GINS2 Promotes Osteosarcoma Tumorigenesis via STAT3/MYC Axis

GINS2 is overexpressed in several cancers, but little is known about its role in osteosarcoma (OS). A series of in vivo and in vitro experiments were conducted to explore the role of GINS2 in OS. In this study, we demonstrated that GINS2 was found to be highly expressed in OS tissues and cell lines, which was associated with poor outcomes in OS patients. GINS2 knockdown hindered the growth and induced apoptosis in OS cell lines in vitro. Furthermore, GINS2 knockdown effectively inhibited the growth of a xenograft tumor in vivo. By using an Affymetrix gene chip and intelligent pathway analysis, it was demonstrated that the GINS2 knockdown could reduce the expression of several targeted genes and reduce the activity of the MYC signaling pathway. Mechanically, LC-MS, CoIP, and rescue experiments revealed that GINS2 promoted tumor progression through the STAT3/MYC axis in the OS. Moreover, GINS2 was associated with tumor immunity and may be a potential immunotherapeutic target for OS.


Introduction
Osteosarcoma (OS) is a type of primary malignant bone cancer that occurs with a high frequency, accounting for 60% of cases in adolescents [1,2]. Te overall incidence of OS in the United States was approximately 4.5 per million [3]. To inhibit tumor growth and metastasis, chemotherapy, and radiotherapy are the main treatment options for OS [4]. Terefore, surgery combined with chemotherapy is the main treatment for osteosarcoma. However, most OS will eventually acquire resistance to these therapies. Moreover, osteosarcoma patients had poor prognoses when occurred with distant metastases. It means the 5-year overall survival rate is less than 25% [5,6]. Tere are still no efective therapies for metastatic osteosarcoma. However, the underlying molecular mechanisms of osteosarcoma progression are not fully elucidated.
GINS is a eukaryotic replication helicase that consists of four subunits including Sld5, Psf3, Psf2, and Psf1 which play a central role in the cell cycle and DNA replication [7,8].
GINS2, also known as Psf2, is a member of the GINS complex. GINS2 was overexpressed in multiple tumors and reported to be involved in tumorigenesis in several types of cancers including breast cancer [9], leukemia [10,11], and lung cancer [12,13]. For example, GINS2 knockdown inhibits cell proliferation and induces apoptosis in a human lung adenocarcinoma cell line and pancreatic cancer cell lines. All these fndings suggest that GINS2 is involved in the progression of multiple cancers. However, the roles of GINS2 in OS progression remain unknown.
In this study, we frst showed that GINS2 was an oncogene and highly correlated with cancer progression as well as patient survival prognosis. Te data from bioinformatics, in vitro, and in vivo studies demonstrated that GINS2 promoted cellular malignancy and may be a potential immunotherapeutic target for OS. Mechanistically, GINS2 activated the MYC pathway and ultimately induces carcinogenicity by upregulating STAT3. Tus, these fndings imply GINS2 promoted tumor progression through the STAT3/MYC axis in the OS. It provides new insights into the development of new treatment strategies using GINS2 as a therapeutic target for the clinical treatment of OS.

Patient Samples and Follow-up.
We selected 59 cases of OS from the databases of the First Afliated Hospital of Nanchang University, China, and obtained their corresponding tissue samples. Before the biopsy stage, the patients had not received radiotherapy and preoperative chemotherapy. When OS was diagnosed, all patients received combined chemotherapy, such as high-dose ifosfamide and doxorubicin, cisplatin, and methotrexate, and then underwent a partial removal of the primary tumor, which was followed by an adjuvant chemotherapy cycle. After the completion of chemotherapy, patients were followed up every 3 months for 6 years.

Immunohistochemical Staining for GINS2 in OS Samples.
Te endogenous peroxidase was inactivated in the parafnembedded tissue sections by the treatment with hydrogen peroxide. Te antigen was recovered by adding 10 mmol/L citrate bufer at pH 6.0 to the sections and microwaving them. Next, the sections were incubated with the anti-GINS2 antibody (Sigma) at 4°C overnight, and protein expression was detected using a secondary antibody (Sigma). During each immunohistochemical test, negative and positive controls were included. Te immunohistochemical staining was systematically and comprehensively assessed by two independent pathologists, and the patient's identity was kept confdential. Te intensity and positive staining rate (0/1+/ 2+/3+) in the cytoplasm and cell membranes of the cancer tissue and adjacent tissue (epithelial) samples were evaluated separately. Samples were grouped based on the product of the "staining intensity score" and the "staining positive rate score" as the total score, and samples with a total score ≤6 were defned as the antibody low expression group and those with a score >6 were designated as the antibody high expression group.

