore, on basis of previous studies, this study intends to provide more theoretical basis in OSAHS clinical treatment by exploring the interaction between mitochondria and miR-17 in genioglossus functions and revealing the molecular mechanismsof miRNAs in the treatment of abnormal metabolism induced muscle dysfunction diseases.Methods1. Establish and assess the hypoxia animal model of SD rats by using intermittent hypobaric hypoxia andblood gas analysis through the abdominal aorta.2. Using HE staining, ATP enzyme staining and transmission electron microscopy(TEM) to detect the effect of hypoxia on genioglossus energy metabolism3. The genioglossus satellite cells(genioglossus muscle satellite cells, GG MuSCs) of SD ratswas primary cultured by using mechanical separation combined with collagenase digestion; GG MuSCs were purified by using the different speed-attaching method; The GG MuSCs were identified by immunofluorescence staining.4. Compared the miR-17 expression between hypoxia group and normoxia group, screening specific miR-17; Comparied the miR-17-5p expression of different hypoxia time.5. Promoted and inhibited miR-17-5p by cell transfection; Detected the GG MuSCs proliferation and apoptosis by MTT assay and flow cytometry.6. Tested the changes of mitochondria in GG MuSCs by the following three aspects: TEM observation of mitochondrial ultrastructure; mitochondrial membrane potential fluorescent probes to detect the changes in membrane potential; confocal microscopy Orbit Ca2+ transport in each group by confocal laser.7. Predicted the target gene of miR-17-5p by bioinformatics analysis software; Identified target genes by RT-PCR and Western Blot. Results1. Improved intermittent hypoxia animal model is successfully established; blood gas analysis showed that oxygen saturation and oxygen partial pressure index have a significant reduction in hypoxia group.2. HE staining, ATP enzyme staining results showed more class Ⅱ fiber of genioglossus in H group than N group and the way of metabolism transfered to glycolytic; The TEM result indicated the mitochondria number decreases and shape swells or cristae in H group.3. The GG MuSCs showed rhythmic contractions after differentiating into myotubule; the immunofluorescence staining indicated that the positive expression of skeletal muscle myosins heavy chain(MHC) in GG MuSCs and the percentage of positive staning was more than 95%, signifying the sufficient purity of the primary culture cells for subsequent studies.4. Detected the expression of miR-17 family gene level by RT-PCR in hypoxia model group, and miR-17-5p is the targeted gene; After different time stimulation by hypoxia, the miR-17-5p in hypoxia 12 h group expressed most.5. The MTT, flow cytometry results indicated: the cell viability of hypoxic group decreased, apoptosis rate increased; It showed that hypoxia can cause cell damage; In transfected pre-miR-17-5p hypoxic group, its proliferation has been restored and the rate of apoptosis decreased; It showed upregulation of miR-17-5p cells may have a protective effect; Transfected anti-miR-17-5p hypoxic cells, its cell proliferation decreased, apoptosis rate increased; it showed inhibition of mi R-17-5p cells produce a certain amount of damage.6. The result from TEM found that GG MuSCs was significantly damaged resulting from mitochondrial swelling, cristae, endoplasmic reticulum swelling, increased autophagy and other changes; suppression performance after miR-17-5p injury is more serious, the number of mitochondria and edema was significantly increased phagocytosis observed mitochondria lysosomes after the injury has been overexpressed miR-17-5p reply, but still normal; phenomenon after Rhod-3 binding assays mean fluorescence intensity of cells showed H and H+anti group compared with the normal activity of the mitochondrial membrane potential group decreased; H+pre group compared with the normal group, although the membrane potential decline, but higher H groups, indicating that over-expression mi R-17-5p post on mitochondrial function has been restored.7. Confocal laser results showed low efficiency of Ca2+ transposition in H group. After the inhibition, Ca2+ transposition efficiency decreased. After over-expressed mi R-17-5p, Ca2+ transposition efficiency recovered than H group although slightly lower than the normal group. These findings showed miR-17-5p can affect the Ca2+ influx efficiency and thus may regulate genioglossus function. Conclusions1. OSAHS animal model is effectively established through intermittent hypobaric hypoxia devices.2. Hypoxia can cause mitochondrial ultrastructure damage, muscle fiber type transformation, the level of energy metabolism of genioglossus reduction.3. High purity genioglossus satellite cells can be obtained by enzyme digestion and differential adhesion method.4. Regulated miR-17-5p has a protective effect in genioglossus mitochondrial function.5. Mfn2 may be the target gene.