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

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.


Introduction
Sarcomas are rare cancers predominantly derived from embryonic mesoderm, accounting for approximately 1% of human malignancies, which primarily affect younger patients and are difficult to treat (1)(2)(3).The histologic and molecular diversity, coupled with limited access to specialized sarcoma centers, often leads to erroneous therapeutic plans (4).Currently, localized sarcomas are typically treated with surgery, and chemotherapy or targeted therapy is employed for metastatic disease (2), while immunotherapy for sarcoma remains largely investigational (2,4,5).Despite numerous clinical trials exploring various chemotherapy regimens, the progression-free survival rates range from 3 to 7 months, with an overall survival (OS) of 12 to 18 months (6,7).Therefore, gaining a comprehensive insight into the mechanism underlying sarcoma tumorigenesis and progression is crucial to develop effective therapeutic approaches against this disease.
MicroRNAs (miRs) are small non-coding RNAs, possessing approximately 22 nucleotides, which can regulate gene expression through binding to the 3' untranslated region (3' UTR) of its target mRNAs, ultimately contributing to mRNA cleavage and translational inhibition (21,22).As post-transcriptional regulators, miRNAs have been implicated in modulating various malignant behaviors, including cancer cell proliferation, apoptosis, motility, adhesion and drug resistance (22).Many miRNAs are directly or indirectly associated with oncogenes and can function as either tumor suppressors or oncomiRs (23).During sarcoma initiation and progression, changes in the expression patterns of certain miRNAs significantly impact sarcoma cell proliferation, invasion, and apoptosis (8).However, the precise molecular mechanism by which miRNAs regulate BATF2 to enhance sarcoma growth remains elusive.
In this study, we demonstrated a downregulation of BATF2 in sarcoma, which was positively correlated with prognosis.In vitro and in vivo experiments revealed that miR-939-3p enhanced sarcoma proliferation through suppressing BATF2 expression via binding to its 3' UTR.These findings highlight the miR-939-3p/ BATF2 axis as a promising therapeutic target for sarcoma patients.

Cell culture
The human fibrosarcoma cell line HT-1080, synovial sarcoma cell line SW-982 and human skin fibroblast cell line HSF were obtained from iCell Bioscience (Shanghai, China).The stable transfection of HT-1080 cells was performed as we described (9).Briefly, HT-1080 cells were transfected with lentivirus-packaged recombinant plasmids expressing miR-939-3p or negative control (Tsingke Biotechnology, Shanghai, China), and resistant cells were selected by using Puromycin (Sigma, St. Louis, MO, USA) to establish stable cell lines.

Human samples
Sarcoma, colorectal cancer (CRC) and the corresponding paratumor tissues from 12 patients were separately collected from the People's Hospital of Xishui County (Zunyi, China).The study was carried out in accordance with the Declaration of Helsinki and approved by the People's Hospital of Xishui County.Written informed consent has been obtained from all patients.

Animal studies
Four-week-old female nude mice were purchased from the Beijing Huafukang Biotechnology (Beijing, China).The mice were subcutaneously injected with 5 × 10 6 HT-1080 cells in 0.2 mL PBS per mouse with or without stable miR-939-3p overexpression (n = 5).After 21 days, the xenografts were excised, measured for volume and weight, and further processed for RNA and protein extraction, which was approved by the Animal Welfare and Ethics Committee of Army Medical University.

qPCR
Total RNA of HT-1080 cells and tumor tissues was extracted by using an RNA Purified Total RNA Extraction Kit (Beyotime), and then reversely transcribed into cDNA with RT Master Mix (MedChemExpress, Monmouth Junction, NJ, USA), as we previously described (24).qPCR was performed using a SYBR Green qPCR kit (MedChemExpress).Specific primer sets were provided in Supplementary Table S1.

miRNA extraction and qPCR
To quantify miR-939-3p, total RNA, including miRNA, was extracted using the RNAiso Plus reagent (TaKaRa, Shiga, Japan), which was reversely transcribed into cDNA by using a miRNA First Strand cDNA Synthesis kit from Sangon (Shanghai, China).qPCR was performed using a SYBR Green qPCR kit (MedChemExpress).The primer sets were listed in Supplementary Table S2.

Western blot
Western blot was performed as we described (24).Briefly, proteins were extracted from sarcoma cells and tissues by using RIPA lysis buffer (Beyotime).Proteins were separated and transferred onto PVDF membranes, which were then incubated with the antibodies against BATF2 (Abcam, Cambridge, MA, USA) or b-actin (Santa Cruz, Dallas, TX, USA), followed by the incubation of secondary antibody.Gray-scale of the bands were quantified by using ImageJ software.

