miR-939-3p induces sarcoma proliferation and poor prognosis via suppressing BATF2

Abstract

Background: Sarcoma is a rare and aggressive malignancy with poor prognosis, in which oncogene activation and tumor suppressor inactivation are involved. Accumulated studies suggested basic leucine zipper transcription factor ATF-like 2 (BATF2) as a candidate tumor suppressor, but its specific role and mechanism in sarcoma remain unclear.

Methods: The expression levels of BATF2 and miR-939-3p were evaluated by using human sarcoma samples, cell lines and xenograft mouse models. Bioinformatics analysis, qPCR, Western blot, cell proliferation assay, overexpression plasmid construction, point mutation and dual luciferase reporter assay were utilized to investigate the role and mechanism of miR-939-3p in sarcoma.

Results: In this study, we demonstrated that the expression of BATF2 was downregulated in human sarcoma tissues and cell lines. The downregulation of BATF2 was negatively associated with the prognosis of sarcoma patients. Subsequent bioinformatic prediction and experimental validations showed that BATF2 expression was reduced by microRNA (miR)-939-3p mimic and increased by miR-939-3p inhibitor. Additionally, miR-939-3p was upregulated in sarcoma tissues and cells, correlating with a poor prognosis of sarcoma patients. Moreover, miR-939-3p overexpression suppressed sarcoma cell proliferation, which was significantly attenuated by the restoration of BATF2, while siRNA-mediated knockdown of BATF2 aggravated the miR-939-3p-induced promotion of sarcoma cell proliferation. Further computational algorithms and dual-luciferase reporter assays demonstrated that miR-939-3p repressed BATF2 expression via directly binding to its 3' untranslated region (3' UTR).

Conclusion: Collectively, these findings identified miR-939-3p as a novel regulator of BATF2, as well as a prognostic biomarker in sarcoma, and revealed that suppressing miR-939-3p or inducing BATF2 expression may serve as a promising therapeutic strategy against sarcoma.

Keywords: BATF2; miR-939-3p; prognosis; sarcoma; therapeutic target.

Figure 1

Downregulation of BATF2 correlates with poor prognosis of sarcoma. (A–D) qPCR and Western blot analysis of the expression level of BATF2 in soft tissue sarcoma and pericarcinoma (A, B) and the human skin fibroblast cell line (HSF), human fibrosarcoma cell line (HT-1080) and synovial sarcoma cell line (SW-982) (C, D). (E) Kaplan-Meier estimates of overall survival time based on BATF2 expression levels from 262 CRC patients by using a GEPIA database. Hazard ratio (HR) = 0.65. P(HR) = 0.031. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

BATF2 expression was repressed by miR-939-3p in sarcoma. (A) miRNAs in the 3′-untranslated region (UTR) region of BATF2 were predicted by using miRDB, miRwalk and targetScan software programs, and 9 common miRNAs were found. (B, C) qPCR screening of the predicted 9 miRNAs in control and sarcoma tissues from sarcoma patients (B) and HSF and HT-1080 cells (C). (D–K) qPCR and Western blot analysis of BATF2 expression in HT-1080 cells or SW-982 cells transfected with the mimic or inhibitor of miR-939-3p or miR-455-5p. ns: not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Upregulation of miR-939-3p associates with poor prognosis of sarcoma. (A, B) qPCR analysis of the expression of miR-939-3p in soft tissue sarcoma and pericarcinoma (A) and the human skin fibroblast cell line (HSF), human fibrosarcoma cell line (HT-1080) and synovial sarcoma cell line (SW-982) (B). (C) Circulating miR-939-3p levels in healthy individuals (n = 275) and sarcoma patients with various histological subtypes, including glioma (n = 30), malignant soft tissue tumor (n = 402), pancreatic cancer (n = 30), gastric cancer (n = 30), intermediate soft tissue tumor (n = 144), lung cancer (n = 30), benign soft tissue tumor (n = 30), esophageal cancer (n = 30), colorectal cancer (n = 30) and hepatocellular carcinoma (n = 30), were analyzed by using a CancerMIRNome database. (D–G) Cell viabilities of HT-1080 or SW-982 cells transfected with control, miR-939-3p mimic or inhibitor separately for two days were analyzed by using cell counting kit 8 or BrdU ELISA kit. (H) Kaplan-Meier estimates of overall survival time based on miR-939 expression levels from 259 sarcoma patients by using a CancerMIRNome database. Hazard ratio (HR) = 2.69. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

miR-939-3p suppresses BATF2 expression via binding to its 3’UTR region. (A) Statistical analysis of the negative correlation between miR-939-3p and BATF2 mRNA expression levels by using linear regression and Pearson correlation analysis. (B) Bioinformatic analysis of the binding sites of miR-939-3p in the 3’ UTR of BATF2 mRNA. (C) A luciferase reporter assay was performed to verify the binding of miR-939-3p in the 3’ UTR of BATF2 mRNA. The wild type (WT) 3’ UTR (GCCCAGG) was mutated into mutant 3’ UTR (ATTTCAA). ns: not significant. **P < 0.01. (D) pAP-1-Luc was co-transfected with Renilla into HT-1080 cells in combined with control or miR-939 mimic or inhibitor for luciferase reporter gene assay. pAP-1-Luc activity was normalized against Renilla activity. (E) qPCR analysis of MET expression in HT-1080 cells transfected with control or miR-939 mimic or inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Overexpression of BATF2 attenuates miR-939-3p-mediated cancer proliferation. (A) Recombinant BATF2 plasmids (pBATF2) or control plasmids (pControl) were separately transfected into HT-1080 cells by using Lipofectamine 3000. (B, C) qPCR and Western blot analysis of BATF2 expression in HT-1080 cells transfected with pControl or pBATF2 treated with control or miR-939-3p mimic. (D, E) Cell viability of cells in (B) was analyzed by using CCK-8 or BrdU ELISA kit. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

miR-939-3p enhances sarcoma growth in vivo. Xenograft nude mouse models were constructed by subcutaneously injecting 5 × 10⁶ HT-1080 cells stably infected with lentivirus expressing miR-939-3p or control separately (n = 5 per group). (A) The tumor volume was calculated every 3 days, and the mice were sacrificed 21 days after cell inoculation. (B, C) The xenografts were excised for the comparisons of tumor size and weight. (D) qPCR analysis of miR-939-3p expression in the xenografts. (E, F) qPCR and Western blot analyses of BATF2 expression in the xenografts. (G, H) Immunohistochemical analyses of the expressions levels of BATF2 and Ki67 in tumor xenografts. n = 5 per group. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

A proposed model elucidating the mechanism of miR-939-3p-induced sarcoma proliferation and poor prognosis through suppressing BATF2 via directly binding to its 3’ UTR.