| Background and ObjectiveGliomas are the most common intracerebral primary brain tumors in adults,accounting for more than 50%of the central nervous system tumors.Currently,surgery,radiotherapy,and chemotherapy are the main approaches for the treatment of gliomas,but the prognosis of patients undergoing treatment is poor.Long non-coding RNA(LncRNA)is a type of RNA that is longer than 200 nucleotides in length and does not encode proteins.Studies have shown that LncRNA plays a role in human neoplastic diseases.There are evidences that the expression of long non-coding RNAs are closely related to neurode’velopment,neurodegenerative and neuroimmunological diseases,mental illness,primary brain tumors,and neuronal tumors.Human glioma is a type of tumor with the highest malignancy and worst prognosis in adult primary brain tumors.Its pathogenesis,invasiveness,metastasis,and prognosis are related to LncRNA in many ways,and has become a research hotspot in recent years.The study found that LncRNAs can regulate the regulation of tissues and cells through multiple pathways,such as the use of siRNAs to silence LncRNA,antisense sequences degrade LncRNAs,corresponding enzymes directly cleave LncRNAs.Feng et al constructed the LncRNA-ROR by up-and down-regulation of LncRNA-ROR by constructing PcDNA-LncRNA-RoR plasmid and LncRNA-RoR shRNA plasmid,and MTS assayed U87 cell proliferation ability,confirming that LncRNA-RoR may be a novel glioma suppressor gene.Liang et al used siRNA transfection experiments to downregulate the expression level of LncRNA-PVT1 and found that the proliferation of bladder cancer cells was reduced.Down-regulation of LncRNA-PVTl expression in gastric cancer cells by siRNA can reduce cell proliferation,and overexpression of LncRNA-PVT13 increases cell proliferation.Our team’s previous research combined with previous studies found that LncRNA-PVT1 is highly expressed in clinical glioma tissue samples and multiple glioma cell lines,and its expression level is consistent with the severity of glioma cells,and has the positive correlation.Based on these,this study transfected by PVT1 silencing and overexpression both in vivo and in vitro,artificially modulate PVT1 expression,to study the proliferation and migration of glioma cells,and further understand the relationship between PVT1 and EZH2,which may lay the laboratory foundation for the treatment of gliomas.MethodsClinical tissue samples collection and Cell culture1.80 clinical glioma tissue samples and 10 normal brain tissue samples came from Nanjing Military Region General Hospital,from 2001.1 to 2009.6.All samples were freshly frokzen in liquid nitrogen and then stored at-80℃ until RNA extraction.All samples were histopathologically examined at the Nanjing Military Region General Hospital(Nanjing,China)and,according to the World Health Organization(WHO)criteria,classified into 4 grades:primary grade pilocytic astrocytoma(WHOⅠ),grade Ⅱ astrocytoma(WHO Ⅱ),grade Ⅲ anaplastic astrocytoma(WHO Ⅲ)and grade IV glioblastoma multiforme(WHO Ⅳ).Of each grade 20 cases were included.The normal brain tissues samples came from intracranial trauma cranial decompression surgery.The normal glial cell line HEB and glioma cell lines HS683,T98MG,U373,SHG44,A172,U251and U87MG were purchased from the Shanghai Cell Collection(Shanghai,China).The cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM)supplemented with 10%fetal bovine serum(FBS)at 370C in a 5%CO2 humidified atmosphere.2.Detection of PVT1 and EZH2 gene expression levels by qRT-PCR.For normal brain tissue,WHOI,WHOI,WHO Ⅲ,WHO Ⅳ,HEB cells,HS683 cells(equivalent to WHO Ⅱ),T98MG cells(equivalent to WHO Ⅱ)U373 cells(equivalent to WHO Ⅲ),SHG44 cells,A172 cells,U251 cells,and U87MG cells(four are glioblastomas,equivalent to WHO IV),qRT-PCR detected Lnc PVT1 and EZH2 genes expression.3.Log-rang(Mantel-Cox)of survival curves.Follow-up of 80 cases of glioma patients included in the study,followed up for 40 months,observed overall survival rate,and plotted Log-rang(Mantel-Cox)survival curve.4.Western blot detection of EZH2 protein expression levels.Take 8 cases of normal brain tissue,WHOI,WHOI,WHO Ⅲ,WHO Ⅳ glioma tissue;and HEB cells,HS683 cells(equivalent to WHO Ⅱ),T98MG cells(equivalent In WHO Ⅱ),U373 cells(equivalent to WHO Ⅲ),SHG44 cells,A172 cells,U251 cells,and U87MG cells(four are glioblastomas,equivalent to WHO Ⅳ)were subjected to Western blot analysis to detect the levels of EZH2 protein.5.Immunofluorescence detection of EZH2 protein expression levels in glioma tissues.Eight normal brain tissues,WHOI,WHOI,WHO Ⅲ,and WHO Ⅳ glioma tissues were used to detect EZH2 protein by immunofluorescence,and EZH2-positive proportion were counted.6.Immunofluorescence detection of EZH2 protein expression levels in glioma cell lines.HEB cells,HS683 cells,T98MG cells,U251 cells,and U87MG cells were cultured separately to