Cells Lines and Cell Culture
Conditions. OS cell lines were obtained from our laboratory. U2OS and HOS cell lines were kept in DMEM, and Saos-2 cells were kept in McCoy's 5A medium. Te medium was supplemented with 10% fetal bovine serum (FBS; Gibco). Te cells were cultured in sterile conditions in a humidifed incubator at 37°C and 5% CO 2 in the air, and the medium was replaced every 2 days. When the cells reached 75% confuence, they were harvested using trypsin for in vivo and in vitro experiments.

Quantitative Real-time Reverse Transcriptase-Polymerase
Chain Reaction. A total RNA was extracted using TRIzol (Superfec TRI, Shanghai, China), and RT-qPCR was performed using a Promega M-MLV RT-qPCR Kit. Te expression of GINS2 was quantifed using M-MLV-R Tase (Promega), and GAPDH was used as an internal control. Te primers used for real-time RT-qPCR were as follows: GINS2: forward primer: CAGAAATGTCGCCTGCTCC, reverse primer: GGATTTCGTCTGCCTTCG. GAPDH: forward primer: TGACTTCAACAGCGACACCCA. Reverse primer: CACCCTGTTGCTGTAG CCAAA. Relative expression levels were calculated using the comparative △Ct method (△CT values � Ct value of target gene − Ct value of reference gene). △CT values less than 12 were considered high abundance expression, 12-16 as medium abundance expression, and greater than 16 as low abundance expression.

Lentivirus-Vector Construction and Cell Transfection.
Te GINS2 gene was used as a template to construct a lentiviral vector (LVpGCSIL-004PSC24135-1) for shRNA interference. Te infectious shRNA lentivirus was delivered into 293T cells, purifed, and used to infect HOS, Saos-2, and U2OS cells to obtain stable clones. Te fuorescence was measured 72 hours after infection in an inverted fuorescence microscope to evaluate the infection efciency. When the infection rate has reached 80%, RT-qPCR is used to measure the expression level of GINS2.
2.6. Cell Proliferation Analysis. In short, OS cells infected with shRNA lentivirus or shGINS2-lentivirus control were trypsinized (Sangon Biotech) in the logarithmic growth phase, resuspended in a standard medium, and then seeded into a 96-well (2 × 10 3 cells/well) plate. After the plating was completed, at room temperature, the plates were scanned under the green fuorescence every day for 5 consecutive days, and a Celigo ® Image Cytometer (Nexcelom) was used to evaluate the number of cells.
In the rescue experiment, we utilized the MTT method to evaluate cell proliferation. Te cells were seeded on 96-well plates at a density of 2 × 103 cells/mL in the STAT3, shGINS2, and shCtrl overexpression groups. Te time period was 1-5 days, and each group was seeded in three wells for cultivation. After the incubation was completed, we added 10 μL of 5 mg/mL MTT to each well and incubated the cells at 37°C for 4 hours. Ten we removed the medium and added 150 μL of dimethyl sulfoxide (DMSO). We used an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 490 nm. We used the absorbance values to draw the cell proliferation curve.

Cell Apoptosis Analysis.
Annexin V-APC was used for staining, and then fow cytometry was used to efectively evaluate cell apoptosis. Te cells were placed at 37°C for 48 hours. After that, the cells were centrifuged at 100 × g, washed twice with PBS, and resuspended in 1 × binding bufer. Te pellet was resuspended in Annexin V-APC and propidium iodide, incubated at room temperature in the dark for 15 minutes, and washed twice with PBS. Te analysis was based on the FACS Calibur fow cytometer (BD Biosciences). All experiments were performed three times.
Te Caspase-3/7 Assay Kit (Promega) was used to detect the activity of caspases 3/7 in the OS cells according to the manufacturer's instructions. Te fuorescence intensity in 2 Journal of Oncology the cells was quantitatively evaluated at the excitation of 499 nm using an ELISA tablet counter.

