Long Non-coding RNA GAS5 Plays Crucial Roles In Proliferation And Apoptosis In Non-small-cell Lung Cancer By Post-transcriptional Regulating The Expression Level Of P53 And E2F1

Background and ObjectiveLung cancer is one of the common human malignancies.Approximately two-thirds of patients are diagnosed at an advanced stage,because lung cancer is difficult to early detection and early diagnosis.In spite of recent advances in surgical,radiotherapy and anticancer drugs,lung cancer remains the leading cause of cancer-associated death both worldwide and in China.The crucial reason for high mortality is sustained proliferation and the tumor’s metastatic potential.Therefore,a better understanding of the molecular mechanisms underlying tumor cell proliferation and apoptosis is needed for more efficient NSCLC management.Lung cancer is classified into a variety of different histological subtypes and the main type is non-small-cell lung cancer(NSCLC),which accounts for 85%of lung cancer cases.In the past few decades,numerous molecular epidemiological studies have indicated that the mutations in certain protein-coding genes or increase in protein-coding genes copy number(EGFR,KRAS,FGFR1)play an important role in the pathogenesis of NSCLC.However,recent advances in both genome and transcriptome sequencing technology have revealed that another important type of genes,non-coding genes,may also be emerging as new players in the cancer paradigm.Long non-coding RNA is an RNA molecule that is longer than 200 nucleotides and lacks open reading frames,so that it is not translated into a protein.Accumulated data have begun to advance the idea that lncRNAs are implicated in a spectrum of cellular processes,for example,proliferation,apoptosis,migration and even carcinogenesis.It is important to notice that misregulated lncRNA expression can cause various types of human tumors.The growth arrest-specific transcript 5(GAS5),which comprises 12 exons,is encoded at 1q25 and is approximately 630 nt in length.It is originally found in growth arrest cells.The subsequent study found that GAS5 was able to compete with DNA GREs for binding to the DNA-binding domain of the glucocorticoid receptor(GR),leading to the suppression of the glucocorticoidmediated induction of several response genes.In addition,it was reported that GAS5 overexpression leads to a slower cell cycle and an increase in apoptosis.Importantly,GAS5 is down-regulated in various types of human tumors.Yet,the expression profile and biological role of GAS5 and its mechanism in NSCLC remains largely unknown.The aim of this study was firstly to investigate the correlation between GAS5 expression and NSCLC patients and to explore the effect and mechanism of GAS5 on proliferation and apoptosis in vivo and in vitro,finally to provide the theoretical basis for NSCLC carcinogenesis.Methods and Materials1.Paired NSCLC and adjacent normal lung tissue were obtained in surgery from 72 patients.RNA was extracted by Trizol and then reversed into cDNA.The expression level of GAS5 and GAPDH were detected in NSCLC tissues and corresponding normal lung tissue by quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR).The relationship between GAS5 expression level and clinicopathological characteristics was also evaluated by statistical methods.2.To understand the role of GAS5 in the development of NSCLC,we used qRT-PCR analysis to assess GAS5 expression levels in six different NSCLC cell lines,compared with the expression level of GAS5 in human bronchial epithelial cells(IHBE).Then,to investigate the potential mechanism leading to decreased GAS5 expression,we focused on the methylation of CpG islands and histone in the GAS5 promoter regions.We exposed H1650 and A549 cells to 5-aza-2-deoxy-cytidine(5-aza-CdR)at different concentrations(0,5μM,IOμM)and analyzed the GAS5 expression in these cells after 72 hours using qRT-PCR.Meanwhile,siRNA specifically targeting the suppressor of zeste-12 protein(SUZ12)and the enhancer of zeste protein-2(EZH2)was also constructed.After 48 hours transfection,qRT-PCR was used to detect the expression level of GAS5.3.In vitro,the full-length sequence of GAS5 was constructed