HIF-1α-mediated augmentation of miRNA-18b-5p facilitates proliferation and metastasis in osteosarcoma through attenuation PHF2

Extensive evidence has explored the involvement of microRNAs (miRNAs) in osteosarcoma (OS). Limitedly, the concrete function of microRNA-18b-5p (miR-18b-5p) in OS remains unexplored and largely elusive. Here, we validated that miR-18b-5p significantly elevated in OS via analyzing the data from GEO database. The results showed that miR-18b-5p was overexpressed in human OS tissues and cell lines. The clinical evidence suggested that high level of miR-18b-5p was negatively correlated with the poor prognosis of OS. Meanwhile, miR-18b-5p upregulation facilitated the proliferation and metastasis of OS cells in vitro and in vivo. The mechanism exploration demonstrated that miR-18b-5p acted as a potential inhibitor of PHF2, a tumor suppressor gene, at post-transcriptional level. Moreover, hypoxia induced gene expression of miR-18b-5p was clarified to be transcriptionally mediated by HIF-1α. The clinicopathological analysis in samples of OS patients further supported that miR-18b-5p had a positive correlation with HIF-1α expression, and negative correlation with PHF2. Collectively, the present study uncovered a new molecular mechanism of OS tumorigenesis and development and miR-18b-5p might be a prognostic biomarker and potential therapeutic target for OS treatment.


Scientific Reports
| (2022) 12:10398 | https://doi.org/10.1038/s41598-022-13660-w www.nature.com/scientificreports/ driver mechanisms of the occurrence, progression, invasion and metastasis of OS cells under hypoxia, so as to provide new ideas and drug targets for the clinical diagnosis and treatment of tumors and effectively improve the quality of life of patients. PHF2 gene, encoding plant homeodomain (PHD) and Jumonji C (JmjC) domain-containing proteins, is identified as transcriptional regulators affecting gene expression by demethylation of histone lysine [11][12][13] . Research has clarified that PHF2 was downregulated in squamous cell carcinoma, Gastric and Colorectal Cancers and other malignant tumors [14][15][16] . Moreover, the deletion/methylation of PHF2 gene leads to downregulation of PHF2 expression in breast cancer 17 . Meanwhile, low expression of PHF2 is related to the aggressiveness and poor prognosis of clear cell renal cell carcinoma (CCRCC) 18 . The overexpression of PHF2 in esophageal squamous cell carcinoma (ESCC) is correlated to decreased overall survival rate of ESCC patients 19 . Mechanism studies have elucidated that PHF2 positively regulated p53 expression via demethylation of histone H3K9 methylation at p53 promoters, indicating that PHF2 acted as a tumor suppressor gene and played a negative regulatory role in tumor genesis and development 16 . Whereas, the function and underlying molecular mechanisms of PHF2 in OS cells are rarely discussed.
MicroRNAs (miRNAs) are a class of endogenous regulatory small non-coding RNA, which identify complementary sequences of its target genes in the 3′ -untranslated region (UTRs), leading to mRNA degradation, translation inhibition or activation 20,21 . miRNA plays a role in regulating cell proliferation, differentiation, apoptosis and ontogenesis 22 . Various studies have elucidated that miRNA is closely related to the occurrence and development of OS 23,24 . Here, we identified miR-18b-5p significantly elevated in OS via analyzing the data from GEO database. Our results suggest that upregulation of miR-18b-5p in OS plays a crucial role in the proliferation and metastasis of OS cells. Additionally, we make sense out of the regulative mechanism of miR-18b-5p in OS. Our results further broaden our understanding of the action and mechanism of miRNAs in OS, and can potentially provide new avenues for OS treatment.
Upregulation of miR-18b-5p accelerates development of OS. To identify the biological function of miR-18b-5p in OS, the effects of upregulated and downregulated miR-18b-5p on cell proliferation and motility were investigated. Compared with other OS cells, miR-18b-5p expression was most elevated in KHOS and U2OS cells and lowest in MG-63 and 143b cells, so we inhibited the expression of miR-18b-5p in KHOS and U2OS cells and up-regulated the expression of miR-18b-5p in MG-63 and 143b cells. MiRNA mimic and inhibitor were applied to elevate or decrease endogenous miR-18b-5p, respectively (Fig. 2a, Fig. S1A). MTT assay indicated significant enhanced proliferation of OS cells with miR-18b-5p overexpression compared with the control group, whereas the opposite effects were found following miR-18b-5p depletion (Fig. 2b, Fig. S1B). In addition, the results of transwell assay suggested that miR-18b-5p overexpression accelerated the invasion and migration of OS cells (Fig. 2c,d). In contrast, miR-18b-5p inhibition repressed cell invasion and migration (Fig. S1C, D).
