The Effects Of SIRT6 On Funtions Of Human Bone Marrow Mesenchymal Stem Cells

Stem cells are cells that have multiple differentiation potentials and self-renewal ability, which maintained the balance of organs and repaired the injured tissuess. There are two main types of stem cells : adult stem cells and embryonic stem cells(ES cells). Compared to ES cells, adult stem cells were widely studied due to their convenient and rich resource. Bone marrow mesenchymal stem cells are one of adult stem cells which have been most widely used in the research. There was remarkable progress in the study of stem cell treatment especially autologous stem cell treatment on many diseases such as diabetes, peripheral vascular disease, cardiomyopathy, neurodegenerative diseases, joint diseases, ect. But in the preclinical studies, we found that the therapeutic effect of stem cells on elderly patients was not well as the younger patients.Aging is one of the most important reasons. It was reported that the amount, proliferative ability and differential potential of stem cells decreased with age. Rejuvenation of senescent stem cells is a feasible method to improve the therapeutic effect of stem cells for elderly patients.SIRT6, a silence regulatory protein, is a NAD dependent histone deacetylase. It regulates the structure of telomere and silences target genes, and is involved in maintaining telomere and DNA damage repair which may be one of mechanisms of anti-aging. Mostoslavsky found that SIRT6-deficient mice showed premature phenotype, and died in approximately 4 weeks after birth. Kanfi,Kawahara, etc. showed extended lifespan by overexpression of SIRT6 in mice. In cellular level, SIRT6 was also reported to improve the proliferative ability, suppress the senescence phenotype and enhance the differentiative ability. These results suggested that SIRT6 may be an anti-aging factor, but the function of SIRT6 on hBM-MSCs(human bone marrow mesenchymal stem cells) was unknown.The hypothesis of this study is that SIRT6 is an anti-aging factor for h BM-MSCs: knockdown of SIRT6 results in impairment in related functions of h BM-MSCs. Based on the hypothesis, this research mainly include four parts as follows:1. The culture and identification of h BM-MSCs of different ages.2. Confirm different expression of SIRT6 in h BM-MSCs from different ages.3. Confirm changes of relative functions of h BM-MSCs by SIRT6-knockdown.Objective: 1. To confirm different expression of SIRT6 in h BM-MSCs from different age. 2. To demonstrate knockdown of SIRT6 enables h BM-MSCs senescence. 3. To elucidate the relationship between the levels of SIRT6 and senescence.Methods: Harvest and culture of h BM-MSCs : Human bone marrow was collected from different age, infant group(I): n=27, mean age=5.39±0.69 years(1-13 years); young group(Y): n=38, mean age=25.92±0.81 years(16-36years); old group(O): n=53, mean age=67.90±0.98 years(51-81years). Human bone marrow was harvested from the posterior superior iliac spine of patients. Briefly, 1ml of human bone marrow was cultured in T-25-flasks with 5ml IMDM with 10% fetal bovine serum(FBS). When the cultured cells reached a confluence of 80-90%, cells were sub-cultured in a density of 5000 cells/cm2. The cells were used for all experiments at fourth passage. Identification of h BM-MSCs: Stem cell markers staining and osteogenetic differentiation, adipogenic differentiation were used to identification of h BM-MSCs. For stem cell markers staining: cultured cells were trypsined and resuspended at 106 cells/ml, CD105-FITC, CD34-PE, CD45-PC5 and CD44-PE was respectively added to 100 μl cell suspension(20 min at room temperature avoiding light), the percentage of CD105-positive cells, CD34-positive cell, CD45-positive cells and CD44-positive cells was quantified byflow cytometry(n=4/group). Cultured h BM-MSCs were respectively added osteogenetic differential medium and adipogenic differential medium according to the manufacturer’s instructions. Alizarin red staining was used to evaluate osteogenetic differentiation and red “O” staining was used to test adipogenic differentiation. Quantification of SIRT6 and p16 m RNA and proteins in h BM-MSCs:SIRT6 and p16 m RNA