Metformin Sensitizes Osteosarcoma Cells to Chemotherapy Through IGF-1R/miR-610/FEN1 Pathway


 Background: Due to constitutive or acquired non-sensitive to cytotoxic agents, the prognosis of osteosarcoma remains unfavorable. It’s has been proved that metformin could enhance the chemosensitivity of cancer cells to anticancer drugs. A novel finding states that IGF-1R involves in cancer chemoresistance, However, whether IGF-1R play a role in metformin-induced osteosarcoma chemosensitivity is incompletely understood. Hence, the current study aimed to elucidate the role of metformin in OS cell chemosensitivity modulation to identify the underlying mechanism of metformin regulating the IGF-1R/miR-610/FEN1 signaling.Methods: Immunohistochemistry and qRT-PCR were used to evaluate the expression pattern of IGF-1R, miR-610 and FEN1 in osteosarcoma and paired normal tissues. Western blot and qRT-PCR were performed to determine changes in expression of key molecules in the IGF-1R/miR-610/FEN1 signaling pathway after various treatments. The direct modulation between miR-610 and FEN1 was monitored by luciferase reporter assay. Osteosarcoma cell sensitivity to chemotherapy was detected by MTS assay. In vivo experiments were conducted to further verify the role of the metformin in the chemosensitivity modulation of OS cells to ADM.Results: We found that IGF-1R, miR-610 and FEN1 were abberently expressed in osteosarcoma, and participated in apoptosis modulation (p < 0.05). We found that this effect was abated by metformin treatment. Luciferase reporter assays confirmed that FEN1 is a direct target of miR-610. Moreover, we observed that metformin treatment decreased IGF-1R and FEN1, but elevated miR-610 expression. Metformin sensitized OS cells to cytotoxic agents, while overexpression of FEN1 compromised the sensitizing effects of metformin partly. Furthermore, metformin was observed to enforce the ADM treatment effect in nude mice xenograft models.Conclusions: Overall, metformin enhanced the sensitivity of OS cells to cytotoxic agents via the IGF-1R/miR-610/FEN1 signaling axis, highlighting the capacity of metformin as an adjunct to the chemotherapy of OS.


Introduction
Osteosarcoma (OS), the most frequent primary malignant bone tumor in children and the most common primary malignancy in adolescents apart from leukemia and lymphoma 1 . Currently, the combination of limb salvage and neoadjuvant chemotherapy are still major treatments for osteosarcoma patients, despite many efforts have contributed to treatment outcomes, its rate of death remains high by now 2 .
Several high doses of anticancer drugs such as Adriamycin (ADM), cisplatin (DDP), and methotrexate (MTX) were most used to treat osteosarcoma 3 , but systemic toxicity and a risk of secondary cancer are concerns [4][5][6] . Moreover, constitutive or acquired non-sensitive compromised the effective of the majority of chemotherapeutics 7 .
Metformin (Met) is the most widely used drug in the treatment of type 2 diabetes with safe effects of insulin resistance reduction and blood glucose decrease 8 , has recently emerged as a potential of tumor prevention and treatment [9][10][11] . It has been reported that varied mechanisms underlying the antitumor effect of metformin, including inhibition of gluconeogenesis and oxidative phosphorylation, affecting cell growth, mobility, apoptosis, stemness and autophagy, etc 9,12,13 . More importantly, several studies have shown that metformin could enhance the cytotoxic effect in combine with various cytostatic drugs 14,15 .
However, the mechanisms of these cooperative effects are still unclear.
Recent study showed that metformin could overcome primary resistance to EGFR-TKIs with EGFR mutation via targeting Insulin-like growth factor 1 receptor (IGF-1R) signaling pathway 16 . IGF-1R is a transmembrane tyrosine kinase receptor in the insulin receptor family, which expression is upregulated in OS, and the upregulation of IGF-1R is associated with surgical stage and metastasis. What's more, IGF-1R blockade increases chemo-sensitivity in multidrug-resistant osteosarcoma cell lines 17 .
The aim of the present study was to elucidate the potential role of metformin in OS cells chemosensitivity modulation and the underlaying mechanisms. We show that metformin enhances the anti-OS agents' cytotoxic effects by modulate IGF1R-miR610-FEN1 signaling pathway.

