The human and mouse hepatocyte nuclear factor 1 homeobox A (HNF1A) gene locus expresses the HNF1A transcript and a long non-coding RNA in the antisense (HNF1A-AS1) direction. HNF1A-AS1 is expressed in numerous types of cancers. Poor clinical outcomes such as higher mortality rates, greater metastatic capacity, and poor prognosis of the disease are associated with this expression. It is also involved in metabolic disorders such as fatty liver disease. Since HNF1A-AS1 is mostly localized to the nucleus, it is thought to interfere with gene expression. We studied the expression level and localization of HNF1A-AS1 RNA, HNF1A RNA, and HNF1A protein in both cancer cells and pancreatic beta cells (insulin-secreting cells of the Islets of Langerhans). We also investigated the association of HNF1A-AS1 with polycomb repressive complex 2 (PRC2). A total of four human colon cancer cell lines, including HT-29, HTC116, RKO, SW480, and a normal colon cell line CCD841 along with a lung cancer cell line A549 were cultured. Additionally, we used Min6 cells, a clonal cell line of pancreatic beta cells. Western Blot was done on all samples to quantify HNF1A protein level. RT-qPCR was done to illustrate the expression level of HNF1A-AS1 and HNF1A genes in total cell lysates as well as subcellular compartments. We used triplex domain finder (TDF) for investigating the probability of triplex formation between HNF1A-AS and DNA as well as predicting DNA binding domains of HNF1A-AS along with possible target genes. We have done RNA immunoprecipitation (RIP) to investigate the association of enhancer of zeste homolog 2 (EZH2) as a member of PRC2 complex and HNF1A-AS1. Then anchorage-independent growth assay was done to investigate the effect of inhibiting EZH2 on colony formation in HT-29 and RKO cell lines. Subcellular localization of HNF1A-AS1 varied in different cell types. In adenocarcinomic human alveolar basal epithelial cells (A549), it was localized chiefly in the cytoplasm, while in colorectal adenocarcinoma cells (HT-29) as well as Min6 cells mainly were localized in the nucleus. HNF1A protein was mostly located in the nuclear compartment, while HNF1A RNA was commonly cytoplasmic. Using TDF, we found that HNF1A-AS1 had potential to form a RNA:DNA:DNA triplex with many genomic sites. We showed the association of HNF1A-AS1 with PRC2 complex and the effect of inhibiting EZH2 on colony formation. Possible function of HNF1A-AS1 might be through forming triplex with genomic sites and recruiting EZH2 to the site. This knowledge about the location and the mechanism will increase insight for planning subsequent experimental investigations and hopefully better preventive and therapeutic modalities and consequently improve clinical outcomes in the future.