Upregulation Of Long Intergenic Noncoding RNA 00673 Promotes Tumor Proliferation Via LSD1 Interaction And Repression Of NCALD In Non-small-cell Lung Cancer

Background and ObjectiveLung cancer is a malignant disease of high morbidity and mortality owing to sustained proliferation and potential metastasis of cancer cells.The vast majority of patients inevitably progressed to advanced stages despite of recent great advance in diagnostic methods and treatment strategies.Lung cancer is divided into NSCLC(non-small cell lung cancer)and SCLC(small cell lung cancer).NSCLC become the focus of research since it accounts for 85%of lung cancer cases.With the advent of techniques such as microarrays and high-throughput sequencing,lncRNAs(long non-coding RNAs)have come to the attention of scientists and became the research hotspot.LncRNAs are defined as RNA molecules with length more than 200 nucleotides and little protein-coding potential because of lacking open reading frames.LncRNAs are involved in a spectrum of physiological and pathological processes,such as cell differentiation,embryo development,cell proliferation and apoptosis,cell metastasis.Previous researches have proved that dysregulated lncRNAs play an oncogenic or anti-tumor function in many human tumors.Linc00673 is encoded in 17q25.1 and approximately 2775nt in length.The analysis of microarray dataset(GSE18842)reveals that linc00673 is the remarkably up-regulated lncRNA in NSCLC tissues compared with matched normal lung tissues.There is a study which has also reported the same expression pattern of linc00673,however,no further studies about biological functions and molecular mechanisms are carried out.The objective of this study is to test the relationship between linc00673 expression and NSCLC clinicopathological features and to investigate the impact and mechanism of linc00673 on NSCLC cells proliferation and apoptosis in vitro and in vivo,eventually provide novel theoretical basis for pathogenesis and therapeutic targets of NSCLC.Methods and Materials1.Collect a total of 80 cases NSCLC tissues and matched normal lung tissues samples.Total RNA were extracted by Trizol and then reversed into cDNA.The expression levels of GAPDH and linc00673 in paired NSCLC tissues and normal lung tissues were tested through quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR).The correlation between linc00673 expression level and clinicopathological parameters was analysis through statistical methods.2.The expression level of linc00673 in eight NSCLC cell lines was tested by qRT-PCR,compare with that in human bronchial epithelial cells(HBE).The subcellular localization of linc00673 was detected by Nuclear/Cytosol Fractionation.The pcDNA3.1-linc00673 plasmid to specially upregulate linc00673 and siRNA that specifically downregulate linc00673(si-linc00673)were constructed.SiRNA-linc00673 and si-NC were transfected into A549,H1975 and SPC-A1,while pcDNA3.1 and pcDNA3.1-linc00673 were transfected into H1703.The expression level of linc00673 was tested using qRT-PCR.3.The effect of linc00673 on cancer cell proliferation and colony-forming ability was evaluated by MTT assay,EDU assay and colony formation assay.Flow-cytometric analysis was used to detected cell cycle and cell apoptosis.4.Tumor formation assay was performed using 4-week olds male BALB/c mice.Twelve mice were divided into two groups,respectively injected on the right axilla with 2 × 106 suspended A549 cells cultured with empty vector or shRNA-linc00673.Four days after injection,tumors were observed and measured every three days.The tumor volumes were calculated using the equation V = 0.5 × D × d2(V,volume;D,longitudinal diameter;d,latitudinal diameter).After 2 weeks,the tumors were removed from all animals and the growth and weight of each tumor were examined and statistically analyzed.Total RNA was extracted from tumor tissue using Trizol reagent and linc00673 expression level was tested by qRT-PCR.At the same time,the tumor tissues were under HE staining and IHC staining against Ki-67.5.To investigate the molecular mechanism of linc00673,RIP assay(RNA binding protein immunoprecipitation)and RNA pull down assay were used to detect proteins that bind with linc00673.To investigate the downstream molecules regulated by linc00673,RNA-seq was performed using A549 cells transfected with si-scrambel or siRNA-linc00673.Bioinformatics analysis and qRT-PCR were used to search the possible target gene.And then,CHIP assay was carried out to test whether the target RBP(RNA binding proteins)bind to the promoter region of target gene.6.The plasmid that upregulates target gene was constructed.MTT assay,EDU assay and colony formation assay were performed to test the