To validate the biological function of miR-18b-5p in vivo, MG-63 cells, transfected with recombinant lentiviral vector LV-miR-18b-5p (LV-miR-NC as control), were utilized to establish xenograft and metastasis models by subcutaneous or tail vein injection. The results showed that compared with LV-miR-NC group, miR-18b-5p overexpression significantly increased the tumor growth ( Fig. 2e-g). Consistent with the results of the in vitro assay, miR-18b-5p overexpression enhanced the metastasis of OS cells in vivo (Fig. 2h,i). Meanwhile, miR-18b-5p upregulation group exhibited shorter survival rate compared with the control group (Fig. 2j). Thus, these results revealed that miR-18b-5p functioned as an oncogenic miRNA in OS. miR-18b-5p modulates the expression of PHF2 through post-transcriptional regulation. According to the reported methodology 30,31 , the bioinformatics databases (miRanda, miRmap, microT and PicTar) were applied to predict potential target genes of miR-18b-5p and explore underlying mechanisms involved. The results of Venn's diagram showed that there were 10 genes, including PHF2, which might be downstream targets of miR-18b-5p (Fig. 3a,b). Since it has been reported that the histone demethylase PHF2 is considered a putative anti-tumor gene, and is associated with p53 as a tumor suppressor in cancer 16 . Thus, we wondered whether miR-18b-5p exerted its function through targeting PHF2. The RT-qPCR results indicated that overexpression of miR-18b-5p decreased the mRNA level of PHF2, while inhibition of miR-18b-5p increased the mRNA level of PHF2 (Fig. 3c,d). Additionally, overexpressed miR-18b-5p dramatically decreased PHF2 www.nature.com/scientificreports/ protein levels, while transfection of miR-18b-5p inhibitors promoted PHF2 protein level in both MG-63 and 143b cells (Fig. 3e,f). These results indicated that miR-18b-5p negatively regulated expression of PHF2 at the post-transcriptional level. In order to validate that miR-18b-5p specifically bound to the 3′UTR of PHF2 mRNA, immuno-precipitation using biotin labeled miR-18b-5p was conducted with biotin-miR-NC served as a negative control. The result showed that PHF2 mRNA was only detected in the biotin labeled miR-18b-5p group (Fig. 3g). Subsequently, we performed the luciferase reporter assay to further prove the direct interaction between miR-18b-5p and PHF2 mRNA. We performed luciferase assays by transfecting MG-63 and 143b cells with luciferase reporters carrying Wild or Mutant type (WT, MUT) of PHF2-mRNA-3′ UTR sequences along with miR-18b-5p overexpression or depletion. The results suggested that miR-18b-5p overexpression significantly decreased luciferase activity of WT-3′ UTR-PHF2 cells, while miR-18b-5p inhibition significantly increased luciferase activity of WT-3′ UTR-PHF2 cells (Fig. 3h,i). Neither miR-18b-5p overexpression nor miR-18b-5p inhibition affect luciferase activity of MUT-3′ UTR-PHF2 cells. Therefore, these results indicated that PHF2 was a downstream target of miR-18b-5p.
PHF2 is responsible for miR-18b-5p-mediated cell proliferation, invasion and migration. To identify the ability of miR-18b-5p to promote tumor progression through inhibiting PHF2, we co-transfected MG-63 and 143b cells overexpressing PHF2 or scrambled vector with miR-18b-5p mimic or miR-NC (Fig. 4a,b). The results of MTT assay indicated that PHF2 overexpression significantly decreased the cell proliferation, which could be rescued by miR-18b-5p upregulation (Fig. 4c). Moreover, transwell assay showed that overexpression of miR-18b-5p rescued the suppressive effects of PHF2 overexpression on OS invasion and migration (Fig. 4d,e). Collectively, these results revealed that miR-18b-5p promoted proliferation, invasion and migration via targeting PHF2.

Hypoxia-driven HIF-1α is critical for upregulation of miR-18b-5p in OS.