in cultured cells were quantified using real-time(RT)-PCR with the following primers(SIRT6 Forward: 5’-GTCTTCCAg Tg Tgg Tg TTCCA-3’; Reverse: 5’-CCCAg TCTAgg ATgg Tg TCC-3’; p16 Forward: 5’-CTCACCATgg ATg ATg ATATCg C-3’; Reverse:5’-Agg AATCCTTCTg ACCCATg C-3’; β-actin(ACTB)Forward: 5’-CCTg AAg TACCCCATCg Ag C-3’; Reverse: 5’-CTCTTg CTCg AAg TCCAggg-3’). RTPCR products were evaluated using Image J software(NIH, Bethesda, USA) and relative levels of SIRT6 m RNA were presented as the ratio of SIRT6 to ACTB(n=4/group). The SIRT6 protein levels were quantified using western blot analysis using antibodies against SIRT6(1:250, Abcam) and ACTB(1:2000, Abcam). The density of protein bands were analyzed with Image J software, and the ratios of SIRT6 to ACTB were then calculated. The p16 protein levels were quantified using immunocytofluorescence(ICF). Cultured cells were fixed in 4% paraformaldehyde in phosphate buffered saline(PBS) and then incubated with mouse antibodies against p16(1:100 in PBS,4 ℃overnight in humidified box). The slides were incubated with Cy3-conjugated secondary antibody(1:200 in PBS for 1 h at room temperature in humidified box avoiding light). The slides were then counter stained with 4’,6-diamidino-2-phenylindole(DAPI) [100ng/ml in PBS,37 ℃for 10 min in the dark]. The percentage of p16-positive cells was quantified by counting cells with red color compared to total cells in the same field(n=4/group). location of SIRT6 in h BM-MSCs: Localization of SIRT6 in cultured h BM-MSCs was evaluated using IFC. The cultured cells were fixed in 4% paraformaldehyde in PBS and then incubated with rabbit anti-SIRT6 antibody(1:200 in PBS overnight in humidified box at4°C). The slides were incubated with FITC-conjugated secondary antibody(1:200 in PBS at 37°Cfor 1h in humidified box avoiding light). The slides were then counterstainedwith DAPI(100ng/ml in PBS, 37℃for 10 min in dark). The SIRT6-positive cells were photographed. Cell growth: To evaluate the proliferative ability of h BM-MSCs, we preformed the Brd U labeling technique and cell growth curve assay. For the Brd U assay, cells were seeded in 24-well plates. When cells reached 40-50% confluence, they were further cultured in Brd Umedium(IMDM with 10%FBS containing 3 μg/ml Brd U) for three days. Cells were then immunostained for Brd U. The Brd U-positive cells were counted by Image J software and the percentage of Brd U-positive cells was calculated. For the cell growth curve counting, 7000 cells were seeded in 12-well plates(there were three replicative wells in each sample). The cell numbers were counted from 0 day to 6 days every 2 days to establish the growth curve. Cell migration assay: To measure the migration ability of h BM-MSCs, cells were cultured in 6-well plates. When the cell cultures reached approximately 100% confluence, a scratch wound was made through the middle of the wells using a 20μl pipette tip. Images were taken using an inverted microscope(Olympus, Tokyo, Japan). The percentage of migration rate was measured and analyzed 24 h after scratch by Image J software(n = 4/group). Cell death and apoptosis assay: h BM-MSCs were exposed to H2O2(1000μM) for 6 h and harvested. Apoptosis was tested by flow cytometry(Beckman Coulter, Brea, California) staining for Annexin-V and PI(Life Technologies). The percentage of dead(PIpositive and Annexin-V-negative cells) and apoptotic cells(Annexin-V-positive cells) was quantified by flow cytometry(n = 4/group). Measurement of SA-β-Gal activity: SA-β-Gal activity was evaluated using a senescence detection kit(Solaribio, China) according to the manufacturer’s instructions. The percentage of positive cells was counted by counting cells with blue color compared to total cells in the same field(n=4/group). Knockdown of SIRT6 in h BM-MSCs: Only one type of si RNA was used to knockdown SIRT6 expression in the h BM-MSCs according to the manufacturer’s protocol.Briefly, 7000 cells were seeded in 24-well plates in IMDM(FBS and antibiotic free). Cells cultured for 24 h were transfected with 3 μl 20 u M Oligo RNA-5 carboxy-fluorescein(FAM) by 2 μl lipofectamin 2000(Invitrogen, USA). The medium was replaced at 6 h after transfection and the