| Background and Objective: The naked mole rat live their all life in subterranean burrow systems whose oxygen content is only 10%~15%,which indicates that the ability of enduring hypoxia in naked mole rat exceeds that of any other living overground species.It is suggested that the injury or pathological changes caused by hypoxia are either attenuated or be completely cleared in this extraordinarily tolerance to hypoxic environment species.However,the mechanisms underlying the tolerance to hypoxia in this species remained poorly understood.Hypoxia factor plays a very important role during the process of the body damage caused by ischemic heart diseases.Moreover,hypoxia often secondarily occurs in the development of some other lethal damage diseases.The latest datas showed that the incidence of ischemic heart disease showed an increasing trend in the worldwide,especially in middle-income countries,which is the main reason of leading to the global burden of disease.Studying the unique mechanism of naked mole rat resistance to hypoxia will help prevent and treat of human ischemic diseases and provide new ideas of taking medical protective measures against hypoxic environment.At present,the datas confirmed that the local hypoxic microenvironment was not only the initial factor of inducing malignant transformation,but it also could induce tumor cells resistance to radiation and chemotherapy.Therefore,the local hypoxic microenvironment of solid tumors was considered closely related with a variety of malignant tumors relapse after treatment,poor treatment and poor prognosis.Therefore,clarifying the molecular mechanism of the naked mole rat resistance to hypoxia will provide new clues to the treatment of malignant tumors.Methods: Using the low-oxygen chamber to control oxygen concentration.And detecting the effect of hypoxia on gene expression profiles after treating adult naked mole rats(8 months old)with acute hypoxia(5% O2)for 1h,4h,8h,12 h by using transcriptome sequencing method on the basic of extracting total RNA from the naked mole rat muscle tissue.Screening of the differentially expressed genes after hypoxic stress on the basic of gene expression level,and using hierarchical clustering method to cluster the differentially expressed genes in each group and then study of the enrichment of differentially expressed genes in GO functional classification and KEGG signaling pathway.According to the result of the enrichment of genes in GO functional classification and KEGG signaling pathways,selecting a significantly different signaling pathway(MAPK signaling pathway)in which STMN1,JIP1/MAPK8IP1,JNK3/MAPK10 expressed significantly different.And then verifying the differentially expressed genes using real-time PCR and Western Blotting methods.Further more,silencing the expression of STMN1,JIP1/MAPK8IP1,JNK3/MAPK10 by si RNA interference methods in the naked mole rat muscle fibroblast cells under the conditions of hypoxia.Studying the effect of silencing the expression of STMN1,JIP1/MAPK8IP1,JNK3/MAPK10 on the apoptosis,cell cycle and cell proliferation using flow cytometry and cell proliferation assay(CCK8).Results: Screening of differentially expressed genes Compared with the control group,there were 243,304,717,527 genes upregulated after hypoxic stimulation for 1h,4h,8h,12 h respectively in naked mole muscle tissue and the number of genes that down-regulated were 344,332,487,250.In any case,as compared with the control group,the total number of genes whose expression levels upregulated was 1450,while the total number of down-regulated genes is 971.The total number of 2337 differentially expressed genes was divided into six functional gene clusters using the hierarchical clustering method and significantly gathered them into four experimental treatment groups.There were 566,566,227,674,108,196 differentially expressed genes in Cluster1,Cluster2,Cluster3,Cluster4,Cluster5,Cluster6 group respectively.Genes of cluster1 were significantly clustering in hypoxic group of 1h,genes of cluster 2 were significantly clustering in hypoxic group of 4h,and genes of cluster3,cluster6 were significantly clustering in hypoxic group of 12 h,genes of cluster4,cluster5 were significantly clustering in hypoxic group of 8h.GO functional enrichment of differentially expressed genes The genes in each cluster were assembled refering the classic gene function classification system named Gene Ontology(GO),and there were 1682,1300,1384,2185,637,592 GO terms significantly enriched in cluster1,cluster2,cluster3,cluster4,cluster5,cluster6 respectively.The most significant GO terms were anatomical structure morphogenesis,single-organism process,cellular component organization,single-organism process,ion transport,cell-cell signaling in cluster1,cluster2,cluster3,cluster4,cluster5,cluster6 respectively. Differentially expressed genes in KEGG signaling pathway enrichment Preliminarily judging the signaling pathways that genes enriched significantly in each cluster refering the KEGG database.Results showed that differentially expressed genes in cluster1,cluster2,cluster3,cluster4,cluster5,cluster6 most significantly enriched in focal adhesion,dilated cardiomyopathy,Circadian rhythm–mammal,MAPK signaling pathway,glycine,serine and threonine metabolism,type II diabetes mellitus signaling pathway respectively.Validation of differentially expressed genes(STMN1,JIP1/MAPK8IP1,JNK3/MAPK10)in vitro experiment Compared with the control group,the STMN1,JIP1/MAPK8IP1 and JNK3/MAPK10 expression levels significantly increased in the naked mole rat muscle fibroblasts after treated with hypoxic culture conditions for 24 h.Silencing the expression of STMN1,JIP1/MAPK8IP1,JNK3/MAPK10 significantly inhibited the naked mole rat muscle fibroblasts proliferation and induced apoptosis.And silencing the expression of STMN1 arrested the naked mole rat muscle fibroblasts in G0/G1 and S phase,while silencing the expression of JIP1/MAPK8IP1 and JNK3/MAPK10 blocked the naked mole rat muscle fibroblasts in S phase.Conclutions: Using the transcriptome sequencing method to high-throughput screening of 2337 genes of different expressed in naked mole rat muscle tissue after hypoxic treatment,and these differentially expressed genes were enriched for GO functional classification and KEGG signaling pathways.Three differentially expressed genes(STMN1,JIP1/MAPK8IP1,JNK3/MAPK10)were verificatied in vitro.These datas suggested that they may play an important role in the regulation of tolerace to low oxygen environment in naked mole rats. |
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