Expression And Functional Role Of HIF-2α Promoter Upstream Long Non-coding RNA (HIF2PUT) In Colorectal Cancer Stem Cells

【Objective】We undertook this study in an attempt to explore the expression of a HIF-2α promoterupstream long non-coding RNA (lncRNA-HIF2PUT) in colorectal cancer (CRC) andpericarcinous tissues. In addition, the regulatory association of lncRNA-HIF2PUT withHIF-2α, the biological function of lncRNA-HIF2PUT in CRC stem cell lines (CSCs)and the possible molecular mechanism involved were investigated.【Methods】The expression levels of lncRNA-HIF2PUT and HIF-2α in CRC and pericarcinoustissues were determined by using the real-time PCR assay. We designed siRNAinterference sequences for lncRNA-HIF2PUT synthesis and transfected them intoDLD-1. The lncRNA-HIF2PUT and HIF-2α expression was evaluated by the real-timePCR. Effects of downregulation of lncRNA-HIF2PUT on CRCs after transfection at48h were detected using a series of in vitro experiments: the expression levels of stemcell markers OCT4, SOX2, NANOG, KLF4, CD44, and CD133were determined by useof real-time PCR and Western-blot method; DLD-1cell proliferation was inspectedusing MTS and clone formation assay; the suspension culture stem cell sphere-formingmethod was utilized to test the sphere-forming ability of DLD-1cells afterlncRNA-HIF2PUT downregulation; the migration of the DLD-1cells was examined byscratch test. We also tested the expression status of HIF-2α in case of dowm-regulatedlncRNA-HIF2PUT. 【Results】The expression levels of lncRNA-HIF2PUT and HIF-2α in CRC tissues were notsignificantly different compared to the adjacent tissues. In cancerous tissues, there was apositive association between lncRNA-HIF2PUT expression and HIF-2α levels. Wemanaged to transfect the siRN sequences into DLD-1cells and found a significantreduction in OCT4, SOX2, and CD44expression when lncRNA-HIF2PUT wasdownregulated. The MTS method revealed a notable reduction in proliferative ability ofDLD-1cells. The same trend was seen in the number of stem cell spherical clone andmigration ability. We also saw a significantly decreased HIF-2α expression in DLD-1cells while downregulating lncRNA-HIF2PUT.【Conclusions】These data suggest lncRNA-HIF2PUT may act as a regulator of HIF-2α levels and anovel cancerous promoter of CRC, and that HIF-2α inhibition by suppressinglncRNA-HIF2PUT could be used as a strategy to inhibit cellular proliferation, migrationand self-renewal ability. Further research is required to confirm or refute the currentfindings and to clarify whether lncRNA-HIF2PUT is a new biomarker and therapeutictarget of CRC.