MicroRNA-140-5p regulates osteosarcoma chemoresistance by targeting HMGN5 and autophagy

Chemotherapy is an important treatment modality for osteosarcoma. However, it often fails because of chemoresistance, especially multidrug resistance. Previously, we found several genes were involved in chemoresistance development. In this report, we used high-throughput microRNA (miRNA) expression analysis to reveal that expression of miR-140-5p was associated with chemosensitivity in osteosarcoma. The exact roles of miR-140-5p in the chemoresistance of osteosarcoma were then investigated, we found that knockdown of miR-140-5p enhanced osteosarcoma cells resistance to multiple chemotherapeutics while overexpression of miR-140-5p sensitized tumors to chemotherapy in vitro. Moreover, in vivo, knockdown of miR-140-5p also increased the osteosarcoma cells resistance to chemotherapy. Luciferase assay and Western blot analysis showed that HMGN5 was the direct target of miR-140-5p which could positively regulated autophagy. Silencing these target genes by siRNA or inhibition of autophagy sensitized osteosarcoma cells to chemotherapy. These findings suggest that a miR-140-5p/HMGN5/autophagy regulatory loop plays a critical role in chemoresistance in osteosarcoma. In conclusion, our data elucidated that miR-140-5p promoted autophagy mediated by HMGN5 and sensitized osteosarcoma cells to chemotherapy. These results suggest a potential application of miR-140-5p in overall survival, chemoresistance prognosis and treatment.


Results
miR-140-5p lowly expressed in osteosarcoma tissues after chemotherapy. To screen miRNAs that are potentially involved in chemoresistance, miRNA microarray analysis was carried out with a chemoresistance cohort (n = 8, T) and tumor stage-matched chemosensitive control cases (n = 7, C). Totally, 24 miRNAs showed significant differential expression, among which 9 miRNAs were down-regulated and 15 miRNAs were up-regulated (Fig. 1A). miR-140-5p, miR-3395, miR-611 and miR-1 was the top four down-regulated miRNAs in these down-regulated miRNAs. To further confirm the miRNA microarray results, qRT-PCRs were performed and showed that miR-140-5p frequently lowly expressed in chemoresistance cohort (Fig. 1B). However, the other three downregulated miRNAs did not consistently lowly expressed in chemoresistance group. These results suggested that miR-140-5p played an important role in chemoresistance and further functional study was conducted to prove the correlation between miR-140-5p and clinical outcomes.  Table 1. We analyzed the associations between clinicopathologic characteristics and miR-140-5p expression levels in 40 patients. Totally, the relative level of miR-140-5p expression in osteosarcoma tissues (mean ± SD: 1.36 ± 0.02) was significantly lower than that in matched adjacent nontumor tissues (ANCTs) (mean ± SD: 2.52 ± 0.03; p < 0.001, Fig. 1C). Furthermore, the median value of miR-140-5p expression was used as the cut-off point for dividing samples into two groups: moderate expression group (>median, n = 20) and low expression group (<median, n = 20). We found a statistically significance between miR-140-5p expression and some clinical features of osteosarcoma (Table 1), including tumor size (p = 0.029), clinical stage (p = 0.016), recurrence (p = 0.023) and distant metastasis (p = 0.016). No significant difference was observed between the expression of miR-140-5p with patients' age (p = 0.945) and gender (p = 0.058).
Kaplan-Meier analysis revealed that patients with moderate miR-140-5p expression survived significantly longer than those with low miR-140-5p expression by using the log rank test (Fig. 1D, p = 0.027). Our results suggested that miR-140-5p is closely correlates with multiple clinical features in osteosarcoma. miR-140-5p regulated the sensitivity of osteosarcoma cells to chemotherapeutic agents in vitro by targeting HMGN5. To confirm the results in Fig. 1, miR-140-5p expression was examined in four osteosarcoma cell lines using quantitative real-time PCR (qRT-PCR). qRT-PCR results showed that compared with hFOB 1.19 cells, miR-140-5p was significantly down-regulated in osteosarcoma cell lines ( Fig. 2A).
