NEK6 Promotes the Progression of Osteosarcoma Through Activating STAT3 Signaling Pathway by Down-Regulation of miR-26a-5p

Abstract

Background: Osteosarcoma is a malignant tumor originating from the skeletal system. There is no effective treatment other than surgery and chemotherapy, which seriously endangers the health of children and adolescents. NEK6 is a novel discovered Serine/Threonine protein kinase that can regulate cell cycle and activate several oncogenic pathways.

Methods: NEK6 expression in pan-cancer including sarcoma was evaluated using analysis tools of TIMER, UALCNA and GEPIA with TCGA database, and its association with overall survival in patients with sarcoma was also analyzed. TargetScan, tarbase, microT-CDS and Starbase online software were used to predict NEK6-targeted miRNAs, including miR-26a-5p. Tumor tissues from patients with osteosarcoma were collected for NEK6 and miRNA detection using RT-qPCR. NEK6 down-regulated by siRNAs or miR-26a-5p in osteosarcoma cells was detected by RT-qPCR, Western blot and Immunofluorescence staining assays. Effects of NEK6 knockdown on proliferation, migration, invasion and apoptosis of osteosarcoma cells were detected by CCK-8, wound healing, transwell and flow cytometry, respectively. The expressions of STAT3, metastasis and apoptosis-related genes were detected by Western blot.

Results: High expression of NEK6 and low expression of miR-26a-5p were lowly expressed in osteosarcoma and they were negative correlation. NEK6 has been confirmed as a direct target for miR-26a-5p. In addition, NEK6 down-regulated by siRNAs or miR-26a-5p led to inhibition of cell proliferation, migration and invasion while promoting cell apoptosis. The levels of phosphorylated STAT3 and metastasis genes (MMP-2, MMP-9) were inhibited, while apoptotic gene Bax was promoted and Bcl2 was inhibited by miR-26a-5p upregulation.

Conclusion: NEK6 can promote osteosarcoma progression via activating STAT3 signaling pathway, which is inhibited by miR-26a-5p, suggesting that NEK6 is a potential oncogene and miR-26a-5p is a suppressor of osteosarcoma. The strategy of inhibiting of NEK6 by miR-26a-5p may be an effective approach for osteosarcoma therapy.

Keywords: NEK6; STAT3 signaling pathway; miR-26a-5p; osteosarcoma.

Figure 1

NEK6 is highly expressed in many human cancers and sarcomas. (A) Expression profile of NEK6 in human different cancers with TIMER dataset. (B) NEK6 expression was higher in sarcoma (SARC) tissues (T, tumor, n=260; N, normal, n=2) in UALCNA dataset. (C) Effect of NEK6 expression level on SARC patients survival (P=0.013) in UALCNA dataset. (D) The transcripts per million (TPM) value of NEK6 are the highest of many human cancer types with GEPIA dataset. (E) NEK6 expression was higher in sarcoma (SARC) tissues (T, tumor, n=262; N, normal, n=2) in GEPIA dataset. (F) The TCGA dataset showed overall survival (P=0.0092) with high NEK6 expression in sarcoma with GEPIA dataset. *P<0.05, ***P<0.001, **P<0.01, *P<0.05 vs normal control tissues.

Figure 2

NEK6 was highly expressed in osteosarcoma tissues and cells. (A) NEK6 expression was higher in osteosarcoma tissues (Tumor tissues, n=13; Normal tissues, n=13). (B) The mRNA levels of NEK6 in osteosarcoma cells MG63 and Saos-2. (C) The protein levels of NEK6 in MG63 and Saos-2 cells. *P<0.05 vs normal tissues, ^#P<0.05 vs hFOB1.19 cells.

Figure 3

NEK6 was downregulated by siRNAs in osteosarcoma cells. (A) The mRNA levels of NEK6 were inhibited by NEK6 specific-targeted siRNAs (si-NEK6-1, si-NEK6-2 and si-NEK6-3) in MG63 cells detected by RT-qPCR. (B) The mRNA levels of NEK6 were inhibited by NEK6 specific-targeted siRNAs (si-NEK6-1, si-NEK6-2 and si-NEK6-3) in Saos-2 cells detected by RT-qPCR. si-NEK6-3 was the best silence of NEK6 expression, and labeled as si-NEK6 for following experiments. (C) The protein levels of NEK6 in human different osteosarcoma cells including MG63, U-2 OS and Saos-2 cells. (D) NEK6 expression in MG63 cells detected by immunofluorescence staining. (E) NEK6 expression in Saos-2 cells detected by immunofluorescence staining, scale bar=50 μm. *P<0.05 vs untreated cells (Un).

