Effects Of Long-chain Non-coding RNA CRNDE On The Occur Rence And Development Of Glioma And Prognostic Analysis

Part One Changes in expression levels of long-chain non-coding RNA CRNDE in glioma and its association with clinicopathological characteristicsObjective: To investigate the expressions of CRNDE in glioma tissues of different pathological grades and adjacent non-tumor tissues,and to explore the association between CRNDE expression levels and clinicopathological characteristics.Methods:1.A total of 164 tumor tissue samples and adjacent non-tumor tissues were collected from patients who were treated with surgical resection of glioma in the Department of Neurosurgery,the Second Hospital of Hebei Medical University from 2010 to 2012.All glioma samples identified by pathological investigation were stored in 2ml sterile cryogenic vials and immediately frozen in liquid nitrogen.2.Quantitative real-time PCR(qRT-PCR)was performed to detect CRNDE expression levels in adjacent non-tumor tissues and glioma tissues of different grades for comparison.Results were expressed as mean±standard deviation.Comparisons between groups were performed using the t-test.3.Chi-square test was used to investigate the relationship between clinicopathological characteristics and CRNDE m RNA expression levels in 164 glioma tissues.SPSS software 17.0(SPSS Inc.,Chicago,IL,USA)was applied for statistical analysis.Results:1.Compared with adjacent non-tumor tissues(1.00±0.06),expression level of CRNDE in glioma tissues(3.49±1.71)was highly up-regulated,and the differences were statistically significant(P<0.05).2.There was a positive correlation between CRNDE expression level and grades of glioma.CRNDE expression level in high-grade gliomas(III-IV)(4.26±1.29)was significantly higher than that in low-grade gliomas(I-II)(1.51±0.86),showing significant differences(P<0.05).3.There was no correlation between CRNDE expression level and gender,age,preoperative symptoms as well as tumor necrosis of the patient,The P values were 0.35,0.59,0.38 and 0.49(P>0.05)respectively,suggesting no significant differences.4.CRNDE m RNA expression level was correlated with tumor size,pathological grades of tumor and tumor recurrence.The P values were 0.01,0.001 and 0.008 respectively,therefore,the difference were statistically significant.The contingency coefficients between CRNDE mRNA expression level and tumor size,tumor grade as well as tumor recurrence were 0.2,0.38 and 0.2 respectively.Part Two Multivariate analysis of CRNDE expression level,related clinicopathological characters and patient prognosisObjective: To explore clinically relevant factors that influence the survival and prognosis of patients with glioma,so as to determine prognosis more accurately,thereby guiding the patient's follow-up treatment.Methods:1.Collection of clinical data of patients: The duration from surgery to death was 3 to 65 months in 164 patients,with a mean follow-up of(43.8± 19.8)months.The age,gender,preoperative symptoms,tumor size,tumor necrosis,WHO grading,tumor recurrence and CRNDE expression level were recorded.2.Univariate analysis was performed by the Kaplan-Meier method and survival curves were constructed.The survival time was used as a nonindependent variable.The effects of various factors including clinicopathologic characteristics and CRNDE expression on survival time were investigated and analyzed using Log-rank analysis.3.For variables with P <0.05 by univariate analysis,multivariate analysis by Cox regression model was performed with a forward selection.Results:1.Univariate analysis: Four factors were related to the prognosis of patients with glioma,including WHO grading,tumor recurrence,tumor necrosis and CRNDE expression level,suggesting significant differences(P<0.05).The hazard ratio(HR)in recurrence group was 2.24 times that of non-recurrence group,The median time in recurrence group is 34 months,The median time in non-recurrence group is 58 months.Compared with low-grade group,HR had increased by 2.87 times in high-grade group,The median time in high-grade group is 32 months,The median time in low-grade group is 57 months.HR in high CRNDE expression group and necrosis group was 2.24 and 1.85 times that of low expression group and non-necrosis group,respectively,The median time in high CRNDE expression group is 32 months,The median time in low expression group is 57 months,The median time in necrosis group is 41 months,The median time in non-necrosis group is 57 months.2.Multivariate Cox regression analysis: The prognosis of patients with glioma was associated with WHO grading,tumor recurrence,tumor necrosis and CRNDE expression level(P<0.05),which were independent prognostic factors.Their HRs were 4.32,3.33,1.76 and 1.59 times,respectively.Part Three Effects of CRNDE on the proliferation,apoptosis and migration of brain glioma cellsObjective: To discuss the effect of CRNDE on proliferation,apoptosis and