Ethics statement
The present study was approved by the Institutional Ethics Committee of The Third A liated Hospital of Kunming Medical University. Informed consent was obtained from each patient for the use of their tissues for research purposes, in accordance with the Declaration of Helsinki. Animal experiments were performed according to a strictly designed protocol, in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All efforts were made to ensure minimal suffering of the animal included in the study.

Tissue samples
Paired tumor and adjacent normal formalin-xed para n-embedded (FFPE) samples were obtained from 66 patients, collected between 2015 and 2019. The miRNeasy FFPE Kit (Qiagen, Germany) was used to extract RNA from para n-embedded specimens. Patients with a diagnosis of relapse and who had received preoperative radiation, chemotherapy, or biotherapy were excluded from the study to avoid changes in tumor marker determination resulting from treatment.

Cell culture, transfection, antibodies and reagents
The human MG-63, U2OS and 143B cell lines were provided by American Type Culture Collection, ATCC.
Cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scienti c, USA.) and maintained at 37 °C in a humidi ed incubator with 5% CO 2 . The miR-610 mimic, miR-610 inhibitor, and negative control were designed and synthesized by Guangzhou RiboBio Co., Ltd (Guangzhou, China). The small interfering RNA (siRNA) targeting FEN1 and IGF-1R (si-FEN1, si-IGF1R) and negative control (NC) siRNA were also synthesized by Guangzhou RiboBio Co., Ltd. All plasmid constructs were veri ed by sequencing. Lipofectamine® 2000 (Invitrogen, USA) was used for miRNA or siRNA transfection according to the manufacturer's protocols. Cells were harvested for subsequent experiments 48 h after transfection or anti-cancer drugs treatment. Metformin, ADM, DDP and MTX were purchased from Sigma, (St. Louis, MO, USA). Working dilutions of all drugs were prepared immediately before use. The antibodies to FEN1, IGF-1R, cleaved caspase-3 and GAPDH were obtained from Abcam.
Luciferase reporter assay FEN1 3′-untranslated region (3′-UTR) containing the wild type or mutated miR-610 binding sequences were cloned into the pGL3-basic luciferase reporter vector (Promega, USA), named FEN1 WT and FEN1 MUT, respectively. Brie y, OS cells were seeded into a 24-well plate at a density of 1 × 10 5 cells/well. The cells were co-transfected with FEN1 WT or FEN1 MUT and the miR-610 mimic or NC miRNA by using Lipofectamine 2000 (Invitrogen, USA). The luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega, USA) and the activity of re y luciferase was normalized to the corresponding Renilla luciferase activity.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted using TRIzol Reagent (Invitrogen, USA) and puri ed with the RNeasy Maxi kit (Qiagen, Germany). The isolated total RNA was reverse transcribed using an miScript II RT Kit (Qiagen, Germany) to quantify both the mRNAs and miRNAs in the total RNA sample in a Roche Lightcycler 480 Real-Time PCR system (Roche Diagnostics, Switzerland). The relative miR-610 expression levels in tissue specimens and cells were calculated using the 2 −ΔΔCt method 18 , mRNA and miRNA expression were normalized to GAPDH and U6.. The primer used are depicted in Table 1. Table 1 Primer sequences for RT-qPCR.

Western blotting
Protein was extracted with RIPA lysis buffer (89900, Pierce, USA) following the protocol. Protein concentrations were measured using the BCA assay (23227, Thermo, USA). For each sample, 50 µg of protein lysate was loaded per well. Samples were electrophoresed on SDS-PAGE gels and transferred onto polyvinylidene uoride (PVDF) membranes by electroblotting. The membranes were pretreated with 5% nonfat milk in TBS-T for 2 h, followed by incubation with primary antibodies at 4 °C overnight. The following primary antibodies were used: anti-FEN1, ab17994; anti-IGF-1R, ab263903; and anti-Cleaved Caspase-3, ab49822, all from Abcam, USA. The membranes were then incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000, #7076, Cell Signaling Technology, USA) for 1 h.
GAPDH was used as the internal loading control (1:1000, ab181602; Abcam). All the samples were assayed three times.

Apoptosis analysis
Cell apoptosis was analyzed using an Annexin V-uorescein isothiocyanate (FITC) Apoptosis kit (BD Biosciences, USA) following the manufacturer's protocol. Cells were seeded into 24-well plates (1 × 10 5 cells/well) and cultured for 24 h. Cells were collected and washed twice with cold PBS, and stained with 1% FITC-labeled Annexin V and propidium iodide. After incubation apoptosis levels were evaluated using the FACS Aria system (BD Immunocytometry Systems, USA) and analyzed by Cell Quest software (Becton Dickinson Ltd). All experiments were performed three times independently.