Figure 4

Effects of NEK6 knockdown on the proliferation, migration, invasion and apoptosis of osteosarcoma cells. (A) The cell proliferation of MG63 was inhibited by NEK6 siRNA (si-NEK6) detected by CCK8 assay. (B) The cell proliferation of Saos-2 was inhibited by si-NEK6 by CCK8 assay. (C) The cell migration of MG63 was inhibited by si-NEK6 detected by wound healing assay, scale bar=50 μm. (D) The cell migration of Saos-2 was inhibited by si-NEK6 detected by wound healing assay, scale bar=50 μm. (E) The cell invasion of MG63 and Saos-2 were inhibited by si-NEK6 detected by Transwell assay, scale bar=20 μm. (F) The cell apoptosis of MG63 and Saos-2 were promoted by si-NEK6 detected by FCM analysis. *P<0.05, vs si-NC treated cells or untreated cells (Un).

Figure 5

NEK6 was directly targeted by miR-26a-5p. (A) Venn diagrams were used to screen out NEK6 from miR-26-5p-targeted mRNAs predicted by bioinformatics tools, including TargetScan, tarbase, microT-CDS and Starbase. (B) The expression levels of miR-141-3p, miR-200a-3p, miR-26a-5p, miR-26b-5p, miR-27a-3p and miR-27b-3p were detected in MG63 and Saos-2 cells. (C) The binding sites of miR-26a-5p on NEK6 were displayed by TargetScan through sequence alignment and DLR report vectors. (D) Inhibition effect of miR-26a-5p on NEK6 was detected by DLR assay in HEK293T cells. *P<0.05 indicates miRNA levels in MG63 vs in hFOB1.19 cells, ^#P<0.05 indicates miRNA levels in Saos-2 vs in hFOB1.19 cells, ^{^}P<0.05 vs pmirGLO-NEK6 3’UTR (WT) and NC co-transfected cells.

Figure 6

miR-26a-5p expression in osteosarcoma and cells. (A) The expression levels of miR-26a-5p in patients with osteosarcoma. (B) The correlation between miR-26a-5p expression and NEK6 expression. (C) miR-26a-5p expression regulated by miR-26a-5p mimics in MG63 and Saos-2 cells. (D) The protein levels of NEK6 were inhibited by miR-26a-5p in MG63 and Saos-2 cells. *P<0.05 vs normal tissues, ^#P<0.05 vs untreated (Un) and si-NC treated cells in MG63 cells, ^{^}P<0.05 vs untreated (Un) and si-NC treated cells in Saos-2 cells.

Figure 7

Effects of miR-26a-5p up-regulation on the proliferation, migration, invasion and apoptosis of osteosarcoma cells. (A) The cell proliferation of MG63 was inhibited by miR-26a-5p mimics detected by CCK8 assay. (B) The cell proliferation of Saos-2 was inhibited by miR-26a-5p mimics by CCK8 assay. (C) The cell migration of MG63 was inhibited by miR-26a-5p mimics detected by wound healing assay, scale bar=50 μm. (D) The cell migration of Saos-2 was inhibited by miR-26a-5p mimics detected by wound healing assay, scale bar=50 μm. (E) The cell invasion of MG63 and Saos-2 were inhibited by miR-26a-5p mimics detected by Transwell assay, scale bar=20 μm. (F) The cell apoptosis of MG63 and Saos-2 were promoted by miR-26a-5p mimics detected by FCM analysis. *P<0.05 vs mimics NC treated cells or untreated cells (Un).

Figure 8

Effects of miR-26a-5p up-regulation on the phosphorylation levels of STAT3, cell apoptosis-related genes (Bcl2 and Bax), and metastasis-related genes (MMP-2, MMP-9) in MG63 and Saos-2 cells were measured by Western blot. (A) Representative blots of the phosphorylation levels of STAT3, cell apoptosis-related genes (Bcl2 and Bax), and metastasis-related genes (MMP-2, MMP-9). (B) The phosphorylation levels of STAT3, Bcl2, Bax, MMP-2 and MMP-9 in MG63 cells. (C) The phosphorylation levels of STAT3, Bcl2, Bax, MMP-2 and MMP-9 in Saos-2 cells. *P<0.05, vs mimics NC treated cells or untreated cells (Un).

Figure 9

Predictive mechanism diagram of NEK6 in osteosarcoma. NEK6 promotes the progression of osteosarcoma through activating STAT3 signaling pathway by down-regulation of miR-26a-5p.