| SIRT6 is a member of the Sirtuin family,has deacetylase activity and single ADP ribosyl transferase activity.Current research shows that SIRT6 is a key gene for cell and individual regulation of aging and longevity in humans and mice,participates in a variety of physiological activities including telomere maintenance,DNA damage repair,glucose metabolism,and lipid metabolism,etc.However,there have been no reports on the function of SIRT6 gene in buffalo.In this study,SIRT6 gene of buffalo was cloned,and the function and role of SIRT6 in the aging of buffalo fibroblasts were preliminarily discussed,which provided experimental basis and theoretical basis for the further application of SIRT6 in buffalo donor choose and culture,gene protection and aging mechanism of buffalo and other related large animals.First,the SIRT6 gene in buffalo was cloned and bioinformatics analysis was performed.The SIRT6 coding region sequence of cattle(NM 001098084.1)was used to design the amplification primer,and the specific amplification was carried out using the skin RNA of buffalo fetal cattle as the template.The sequencing results showed that the buffalo SIRT6 gene sequence(1080 bp)was successfully obtained.The bioinformatics analysis of the buffalo SIRT6 sequence showed that the buffalo SIRT6 gene could encode 359 amino acids,with the highest leucine and proline content(10.3%)and the lowest tyrosine content(1.1%).The similarity of the buffalo SIRT6 nucleic acid sequence to bovine and human sequences was 98.4%and 85.9%,respectively,and contained the catalytic structure of Sir2 deacetylase specific to the Sirtuin family.By analyzing the expression profile of buffalo SIRT6 tissue,SIRT6 was expressed in heart,liver,spleen,lung,kidney,intestine,stomach,brain,genital warts and skin.The expression level of genital warts was the highest,followed by skin and heart.Buffalo fibroblasts were successfully obtained from buffalo fetal calf,3 year old buffalo and 10 year old buffalo skin tissue by tissue explants adherent method.The third generation of fibroblasts from different ages were analyzed for biological characteristics.As the buffalo ages,the result showed that a significant decrease in S-phase cell and cell proliferation index(P<0.05),β-Gal-positive cells(P<0.05).The expression level of senescent protein p16,p21,aging p53 mRNA were significantly increased(P<0.05),and the expression level of SIRT6 was also significantly decreased(P<0.05).The DNA damage sites and telomere damage degree were observed by immunofluorescence technique.The results showed that DNA damage sites and telomere damage degree increased in fibroblasts of 10 years old buffalo.The above results indicate that the fibroblasts isolated from aged buffaloes have a slower proliferation rate than that of the buffalo fetal fibroblasts(BFF)cells,and the aging phenotype is more significant.The SIRT6 gene is inversely related to the degree of cellular senescence.To further investigate the role of SIRT6 in buffalo fibroblasts,three interfere shRNA lentiviral vectors of SIRT6 gene were constructed,and then the interference vectors were screened by virus infection in the fourth-generation of BFF.The quantitative results showed that the interference efficiency of psi-sh3-LVRU6GP was the highest(P<0.01).The function of SIRT6 gene was studied by using BFF P5(PN-BFF P5)infected with Psi-LVRU6GP blank plasmid virus as control group,and using BFF P5 infected with Psi-sh3-LVRU6GP virus solution was.used as experimental group(Ps-BFF P5).The results showed that after interfering with the expression of SIRT6 gene,the cells became coarse,flat,weakly refractive,and the cell edges became unsmooth.The proliferation rate is slow,and the cell confluence is difficult to grow to 100%after passage,and a large number of cell deaths accompany the culture process,and the growth curve is no longer a S-type of normal cell proliferation curve.The results showed that after interfering SIRT6 gene expression,the cells became larger,flatter,less refractive,and the cell edges became not smooth.The proliferation rate slowed down,and the cell confluence could not reach 100%after passage.In addition,a large number of cells died during the culture process,and the growth curve no longer showed the s-type of normal cell proliferation curve.Cycle results showed that the Ps-BFF P5 S phase and G2 cells and cell proliferation index were significantly lower than that of the PN-BFF P5(P<0.05),protein p16,p21,aging p53mRNA expression level increased significantly(P<0.05),beta Gal cell positive rate increased significantly(P<0.01).The normal karyotype rate of Ps-BFF P5 was 47%,the normal karyotype rate of PN-BFF P5 was 83%,which showed that the normal karyotype rate of was significantly decreased after SIRT6 down-regulation(P<0.05).Furthermore,qRT-PCR analysis was performed on the telomere length of SIRT6 interference-positive cells and the components of telomere-protected protein complex gene.The results showed that the telomere damage site and DNA damage site increased after the interference of SIRT6,and the damage area increased,but the telomere length was not significantly different from the control group(P>0.05).The relative quantification of telomere-protected protein complex gene showed that the five components of TRF1,POT1,RAP1,TPP1 and TIN2 decreased with the decrease of SIRT6 expression,but the expression of TRF2 increased with the decrease of SIRT6 expression.The experiments indicated that SIRT6 is a key gene regulating the aging of buffalo fibroblasts,and interfering with SIRT6 expression will cause irreversible DNA damage and telomere damage.In summary,the conclusion of this paper are as follows.The sequence of the coding region of the buffalo SIRT6 gene is 1080p long and encodes 359 proteins.With the aging of the individual,the isolated fibroblasts have a shorter culture life in vitro,showing a more obvious aging phenotype and lower SIRT6 expression.SIRT6 is a key gene regulating the senescence of buffalo fibroblasts.Interfering with SIRT6 expression can cause irreversible DNA damage and telomere damage.However,SIRT6 can not regulate the telomere length of buffalo fibroblasts.It is speculated that it may regulate cell genomic stability by regulating DNA damage and telomere damage,thereby regulating cell senescence... |
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