change of cancer cells proliferation.Furthermore,rescue experiment was performed to determine whether target gene overexpression could partially counteract the oncogenic function of linc00673.7.The expression level of target gene was quantified by qRT-PCR and its correlation with linc00673 was also analyzed.In addition,the correlation between target gene and patients prognosis was analyzed by tissue microarray.Results1.Linc00673 expression was upregulated in NSCLC tissues compared with matched normal lung tissues and correlated with tumor size,lymph nodes metastasis and TNM stage:The expression level of linc00673 was quantified by qRT-PCR in 80 paired clinical NSCLC tissues and corresponding normal tissues and was upregulated in 62 NSCLC tissues relative to corresponding normal tissues.According to the linc00673 expression,the patients were divided into high linc00673 expression group and low linc00673 expression group.Results showed that larger tumors,with lymph node metastasis,or more advanced tumors,had higher linc00673 expression levels(P=0.015,P=0.047,P=0.033).Nevertheless,there was no significant relationship between linc00673 expression and other clinical characteristics,such as age,gender,differentiation and smoking history.Additionally,we produced a receiver operating characteristic curve(ROC curve),using corresponding normal tissues as control.The cutoff value for distinguishing NSCLC tissues from normal tissues was 7.75(△Ct).The sensitivity and specificity were 68.75 and 66.25,respectively,and the area under the ROC curve was 0.683(95%confidence interval:0.605 to 0.755,P<0.0001,Figure 1h).These data demonstrate that linc00673 might be an oncogene in the context of NSCLC progression and may potentially serve as diagnostic biomarker for NSCLC.2.The expression level of linc00673 in NSCLC cell lines and its subcellular location:Compared with human bronchial epithelial cells(HBEs),linc00673 expression was elevated to in 6 lung cancer cell lines(A549、SPC-A1、PC9、H1299、H520、H1975),whereas lower in H1 703 and H226.Furthermore,result showed that linc00673 was localized both in the nucleus and cytosol of A549,H1975,SPC-A1 and H1703,which implied that it may exert diverse regulatory functions.Three chemically synthesized siRNAs were used to knock down endogenous linc00673 in A549,H1975 and SPC-A1 cells,and pcDNA3.1-linc00673 was used to develope a linc00673 overexpression model in H1703.Total RNAs were extracted at 48h post-transfection and linc00673 expression levels were knocked down more than 80%in A549,H1975 and SPC-A1 and substantially 210-fold up-regulated via pcDNA3.1-linc00673 transfection relative to control cells.3.siRNA mediated Iinc00673 downregulation could repress NSCLC cells proliferation and promote G1 arrest:MTT assay and EDU assay both showed that linc00673 knockdown in A549,H1975 and SPC-A1 resulted in a significant decrease in cell proliferation rate compared with control cells,consistently,linc00673 overexpression in H1703 displayed a cell proliferation rate relative to negative control.In addition,the colony-formation assay showed that linc00673 knockdown also greatly attenuated the cell colony-forming ability while linc00673 overexpression enhanced cell colony-formation ability.These results together validated a positive role of linc00673 in promoting NSCLC cell proliferation.The results of flow cytometric analysis revealed that linc00673 knockdown bring in an increase in cells arrested in G0/G1 phase.On the contrary,linc00673 overexpression led to a decrease in cells arrested in G0/G1 and an accumulation of cells at S phase.Furthermore,we detected the expression levels of CDKs(cyclin-dependent kinases)after transfection.Compared to control cells,linc00673 knockdown significantly reduced CDK6(cyclin-dependent kinase 6)transcript and protein levels rather than CDK2 and CDK4.The results of flow cytometric analysis indicated that the proportion of apoptotic H1975 cells have not changed following linc00673 knockdown.Thus,linc00673-mediated acceleration of NSCLC cell proliferation appears to be mediated by affecting the G1-S checkpoint,rather than by apoptotic pathways.4.Linc00673 downregulation suppressed NSCLC tumor growth in vivo:Results showed that linc00673 downregulation repressed the volume and weight of tumors compared with control group.The expression levels of linc00673 in xenografts were validated by qRT-PCR.In addition,the HE staining and immunohistochemical staining showed the typical characteristics of tumor cells and the increased proliferation index Ki-67 in the control group.Taken together,these results clearly showed that linc00673 downregulation inhibited NSCLC tumor growth in vivo.5.Linc00673 silences NCALD transcription