prepare cell slides for immunofluorescence detection of EZH2 protein,and EZH2-positive proportion were counted.In vitro study experiments:1.Selection of glioblastoma cell lines U87MG and U251 for in vitro experiments2.The siRNA-PVT1 and OEPVT1 plasmids were synthesized separately,and U87MG and U251 cells were transfected with Lipofectamine 2000.Silencing negative control group(Silence-NC group)and overexpression negative control group(Over-NC group)were set.3.qRT-PCR and Western blot detection of transfection efficiency.After transfection 48h,qRT-PCR detected the Lnc PVT1 and EZH2 gene expression levels to determine transfection efficiency;Western blot detected the EZH2 protein expression levels.4.Cell proliferation activity assayed by MTT assay.The absorbance values of cells in each group were determined at time points of 0h,24h,48h,and 72h,and absorbance curves were plotted to compare the proliferation activity of the cells in each group.5.Annexin V-FITC/PI double staining flow cytometry to detect apoptosis.After transfection 48h,cells were collected by trypsin digestion,and Annexin V-FITC/PI double staining was performed.Flow cytometry was used to detect the apoptosis rates in each group.6.PI double staining flow cytometry to detect cell cycle.After transfection 48 h,cells were harvested by trypsin digestion,75%alcohol was fixed overnight,and then stained with PI,flow cytometry was used to measure cell cycle changes in each group,and the cell proliferation index was calculated according to the formula PI =(S+G2/M)/(G0/G1+S+G2/M).7.Transwell assay for cell migration.After transfection 48 h,cells were harvested by trypsin digestion,and Transwell chambers were used for detection of migration ability.8.Transwell assay for cell invasion.After transfection 48 h,cells were harvested by trypsin digestion,and Transwell chambers were used for detection of invasion.9.RIP was used to detect the relationship between PVT1 and EZH2.In vivo study experiments:1.U251 cells were stably transfected,and Silenced and overexpressed stable cell lines were screened.2.Tumor formation experiment.Stably transfected U251 cell line was selected.BALB/c nude mice aged 6-8 weeks were selected,and the stable cell line was injected subcutaneously in the right subphrenic fossa to establish a transplanted tumor model.3.Measurement of growth rate of xenograft.Tumor size was measured every 4 days with a vernier caliper and recorded according to the formula:tumor volume(V)=(L x S2)/2,where L is the longest diameter of the tumor and S is the shortest diameter of the tumor,and then calculated tumor volume.4.Weight of transplanted tumors.When tumors grow to the longest diameter of 1.5 cm,the nude mice were sacrificed and the tumors were harvested.The adhesion tissue was separated,the weight of the tumor tissue was weighed and recorded.5.qRT-PCR detection of apoptosis-related protein expression in xenografts.qRT-PCR experiments were performed to detect the expression levels of PVT1,EZH2,Caspase3,Bax and Bcl-2 mRNA.6.Western blot detected the expression of apoptosis-related proteins in xenografts.Total protein was extracted from all groups,and Western blot was performed to detect the expression of PVT1,EZH2,Caspase3,Bax and Bcl-2.7.TUNEL detection of apoptosis in xenografts.Tumor tissues from each group were used for TUNEL experiments to analyze the overall apoptotic status of the transplanted tumors.8.Immunohistochemical detection of PCNA protein expression in xenografts.PCNA immunohistochemistry experiments was performed to analyze the positive rate of PCNA protein expression in all groups of xenografts tumors.Statistical methodStatistical analysis was performed using SPSS 20.0 software.The expression levels of Lnc PVT1 and EZH2 in glioma tissue and normal brain tissue were compared using the Paired Wilcoxon test.Log-rang(Mantel-Cox)method was used to draw the survival curves.All data were expressed as mean ± standard deviation(x ±s).One-way analysis of variance(One Way ANOVA)was used for comparison between groups.Dunnett-t method or SNK method was used for pairwise comparison.P<0.05 indicates that the difference was Statistical significance.ResultsGliomas Clinical Samples1.The results of qRT-PCR experiments showed that compared with the normal brain tissue,the expression levels of PVT1 increased with the increase of glioma grade,and showed a positive correlation with the malignant degree of glioma.Compared with normal brain cell HEB cells,the expression levels of PVT1 gene were significantly different among HS683,T98MG,U373,SHG44,A172,U251 and U87MG There was also a positive correlation between the expression of PVT1 protein and the malignant degree of glioma.2.The results of qRT-PCR and Western blot showed that compared with normal brain tissue,the expression level of EZH2 increased with the increase of glioma grade,and it was positively