LINK-A promotes cell proliferation through the regulation of aerobic glycolysis in non-small-cell lung cancer

Abstract

Purpose: Non-small-cell lung cancer (NSCLC) is the one of the most common malignancies worldwide, and occurs at a higher frequency in male individuals. Little is known about the role of the long intergenic noncoding RNA for kinase activation (LINK-A) in NSCLC, so in the present study we assessed its potential role on cell proliferation in NSCLC.

Methods: Expression levels of LINK-A in NSCLC tissues and cell lines were detected by quantitative reverse-transcription polymerase chain reaction. LINK-A was knocked down and overexpressed separately in A549 cells and NCI-H1299 cells. The effect of LINK-A expression on cell proliferation was determined by MTT assay. The correlation between LINK-A and hexokinase II (HKII) expression was investigated by Western blot and HKII Activity Assay. Glucose consumption and lactate production assay were used to investigate the aerobic glycolysis in NSCLC cells. The effect of LINK-A in vivo was determined by xenograft assay.

Results: LINK-A expression levels were increased in NSCLC tissues compared with normal tissues. Moreover, LINK-A expression was positively correlated with NSCLC clinicopathological characteristics and survival rate, while knockdown of LINK-A reduced NSCLC cell proliferation. LINK-A expression was also positively correlated with HKII, and NSCLC cells with low LINK-A expression were found to have significantly reduced HKII protein expression, accompanied by a reduction in enzyme activity levels. Both in vitro and in vivo experiments showed that LINK-A expression affected glucose consumption and lactate production through regulation of HKII expression.

Conclusion: These data suggest that the functions of LINK-A in NSCLC might play a key role in tumor progression and that LINK-A could be a promising predictive biomarker and potential therapeutic target for NSCLC.

Keywords: LINK-A; aerobic glycolysis; hexokinase II; non-small-cell lung cancer; prognosis; proliferation.

Figure 1

LINK-A is overexpressed in NSCLC and correlates with patient outcome. Notes: (A) qRT-PCR evaluation of LINK-A expression in NSCLC samples compared with adjacent normal tissues. (B) Expression levels of LINK-A expression in different NSCLC cell lines, including A549, H1703, SK-MES-1, and NCI-H1299 cells. (C) Overall survival Kaplan–Meier analysis of NSCLC patients. **P<0.01. Abbreviations: LINK-A, long intergenic noncoding RNA for kinase activation; NSCLC, non-small-cell lung cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction.

Figure 2

Overexpression of LINK-A promotes NSCLC cell proliferation. Notes: qRT-PCR evaluation of LINK-A expression in LINK-A-knockdown A549 (A) and LINK-A-overexpressing NCI-H1299 cells (B). Cell proliferation assay was performed in LINK-A-knockdown A549 (C) and LINK-A-overexpressing NCI-H1299 cells (D). **P<0.01; ****P<0.001. Abbreviations: LINK-A, long intergenic noncoding RNA for kinase activation; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; CTRL, control.

Figure 3

Expression of LINK-A affects the aerobic glycolysis of NSCLC cells. Notes: (A) Analysis of the expression correlation between LINK-A and HK II in the GEO profile database (GDS1479). (B) Expression of HK II in LINK-A-knockdown A549 and LINK-A-overexpressing NCI-H1299 cells. (C) HKII enzyme activity changes were determined in LINK-A-knockdown A549 and LINK-A-overexpressing NCI-H1299 cells. (D) Lactate production and glucose consumption were measured in LINK-A-knockdown A549 and LINK-A-overexpressing NCI-H1299 cell media. (E) Test of HK II enzyme activity in LINK-A-overexpressed-NCI-H1299 cells after 3-BrPA and HK II specific siRNA treatment. (F) Treatment with 3-BrPA and siRNA inhibit NSCLC cancer cell proliferation in cells overexpressing LINK-A. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Abbreviations: LINK-A, long intergenic noncoding RNA for kinase activation; NSCLC, non-small-cell lung cancer; HK II, hexokinase II.

Figure 4

Expression of LINK-A affects NSCLC tumorigenicity in vivo. Notes: (A) In vivo tumor growth curve of LINK-A-knockdown and control xenografts over 4 weeks. (B) Photograph and average tumor weight of control and LINK-A-knockdown xenografts at the endpoint. (C) HKII protein levels and enzyme activity in LINK-A-knockdown xenografts. (D) Glucose consumption and lactate production in LINK-A-knockdown xenografts. (E) IHC detected Ki-67 and HK II expression in xenograft groups. (F) Evaluation of Ki-67 and HK II protein level by IHC data. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: LINK-A, long intergenic noncoding RNA for kinase activation; NSCLC, non-small cell lung cancer; HK II, hexokinase II; IHC, Immunohistochemistry; CTRL, control.