Long noncoding RNA CASC7 is a novel regulator of glycolysis in oesophageal cancer via a miR-143-3p-mediated HK2 signalling pathway

Figures

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  Fig. 1. lncRNA CASC7 is upregulated and correlated with poor prognosis in oesophageal cancer. A Expression of lncRNA CASC7 in a series of tumours and their adjacent tissues from GEPIA. B Expression of lncRNA CASC7 in oesophageal cancer tissues and normal tissues from TCGA analysis. C Survival analysis of high-expression and low-expression lncRNA CASC7 in oesophageal cancer from the database. D qRT–PCR assay for mRNA expression of lncRNA CASC7 in tumour and normal tissues from surgery patients. (n = 10) (E). RNA-FISH of lncRNA CASC7 in tumour and normal tissues from surgery patients. Scale bar: 20 μm. **P < 0.01.

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  Fig. 2. Knockdown of lncRNA CASC7 inhibits proliferation of oesophageal cancer. A Relative mRNA expression of lncRNA CASC7 in different oesophageal cancer cell lines (n = 3). B qRT–PCR assay for lncRNA CASC7 expression in TE2 and TE7 cells after transfection with shCASC7 (n = 3). C RNA-FISH assay for lncRNA CASC7 expression in TE2 and TE7 cells following treatment with shCASC7. Scale bar: 25 μm. D, E A cell colony formation assay was performed to analyse the proliferation of TE2 and TE7 cells after knockdown of CASC7. F CCK8 was performed to detect the viability of TE2 and TE7 cells after knockdown of CASC7. G. A cell cycle assay was performed to determine the effect on the cell cycle following treatment with shCASC7 (n = 3). **P < 0.01, *P < 0.05.

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  Fig. 3. Silencing of lncRNA CASC7 attenuates tumour glycolysis in oesophageal cancer. A Relative abundance of glucose secretion levels in the medium of shNC- and shCASC7-transfected TE2 cells (n = 3). B Relative abundance of glucose secretion levels in the medium of shNC- and shCASC7-transfected TE7 cells (n = 3). C Lactate concentration level in the medium of shNC- and shCASC7-transfected TE2 cells. D Lactate concentration level in the medium of shNC- and shCASC7-transfected TE7 cells (n = 3). E Extracellular acidification rate (ECAR) analysis presented the glycolytic capacity of shNC- and shCASC7-transfected TE2 and TE7 cells. F Extracellular acidification rate (ECAR) analysis presented the glycolytic capacity of shNC- and shCASC7-transfected TE7 cells (n = 3). ***P < 0.001, **P < 0.01.

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  Fig. 4. lncRNA CASC7 affects tumour glycolysis and proliferation by regulating HK2 expression. A Correlative expression of lncRNA and HK2 in the oesophageal cancer database (cohort 1). B qRT–PCR analysis of the relative HK2 mRNA expression in shNC- and shCASC7-transfected TE2 and TE7 cells. C Western blotting for HK2 expression in shNC- and shCASC7-transfected TE2 and TE7 cells. D Relative abundance of glucose secretion levels in the medium of shNC-transfected, shCASC7-transfected and shCASC7 + HK2-transfected TE2 and TE7 cells (n = 3) E Lactate concentration level in the medium of shNC-transfected, shCASC7-transfected and shCASC7 + HK2-transfected TE2 and TE7 cells (n = 3). F Extracellular acidification rate (ECAR) analysis revealed the glycolytic capacity of shNC-transfected, shCASC7-transfected and shCASC7 + HK2-transfected TE2 and TE7 cells. G CCK8 assay of shNC-transfected, shCASC7-transfected and shCASC7 + HK2-transfected TE2 and TE7 cells. (n = 3) **P < 0.01, *P < 0.05, NS, not significant.

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  Fig. 5. lncRNA CASC7 regulates HK2 expression by competitively binding to miR-143-3p. A miR-143-3p has a binding site in CASC7 and HK2 based on analysis of the Diana-lncbase V2 and StarBase databases, and MUT-HK2 and MUT-CASC7 with alterations in the base sequence on the binding sites were designed for further experiments. B RNA-FISH assay of lncRNA CASC7 and miR-143-3p colocalization in TE2 cells. Scale bar: 20 μm. C A dual-luciferase reporter gene assay was conducted to verify the targeted relationship between miR-143-3p and lncRNA CASC7, as well as miR-143-3p and HK2. D qRT–PCR analysis of the relative miR-143-3p mRNA expression in shNC- and shCASC7-transfected TE2 and TE7 cells (n = 3). E qRT–PCR analysis of the relative HK2 mRNA expression in shNC-transfected, shCASC7-transfected and shCASC7+miR-143-3p inhibitor-transfected TE2 and TE7 cells (n = 3). F qRT–PCR analysis of relative HK2 mRNA expression in NC mimic- and miR-143-3p mimic-transfected TE2 and TE7 cells (n = 3). G Western blotting for HK2 expression in shNC-transfected, shCASC7-transfected and shCASC7+miR-143-3p inhibitor-transfected TE2 and TE7 cells (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05, NS, not significant.

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  Fig. 6. Clinical significance of lncRNA CASC7 and HK2 in oesophageal cancer. A Correlative mRNA expression of lncRNA CASC7, HK2 and miR-143-3p in the oesophageal cancer database (cohort 2). B Immunohistochemical analysis of the expression of HK2 in low CASC7 expression tumour tissue and high CASC7 expression tumour tissue. Scale bar: 100 μm. C Correlative mRNA expression between lncRNA CASC7 and HK2 in tumour tissues (cohort 2) (n = 87). D, E Five-year overall survival assay for different levels of CASC7 and HK2 expression in the oesophageal cancer database (cohort 2) (n = 87) ***P < 0.001, *P < 0.05.