miR-491-5p inhibits osteosarcoma cell proliferation by targeting PKM2

Abstract

Increasing evidence has indicated that microRNAs (miRNAs/miRs) are associated with tumorigenesis and the development of numerous cancer types. Previous studies have suggested miRNA-491-5p is downregulated in osteosarcoma (OS) and functions as a tumor suppressor. However, the biological roles and underlying mechanisms associated with miR-491-5p function in OS require further exploration. In the present study, it was demonstrated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) that miR-491-5p was downregulated in 36 pairs of OS tissues, compared with in adjacent normal bone tissues. Furthermore, CCK-8 and colony formation assays indicated that miR-491-5p mimics suppressed OS cell proliferation. However, an miR-491-5p inhibitor enhanced cell proliferation. In addition, luciferase reporter assays, RT-qPCR and western blot analysis demonstrated that PKM2 was a direct target of miR-491-5p. The miR-491-5p mimic inhibited the mRNA and protein expression of PKM2, while the miR-491-5p inhibitor promoted PKM2 mRNA and protein expression. In addition, PKM2 overexpression reversed the proliferation-inhibiting effects of miR-491-5p in OS cells. Therefore, these results indicated that miR-491-5p serves as a tumor suppressor in OS cells, which may be important in OS treatment.

Keywords: cell proliferation; microRNA; microRNA-491-5p; muscle 2; osteosarcoma; pyruvate kinase.

Figure 1.

Expression of miR-491-5p is significantly downregulated in OS tissues and cells. (A) Expression of miR-491-5p was downregulated in OS tissues compared with adjacent normal tissues (n=36) by RT-qPCR analysis. U6 was used as the internal control. (B) Expression of miR-491-5p was downregulated in 4 different OS cell lines (KHOS, LM7, U2OS, and MG-63) compared with a normal osteoblast cell line (Nhost). U6 was used as the internal control. *P<0.05, **P<0.01 compared with Nhost. miR, microRNA' OS, osteosarcoma.

Figure 2.

miR-491-5p suppresses proliferation in osteosarcoma cells. CCK8 assay was used to evaluated the cell proliferation rate following transfection with miR-NC, miR-491-5p mimics or miR-491-5p inhibitors in (A) U2OS and (B) MG-63 cells. The miR-NC group was used as the control at 1, 2, 3, 4 and 5 days, *P<0.05 compared with control. Cell colony formation assays were performed and cell colony number was analyzed following transfection with miR-NC, miR-491-5p mimics or miR-491-5p inhibitors into (C) U2OS and (D) MG-63 cells, *P<0.05. miR, microRNA; NC, negative control; OD, optical density.

Figure 3.

PKM2 is a direct target of miR-491-5p. (A) The wild-type 3′-UTR region of the PKM2 mRNA (3′UTR-PKM2-WT) containing a putative miR-491-5p-binding site and the corresponding mutant 3′-UTR region of the PKM2 mRNA (5′-CCCCAC-3′ to 5′-GGGGUG-3′) (3′UTR-PKM2-MUT). The 198 was the location in the genome of PKM2. (B) U2OS cells were co-transfected with 3′UTR-PKM2-WT/MUT plasmid, along with miR-491-5p or miR-NC using Lipofectamine 2000. The relative luciferase activity is presented. The relative mRNA expression was detected using reverse transcription polymerase chain reaction following transfection with miR-NC, miR-491-5p mimics or miR-491-5p inhibitors into (C) U2OS or (D) MG-63 cells. *P<0.05. UTR, untranslated region; PKM2, pyruvate kinase, muscle 2; WT, wild type; miR, microRNA.

Figure 4.

miR-491-5p regulates PKM2 protein expression. (A) The relative PKM2 mRNA expression was detected using western blot analysis following transfection with miR-NC, miR-491-5p mimics or miR-491-5p inhibitors into U2OS cells. (B) Quantification of protein bands. *P<0.05. (C) The relative PKM2 mRNA expression was detected using western blot analysis following transfection with miR-NC, miR-491-5p mimics or miR-491-5p inhibitors into MG-63 cells. (D) Quantification of protein bands. *P<0.05. PKM2, pyruvate kinase, muscle 2; miR, microRNA; NC, negative control.

Figure 5.

PKM2 overexpression reverses the cell proliferation-inhibiting effects of miR-491-5p in OS cells. (A) PKM2 expression was demonstrated to be increased in OS tissues compared with adjacent normal tissues (n=36) by reverse transcription quantitative polymerase chain reaction analysis. (B) Increased PKM2 expression was negatively associated with miR-491-5p by Spearman correlation analyses. (C) CCK8 assay was used to evaluated the cell proliferation rate following transfection with miR-NC, miR-491-5p mimics or miR-491-5p mimics plus pcDNA-PKM2 plasmids into U2OS cells. *P<0.05. (D) CCK8 assay was used to evaluate the cell proliferation rate following transfection with miR-NC, miR-491-5p mimics or miR-491-5p mimics plus pcDNA-PKM2 plasmids into MG-63 cells. *P<0.05. PKM2, pyruvate kinase, muscle 2; miR, microRNA; NC, negative control; OD, optical density.