Immunohistochemistry staining
Immunohistochemistry (IHC) staining was performed to detect the expression levels of BATF2 (Abcam) and Ki67 (Santa Cruz) in tumor xenografts as we previously described (9).

Gene expression profiling interactive analysis
GEPIA ( 25) is a publicly available platform integrating data based on The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) project.It provides comprehensive pan-cancer analysis of RNA-sequencing data, including gene expression, correlation, and survival analysis.

CancerMIRNome
The correlation between miR-939-3p expression levels and the prognosis of sarcoma patients, as well as the circulating miR-939-3p expression levels, were analyzed by using CancerMIRNome online database (26).CancerMIRNome is publicly available that enables analysis of miRNAs from TCGA projects and circulating miRNome datasets.Data are available from the Gene Expression Omnibus (GSE124158).

miRNA binding sites prediction
Potential miRNAs targeting BATF2 were predicted using miRDB, miRwalk and targetScan software programs.The potential binding sites of miR-939-3p in BATF2 3′ UTR were analyzed by using targetScan online software.

miRNA overexpression and knockdown
MiR-939-3p mimics or inhibitors, and negative controls were purchased from Tsingke Biotechnology, which were listed in Supplementary Table S3.HT-1080 cells were harvested 48 h after transfection using Lipofectamine 3000 (Invitrogen).

Cell proliferation assay
HT-1080 cells were seeded into a 96-well plate and cotransfected with vector control or BATF2 overexpression plasmids and miR-939-3p mimic or inhibitor.Cell viability was determined by using Cell Counting Kit 8 (CCK-8) (Dojindo, Kumamoto, Japan) after 48 h or a Bromodeoxyuridine (BrdU) ELISA kit (Roche, Burgess Hill, UK) according to the manufacturer's instructions.

Statistical analysis
Data were presented as mean ± SD and analyzed by using Graphpad Prism 8 software.Comparisons were statistically significant at P < 0.05 by using unpaired t-test or ANOVA.

Downregulation of BATF2 correlates with poor prognosis of sarcoma
We first explored the expression levels of BATF2 in human soft tissue sarcoma.It was found that BATF2 mRNA and protein levels were significantly decreased in 12 cases of sarcoma tissues, compared to the corresponding adjacent tissues (Figures 1A, B).Besides, BATF2 mRNA and protein expression was also downregulated in human fibrosarcoma cells (HT-1080) and synovial sarcoma cells (SW-982), compared with that in human skin fibroblast cells (HSF) (Figures 1C, D).Further, sarcoma patients were divided into two groups according to BATF2 expression levels.Compared with patients with high BATF2 expression (n = 131), patients with low BATF2 expression (n = 131) had obviously worse survival (P = 0.031) with a hazard ratio of 0.65 (P = 0.031) (Figure 1E).These findings indicated that BATF2 was downregulated in sarcoma and negatively associated with the prognosis of sarcoma patients.

miR-939-3p inhibits BATF2 expression in sarcom
Since miRNAs play an extensive role in modulating gene expression in sarcoma (21,22), we hypothesized that BATF2 might be regulated by miRNA.To test this hypothesis, we utilized miRDB, miRwalk and targetScan software programs to identify miRNAs that had potential binding sites in the 3′ UTR of BATF2 (Figure 2A).We then screened the predicted nine miRNAs using quantitative PCR (qPCR) in both sarcoma tissues (Figure 2B) and cell lines (Figure 2C), which showed that miR-939-3p and miR-455-5p were upregulated in sarcoma tissues and cells compared to controls.Further, BATF2 expression was reduced by miR-939-3p mimic and increased by miR-939-3p inhibitor in both HT-1080 and SW-982 cells (Figures 2D-G).However, neither the mimic nor the inhibitor of miR-455-5p could regulate BATF2 expression (Figures 2H-K), suggesting that BATF2 expression was repressed by miR-939-3p in sarcoma.