correlated with the malignant degree of glioma.In glioma cell lines,compared with HEB cells,there were significant differences in the expression of HS683,T98MG,U373,SHG44,A172,U251,and U87MG,and EZH2,and there was also a positive correlation with the degree of malignancy of gliomas.3.Patients with gliomas were divided into three groups according to PVT1 mRNA expression levels(low:<3,n = 18;medium:3-6,n=19;high:>6,n = 34)and then subjected to Log-rang(Mantel-Cox)analysis.The results showed that the median survival time for high PVT1 expression was 9 months,while was 20 months for medium PVT1 expression,and 31 months for low PVT1 expression.We divided the patients into three groups based on EZH2 mRNA expression levels(Low:<2,n =16;medium:2-4,n = 17;High:>4,n = 38)and then conducted Log-rang(Mantel-Cox)analysis.The survival curves showed that the median survival time for high PVT1 expression was 9.5 months,while was 21 months for medium EZH2 expression,and 33 months for low EZH2 expression.The clinical results showed that the expression levels of Lnc PVT1 and EZH2 were correlated with the survival of glioma patients.The higher the Lnc PVT1 and EZH2 expression levels,the shorter the survival time of glioma patients.4.Immunofluorescence staining showed that EZH2 protein was expressed on the nucleus in glioma tissue.Compared with normal brain tissues,the expression of EZH2 protein in gliomas was significantly upregulated.With the increase of malignant degree of gliomas,the positive expression rate of EZH2 was significantly increased,that is,the expression level of EZH2 was positively correlated with the degree of malignancy of gliomas.The higher the degree of malignancy,the higher the expression level of EZH2.5.Immunofluorescence staining showed that in the glioma cell line,EZH2 protein was expressed on the nucleus.Compared with normal brain cell HEB cells,the expression level of EZH2 protein in glioma cell lines was significantly upregulated;with the increase of malignant degree of gliomas,the positive expression rate of EZH2 was significantly increased,namely the expression level of EZH2 and glioma malignant degree showed a positive correlation.The higher the degree of malignancy,the higher the expression level of EZH2.In vitro study results:1.The results of qRT-PCR and Western blot showed that compared with the NC negative group,transfection with PVT1-siRNA significantly decreased the expression of PVT1(P<0.05),and transfection with OEPVT1 significantly increased the expression of PVT1(P<0.05).It shows that PVT1-siRNA and OEPVT1 have higher transfection efficiency and can be used in subsequent experiments.2.Compared with the control group,the proliferation activity of cells transfected with PVT1-siRNA was significantly decreased compared with the control group.Compared with the control group,the overexpression of OEPVT1 group was significantly increased in cell activity,and the data was statistically significant(P<0.05)..3.Annexin V-FITC/PI double-staining flow cell apoptosis.The apoptosis rate of U87MG cells was(38.26±1.53)%in the silencing group and(1.53±0.63)%in the control group.The apoptosis rate was(31.46±2.15)in the U251 cell silencing group and(1.97 ± 0.68)%in the control group.Compared with the control group,the apoptotic rate of U87MG and U251 cells in the silencing group was significantly decreased(P<0.05),while no obvious apoptotic rate was observed in the overexpressed group(Figure 2-4,P>0.05).4.PI flow cell cycle.Compared with Silence-NC control group,the percentage of G0/G1 phase increased in the silencing group,and the percentage in S+G2/M phase decreased,showing that the cell proliferation index PI decreased from(0.40±0.08)in the control group(0.27±0.07)for the silent group(P<0.05).U251 cells also showed an increase in the percentage of G0/G1 phase,a decrease in the percentage of S+G2/M phase,and a decrease in cell proliferation index PI.Compared with the Over-NC control group,the percentage of cells in the overexpression group decreased at the G0/G1 phase,and the percentage at the S+G2/M phase increased,indicating that the cell proliferation index PI increased from(0.41±0.09)in the control group to the overexpression group(0.58± 0.10)(P<0.05).U251 cells also showed a decrease in the percentage of G0/G1 phase,an increase in the percentage of S+G2/M phase,and an increase in cell proliferation index PI(P<0.05).5.Transwell invasive ability.Observed and counted in three fields,the number of invasive cells in U87MG and U251 cells was 41.23 ± 4.06 and 31.33 ± 4.28 in the PVT1 group,and 175.00 ± 8.52 and 184.67 ± 7.77 in the overexpression group,respectively.In contrast,the number of invasive cells in the Silence group was significantly reduced,and the number of cells in the overexpressing group was significantly higher(P<0.05).6.Transwell migration capabilities.The results of observation and