Cell Cycle Distribution.
To assess cell cycle distribution, U-2OS cells were trypsinized, resuspended in 70% ethanol, and incubated overnight at 4°C. Te fxed cells were centrifuged, washed in ice-cold PBS, and then incubated in RNase A at 37°C for 30 min and in 400 μL of propidium iodide (BestBio) at 4°C for 30 min. Annexin V/propidium iodide (PI) apoptosis detection kit (Sigma) was used to assess the cell cycle according to the manufacturer's instructions. A FACS Calibur fow cytometer (BD Biosciences, San Jose, USA) and ModFit 3.0 software (Verity Software House, Topsham, USA) were used for analysis.
2.9. Transwell Assay. Te cells were resuspended in 100 μL of serum-free medium and then inoculated into the 8 μm chamber coated with Matrigel. Te chamber was incubated in 500 μL of complete medium for 24 hours. We use a cotton swab to remove the remaining Matrigel and stain the cells in the upper chamber. Te chamber was immersed in 4% paraformaldehyde (PFA), and then the cells were stained with crystal violet dye. Six microscope felds were imaged, and the number of cells in these felds was counted (100× magnifcation).

Wound
Healing Assay. For this assay, 5 × 10 4 cells were plated in 6-well plates and grown until they reached 90% confuency. We used a 10 μL plastic pipette tip to make a scratch. Wound healing images were taken at 0, 4, and 8 hours. At the same time, we calculated the cell migration distance and compared it with T � 0 hours.

In Vivo Xenograft Mouse Model. Animal care procedures and mouse experiments have been approved by the relevant units. Experiments were performed in female Fox
Chase severe combined immunodefciency 6-week-old mice. We split mice into control and experimental groups. HOS cells stably expressing shGINS2 or GINS2 were suspended in PBS (4 × 10 6 cells in 200 μL) and inoculated subcutaneously in the right axillary position (n � 10 per group). Data collection began 12 days after the mice were subcutaneously injected with OS cells (weigh the nude mice and measure the length and short diameter of the tumor) and then collected once every 2 days for a total of 5 times. After 4 weeks, the mice were sacrifced, and their internal tumors were analyzed.

Microarray Gene Expression Analysis.
After the infection with shGINS2, a total of RNA was extracted from HOS cells. We used the NanoDrop 2000 spectrophotometer and Agilent 2100 bioanalyzer system to evaluate the concentration and quality of RNA. Qualifed samples were analyzed using the GeneChip PrimeView human gene expression array (Afymetrix, USA). Te analysis results were selected according to the critical P value <0.05 for the selection of diferentially expressed genes. At this time, the critical value of the fold change was equal to 2. We used "subtle path analysis" to analyze the diferentially expressed genes obtained in microarray analysis. Specifcally, we evaluated the molecular pathways and core organisms in which the diferentially expressed genes were involved.
2.13. Western-blot. Total protein was extracted using a lysis bufer and protease inhibitor (Beyotime Biotechnology). Equivalent protein amounts were denatured in an SDS sample bufer and then separated by SDS-PAGE and transferred onto a polyvinylidene difuoride membrane. After being blocked with 5% nonfat dry milk in TBST, the blotted membranes were incubated with anti-GINS2 antibodies (1 : 500, SIGMA) and then incubated with a secondary antibody (1 : 2000, Santa Cruz, USA). GAPDH protein levels were also determined by using the specifc antibody (1 : 2000, Santa Cruz, USA) as a loading control.

LC-MS Analysis and Coimmunoprecipitation.
Te fusion GINS2 gene was amplifed by adding a 3 × FLAG tag sequence (Sigma) using PCR and inserted into the GV208 lentiviral vector. Te construct is called 3 × FLAG-GINS2. Lentiviral empty vector and 3 × FLAG-GINS2 plasmid were cotransfected along with the helper plasmid into 293T cells, and the lentiviral particles (lenti-control (NC) and lenti-3 × FLAG-GINS2 (OE)) were harvested. We lysed the NC and OE stable cell lines based on the radioimmunoprecipitation assay, and the total protein in the supernatants was collected by centrifugation to quantify proteins using the bicinchoninic acid assay (BCA). Equal amounts of total protein from the two groups were used for coimmunoprecipitation (Co-IP) with FLAGbeads (Sigma), and the subsequent in-depth SDS-PAGE and Coomassie brilliant blue staining. Proteins in the gel bands were further hydrolyzed using trypsin to generate peptides for liquid chromatography−mass spectrometry (LC−MS) identifcation. Te protein identifcation results in each sample were obtained by performing a database search using the PD/ MASCOT software. Finally, bioinformatics analysis of the proteins specifcally identifed in the OE group was performed, and the gene network map was drawn.

