The Role And Relative Mechanism Of Ykl-40 Involved In The Growth Of Brain Gliomas And Intervention Research

Gliomas are the most malignant brain tumors with the worst clinical prognosis in both adults and children. They exhibit aggressive and invasive growth characteristics which are responsible for the patients'short recurrence-free interval, poor prognosis and short survival. Despite advances in surgical techniques, combined with radiation and adjuvant chemotherapy, the quality of life and the life expectancy of patients with gliomas has remained essentially unchanged over the last several decades. Hence, understanding its molecular mechanism of tumorigenesis is imperative for neuroncologists.Recently, several retrospective and prospective studies of patients with different types of primary or advanced solid tumors reported that YKL-40 might be a biomarker in these patients used as a"prognosticator". As a member of"mammalian chitinase-like proteins", the elevated serum YKL-40 is found in a subgroup of patients with different types of localized or advanced solid cancers and reflects poor prognosis. In patients with gliomas, serum YKL-40 was highly correlated with tumor grade as well as tumor burden. However, most of the studies were focused on the correlation of serum YKL-40 level with prognosis, resistance to radiotherapy and survival. The exact role as well as the precise mechanism of YKL-40 involved in the progression of gliomas remains largely unknown. To achieve this goal, our study is designed to investigate the mechanism of YKL-40 in the proliferation of glioma cells, furthermore, to explore the possibility of inhibiting YKL-40 as a targeted therapy in clinic.1. The expression and significance of YKL-40 protein in WHO I- IV brain gliomas210 human primary astrocytomas were collected from specimens resected during operation, including 11 WHO I grade pilocytic astrocytoma ( PA ); 69 WHO II grade diffuse astrocytoma ( DA ); 62 WHO III grade anaplastic astrocytoma (AA ) and 68 WHO IV grade glioblastoma multiforme ( GBM ). The expression of YKL-40 protein was detected by using immunohistochemical staining. The correlation between the protein expression and the malignancy grade of astrocytomas was analyzed. The results showed that YKL-40 protein was highly expressed in human astrocytomas. The positive rate was significantly different among WHO I~IV grades (χ~2=69.151,P=0.00<0.01 ); The difference of positive rate between grade I and IV (χ~2=66.211,P=0.00<0.01 ) ; grade II and IV (χ~2=61.226,P=0.00<0.01 ) ; grade III and IV (χ~2=26.055,P=0.00<0.01 ) ; grade II and III (χ~2=9.651,P=0.002<0.01 ) ; grade I and III (χ~2=9.252,P=0.002<0.01 ) was statistically significant. The immunoreactivity score (IRS) was significantly different among WHO I~IV grade ( P=0.000<0.01 ); The difference of IRS between grade I and IV, grade II and IV , grade III and IV, grade II and III, grade I and III was statistically significant ( P=0.000<0.01 ); The difference of IRS between grade I and II was statistically different ( P=0.029<0.05 ). Thus, the expression of YKL-40 protein increased in a grade-dependent manner among human astrocytomas. Furthermore, the positive rate as well as the IRS in GBM was statistically significant ( P<0.01 ) when compared with the other types of astrocytomas ( PA, DA, AA). Based on the result of immunohistochemistry analysis for 210 human astrocytomas, YKL-40 protein is not only related with the initiation of brain gliomas but also closely associated with tumor's malignancy.2. The impact of YKL-40 SiRNA on the biological function of human glioma cells in vitroFour types of human glioma cell lines ( U373, U87, A172, T98 ) and Five human glioma primary cells ( Case 1, Case 2, Case 3, Case 4, Case 5 ) were screened for YKL-40 expression. Among them, the highest level was observed in U87, Case 2 and Case 5 cells. SiRNA method was used to specifically silencing YKL-40 gene. WST-1 assay, PI staining for flow cytometry and Matrigel invasion assay were performed to observe the proliferation rate, cell cycle progression and invasion ability of U87 cells after YKL-40 gene silencing, respectively. The results of WST-1 cell proliferation assay showed that the cell growth of U87-Si, Case 2-Si and Case 5-Si was suppressed significantly on 48 h, 72 h and 96 h ( P<0.01 ) compared with the control groups on corresponding time points. The PI staining of flow cytometry indicated that 86.59%±0.56% U87-Si cells were arrested in G0/G1 phase, wherease, the counterpart of U87 cells and U87-Co cells were 72.58%±0.82% and 74.09%±0.60%, respectively. After HE staining and cell counting, results from Matrigel invasion assay showed that the invasion ability of U87-Si cells decreased compared with the control group. On 96 h, cells distributed sparsely and lost their normal morphology by exibiting typical apoptotic features, such as karyopyconosis, nuclear intense staining, karyorrhexis. Our study demonstrated for the first time that YKL-40 gene silencing could inhibit U87 cell growth by arresting cell cycle in G0/G1 phase and impair their invasion