Expression And Clinical Significance Of LncRNA AFAP1-AS1in Non-small Cell Lung Cancer

Background and objectLung cancer is one of the most frequent cancers in the world. Around the world, theincidence of lung cancer significantly increased. Lung cancer is expected to account for26%of all female cancer deaths and28%of all male cancer deaths in2013. So far, lungcancer has become the leading cause of cancer-related death around the word. Theincidence of lung cancer in China has been increasing rapidly, in the past few decades,lung cancer mortality increased465%and the mortality rate is the leading one in urbanareas and the second in rural areas. According to histological type, non-small cell lungcancer (NSCLC) accounts for approximately75-80%of all lung carcinomas. Theprognosis of NSCLC remains poorly in spite of recent progresses of clinical andexperimental oncology. The5-year survival rate of NSCLC is only about11%. Themain cause of high mortality of lung cancer is that tumor cell invasion and metastasishas occurred when diagnosed, lacking of effective treatment. Therefore, in order toprovide effective treatment strategies for lung cancer patients and to improve thesurvival rate of lung cancer patients, we must actively seek effective early diagnosticand prognostic markers for lung cancer.The genome sequencing projects revealed that the human genome is comprised ofless than2%protein coding genes and more than90%of the genome is transcribed as noncoding RNAs (ncRNA). These ncRNAs are classified into two groups depending onthe nucleotide size: short ncRNAs and long ncRNAs (lncRNAs). LncRNA is a RNAmolecule that is longer than200nucleotides and is not translated into a protein product.It is widely known that lncRNAs play an important role in cancer progression; they arerelated to many biological processes of various cancers, such as the process of invasionand metastasis of cancer. Although genome-wide transcriptome studies have identifieda lot of lncRNAs, only a few lncRNAs have been well characterized. For example, thefunction and molecular mechanisms of lncRNA MALAT-1and HOTAIR have beenwell charaterized. Meanwhile, clinical studies have found that these two lncRNAs maybe acted as the potential markers in cancer treatment because their expression levels arerelated to clinicopathological features and prognosis.NATs are a class of lncRNA molecules that are transcribed from the opposite DNAstrand of other RNA stranscripts with which they share sequence complementarity.Now, studies have found NATs play an important role in tumorigenesis and cancerprogression. AFAP-AS1, as a NAT of AFAP1, is recently discovered in Battett’sesophagus (BE) and esophageal adenocarcinoma (EAC). AFAP1-AS1was extremelyhypomethylated and overexpressed in BE and EAC. Subsequent experiments revealedthat AFAP1-AS1knockdown inhibited EAC cell proliferation, migration and invasion.The gastric tissue microarray of our lab revealed that AFAP1-AS1was overexpressedin gastric cancer. However, the study of AFAP1-AS1related to non-small cell lungcancer clinicapathological features has not been reported to date. Therefore, this studywas designed to detect the expression of AFAP1-AS in NSCLC tissues andcorresponding normal lung tissues by qRT-PCR. The correlation between AFAP1-AS1expression level and clinicopathological characteristics was analyzed to reveal thepotential role of AFAP1-AS1in NSCLC.Methods1. Patients and tissue samples: Paired NSCLC tissues and adjacent normal tissues(≥5cm away from tumor) were obtained from65patients who received surgicalresection of NSCLC between2012and2013at Department of Thoracic Surgery,General Hospital of Guangzhou Military Command of PLA. All clinicopathologicaldata were obtained from clinical and pathologic records. 2. Cell lines and cell culture:16HBE, A549, H1299, H446and95-D cells wereculruted in RPMI-1640medium, supplemented with10%fetal bovine serum100U/mlpenicillin, and100mg/ml streptomycin in humidified air at37℃with5%CO2.3. Total RNA extraction and qRT-PCR analysis: Total RNA was isolated withTRIzol reagent according to the manufacturer’s protocol. RNA was reverse transcribedinto cDNA. Then, the quantitative real-time polymerase chain reaction (qRT-PCR) wasperformed on ABI7500system.4. Statistial analyses: All statistical analysis was performed with SPSS16.0software. α=0.05as the level of inspection. Differences of AFAP1-AS1expressionbetween NSCLC tissues and corresponding normal tissues were compared by pairedt-test. The relationship between AFAP1-AS1expression, AFAP1expression andclinicopathological characteristics was tested by χ2analysis. The relationship betweenAFAP1-AS1expression and AFAP1expression was tested by Spearman test.Results1. AFAP1-AS1expression in lung cancer tissues and cellsIn a cohort of65NSCLC patients, we found that AFAP1-AS1expression levels incancer tissues were significantly higher than those in corresponding normal tissues (P＜0.05). The expression ofAFAP1-AS1was overexpressed in50.8%(33/65) NSCLCtissues compared with corresponding adjacent normal tissues. Meanwhile，AFAP1-AS1was overexpressed in H1299and H460cells.2. The relationship of AFAP1-AS1expression levels with clinicopathologicalcharacteristics in NSCLC tissueAFAP1-AS1expression level was specifically associated with lung adenocarcinomaof NSCLC (P＜0.05), but was not correlated with gender, age, smoking, tumor size,lymph node metastasis or TNM stage (P＞0.05).3. The relationship between AFAP1-AS1expression level and AFAP1expressionlevel in NSCLCAFAP1-AS1expression level and AFAP1expression level was significantlypositively correlated (r=0.548, P＜0.05). AFAP1expression level was specificallyassociated with lung adenocarcinoma of NSCLC (P＜0.05), but not correlated withgender, age, smoking, tumor size, lymph node metastasis or TNM stage (P＞0.05). Conclusion1. AFAP1-AS1may be associatied with the occurrence and development of lungadenocarcinoma. It maybe act as an oncogene in the process of lung adenocarcinoma.2. AFAP1-AS1expression level and AFAP1expression level was significantlypositively correlated in NSCLC.3. Both AFAP1-AS1expression level and AFAP1expression level are associatedwith lung adenocarcinoma, this result suggests a potential interrelated role ofAFAP1-AS1and AFAP1in lung adenocarcinoma.