Prediction of Immunotherapy Response. Te Tumor
Immune Dysfunction and Exclusion (TIDE) algorithm is a computational mechanism that uses gene expression profles to forecast immune checkpoint inhibitor responses. We collected gene expression profles of 47 osteosarcoma samples from the GSE39058 cohort. And TIDE algorithm was used to predict the immunotherapy response of patients with osteosarcoma. Te deconvolution consequences for the tumor-infltrating immune cells were analyzed through the CIBERSORT algorithm.

Statistical Analysis.
Tree experiments were performed, and the data was calculated according to the mean ± standard deviation (SD), and the analysis was Journal of Oncology 3 performed with the help of SPSS 23.0 and GraphPad Prism 7.0. We used the Student's t-test and analysis of variance (ANOVA) to assess the diferences between the two groups. P < 0.05 was used to indicate statistical signifcance.

GINS2 Level Correlates with Tumor Progression and Predicts Clinical Outcomes of OS Patients.
To systemically study the role of GINS2 in osteosarcoma (OS), we analyzed the protein levels of GINS2 in 59 OS tissues with 8 normal tissues by immunohistochemistry (IHC) and found that the protein levels of GINS2 are signifcantly elevated in OS tissues (P < 0.01, Figures 1(a) and 1(b)). According to the receiver operating characteristic (ROC) curve (Figure 1(c)) with an optimal cutof point of 6.0, we found that GINS2 is overexpressed in 64.4% (38 of 59) of human OS specimens (Table 1). To determine the clinical importance of GINS2 in human OS, correlation analysis revealed that elevated GINS2 isn't correlated with the characteristics of OS patients (P > 0.05) ( Table 1). However, Kaplan-Meier analysis showed that patients with a higher level of GINS2 were associated with overall survival (P < 0.01, Figure 1(d)).
Together, these data demonstrated that GINS2 was a prognostic marker, suggesting that GINS2 might possess a promoting role in OS malignancy.

GINS2 Promotes OS Cell Growth and Metastasis.
In accordance with GINS2 expression in OS patients, the RT-qPCR assay demonstrated that GINS2 was highly expressed in the three OS cell lines. To gain insights into the potential role of GINS2 in OS progression, we constructed stable GINS2 knockdown (shGINS2) cells using Saos-2, U2OS, and HOS, respectively (Figures 2(a)-2(e)). Cell proliferation monitored by high-content screening (HCS) analysis showed that depletion of GINS2 obviously inhibited cell proliferation in all three OS cells (Figures 3(a) and 3(b)). To confrm the relationship between GINS2 and cell metastasis, a transwell assay and wound healing analysis were performed. Te results showed that GINS2 silencing markedly reduced the migration and invasion of OS cells (Figures 3(c)  and 3(d)). Tese observations are in line with the fact that elevated levels of GINS2 in OS were accompanied by aggressive progression and poor outcomes.

Knockdown of GINS2 Promotes Apoptosis and Induces Cell
Cycle Arrest. Subsequently, we evaluated the caspase-3/7 activity in GINS2 knockdown cells (Saos-2, U2OS, and HOS). Compared to the control group, caspase 3/7 activity was signifcantly increased in the GINS2 knockdown cells (Figures 4(a)-4(c)). In addition, Annexin V-APC staining was evaluated to analyze cell apoptosis by fow cytometry assays. Te results suggested that silenced GINS2 promoted cell apoptosis (Figures 4(d)-4(f )). In order to further analyze the efect of GINS2 on cellular behaviors, a fow cytometry assay was used to monitor cell cycle phases in U2OS cells. It showed that the percentage of cells in the S phase and G2/M phase was signifcantly increased, while the percentage of cells in the G1 phase was reduced in the GINS2 knockdown group. It suggested that the cell cycle was blocked in the S and G2/M phases (P < 0.05, Figure 4(g)). Tese data support the notion that GINS2 may be involved in the regulation of OS progression.

Silencing of GINS2 Inhibits Tumourigenicity In Vivo.
To investigate the role of GINS2 on tumorigenicity in vivo, we inoculated U2OS-shCtrl and U2OS-shGINS2 cells into the right fanks of nude mice. Te animals were sacrifced at the end of the experiment, and the tumors were dissected and weighed. Te tumors derived from the cells with GINS2-depleted tumors were smaller and lighter (Figures 5(a)-5(c)). Tis was consistent with the result of our in vitro experiments demonstrating that GINS2 knockdown impaired the proliferation of OS cells.

