microRNA-216b enhances cisplatin-induced apoptosis in osteosarcoma MG63 and SaOS-2 cells by binding to JMJD2C and regulating the HIF1α/HES1 signaling axis

Although cisplatin-based chemotherapy represents the standard regimen for osteosarcoma (OS), OS patients often exhibit treatment failure and poor prognosis due to chemoresistance to cisplatin. Emerging research has highlighted the tumor suppressive properties of microRNAs (miRNAs or miRs) in various human cancers via the inhibition of the histone demethylase jumonji domain containing protein 2C (JMJD2C). As a coactivator for hypoxia-inducible factor 1α (HIF1α), JMJD2C targets hairy and enhancer of split-1 (HES1) gene. Hence, the current study aimed to elucidate the role of miR-216b in OS cell cisplatin resistance to identify the underlying mechanism of miR-216b regulating the JMJD2C//HIF1α/HES1 signaling. Tumor and paracancerous tissues were collected from OS patients to determine the expression patterns of miR-216b and JMJD2C. After ectopic expression and knockdown experiments in the OS cells, CCK-8 assay and flow cytometry were employed to determine cell viability and apoptosis. The interaction of miR-216b, JMJD2C, HIF1α and HES1 was subsequently determined by dual luciferase reporter, co-immunoprecipitation (IP) and ChIP-qPCR assays. In vivo experiments were conducted to further verify the role of the miR-216b in the resistance of OS cells to cisplatin. miR-216b expression was reduced in the OS tissues, as well as the MG63 and SaOS-2 cells. Heightened miR-216b expression was found to be positively correlated with patient survival, and miR-216b further enhanced cisplatin-induced apoptosis of MG63 and SaOS-2 cells. Mechanistically, miR-216b inhibited JMJD2C expression by binding to its 3’UTR. Through interaction with HIF1α, JMJD2C removed the H3K9 methylation modification at the HES1 promoter region, leading to upregulation of HES1 in vitro. Furthermore, miR-216b was observed to increase the tumor growth in nude mice in the presence of cisplatin treatment. HES1 overexpression weakened the effects of miR-216b in MG63 and SaOS-2 cells and in nude mouse xenografts. Overall, miR-216b enhanced the sensitivity of OS cells to cisplatin via downregulation of the JMJD2C/HIF1α/HES1 signaling axis, highlighting the capacity of miR-216b as an adjunct to cisplatin chemotherapy in the treatment of OS.


Background
Osteosarcoma (OS) represents one of the most common primary bone malignancies, predominantly affecting children and adolescents [1]. In addition to the highest incidence of OS in adolescence, a second incidence peak has been reported in the elderly, a high-risk population group [2]. The incidence of OS is estimated as '4' for the 0-14 years age range and '5' for 15-19 years age range per million people worldwide [3]. Moreover, the 5-year survival rate worldwide for OS remains to be largely appalling, with studies estimating a survival rate between 40 and 70% [3,4], a statistic which has failed to improve for decades [5]. Young age, first primary tumor, localized stage, low grade, and surgical treatment are factors that have all been positively linked with overall survival in OS [6]. Currently, a combination of surgical intervention as well as chemotherapy is regarded as the first line treatment for OS. However, it is well-documented that the advent of chemotherapy resistance is a big obstacle in the treatment of OS [7][8][9][10], which may, at least partly, contribute to the unfavorable outcomes seen in OS patients. Hence, the current study set out to identify the potential mechanism by which chemotherapy drug resistance occurs during the course of OS treatment. microRNAs (miRNAs or miRs) are known to possess the ability to regulate cell proliferation and apoptosis, and as a result, are implicated in a wide variety of human cancers as oncogenes or tumor suppressors [11]. MiRNAs refer to small non-coding RNA molecules, which serve as regulators of protein expression via the degradation of targeted mRNA or blockade of protein translation [12]. More notably, one such miRNA, miR-216b has been recently shown to potentially exert a tumor inhibitor function in OS by targeting FoxM1 as it inhibits OS cell proliferation, migration, and invasion [13]. Moreover, the over-expression of miR-216b has also been previously demonstrated to significantly enhance the sensitivity of non-small cell lung cancer cells to cisplatin-induced apoptosis by targeting c-Jun [14]. In addition, miR-216b can diminish cell proliferation, while promoting the sensitivity of colorectal cancer cells to oxaliplation by suppressing PDZ-binding kinase [15]. These findings lead the authors to hypothesize that miR-216b may confer a similar role in OS to affect the chemosensitivity of OS cells.
Histone demethylase jumonji C domain-containing 2C (JMJD2C) is known to demonstrate regulatory potentials in the epigenetic mechanism in malignant diseases, particularly in regard to moderating the influence on the promoter activity of target genes which are strongly associated with tumor development [16]. Prior evidence has proposed that JMJD2C is highly-expressed in OS, and even confers a regulatory role in the context of OS [17]. Moreover, JMJD2C has also been shown to serve as a co-activator for hypoxia-inducible factor 1 (HIF1α) for cancer progression [18]. Meanwhile, as a widelydocumented regulator of cellular metabolism, HIF1α performs essential functions in the survival and differentiation of mesenchymal stem cells [19]. HIF1α has been reported to be highly-expressed in OS [20], and to further function to activate downstream genes through its transcriptional activation to promote OS [21]. More importantly, HIF1α has been reported to induce drug resistance through the hairy and enhancer of split-1 gene (HES1) in breast cancer [22]. HES1 represents a critical factor for the maintenance of stem cells, quiescent cells or cancer cells, and has also been demonstrated to elicit drug resistance and metastasis of tumor cells [23]. Furthermore, HES1 has been reported to be highly-active in OS [24], with studies suggesting that HES1 promotes the critical phenomenon of chemotherapy resistance in cancer [25,26]. In order to effectively inhibit HES1 to reduce chemotherapy resistance, one method is to modify H3K9 methylation at its gene promoter [27]. Hence, the current study set out to examine whether miR-216b inhibits cancer growth in OS by reducing the levels of JMJD2C and HES1. We further aimed to determine the involvement of HIF1α and H3K9 methylation modification in the underlying mechanism associated with the effects of miR-216b.

