| Objectives:The TPD52 gene is located in chromosome 8q.21.13.Previous studies have found that the expression of TPD52 is abnormally increased in several malignant tumors,such as breast cancer and prostate cancer,and plays an important role to the tumor progressions.However,the expression of TPD52 in gliomas and its regulatory mechanism have not been reported.The biological function and the role of abnormal expression of TPD52 in the occurrence and development of glioma are still unclear.As an epigenetic regulatory mechanism,miRNA-mediated gene silencing can regulate gene expression at the post-transcriptional level.Previous studies have shown that the abnormal expression and structure of miRNA are important factors leading to abnormal expression of proteins in tumor cells.However,the miRNAs that regulate the expression of TPD52 in glioma cells have not been determined,and whether the miRNA regulation disorder is the molecular mechanism that leads to the abnormal expression of TPD52 in glioma cells remains to be explored.We design the study for: 1.To detect the expression of TPD52 in the different grades of gliomas,and to analyze the correlation between its expression and the grades of gliomas or the prognosis of patients.2.To observe the effect of TPD52 on proliferation and invasion of glioblastoma by in vitro and in vivo experiments,and to explore its molecular mechanism.3.Through bioinformatics prediction and CGGA database analysis to screen candidate miRNA which is closely related to TPD52 expression in glioma cells,to confirm TPD52 is a target gene of the candidate miRNA in the glioma cells by luciferase assay.4.To detect the expression of miRNA in gliomas,which regulating TPD52 expression,and to analyze its relationship between TPD52 expression,the grade of gliomas and the prognosis of patients.5.To observe the expression level of TPD52 and the influence of cell proliferation and invasion of glioma cells after correcting the miRNA abnormal expression.6.Through the pathway research to further explore the regulation mechanism of TPD52 expression in glioma cells.Methods:1.IHC was used to detect the expression levels of TPD52 and Ki-67 in non-tumoral brain tissue and different grades of glioma tissues(tissue microarray),and analyze their expression changes in different grade of gliomas.Pearson method was used to analyze the relationship between TPD52 expression level and Ki-67 labeling index.The relationship between TPD52 expression level and patients' overall survival and disease-free survival was analyzed by Kaplan-Meiers.The Cox's proportional hazards regression model was established to screen out independent survival predictors for glioma patients.2.Using recombinant lentivirus to construct a sub-cell line of human glioblastoma cells U251 and LN229 that stably knockdown TPD52(U251/LN229-sh-TPD52#1,U251/LN229-sh-TPD52#2)and control sub-cell lines(U251/LN229-sh-NC).The expression efficiencies is confirmed by Western blot.3.EdU,MTS and flow cytometry cell cycle assay were used to detect whether knockdown of TPD52 can effectively inhibit proliferation activity and the cell cycle distribution of glioma cells.Transwell cell invasion assay was used to detect the effect of TPD52 on the invasion ability of glioma cells.F-actin fluorescence staining assay was used to observe the effect of TPD52 on the distribution of F-actin in glioma cells.4.Athymic nude mice were orthotopically implanted with LN229 stable sub-cell lines(LN229-sh-NC,sh-TPD52#1,sh-TPD52#2).The weight and survival of each group of mice were observed and recorded.The bioluminescence imaging was used to detect the transplanted tumor growth regularly every 2 weeks.After the transplanted tumor was removed,the tumor size and invasion were observed by HE;the expression levels of TPD52 and Ki-67 in the tumor were detected by IHC.5.Phospho-proteomics was used to screen the signaling pathway closely related to TPD52 promoting glioma cells proliferation and invasion,and further verified the effector pathway and molecular mechanism by Western blot.6.Bioinformatics prediction results were used to screen miRNAs regulating TPD52 expression,Combined with that miRNAs in the CGGA database are significantly negatively correlated with TPD52 expression,it was found that miRNA(miR-449b)regulates TPD52 expression in gliomas.Dual-luciferase assay was used to verify whether TPD52 is a target gene for miR-449 b in gliomas.7.ISH with the LNA-modified probe was used to detect the expression level of miR-449 b in the same sample of non-tumoral brain tissues and glioma tissues,and analyze its expression changes in different glioma grades;Pearson's method was used to analyze the relationship between the expression levels of