Effects Of TAG1Signal On Proliferation And Differentiation Of Glioma Stem-cells And Its Genetic Regulatory Mechanism

Background:Glioma is the most common intracranial tumors, account for approximately29%of primary tumors in the central nervous system, and approximately80%of malignant brain tumors. Glioma has been one of the least treatable neurol ogical malignant disorders and is associated with the rapid proliferation, high invasion degree, and high mortality. Although a lot of progress have made for the comprehensive methods of clinical therapy of positive surgery, routine r adiotherapy, and chemotherapy, but the outcome and prognosis for patients wi th glioma is still unsatisfactory now. With more profound researches for gliom a, the concept of glioma stem cells (GSCs) has gained more and more attent ion. Through the understanding the cellular heterogeneity of glioma cells, a s mall cellular subpopulation of glioma is defined as glioma stem cells or glio ma stem-like cells, by considering having the "sternness" characteristics of se1f-renewal, unlimited proliferation, differentiation of tumor, and resulting to g1ioma initiation, progess, and recurrence. Therefore, researches related to biolo gical and mechanism of GSCs is thought to probably promote diseovery of new therapy. Researches suggest that glioma-genesis may be involved with some abno rmal expression of related genes, receptors, signaling molecules and its abnor mal transduction. Changes of signaling molecules and its signaling transductio n within the niche may also has impact on the biological behavior of cell pro liferation and differentiation, etc.. Therefore, studies of finding and thorough u nderstanding signaling pathways of glioma stem cell have become the hotspot.TAG1(transient axonal glycoprotein1, TAXI, Contactin2, CNTN2, Axoni n1) is a cell surface adhesion molecules, which belongs to the subfamily Contact in/F3immunoglobulin superfamily, and express mainly in the developing central nervous system. Previous studies about the physiological function of TAG1are indicated as a cell adhesion molecule guided neuronal migration and axonal gr owth. But the definite mechanism is unclear yet. Studies have found that TA G2may be related with glioma. The expression of TAG1was detected in glio ma cells from clinical tumors or cell lines. Study from our laboratory has als o found TAG1expression in glioma cells and proved that could participate in t he occurrence and development of glioma by promote the proliferation of glio ma cells. In addition, the TAG1signal promote proliferation and inhibite differ entiation in glial precursor cells through the interaction with of SHH signal. B ut the functions of TAG1in glioma stem cells are still unclear.Amyloid β precursor protein (APP) as a TAG1receptor molecular has be en found recently. APP widely express in the nervous system, particularly neu ral stem cells. Researches show that APP related to the signal maintaining of the cell-cell and cell-extra. Physiological function involved of APP is very extensive, such as migration, differentiation and regeneration of neural cell, syn aptic development, and neural protection. Secondly, APP drived regulated intram embrane proteolysis (RIP) product, the APP intracellular domain (AICD), can directly transducte into the nucleu and involved in transcriptional regulation fu nction. The study found that both APP and AICD may be associated with glio ma. APP is observed overexpression in glioma cell line cells, which involves t he abnormal proliferation and abnormal morphology of glioma cells. APP may also increase glial differentiation direction in exogenous neural stem cells or n eural progenitor cells. And, AICD promote gliaogenesis in neural progenitor c ells by increase expression of NICD and Hesl through the directly transcripio nal regulation or interaction with NICD. However, the mechanism function of APP and AICD in gliomas remains to be elucidated.Since TAG1/APP signal is first found expression in neural stem cells, and the expression of TAG1and APP signal molecules are also observed in glioma cells. Therefore, we selected glioma stem cells as the object of this research. In order to provide new clues for the therapeutic strategies of glioma, the expression of TAG1/APP signaling in glioma stem cells and underlying mechanisms possibly involved