HMGN5 is a member of the HMG box family and acts as damage associated molecular pattern molecule. HMGN5 is also involved in autophagy in osteosarcoma as shown by our previous report 19 . To further explore the function of miR-140-5p, the markers of autophagy were also assessed in U2-OS/miR-140-5p cells. BECN-1, ATG5 and LC3-II/I proteins expression levels were significantly decreased in U2-OS/miR-140-5p cells compared with U2-OS cells, Whereas p62 were significantly increased in U2-OS/miR-140-5p cells. Interestingly, we found that the overexpression of HMGN5 led to the increased expression level of BECN-1, ATG5, LC3-II/I proteins and a decrease of p62 protein, whereas the expression of Atg12 and ATG7 was not markedly altered by HMGN5  revealed a marked accumulation of autophagosomes in the cytoplasm of U2-OS/KD cells compared with U2-OS cells (Fig. 4A). Autophagic vacuoles are denoted by arrows.
Next, we investigated the effect of miR-140-5p on the expression of LC3-I and LC3-II. Results showed that miR-140-5p overexpression significantly reduced the conversion of LC3-I to LC3-II, while knocking down of miR-140-5p induced autophagy, with an increase in the conversion of LC3-I to LC3-II (Fig. 4B). To further confirm the increased autophagic flux, we examined changes in autophagic flux by comparing the levels of LC3-II in the presence and absence of the lysosome inhibitor chloroquine (CQ) and Bafilomycin A1 (Baf A1). Increased LC3-II expression and an accompanying increase in the conversion of LC3-I to LC3-II were clearly detected in U2-OS/KD compared with U2-OS cells (Fig. 4C). More importantly, CQ or Baf-A1 treatment significantly increased endogenous LC3-II accumulation (Fig. 4C). Therefore, the conversion of LC3-I to LC3-II was up-regulated after CQ or Baf-A1 treatment, confirming increased autophagic flux in U2-OS/KD cells. To further validate these results, we established a U2-OS/KD cell model that stably expresses an mRFP-GFP-LC3 fusion protein. U2-OS/KD cells showed a higher mRFP-GFP-LC3 signal than parental U2-OS cells, indicating that autophagy is enhanced when osteosarcoma cells knocking down of miR-140-5p (Fig. 4D). Inhibition of autophagy restored the chemosensitivity of U2-OS/KD. We have confirmed that overexpression of HMGN5 decreased the sensitivity of U2-OS/miR-140-5p and MG63/miR-140-5p cells to anticancer agents (Fig. 3C,D), as well as increased autophagy (Fig. 3E). The next question was to investigate whether autophagy truly contributed to miR-140-5p down-regulation mediated chemoresistance in osteosarcoma cells. Autophagy was inhibited by knocking-down of ATG5 (Fig. 5A, left panel) or BECN-1 (Fig. 5A, right panel), and then the effects of chemotherapy were assessed. As shown in Fig. 5B, MDC staining indicated that autophagy was markedly decreased in both U2-OS/KD and MG63/KD cells that transfected with siRNAs targeting BECN-1 or ATG5 (Fig. 5B). Consistently, decreased conversion of LC3-I to LC3-II proteins were also observed in siATG5 or siBECN-1 cells (Fig. 5C). Fig. 5D showed that either knocking down BECN-1 or ATG5 enhanced the sensitivity of U2-OS/KD and MG63/KD cells to doxorubicin (Dox) (Fig. 5D). Consistently, knocking down of BECN-1 or ATG5 decreased the IC50 values for the three chemotherapeutic agents (Fig. 5E). These data suggested that U2-OS/KD and MG63/KD cells exhibit chemoresistance by up-regulating autophagy. miR-140-5p was associated with chemoresistance and increased the chemoresistance. To further verify the function of miR-140-5p in clinical samples, qRT-PCR and immunohistochemistry were used to detect miR-140-5p and the expression of HMGN5 and BECN-1 expression was detected in tissues from 15 cases of patients with relapsed osteosarcoma by immunohistochemistry (chemoresistant) and 15 cases of chemosensitive patients. We found that the Expression levels of miR-140-5p were decreased in 15 patients with chemoresistant, and the expression of HMGN5 and BECN-1 were significantly up-regulated in these chemoresistant patients compared with chemosensitive patients (Fig. 6A,B).