GINS2 Promotes Tumor Progression via the MYC
Pathway. Since GINS2 has been reported to be involved in multiple signaling pathways in a wide spectrum of cancer types, the core mechanism of GINS2-mediated tumor progression is still unclear. We decided to conduct Afymetrix gene chips and Intelligent Pathway Analysis (IPA) to investigate the role of GINS2 in U2OS cells (Figure 6(a)). A comparison with shCtrl cells revealed that 265 genes were upregulated and 755 genes were down-regulated in GINS2 knockdown cells. IPA revealed that multiple signaling pathways related to cancer development and apoptosis, such as the mTOR and IL-8 signaling pathways, were inhibited by GINS2. In addition, the gene list obtained from the microarray analysis was uploaded to the IPA system, and the key biological pathways were analyzed and processed to complete the identifcation of the related molecular networks. It was found that the expression of target genes in the MYC pathway including YAP1, SKP2, TFDP1, and THBS1 was signifcantly suppressed by GINS2 knockdown (Figure 6(b)). Tese results were confrmed by RT-qPCR and western blot (Figures 6(c) and 6(d)). In order to further examine the potential mechanisms between GINS2 and MYC, we conducted RT-qPCR and Western blot assays, which showed that MYC was signifcantly down-regulated by GINS2 depletion (Figure 6(e)). Together, the mechanism of GINS2-mediated tumorigenicity may be involved in the MYC signaling pathway.

STAT3 is Required for GINS2-Mediated OS Malignancy.
To determine the role of GINS2 in tumorigenicity, we used SDS-PAGE to separate the coimmunoprecipitated proteins and stain them (Figure 7(a)). Te Coomassie brilliant blue staining of the gel with GINS2-IP identifed several bands that were not present in the vector or IgG controls. Te immunoprecipitated protein was extracted from the gel, digested by trypsin, and then subjected to the LC-MS analysis. Following coimmunoprecipitation and western blot, we identifed STAT3, a core MYC interactor that existed as a candidate interaction partner of GINS2 (Figure 7(b)). We further verifed the interaction between GINS2 and STAT3 in functional experiments in vitro. STAT3 was overexpressed in the GINS2 knockdown cell lines by lentivirus infection. A series of experiments showed that STAT3 overexpression restored cell proliferation and metastatic capacity in GINS2 knockdown cells (Figures 7(c)-7(e)).

Role of GINS2 in Immunotherapy Response.
Given that patients with a higher tumor immune dysfunction and exclusion (TIDE) score have a higher chance of immune escape, they have a lower response rate to immunotherapy.    27 16 All factors were well balanced between the two groups (P > 0.05).

6
Journal of Oncology    All data were presented as the mean ± SD, statistical signifcance was analyzed by Student's t-test and one-way ANOVA, * P < 0.05, * * P < 0.01. (g) the cell cycle of USO2-shGINS2 cells was monitored by FACS analysis. All data were presented as the mean ± SD, statistical signifcance was analyzed by Student's t-test and one-way ANOVA, * P < 0.05, * * P < 0.01. (e) inhibit GINS2 on HOS cells to detect MYC expression. All data were presented as the mean ± SD, statistical signifcance was analyzed by Student's t-test and one-way ANOVA, * P < 0.05, * * P < 0.01.
Te TIDE algorithm result of GSE39058 showed that TIDE scores and exclusion scores were lower in the low GINS2 group (P < 0.05, Figures 8(a)-8(c)). It indicated that higher GINS2 did have a lower rate of immunotherapy response (Figure 8(d)). Moreover, immune checkpoints are important predictors of immunotherapy response. We evaluated the correlation between GINS2 and 6 immune checkpoints. Figure 8(e) showed that GINS2 was positively correlated with TNFSF9 (P < 0.05), but negatively correlated with TIGIT, IL10, IDO2, CTLA4 and CD80 (P < 0.05). Te immune cell infltration was signifcantly diferent in the two groups, with more T cells, follicular helper cells,   macrophages M1, and neutrophils in the low GINS2 group (Figure 8(f ), P < 0.05). Tese data together suggest that GINS2 plays an important role in tumor prognosis and may be an immunotherapy response prediction in osteosarcoma.

