The Mechanisms Study Of TNFAIP8L2 On Senescence Through Regulating Tolemerase

Cell senescence is the process of DNA damage which leads to cell death by telomere shortening or others stimulation when the cell division and proliferation.Cell senescence is a relatively stable condition.While the cells senescence,the DNA damage activates the p53 gene,which in turn affects the cell cycle and eventually leads to cell death.In this process,if the oncogene is mutated,the cell becomes a permanent biochemical cell avoiding death,which eventually evolves into a tumor.The process of cell senescence is ubiquity in normal cells,and cell senescence also occurs in cancer cells.When the cell is going on senescence,the cell cycle is blocked,and the cell proliferationis suppressed.Because of inhibiting tumor growth,the cell senescence is also regarded as one of the suppression mechanism for the development of tumorigeneis.Senescent cells produce a variety of inflammatory cytokines,which recruit macrophages,NK cells,and neutrophils to remove to senescent cells toinduce a series of inflammatory responses.Telomeres are short tandem repeats of DNA at the ends of chromosomes.The telomere is the DNA containing double chain repeating sequence TTAGGG.It has a single chain of 3 ’telomere ring part,which can prevent the end of the terminal double helix and the base pair structure.The telomere acts as a "hat" function by reducing the degradation and fusion of the ends of chromosomes.In the process of cell replication,the telomere repetition sequence is shortened continuously.When the telomere is shortened to the critical point,the function of chromosome protection disappears.DNA damage accelerates aging,and the cell dies.Studies indicate that telomerase can maintain or even lengthen the telomerase.Telomerase is a nucleoprotein(RNP),which is composed of RNA repeating sequences and protein catalytic subunits(TERT).Telomerase is not detectable in normal somatic cells except for stem cell or germ cells.Telomerase activity also is not detectable in some benign lesion cells,but has high activity in malignant tumor cells.Therefore,telomerase,as a specific substance of tumor cells,also affects the biological characteristics of the tumor.Cell senescence is the last stage of cell life,and tumor cells also go through the aging process.Telomerase as a telomere regulating enzyme,which plays an important role in the occurrence of aging.TIPE2,a fectorthat inhibits inflammation and tumorigenesis,is likely to regulating telomerase activity and affecting the occurrence of cell senescence thereby affecting the growth of tumor.Our early studies showed that the expressionof TIPE2 increased in elder mice,indicating that TIPE2 might be associated with cell senescence.The paper mainly focuses on the roles and mechanism of TIPE2 in aging.Objective1.To explore the roles of TIPE2 in cell senescence;2.To elucidate the molecular mechanism of TIPE2 in regulating telomerase activity to participate in cell senescence;3.To explore the role and mechanism of cell senescence regulated by TIPE2 in tumorigenesis.Methods1.In vivo study1.1 Animal modelMale C57 wild type mice(WT)and TIPE2-/-were sacrificed at the age of 3 months and 24 months.Fresh serawere collected to determine albumin(ALB),alkaline phosphatase,alanine aminotransferase(ALT),aspartate aminotransferase(ASTL),gamma glutamyl transpeptidase(GGT),and total cholesterol(CHO).One part of the tissues such as liver,kidney,and spleen were frozenin-80 ℃to prepare to do western blot,others were embedded to prepare sections to test TIPE2 expression.1.2 The detection of the expression of TIPE2The splenic tissues were cut into small pieces to extract the protein by the lapping bar.Anti-TIPE2 was used to test the expression of the TIPE2 protein with Western blot or IHC.2.The studies of roles of TIPE2 on telomeraseactivity in vitro2.1 Cell cultureColon cancer cell line(HT-29)andprimary lung airway smooth muscle cells in mice(ASMC)were cultured in the RIPA1640 medium containing 10%serum,colon cancer cell line SW480 was cultured in L-15,containing 10%serum Cells were incubated in wet cell incubator with 5%CO2 at 37 ℃.2.2 To explore the relationship between the levels of TIPE2 expression and cell senescence in vitro.The expression of TIPE2 was detected using Western blot in the 7th generation ASMC cells and 14th generation cells.TIPE2-pRK5 plasmid was transfected into ASMC or HT-29 cells treated with or without HzOz.SA-(3-Gal staining was used to detect cell senescence.The relationship between the levels of TIPE2 protein and cell senescence was analysed.2.3 Cells proliferation and cell cycle analysis.The cleaved protein of caspase-3 and P21 weredetected using Western blot,the cells proliferation was detected using CCK-8 kit,and cell cycle,apoptosis were deteemined by flow cytometry in HT-29 and ASMC cells transfectedwith TIPE2-pRK5 or vector control.3.The mechanism of TIPE2 on telomerase3.1 The effect of TIPE2 overexpression on hTERT expression and activityThe protein levels of hTERT transfected with TIPE2 plasmid were analyzed in HT-29 and SW480 cells using Western blot.The activity of telomerase was detected by telomerase TERT activity detection kit.The relationship between the levels of TIPE2 protein and hTERT activity is analysed.3.2 The mechanism of TIPE2 onhTERTWestern blot was used to