SETDB1 Promotes the Growth of Glioma via CSF-1-dependent Macrophage Recruitment by Activating the AKT/mTOR Signaling Pathway

doi:10.21203/rs.3.rs-35583/v1

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SETDB1 Promotes the Growth of Glioma via CSF-1-dependent Macrophage Recruitment by Activating the AKT/mTOR Signaling Pathway

https://doi.org/10.21203/rs.3.rs-35583/v1

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published 15 Oct, 2020

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Background: Glioma is a common disease of the central nervous system (CNS), with high morbidity and mortality. Among the infiltrates in the tumor microenvironment, tumor-associated macrophages (TAMs) are abundant and they are significant factors in glioma progression. However, the exact details of disease progression have yet to be determined.

Methods: The clinical relevance of SETDB1 was analyzed by immunohistochemistry, real-time PCR and Western blotting and of glioma cancer tissues. Tumor cell proliferation, migration and invasion were investigated by MTS assay, colony formation assay, xenograft, wound healing and Transwell assay. The relationship between SETDB1 and CSF-1, as well as TAMS was examined by Western blotting, real-time PCR and syngeneic mouse model.

Results: This work shows the presence and upregulation of SETDB1 in gliomaand its relationship with disease prognosis. Gain and loss of function approaches showed the inhibition of apoptosis and the promotion of growth, migration and invasion of the disease with SETDB1 overexpression and converse effects with SETDB1 silencing in vitro. Mechanistically, SETDB1 promotes CSF-1 expression by activating the AKT/mTOR signaling pathway. This leads to macrophage recruitment in the tumor, leading to tumor growth.

Conclusion: This studyclarifies the modulation of tumor functions by SETDB1 and hence presents a future avenue for treating glioma.

Neurosurgery

SETDB1

glioma

AKT/mTOR

TAMs

CSF-1

Glioma isa tumor type that derived from glial cells,with a high incidence, high recurrence rate, and poor prognosis[1]. Previousresearch has demonstrated that gliomas account for 47.1% of primary malignant brain and other central nervous system tumors, of which glioblastoma is the main type of gliomas, accounting for about 56.1% of cases[2, 3]. The treatment process for glioma includes surgeryfollowed by radiotherapy, with or without temozolomide (TMZ) chemotherapy[4, 5].

Tumor progression and metastasis involve the several stromal cells that are not malignant, such as tumor-associated macrophages (TAMs) of the hematopoietic lineage,which areabundant migratory cells[6, 7]. The density of these cells has been shown to be related to the prognosis of several typesof cancer, such as glioma[8, 9]. The heterogeneous nature of these cells and their ability to show different responses to cues from the environment is indicative of their roles in cancer progression[10, 11].This microenvironment is modulated by the chemokine profile at the tumor site, as this influences macrophage differentiation to hence affect the advancement of glioma[12, 13]. Among these chemokines, colony stimulating factor 1 (CSF-1) is a ubiquitously produced factor seen in many tumors (includingglioma) that is vital for metastasis[14]. This factor causes the recruitment of TAMs and other cell subsets to influence the processes of inflammation, angiogenesis, proliferation and evasion of the immune response[15, 16]. The use of anti-CSF-1 antibodies has been shown to decrease the in vivo tumor burden by 96%, according to preclinical cancer models[17].

SETDB1 (SET domain bifurcated 1) is encoded by the approximately 38.6 kb long SETDB1gene located on human chromosome 1q21.3[18]. This protein is a member of the methyltransferase family of SET-domains (Su(var)3-9, E(z), Trithorax) that function by silencing genes or inhibiting transcription via H3K9 trimethylation[19]. SETDB1 is linked to embryonic development and is also a candidate for early Huntington disease therapy[20]. Recently, research pointed out that SETDB1 is expressed at abnormal and high levels in melanoma, ovarian cancer, lung cancer, and breast cancer[20-24]. The involvement and function of SETDB1 in gliomahave yet to be well studied, which calls for studies in this direction.

In the current study, we identiﬁed that SETDB1was markedly upregulated in glioma and displayed a significant association with the clinicopathological characteristics and survival of glioma patients. Overexpressing SETDB1 boosted the transcription of CSF-1 by activating the AKT/mTOR signaling pathway. Furthermore, SETDB1 induced CSF-1 expression in glioma cells leading to the recruitment of TAMs and subsequent tumor growth. These finding indicate the role of SETDB1 in both oncogenesis and TAM recruitment in glioma pathogenesis.