were explored.There are three parts of experiment in this research.Chapter I:Isolation, cultivation and identification of glioma stem cells derived from U87human glioma cells lineObjective:In this part of experiment, we aimed to isolate and amplify gl ioma stem cells from U87human glioma cells line, and then to identify its st emness characteristics.Methods:Serum-free medium (DMEM/F12+20ng/mLbFGF+20ng/mLEGF+2%B27) and hypoxia culture condition (1%O2,5%CO2,94.5%N2,98%h umidity,37℃) was utilized to isolate and amplify the glioma stem cells from U87human glioma cells line. Cell morphology of the U87glioma stem cells was observed. Stem cell surface markers were detected by immunohistochemis try to identify the sternness characteristics. Serum-containing medium (DMEM/F12+10%FBS) was replaced to induce differentiation of tumorsphere cells. Phe notypes of differentiated cells were identified to detect the multiple differentiat ion ability of the tumorsphere cells. Tumorigenicity potential of U87tumorsphe res was confirmed in nude mice by U87tumorsphere cells subcutaneous impla ntation. Cell proliferation test using CCK-8(Cell Counting Kit-8) were measu red and tumor growth curve was drawn to show the proliferation ability of U87tumorsphere cells.Results:Human U87glioma cells grew adherently in serum-containing m edium. U87glioma cells have long protuberances and multiple forms, such as long-spindle, star or polygonal pattern. After incubation in Serum-free medium and1%O2culture condition, the suspending tumor cell spheres would be o bserved. These tumorspheres could be digested to single-cell suspensions and reformed tumorspheres repeatedly at least a month or more. This showed the strong self-renewal ability of U87tumorspheres. Using immunohistochemistry s taining method, U87tumorsphere cells expressed positive stem cell markers CD133and Nestin. Using flow cytometry method, the positive rate of CD133and N estin in U87tumorsphere cells reached above90%. When these U87tumorsph ere cells transferred into serum-containing medium for differentiation, morpholo gy of the reformed adherent cells was similar to their parental U87cells. Im munohistochemistry of reformed adherent cells showed positive staining for G FAP (astrocytes), Ⅲβ-tubulin (neuron) and O1(immature oligodendrocytes), which represented multilineage differentiation potential. Xenograft tumor could be observed after the U87tumorsphere cells were injected subcutaneously in nude mice, which represented the ability of tumorigenic capacity. Quantitative analysis of OD ratios of U87glioma cells are1day:0.520±0.006,3days:0.690±0.010,5days:0.964±0.014, and U87tumorsphere cells are1day:0.855±0.049,3days:1.452±0.139,5days:1.925±0.051. Statistical analysis sh owed that U87tumorsphere cells show better cell viability when compared to U87glioma cells (F=37.359, P<0.05). These results suggested that the turn orsphere cells could be used as GSCs.Conclusion:U87glioma stem cells which showed unlimited proliferation, self-renewal and multi-directional differentiation potential exist in U87huma n glioma line cells. Serum-free medium and hypoxia culture condition can effe ctively isolate and amplify U87glioma stem cells from U87glioma cells.Chapter II:Expression of TAG1/APP signal in proliferation and differentiation of U87glioma stem cellsObjective:In this part of experiment, we aimed to observe the expressi on of TAG1/APP signal when the U87glioma stem cells proliferation and diffe rentiation in vitro. And try to analysis the relationship between cell proliferatio n, differentiation and TAG1/APP signal in glioma stem cells.Methods:The fifth-generation U87glioma stem cells were used as glioma stem cell model. U87glioma stem cells cultured with serum-free medium (D MEM/F12+bFGF20ng/ml, EGF20ng/ml, B270.2%) in normal oxygen concentr ation,37℃,5%CO23days for proliferation, and then cultured with DMEM/F12contained10%fetal bovine serum medium for0,1,5days for U87gliom a stem cells differentiation. Expressions of TAG1and APP were detected using by qRT-PCR and Western blot method. Results:U87glioma stem cells propagated as tumorspheres, the fifth-gen eration and above were gathered to detect the expression of TAG1and APR Compared with the U87glioma cells, expressions of TAG1and APP mRNA in U87glioma stem cells when cells proliferation were significantly enhanced (t=-3.581, P=0.023, and t=-4.913, P=0.039). And protein levels of TAG1and APP were significantly increased (t=-3.560, P=0.024, and t=-9.839, P=0.001). U87glioma stem cells differentiated in culture medium containing10%fet al bovine serum. After1day differentiation, expressions of TAG1and APP wer e not reduced. When the proportion of differentiated glioma cells increased, exp ression of TAG1and APP mRNA and protein were declined after five days diff erentiation (mRNA level:P=0.003, and P=0.004, protein level:P=0.002, an d P=0.000).Conclusion:Abnormal high expression of TAG1and APP signal was obse rved in U87glioma stem cells. These abnormal expression patterns of TAG1/APP signal probably involved in U87glioma stem cell proliferation maintenanc e and differentiation initiation.Chapter Ⅲ:The regulatory effect of TAG1/APP signal on proliferation and differentiation in U87glioma stem cellsObjective:In this part of experiment, we aimed to investigate the chang es of cells proliferation and differentiation behavior and the changes of express ions of EGFR and Hesl signal after RNAi TAG1expression in U87glioma st em cells. In order to explore the probable potential pathogenic mechanisms thro ugh TAG1/APP/EGFR and TAG1/APP/Hesl signal in glioma stem cells.Methods:According to the human gene sequence of CNTN2(TAG1), we designed and constructed four miRNA interference vector (named CNTN2-1,2,3,4, respectively), and we identified their gene sequences by sequencing. After transiently transfected to U87glioma stem cells, we screened the interf erence efficiency by Western blot method.In order to improve the efficiency of gene interference, the best interference fragment subcloned into the lentiv iral vector.The best inference CNTN2-2was selected to lentivirus constructed. According to MOI=100, transfecting u87glioma stem cells, observe the expre ssion of cell growth state and green fluorescence after72hours. CCK-8was used to detect the proliferation activity. To evaluate the proliferation effect, th e number and size of U87tumorsphere were detected after RNAi. GFAP ex pression of the differentiated U87glioma stem cells and the sizes of xenogr afts of the U87glioma stem cell transplantation were detected to evaluate th e differentiation effect of RNAi. The expression levels of AICD, EGFR and HES1were detected using qRT-PCR and Western blot methods to evaluate the possible role of RNAi on EGFR and HES1expression in proliferation and differentiation.Results:The interference effect is best in CNTN2-2detected by Western blot, and CNTN2-2build as Lenti-EGFP-CNTN2-miR, Lenti-EGFP-NC-miR was empty virus control.Glioma stem cells differentiated were inhibited by RN Ai of TAG1signal (RNAi-CNTN2group), compared with the Lenti-EGFP-NC-miR (Negtive group), cell proliferation activity of Lenti-EGFP-CNTN2-miR (RNAi-CNTN2group) were significantly decreased in CCK-8growth curve (F=3.202, P<0.05), the number of U87tumorsphere significantly reduced (F=11.773, P=0.015), the diameter of U87tumorsphere significantly reduced (F=6.753, P=0.008). After TAG1signal interferenced by RNAi, differentiati on of glioma stem cells was also restrained. Compared with negative control group of Lenti-EGFP-NC-miR empty virus, After5day differentiation, expre ssions of GFAP in Lenti-EGFP-CNTN2-miR group were significantly decrease d (F=1651.841, P=0.000). Subcutaneous tumor volume of nude mice signi ficantly smaller in20days (F=9.817, P=0.008). Western blot results sho wed that, compared to the control group, RNAi-TAG148hours after transfect ion, expression of APP in U87glioma stem cells was no significant change (F=9.719, P=0.131). However, expression of AICD showed decreas (F=121.417, P=0.006). After differentiation3days, protein expression of GFAP in RNAi-CNTN2group was significantly reduced (F=1651.841, P=0.000). By qRT-PCR and Western blot test, statistical analysis showed that, gene exp ressions of EGFR and Hesl significantly lower (F=26.967, P=0.048, and F=1598.701, P=0.000), protein expressions of EGFR and Hesl also decre ased correspondingly (F=765.277, P=0.000and F=3580.478, P=0.000).Conclusion:RNAi-TAG1signal probably inhibit proliferation and differenti ation in U87glioma stem cells through TAGl/APP/AICD/Hesl signal and TAG1/APP/AICD/EGFR signal pathway. Inhibition of TAG1signal may provide ne w clues for the treatment of glioma.