The long noncoding RNA HOXA11-AS promotes lung adenocarcinoma proliferation and glycolysis via the microRNA-148b-3p/PKM2 axis

Abstract

Background: Lung cancer is the most common malignancy in the world and a growing number of researches have focused on its metabolic characteristics. Studies have shown that the long non-coding RNA (lncRNA) HOXA11-AS is aberrantly expressed in many tumors. However, the role of HOXA11-AS in lung adenocarcinoma (LUAD) glycolysis and other energy metabolism pathways has not been characterized.

Method: The mRNA levels of HOXA11-AS, microRNA-148b-3p (miR-148b-3p), and pyruvate kinase M2 (PKM2) were detected using qRT-PCR. The expression levels of proteins were measured using immunohistochemistry and western blot. The CCK-8, EdU, and colony formation assays were used to assess proliferation. Glycolytic changes were assessed by measuring lactate production, ATP production, and ¹⁸ F-FDG uptake. Bioinformatics analysis and dual-luciferase reporter assays were used to characterize the relationship between HOXA11-AS, miR-148b-3p, and PKM2. Proliferation and glycolytic changes were analyzed in xenograft tumor experiments using Micro-PET imaging after downregulation of HOXA11-AS in vivo.

Results: The expression of HOXA11-AS was markedly increased in LUAD, and was strongly associated with a poor prognosis. In addition, HOXA11-AS promoted proliferation and glycolysis in LUAD, and miR-148b-3p inhibited proliferation and glycolysis in LUAD. Mechanistically, HOXA11-AS positively regulated PKM2 expression by binding to miR-148b-3p, thereby promoting LUAD proliferation and glycolysis. In addition, HOXA11-AS inhibited LUAD xenograft growth and glycolysis via upregulation of miR-148b-3p expression and downregulation of PKM2 expression in vivo.

Conclusions: These results showed that HOXA11-AS enhanced LUAD proliferation and glycolysis via the miR-148b-3p/PKM2 axis. The findings in this paper expanded our understanding of the molecular mechanisms of LUAD tumorigenesis and glycolysis and showed that HOXA11-AS could be useful as a diagnostic and prognostic marker for LUAD. ¹⁸ F-FDG PET/CT can be used to visually evaluate the therapeutic effect of targeting HOXA11-AS.

Keywords: HOXA11-AS; aerobic glycolysis; lung adenocarcinoma; microRNA-148b-3p; pyruvate kinase M2.

FIGURE 1

HOXA11‐AS was overexpressed in LUAD and predicted poor prognosis. (A) Analysis of HOXA11‐AS expression in LUAD tissues and normal tissues in TCGA database. (B) Survival analysis using Kaplan–Meier Plotter. The high expression group survival time was lower than that of the low expression group. (C) HOXA11‐AS expression in A549, HCC827, H1299, and HBE cells (qRT‐PCR). (D) HOXA11‐AS up‐regulation efficiency. (E) HOXA11‐AS down‐regulation efficiency. *p < 0.05.

FIGURE 2

Overexpression or knockdown of HOXA11‐AS affected tumor proliferation and glycolysis in vitro. (A) EdU assay showed that HOXA11‐AS promoted proliferation (magnification 400×). (B and C) CCK‐8 assay. (D) Colony formation assay. (E) Lactate production assay indicated that HOXA11‐AS overexpression increased lactate production, and knockdown of HOXA11‐AS inhibited lactate production. (F) ATP production assay showed that ATP production increased after overexpression of HOXA11‐AS, and knockdown of HOXA11‐AS inhibited ATP production. (G) ¹⁸F‐FDG uptake assay showed that ¹⁸F‐FDG uptake increased after overexpression of HOXA11‐AS, and knockdown of HOXA11‐AS inhibited ¹⁸F‐FDG uptake. (H) Western blot showed that PKM2, GLUT1, HK2, and LDHA expression increased after overexpression of HOXA11‐AS, and knockdown of HOXA11‐AS inhibited PKM2, GLUT1, HK2, and LDHA expression. *p < 0.05.