Cell lines and culture

American Type Culture Collection (ATCC, Manassas, VA, USA) was the source of glioma cell lines: U87, U251, H4, A172, U118, LN229, SHG-44 and GL261. Cell culture was performed in Dulbecco’s modiﬁed Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, Thermo, USA) at 37°C and 5% CO₂. NHAs (Normal human astrocytes) were procured from Lonza (Switzerland) and cultured as per supplied instructions.

Human glioma samples

Glioma tissues (n = 40) and neighboring healthy tissues (n = 40) were surgically excised at The People’s Hospital of China Medical University and the First Hospital of China Medical University. Table 1displays the clinicopathological features of the patients. Regular follow-up was performed for patients along with informed consent. Other data included relapse-free survival and overall survival (OS). This work received approval from the ethics committee of The People’s Hospital of China Medical University and the First Hospital of China Medical University.

Immunohistochemistry(IHC)

In accordance with previous works[25, 26], staining of tissues was performed with a SETDB1 antibody (Sigma-Aldrich, USA). The scoring of cells was performed in accordance with the following guide: 0 (no positive staining); 1 (1 to 25% positive cells); 2 (26 to 50% positive cells); 3 (51 to 75% positive cells); and 4 (> 75% positive cells). The scoring for staining intensity was performed as follows: 0, negative; 1, weak; 2, moderate; and 3, high. The formula for SETDB1-positive cells was as follows: staining intensity score ×percentage. This score involved both the nucleus and cytoplasm and was analyzed by two pathologists.

Cell migration and invasion assay

The migration assay used Falcon cell culture inserts from BD (Franklin, USA). The invasion assay utilized a BioCoat™ Matrigel™ Invasion Chamber (BD) in accordance with the instructions of the manufacturer. A phase contrast microscope was utilized to count cells; the average of 5 various fields/well was considered.

MTS analysis

A 96-well plate seeded with cells at 5000 cells/well. AMultiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientiﬁc) was employed to measure the absorbance at 570 nm for three independent assays.

RNA extraction and real-time PCR

Real-time PCR was performed as previously described[27, 28]. Briefly, TRIzol (Invitrogen, USA) was utilized for the extraction of total RNA. cDNA was synthesized with thePrimeScript™ RT reagent kit (Takara, Dalian) in accordance with the instructions of the manufacturer. SYBR Premix ExTaq II (Takara, Dalian) was utilized for real-time PCR of this cDNA on an ABI PRISM 7300 (Applied Biosystems, USA) to analyze the chemokines of the immune system. GAPDH was used to normalize mRNA levels. The primers used are listedin Table 2.

Western blotting

Western blotting was performed as previously described[29, 30]. Briefly, cell lysis was performed with the RIPA buffer protein extraction reagent (Pierce, Rockford, IL, USA) containing a protease inhibitor cocktail (Roche, USA). The proteins were resolved by 10% SDS-PAGE followed by transfer to polyvinylidene ﬂuoride (PVDF) membranes (Sigma-Aldrich). Next, the membranes were blocked using 5% bovine serum albumin (BSA) and incubated with primary antibodies at 4°C overnight. Appropriate secondary antibodies were later added and then visualizedby using an ECL chemiluminescence kit. The primary antibodies used are listed as follows: SETDB1 (HPA018142, Sigma-Aldrich), cleaved caspase 3 (9661, Cell signaling technology), cleaved caspase 8 (9748, Cell signaling technology), slug (9585, Cell signaling technology), vimentin (5741, Cell signaling technology), E-cadherin (14472, Cell signaling technology), mTOR (2983, Cell signaling technology), p-mTOR (5536, Cell signaling technology), AKT (4685, Cell signaling technology), p-AKT (4060, Cell signaling technology), CSF-1 (3155, Cell signaling technology), β-actin (3700, Cell signaling technology).

Cell isolation and differentiation

The preparation of human monocytes from buffy coats of healthy volunteers was performed using Ficoll-Hypaque (Pharmacia Corporation) for 50 min at 400 g. Twenty-four-well plates were seeded with 2×10⁶ cells/mL in RPMI 1640 medium containing 10% heat inactivated human AB serum, 50 U of penicillin/mL, 50 U of streptomycin/mL, 2 mM L-glutamine, and 100 ng/mL human M-CSF (which allows differentiation into macrophages). Warm medium was used to gently wash away non-adherent cells 6 days post-culture. CD14⁺ macrophages were found to account for greater than 95% of the adherent cells. The activation of these monocytes to macrophages in vitro involved the treatment of 2×10⁶ cells/L with 25 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) to produce M1-polarized macrophages and 45 ng/mL recombinant human interleukin-4 (IL-4; R&D) to produce M2-polarized macrophages. Flow cytometry was employed to detect the formation of macrophages. For the following in vitro assays, cells were cultured for 24 hours with RPMI media minus supplements and meticulously washed with PBS prior to the experiments.