Moreover, to further determine the effects of miR-140-5p on tumor chemoresistance, nude mice were subcutaneously injected with U2-OS/KD cells or control cells (Blank) and the animals were monitored closely for tumor growth. As shown in Fig. 6C, there was no significant difference in tumor size between miR-140-5p-knocking-down cells (U2-OS/KD) and those in cells expressing the control vector within 2 weeks. Importantly, after expose to Dox for 3 weeks, miR-140-5p-knocking-down tumors were bigger in size and heavier in weight compared to the controls (Fig. 6D).

Discussion
In this study, by using miRNA microarrays analysis, we identified 23 differentially expressed miRNAs in human osteosarcoma samples compared with normal bone samples. We found that miR-140-5p frequently highly expressed in human osteosarcoma samples. Moreover, the correlation between the expression of miR-140-5p and the tumor size, clinical stage, recurrenceand distant metastasis of osteosarcoma was also analyzed. To further investigate the role of miR-140-5p, gain-and loss-of-function approaches revealed that miR-140-5p in osteosarcoma enhanced the chemoresistance by regulating autophagy. The function of miR-140-5p was further confirmed in mouse tumor models. In addition, we discovered a novel mechanism of autophagy regulated by non-coding miR-140-5p in osteosarcoma through direct binding at the 3′-UTR of the HMGN5 mRNA. HMGN5, which widely expressed in eukaryotes, regulates the growth, and differentiation of various types of cells 31 and our previous results demonstrated that HMGN5-mediated autophagy contributed to chemoresistance in osteosarcoma cells. Our results extended previous observations and found that miR-140-5p in osteosarcoma enhanced the chemoresistance by up-regulating HMGN5-mediated autophagy, suggesting that miR-140-5p is a potential target of an adjuvant therapy for osteosarcoma patients.
Previous studies demonstrated that miR-140-5p functions as both a tumor suppressor and a tumor promoter in cancers. Recent research found that miR-140-5p serves as a tumor suppressor by inhibiting ovarian cancer via repression of PDGFRA 25 . However, our studies found that miR-140-5p facilitated the chemoresistance of osteosarcoma through regulating HMGN5-mediated autophagy, promoting tumor in osteosarcoma. This result suggests that the function of miR-140-5p may be disease specific. Consistent with our findings, miR-140-5p has also been reported to participate in the regulatory network for cancer invasion in other cancer types 28,32 . We showed that HMGN5 is the direct target of miR-140-5p and overexpression of miR-140-5p in osteosarcoma cell lines increased the osteosarcoma cell chemoresistance. Our studies further revealed that the increase of miR-140-5p-mediated chemoresistance was directly due to the up-regulation of HMGN5. By utilizing siRNA against HMGN5, we found that i depletion of HMGN5 could reverse the effect of miR-140-5p and lead to increase of chemosensitivity in osteosarcoma cell lines. Consistently, miR-140-5p has been reported to participate in the regulatory network of autophagy in colorectal cancer cells 27 .
The clinical relevance of miR-140-5p was clearly demonstrated by quantification of the miR-140-5p' expression levels in a set of 40 osteosarcoma patient samples of primary tumor tissues and ANCT. As expected, we found that miR-140-5p was progressively up-regulated in osteosarcoma tumor tissues and further increased in the chemoresistance osteosarcoma patients, suggesting that miR-140-5p is significantly correlated with osteosarcoma chemoresistance. In addition to HMGN5 and chemoresistance, our work revealed a novel regulatory function of miR-140-5p in osteosarcoma and autophagy.