MTS assay
To determine the cytotoxicity of metformin and the combined effect of metformin and chemotherapeutic agents. 5×10 3 cells were plated per well in 96-well culture plates in 150 µl of medium with serial doses of ADM, DDP or MTX, and 6 parallel wells were assigned to each group, as well as a negative control (without cells). 30 µl of MTS substrate was added to each well, and then incubated for 2 hrs in the dark, followed by ultraviolet irradiation for 15 min. The absorbance at 490 nm was measured using plate reader (BMG Labtech, Germany). The concentrations required to inhibit growth by 50% (IC50) were calculated using the Bliss method 19 . All experiments were performed three times independently.

Immunohistochemical (IHC) analysis
Samples were processed for IHC analysis to determine FEN1 and IGF-1R expression levels and distribution patterns. 4 µm para n-embedded tissue sections were mounted on charged glass slides and baked at 60 °C for 2 h. After depara nize in xylene, sections were microwave-treated for 10 min in citrate buffer (pH 6.0) for antigen retrieval, and endogenous peroxidase activity was blocked by incubation in 0.3% H

Statistics
The correlation between immunocytochemical labeling of FEN1, IGF-1R and other clinical pathology parameters was analyzed by the χ 2 test. Quantitative data are expressed as means ± SD relative to the control value unless indicated otherwise. A p-value < 0.05 was considered signi cant. All statistical analyses were performed in SPSS for Windows version 18.0 (SPSS Inc., Chicago, IL, USA). All assays were performed in triplicate.
Then we examined the expression levels of IGF-1R and FEN1 in 66 OS and paired normal tissues, IGF-1R positive staining was con ned mainly to the membrane and cytoplasm in OS tissues compared to a negatively stained normal tissue, and FEN1 expression mainly located in nucleus (Fig. 1C). Statistic results also con rmed the positive correlation between IGF-1R and FEN1 (p = 0.009, r = 0.319, Table. 2, 3).
In order to study the underlaying mechanisms between IGF-1R and FEN1 interaction, miRTarBase and TargetScan predict miR-610 might bind to FEN1 3'-untranslated regions (3'-UTR). Besides, dbDEMC 2.0 database analysis showed that miR-610 expression was decreased in sarcoma, osteosarcoma subtype. More importantly, we examined the expression level of miR-610 in 20 OS and paired normal tissues, con rmed a lower expression of miR-610 in OS tissues (Fig. 1D). Based on these results, we suspect IGF-1R/miR-610/FEN1 axis might participates in OS cells apoptosis and chemo-sensitivity modulation.

IGF-1R/miR-610/FEN1 axis modulates OS cells apoptosis
To further study the function of IGF-1R in OS, MG-63 and U2OS cells were transfected with IGF-1R si-RNA, both cell lines showed increased miR-610 and decreased FEN1 ( Fig. 2A-D). Then we investigated the impact of IGF-1R silencing on cell apoptosis, FACS results showed that knockdown of IGF-1R induced OS cells apoptosis signi cantly (Fig. 2E).
miRTarBase and TargetScan prediction indicated that FEN1 was a potential target for miR-610. OS cell lines were transfected either with an miR-610 mimic or with an inhibitor. miRNA RT-PCR was utilized to assess miR-610 expression following transfection (Fig. 3A). While western blotting and real-time PCR showed that the miR-610 inhibitor increased FEN1 expression. In contrast, OS cells treated with the miR-610 mimic exhibited the opposite trend (Fig. 3B, C).
In the dual-luciferase reporter assays, both FEN1 WT and MUT 3'-UTR sequences were structured based on potential binding sites. miR-610 overexpression greatly decreased luciferase activity when FEN1 WT was co-transfected into 293T cells. Whereas no alterations were detected upon co-transfection with MUTt-FEN1-3'UTR in cells. These data suggest that FEN1 is a direct target of miR-610 in OS cells (Fig. 3D).
Next, FEN1 si-RNA were transfected into OS cells (Fig. 4A). Then FACS results showed that knockdown of FEN1 induced OS cells apoptosis and elevated the expression of cleaved caspas-3 (Fig. 4B, C). These results indicated that inhibition of IGF-1/miR-610/FEN1 axis could induce OS cells apoptosis.