Clone formation assay

Six-well plates were used to plate single-cell suspensions at a density of 1,000 cells per plate. Every 3 days, the culture medium was replaced. After allowing 2 weeks for clone formation, fixation and staining of the clones was performed using 0.1% crystal violet/40% methanol. Microscopy was performed to countcolonies with >50 cells/colony.

Animal experiments

The syngeneic glioma mouse model was generated in accordance with previous reports[31, 32]. Briefly, 2% isoflurane in O₂was used to sedate C57BL/6J mice (4-6weeks old). the addition of 5 × 10⁵GL261-EV andGL261-SETDB1cells in 100 μL of PBS was performed followed by subcutaneous injection in the flanks of C57BL/6J mice.The mice were sacrificed after 6 weeks and tumor weights were assessed.

For the subcutaneous model, the addition of 5 × 10⁵U251-EV, U251-SETDB1, U87-shCon or shSETDB1 cells in 100 μL of PBS was performed followed by subcutaneous injection in the flanks of nude mice. The mice were sacrificed 15 days and tumor weights were assessed. The mice were kept at the Mouse Experimentation Core premises of the China Medical University.

Statistical analysis

The mean ± standard deviation (SD) was used to represent the data of triplicate assays. Student’s t-test was applied to assess signiﬁcant differences between groups. Repeated measures analysis of variance was performed to assess variations between tumor parameters (growth rate and cell growth) of the animals.

SETDB1 is frequently overexpressed and predicts poor glioma prognosis

To investigate the role of SETDB1 inglioma cells, the level of SETDB1 was assessed in glioma patients. IHC and real-time PCR results demonstrated that SETDB1 was evidently increased in tumor samples compared with surrounding healthy tissues (Figure 1A and 1B). Next, real-time PCR was employed to assess the expression of SETDB1 mRNA in cell lines. Higher levels of SETDB1 were detected in the glioma lines in comparison with NHAs (Figure 1C). In addition, Western blotting showed that SETDB1 levels were higher in glioma cells than inNHAs cells (Figure 1D).

This was followed by studying the effect of this SETDB1 overexpression in the clinic. Increased mRNA expression of SETDB1 was evidently linked to shorter relapse-free glioma survival, as shown by Kaplan-Meier curves (Figure 1E). Our findings indicate that age and sex had no association with SETDB1 expression and TNM stage were found tobe linked with the protein expression (Table 1).SETDB1 was also found to serve as an independent factor for prognosis and shorter relapse-free survival, as shown by the multivariate Cox regression analysis (Table 3). Overall, high expression of SETDB1 was found to serve as a prognostic factor for glioma.

SETDB1 boosts the growth of glioma and inhibits apoptosis

The examination of the biological functions of SETDB1 involved overexpressing the protein using cDNA in or silencing it using SETDB1-speciﬁc small hairpin RNAs (shSETDB1) using lentiviral vectors in U87 and U251 cells. Lentiviruses with empty vector (EV) or negative control shRNA (shCon) served as controls in the transduction assays. The following changes in expression were assessed using real-time PCR and Western blotting (Figure 2A, 2B, S1A and S1B). Increased SETDB1 expression caused an evident increase in viability and clone formation in the cell lines studied compared withthose in the controls (Figure 2C and 2D). SETDB1 silencing caused the opposite results:decreased cell division and clone formation (Figure S1C-S1F). Next, the effect of this ectopic expression on apoptosis was studies; increased expression of SETDB1 noticeably reduced the levels of the active (cleaved) forms of caspase-8 and caspase-3 and hence decreased apoptosis in the cell lines mentioned previously in the presence or absence of TRAIL (Figure 2E). The opposite results were seen when silencing SETDB1, as indicated by increased sensitivity to TRAIL treatment and increased apoptosis and expression of its markers in U251 cells (Figure S1G).