Ethics statement
The current study was performed with the approval of the Ethics committee of Shanghai Tenth People's Hospital (approval number: 2010-0017) and in strict accordance with the Declaration of Helsinki. Signed informed consents were obtained from all participants or their guardians prior to the study. 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.

Sample collection from OS patients
Cancer tissues and paracancerous tissues were collected from a total of 60 OS patients (aged 10-58 years, with an average age of 21.63 ± 9.70 years old) at the Shanghai Tenth People's Hospital from April 2010 to April 2013. All included patients had not undergone chemotherapy or radiotherapy prior to specimen collection. Patient follow-up was conducted over a period of 60 months. The time from specimen collection to tumor recurrence or death (overall survival) was recorded. The last followup date was recorded if no tumor recurrence or death occurred. All included patients received the same treatment regimen at the Shanghai Tenth People's Hospital, with no metastasis occurring during the follow-up period.

RNA quantitation by reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA content was extracted from the fresh tissues using RNeasy Mini kits (Qiagen, Valencia, CA, USA). For mRNA detection, the extracted total RNA was reverse transcribed into complementary DNA (cDNA) with the help of reverse transcription kits (RR047A, Takara, Japan). For miRNA detection, cDNA was obtained using miRNA First Strand cDNA Synthesis (Tailing Reaction) kits (B532451-0020, Sangon Biotech Co., Ltd., Shanghai, China). The samples were then loaded using a SYBR® Premix Ex TaqTM II (Perfect Real Time) kit (DRR081, Takara, Japan). RT-qPCR was performed on an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA), and each sample was evaluated in triplicate. The universal negative primers for miRNA, while the internal reference U6 were provided by miRNA First Strand cDNA Synthesis (Tailing Reaction) kit. The remaining primers were provided by Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences are depicted in Table 1. The Ct value of each target gene was recorded and normalized to an internal reference, namely, β-actin or U6. Relative expression of all target genes was calculated by means of relative quantification (2 -ΔΔCt method) [29].
Binding of JMJD2C to HIF1α determined by coimmunoprecipitation (co-IP) assay The cells were lysed for 30 min at 4°C in RIPA buffer (Thermo Scientific, Waltham, MA, USA), and then centrifuged at 13,000 g for 30 min at 4°C. The supernatant was subsequently collected and incubated with specific antibody at 4°C overnight, followed by the addition of pierce protein A/G Magnetic Beads (#88803, Thermo Scientific) and 4 h of incubation by shaking at 4°C. The beads were then centrifuged, washed three times, and mixed with the loading buffer, followed by protein evaluation using SDS-PAGE and immunoblotting [31].