Since researchers had elucidated that hypoxic microenvironment played a vital role in mediating multiple processes of the malignant progression of OS 32,33 . Therefore, we ought to validate whether hypoxia could induce upregulation of miR-18b-5p in OS. To validate, we designed luciferase reporters containing the promoter sequence of miR-18b-5p host gene, miR-18b, or negative control sequence. The result indicated that the activity of miR-18b promoter was remarkably elevated under hypoxia (Fig. 5a). Meanwhile, RT-qPCR assay indicated that the expression of miR-18b-5p was remarkably increased with the extension of hypoxic culture time (Fig. 5b, Fig. S2A). Next, we found a HRE element in the miR-18b promoter via analyzing the promoter sequence of miR-18b promoter. Chromatin immunoprecipitation (ChIP) assay suggested that HIF-1α could bind to the promoter of miR-18b under hypoxia (Fig. 5c). In order to further demonstrate that HIF-1α regulates miR-18b-5p by binding to the HRE site of miR-18b promoter, the MG-63 and 143b cells were transfected with WT or MUT plasmid carrying promoter sequence of miR-18b. The luciferase assay revealed that hypoxia significantly enhanced the activity of miR-18b promoter in WT cells, which was reversed by HIF-1α silencing (Fig. 5d,e). Additionally, RT-qPCR results indicated that HIF-1α inhibition reversed the miR-18b-5p expression induced by hypoxia (Fig. 5f). Consistently, the expression of PHF2 was remarkably decreased under hypoxia, which was restored by HIF-1α inhibition   . 5g). Therefore, the results above revealed that hypoxia induced HIF-1α is responsible for the upregulation of miR-18b-5p in OS under hypoxic microenvironment.
Hypoxia induced miR-18b-5p promotes OS via miR-18b-5p-PHF2 axis. In order to delineate the correlation of HIF-1α-miR-18b-5p-PHF2, we evaluated the expression of HIF-1α, miR-18b-5p and PHF2 in our clinical specimens. The results showed that expression of miR-18b-5p had a positive correlation with HIF-1α, while negative correlation with PHF2 ( Fig. 6a,b). Meanwhile, expression of HIF-1α had a negative correlation with PHF2 (Fig. 6c). In summary, our results depicted that hypoxia driven HIF-1α enhanced the expression of www.nature.com/scientificreports/ miR-18b-5p in transcriptional level via binding to the HRE site of pre-miR-18b-5p promoter. The upregulated miR-18b-5p decreased PHF2 expression in post-transcriptional level by binding to the 3′UTR of PHF2 mRNA, leading to the increased proliferation and metastasis of OS cells (Fig. 6d).

Discussion
Rapid proliferation and pulmonary metastasis were validated as the main cause of poor outcomes in OS patients.
Although great progress has been achieved in the options of treatment, the 5-year survival rate of OS is still low. Early and correct diagnosis is not only the key to treating the disease successfully, but also an important guarantee for improving the prognosis and survival rate of patients. Since thousands of functional miRNAs have been identified in cancer, these small molecules can be utilized as biomarkers to diagnose the diseases and prognosis. In our present study, we identified miR-18b-5p was upregulated in OS cell lines compared with normal bones via GEO datasets. The in vitro and vivo assays validated that enhanced miR-18b-5p expression contributed to the increased proliferation and metastasis of OS. Mechanically, miR-18b-5p repressed the expression of tumor suppressor gene-PHF2 in post-transcriptional level. Furthermore, the upregulation of miR-18b-5p under hypoxia is due to the transcriptional regulation by HIF-1α. In this study, our results have enriched the mechanisms by which miRNA plays a carcinogen-promoting role in OS. Increasing evidence reported that dysregulated miRNA exerts a vital role in increasing the ability of proliferation and motility of cancer cells. Here, we identified abnormal expressed miR-18b-5p in OS cells compared to normal bones through screening the GEO dataset. Reports have elucidated the involvement of miR-18b-5p in several other types of cancers, including ovarian cancer 34 , colorectal cancer 35 , breast cancer 36 , hepatocellular carcinoma 37 , gallbladder cancer 38 . Among these studies, miR-18b-5p were mainly absorbed by lncRNA, and acted as a tumor suppressor or promotor. In our exploration, we identified that miR-18b-5p exerted its function as a tumor promotor. Survival analysis suggested that high miR-18b-5p level was associated with poor prognosis in OS patients. Therefore, miR-18b-5p is a promising therapeutic target for cancer treatment in OS. While, miR-18b-5p can act on multiple target genes and signaling pathways, and the interference of miR-18b-5p may  miR-18b-5p played opposite roles in different tumors, suggesting that tumor microenvironment may have a certain influence on the expression and activity of miR-18b-5p. In our studies, we clarified that miR-18b-5p was upregulated by hypoxia induced HIF-1α at transcriptional level in OS. Meanwhile, miR-18b-5p medicated the enhanced proliferation and metastasis of OS cells via targeting PHF2 at post-transcriptional level. HIF-1α is a major transcription factor involved in initiating a variety of cellular processes required to restore oxygen homeostasis by regulating the expression of various genes 39 . Some JmjC demethylases has been revealed to be direct targets of HIF transcription factors, suggesting that they are involved in the function of hypoxia response 40 . Meanwhile, the research reported that PHF2 was significantly increased in HepG2 cells under hypoxic conditions 40 . In our studies, we found that PHF2 was remarkably downregulated by HIF-1α-driven miR-18b-5p In summary, our results depicted that hypoxia driven HIF-1α enhanced the miR-18b-5p expression in transcriptional level via binding to the HRE site of miR-18b-5p promoter. The upregulated miR-18b-5p decreased PHF2 expression in post-transcriptional level by binding to the PHF2-mRNA -3′UTR, leading to the increased proliferation and metastasis of OS cells.