Discussion
Osteosarcoma (OS), which occurs frequently in adolescents and adults, is a primary malignant bone tumor [6]. Although the prognosis in patients with OS has improved due to efective therapeutic strategies, patients with distant metastasis or local recurrence still pose an extremely difcult treatment challenge [14]. Terefore, a better understanding of the molecular biology of OS is required in order to improve therapeutic efciency.
As an important subunit of the GINS complex, GINS2 can mediate the initiation of DNA replication in eukaryotic cells [15]. Relevant research reports pointed out that high expression of GINS2 had a clear connection with the occurrence and development of many malignant tumors, and it could also play an efective role in the tumorigenesis stage by regulating tumor cell apoptosis, signaling pathways, and the cell cycle [16,17]. However, the role of GINS2 in OS remains unclear to date. Studies have found that GINS2 played a central role as an oncogene in the development stage of OS. When we compared OS with normal bone tissues, we found that the expression of GINS2 in OS samples was upregulated, which was related to a poor prognosis. Trough a series of in vitro studies using three OS cell lines, we have demonstrated the core role of GINS2 in cell proliferation and apoptosis. Ten, nude mice were injected subcutaneously with HOS cells to study tumor growth in vivo in a xenograft model. Tis experiment verifed the in vitro results, showing that there was a positive correlation between GINS2 expression and OS growth. In general, our study results revealed sufcient evidence that GINS2 is a prognostic marker and a potential new therapeutic target in OS.
Previous studies reported that GINS2 promoted tumor growth by regulating specifc downstream signaling pathways [13,18]. Te regulation of GINS2 by the downstream signaling pathways in tumor cells is not yet fully understood. To examine the potential mechanism by which GINS2 regulated the progression of OS, we performed microarray analysis to investigate the diferences in cancer-related genes between ordinary OS cells and GINS2-depleted cells. IPA and WB data indicated that GINS2 knockdown cells downregulated the expression of MYC and multiple target genes in the MYC axis. MYC is a proto-oncogene that can improve the performance of oncogenic transcription amplifcation, is one of the most highly amplifed oncogenes in numerous human cancers, and is a signifcant target of cancer therapy [19][20][21]. In addition, misregulated expression of MYC is constantly associated with OS oncogenesis and progression [22][23][24]. In summary, GINS2 might exert an accelerating efect on the activity of the MYC signaling pathway and thus impact OS tumorigenesis and development.
Te MYC signal transduction pathway includes the corresponding gene family related to cell proliferation. It is still not clear whether GINS2 is directly or indirectly regulated by the downstream MYC signaling pathway in OS. By performing CoIP-MS analysis and rescue experiments, we obtained potential evidence that the interplay between STAT3 and GINS2 was associated with OS growth. As a transcription factor, STAT3 directly regulates the expression of oncogenes, thereby triggering tumor development [25][26][27]. Several previous reports have addressed its role as a potential therapeutic target for cancer treatment [28]. A variety of STAT3 inhibitors have also been successfully discovered [29]. In addition, various studies have shown that STAT3/MYC signaling can regulate tumor energy metabolism and the microenvironment to impact tumor cell proliferation and metastasis [30,31]. Tese studies have enriched our understanding of how STAT3 can exert an efect on cell proliferation in OS. Terefore, we surmised that GINS2 might regulate the MYC signaling pathway via possible interactions with STAT3 during the carcinogenesis of OS, and utilization of GINS2-STAT3-MYC interaction axis inhibitors might be an efective approach in OS therapy. However, the exact mechanism of MYC pathway regulation by GINS2-STAT3 interaction requires further research. Meanwhile, the mechanism of GINS2 as an immunotherapy response predictor also requires further research in followup studies.

Conclusions
In summary, we have found that high expression of GINS2 is signifcantly associated with OS tumorigenesis. Furthermore, GINS2 was involved in the regulation of the STAT3/ MYC signaling pathway to trigger the growth of OS cells. From a bioinformatics study, GINS2 may be an immunotherapy response prediction in osteosarcoma. Terefore, our fndings suggest that GINS2 possesses great potential strategies for treating OS.

Data Availability
Te underlying data supporting the results of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Tis study was approved by the Ethics Committee of the First Afliated Hospital of Nanchang University. According to the content of the ethics committee guidelines, informed written consent was obtained from all patients or their consultants.
Bingkai Ren, Yibin Zheng, Leiwen Huang, and Jingdu Wei analyzed the data. Bingkai Ren drafted the manuscript. All authors have read and approved the fnal manuscript.