Quantity of the HES1 promoter determined by chromatin immunoprecipitation (ChIP)-qPCR
ChIP was performed using EZ-Magna ChIP A/G Chromatin Immunoprecipitation kits (17-371, Millipore, Billerica, MA, USA). Briefly, the cells were sonicated and centrifuged at 12,000 g for 10 min at 4°C to remove insoluble precipitates. The cells were subsequently incubated with protein G agarose beads at 4°C for 1 h, and centrifuged at 5000 g for a total of 1 min. Next, 10 μL (1%) of the supernatant was collected as the 'Input', while the remaining supernatant was divided into 2 portions and incubated with antibodies against H3K9me3 (ab8898, dilution ratio of 1:20, Abcam Inc., Cambridge, UK), H3K9me2 (ab1220, dilution ratio of 1:10, Abcam Inc., Cambridge, UK), H3 (ab213257, dilution ratio of 1: 5, Abcam Inc., Cambridge, UK), and NC rabbit antihuman IgG (ab2410, dilution ratio of 1:25, Abcam Inc., Cambridge, UK) at 4°C overnight. The precipitated protein-DNA complex was then incubated with protein G agarose beads for 1 h at 4°C. After centrifugation at 5000 g for 1 min, the supernatant was discarded. The protein-DNA complex was fragmented overnight at 65°C. The DNA fragment was recovered and used as an amplification template for RT-qPCR. The primers employed to detect the quantity of HES1 promoter were agomir NC (5′-UUCUCCGAACGUGUCACGUTT-3′) and miR-216b agomir (5′-AAAUCUCUGCAGGCAAAU GUGA-3′).

Cell viability assays
Cell proliferation was measured using a cell counting kit-8 (CCK-8) assay (Kumamoto, Japan). MG63 and SaOS-2 cells were firstly seeded in 96-well plates. After 24 h of transfection, various concentrations of cisplatin (0, 0.2, 0.4, 0.8, 1.6 μM) were added to the cells and incubated for 72 h. The CCK-8 reagent (10 μL) was then added to each well and incubated for 1 h. Absorbance was finally measured at a wavelength of 450 nm using a microplate reader.

Apoptosis assays
After 24 h of transfection, the cells were treated with 1 μM cisplatin [32] for 72 h. An apoptosis assay was subsequently performed using Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) kits (KeyGEN Biotech. Co., Ltd., Nanjing, China). The cells were subsequently observed and analyzed using a flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NY, USA). The experiment was performed in triplicate, with the respective average values obtained and recorded.

Tumor xenograft in nude mice
A total of 30 BALB/c nude mice (aged 5-6 weeks old, weighing 15-18 g, acquired from the Shanghai Experimental Animal Center of Chinese Academy of Sciences) were randomly divided into the 5 following groups (6 mice per group): control group, Lv-oe-NC group, Lv-oe-NC + Cis group, Lv-oe-miR-216b + Lv-oe-NC + Cis group, and Lv-oe-miR-216b + Lv-oe-HES1 + Cis group. The lentiviruses carrying oe-NC, oe-miR-216b and oe-HES1 were purchased from Shanghai Gene Pharma Co., Ltd. (Shanghai, China). Following lentivirus infection, the stablytransfected MG63 cells were subcutaneously injected into the axillary region of nude mice at a density of 1 × 10 6 cells. From the 7th day of inoculation, cisplatin (8 mg/kg, [33]) was intraperitoneally administered to the mice in the Lv-oe-NC + Cis, Lv-oe-miR-216b + Lv-oe-NC + Cis, and Lv-oe-miR-216b + Lv-oe-HES1 + Cis groups twice a week. On the 14th day, the mice were euthanized using carbon dioxide. The tumors were subsequently removed, after which the tumor volume was measured using the following formula: (a × b 2 )/2 [34], where a represented the length of the tumor and b was the width of the tumor. The volume of the tumor was calculated and a tumor growth curve was then plotted [35].