FIGURE 3

HOXA11‐AS acted as a ceRNA for miR‐148b‐3p. (A) The binding sites between HOXA11‐AS and miR‐148b‐3p predicted by starBase. (B) Luciferase activity detected after co‐transfection with miR‐148b‐3p mimic or miR‐NC and HOXA11‐AS‐WT or HOXA11‐AS‐MUT. (C) Overexpression of HOXA11‐AS resulted in decreased expression of miR‐148b‐3p, and knockdown of HOXA11‐AS resulted in increased miR‐148b‐3p expression. *p < 0.05.

FIGURE 4

MiR‐148b‐3p inhibited proliferation and glycolysis in LUAD. (A) miR‐148b‐3p expression in A549, HCC827, H1299, and HBE cells (qRT‐PCR). (B) EdU assay showed that miR‐148b‐3p inhibited proliferation (magnification 400×). (C) CCK‐8 assay. (D) Colony formation assay. (E) Lactate production assay showed that miR‐148b‐3p overexpression reduced lactate production, and knockdown of miR‐148b‐3p promoted lactate production. (F) ATP production assay showed that ATP production was reduced after miR‐148b‐3p overexpression, and knockdown of miR‐148b‐3p promoted ATP production. (G) ¹⁸F‐FDG uptake assay showed that miR‐148b‐3p overexpression reduced ¹⁸F‐FDG uptake, and knockdown of miR‐148b‐3p promoted ¹⁸F‐FDG uptake. *p < 0.05.

FIGURE 5

MiR‐148b‐3p targeted PKM2, a key enzyme in glycolysis. (A) Predicted PKM and miR‐148b‐3p binding sites by starBase. (B) Luciferase activity detected after co‐transfection with miR‐148b‐3p mimic or miR‐NC and PKM‐WT or PKM‐MUT. (C) qRT‐PCR showed that miR‐148b‐3p overexpression resulted in decreased PKM2 expression, and knockdown of miR‐148b‐3p resulted in increased PKM2 expression. (D) Western blot showed that miR‐148b‐3p overexpression resulted in decreased PKM2 expression, and knockdown of miR‐148b‐3p resulted in increased PKM2 expression. *p < 0.05.

FIGURE 6

HOXA11‐AS regulated PKM2 through miR‐148b‐3p to promote proliferation and glycolysis in LUAD. (A) Down‐regulation of miR‐148b‐3p reversed PKM2 mRNA levels inhibited by si‐HOXA11‐AS. (B) Down‐regulation of miR‐148b‐3p reversed PKM2 protein levels inhibited by si‐HOXA11‐AS. (C) Down‐regulation of miR‐148b‐3p reversed the inhibitory effect on proliferation induced by si‐HOXA11‐AS (CCK‐8 assay). (D) EdU assay (magnification 400×). (E) Colony formation assay. (F) Down‐regulation of miR‐148b‐3p reversed si‐HOXA11‐AS‐induced inhibition of lactate production. (G) Down‐regulation of miR‐148b‐3p reversed si‐HOXA11‐AS‐induced inhibition of ATP production. (H) Down‐regulation of miR‐148b‐3p reversed si‐HOXA11‐AS‐induced inhibition of ¹⁸F‐FDG uptake. *p < 0.05.

FIGURE 7

Downregulation of HOXA11‐AS inhibited proliferation and glycolysis of LUAD xenografts in vivo via miR‐148b‐3p/PKM2. (A) Picture and weights of tumor xenografts. (B) Tumor xenograft volumes. (C, D) The levels of HOXA11‐AS decreased, the levels of miR‐148b‐3p increased, and PKM2 mRNA and protein levels decreased in the si‐HOXA11‐AS group. (E) ¹⁸F‐FDG micro‐PET imaging of the si‐HOXA11‐AS and si‐NC groups. White arrows indicate the tumor xenografts. (F) Tissue sections from xenograft experiments were tested with H&E staining and IHC staining to detect Ki67, PKM2, GLUT1, HK2, and LDHA expression (magnification 400×). *p < 0.05.