into pcDNA3.1 vector plasmid to obtain the pcDNA3.1-GAS5 plasmid.SiRNA specifically targeting GAS5(siGAS5)was also constructed.According to the expression level of GAS5 in NSCLC cell lines,pcDNA3.1-GAS5 and pcDNA3.1 vector were transfected into H1650 and A549,while siRNA GAS5 and si-NC were transfected into SPC-A1.The expression of GAS5 was confirmed using qRT-PCR.4.Cell proliferation and colony-forming ability of GAS5 overexpressed H1650 cells and A549 cells and GAS5 knockdown SPC-A1 cells and respective control cells were detected by both MTT assay and colony formation assay.Cell cycle was detected via PI staining in each H1650 and A549 cells to explore the mechanism involved in cell proliferation.Both Flow-Cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)were used to detect the effect of GAS 5 on apoptosis.The impact of GAS5 expression level on cell migration and invasion in A549 cell lines were furthermore observed in transwell experiment without and with matrigel matrix respectively.The underlying molecular mechanisms and detailed signaling pathways for GAS5 were also explored by qRT-PCR and western blot analysis.5.Age(4 weeks)-and sex(male)-matched BALB/c mice were used for the tumor formation assay.A549 cells were transfected with pcDNA3.1-GAS5 or empty vector were cultured in six-well plates for 48 hours.Then,the cells were washed with PBS and resuspended at a concentration of 2 × 107 cells/ml.Each mouse was injected on the right axilla with 2 × 106 suspended cells.Tumor growth was measured by calipers every 3 days.The tumor volumes were calculated using the equation V = 0.5 × D × d2(V,volume;D,longitudinal diameter;d,latitudinal diameter).The tumors were removaed from all of the animals after 15 davs.and the subcutaneous weight of each tumor was examined and finally entered into the statistical analysis.Total RNA was extracted from tumor tissue using Trizol reagent and lncRNA GAS5 expression level was quantified by qRT-PCR.Meanwhile,the tumor tissues were also under HE staining and IHC staining which uses antibodies against Ki-67.Results1.Compared to the corresponding normal lung tissues,the expression level of lncRNA GAS5 in NSCLC tissues was down-regulated and was highly related to tumor size and TNM stage:The relative expression level of GAS5 in 65 NSCLC tissues was reduced compared to the normal tissue in total 72 NSCLC specimens.The mean GAS5 expression level in all pairs was 0.578±0.594.Furthermore,we evaluated the correlation of GAS 5 expression with clinicopathological parameters and the results indicated that larger tumors which represent a higher tumor burden had lower GAS5 expression,compared with smaller tumors(P=0.0065).What’s more,according to the result of qRT-PCR,NSCLC patients were further divided into two groups,GAS5 high expression group and low group.Statistical analysis was found that there were significant relationships between GAS5 expression and tumor size and TNM stage(P=0.014 and P=0.013).These analyses demonstrate that GAS5 may be a good diagnostic biomarker for early-stage NSCLC.2.DNA methylation might partially contribute to the down-regulation GAS5 expression:The expression levels of GAS5 in 6 NSCLC cell lines and human bronchial epithelial cell lines were assessed by qRT-PCR.We found that GAS5 expression was at a comparatively low level in 5 lung cancer cell lines,including A549,H1650,H1299,H1975,and SK-MES,compared to HBE cells,whereas GAS5 expression in SPC-A1 was slightly higher than in HBEs.To investigate the potential mechanism leading to decreased GAS5 expression,we focused on the methylation of CpG islands in the GAS5 promoter regions.QRT-PCR was used to analyze the GAS5 expression level and GAS5 expression was significantly increased after exposing H1650 and A549 cells to 5-aza-2-deoxy-cytidine(5-aza-CdR)at different concentrations.However,siRNA-mediated knockdown of SUZ12 or EZH2,two main components of PRC2,indicated that there was no statistical correlation between histone methylation and GAS5 expression.Therefore,DNA hypermethylation may partially contribute to the down-regulation GAS5 expression in NSCLC tissues and cell lines.3.GAS5 overexpression