Objective ·To investigate the biological roles and associated mechanisms of the hypoxia-induced long non-coding RNA 68 (HILRNA68) in hepatocellular carcinoma (HCC) cell lines. Methods ·Long non-coding RNA (lncRNA) microarray analysis was conducted to study the differential expression of lncRNAs in the HCC cell lines cultured under hypoxia treatment and normoxia treatment separately for 12 h, and DEseq2 R package was used for the analysis of differentially expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the differential lncRNAs. Short hairpin RNAs (shRNAs) were used to knock down hypoxia-inducible factors (HIFs) to investigate whether HILRNA68 transcription was regulated by HIFs under hypoxia. Nucleus-cytoplasmic isolation combined with qRT-PCR and RNA fluorescence in situ hybridization (RNA-FISH) experiments were used to investigate the subcellular localization of HILRNA68. HILRNA68 was knocked down in SMMC-7721 and MHCC-97H cells by small interfering RNA (siRNA) to investigate its cellular function under hypoxia. The impact of HILRNA68 on the cell proliferation and invasion capabilities of HCC cells under hypoxia was examined by cell counting and Transwell assays. Dual-luciferase reporter assay was employed to identify how HILRNA68 regulated the transcriptional activity of HIFs under hypoxia. Results ·By differential expression analysis of lncRNAs, a total of 247 and 17 significantly (defined as fold change≥4, FDR≤0.05) up- and down-regulated lncRNAs, respectively, were identified. Among these differentially expressed genes, lncRNA HILRNA68 was up-regulated about 10-fold in multiple HCC cell lines when cultured under hypoxia for 12 h. Knockdown of HIF1α, HIF2α, and HIF1β significantly suppressed (all P<0.05) the upregulation of HILRNA68 under hypoxia. Luciferase reporter assay suggested that the transcription of HILRNA68 was regulated by HIFs. Subcellular localization studies revealed that HILRNA68 was mainly localized in the nucleus. Biological function experiments showed that silencing of HILRNA68 significantly inhibited the proliferation and invasion of HCC cells under hypoxia (all P<0.05). Mechanistic studies demonstrated that knock-down of HILRNA68 significantly suppressed the transcriptional activity of HIF1α under hypoxia (P<0.05) and the up-regulation of these canonical HIFs targets under hypoxia was also significantly inhibited after HILRNA68 knockdown (P<0.05). Conclusion ·The current study identifies a series of differential hypoxia-regulated lncRNAs and functionally annotates the upregulated HILRNA68. HILRNA68 is directly up-regulated by HIFs which promotes cell proliferation and invasion under hypoxia. Mechanistically, the upregulation of HILRNA68 under hypoxia enhances the transcriptional activity of HIF1α.