ability.3. The mechanism involved in the impact of YKL-40 SiRNA on the biological function of human glioma cells in vitroWestern blot was conducted to measure the expression of phosphorylation and total protein of pivotal members in MAPK family, including Phospho-ERK1/2, ERK1/2; Phospho-p38,p38; Phospho-JNK1/2, JNK1/2, the expression of Phospho-AKT and AKT were measured as well. Moreover, gel shift assay was performed to explore the possibility of nuclear factor NF-κB ( Nuclear factor kappa B, NF-κB ) and AP-1 ( Activator protein-1, AP-1 ) involving in cell growth inhibition induced by YKL-40 SiRNA. Electrophoresis result and relative density analysis showed that the expression of p-ERK1/2 and p-AKT which were closely associated with cell proliferation and cell cycle regulation decreased significantly ( P<0.01 ), meanwhile, the expression of p-p38 and p-JNK1/2 which were highly correlated with cell apoptosis increased significantly ( P<0.01 ). Thus, the inhibited growth of U87-Si cells was carried out through the downregulation of ERK1/2 and AKT pathway. Nevertheless, the upregulation of p-p38 and p-JNK1/2 was a clue to apoptosis activation. But there was no significant difference of the expression of nuclear factor NF-κB and AP-1 between U87-Si cells and U87-Co cells. It is probably that YKL-40 promoter sequence has binding sites with some other specific nuclear factors. Consequently, our study verified that MAPKs and PI3K/AKT pathway were involved in the inhibition of U87 cells growth by YKL-40 gene silencing, in turn, it may reflect the potential role of YKL-40 in stimulating cell proliferation and protecting cell from undergoing apoptosis in gliomas.4. The intervention study of downregulation YKL-40 expression by human recombinant INF-αand resveratrolBy using the luciferase reportor gene vector PGV-B2, YKL-40 promoter was successfully synthesized and transfected into U87 cells. Several wildly used chemotherapy drugs in clinic were choose to investigate their effect on the activity of YKL-40 promoter, including: TNF-α( Tumor necrosis factor-α, TNF-α, 10 ng/ml ),MIP-1 ( Macrophage inflammatory protein-1, MIP-1, 10 ng/ml ),INF-α( 2000 u/ml ) and a kind of anti-cancer plant extraction - resveratrol ( Res ). The result of luciferase assay suggested that all of the drugs could attenuate the activity of YKL-40 promoter, however, INF-αand Res owned the most significant effect ( P<0.01 ). Real time RT-PCR was used to measure the mRNA transcriptional level of YKL-40 by treating with INF-αand Res. Results showed that after U87 cells treated with 2000u/ml INF-αfor 24 h, 48 h and 72 h, the mRNA transcriptional level of YKL-40 was decreased, especially for 48 h and 72 h, the difference was statistically significant compared with the control group ( P<0.01 ). By treating with 100μM Res for 24 h , 48 h and 72 h, the mRNA transcriptional level of YKL-40 was decreased significantly in U87 cells ( P<0.01 ). When the protein level was concerned, western blot as well as ELISA assay was performed. Results showed that after U87 cells treated with 2000u/ml INF-αfor 24 h, 48 h and 72 h, the protein level of YKL-40 was decreased, especially for 48 h and 72 h, the difference was statistically significant compared with the control group ( P<0.01 ). By treating with 100μM Res for 24 h, 48 h and 72 h, the protein level of YKL-40 was decreased significantly in U87 cells ( P<0.01 ). WST-1 assay and Matrigel invasion assay indicated that 100μM Res induced the inhibition of cellular proliferation and decreased cell invasiveness in U87 cells. More interestingly, Res reduced the expression of p-ERK1/2 in a time - dependent manner and ERK1/2 pathway was involved in the Res - induced repression of YKL-40 expression. Therefore, our experiment demonstrated for the first time that human recombinant INF-αand Res, especially for Res, could significantly decrease the expression and secretion of YKL-40 in U87 cells.5. Resveratrol inhibits cell growth and induces apoptosis in glioma cellsMTT assay revealed that Res inhibited C6 cell growth. There was statistically significant of cell growth inhibition between 90μM and 210μM groups ( P<0.05 ). Flow cytometry result showed that C6 cells were significantly arrested in S phase by treating with 210μM Res for 24 h. RT-PCR and western blot were performed to detect mRNA and protein level of caspase 3 treated with various dose of Res for 24 h and 48 h. The results indicated that the mRNA level and protein expression of caspase 3 increased with Res in a dose- and time- dependent manner. Therefore, the study confirmed that Res inhibited C6 cell growth by arresting cells in S phase and Res induced apoptosis of C6 cells by activating caspase 3. Normal fibroblast 3T3 cells were not suppressed by Res and there was no apoptosis-inducing effect by Res on 3T3 cells.In summary, based on the results above, we believe that YKL-40 plays a critical role in initiation and progression of human brain gliomas; Hopefully, the adjuvent therapy of inhibiting YKL-40 might be an attractive and prospective field in the treatment of human gliomas.