could post-transcriptional regulated P53 and E2F1,leading to cell growth arrest and apoptosis:According to the result of qRT-PCR,we selected A549 and H1650 cells as the overexpressed experimental model and SPC-Al cells as the loss of function experimental model.The result of MTT assay showed that compared to control cells,cell proliferation rates were decreased in GAS5 overexpressed cells(P<0.05).While,siRNA-mediated knockdown of GAS5 promoted tumor cell growth(P<0.05).The results of colony formation assay revealed that both the number and size of colonies were lower in GAS5 overexpressed cells(P<0.05),which indicated that GAS5 could attenuate the dependent and colony-forming ability of the population.In addition,cell cycles in A549 and H1650 cells demonstrated that GAS5 also decrease in the proportion of cells in the S phase and a clear increase in the proportion of cells in G1,leading to growth retardation(P<0.05).The result of flow cytometric analysis showed that GAS5 overexpression significantly induced apoptosis,especially early apoptosis(P<0.05).Consistently,microscopic analysis of TUNEL staining of H1650 and A549 cells also showed that the number of apoptotic cells was greater in the GAS5 overexpression cells than the controls(P<0.05).In sharp contrast,transwell analysis demonstrated that GAS5 overexpression had no effect on migration and invasion,compared to the control cells(P>0.05).To further investigate the underlying molecular mechanisms responsible for the growth arrest and apoptosis induced by GAS5 in NSCLC,we focused on associated signaling pathways.Compared with control cells,transfection of pcDNA3.1-GAS5 led to a significant increase in P53 and P21 protein expression and a decrease in E2F1 and cyclinDl protein expression(P<0.05).However,P53 and E2F1 mRNA levels remained unaltered in the GAS5 overexpression cells as confirmed by qRT-PCR(P>0.05).What’s more,qRT-PCR results revealed that GAS5 overexpression or GAS5 knockdown did not statistically affect the expression of the cellular inhibitor of apoptosis 2(cIAP2)and the serum/glucocorticoid-regulated kinase 1(SGK1)(P>0.05).4.GAS5 overexpression suppressed NSCLC tumor growth in vivo:2 weeks after injection,GAS5 overexpression treatment dramatically decreased tumor growth,which was demonstrated by significantly reduced tumor size(0.0323 cm3 vs 0.727 cm3,P=0.00048)and weight(0.085g vs 0.457g,P=0.00118),compared to the control treatment.The up-regulation of GAS5 in tumors(22.7-fold)after pCDNA-GAS5 transfection was confirmed by qRT-PCR analysis.In addition,the HE staining showed the typical characteristics of tumor cells,and the proliferation index Ki-67 determined by immunohistochemical staining significantly decreased in the GAS5-transfected tumors.Taken together,these results clearly showed that GAS5 inhibited NSCLC tumor growth ability.Conclusion1.GAS5 is an important factor to NSCLC development and could serve as a potential diagnostic marker for NSCLC:The expression level of GAS5 in NSCLC specimens detected by qRT-PCR demonstrated that the expression level of GAS5 correlated with tumors size and TNM stage.Reduced GAS5 expression could be an important factor to NSCLC development.Since the expression level of GAS5 was lower in more advanced tumors,GAS 5 may be a good diagnostic biomarker for early-stage NSCLC.2.It is demonstrated that GAS5 increased tumor cell growth arrest and induced apoptosis through post-transcriptional regulating tumor suppressor gene P53 and transcription factor E2F1 expression:GAS5 overexpression could effectively increased in the proportion of cells in G1,tumor cell growth arrest,attenuated the colony-forming ability and induced apoptosis through P53-drpendent and P53-independent pathways in vitro.This provides a new theoretical foundation for elucidating the molecular mechanism of NSCLC.3.The effect of GAS5 expression on inhibiting tumorigenicity in vivo:Compared to the control treatment,GAS5 overexpression could dramatically decreased tumor growth,reduced tumor tumor and the proliferation index Ki-67.It provides a novel therapeutic target in patients with NSCLC,with prospects of clinical translation and industrialization.