Autophagy, a mechanism that delivers aging organelles and damaged components to lysosomes for degradation, could facilitate cell survival by supplying nutrients to stressed cancer cells 33 , and promote cancer invasion through the TGF-β/Smad2 signaling pathway 27,34,35 . Furthermore, mounting evidences had suggested that activation of autophagy promoted chemoresistance 8,36,37 . On the other hand, we found that siRNA knocking down of BECN-1 or ATG5 could reduce the chemoresistance and reversed the effect of miR-140-5p, suggesting an important role of autophagy on chemoresistance and miR-140-5p increased the chemoresistance through miR-140-5p/ HMGN5/autophagy regulatory loop.
Many studies have been carried out to investigate the potential role of miRNAs in osteosarcoma, which provides new anti-osteosarcoma therapeutic regimens. For example, researchers found that low expressions of miR-133b/miR-503 were correlated with shorter overall survival of osteosarcoma patients 38 . miR-20a promotes the proliferation of human osteosarcoma cells by suppressing growth response 2 expression 39 . More importantly, it is reported that transfection of miR-202 mimics into osteosarcoma cells significantly promotes chemoresistance by targeting PDCD4 while transfection of miR-202 inhibitor enhances the chemosensitivity. In addition, it had been shown that several miRNAs regulated cancer progression through autophagy, such as miR-502 40 , miR-30a 41 , miR-34a 42 , miR-4487 and miR-595 43 .
Our research is the first evidence demonstrating that miR-140-5p has an effect on chemoresistance of osteosarcoma cells. The Kaplan-Meier analysis in our study revealed that the prognosis of osteosarcoma patients was significantly related to the miR-140-5p expression level. However, these results just provide very limited evidence due to a high variability in miRNA expression levels, inter-individual and race variation. Furthermore, the use of osteosarcoma cells from two cell line still very limited. Similarly, the lentiviral based delivery approach is also not ideal for therapeutic development., To confirm the role of miR-140-5p, we should use more cell lines and primary tumor in the future. On the other hand, we need to develop a more effective and optimized method to deliver miR-140-5p inhibitor in vivo. In addition, much more work need to be performed to elucidate the reason that promoted down-regulation of miR-140-5p. Our further study aimed to clarify the underlying mechanism of dysregulation of miR-140-5p in osteosarcoma chemoresistance. Further study, which using more cell lines and primary tumor is therefore indispensable to confirm the findings of this study.
In conclusion, we found that miR-140-5p promoted chemoresistance through the up-regulation of HMGN5, which is a key player in transcriptional activation by interacting with nucleosomes 44 . miR-140-5p promoted chemoresistance by regulating HMGN5-mediated autophagy in vitro and vivo. We identified a novel mechanism of miR-140-5p as a critical regulator of autophagy in osteosarcoma. As many miRNA based therapeutics are currently in clinical trials, our results suggest that hsa-miR-140-5p could be a valuable chemotherapy strategy for treating osteosarcoma patients.

Materials and Methods
Ethics approval. All research involved human tissue samples was approved by the Ethics Review Committee of Second Military Medical University Shanghai and written informed consent was obtained from all participating patients. All participants provided written informed consent for participation in this study and the methods were carried out in accordance with the approved guidelines. All animal experiments were carried out in "accordance" with the relevant guidelines.