by binding with LSD1(Histone demethylase lysine specific demethylase 1):In order to analyze the linc00673-associated gene transcriptional changes,we applied RNA transcriptome sequencing to assess the gene expression profiles of linc00673-depleted A549 cells and control cells.This unbiased genome-scale analysis identified 988 differentially expressed transcripts(|log2(FoldChange)|>1 and P<0.05)in NSCLC cells after linc00673 knockdown compared to controls,including 499 downregulation transcripts and 489 upregulation transcripts.KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis determined that cell cycle progression was involved in the affected functional processes in linc00673-depeleted cells,which was consistent with our results.Using qRT-PCR,we confirmed representative genes which were identified as oncogenes or tumor suppressor genes in A549,H1975 and SPC-A1 and that NCALD was the most up-regulated in response to linc00673 downregulation.Western blot also determined the elevated protein level of NCALD in response to linc00673 downregulation.Therefore,NCALD might be the downstream gene involved in oncogenic role of linc00673.Previous studies found that IncRNAs silence target genes through binding with RBPs(RNA binding proteins).By RNA-Protein interaction prediction website(http://pridb.gdcb.iastate.edu/RPISeq/),we predicted that the probabilities interaction of linc00673 with LSD1,SUZ12 and EZH2 were 0.7,0.85,0.6 using RF classifier and 0.83,0.85,0.88 using SVM classifier.In addition,qRT-PCR and western blot analyses showed that LSD1 knockdown not EZH2 or SUZ12 knockdown induced NCALD mRNA and protein expression.Thus,we proposed a hypothesis that linc00673 might repress NCALD transcription through binding LSD1 to the NCALD promoter region.To validate this assumption,we first performed RIP analysis and results indicated that linc00673 could directly bind LSD1 in A549 and H1975 cells.RNA pull down assay further confirmed this binding of linc00673 and LSD1.Next,ChIP assay proved that LSD1 could directly bind the promoter region of the NCALD gene,and knockdown of linc00673 reduced LSD1-mediated H3K4me2 demethylation.6.NCALD overexpression could repress NSCLC cells proliferation and partially counteract the oncogenic role of linc00673:The results of MTT,EUD and colony-formation assays demonstrated that compared with control cells,NCALD overpression in A549,H1975 and SPC-A1 could repress cells proliferation.We further performed rescue experiments and results showed that NCALD overexpression could partially repress the pro-proliferation function of linc00673.7.NCALD expression was downregulated in cancer tissues and low NCALD level was associated with poor prognosis in NSCLC patients:Two microarray data sets from Garber and Bhattacharjee lung cancer cohorts downloaded from Oncomine displayed that the expression of NCALD mRNA was significantly lower in NSCLC tissues compared to normal tissues.We also detected the same expression tendency of NCALD mRNA in paired clinical NSCLC tissues and adjacent normal tissues,which was in agreement with the data from Oncomine.And the NCALD mRNA expression level was inversely correlated with linc00673 in these samples(Pearson’s correlation coefficient r2=0.3205,P= 0.0006).High NCALD protein expression was significantly associated with increased survival overall through tissue microarrays(TMAs)containing 90 primary operable NSCLC cases(OS;P=0.0449).Conclusion1.The dysregulation of linc00673 was involved in the progression of NSCLC and could act as a potential diagnostic marker of NSCLC:Linc00673 was upregulated in clinical NSCLC tissues relative to corresponding normal lung tissues and positively related with tumor size,lymph node metastasis and TNM stages,which implied that elevated linc00673 expression was an important factor of NSCLC development.In addition,ROC curve demonstrated that linc00673 could be a diagnostic marker of appropriate sensitivity and specificity.2.Linc00673 silences NCALD transcription by binding with Histone demethylase lysine specific demethylase 1(LSD1),leading to NSCLC cells proliferation:LSD1 was recruited by linc00673 to the promoter region of NCALD,and repressed NCALD transcription through H3K4me2 demethylation.Linc00673 acted as an oncogenic IncRNA that could promote cance rcells proliferation,which provided a novel molecular basis for NSCLC.3.The down-regulated expression of linc00673 inhibited tumor growth in vivo:The in vivo experiment showed that linc00673 downexpression significantly reduced the growth rate,weight and proliferation index ki-67 expression of tumors.It provides a potential therapeutic target for NSCLC patients,with prospects of clinical translation and industrialization.