Upregulation of miR-939-3p associates with poor prognosis of sarcoma
To explore the role of miR-939-3p, we collected sarcoma tissues and observed an elevated expression of miR-939-3p compared to pericarcinoma tissues (Figure 3A).Similarly, sarcoma cell lines (HT-1080 and SW-982) also exhibited higher levels of miR-939-3p compared to a human skin fibroblast cell line (HSF) (Figure 3B).Moreover, circulating miR-939-3p levels were found to be upregulated in sarcoma patients with various histological subtypes, including glioma, malignant soft tissue tumor, pancreatic cancer, gastric cancer, intermediate soft tissue tumor, lung cancer, benign soft tissue tumor, esophageal cancer, colorectal cancer and hepatocellular carcinoma, compared to healthy individuals (Figure 3C).
Functional experiments further revealed that the viabilities of HT-1080 and SW-982 cells were enhanced by miR-939-3p mimic, while suppressed by miR-939-3p inhibitor, as evidenced by CCK-8 and BrdU incorporation assay (Figures 3D-G).Consistent with our hypothesis, Kaplan Meier survival analysis showed a negative correlation between miR-939-3p expression levels and the prognosis of sarcoma patients, as analyzed using both the CancerMIRNome database (P = 0.014, with a hazard ratio of 2.69) (Figure 3H) and UALCAN databases (P = 0.0036) (Supplementary Figure S1).These data revealed that miR-939-3p was significantly upregulated in sarcoma and was inversely associated with the prognosis of sarcoma patients.

Overexpression of BATF2 attenuates miR-939-3p-mediated sarcoma proliferation
To investigate whether miR-939-3p-mediated regulation of BATF2 is involved in sarcoma proliferation, HT-1080 cells were transfected with BATF2 overexpression plasmids (pBATF2) or control plasmids (pControl) (Figure 5A).It was found that overexpression of BATF2 significantly alleviated the miR-939-3pmediated suppression of BATF2 expression and promotion of sarcoma cell proliferation (Figures 5B-E), as well as cell migration and invasion (Supplementary Figure S3).Besides, miR-939-3p could neither further enhance sarcoma cell proliferation nor regulate AP-1 activity or MET expression, when BATF2 was knocked down by a large amount of in HT-1080 cells (Supplementary Figures S4A-E).These findings collectively suggest that miR-939-3p could induce sarcoma cell proliferation via inhibiting the expression of BATF2.

miR-939-3p enhances sarcoma growth in vivo
To further examine the role of miR-939-3p, in vivo experiments utilizing xenograft nude mouse models were conducted.HT-1080 cells stably infected with lentivirus expressing miR-939-3p or control were injected into the mice separately.Compared to the control mice, the miR-939-3p group exhibited larger tumor volumes and weights (Figures 6A-C).As anticipated, miR-939-3p expression was significantly higher in the xenografts of miR-939-3p group, whereas the expression levels of BATF2 mRNA and protein were much lower, compared to the control mice (Figures 6D-G), while immunohistochemical staining of Ki67, a biomarker of cell proliferation, showed that Ki67 expression was much higher in tumor xenografts from miR-939-treated mice than that in control mice (Figure 6H).These data revealed that miR-939-3p enhanced sarcoma growth in vivo.