Statistical analysis
Data analyses were processed using the SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. Data with normal distribution and homogeneity of variance between two groups were compared using paired ttest (paired data) or unpaired t-test (unpaired data). Data comparisons between multiple groups were performed using one-way analysis of variance (ANOVA), followed by a Tukey's test. Data comparison between groups at different time points was performed by repeated measures ANOVA with Bonferroni post-hoc test. Correlation of two variants was analyzed by Pearson correlation coefficient. Patient survival rate was analyzed using the Kaplan-Meier method followed by log-rank test. A p < 0.05 value was considered to be indicative of statistical significance.

miR-216b is poorly-expressed in OS and its high expression is positively-correlated with patient survival
Firstly, the expression of miR-216b was found to be markedly lower in OS tissues compared to that in paracancerous tissues (fold = 0.315) (Fig. 1a). The median miR-216b relative expression pattern in cancer tissues from 60 patients was subsequently determined, after which the patients with values above the median were placed into the high miR-216b expression group, while those below the median were regarded as the low miR-216b expression group. In addition, Kaplan-Meier survival analysis revealed that elevated expression of miR-216b was correlated with higher overall survival in OS patients (Fig. 1b). However, no significant correlation was identified between the miR-216b expression and factors such as age, sex, location of the tumor, and metastasis (Table 2). Additionally, results of multivariate analysis demonstrated that miR-216b served as an independent prognostic factor for OS (Table 3). Lastly, the expression of miR-216b was determined to be significantly lower in the MG63 and SaOS-2 cell lines relative to other OS cell lines and control (hFOB1.19 cell line) (U2OS: fold = 0.436, HOS: fold = 0.614, SaOS-2: fold = 0.248, MG63: fold = 0.161) (Fig. 1c). Hence, MG63 and SaOS-2 cells were selected for subsequent experimentation.

miR-216b improves the effects of cisplatin on OS cells in vitro
We subsequently set out to evaluate the sensitivity of  (Fig. 2a). Cisplatin was found to also augment cell apoptosis, which was further elevated following miR-216b agomir treatment in cells (MG63: miR-216b agomir fold = 1.39; SaOS-2: miR-216b agomir fold = 1.26) (Fig. 2b, c). These results suggested that miR-216b enhanced the promotive effects of cisplatin on OS cell apoptosis, while inhibiting cell viability in vitro.
b Pearson correlation of miR-216b expression with JMJD2C expression in OS tissues (n = 60). c miR-216b expression and JMJD2C mRNA expression were determined by RT-qPCR in cells, relative to U6 and β-actin, respectively, and representative Western blots of JMJD2C protein and its quantitation in cells, relative to β-actin.

