ABSTRACT: Background:Long non-coding RNAs (lncRNAs) participate in a series of pathological processes in tumorigenesis. Reports show that C5orf66-AS1, an antisense lncRNA, is expressed in various tumors. However, the role of C5orf66-AS1 in gastric cancer (GC) has not been fully clarified. The study focused on the expression patterns and serum level of C5orf66-AS1 in GC to explore its potential application in GC screening and diagnosis. The effects of C5orf66-AS1 on GC cells were also analyzed. Methods:Tissue and serum samples were used for RNA isolation. Expression levels of C5orf66-AS1 in GC tissues, serum, and cell lines were detected using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). CCK-8, transwell, and wound healing assays were performed to determine the effects of C5orf66-AS1 on GC cell behavior. Results:C5orf66-AS1 expression was downregulated in GC cells compared to that in adjacent normal tissues. Serum C5orf66-AS1 levels were significantly lower in GC patients than in superficial gastritis (GS) and atrophic gastritis (GA) patients. Low serum expression of C5orf66-AS1 was associated with an increased risk of gastric dysplasia (GD) and GC. Receiver operating characteristic curve results showed that the area under curve (AUC) for GC was 0.688, with a sensitivity and specificity of 77.5% and 53.6%, respectively. For the GD + early gastric cancer (ECG) group, the AUC was 0.789, with a sensitivity and specificity of 85.15% and 62.86%, respectively. Correlation analyses of clinicopathological parameters showed that serum C5orf66-AS1 was predominantly associated with Lauren type, TNM stages, pTNM stages, and vessel tumor emboli. Additionally, in vitro overexpression of C5orf66-AS1 in AGS cells inhibited cell proliferation, migration, and invasion. Conclusion:Decreased expression levels of serum C5orf66-AS1 can be utilized for diagnosis of GC, especially for early diagnosis. The low level of serum C5orf66-AS1 indicated poor biological behavior of tumors in GC patients. In addition, C5orf66-AS1 can inhibit GC cell proliferation, migration, and invasion.