Discussion
Sarcoma is a type of mesenchymal neoplasm exhibiting distinct prognosis, clinical behavior and treatment options (1,28,29).Sarcoma is regarded as a formidable type of malignancy to treat, as conventional chemotherapies have yielded frustratingly slow while immunotherapy remains an investigational approach, making sarcoma one of the most difficult tumors to treat (4).Due to its rarity and histologic heterogeneity, the understanding of sarcoma biology is also relatively limited, compared with that of common epithelial cancers (1).Notably, the clinical behavior of sarcoma results from interactions at many levels, among which the oncogenes and tumor suppressor genes are crucial driving force for tumorigenesis and progression (8,30).We and others have previously found that BATF2 is an important tumor suppressor in many cancers (9)(10)(11)(12)(13)(14)(15)(16)(17)(18), although its role in sarcoma remains unknown.In this study, we found that BATF2 was downregulated in sarcoma tissues and cells, correlating with a poor prognosis of sarcoma patients.Further investigation revealed that BATF2 inhibited the proliferation of sarcoma cells, although the regulatory mechanism and its role in sarcoma remain unclear.
BATF2 belongs to the activator protein 1 (AP-1) family and inhibits AP-1 activity mainly through interacting with AP-1 via its bZIP domain, thereby leading to tumor repression (10-13).It has been reported that BATF2 is primarily expressed in normal cells but not in the corresponding tumor cells, while the upregulation of BATF2 has been shown to inhibit cancer cell proliferation, angiogenesis, invasion and metastasis (13,14), underscoring its importance in cancer therapy.Because of the myriad of tumor suppressor functions of BATF2, it is imperative to elucidate the mechanisms underlying how BATF2 is regulated during tumorigenesis and development.We and others have recently explored the regulators of BATF2, including transcriptional factors, N6-methyladenosine modification and nuclearcytoplasmic translocation (9,10,15).Our previous studies have reported glucocorticoid receptor, a transcriptional factor, which suppresses lymphoma cells through inducing BATF2 expression (10).We have also found that repressing the nuclear export of BATF2 by mutating its nuclear export sequence and suppressing the expression of chromosome region maintenance 1 (CRM1) might serve as potential therapeutic approaches for patients with colorectal cancer (9).Nevertheless, these studies have still not clarified the reason for the downregulation of BATF2, especially in sarcomas.
In this study, the bioinformatic analysis and experimental verification identified miR-939-3p as a candidate regulator of BATF2 in sarcoma tissues and cell lines, and upregulation of miR-939-3p was associated with a poor prognosis of sarcoma patients, suggesting its role as a potent prognostic biomarker and therapeutic target during sarcoma progression.As reported, play a vital role in gene regulatory networks, and have gradually been demonstrated as promising diagnostic and prognostic biomarkers in human cancers, as well as potential targets for cancer therapy (22,23,31).More importantly, accumulated studies have established the therapeutic potential of miRNA mimics or antagomirs against cancer (32)(33)(34)(35)(36). Specifically, miR-939-3p is involved in the pathological processes of tumorigenesis and progression (37-39).In ovarian cancer, miR-939 was demonstrated to promote tumor proliferation by repressing the expression of adenomatous polyposis coli 2 (APC2) (37).In hepatocellular carcinoma, miR-939-3p induced epithelialmesenchymal transition via targeting estrogen receptor 1 (ESR1) (38).Besides, miR-939-3p was identified as a potential independent prognostic marker in lung cancer (39).Although miR-939-3p has been studied in several cancers, its contribution to sarcoma proliferation had not been investigated.This work demonstrated for the first time that upregulation of miR-939-3p promoted sarcoma cell proliferation in vitro and enhanced tumor growth in vivo.Notably, clinical survival analysis revealed a negative correlation between miR-939-3p and BATF2 expression levels, and subsequent mechanistic studies illustrated that miR-939-3p suppressed BATF2 expression via binding to its 3' UTR region.It has also been reported that miR-939 could induce mRNA degradation of its target gene (40), further supporting our results.These findings may shed new light on the oncogenic role of miR-939-3p targeting BATF2 in sarcomas.
Moreover, this study also found that circulating miR-939-3p levels were higher in tumor patients than that in healthy individuals.Due to their stable existence in circulation and enormous potential as non-invasive early detection biomarkers for cancer (22,23), exploring the role of circulating miR-939-3p as a biomarker for sarcomas is also an interesting topic.Besides, it has been reported that miRNA-765 mediated multidrug resistance (MDR) via targeting BATF2 in gastric cancer cells (19).To investigate the roles of miRNA in chemo-resistance, a previous report have examined the miRNA expression profiles on 4 responder and 4 non-responder colon cancer to chemotherapy (41).The miRNA profiling data indicated that miR-939 was one of the most upregulated miRNAs in responder, compared with nonresponder.However, subsequent screening and validation experiments excluded miR-939, since there was no significant difference in the expression level of miR-939 between nonresponder and responder colon cancer patients (41).Therefore, it would be also interesting to explore the association between miR-939-3p and MDR in the future studies.
In summary, this study identified BATF2 as the target gene of miR-939-3p in sarcoma, and revealed that both upregulated miR-939-3p and downregulated BATF2 expression levels were significantly associated with a poor prognosis of sarcoma patients.Further mechanistic studies demonstrated that miR-939-3p promoted sarcoma cell proliferation via binding to the 3' UTR of BATF2 (Figure 7).These findings suggest that suppressing miR-939-3p or inducing BATF2 expression may represent a new therapeutic strategy for sarcoma treatment, which may provide novel insights for the development of potent therapeutic approaches for sarcoma patients.A proposed model elucidating the mechanism of miR-939-3pinduced sarcoma proliferation and poor prognosis through suppressing BATF2 via directly binding to its 3' UTR.
FIGURE 6 miR-939-3p enhances sarcoma growth in vivo.Xenograft nude mouse models were constructed by subcutaneously injecting 5 × 10 6 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.
The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This work was supported by Natural Science Foundation of Chongqing Science & Technology Commission (CSTB2022NSCQ-MSX0220) and National Natural Science Foundation of China (No. 82322012, 82170705, 81802783).