Tissue specimens. Forty osteosarcoma patients who received neoadjuvant chemotherapy and underwent surgery were included in Shanghai Changzheng Hospital between January 2011 and January 2014. Eligibility criteria for entry onto the study were diagnosis of primary, central, high-grade osteosarcoma of the extremity who received neoadjuvant chemotherapy and underwent surgery; and normal hepatic, renal, bone marrow, and cardiac function. The TNM and histological classification were performed according to World Health Organization criteria. After screening, the chemoresistance cohort consists of eight osteosarcoma patients who showed <90% tumor necrosis (mean 58.2% ±3.2%) after neoadjuvant chemotherapy and were defined as poor responders. Another tumor stage-matched osteosarcoma patients, who showed ≥90% tumor necrosis (mean 96.1% ± 4.1%) Scientific RepoRts | 7: 416 | DOI:10.1038/s41598-017-00405-3 as good responders 45 , were enrolled as chemosensitive controls (n = 7). All patients were treated preoperatively with neoadjuvant chemotherapy, the cumulative doses of medicines as follows: doxorubicin 420 mg/m 2 , methotrexate 120 g/m 2 , cisplatin 600 mg/m 2 . All tumor specimens and the adjacent non-cancer tissues (ANCT) were immediately frozen in liquid nitrogen after resection and stored at −80 °C. All 40 osteosarcoma patients received follow-up and the median follow-up was 32 months (range, 6-38 months). miRNA Microarrays analysis. miRNA microarrays analysis was outsourced to RiBio Cor (Ribobio, Guangzhou, China). Briefly, total RNA extract was purified using mirVana ™ miRNA Isolation Kit (Ambion, USA) from the tumor specimen before and after chemotherapy and then labeled and hybridized using the Agilent miRNA Complete Labeling and Hybridization Kit (Agilent Technologies, San Diego, USA). The labeled RNA was performed with the Axon GenePix 4000B microarray scanner. miRNA signal intensities were log2 transformed, and analyzed for differentially expressed miRNAs by using the significance analysis of microarrays (SAM, version 3.01). We selected miRNAs whose expression levels changed at least two fold between the tumor specimen before and after chemotherapy for further analysis.

The 50% cytotoxic concentration (IC50) values in osteosarcoma cells. The four osteosarcoma
cell lines (mimic NC, miR-140-5p mimics, anti-NC or anti-miR-140-5p) were seeded into wells of a 96-well plate at a density of 1 × 10 4 cells/well in medium containing 10% FBS. After 24 hrs incubation, cells were washed twice with the medium and exposed to a range of chemotherapeutic agents for 48 hrs. Subsequently, cells were detached by trypsinization and the viable cell population was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide. The 50% cytotoxic concentration (IC50) values were defined as the chemotherapeutic agents concentration producing 50% inhibition of cell growth. Similarly, the IC50 values of transfected cell lines (miR-140-5p, HMGN5 and miR-140-5p/HMGN) and controls (untransfected) were also determined as described above.
Quantitative real-time PCR (qRT-PCR). Total RNA was extracted from cells or surgical specimens using TRIzol reagent (Life Technology, CA, USA). To detect the expression of miR-140-5p, qRT-PCR was performed using a Reverse Transcription Kit Ribobio (Ribobio, Guangzhou, China) according to the manufacturer's instructions. Specific qRT-PCR primers for miR-140-5p and U6 snRNA were purchased from Ribobio (Ribobio, Guangzhou, China). Each sample was analyzed in triplicate. U6 snRNA was used as the endogenous control. The fold-change in gene expression was calculated by 2 −ΔΔCT method.
Luciferase reporter assay. Luciferase reporter assay was performed as described previously 47 . The PCR fragment of the 3′UTR of HMGN5 containing predicted binding sites of miR-140-5p was cloned from human genomic DNA and inserted into pGL3-control vector (Promega Corporation, WI, USA). 24 hrs before transfection, 1.5 × 10 4 U2-OS cells were plated in a 96-well plate. 10 pmole of miR-140-5p mimics, mimic NC, miR-140-5p inhibitor, Anti-miR-140-5p or Anti-NC was transfected into cells together with 100 ng of pGL3-3UTR HMGN5 and 1 ng of Renilla luciferase plasmid pRL-SV40 (Promega Corporation, WI, USA). Luciferase assays were performed 24 hrs after transfection by the dual-luciferase reporter assay system. Western blots. Cells were collected and lysed in 1 × RIPA buffer (Sigma, MO, USA) and then subjected to Western blot as described previously 46 . 80 µg of each protein extract was separated by 6~15% SDS-PAGE gels and transferred to nitrocellulose membrane. The immunoreactive bands were firstly incubated with the primary antibodies, including HMGN5 Electron microscopy. Cells were fixed in 2.5% glutaraldehyde for 2 hrs at room temperature, then incubated in 1% osmium tetroxide for 1 hr. After dehydration in an increasing concentration gradient of ethanol, the samples were embedded in Durcopan ACM for 6 hrs, then samples were cutted into 50-nm sections and stained with Scientific RepoRts | 7: 416 | DOI:10.1038/s41598-017-00405-3 3% uranyl acetate and lead citrate. Images were generated using a transmission electron microscope (Philips CM, Amsterdam, Netherlands).
