MiR-489-3p Reduced Pancreatic Cancer Proliferation and Metastasis By Targeting PKM2 and LDHA Involving Glycolysis

Abstract

Introduction: Malignant proliferation and metastasis are some of the causes of high mortality in pancreatic cancer. MicroRNAs have been a hot spot in cancer research and are involved in tumor formation and metabolic stress responses. However, the biology function and underlying mechanism of miRNA regulating pancreatic cancer progress is remained uncleared.

Methods: RNA-seq analysis the glycolysis associated miRNAs and verified miRNA-489-3p was involving in glycolysis. We used RNA in situ hybridization (ISH) and qRT-PCR to analyze the differential expression of miR-489-3p in pancreatic cancer tissues and adjacent tissues and cell lines. Then the function assay of in vivo and in vitro were used to evaluated the role of miR-489-3p in the proliferation, metastasis and glucose metabolism of pancreatic cancer. Furthermore, dual luciferase reporter and rescue experiments were performed to explore the mechanism underlying in the role of miRNA-489-3p.

Results: We determined that glycolysis associated miRNA miR-489-3p was downregulated in pancreatic cancer tissues and cell lines. The gain and loos of function experiments confirmed that miR-489-3p could inhibit the proliferation, metastasis and glucose metabolism of pancreatic cancer. Further, we found that miR-489-3p could target regulating LDHA and PKM through the luciferase report experiment. Finally, in vivo experiment confirmed that highly expressed miR-489-3p inhibited the growth of pancreatic cancer.

Conclusion: In short, this study identified miR-489-3p as a novel therapy target for pancreatic cancer which was involving in the proliferation, metastasis and glycolysis, but its diagnostic value deserves further study.

Keywords: glycolysis; metastasis; miR-489-3p; pancreatic cancer; proliferation.

Figure 1

The expression of miR-489-3p and its relationship with clinical prognosis of patients. (A) RNA-seq analysis the different expressed miRNAs associated with glycolysis. (B) q-RT-PCR analysis the upregulated miRNA in the exposure with glycolysis inhibitor 2-DG. (C, D) RNA in situ hybridization experiments showed the expression of miR-489-3p in paracancerous and cancerous tissues. The bar stands for 50 microns (E) qRT-PCR analysis of the relative expression of miR-489-3p in adjacent tissues and PC tissues. (F) qRT-PCR showed the relative expression of miR-489-3p in PC cell lines and pancreatic normal duct epithelial cells (HPDE). (G) Kaplan-Meier curve was divided into survival periods by miR-489-3p expression. Among them, patients were divided into high expression group (red) and low expression group (blue) by median expression of miR-489-3p. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

MiR-489-3p inhibits proliferation and invasion of pancreatic cancer. (A) qRT-PCR showed the relative expression of miR-489-3p after transfection of miR-489-3p mimic and inhibitor in PC cells. (B–D) CCK8 and plate cloning assays showed the cell proliferation ability of PC cells transfected with miR-489-3p mimics and miR-489-3p inhibitors. (E, F) Transwell migration and invasion assays showed the cell migration and invasion ability of PC cells transfected with miR-489-3p mimics and inhibitors. (**P < 0.01, ***P < 0.001).

Figure 3

MiR-489-3p targets LDHA and PKM2. (A, B) qRT-PCR and western blot assays showed relative expression of LDHA and PKM2 after PC cells transfected with miR-489-3p mimics and miR-489-3p inhibitor. (C) Spearman rank correlation analysis showed correlation between miR-489-3p and LDHA and PKM. (D) The predicted binding site of miR-489-3p in human LDHA and PKM gene 3’ UTR, and the corresponding sequence in the mutated type. (E) Luciferase reporter gene assay analysis of the relationship between miR-489-3p and LDHA and PKM. (*P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant).

Figure 4

MiR-489-3p regulates PC glycolysis. (A–C) Cell metabolism experiments showed glucose uptake, lactic acid production, and ATP production of miR-489-3p mimics and miR-489-3p inhibitors in PC cells. (D) The hippocampal XF extracellular flux analyzer analysis of the PC ECAR in miR-489-3p mimics and inhibitors group. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

LDHA and PKM2 restored the function of miRNA-mediated proliferation and metastasis ability. (A) Western blot experiments showed that the relative expression of LDHA and PKM2 in PC cells transfected with miR-489-3p mimic, inhibitor, LDHA, PKM2 overexpressed plasmid or shRNA. (B, C) CCK8 and plate cloning and transwell migration assays showed that the proliferation ability of PC cells transfected with miR-489-3p mimic, inhibitor, LDHA, PKM2 overexpressed plasmid or shRNA. (D) Transwell assays show that the migration and invasion ability of PC cells transfected with miR-489-3p mimic, inhibitor, LDHA, PKM2 overexpressed plasmid or shRNA. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 6

MiR-489-3p suppresses glycolysis through LDHA and PKM2. (A–C) Cell metabolism experiments showed that glucose uptake, lactic acid production, and ATP production of PC cells transfected with miR-489-3p mimic, inhibitor, LDHA, PKM2 overexpressed plasmid or shRNA. (D) The hippocampal XF extracellular flux analyzer analysis of the PC ECAR after transfected with miR-489-3p mimic, inhibitor, LDHA, PKM2 overexpressed plasmid or shRNA. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 7

Effects of overexpression of miR-489-3P on PC proliferation and metabolism in vivo. (A) Typical images of nude mice tumors (n = 5), (B) subcutaneous tumor weight, (C) subcutaneous tumor volume, (D) miR-489-3p expression in xenografts by qRT-PCR. (E) Typical IHC staining images of xenografts show Ki-67 and PCNA expression. The bar stands for 50 microns. (F) Typical IHC staining images of xenografts show the expression of metabolic indicators (LDHA, GLUT1, HK2, PKM2). The bar stands for 100 microns. (**P < 0.01, ***P < 0.001).