identification of transfection was performed by a fluorescence microscope(Olympus, Tokyo, Japan) and the transfection efficiency was quantified by flow cytometry for FAM. The SIRT6 knockdown efficiency in h BM-MSCs was evaluated by RT-PCR(24 h after transfection) and Western blotting(72 h after transfection)(n=4/group). Statistics: Data are presented as mean ± standard deviation(SD). Statistical analyses were performed using Graph Pad Prism 5(Graph Pad, La Jolla, CA). A Two-tailed Student’s t-test was used for two-group comparisons. All other comparisons among three or more groups were analyzed by one-way ANOVA followed by Tukey post-hoc tests. Differences were considered statistically significant at P<0.05.Results: Identification of h BM-MSCs: The cultured h BM-MSCs were identified using flow cytometry to examine the cell markers. We found that greater than 99% of cells were CD105 and CD44 positive and there was no significant difference among infant, young and aged groups. The percentage of both CD34-and CD45-positive cells was lower than 1% and there was also no significant difference among the 3 groups. After osteogenetic and adipogenic differentiation, alizarin red and oil red-O staining were positive. h BM-MSCs were capable of differentiating into adipocytes and osteogenic cells under specific induction. These data suggest that the cells cultured and used in the subsequent experiments were h BM-MSCs. Age-dependent increase of SIRT6 expression: The expression of SIRT6 in h BMMSCs among the different age groups was evaluated.SIRT6 is a predominantly nuclear protein as shown by immunocytofluorescent staining.The m RNA and protein(Fig. 1D)expression of SIRT6 wersignificantly higher in the old group compared to the infant and young groups(P<0.01). si RNA knockdown of SIRT6 expression: The positivity of si RNA transfection was detected by immunofluorescent analysis for FAM at 6 h post transfection. The untransfected cells were negative for FAM staining, but cells in the si RNA control(NC) and si RNA SIRT6 groups were positive.When one type of si RNA was transfected into cells from both age groups, we found that the percentage of FAM-positive cells in the old group was significantly lower compared to the young group. si RNA was found to effectively knockdown SIRT6 gene expression. We found that m RNA expression of SIRT6 was significantly decreased in young and old si RNA groups(using the same si RNA) compared to their respective NC control groups(P<0.01). The SIRT6 protein levels were also significantly decreased in young and old si RNA groups compared to their respective NC groups Decreased proliferation of h BM-MSC after SIRT6 knockdown: To determine the impact of age and SIRT6 on the proliferative ability of h BM-MSCs, Brd U staining and cell growth curve counting methods were employed. Brd U-labeled cells were observed in the h BM-MSCs of all three age groups. The percentages of Brd U-positive cells were twofold higher in the infant and young groups compared to the aged group(Fig. 3B, P<0.01). We also found that the growth rates in the infant and young groups were significantly higher compared to the aged group(Fig. 3C, P<0.01).These data indicate that proliferative ability decreased with aging. The SIRT6 levels in young and aged h BM-MSCs decreased after SIRT6 si RNA transfection. The growth rates in the si RNA groups of young and aged h BM-MSCs were significantly lower compared with the NC control groups(P<0.01). Although the proliferative rate in aged cells was significantly lower than young cells, we observed almost no cellular growth in aged si RNA knockdown cells. These results suggest that SIRT6 plays a significant role in cell growth and proliferation.Impaired h BM-MSC migrative ability after SIRT6 knockdown: To determine the impact of age and SIRT6 on the migration ability of h BM-MSCs,the scratch assay was performed. The cellular migration rate was significantly decreased in aged cells. The migration rates were three-fold higher in infant and young cells compared to aged cells(P<0.01). The migration rate of SIRT6-knockdown cells was also significantly decreased relative to control cells, but independent of age. The migration