Expression Of Long Non-coding RNA ZXF2 In Lung Adenocarcinoma And The Effect On Cell Growth

Part Ⅰ Changes of lncRNA expression profiling between pulmonary adenocarcinoma tissue and paracancerous tissueObjective To test the differentially expressed lncRNAs in the human pulmonary adenocarcinoma tissue compared with the paracancerous normal lung tissue with lncRNA Expression Microarray.Methods Total RNA from the pulmonary adenocarcinoma tissue and normal lung tissue of 6 patients with pulmonary adenocarcinoma were prepared respectively, ds-cDNA was synthesized and labeled. Hybridization was performed with the profile chip containing more than 22000 human lncRNAs.Data analysis was performed using the Agilent GeneSpring GX software.Results Within the 22000 lncRNAs, about 1025 lncRNAs were differentially expressed in pulmonary adenocarcinoma tissue, in which 464 lncRNA were up-regulated and 561 lncRNA were down-regulated compared with the normal lung tissue. According to the filter condition, chose SEQ ID for AA311918 lncRNA (temporarily named ZXF2) as a follow-up study.Conclusions Obvious changes of lncRNA expression profiling were observed in the pulmonary adenocarcinoma occurrence, lncRNA may be related to the progress of pulmonary adenocarcinoma.Part Ⅱ lncRNA ZXF2 expression in 40 lung adenocarcinoma and clinical data analysisObjective Validate the high expression of lncRNA ZXF2 in 40 lung adenocarcinoma specimens, explore the relationship between the high expression of lncRNA ZXF2 and clinical pathological data.Methods Collected 40 pairs of cancerous and adjacent non-cancerous lung adenocarcinoma specimens. Quantitative real-time polymerase chain reaction was used to detect the expression of ZXF2 in these specimens. The relationship between the expression of ZXF2 and clinical pathology was explored.Results 27 cases (67.5%) of all lung adenocarcinoma specimens had 2 fold or higher ZXF2 expression in cancer tissues than paracancerous tissues (P<0.05). High expression of ZXF2 was positively correlated with histological grade (p=0.024), T stage classification (p=0.016), lymph node metastasis (p=0.014) and advanced clinical staging (p=0.006). The relative expression level of ZXF2 was positively correlated with tumor lymph node metastasis (x2=8.485, P<0.05) and poor prognosis of the patients (p=0.0217).Conclusions The expression of lncRNA ZXF2 was significantly high, high expression of lncRNA ZXF2 was closely related to tumor stage, metastasis and prognosis.PartⅢ SiRNA targeting silences A549 and suppresses lncRNA ZXF2 expressionObjective Select the lung adenocarcinoma cell line with high lncRNA ZXF2 expression, design and select the siRNA which has the strongest interference efficiency to ZXF2.Methods Tested the expression of lncRNA ZXF2 in A549, H1299, and H1395 lung adenocarcinoma cell lines by qRT-PCR. Designed three different interference sequences, tested the expression of IncRNA ZXF2 after transfected by qRT-PCR.Results A549 had highest level of ZXF2 among three cell lines, transfecting of siRNA-65 (sequence:5’-GCUGUUGGAUGUCACACACTT-3’) led to about 70% reduction of ZXF2 expression, which showed the most inhibition efficiency among these three siRNAs.Conclusions A549 had the highest level of ZXF2 expression, siRNA-65 had the most inhibition efficiency.Part IV The role of lncRNA ZXF2 on lung adenocarcinoma cancer cell growthObjective To explore the role of lncRNA ZXF2 on lung adenocarcinoma cell growth.Methods After siRNA interference, CCK-8 was used to detect cell proliferation, FACS was used to detect cell cycle and cell apoptosis, western blot was used to detect the expression level of related protein, transwell assay was used to detect cell migration and invasion.Results SiRNA-ZXF2 treatment inhibited cell proliferation (P<0.01) leading to cell cycle arrest (P<0.01). The cell migration and invasion were suppressed by siRNA-ZXF2 treatment (P<0.01). Further biochemical studies revealed that the knockdown of ZXF2 led to down regulation of c-Myc signaling.Conclusions High expression of ZXF was closely related to tumor progression through c-Myc related pathway, ZXF2 might be an important molecular target for lung adenocarcinoma treatment.