The Expression Of LncRNA DILC In Glioma And Its Affection On Cell Proliferation

Objective: Glioma is the most common and lethal primary malignant cancer of the central nervous system,it originated from neuroectodermal and accounting for about 33.3-58.6% of all sorts of intracranial tumors.Recent statistics reports that there will be 3-10 people in 100,000 who developed glioma.The first line setting of glioblastoma patient treatment encompasses surgery followed by tumor bed irradiation and concomitant and then adjuvant chemotherapy.Despite such a complex therapy,recurrence is unavoidable,due to the resistance of radiochemotherapy of glioblastoma cell,the median survival is nowadays around 14 months.Thus glioma research worth more attention and more human and material resources need to be invested in.With the progress of such researches,the mechanisms underlied the initiation and progression of glioma will be clarified and more diagnostic biomarkers will be identified,which could provide novel targets and theoretical basis for treatment.Evidence from whole genome and transcriptome sequencing suggests that the complexity of an organism might be regulated by noncoding RNAs(ncRNAs)which is part of the noncoding region of the genome.Thus,the attentions of researchers are now shifting to one of the most common but least well-studied RNA species: LncRNAs.LncRNA is most commonly defined as a non-protein-coding RNA molecule longer than 200 nucleotides.Thanks to the identification of an enormous number of lncRNAs,the cellular function of lncRNAs needs to be revealed.In the past few years,common knowledge has pointed out that these long noncoding transcripts are simply transcriptional noise or cloning coproducts.Nonetheless,rigid biological regulation and explicit evolutionary conservation has been discovered in several lncRNAs,supporting the assumption that transcripts are of functional significance.Lnc-DILC(lncRNA down-regulated in liver cancer stem cells)was identified by microarray and validated by real-time PCR.Studies showed that lncRNA DILC repress self-renewal of liver cancer stem cell(LCSC)via inhibiting autocrine IL-6/STAT3 axis,lncRNA DILC expression was down regulated in LCSCs and lncRNA DILC repressed the expansion of LCSC.Experiments conducted in vivo suggested that the depletion of lncRNA DILC will facilitate the initiation and progression of hepatoma.Further studies showed that lncRNA DILC suppressed LCSC expansion via inhibiting JAK2/STAT3 activation and lncRNA DILC regulated the autocrine signaling of IL-6/JAK2/STAT3 in LCSCs,meanwhile,the knocking down of lncRNA DILC increased IL-6 autocrine signaling,and thus enhanced the activation of IL-6/STAT3 pathway in LCSCs.In recent studies,IL-6 has been closely associated with the development of many malignancies.Gliomas are not an exception as the elevated levels of IL-6 which were observed in a mass number of patients are correlated with poor prognosis and increased aggressiveness.Furthermore,IL-6 gene depletion is able to suppress the progression of tumor in glioma cells which highlights its significance as a cancer regulator.Up to date,there is no report about the expression and function of lncRNA DILC in glioma.Given that the report about IL-6/STAT3 signaling cascade made the prospection of lncRNA DILC were differentially expressed in all grades of glioma and the role as a regulatory a promising one.Therefore,present study aimed to getting the expression profile of DILC in glioma and normal brain tissue through the technology of Q-PCR,elucidate the expression of all grades of glioma and normal brain tissue,analyze the relevance of differentiation of expression and grades of glioma.At the meantime,design the sequence of siRNA which could knock down the expression of lncRNA DILC,transfect glioma cell U87 and U251,using colony formation assay and CCK-8 cell viability analysis to explore the influence of lncRNA DILC on the glioma cell biological function and the possible mechanism.Methods:1 Collecting of clinical samples: over the last year,tissue samples were collected from different levels of glioma and normal brain tissue from the Second Hospital of Hebei Medical University.According to the classification standards published by WHO in 2007,we collected a total of 43 samples of glioma and normal brain tissues,23 for grade I-II,7 for grade III,6 for grade IV,and the rest 7 samples were from normal brain tissue.2 Analyzation of clinical samples: the full-length sequence was obtained from GeneBank,then design the forward primer and reverse primer.The expression of lncRNA DILC in tissue samples were detected by quantitative PCR.3 Design the sequence of siRNA,which could knock down the expression of lncRNA DILC,transfect glioma cell U87 and U251,using colony formation assay and CCK-8 cell viability analysis to explore the influence of lncRNA DILC on the glioma cell biological function and the possible mechanism.4 Using spss 13.0 to analyze experimental data.Results:1 After the RNA extraction,purity testing(1.8<A260/A280<2.1),integrity assessment,amplification of target gene lncRNA DILC,tab them with flurescent marker and then use spss13.0 to analyze the results in a statistical way.Results shows that expression of lncRNA DILC had a significant difference among low-grade gliomas(WHO grade I-II),high-grade gliomas(WHO grade III,IV)and normal brain tissue.Taken together,these data demonstrated that with the rises of grade of glioma,the relative expression of lncRNA DILC decreases;2 Right after the successful synthesis of siRNA,it is transfected into glioma cell line U87 and U251,and then assess the cell model with the technology of Q-PCR,with the application of colony formation assay and CCK-8 cell viability analysis.It comes to a result that,compared with control cell group,the forming ability of glioma cell in experimental group was increased by the knocking down of lncRNA DILC.CCK-8 detection showed that,the knocking down of lncRNA DILC might accelerate the proliferation of U87 and U251.Conclusions:1 The relative expression level of lncRNA DILC declined in glioma tissues and showed a notable differentiation in various grades of glioma.With the rises of grade of glioma,the relative expression of lncRNA DILC decreases,it is closely associated with the malignancy.This suggested the potential of lncRNA DILC as a diagnostic biomarker and a novel therapeutic targets.2 DILC might have strong impacts on proliferation of glioma cell,the knocking down of lncRNA DILC could enhance the cellular function of cell proliferation.