Material and methods
Human tissue specimens. The histologically conformed cancer tissues and adjacent cancerous tissues of 60 OS patients were collected from Huazhong University of Science and Technology Union Shenzhen Hospital between 2016 to 2018. All specimens were obtained during surgery and immediately frozen in liquid nitrogen in accordance with the agreement of the ethics committee. And 60 patients were enrolled in clinical follow-up data for 3 years. OS patients with miR-18b-5p expression above or below the 50th percentile were classified as high or low miR-18b-5p group. All experiments were approved by the medical ethics committee affiliated to Huazhong University of Science and Technology Union Shenzhen Hospital and were carried out in accordance with relevant guidelines and regulations. All patients signed informed consent before recruitment.  Transwell assay. Transwell assay was performed to evaluate cell invasion and migration. Firstly, the treated cells (5 × 10 4 ), suspended in 200 μl of serum-free medium, were added on Matrigel-coated and uncoated upper chambers for invasion assay and migration assay, respectively (BD Bioscience, USA). Culture medium containing 10% FBS was added to the lower wells. After incubated at 37 °C for 24 h, the cells were fixed using 4% paraformaldehyde and stained using crystal violet. Then the cells on the membrane of transwell chambers were imaged using a microscope (Olympus, Japan). The number of invaded and migrated cells was observed in 5 random fields.
Transfection. The miR-18b-5p mimic, mimic negative control (NC), miR-18b-5p inhibitor, inhibitor NC, small interfering RNAs targeting HIF-1α and scrambled NC siRNA were brought from GenePharma (Shanghai, China). For stable transfection, miR-18b-5p was constructed into lentiviral vector (LV), with empty vector as a negative control. The LV-miR-18b-5p and LV-miR-NC were purchased from GeneChem (Shanghai, China) and transfected into MG-63 cells according to the given instruction. The transfection efficiency was shown in Table S1. The overexpression plasmid of PHF2 and scrambled vectors (Table S2) were purchased from Gene-Chem (Shanghai, China). Lipofectamine 2000 (Invitrogen) was utilized for the transfection. For the co-transfection of miR-18b-5p mimic and PHF2 overexpression plasmid, appropriate number of cells (20 × 10 4 /well) were inoculated in 6-well plates 1 day before transfection. And 4 μg PHF2 overexpression plasmids and 50 nM miR-18b-5p mimic were co-transfected into MG-63 and 143b cells. And 48 h later, the cells were harvested for further analysis. qRT-PCR and Western blotting was conducted to assess the transfection efficiencies. The corresponding sequences were listed in Table 1. www.nature.com/scientificreports/ U6 snRNA served as control to normalize the expression of miRNA. β-actin acted as control for the expression of mRNA. The primers for genes used in the study were listed in Table 2.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR
Luciferase assay. The procedure was performed as previously described 41 . PGL3 vector (Table S2) wild and mutant reporter plasmids carrying the predicted binding sites of miR-18b-5p in PHF2 mRNA-3′UTR sequence and mutant (PHF2-WT: Table S3, PHF2-MUT: Table S4) were transfected in MG-63/143b cells with miR-18b-5p overexpression or inhibition to identify the interaction between miR-18b-5p and 3′UTR of PHF2 mRNA. PGL3-basic plasmids containing the promoter sequence of miR-18b-5p (a 2-kb sequence upstream of miR-18b, Table S5) were transfected in MG-63 and 143b cells culture under normoxia or hypoxia to evaluate the transcriptional activity of miR-18b. The plasmid pRL-TK carrying the Renilla luciferase was co-transfected into the cells as internal reference. Lipofectamine 2000 (Invitrogen) was applied to conduct the transfection. 48 h later, the luciferase activity was quantified using a Dual-Luciferase Reporter Assay System (Promega, USA) based on the manufacturer's instructions. Specifically, the 5 × passive lysis buffer (PLB) was provided in the system and diluted to 1 × PLB as the given instructions. 48 h after cells co-transfection, each well was cleaned with PBS and added 1 × PLB. Subsequently, the culture plate was placed in a shaker for 25 min to allow the cells to be completely lysed. The lysate was then transferred to a 96-well plate protected from light, and the pre-mixed Luciferase Assay Reagent II was added to each well for 2 s to detect the first luminescence value (firefly luciferase activity). After determination, pre-mixed Stop&Glo Reagent was added to each well for 2 s, and the second luminescence value was Renilla luciferase. Finally, the data was presented as the ratio of firefly luciferase activity to Renilla luciferase activity.