rate of young si RNA cells was significantly lower than their respective NC control cells(P<0.05). Similarly, the migration rate of old si RNA cells was also significantly lower than their respective NC control cells(P<0.05). These data indicate that aged h BM-MSCs have lower migration ability and that SIRT6 is involved in cell migration capacity. Effect of SIRT6 on cellular apoptosis: Apoptosis of h BM-MSCs was quantified by flow cytometryto stain for Annexin-V and PI. We found that the percentage of apoptotic cells(Annexin-V-positive cells)was more than8-fold higher in aged cells compared to infant and young cells(P<0.05). Despite the higher percentage of dead cells in aged cells compared to infant and young cells, there was no statistical difference among three groups. To determine the anti-apoptosis effect of SIRT6, h BM-MSCswere knocked down with si RNA. We observed no significant difference in cell apoptosis levels between young si RNA and NC control cells. Similarly, there was also no significant difference in the percentage of dead cells between si RNA and NC control cells. These results indicate that the level of apoptosis in h BM-MSCs increased with age; however, knockdown of intracellular SIRT6 did not affect cellular apoptosis. To investigate the effect of age on cellular oxidative stress resistance, h BM-MSCs from different age groups were treated with H2O2 for 6 h and cell viability was examined by flow cytometrystaining with Annexin-V and PI. The percentage of dead cells was significantly increased in old cells compared to infant and young cells(P<0.05), and the percentage of apoptotic cells was 8 times greater in aged cells compared to infant and young cells(P<0.01).To determine the effect of intracellular SIRT6 on cellular oxidative stress resistance, the young h BM-MSCs were first transfected with SIRT6-si RNA or NC-si RNA for 72 h which was followed by 6 h of H2O2 treatment. The percentage of dead cells was more than two times higher(P<0.05) in the si RNA group compared to the NC control group. However, there was no significant difference in the percentage of apoptotic cells between the NC and si RNA groups. These data suggest that aging increased cellular sensitivity to oxidative stress; however, intracellular SIRT6 levels did not alter cellular sensitivities. Increased activity of SA-β-Gal after SIRT6-knockdown in h BM-MSCs: SA-β-Gal activity is a characteristic feature of cell senescence. Compared to aged h BM-MSCs in the aged group, the percentage of SA-β-Gal-positive cells in the infant and young groups was 16 times lower(P<0.01). After knocking down SIRT6 in young h BM-MSCs, the percentage of SA-β-Gal-positive cells in the si RNA group was more than two times higher than the control group(P<0.05). These results indicate that aging up-regulates h BM-MSC senescence and that SIRT6 down-regulates cell senescence. Increased p16 expression after SIRT6-knockdown in h BM-MSCs: p16 expression is an important parameter of cell aging. Compared to infant and young h BM-MSCs, the m RNA expression of p16 in the aged group was more than 2 times higher(P<0.01). After knocking down SIRT6 in young h BM-MSCs, the m RNA expression of p16 in the si RNA group was also more than two times higher than the control group(P<0.05). Similarly, the percentage of p16-positive h BM-MSCs in the aged group was significantly higher compared with the infant and young groups(P<0.01), and significantly higher in SIRT6-knockdown young h BM-MSCs compared with control cells(P<0.01). These results again suggest that aging up-regulates h BM-MSC senescence and that SIRT6 down-regulates cell senescence.Conclusion: 1. SIRT6 predominantly located in nucleus of h BM-MSCs and the expression level increased with age both in m RNA and protein levels.2. The proliferative ability, migrative potential, anti-apoptisis ability and oxidative stress resistance ability decreased but SA-β-Gal activity and p16 expression increased in h BM-MSCs with age. 3. Down-regulation of SIRT6 in h BM-MSCs resulted in less proliferation and migration, but increased SA-β-Gal activity and p16 expression. 4. Aging up-regulates h BM-MSC senescence and that SIRT6 down-regulates cell senescence.