RÉSUMÉ
Objective To construct plasmids and knock out HIF-1α gene expression in an naked mole rat skin fibroblasts(NSF)cell line using CRISPR/Cas9 genomic editing technology,to provide an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia-related diseases in naked mole rats.Methods We designed four pairs of single guide RNA(sgRNA)sequences targeting exons 1～4 of the NSF HIF-1αgene and successfully constructed an expression plasmid.The plasmid with the optimal sgRNA was identified and transfected into 293T cells,and the supernatant was used for detecting the virus titer.Lentivirus particles carrying sgRNAs of HIF-1α were transfected into NSF cells which express Cas9 protein,based on a previous protocol.After transfection,fluorescence signals were observed under a fluorescence microscope,and HIF-1α expression in NSF cells was detected by Western Blot and T7 endonuclease 1(T7E1)analysis.Results Sanger sequencing showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors,demonstrating successful construction of a recombinant plasmid for transfection.T7E1 digestion successfully removed three bands and the target efficiency of sgRNA was 54％.Western Blot showed that the HIF-1α gene was successfully knocked out and its protein level was significantly reduced in NSF cells from naked mole rats(P=0.0019).There were no obvious morphological changes in HIF-1α-knockout cells under the microscope,and gene knockout had no obvious effect on cell proliferation.Conclusions We successfully constructed an HIF-1α-knockout cell line using CRISPR/Cas9 technology,to provide an experimental basis for further studies of the biological function of HIF-1α,as well as the mechanism of hypoxia tolerance in naked mole rats.The result also provide a theoretical foundation for the prevention and treatment of hypoxia-related diseases.
RÉSUMÉ
Objective To investigate the effects of down-regulation of Beclin 1, which is an autophagy regulatory molecule, expression induced by RNA interference on the proliferation and apoptosis in skin fibroblasts of naked mole rat. Methods The expression levels of Beclin 1 were detected after starvation or H2 O2 treatment.The fibroblasts were transi-ently transfected with specific siRNA targeting Beclin 1 and then screened by real-time PCR and Western blot.Cell prolifer-ation and apoptosis were determined using CCK-8 detection kit and flow cytometry ( FCM ) .The expressions of related genes were detected by Western blot.Results The expression of Beclin 1 gene at mRNA and protein levels was signifi-cantly lower in fibroblasts of the naked mole rat.Starvation and H2 O2 treatment induced changes of the Beclin 1 expression. Inhibition of Beclin 1 gene expression can inhibit cell proliferation and induce early and late apoptosis.The protein levels of p53, BAX, Bcl2, LC3B, p-AKT and mTOR were reduced after transient transfection with Beclin 1-siRNA.Conclusions The expression of Beclin 1 in fibroblasts of naked mole rat are changed in response to starvation or H2 O2 stimulation.Inhi-bition of Beclin 1 gene expression can inhibit cell proliferation and induce apoptosis.Therefore, Beclin1 gene may play a regulatory role in autophagy, proliferation and apoptosis in the skin fibroblasts of naked mole rat.