counting in three fields showed that the number of migrating cells in the Silence group U87MG and U251 cells were 101.23±4.25 and 91.33±4.08,respectively;and were 195.00±9.52 and 204.67 ± 9.87 in the overexpression group,respectively.In contrast,the number of migrated cells in the Silence group was significantly decreased,and the number of cells in the overexpression group was significantly increased(P<0.05).6.The regulatory relationship between PVT 1 and EZH2.RNA co-immunoprecipitation(RIP)showed that the expression of PVT1 in anti-EZH2 group was 50%higher than that in total RNA group,and the expression of PVT1 in anti-EZH2 group was significantly higher than that in IgG group,indicating that PVT1 can be specifically and tightly combined with EZH2.RIP experiments show that PVT1 exerts its biological functions by binding to EZH2.In vivo study results:1.Transplant tumor volume.Silence and its Silence-NC group,U251 cells were injected into nude mice.After tumor formation(8 days),the length and width of the tumor were measured with a vernier caliper every four days.Compared with Silence-NC group,the volume of transplanted tumor in Silence group was significantly decreased(P<0.05);U251 cells from Over and Over-NC group were injected into nude mice,after tumor formation(4 days),the length and width of the tumor were measured once a four-day vernier caliper.Compared with the Over-NC group,the tumor volume in the Over group was significantly increased,with a statistically significant difference(P<0.05).2.Transplant tumor weight.Silence and its Silence-NC group,U251 cells were injected into nude mice for 28 days.The tumor tissues were isolated;Over and NC group U251 cells were injected into nude mice for 20 days and the tumor tissues were isolated.Compared with the Silence-NC(1.03 ± 0.095)group,the weight of transplanted tumors in the Silence group(0.664±0.148)was significantly reduced(P<0.05);compared with the Over-NC group(1.024 ± 0.113),The weight of transplanted tumors in the Over group(1.71± 0.269)was significantly increased,with a statistically significant difference(P<0.05).3.qRT-PCR analysis of EZH2,Caspase3,Bax,and Bcl-2 gene expression in the transplant tumor.Compared with Silence-NC group,after silencing PVT1 gene,the expression of EZH2 gene was decreased,and the proapoptotic protein caspase3 was induced.The level of Bax gene expression was significantly higher(P<0.05),and the expression level of the anti-apoptotic protein Bcl-2 gene was significantly lower(P<0.05).Compared with the Over-NC group,over expression of the PVT1 gene was accompanied by an increase in the expression of the EZH2 gene;the expression levels of the pro-apoptotic proteins caspase3 and Bax were significantly decreased(P<0.05),and the anti-apoptotic protein Bcl-2 was significantly increased(P<0.05).The difference was statistically significant.4.Western blot analysis of EZH2,Caspase3,Bax and Bcl-2 protein expression in the transplant tumor.Compared with Silence-NC group,after silencing PVT1 gene,the expression of EZH2 protein was decreased,causing proapoptotic protein caspase3.Significant increase in expression levels of Bax and Bax(P<0.05)and significant decrease in Bcl-2 expression(P<0.05).Compared with the Over-NC group,overexpression of PVT1 gene was accompanied by an increase in the expression of EZH2 protein;the pro-apoptotic protein caspase3 and Bax expression levels were significantly decreased(P<0.05),and the anti-apoptotic protein Bcl-2 was significantly higher(P<0.05).The difference was statistically significant.5.TUNEL assay analysis of the overall apoptosis of transplanted tumors.TUNEL results showed that compared with the Silence-NC control group(1.23 ±0.21%),the apoptotic index of the transplanted tumors in the Silence group(12.64±1.24%)was significantly higher(P<0.05);Compared with Over-NC control group(1.33±0.25%),the apoptotic index of transplanted tumors in the Over group(1.26±0.24%)did not change significantly,and there was no significant difference(P>0.05).6.Immunohistochemical assay analysis of PCNA protein expression.The results showed that PCVA protein was expressed in the nucleus,showing diffusive or granular expression.Compared with the Silence-NC group(30.2±2.6%),the positive rate of PCNA in the Silence group(12.6±2.24%)was significantly lower(P<0.05);and compared with the Over-NC group(32.5± 4.6%),the positive rate of PCNA(62.5± 5.4%)was significantly higher in the Over group,with significant difference(P<0.05).Conclusion1.In glioma clinical tissues and their cell lines,long non-coding RNA PVT1 and EZH2 are highly expressed,and their expression levels are positively correlated with the degree of malignancy of gliomas.2.Long non-coding RNA PVT1 regulates the proliferation and migration of glioma cells by targeting the expression of EZH2 protein. |
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