Discussion
Although not as prevalent as many other malignancies, OS represents the foremost primary bone cancer with the vast majority of cases seen among younger populations [1]. In addition, the 5-year survival rate worldwide has failed to improve for decades, remaining between 40 and 70% [3][4][5]. This can be partly attributed to the emergence of chemotherapeutic resistance in the treatment of OS [37,38]. Meanwhile, the aberrant regulatory interactions between miRNA-mRNA can potentially mediate the malignant phenotypes of various cancer cells, some of which have shown promise as potential novel targets and therapies aimed at limiting the angiogenesis of malignancies, including OS [39]. Thus, identification of novel non-invasive biomarkers with miRNA-mRNA interaction for the prevention and treatment of OS might prove highly-beneficial. Findings obtained in the current study indicated that miR-216b could augment the effect of the chemotherapeutic agent cisplatin on cell viability and apoptosis in OS cells, which might be attributed to its binding to the JMJD2C gene and the subsequent inhibition of JMJD2C expression, leading to the regulation of the HIF1α/HES1 signaling axis. Overall, our study demonstrated the role of the JMJD2C/HIF1α/ HES1 signaling axis in the miR-216b-mediated enhancement of the anti-cancer effects of cisplatin. Firstly, during the course of the current study, we identified that the expression of miR-216b was markedly reduced in OS, whereas a positive correlation was detected between high expression levels of miR-216b and patient survival. Furthermore, our findings revealed that miR-216b could heighten the effect of cisplatin, resulting in reduced OS cell viability and elevated cell apoptosis. Existing literature has demonstrated that miR-216b possesses the ability to directly reduce cancer cell proliferation, migration, and invasion in OS [13]. In addition, another study proposed the use of miR-216b as a Fig. 6 The miR-216b/JMJD2C/HIF1α/HES1 signaling axis participates in cisplatin-induced apoptosis in vivo. a Representative images of transplanted tumors from nude mice. b The growth of OS xenograft tumor in nude mice was measured every 7 days. c Tumor weight in nude mice. d miR-216b expression was determined by RT-qPCR in tumor tissues of nude mice, relative to U6. e Representative Western blots of Ki67, Bcl-2, JMJD2C, HIF1α and HES1 proteins and their quantitation in tumor tissues, relative to β-actin. * p < 0.05 vs. mice treated with Lv-oe-NC; # p < 0.05 vs. mice treated with Lv-oe-NC + Cis; & p < 0.05 vs. mice treated with Lv-oe-miR-216b + Lv-oe-NC + Cis. Data are expressed as mean ± standard deviation. Data in panel (a) and (c-e) were compared using one-way ANOVA with Tukey's test, while data in panel (b) were compared by repeated measures ANOVA with Bonferroni test. n = 10 for mice upon each treatment potential sensitizer in cisplatin chemotherapy owing to its ability to reduce cell viability and promote the apoptosis of cisplatin-resistant ovarian cancer cells [40]. Diminished levels of miR-216b have been detected in nonsmall cell lung cancer cells while enhanced miR-216b expression has been demonstrated to elevate cisplatininduced apoptosis by targeting Beclin-1 [41]. All in all, these findings and evidence suggest that miR-216b could serve as a promising target to enhance the effects of cisplatin on OS cells.
Moreover, miRNAs are known to possess the capacity to modulate gene expressions at a post-transcriptional level by means of interacting with the 3'UTR of specific target mRNAs [42]. During the current study, online biological prediction and luciferase reporter assay were employed, which revealed data indicating that miR-216b could bind to the 3'UTR of the JMJD2C mRNA and negatively-regulate its expression. In line with our findings, JMJD2C has been previously reported to be highlyexpressed in OS, and even capable of elevating the aggressiveness of OS [17]. Therefore, it would be reasonable to speculate that miR-216b could elicit an inhibitory effect on the JMJD2C expression, consequently delaying the progression of OS. Moreover, recent data have evidenced that knockdown of JMJD2A decreases the proliferation of ovarian cancer cells, while simultaneously acting to improve the sensitivity of ovarian cancer cells to cisplatin [43]. The aforementioned findings support the notion that miR-216b binds to JMJD2C and subsequently inhibits its expression, ultimately enhancing cisplatin-induced apoptosis of OS cells.
Furthermore, our findings revealed that JMJD2C upregulated the expression of the HES1 gene via HIF1α interaction to diminish H3K9 methylation modification. Several studies have stated that HIF1α is an essential component of JMJD2C functioning [18,20]. Moreover, HIF1α and HES1 have been reported to work in tandem to decrease the effects of cisplatin on OS cells, which is very much in line with our findings [22]. As a result, we highlighted JMJD2C and HIF1α as downstream signaling molecules of miR-216b, wherein miR-216b reduced JMJD2C expression, likely interfering with mRNA stability and JMJD2C action in the demethylation of HES1 gene at the promoter region. More importantly, high expression levels of HES1 have been previously correlated with enhanced OS cell proliferation, migration and invasion as well as boosted chemoresistance [44]. What's more, our findings revealed that miR-216b diminished the expression of HES1 via the removal of H3K9 methylation at the gene promoter site of HES1 in OS cells, which has been previously emphasized as a critical factor for HES1 activation [27]. In contrast, HES1 overexpression weakened the effects of miR-126b, as evidenced by suppressed tumor growth and enhanced cell apoptosis in OS. These results are largely consistent with the previous evidence indicating that HES1 promotes chemotherapy resistance in different cancers [25,26]. In addition, we uncovered that HES1 over-expression brought about elevations in the expression of Ki67 and Bcl-2. This is particularly important as Ki67 is recognized as a marker of cancer cell proliferation in OS [45]. Similarly, Bcl-2 is well-documented as a suppressor of apoptosis in various cancers including OS [46]. Consequently, we are convinced that miR-216b may have the potential to enhance cisplatin-induced apoptosis via regulation of the JMJD2C/HIF1α/HES1 signaling axis in OS.

Conclusion
Overall, the current study highlighted that miR-216b augmented cisplatin-induced apoptosis through regulation of the JMJD2C/HIF1α/HES1 signaling axis. The newly found miR-216b/JMJD2C/HIF1α/HES1 signaling axis might pave the way for potential therapeutic mechanisms to reduce chemoresistance in OS. Nevertheless, a few OS cell lines were initially screened in the study, but only MG-63 and SaOS-2 cells were chosen due to highest expression profiles of miR-216b. This selection criterion may pose a selection bias. Therefore, results from our study should be confirmed by future studies incorporating other types of OS cell lines. The current study lends support to the notion that miR-216b may be employed as a chemotherapy adjunct alongside cisplatin for the efficacious treatment of OS.