Stably expressing of mRFP-GFP-LC3. Adenovirus vector containing the mRFP-GFP-LC3 reporter was purchased from Hanbiology (Hanbio, Shanghai, China). Autophagy level was examined as previously described using confocal microscopy (Olympus, Tokyo, Japan) 19 . Briefly, up to 24 hrs recovery after transfection, the cells samples were treated according to the protocol and then analyzed using confocal microscopy.
Immunofluorescence staining. Immunofluorescence staining was performed following standard protocol. Briefly, U2-OS cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 and then blocked with 10% goat serum. Subsequently, the cells were then incubated with primary antibodies recognizing HMGN5 (1:100; Abcam, Cambridge, UK) overnight, and then incubated with AffiniPure donkey anti-rabbit IgG (1:200, red fluorescence) for 1 hr at room temperature. 4′,6-diamidino-2-phenylindole (DAPI, Beyotime Institution of Biotechnology, Haimen, China) was applied to stain the nuclei for 5 mins. The gene expressions were analyzed using confocal microscopy. Apoptosis assays. Cells in each group were treated with Dox (1 µM), Cis (20 mmol/L), or Mtx (50 mmol/L) for 12 hrs and the apoptosis level was analyzed by Flow cytometry. Flow cytometry was performed as described previously 46 . Harvested cells were resuspended in 500 µL binding buffer and then stained with 5 µL FITC-Annexin-V (BD Biosciences, CA, USA) and 5 µL propidium iodide (BD Biosciences, CA, USA) for 30 mins in the dark at 4 °C. Cells were analyzed by flow cytometry within 1 hr.
Immunohistochemical analysis (IHC). Tissue specimens were immediately obtained from patients with relapsed osteosarcom at Shanghai Changzheng Hospital and sent to a pathology department for diagnosis by three pathologists based on histopathological evaluation. As a result, 15 cases of chemoresistance and 15 cases of chemosensitive patients were enrolled in this program. For IHC analyses, paraffn-embedded samples were deparaffnized with xylene and rehydrated through addition of graded ethanol. The samples were incubated with the antibody at a dilution of 1:100 overnight, followed by incubation with biotinylated secondary antibody (1:500 dilutions). Finally, the immunohistochemistry results were scored by both the percentage of positive detection and intensity of the staining.
In vivo Chemosensitivity assay. 6-week-old female nude mice were maintained under specifc pathogen-free conditions at Second Military Medical University Shanghai. Animal experiment was performed to evaluate the tumor growth and the sensitivity of osteosarcoma cells to chemotherapy in vivo. In brief, 1.0 × 10 7 U2-OS cells stably transfected with Lenti-miR-140-5p (U2-OS/KD) or lenti-NC (Blank) were harvested by treatment with trypsin-EDTA (Invitrogen) and resuspended in PBS. Then osteosarcoma cells suspension (U2-OS/KD group) were subcutaneously transplanted in the left proximal tibia of each anesthetized nude mice (n = 6 animals/ group) and the Blank cells group were injected into in the right proximal tibia (n = 6). Two weeks later, the mice were intraperitoneally injected with PBS containing doxorubicin (5 mg/kg) twice per week according to a previous study 49 . After inoculation, tumor size was measured every 7 days, and tumor volumes (mm 3 ) were calculated using the formula: V = length × width 2 /2. The mice were sacrificed on day 35, and the tumors were measured and photographed 35 days after inoculation. All animal experiments were approved by the Animal Care and Use Committee of Second Military Medical University Shanghai.