To Study The Regulation Of SNORD47 In Biological Behaviour Of Glioma

ObjectiveTo investigate the expression of SNORD47 in 124 clinical glioma specimens and adjacent normal tissues.To measure the expression of SNORD47 in 124 clinical glioma specimens of different grades.To explore the relationship between SNORD47 and HOTAIR in WHO grade IV glioma specimens and the effect of SNORD47 on the survival of glioma patients.To further study the effects of SNORD47 on the proliferation,cell cycle,invasion of glioma cells and provide a new insight into suppressing the proliferation and invasion in glioma.MethodsThe first step: Q uantitative Real-Time PCR was used to measure the expression level of SNORD47 in 124 clinical glioma specimens of different grades and adjacent normal tissues.The second step: The expression of SNORD47,GAS5,HOTAIR in 124 clinical glioma specimens of different grades was detected by Q uantitative Real-Time PCR.We study the correlation between HOTAIR and SNORD47,the relationship between the expression of SNORD47 and clinicopathological parameters in glioma patients and the effect of SNORD47 on the survival of glioma patients.The third step: To explore the potential effects of SNORD47 on the biological behavior in glioma cel s after transfected with lenti-SNORD47.a.After transfected with lenti-SNORD47,the expression levels of SNORD47 and GAS5 was detected by Quantitative Real-Time PCR.b.The CCK-8 assay,colony formation assay and Edu assay were used to evaluate the proliferation ability of glioma cells Cell after transfected with lenti-SNORD47.c.The cell cycle was analyzed by flow cytometry and immunofluorescence assay.Western blot was used to detected the expression of cell cycle related proteins after transfected with lenti-SNORD47.d.Transwell assay and wound-healing assay were used to evaluate the change of invasion ability and migration ability of glioma cells respectively after the transfection.Immunofluorescence assay was used to study the change of cytoskeleton and Epithelial-Mesenchymal Transition(EMT).EMT-related proteins was measured by western blot after the transfection.The fourth step: To study the effect of SNORD47 on the sensitivity of glioma cel s to temozolomidea.We treated U87-MG and U251 cells with increasing concentrations of temozolomide for 72 h to screen optimal concentration.b.The changes of invasion ability and cell cycle in lenti-NC?lenti-SNORD47?lenti-NC+TMZ?lenti-SNORD47+TMZ group were explored by CCK-8 assay and flow cytometry respectively.The fifth group: to determine whether SNORD47 had an antitumor effect and whether SNORD47 could strengthen the temozolomide-induced suppression of tumorigenesis in vivo.Results1 ? The expression of SNORD47 in glioma specimens and the effect of SNORD47 on the survival of glioma patientsa.The results showed that the expression of SNORD47 was significantly lower in glioma specimens compared to the adjacent normal tissues(P<0.01).b.The expression of SNORD47 was significantly decreased in grade IV glioblastoma compared with the lower-grade glioma specimens(P<0.05).However,there was no significant difference in the expression of SNORD47 between the grade I/II and grade III glioma specimens.There were no significant differences in the expression of GAS5 among the different grades of glioma specimens.The expression level of HO TAIR was positively associated the grades of the glioma specimens.And the correlation between HOTAIR and SNORD47 was significantly inverse(R=-0.5819,P<0.01).c.The results of clinicopathological parameters in glioma patients showed that the lower expression of SNORD47 was significantly associated with the advanced WHO grade(III/IV).d.Kaplan-Meier survival analysis indicated that patients with higher SNORD47 expression in glioma tissues had better survival than those with lower SNORD47 expression(P=0.0262).2?After transfected U87-MG and U251 cells with lenti-SNORD47 for 48 h,qRT-PCR results showed that the expression of SNORD47 was significantly increased.The results of CCK-8 assay,colony formation assay and Edu assay indicated that SNORD47 significantly inhibited the proliferation ability of glioma cel s.3?After transfected glioma cells with lenti-SNORD47,the percentage of cells in G2/M phase in the treatment group was markedly increased.The results of Immunofluorescence assay indicated a lower proportion of p H3-positive cells in SNORD47-treated cells.The expressions of cell cycle related proteins were significantly changed.4?After transfected with lenti-SNORD47 in glioma cells,the invasion and migration ability were significantly inhibited.The results of Immunofluorescence assay and western blot indicated that the EMT were significantly inhibited.5?After co-treatment with lenti-SNORD47 and temozolomide,the proliferation ability,as measured by CCK-8 assay,was markedly inhibited compared with other groups,including both the SNORD47 overexpression treated and temozolomidetreated groups,but especially compared to the control group.Cells treated with either the SNORD47 overexpression lentivirus or temozolomide alone also exhibited an attenuated capacity of proliferation in comparison with the control group cel s.6? The result of intracranial nude mouse model revealed that SNORD47 represses tumorigenicity and strengthens the antitumor effect of temozolomide in glioma in vivo.Conclusion1?The expression of SNORD47 was decreased in glioma specimens especially in WHO grade IV glioma specimens.The correlation between HOTAIR and SNORD47 was significantly inverse.And SNORD47 could prolong the survival of glioma patients;2?SNORD47 could significantly inhibited the proliferation and invasion ability of glioma cel s;3?SNORD47 enhanced the sensitivity of glioma cel s to temozolomide;4?SNORD47 could function as a potential target in glioma treatment.