Jianyong Sun1,2, Yanlu Xiong2, Kuo Jiang3, Bo Xin4, Tongtong Jiang1, Renji Wei1, Yuankang Zou5, Hong Tan1, Tao Jiang2, Angang Yang6, Lintao Jia7, Lei Wang8. 1. State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China. 2. Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, Shaanxi, China. 3. Department of Spine Surgery, Honghui Hospital, Xi'an Jiaotong University, Xi'an, 710054, Shaanxi, China. 4. Department of Oncology, The 960th Hospital of PLA, Tai'an, 271000, Shandong, China. 5. The Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, Department of Occupational and Environmental Health, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China. 6. Department of Immunology, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China. 7. State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China. Jialth@fmmu.edu.cn. 8. State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China. rnawangl@yeah.net.
Abstract
BACKGROUND: Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) are involved in the hypoxia-related cancer process and play pivotal roles in enabling malignant cells to survive under hypoxic stress. However, the molecular crosstalk between lncRNAs and hypoxia signaling cascades in non-small cell lung cancer (NSCLC) remains largely elusive. METHODS: Firstly, we identified differentially expressed lncRNA cancer susceptibility candidate 15 (CASC15) as associated with NSCLC based on bioinformatic data. The clinical significance of CASC15 in lung cancer was investigated by Kaplan-Meier survival analysis. Then, we modulated CASC15 expression in NSCLC cell lines by RNAi. CCK-8 and transwell assays were carried out to examine the effects of CASC15 on proliferation and migration of NSCLC cells. Upstream activator and downstream targets of CASC15 were validated by luciferase reporter assay, qRT-PCR, Western blotting, and chromatin immunoprecipitation (ChIP). Lastly, RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) were performed to confirm the genetic relationships between CASC15 and related genes in clinical samples. RESULTS: CASC15 was highly expressed in NSCLC tissues and closely associated with poor prognosis. Loss-of-function analysis demonstrated that CASC15 was essential for NSCLC cell migration and growth. Mechanistic study revealed that CASC15 was transcriptionally activated by hypoxia signaling in NSCLC cells. Further analysis showed that hypoxia-induced CASC15 transactivation was mainly dependent on hypoxia-inducible factor 1α (HIF-1α) and hypoxia response elements (HREs) located in CASC15 promoter. CASC15 promotes the expression of its chromosomally nearby gene, SOX4. Then SOX4 functions to stabilize β-catenin protein, thereby enhancing the proliferation and migration of NSCLC cells. HIF-1α/CASC15/SOX4/β-catenin pathway was activated in a substantial subset of NSCLC patients. CONCLUSIONS: HIF-1α/CASC15/SOX4/β-catenin axis plays an essential role in the development and progression of NSCLC. The present work provides new evidence that lncRNA CASC15 holds great promise to be used as novel biomarkers for NSCLC. Blocking the HIF-1α/CASC15/SOX4/β-catenin axis can serve as a potential therapeutic strategy for treating NSCLC.
BACKGROUND: Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) are involved in the hypoxia-related cancer process and play pivotal roles in enabling malignant cells to survive under hypoxic stress. However, the molecular crosstalk between lncRNAs and hypoxia signaling cascades in non-small cell lung cancer (NSCLC) remains largely elusive. METHODS: Firstly, we identified differentially expressed lncRNA cancer susceptibility candidate 15 (CASC15) as associated with NSCLC based on bioinformatic data. The clinical significance of CASC15 in lung cancer was investigated by Kaplan-Meier survival analysis. Then, we modulated CASC15expression in NSCLC cell lines by RNAi. CCK-8 and transwell assays were carried out to examine the effects of CASC15 on proliferation and migration of NSCLC cells. Upstream activator and downstream targets of CASC15 were validated by luciferase reporter assay, qRT-PCR, Western blotting, and chromatin immunoprecipitation (ChIP). Lastly, RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) were performed to confirm the genetic relationships between CASC15 and related genes in clinical samples. RESULTS:CASC15 was highly expressed in NSCLC tissues and closely associated with poor prognosis. Loss-of-function analysis demonstrated that CASC15 was essential for NSCLC cell migration and growth. Mechanistic study revealed that CASC15 was transcriptionally activated by hypoxia signaling in NSCLC cells. Further analysis showed that hypoxia-induced CASC15 transactivation was mainly dependent on hypoxia-inducible factor 1α (HIF-1α) and hypoxia response elements (HREs) located in CASC15 promoter. CASC15 promotes the expression of its chromosomally nearby gene, SOX4. Then SOX4 functions to stabilize β-catenin protein, thereby enhancing the proliferation and migration of NSCLC cells. HIF-1α/CASC15/SOX4/β-catenin pathway was activated in a substantial subset of NSCLCpatients. CONCLUSIONS: HIF-1α/CASC15/SOX4/β-catenin axis plays an essential role in the development and progression of NSCLC. The present work provides new evidence that lncRNA CASC15 holds great promise to be used as novel biomarkers for NSCLC. Blocking the HIF-1α/CASC15/SOX4/β-catenin axis can serve as a potential therapeutic strategy for treating NSCLC.
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