detect the expression of TGF-β,c-myc,p-Smad3 and Smad3 in HT-29 cells transfected with TIPE2-pRK5 or control.And the expression of c-myc and c-Ets-2 in the cytoplasm or nuclear was distributed by the method of nuclear separation.The Co-IP method was used to detect the binding of TIPE2 to the transcription factor c-myc and c-Ets-2.3.3 TIPE2 regulation of telomerase in the nucleus.Immunofluorescence method was used to detect the location of TIPE2 in the cell nucleus in HT-29 cells with TGF-β stimulation.After transfection with Flag-TIPE2 or Flag-R24 in ASMC cells,these cells were stimulated with 2ng/ml TGF-β to determine the localization of TIPE2 with anti-Flag monoclonal antibody or anti-TIPE2 antibodies.Results1.The expression levels of TIPE2 prtein increased in the aging mice.The levels of TIPE2 protein inspleen tissues from 24 months C57 mice were significantly higher than 3 months mice.The sera levels of the liver function index such as alkaline phosphatase,alanine aminotransferase(ALT),aspartate aminotransferase(ASTL),gamma glutamyl transpeptidase(GGT),total cholesterol(CHO)in 24 months TIPE2-/-mice were significant higher than that in 3 months TIPE2-/-mice,but were much lower than the same aged.C57 mice.While the level of albumin(ALB)in 24 months TIPE2-/-mice was lower than that in 3 months mice,but was higher than that in the same aged C57 mice.These data suggest that the expression of TIPE2 increase with the aging in mice and the liver function significantly decreased at the same time.2.TIPE2 can promote cell aging and death in vitro.2.1 Higher expression level of TIPE2 accelerates cell senescence.The senescent cells increased markedly in cells transfected with TIPE2-pRK5 using SA-β-Gal staining compared to control.Meanwhile H2O2 stimulation could accelerate cell senescence and the expression of TIPE2 was up-regulated.2.2 TIPE2 can promote apoptosis and inhibit cell proliferation.The P21 and cleaved Caspase3 were up-regulate after overexpression of TIPE2 in HT29 and ASMC cells.As a result cell apoptosis increased while cell proliferation was inhibited and the cell cycle was blocked in the G1 phase.3.TIPE2 promotes cell senescence by regulating hTERT activity.3.1 TIPE2 inhibits hTERT activity.Both the level of hTERT protein and the activity of telomerase significantly decreased in HT-29 and SW480 cells transfected with TIPE2-pRK5 compared to control,indicating that TIPE2 could inhibit the activity of hTERT.3.2 TIPE2 regulates hTERT through Erk/c-myc pathway.Smad,c-myc,and c-Ets-2 are important cis-acting elements in the regulation area of hTERT promotor.We found that the expression of nuclear transcription factor c-myc was downregulated significantly in TIPE2 overexpressing HT-29 cells compared to control cells.Importantly,both thelevel of the nucleus c-myc and cytoplasm c-myc significantly decreased,indicating that TIPE2 not only reduced the c-myc expression,but also inhibited c-myc to transfer into the nuclearto combine with hTERT promoter.In addition,Erk phosphorylation was significantly reduced in TIPE2 overexpressing cells.Erk phosphorylation inhibitors could rescuethe inhibition effect of TIPE2 overexpression on c-myc expression,but could not completely eliminatetheinfluence on hTERT activity,suggesting that TIPE2 might reduce telomerase activity through other pathway.3.3 TIPE2 inhibits hTERT by combining to c-Ets-2.We also found that the level of c-Ets-2protein was significantly lower inTIPE2 overexpressing HT-29 cells than control cells.The result of nuclear separation experiment showed that c-Ets-2 protein in TIPE2 overexpression cells only reduced in the nucleus,indicating that TIPE2 might block c-Ets-2 to transfer into the nuclear.Theresult ofCo-IP experiment showed that TIPE2 could combine with c-Ets-2.3.4 TIPE2 regulates hTERT through TGF-β/Smad3 pathway.The levelof TGF-β proteinin HT-29 cells transfected with TIPE2-pRK5 was significantly higher than control cells,and the level of p-Smad3 was also significantly higher than control.These data indicated that TIPE2 overexpression in HT-29 cells could significantly up-regulate TGF-P expression and activating Smad3 pathway.TGF-β is an important regulatory factor in Smad signaling pathway.We found thatthe level of TIPE2 protein significantly increased in ASMC cells treated with TGF-β.We also found thatthe levels of nuclear TIPE2 andtruncated TIPE2 in ASMC transfected with TIPE2-pRK5 or truncated TIPE2 increased compared to control indicating that truncated TIPE2 could transfer into nuclear.Further studies needed to be done to confirm the function of nuclear TIPE2.Conclusions1.TIPE2 promotes cell senescence by inhibiting the expression and activity of hTERT;2.TIPE2 regulates the transcription of hTERT mainly at the transcription level.TIPE2 inhibit the c-myc and binding of c-Ets-2 to the promoter region of hTERT by down-regulating the expression level of c-myc and c-Ets-2.3.TIPE2 regulates telomerase activity through Smad and Erk signaling pathways;4.TIPE2 can inhibit tumor cell growth by inhibiting telomerase activity.Innovations and significances1.This is the first report that TIPE2 promotes cell senescence by regulating tolemerase.2.The results show that TIPE2 inhibits telomerase activity by regulating gene transcription of hTERT.3.The effect of TIPE2 on cell senescence may be a potential anti-tumor therapy.