Further investigation involved the subcutaneous injection of U251-EV and U251-SETDB1 into nude mice. This ectopic SETDB1 expression caused a conspicuous growth increase in tumors in the animals (Figure 2F). SETDB1 overexpression was confirmed by Western blotting (Figure 2G). Ki-67 assays showed that U251-SETDB1 xenografts displayed increased numbers of proliferating cells (Figure 2H). However, knockdown of SETDB1 caused an evident decrease in xenograft size (Figure S1H and S1I). Markedly decreased cell proliferation was observed in U87-shSETDB1 xenografts according to Ki-67 staining results (Figure S1J). Overall, SETDB1 is vital in the oncogenesis and advancement of glioma.

SETDB1 increases migration and invasion in glioma cells

Assays to assess the abilities of cells to migrate and invade were performed to examine the metastasis potential in vitro. The ability of cells to migrate and invade was augmented in the presence of ectopic SETDB1 expression in the U87and U251cell lines compared with the control (Figure 3A and 3C). Upregulation of Slug and Vimentin (mesenchymal markers) and downregulation of E-cadherin (an epithelial marker) were shown by Western blotting (Figure 3E). The opposite results were observed for the same cell lines that were subjected to silencing of SETDB1, which decreased the ability of the cells to invade (Figure 3B and 3D), decreased Slug and Vimentin and increased E-cadherin (Figure 3F). The above data suggest regulation ofglioma metastasis by SETDB1.

Increased SETDB1 promotes CSF-1 secretion from glioma

The levels of cytokines and chemokines CSF-1, TGF-β, IL-4, IL-13, and VEGF involved in the infiltration of TAMs were assessed through real-time PCR and enzyme-linked immunosorbent assay (ELISA) for SETDB1 in glioma lines. U87 and U251glioma cells displayed elevated profiles of CSF-1 mRNA and proteins in the presence of SETDB1 overexpression (Figure 4A, 4B and 4E), while knockdown caused the opposite effects (Figure 4C, 4D and 4F). As expected, CSF-1 expression signiﬁcantly correlated with CD163 and SETDB1 expression in glioma samples (Figure 4G and 4H). These observations are indicative of higher CSF-1 secretion due to elevated levels of SETDB1, which is in turn connected to increased TAMs in glioma.

SETDB1 promoted the secretion of CSF-1 by activating the AKT/mTOR signaling pathway

Next, the potential involvement of the AKT/mTOR signaling pathway in the CSF-1 secretion caused by SETDB1was assessed. Increased AKT and mTOR phosphorylation was shown by Western blotting when SETDB1 was overexpressed in glioma lines, and this effect was reversed by administration of MK-2206 (Figure 5A and 5B). Similarly, knockdown of AKT expression using siRNA attenuated the increase of CSF-1 due to SETDB1 overexpression (Figure 5C and 5D). To decipher the SETDB1/AKT/mTOR/CSF-1 axis in vivo, we performed Western blotting analysis on the tumor tissue from the xenograft model. We found that SETDB1 knockdown significantly reduced AKT activation and CSF-1 protein expression in the xenograft model, while SETDB1 overexpression remarkably promoted AKT activation and CSF-1 protein levels in xenograft tumor tissues (Figure 5E and 5F). Overall, these observations suggest the involvement of AKT/mTOR signaling in the increase in SETDB1-mediated CSF-1 synthesis.Hence, this axis can serve as a prognosticmarker in patients.

SETDB1 promotes recruitment and polarization of macrophages via CSF-1

The involvement of this SETDB1-CSF-1 axis in macrophage inﬁltration was assessed by an in vitro migration assay using conditioned media (CM) from glioma cells overexpressing SETDB1. Compared with control CM, CM from these cells was found to clearly augment the recruitment of macrophages (Figure 6A). Further, this recruitment was evidently suppressed when a CSF-1 antagonist was administeredto macrophages. Overall, these results suggest that macrophage recruitment is mediated by SETDB1 via CSF-1 induction.

Next, the effect of SETDB1 on macrophage polarization via CSF-1 was assessed. The CM from gliomacell lines overexpressing SETDB1 caused elevated expression of CD126 (a TAM marker) mRNA compared tothat in cells cultured with control CM, while there was no change in the expression of CD68 (a macrophage marker) (Figure 6B). Flow cytometry showed a similar protein profile (Figure 6C). The cytokine mRNAs and proteins that are representative of TAMs, IL-10, CCL17, and CCL22, were noticeably higher in TAMs incubated with CM from gliomacells with high SETDB1 expression than in TAMs incubated with CM from control cells (Figure 6D and 6E). Overall, these data suggest a role for CSF-1 and SETDB1 in promoting macrophage entry and differentiation.