Metformin regulates IGF-1R/miR-610/FEN1 signal and chemosensitivity of OS cells
The mechanisms of metformin in cancer therapy are still not fully elucidated. When the OS cell lines were subjected to metformin treatment, their apoptosis rates were elevated by metformin (p < 0.05 for both MG-63 and U2OS. (Fig. 5A). Moreover, we proved that metformin treatment decreased the expression of IGF-1R and FEN1, but increased miR-610 expression (Fig. 5B, C).
To explore the effects of FEN1 in the anti-cancer activities of metformin, we constructed plasmids expressing FEN1. Transient transfection of the plasmids led to ectopic expression of FEN1 in OS cells. Then, we evaluated the sensitivity of MG-63 and U2OS cells to ADM, DDP and MTX when in addition with metformin. We found that anti-cancer drugs decreased the cell viability in OS cells but much more so in cells co-treated with metformin. More importantly, FEN1 overexpression compromised the sensitizing effects of metformin partly (Fig. 6A-C, Table 4). Collectively, these data suggest that IGF-1R/miR-610/FEN1 signal involved in metformin mediated chemosensitivity of OS cells.

Metformin elevates chemo-sensitivity of OS cells in vivo
In order to examine the effect of metformin on OS cell tumorigenicity in an in vivo model, we inoculated 143B cells subcutaneously into the right ank of female athymic nude mice. When tumor volumes reach 0.1 cm 3 , mice were injected intraperitoneally with ADM, metformin, or ADM in combined with metformin for 21 days. Metformin combined therapy resulted in a markedly reduced growth rate (p < 0.01) and tumor weight (p < 0.05) compared with chemotherapy alone. (Fig. 7A, B) These data provide in vivo evidence that metformin contributes signi cantly to the chemo-sensitivity of OS cells.

Discussion
Despite of the success of chemotherapy for osteosarcoma, a majority of patients with OS experience recurrence and poor prognosis 24 . In recent years, several targeted-or immune-therapies have been evaluated in clinical trials, but these efforts did not prolong patients' lives signi cantly [25][26][27] . Preclinical studies aimed to uncover the underlaying biological pathways implicated in chemo-sensitivity are need [28][29][30] . It's considered that IGF binding proteins are important regulators of bone metabolism and homeostasis 31 . Recently, researchers have con rmed that IGF-1R is involved in chemo-sensitivity modulation in osteosarcoma cell lines 17,32 . However, the underlaying mechanisms are still not fully demonstrated.
In present study, we predicted that FEN1 is co-expressed with IGF-1R. Then we showed that IGF-1R expression was positively correlated with FEN1. Further study con rmed that IGF-1R modulates FEN1expresison by downregulates miR-610, which bind to FEN1 5'-UTR directly. Functional experiments demonstrated that IGF-1R/miR-610/FEN1 axis is involved in OS cells apoptosis modulation. In our previous study, we have shown that FEN1 is a key regulator of OS cells chemosensitivity 33 . Moreover, it's has been con rmed that metformin could increase TKIs sensitivity by targeting IGF-1R 16 . Thus, we suspect whether metformin impacts OS cells chemosensitivity via IGF-1R pathway.
As expected, we proved that metformin decreased the expression of IGF-1R and FEN1, but increased miR-610. What's more, metformin increased OS cells chemosensitivity, FEN1 overexpression compromised the sensitizing effects of metformin partly. Finally, in vivo study also con rmed the chemo-sensitization effects of metformin.
Cytotoxic chemotherapy causes severe DNA damage directly by induction of breaks or indirectly due to the formation of reactive oxygen species (ROS) and ultimately induce cell death 34,35

Conclusions
Consequently, we are convinced that metformin enhances chemotherapy agents induced apoptosis via regulation of the IGF-1R/miR-610/FEN1 signaling axis in OS. More importantly, combine metformin with anticancer compounds is a tempting therapeutic strategy to enhance the anticancer effects, hopefully with clinical bene ts to patients.

Declarations Ethical Statement
The study was approved by the ethics committee of the Third A liated Hospital of Kunming Medical University, and all patients gave written informed consent and authorization for use of biological specimens, in accordance with the Declaration of Helsinki.

Availability of data and materials
We declare that the materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant con dentiality. expression. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.