ABSTRACT
BACKGROUND: Increasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins. METHODS: The GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms. RESULTS: PDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription. CONCLUSIONS: Our research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.
Subject(s)
Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Neoplastic Stem Cells , Octamer Transcription Factor-3 , RNA, Long Noncoding , Humans , Esophageal Neoplasms/genetics , Esophageal Squamous Cell Carcinoma/genetics , RNA , Ubiquitin-Protein Ligases , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Neoplastic Stem Cells/metabolism , Receptors, Nerve Growth FactorABSTRACT
BACKGROUND: This paper examines the expression, function, and molecular mechanism of long non-coding ribonucleic acid (lncRNA) ARAP1 antisense RNA 1 (ARAP1-AS1) in lung cancer. Specifically, it aims to clarify the molecular mechanism of lncRNA ARAP1-AS1 that affects the occurrence and development of lung cancer, and provide a theoretical basis and molecular targets for targeted therapy or early diagnosis of lung cancer. METHODS: Fluorescence quantitative detection of lncRNA ARAP1-AS1 expression in lung cancer tissues and cell lines, and methylthiazolyldiphenyl-tetrazolium (MTT), plate cloning experiment, and flow cytometry were used to detect the effect of knockdown of lncRNA ARAP1-AS1 on cell proliferation, clone formation, and the cell cycle, respectively. Western blotting was used to detect the expression of cell cycle-related proteins as well as the effect of knockdown of lncRNA ARAP1-AS1 on lung cancer. Cell proliferation was assessed by a nude mouse subcutaneous tumor formation experiment. RESULTS: LncRNA ARAP1-AS1 is highly expressed in lung cancer tissues and cells. Knockdown of LncRNA ARAP1-AS1 can significantly inhibit the proliferation and clonal formation of lung cancer cells and induce G0/G1 cell cycle arrest. Knockdown of ARAP1-AS1 can markedly inhibit the expression of cell cycle-related protein cyclin D1, but has no significant effect on the expression of cyclin-dependent kinase (CDK)4 and CDK6. Furthermore, knockdown of ARAP1-AS1 can also notably inhibit the growth of lung cancer cells and substantially reduce the expression of Ki-67 in tumor-bearing tissues in nude mice. CONCLUSIONS: LncRNA ARAP1-AS1 is highly expressed in lung cancer. Knocking down of this gene can significantly inhibit cell proliferation in vitro and in vivo, and can also cause G0/G1 cell cycle arrest by inhibiting the expression of cyclin D1.