ChIP assay. The ChIP assay was conducted using Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore, Catalog # 17-295) according to the provided instructions 42 . Briefly, 1 × 10 6 MG-63 cells were cultured under normoxia or hypoxia on a 10 cm dish. And 24 h later, the cells were treated with 1% formaldehyde for DNA-protein crosslinking. After removing the formaldehyde and washing cells twice with protease inhibitors containing ice cold PBS, the cells were scraped and collected through centrifugation (4 min, 2000 rpm, 4 °C).
The cells were lysed with 200 µLSDS Lysis Buffer for 10 min on ice. The DNA of cells was sheared by ultrasonic treatment to lengths between 200 and 1000 bp. The ultrasonically treated cell lysates were centrifuged at 4 °C at 12,000×g for 10 min. One percent of supernatant was kept as input. The rest of supernatant was divided into two parts and incubated with anti-HIF-1α (cell signaling technology, #36169) or rabbit-IgG (cell signaling technology, #2729) antibodies at 4 °C overnight, respectively. The DNA-protein complex was precipitated with Protein A Agarose for 1 h at 4 °C. Then the pellet agarose was collected via gentle centrifugation (1 min, 1000 rpm, 4 °C).
The supernatant was removed and the sediment was washed. The DNA fragments were purified and extracted using phenol/chloroform after reversing the cross-linking overnight at 65 °C. Finally, the purified DNA was detected and analyzed by qPCR. The gel electrophoresis on 2% agarose gel was utilized to analyze the PCR products. The sequence of PCR primers are indicated in Table 2.
Biotin miRNA pull-down assay. The method was the same as the literature reported 43 . The 4 × 10 6 MG-63 and 143b cells were transfected with 50 μM single-stranded biotinylated miR-18b-5p or miR-NC, designed and synthesized by the GeneChem (Shanghai, China). And 36 h later, the transfected cells were harvested and lysed by the freeze-thaw lysis method. Freshly prepared cell lysate and the blocked streptavidin-coated magnetic beads were incubated on a bench-top nutating mixer at room temperature for 1 h. After that, the beads were treated and washed with freshly prepared ice-cold pull-down wash buffer on the magnetic separator. Finally, the beads were resuspend in 100 µL of nuclease-free water for further analysis. Extract the total RNA from the resuspended beads. Finally, the purified RNA was further analyzed by RT-qPCR analysis.
In vivo assay. BALB/c nude mice (male, 4 weeks old) were brought from Shanghai Laboratory Animal Research Center (Shanghai, China). For the xenograft model, 16 mice (8 each group) were subcutaneously injected with MG-63 (100 μl, 2 × 10 6 ) transfected with LV-miR-18b-5p or LV-miR-NC in the right hip. The size of the tumor was recorded every 3 days. And 33 days later, the mice were sacrificed and the tumors were harvested  www.nature.com/scientificreports/ and weighted. The tumor volume was detected every 3 days based on the equation V = 0.5 × L (length) × W 2 (width). For the metastasis model, 16 mice (8 each group) were injected with MG-63 (100 μl, 2 × 10 6 ) transfected with LV-miR-18b-5p or LV-miR-NC from the tail vein. The survival time of the mice was recorded. Lung metastasis was quantified by H&E staining. The animal experiments were reviewed and approved by the Animal Care and Use Committee affiliated with Huazhong University of Science and Technology Union Shenzhen Hospital and were carried out in accordance with relevant guidelines and regulations in compliance with the ARRIVE guidelines.