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. 2019 Jan;17(1):1363-1371.
doi: 10.3892/ol.2018.9712. Epub 2018 Nov 15.

miR-142-5p suppresses proliferation and promotes apoptosis of human osteosarcoma cell line, HOS, by targeting PLA2G16 through the ERK1/2 signaling pathway

Deliang Cheng  1 , Jiageng Li  1 , Lijun Zhang  1 , Leiming Hu  1
Affiliations

miR-142-5p suppresses proliferation and promotes apoptosis of human osteosarcoma cell line, HOS, by targeting PLA2G16 through the ERK1/2 signaling pathway

Deliang Cheng et al. Oncol Lett. 2019 Jan.

Abstract

Previous studies have revealed that miR-142-5p serves a critical role in human cancer progression. However, the biological function of miR-142-5p in osteosarcoma (OS) development remains unclear. In the present study, the role of miR-142-5p in human OS HOS cells was determined, and the underlying mechanism involved was examined. Compared with the adjacent healthy tissues, the expression level of miR-142-5p was downregulated and the expression level of group XVI phospholipase A2 (PLA2G16) protein was upregulated in human OS tissues. The aforementioned results were also indicated in human OS HOS cells when compared with human fetal osteoblastic hFOB1.19 cells. Additionally, the results demonstrated that PLA2G16 was a direct target of miR-142-5p. miR-142-5p transfection upregulated the expression level of miR-142-5p and suppressed the expression level of PLA2G16 protein in HOS cells. MTT assays indicated a time-dependent decrease by miR-142-5p transfection in the proliferation of HOS cells. 5-bromo-2'-deoxyuridine incorporation assays confirmed that miR-142-5p transfection inhibited DNA synthesis in HOS cells. In addition, miR-142-5p transfection increased the Caspase-3 (CASP3) activity and apoptotic rate. Western blot analysis indicated that miR-142-5p transfection reduced BCL2, apoptosis regulator expression and upregulated the expression of CASP3 and BCL2 associated X, apoptosis regulator in HOS cells. Furthermore, miR-142-5p transfection decreased the expression levels of phosphorylated (p)-proto-oncogene, serine/threonine kinase, p-mitogen-activated protein kinase kinase, and p-extracellular signal-regulated kinase (ERK) 1/2 proteins in HOS cells. PLA2G16 overexpression restored the expression level of p-ERK 1/2 protein, which was reduced by miR-142-5p overexpression. MTT and CASP3 activity assays indicated that restoration of PLA2G16 reversed the tumour-suppressive role of miR-142-5p transfection in HOS cells. In conclusion, the results of the present study indicated that miR-142-5p suppressed proliferation and promoted apoptosis in human OS HOS cells by targeting PLA2G16 through ERK1/2 signaling pathway.

Keywords: Group XVI phospholipase A2; apoptosis; miR-142-5p; osteosarcoma; proliferation.

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Figures

Figure 1.
Figure 1.
The expression level of PLA2G16 protein and miR-142-5p in human OS tissues and adjacent healthy tissues, in addition to human hFOB1.19 cells and HOS cells. (A) miR-142-5p expression level in OS tissues compared with adjacent healthy tissues. (B) miR-142-5p expression level of human hFOB1.19 cells compared with HOS cells. (C) PLA2G16 protein expression level in OS tissues compared with adjacent healthy tissues. (D) PLA2G16 protein expression level of human hFOB1.19 cells compared with HOS cells. *P<0.05 compared with normal tissues or hFOB1.19 cells. OS, osteosarcoma; miR, microRNA; PLA2G16, group XVI phospholipase A2.
Figure 2.
Figure 2.
PLA2G16 is a direct target gene of miR-142-5p. (A) A microRNA database search predicted an miR-142-5p binding site on the PLA2G16 3′-UTR. (B) miR-142-5p overexpression in 293T cells reduced the luciferase activity of cells transfected with pMIR-PLA2G16-wt 3′-UTR, whereas pMIR-PLA2G16-mut 3′-UTR blocked this effect. (C) Following transfection with miR-142-5p mimics or miR-NC, miR-142-5p transfection induced an elevation in the expression level of mature miR-142-5p in HOS cells. (D) The expression level of PLA2G16 protein in HOS cells was detected and measured 72 h following transfection with miR-142-5p and miR-NC mimics. *P<0.05 compared with control. miR, microRNA; PLA2G16, group XVI phospholipase A2; NC, negative control; UTR, untranslated region; wt, wild type; mut, mutated.
Figure 3.
Figure 3.
Effects of miR-142-5p on the proliferation in HOS cells. (A) Proliferation of HOS cells transfected with miR-142-5p mimics or miR-NC at 24, 48 and 72 h by MTT assay. (B) BrdU incorporation assays confirmed that miR-142-5p overexpression inhibited DNA synthesis in HOS cells transfected with miR-142-5p and miR-NC mimics. *P<0.05 compared with control. BrdU, 5-Bromo-2′-deoxyuridine; miRNAs, microRNAs; NC, negative control; OD, optical density.
Figure 4.
Figure 4.
Effect of miR-142-5p on apoptosis of HOS cells. (A) Increased CASP3 activity was observed following miR-142-5p overexpression in HOS cells transfected with miR-142-5p and miR-NC mimics. (B) Apoptotic percentage measured by flow cytometry in HOS cells transfected with miR-142-5p and miR-NC mimics. (C) The expression level of CASP3, BCL2, Bax and β-actin proteins were detected and measured. miR-142-5p overexpression promoted apoptosis, reduced BCL2 expression and elevated CASP3 and Bax expression levels in HOS cells. *P<0.05 compared with control. Bax, BCL2-associated X, apoptosis regulator; BCL2, BCL2, apoptosis regulator; CASP3, Caspase-3; miR, microRNA; Annexin V-FITC, Annexin V-fluorescein isothiocyanate; PI, propidium iodide; NC, negative control.
Figure 5.
Figure 5.
Effects of miR-142-5p and PLA2G16 on p-ERK1/2 protein expression, proliferation and apoptosis of HOS cells. HOS cells were transfected with miR-142-5p, miR-NC mimics and pcDNA3.1-PLA2G16 plasmids. (A) The expression level of p-Raf-1, Raf-1, p-MEK, MEK, p-ERK1/2, ERK1/2 and β-actin proteins. miR-142-5p transfection decreased the expression levels of p-Raf-1, p-MEK, and p-ERK1/2 proteins in HOS cells. (B) The expression levels of PLA2G16, p-ERK1/2, ERK1/2 and β-actin proteins were detected and measured. The PLA2G16 expression level was enhanced in HOS cells following transfection with pCDNA3.1-PLA2G16 and PLA2G16 overexpression rescued the reduced expression level of p-ERK1/2 protein associated with miR-142-5p transfection in HOS cells. (C) Apoptotic rate of HOS cells following transfection with miR-142-5p mimics either with or without pCDNA3.1-PLA2G16 at 24, 48 and 72 h. (D) CASP3 activity assays were conducted in HOS cells following transfection with miR-142-5p mimics either with or without pCDNA3.1-PLA2G16. *P<0.05 compared with control. #P<0.05 compared with miR-142-5p mimics. OD, optical density; p, phosphorylated; Raf-1, proto-oncogene, serine/threonine kinase 1, CASP3, Caspase-3; ERK1/2, extracellular signal-regulated kinase 1/2; MEK, mitogen-activated protein kinase kinase; NC, negative control; PLA2G16, group XVI phospholipase A2.
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