migration in brain glioma cells,as well as on related genes including Raf-1,MEK-2,ERK-1,ERK-2,c-Myc,NF-?b.Methods:siRNAs(si783 and si809)targeting CRNDE were designed and transfected into human glioma U251 cell lines.The tests were performed as follows:1.Cell proliferation detection: siRNA(si783 and si809)were transfected into human glioma U251 cell lines,and in the meantime,the non-transfection(NT)group and negative control(NT)group were set.Cell proliferation activity was detected by CCK-8 kit at 12 h,24h and 48 h after transfection.Moreover,the proliferation curve was delineated.2.Cell apoptosis detection: Apoptosis of U251 cells was detected by flow cytometry using PE Annexin-fict staining in siRNA(si783 and si809)group,NC group and NT group.3.Cell migration detection: Cell migration assay was performed using Transwell chambers,to analyze the effect of down-regulated CRNDE expression on migration of glioma cells.4.SYBR GreenI Real-time PCR was used to detect changes in mRNA levels of the related genes(Raf-1,MEK-2,ERK-1,ERK-2,c-Myc,and NF-?B)of human glioma U251 cells transfected with interfering siRNA(si783 and si809).Results:1.CCK-8 results: The 450 nm OD values were identical among NC group,NT group and siRNA group at 0h.At 12 h after transfection,there was statistically significant difference in 450 nm OD value between NC group(0.52±0.006)and si783 group(0.43±0.02,P<0.05),but no significant difference was found between si783 group(0.43±0.02)and NT group(0.52±0.02,P>0.05).At 24 h after transfection,450 nm OD value in si783 group(0.49±0.01)was significantly lower than that in NC group(0.65±0.016)and NT group(0.65±0.04,P<0.05).No significant difference was found beween NC group and NT group.At 48 h of transfection,450 nm OD value was remarkably lower in si783 group(0.59±0.01)than in NC group(0.72±0.01)and NT group(0.72±0.006,P<0.05).No significant difference was noted between NC group and NT group.At 12 h of transfection,there was statistically significant difference in 450 nm OD value between NC group(0.52±0.006)and si809 group(0.46±0.004)(P<0.05),whereas there was no significant difference between si809 group(0.46±0.004)and NT group(0.52±0.02)(P>0.05).At 24 h after transfection,450 nm OD value was notably lower in si809 group(0.52±0.02)than in NC group(0.65±0.016)and NT group(0.65±0.04,P<0.05).No significant difference was observed between NC group and NT group(P>0.05).At 48 h after transfection,450 nm OD value was significantly higher in si809 group(0.62±0.01)than in NC group(0.72±0.01)and NT group(0.72±0.006)(P<0.05).Significant difference was not found between NC group and NT group(P>0.05).2.Flow cytometry detection of apoptosis: The apoptosis rates were(19.2±1.6)% and(14.5±1.4)% in si783 and si809 group respectively,which were significantly higher than that in NT group(1.6±0.15)% and NC group(2.7±0.25)%(P<0.05).Nevertheless,there was no significant difference in apoptosis rates between NT group and NC group(P >0.05).3.Cell migration results: Cell migration experiment showed that after transfection,cell counts in si783(287.2±28.6)and si809(351.6±16.9)group were lower than those in NT group(518.2±29.9)and NC group(471.6±29.4)(P<0.05).However,there were no significant differences between NT group and NC group(P >0.05).4.The down-stream related genes expression after RNA interference: The expression levels of Raf-1,MEK-2,ERK-1,ERK-2,c-Myc,and NF-?B had decreased to different degrees.Raf-1 expressions and MEK-2 expressions in si783 and si809 groups were decreased to 31.7% vs.72.2% and 31.3% vs.41.9% of NC group respectively.ERK-1 and ERK-2 expression levels in si783 and si809 groups were reduced to 60.4% vs.48.2%,and 75.3% vs.70.4% of NC group respectively.The NF-?B and c-Myc expression levels in si783 and si809 groups were decreased to 39.9% vs.69.4% and 36.1% vs.47.3% of NC group.Conclusions:1.Expression levels of CRNDE mRNA were significantly higher in human glioma tissues than in adjacent non-tumor tissues,which were associated with pathological grades of glioma.CRNDE expression levels in high-grade gliomas(III-IV)were higher than those in low-grade gliomas(I-II),suggesting significant differences.2.CRNDE expression level was positively correlated with WHO grading,tumor size,and tumor recurrence,other than with gender and age and so on.3.Overall survival time was shorter in those with tumor recurrence,high-grade tumor,high expression of CRNDE and tumor necrosis.4.Four factors were independent risk factors for the prognosis of patients with glioma,including postoperative recurrence,pathological grade,CRNDE expression,and tumor necrosis.5.Inhibition of CRNDE contributes to suppression of the proliferation and migration of glioma cells and promotes the apoptosis of glioma cells.6.CRNDE could affect the proliferation,apoptosis and migration of glioma cells by regulating expressions of related genes(Raf-1,MEK-2,ERK-1,ERK-2),c-Myc and NF-?B in MAPK/ERK pathway.