SETDB1 promotes tumor growth in a macrophage-dependent manner

To demonstrate whether the oncogenic roles of SETDB1 in glioma are macrophage-dependent, we investigated the susceptibility of macrophages to liposomal clodronate treatment. For this, we overexpressed SETDB1 in the mouse glioma cell line GL261, which showed faint SETDB1 protein expression (Figure 7A). Next, we performed asyngeneic mouse model study with injection of SETDB1-overexpressing and control cell lines into the C57BL/6J mice. Prior to injection, liposomes containing clodronate or PBS were administered for two weeks and continued for three additional weeks after tumor implantation (Figure 7B). The former caused a decrease in F4/80+ macrophages in the glioma tumors of the animals (Figure 7Cand 7D). In addition, we noticed more macrophage infiltration in the SETDB1-overexpressing groupthan in the control group (Figure 7E). The increased tumor burden induced by SETDB1 overexpression was largely attenuated in mice that received clodronate liposomes (Figure 7F), suggesting that the tumor-promoting effects of SETDB1 in glioma are, at least in part, mediated by macrophage infiltration of the tumor microenvironment.

The progression of glioma involves the role of interconnected glioma cells and TAMs in the tumor microenvironment[33]. This increase in infiltrates is connected to the poor prognosis ofglioma[5]. The function of this system is yet to be characterized; thus, further studies are warranted to identify such patterns to rapidly unearth potential molecules that may serve as therapeutic tools[9]. Herein, for the ﬁrst time, wereveal that SETDB1 is involved in the modulation of the tumor microenvironment and, hence,gliomaprogression. SETDB1 was found to promote CSF-1 expression by activating the AKT/mTOR pathway. Moreover, SETDB1-induced CSF-1 modulated the tumor microenvironment by recruiting TAMs toglioma tissues, leading to tumor growth. These findingsreveal opportunities for research on the role of SETDB1 in disease progression.

SETDB1 functions as a histone methyltransferase to cause histone H3K9 trimethylation, which is involved in the formation of heterochromatin[34]. These H3K9 and H3K27 sites are connected to transcriptional regulation and epigenetics[35]. This presents an opportunity to target epigenetic modifiers such as SETDB1 to treat malignancies. Research has identified the overexpression of SETDB1 in many malignancies, such asglioma, melanoma, prostate cancer, glioma, and breast cancer (BRC), which was linked to cancer cell division as well as metastasis[19, 22, 34]. However, a complete picture is lacking in this area of cancer studies.

The association of TAMs with cancer cells in the tumor microenvironment has been the focus of studies[36, 37]. These cells have been shown to synthesize several factors that modulate cancer cell division and angiogenesis according to recent studies[38]. Particularly, the presence of symbiosis between macrophages and tumor cells has been shown by experiments where coculture of these cells caused the degradation of collagen[39]. The results from this work highlight several novel features of the mechanisms underlyingglioma.

Such features of TAMs have facilitated research on targeting these cells to address disease. CSF-1 and its receptor, colony-stimulating factor 1 receptor (CSF-1R), is aspect that has been in the spotlight and is seeing developments in clinical research[40]. One feature that is encouraging here is identification of safeapplicationsof immunotherapeutic or standard treatment tools[41-43]. Such promising activity has been demonstrated in autocrine CSF-1-based benign diffuse-type tenosynovial giant cell tumors[44, 45]. In the case of malignant disorders, reports from clinical perspectiveshave yet to be explored.

Continuous research facilitates the understanding that distinct macrophage features, such as functions and phenotypes,are a reflection of various signals (for differentiation, polarization, survival or recruitment) in a tissue-specific environment[46]. The implication here is that targeting TAMs for therapy would show variation according to the organ in which the cells are present[47, 48]. This work showed that increased CSF-1 was caused by SETDB1. The former molecule is synthesized by tumor cells and is involved in tumor progression and TAM inﬁltration;the origin of CSF-1 can be traced back to circulating monocytes in the blood vessels of the tumor. This provides the possibility that CSF-1 and its sustained production could serve as a target for efficient disease treatment. This work showed that the increasein CSF-1 was mediated by SETDB1 via AKT/mTOR activation. Hence, this work proposes a molecular mechanism for CSF-1 overexpression in glioma, opening up the possibility for this molecule or its receptor to be a target in patients with SETDB1-overexpressingglioma.

