MiR-1286 inhibits lung cancer growth through aerobic glycolysis by targeting PKM2

Abstract

Introduction: This study aims to explore the effects of microRNA-1286 (miR-1286) on the development of non-small cell lung cancer (NSCLC) via the aerobic glycolysis pathway by targeting pyruvate kinase muscle isozyme M2 (PKM2).

Material and methods: The mRNA levels of miR-1286 in NSCLC tissues and mouse tumor tissues were detected by q-PCR. MiR-1286 was knocked down and overexpressed separately in A549 cells. The effect of miR-1286 on cell proliferation was determined by CCK8 assay. Western blotting was used to measure the expression of PKM2 protein. Lactate production assay was used to detect the aerobic glycolysis in A549 cells. The effect of miR-1286 in vivo was determined by xenograft assay.

Results: The mRNA level of miR-1286 decreased in NSCLC tissues compared with paired, tumor adjacent normal tissues. In addition, miR-1286 inhibited A549 cell proliferation in vitro. Moreover, knockdown of miR-1286 increased PKM2 expression and lactate production. Thus, miR-1286 expression negatively correlated with PKM2 in A549 cells. At the same time, in vivo experiments also showed that miR-1286 suppressed the growth of A549 cells and PKM2 was the target gene of miR-1286.

Conclusions: These data show that miR-1286 inhibits lung cancer proliferation via aerobic glycolysis by targeting PKM2, which suggests that the functions of miR-1286 in NSCLC may play a key role in tumor progression and that miR-1286 can be a promising predictive biomarker and potential therapeutic target for NSCLC.

Keywords: PKM2; aerobic glycolysis; miR-1286; non-small cell lung cancer.

Figure 1

miR-1286 was downregulated in NSCLC tumor tissues. The mRNA expression levels of miR-1286 in NSCLC tumor samples and normal lung tissues were evaluated by q-PCR **P < 0.01.

Figure 2

MiR-1286 overexpression inhibited the proliferation of NSCLC cells. A – CCK8 assay was performed to detect A549 cell proliferation ability at indicated time points after transfection of miR-1286 mimic or control mimic and miR-1286 inhibitor or control inhibitor. B – Cell number was determined by cell counting after transfection of miR-1286 mimic or control mimic and miR-1286 inhibitor or control inhibitor for 48 h in A549 cells *P < 0.05, **p < 0.01.

Figure 3

PKM2 was a target gene regulated by miR-1286. A – PTIA database was used to scan the matched sequence, and then PKM2 gene was predicted as a direct target of miR-1286. B, C – A549 cells were either transfected with miR-1286 mimic and control mimic or treated with miR-1286 inhibitor or control inhibitor respectively. The mRNA expression of PKM2 was analyzed by qRT-PCR after transfection for 24 h (B) and the protein level of PKM2 was measured by western blotting after transfection for 36 h (C). D – After A549 cells were either transfected with miR-1286 mimic and control mimic or treated with miR-1286 inhibitor or control inhibitor for 24 h, A549 supernatants were collected to detect the production of lactate. E – The expression of several glycolysis-related enzymes was evaluated by qRT-PCR, including hexokinase 1 (HK1), hexokinase 2 (HK2), phosphofructokinase 1 (PFK1) and lactate dehydrogenase (LDH). F – The correlation between expression of PKM2 and miR-1286 in A549 cells was analyzed by Spearman correlation analysis *P < 0.05, **p < 0.01.

Figure 4

MiR-1286 inhibited NSCLC cell proliferation via targeting PKM2. A – Cell proliferation was determined by CCK8 assay after transfection of miR-1286 mimic or control mimic, together with pcDNA3.1-PKM2 or pcDNA3.1 for 48 h. B – Cell proliferation was determined by CCK8 assay after transfection of miR-1286 inhibitor or control inhibitor, together with shPKM2 or sh-Scramble for 48 h. C – Lactate production was measured by using the Nova Bioprofile 100 analyzer after transfection miR-1286 mimic or control mimic, together with pcDNA3.1-PKM2 or pcDNA3.1 for 24 h. D – Lactate production was measured after transfection miR-1286 inhibitor or control inhibitor, together with shPKM2 or sh-Scramble for 48 h *P < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

MiR-1286 affected NSCLC development in vivo. A – Nude mice were subcutaneously injected with 2 × 10⁶ A549-miR-1286 or A549-control cells. Tumor size was monitored over 3 weeks and the tumor volume was calculated. B, C – Expression of miR-1286 (B) and PKM2 (C) in the tumor tissues of A549-miR-1286 or A549-control mice xenograft was detected by q-PCR. D, E – Correlations between expression of PKM2 and miR-1286 (D) or PKM2 and tumor volume (E) in mice tumor tissues were analyzed by Spearman correlation analysis using GraphPad Prism 7 *P < 0.05, **p < 0.01.