miR-449 b and TPD52 and Ki-67 labeling index.Kaplan-Meiers survival analysis was used to analyze the expression level of miR-449 b and the overall survival and disease-free survival of patients.To establish the Cox's proportional hazards regression model to determine whether miR-449 b is a predictor of prognosis in glioma patients.8.EdU cell proliferation assay and MTS were used to evaluate the effect of miR-449 b on glioma cells proliferation activity,Transwell cell invasion assay was used to detect the effect of miR-449 b on glioma cells invasion,F-actin fluorescence staining experiment to observe the effect of miR-449 b on the distribution of F-actin in glioma cells.Exogenous TPD52 was supplemented to observe whether it could reverse the effect of miR-449 b on proliferation,invasion and F-actin distribution of glioma cells.9.qRT-PCR was used to detect the expression of miR-449 b after knockdown of TPD52 and inhibition of ERK activity,and chromatin immunoprecipitation(ChIP-PCR)was used to further explored the molecular mechanism leading to this change.Results:1.IHC showed that the expression level of TPD52 in glioma tissues was generally abnormally increased,and the labling index(LI%)was progressively increased with the elevation of glioma grade(P<0.05~0.001).Pearson correlation analysis showed that TPD52 LI% was significantly positively correlated with Ki-67 LI%(r=0.799;P<0.0001).Kaplan-Meiers survival analysis showed that glioma patients with lower TPD52 level had longer disease-free survival and overall survival than higher TPD52 level.The Cox's proportional hazards regression showed that TPD52 was a risk factor for glioma patients,and TPD52 LI% was an independent prognostic predictor for glioma patients.2.EdU and MTS showed that the cell proliferation activities of the U251 and LN229 sh-TPD52#1 and sh-TPD52#2 sub-cell lines were significantly lower than that in the corresponding sh-NC sub-cell lines(P<0.05~0.001).The FCM cell cycle assay showed that the percentage of G0/G1-phase cells in the U251 and LN229 sh-TPD52#1 and sh-TPD52#2 sub-cell lines were significantly higher than that in the corresponding sh-NC sub-cell lines(P < 0.001).Transwell cell invasion assay showed that the cell invasive ability of sh-TPD52#1 and sh-TPD52#2 sub-cell lines was significantly lower than that of sh-NC sub-cell lines(P<0.001).F-actin fluorescence staining showed that F-actin was diffused in the cells of U251 and LN229 sh-TPD52#1 and sh-TPD52#2 sub-cell lines,whereas F-actin was centralized mostly on cell cortex or lamellae in the cells of the corresponding sh-NC sub-cell lines.3.The in vivo assay showed that the xenografted tumours growth rate and the nude mice weight loss rate in sh-TPD52#1 and sh-TPD52#2 groups were significantly lower than those in sh-NC group,and the nude mice survival rate in sh-TPD52#1 and sh-TPD52#2 groups were higher than that in sh-NC group.HE showed that the size and invasiveness of the xenografted tumours in the sh-TPD52#1 and sh-TPD52#2 groups were significantly lower than those in the sh-NC group.IHC showed that the TPD52 LI% and Ki-67 LI% in the sh-TPD52#1 and sh-TPD52#2 groups were significantly lower than those in the sh-NC group.4.Phospho-proteomics detection functional enrichment analysis shows that in glioma cells TPD52 is mainly involved in cell differentiation,cell cycle,cell cycle regulation,cytoskeleton formation,microtubule cytoskeleton formation,actin cytoskeleton formation and others related signal pathway regulation.Kinase analysis showed that TPD52 regulates these functions mainly related to ERK and GSK kinases.Western blot showed that in glioma cells knockdown of TPD52 could effectively inhibit the expression levels of cyclin D1 and c-Myc,the phosphorylation level of GSK-3?,Erk1/2,MSK1,RSK1,LIMK1/2 and Cofilin,by increasing the phosphorylation level of ?-catenin.5.Bioinformatics predictions showed that there were 8 miRNAs that specifically bind to the 3'-UTR region of TPD52 mRNA;CGGA database result showed that there were 98 miRNAs that significantly negatively correlated with TPD52 expression(p<0.001).Above all,miR-449 b is a candidate miRNA to regulate TPD52 expression in glioma cells.The TPD52 mRNA 3'-UTR has two target sequences complementary to the seed sequence of miR-449 b.Dual-luciferase reporter assay demonstrated that in the U251 and LN229 cell lines,the wild-type TPD52 mRNA 3'-UTR(TPD52-3'-UTR-WT1 and TPD52-3'-UTR-WT2)could effectively suppress the luciferase activity by binding to miR-449 b.Whlie the mutant 3'-UTR(TPD52-3'-UTR-MT1 and TPD52-3'-UTR-MT2),without the binding site,failed to suppress the above luciferase activity.6.ISH showed that the expression level of miR-449 b was lower in gliomas than in