Research On The Role And Mechanism MicroRNA-17 In Cellular Senescence Of Human Foreskin Fibroblasts

Cellular senescence, an irreversible cell cycle arrest, is broadly defined as a state of permanent growth arrest in which cells are refractory to mitogenic stimuli. The classical cellular senescence pathways include p53-p21 and p16-pRb. When the expression of p21 and cyclinD were downregulated and upregulated respectively, cellular senescence would be delayed.MiR-17 family, which is also known as miR-106b family, is made up of 6 family members. Experiments confirmed that miR-17 regulated transcription factors of E2F family and cell cycle regulators pRb to influence cell proliferating, whereas E2F and pRb regulate cell cycle through affecting cell growth and proliferation, which suggest that miR-17 plays an important role in cellular senescence. But its upstream and downstream regulatory factors and mechanisms are poorly understood. So the research of miR-17 on cell senescence and its mechanism can promote the study and treatment of aging related disorders, which also shed light on the molecular mechanism of tumor occurrence and development.Objectives:1. Explore the separation, culture and passage of HFF in vitro.2. The research of miR-17 regulating effect on cell cycle of HFF.3. To study the regulation of miR-17 on cell cycle related proteins of HFF.Methods:1. Take sterility surgical removal of the foreskin from 7-year-old patient. Obtain high purity HFF by dispase Ⅱ enzymatic digestion method, and subcultured. Take the 3rd generation HFF, with rabbit anti-human vimentin monoclonal antibody as an antibody, using immunohistochemical SABC method for cell identification.2. Expression of GFP in HFF-miR-17 and HFF-NC was examined 72h later by fluorescence inverted microscope after the 3rd generation HFF infected by Recombinant lentivirus. Also real-time quantitative PCR was carried out to examine the expression of miR-17 in HFF-miR-17 and HFF-NC.3. The proliferation and senescence of HFF-miR-17 and HFF-NC were detected by CCK-8 and SA-β-Gal kit, respectively.4. Flow cytometry was carried out to examine the cell cycle profile. The protein levels of cyclinD1 and p21 were detected by Western Blotting.Results:1. Only the nuclei stained blue and cytoplasm uncolored in the control group with PBS buffer as an anti-control cells; while anti-Vimentin experimental group cells were spindle or polygonal, cytoplasmic brown staining, suggesting that were HFF cells.2. The green fluorescence distribution in the cells was detected 72h later by fluorescence inverted microscope after the 3rd generation HFF infected by recombinant lentivirus. Comparing with the same vision of contrast group cells, fluorescence in HFF-miR-17 shows the infection efficiency of 90%, suggesting that the recombinant lentivirus infection was successful. Compared with the negative control cells, expression of miR-17 in HFF-miR-17 examined by real-time quantitative PCR was significantly increased, and the difference was statistically significant (P<0.05).3. We observe changes in HFF cell growth by CCK-8 method. Compared with the negative cells HFF-NC, absoPRbance at 450nm in HFF-miR-17 cells was significantly increased for six consecutive days, the difference was statistically significant (P<0.05), which shows miR-17 can promote the proliferation of HFF cells.4. HFF-miR-17 cells showed weak β-galactosidase staining by less than 1% of the cells stained blue, suggesting the cells was normal cells; while up to 20% of the control cells stained blue, suggesting that cells was senescent cells at this time.7. HFF-miR-17 cell arrested in G1 phase, compared with the control HFF-NC, was significantly reduced (81.5% vs 72.6%) by flow cytometry; 72.9% of HFF-NC cells treated by DNA-damaging substance Bleomycin arrested at the G1 phase. Only 2.5% of HFF-NC cells were able to cross the G1 to S phase. And HFF-miR-17 cells at the G1 phase was significantly reduced (54.6%), with 10.2% cells in the S phase. These results demonstrate miR-17 can promote cell proliferation and abrogate the DNA damage-induced G1 arrest. That no apoptotic peak figure appeared in any results suggests miR-17 promoting cell proliferation, not by inhibiting apoptosis.5. Western blotting analysis showed that, compared to the control HFF cells and negative control cells HFF-NC, the expression level of p21 protein in HFF-miR-17 down-regulated, and cyclinDl protein expression levels were significantly increased, which suggests HFF can accelerate cell proliferation.Conclusions:1. High purity HFF cells were obtained by Dispase Ⅱ digestive enzyme, and for the first time with recombinant lentivirus system we successfully constructed miR-17 overexpressing cell lines of human foreskin fibroblasts.2. MiR-17 can inhibit apoptosis of HFF.3. MiR-17 promotes HFF cells exit from G1 and entry into S phase, which means that miR-17 can accelerate cell proliferation.4. MiR-17 regulates expressions of p21 and cyclinDl in HFF, thereby promoting the proliferation of primary cells and inhibiting primary cells senescence.