Mechanism Of Long Non-coding RNA CRNDE Promoting Celler Proliferation In Non-small Cell Lung Carcinoma

BackgroundLung cancer has been the highest mortality malignant tumour in the word.The diagnosis,treatment,and prognosis of NSCLC patients remains elusive,These issues highlight the urgent need of accurate methods to discover the molecular mechanism of NSCLC carcinogenesis,in order to facilitate development of novel cancer biomarkers and more effective therapeutic strategies.Recently,emerging studies have indicated that long non-coding RNAs?lncRNAs?exert their function in every stage of tumour progression.Nevertheless,the biological functions of lncRNAs in the progression of NSCLC remained largely unknown.Colorectal neoplasia differentially expressed?CRNDE?plays an important role in the proliferation,migration,and invasion of cancer cells.CRNDE might also function as a promoter in NSCLC tumourigenesis,but so far,this assumption has not been well studied.Part One:Expression and clinical significance of long non-coding RNA CRNDE in Non-Small Cell Lung Carcinoma1.Methods1.1 Clinical specimensHuman tissues samples,including 30 cases of NSCLC cancer patients and 30untreated specimens were provided by the Department of Cardiothoracic Surgery,the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine and the Second Affiliated Hospital of Nanchang University.1.2 RNA isolation and quantitative real-time PCR?qRT-PCR?Total RNA was extracted from NSCLC tissues samples,including 30 cases of NSCLC cancer patients,and was subjected to reverse transcription-PCR?40 times?.qRT-PCR assays were performed to quantify the expression level of CRNDE.Results were normalized to GAPDH expression.The relative amount of CRNDE was calculated using the equation 2^-??CT.2.RESULTSExpression of CRNDE in NSCLC tissues?1.64±0.4?was significantly increased when compared to the corresponding adjacent non-tumour tissues?0.96±0.28?.The level of CRNDE was obviously enhanced at the point of>3 cm proximal to the necrosis,as compared to the corresponding segments in normal patients3.Conclusion3.1 The high expression of 1ncRNA CRNDE has been observed in the non-small cell lung tissue collected from patients,These data indicate that CRNDE might act as a cancer promoter gene in NSCLC.3.2 The expression level of 1ncRNA CRNDE observed from the collected non-small cell lung tissue is related to size of tumor?tumor size bigger 3cm?,as they have a p-value less than 0.05,which is considered as statistically significant.But it is not related to patient's gender?age and histological type,as they were not statistically significant?p>0.05?.Part Two:CRNDE effects the Cell viability and Cell cycle of in Non-Small Cell Lung Carcinoma cell lines1.Methods1.1 Cell cultureThree NSCLC cell lines?A549,PC-9 and SK-MES-1?,along with the normal bronchial epithelial cell line?16HBE?were maintained in Dulbecco's Modified Eagles Medium?DMEM?at the atmosphere of 5%CO₂ and 37°C.1.2 RNA isolation and quantitative real-time PCR?qRT-PCR?Total RNA was extracted from NSCLC cells and The next same as the part one:1.2.1.3 Cell transfection with plasmid and siRNAPlasmids for CRNDE over-expression were constructed by inserting the amplified CRNDE cDNA into pCDNA3.1 vector,resulting in pcDNA CRNDE.Two small interference RNAs?siRNAs?targeting CRNDE,si-CRNDE#1 and si-CRNDE#2,were both designed and synthesized by Ribo Bio,Cells were transfected with plasmids or si-NC following the manufacturer's protocol,and stable NSCLC cell lines were established as a selection marker.1.4 Cell viability assayCell viability was measured using the MTT assay.At 0?24?48?72 and 96 hours after transfection.The absorbance in each well was measured by a microplate reader and caculate the rate of cell surviver.The experiment repeated 3 times.1.5 Cell cycle assayCell cycle was assessed by FACS Calibur flow cytometry and analysis.Required at least 20-30 thousand cells.1.6 Tumour xenograftsFour-week-old BALB/c nude mice were subcutaneously injected with sh-CRNDE or empty vector?n=6 in each group?.The subcutaneous tumour-bearing mice were killed eighteen days after injection.The tumour weight and volume were detected,and the tumour volume was calculated by the formula:volume?mm³?=?length×width?²/2.2.RESULTS2.1 Highly CRNDE expressed in NSCLC cellsA significant increase of CRNDE level in NSCLC cells?A549:4.89±0.59,PC-9:3.68±0.50 and SK-MES-1:2.25±0.21?compared with normal control?16HBE?are showed.2.2 Knockdown CRNDE inhibited NSCLC cell proliferation and the cell cycleCRNDE depletion significantly reduced cell viability in A549 and PC-9,which transfection with si-CRNDE#1 or si-CRNDE#2,compared with control?si-NC?cells.Silencing CRNDE induced cell cycle arrested in G₀/G₁ stage and decreased the NSCLC cell number in S and G₂/M stage.2.3 CRNDE over-expression enhanced NSCLC cell proliferation and growthCRNDE expression was verified to be significantly upregulated in SK-MES-1cells when compared with the controls?pcDNA?.In addition,CRNDE over-expression dramatically increased NSCLC cell viability than that in the control group.Flow cytometry analysis revealed that CRNDE over-expression significantly enlarged the distribution of G₂/M stage cells,along with decreasing the number of G₀/G₁ stage cells.2.4 CRNDE promoted NSCLC tumour formation and growth in vivoKnockdown CRNDE significantly inhibited tumour growth in vivo,including reduced tumour volume and weight,as compared with empty vector controls.3.Conclusion3.1 Knockdown CRNDE inhibited NSCLC cell proliferation and interfered with the cell cycle and CRNDE over-expression enhanced NSCLC cell proliferation and growth.3.2 CRNDE promoted NSCLC tumour formation and growth in vivo.Part Three:CRNDE regulated the protein of the cell cycle and PI3K/AKT signalling in NSCLC cells1.MethodsWestern blot:Total protein was extracted from cultured cells,and were transferred to polyvinylidene difluoride?PVDF?membranes.the membranes were incubated with various antibodies?phosphorylated AKT,total AKT,PI3Kp85?,CDK4,CDK6,CCNE1?.GAPDH acted as internal control and the protein transferred to membranes was defined with chemiluminescence detection kit and quantified by Image Lab 4.0 software.2.RESULTS2.1 CRNDE expression was corrected with the level of CDK4,CDK6 and CCNE1CRNDE depletion significantly inhibited the expression of CDK4,CDK6 and CCNE1 in A549 cells compared with control?si-NC?group.Inversely,CRNDE over-expression dramatically augmented the expression of CDK4,CDK6 and CCNE1in SK-MES-1 cells as compared with control?pc DNA?group.2.2 CRNDE regulated the activation of PI3K/AKT signalling in NSCLC cellsThe depletion of CRNDE significantly reduced the level of p-AKT/T-AKT and PI3Kp85?in A549 cells compared with the si-NC control group.However,over-expression of CRNDE could obviously heighten p-AKT/T-AKT and PI3Kp85?production in SK-MES-1 cell lines when compared with the control?pcDNA?group.3.ConclusionCRNDE expression was corrected with the level of CDK4?CDK6?CCNE1and CRNDE regulated the activation of PI3K/AKT signalling in NSCLC cells.