non-tumoral brain tissues,and decreased with the elevation of glioma grades(P<0.001).Pearson correlation analysis proved that miR-449 b LI% was a negative correlation with Ki-67 LI%(r=-0.692;P<0.0001)and TPD52 LI%(r=-0.660;P<0.0001).Kaplan-Meiers survival analysis showed that the disease-free survival and overall survival of miR-449 b high-expression group were higher than those of miR-449 b low-expression group in glioma patients(P< 0.0001).Cox's proportional hazards regression showed that miR-449 b is a protective factor for gliomas,and miR-449 b LI% is an independent predictor for disease-free survival and overall survival of glioma patients.7.EdU,MTS and Transwell demonstrated that the cell proliferation activity and invasion ability of U251 and LN229 miR-449 b sub-cell lines were significantly lower than those of the corresponding Con sub-cell lines(P<0.01~0.001).While the effect of miR-449 b in glioma cell proliferation and invasion could be effectively reversed by exogenous TRAF5(P<0.05~0.001).Immunofluorescence experiments showed that miR-449 b significantly reduced the expression of TPD52 in U251 and LN229 cytoplasm.F-actin fluorescence staining showed that F-actin was centralized mostly on cell cortex or lamellae with stronger fluorescence signal in the Con and miR-449b+TPD52 groups,F-actin was dispered in glioma cells with lower fluorescence signal and disordered protein skeleton in the miR-449 b group.8.Database analysis showed that the miR-449 b gene transcription initiation site are enriched with numerous histone H3 lysine 27 trimethylation(H3K27me3)modification.qRT-PCR results showed that both knockdown TPD52 and inhibition of ERK pathway could increase the expression level of miR-449b(P<0.05-0.001).ChIP-PCR showed that H3K27me3 enriched with the miR-449 b gene transcription initiation site was significantly reduced in the ERK inhibitor group compared with the DMSO control group(P<0.01-0.001).Conclusions:1.The aberrant increase of TPD52 expression level is a common feature of human gliomas,which is positively correlated with the elevation of glioma grade,and negatively correlated with the disease-free survival and overall survival of glioma patients.TPD52 LI% could be used as a biomarker to assess the malignant degree of gliomas and an independent predictor to evaluate the prognosis of glioma patients.2.TPD52 is a tumorigenic factor of glioma,its abnormal increase expression may be an important causes leading to the occurrence and development of gliomas.Knockdown of TPD52 can significantly inhibit the proliferation and invasion of gliomas,which could be used as an effective target for gene intervention and molecular targeted therapy of malignant gliomas.3.TPD52 can promote the proliferative activity and invasion ability of glioma cells by activating ERK and GSK pathways.4.TPD52 is a direct target gene of miR-449 b in glioma cell lines,miR-449 b can reduce the expression level of TPD52 protein by regulating post-transcriptional level.5.Abnormal decrease of miR-449 b is common in glioma,its expression level is negatively correlated with the elevation of glioma grade,and positively correlated with the disease-free survival and overall survival glioma patients.miR-449 b LI% could be used as a reference indicator for the malignant degree of gliomas and an independent predictor to evaluate the prognosis of glioma patients.6.miR-449 b is a tumor suppressor of gliomas,its abnormal expression may play an important role in glioma progression.miR-449 b could effectively inhibit the proliferation and invasion of glioma cells,which could partially reversed by increasing the expression level of TPD52.Suggested that the decreased expression of miR-449 b may be one of the reasons for the overexpression of TPD52 in gliomas.7.TPD52 can promote cell cycle progression,proliferation and invasion by activating ERK and GSK signaling pathways in malignant glioma cells.In addition,TPD52 can promote the formation of H3K27me3 by activating the ERK signaling pathway,increasing the amount of H3K27me3 binding the miR-449 b gene transcription initiation site,thereby inhibiting the transcription of miR-449 b and abolishing the targeted inhibition effect of miR-449 b on TPD52.Therefore,there is a TPD52 /ERK/ H3K27me3/miR-449 b abnormal feedback regulation loop in glioma cells.The activation of this loop can further inhibit the expression of miR-449 b and increase the expression level of TPD52 through signal amplification.The overexpression of TPD52 improving proliferation and invasion ability of malignant glioma cells by activating ERK and GSK pathways.Thereby,it plays an important role in the development of malignant glioma. |
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