The Characteristics And Roles Of Long Non-coding RNA AFAP1-AS1 On Gastric Cancer Cells Proliferation, Migration And Invasion

Gastric cancer (GC) is the second and fourth commonly diagnosed cancer among men and women, respectively, and is the third leading causes of cancer death in China. An estimated 680,000 new stomach cancer cases and 500, 00 deaths occurred in 2015 based on Cancer Statistics in china. Although the incidence and mortality for GC have a significantly decrease, a large and rising number of new cases occurred because of population growth and aging. Tumor cell metastasis is the major cause of GC death and markedly decreases the therapeutic effect. An increasing number of oncogenes and tumor suppressors were observed to be involved in the progress of GC, but the mechanism underlying GC metastasis still remains largely unclear.Recent advances in sequencing technologies enabling more indepth genomic and transcriptomic analyses have revealed that as much as 85% of the human genome is transcribed. This was surprising, as studies of mammalian genomes have shown that a drastically low population of RNA transcripts code for protein products. The generation of such a large population of non-coding RNA (ncRNA) transcripts indicates that RNAs have a larger and more diverse role in biological processes than initially anticipated. NcRNAs can be roughly classified into two groups based on their size. One group includes short RNAs less than 200 nucleotides (nt) in length, the other group includes long ncNAs(lncRNAs) of around 200 nt or more. While miRNAs have been heavily studied and are well understood for their function in gene regulation, lncRNAs in contrast are less understood.The aberrant expressions of lncRNAs are believed to be associated with the aggressive development of cancer. LncRNAs beyond doubt participate in cancer pathways. Although increasing number of IncRNAs has been reported to functionally regulate the metastatic behavior of GC cells, a large number of IncRNAs need to be identified, because 70%-90%of the human genome transcribes RNA products but protein-coding genes account for only 2%. Herein, we focus on a lncRNA, AFAP1-AS1, which was first reported in esophageal cancer and acted as an oncogene involved in cell proliferation, migration and invasion.AFAP1-AS1 was also found to have crucial role in various cancers such as colorectal cancer, breast cancer, hepatocellular cancer, lung cancer, etc. However, the expression and function of AFAP1-AS1 in GC development remains largely unknown.Metastasis is a key process in cancer progression, wherein cells in a primary tumor acquire invasive properties and disseminate to other sites in the body to initiate secondary lesions. This complex process involves most of the trans-differentiation steps unique to embryonic development. While aberrant proliferation is a necessity for tumor initiation, a second hit which changes the migratory properties and cell-cell contacts is crucial for the spread of tumor cells in the body. Epithelial-mesenchymal transition (EMT) plays essential roles in carcinogenesis and tumor metastasis. In EMT process, the determinant step is a local invasion through the epithelial basement membrane and modifies cell-cell and cell-matrix interactions. Previous study showed that IncRNA also regulates the EMT progress in GC, and AFAP1-AS1 was showed to participate in the EMT progress and facilitate tumor metastasis in colorectal cancer. However, it is unclear whether AFAP1-AS1 associated with the EMT progress in GC is.In the present study, we found that the expression of AFAP1 -AS 1 was increased in GC Tissues and associated with patients’ poor survival. The down-regulation of AFAP1-AS1 Inhibited the proliferation, migration and invasion of GC cells in vitro, and altered the expression of EMT-related genes.Objectives:1. Screen and identify the gastric cancer associated long non-coding RNAs and study the characteristics and roles.3. Investigate the roles of AFAP 1-AS 1 in the epigenetic regulation of gastric cancer cells proliferation, invasion and metastasis.Methods:Screen and identify the gastric cancer associated long non-coding RNAs by high-throughput tiling microarrays. The expression of AFAP1-AS 1 in GC tissues and cell lines was measured via quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR). The knock-down of AFAP1-AS1 was performed using a lentivirus vector. The proliferation of GC cells was measured by using CCK8 reagent. The migration and invasion of GC cells were analyzed via trans-well assays, respectively. The protein levels of EMT-related genes (ZEB1, ZEB2, E-cadherin, N-cadherin and Vimentin) were detected by using western blot assays. The cut-off value of the expression of AFAP1-AS1 was evaluated by using receiver operating characteristics curve (ROC), and Kaplan-Meier was used to analyze the patients’ survival.Results:The expression of AFAP1-AS 1 was significantly increased in the primary tumor tissues of GC patients with lymph node metastasis or tumor node metastasis (TNM) stage(III or IV) (P < 0.01). ROC curve analysis revealed that the expression of AFAP-AS1 at the cut-off value of 0.5040 could distinguished the GC tissues from the matched normal tissues,with the AUC of 0.8802, the sensitivity of 81.25% and the specificity of 83.75%. The overexpression of AFAP1-AS1 was positively correlated with the poor survival rates of GC patients. Furthermore, the down-regulation of AFAP1-AS1 significantly inhibited the proliferation, migration and invasion of GC cells in vitro (P < 0.01). Down-regulation of AFAP1-AS1 inhibited the mRNA and protein levels of ZEB1 and ZEB2 genes.The decrease of AFAP1-AS1 significantly suppressed the protein level of N-cadherin and increased that of E-cadherin in GC cells. the expressions of lncRNA AFAP1-AS1 correlated with ZEB1 mRNA in gastric tumor tissues ( R2=0.4388; P<0.0001 )Bioinformatics prediction and luciferase activation assays demonstrated AFAP1-AS1 could bind miR-200a and act as miRNAs sponges.Conclusions:We identified that AFAP1-AS1 is a potent GC associated oncogene lncRNA. Our study demonstrated that the expression signature of AFAP1-AS1 may serve as a biomarker for the diagnosis and prognosis of GC, and its down-regulation can repress the aggressive progression of GC partially trough inhibiting the EMT progress. AFAP1-AS1 promotes GC proliferation, invasion and metastasis by binding miR-200a, and then inducing GC EMT.This is the first study to demonstrate that the AFAP1-AS1/miR-200a/ZEB1 axis regulates the proliferation, migration and invasion of GC cells. Potentially, the findings of this study implicate the relevance of AFAP1-AS1 as a possible therapeutic target for aggressive and metastatic gastric cancers.