This work shows the overexpression of SETDB1 and its relevance in gliomavia activating the synthesis of CSF-1 and modulating the tumor microenvironment. The study of the precise function of this SETDB1/CSF-1/TAM axis in gliomacould support research on identifying appropriate therapies to target glioma.

ATCC: American Type Culture Collection; BSA: bovine serum albumin; CM: conditioned media; CNS: central nervous system; CSF-1: colony stimulating factor 1; CSF-1R: colony-stimulating factor 1 receptor; DMEM: Dulbecco’s modiﬁed Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; EV: empty vector; FBS: fetal bovine serum; IHC: Immunohistochemistry; NHAs: Normal human astrocytes; OS: overall survival; PVDF: polyvinylidene ﬂuoride; SD: standard deviation; SETDB1: SET domain bifurcated 1; TAMs: tumor-associated macrophages; TMZ: temozolomide.

Acknowledgements

None

Authors’ contributions

SH, WZ, TG, JZ and FL were involved in the experimental design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. SH, WZ and FL were involved in the experimental design and acquisition of xenografts. SH and FL were involved in the study conception and design, analysis and interpretation of data, revision of the manuscript, and study supervision.

Funding

None

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

This study was approved by the Ethical Committee of The People’s Hospital of China Medical University (The People’s Hospital of Liaoning Province).

Consent for publication

Informed consent was obtained from all individual participants included in the study.

Competing interests

The authors declare that they have no conflicts of interest.

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Table 1. Clinicopathologic Features of SETDB1 expression in glioma
	Low SETDB1 level (n=40)	High SETDB1 level (n=40)	p value
Age	55 ± 13.33	57 ± 12.65	0.432
Gender			0.469
Male	24	29
Female	16	11
TNM stage			0.001
I, II	32	7
III, IV	8	33

Table 2. Real-time PCR primers
SETDB1	Forward	5′-GGAGGAACTTCGTCAGTACATTG-3′
SETDB1	Reverse	5′-TCTTTCTGTAGTACCCACGTCTC-3′
CSF-1	Forward	5′-AGTATTGCCAAGGAGGTGTCAG-3′
CSF-1	Reverse	5′-ATCTGGCATGAAGTCTCCATTT-3′
TGF-β	Forward	5′-AAGAAGTCACCCGCGTGCTA-3′
TGF-β	Reverse	5′-TGTGTGATGTCTTTGGTTTTGTCA-3′
IL-8	Forward	5′-GTGCAGTTTTGCCAAGGAGT-3′
IL-8	Reverse	5′-TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC-3′
IL-4	Forward	5′-CCGTAACAGACATCTTTGCTGCC-3′
IL-4	Reverse	5′-GAGTGTCCTTCTCATGGTGGCT-3′
IL-13	Forward	5′-CCTCTGACCCTTAAGGAGCTTAT-3′
IL-13	Reverse	5′-CGTTGCACAGGGGAGTCTT-3′
VEGF	Forward	5′-CAAGCCAAGGCGGTGAGCCA-3′
VEGF	Reverse	5′-TCTGCCGGAGTCTCGCCCTC-3′
CCL2	Forward	5′-AGGTGTCCCAAAGAAGCTGTA-3′
CCL2	Reverse	5′-ATGTCTGGACCCATTCCTTCT-3′
CCL20	Forward	5′-TCCTGGCTGCTTTGATGTCA-3′
CCL20	Reverse	5′-CAAAGTTGCTTGCTGCTTCTGA-3′
CD86	Forward	5′-TCTCCACGGAAACAGCATCT-3′
CD86	Reverse	5′-CTTACGGAAGCACCCATGAT-3′
CD163	Forward	5′-TCCACACGTCCAGAACAGTC-3′
CD163	Reverse	5′-CCTTGGAAACAGAGACAGGC-3′
IL-10	Forward	5′-ATGCTGCCTGCTCTTACTGACTG-3′
IL-10	Reverse	5′-CCCAAGTAACCCTTAAAGTCCTGC-3′
CCL17	Forward	5′-AGGGACCTGCACACAGAGAC-3′
CCL17	Reverse	5′-AGGTAGTCCCGGGAGACAGT-3′
CCL22	Forward	5′-TGCCATCACGTTTAGTGAAGG-3′
CCL22	Reverse	5′-CGGCAGGATTTTGAGGTCCA-3′
GAPDH	Forward	5′-AATGGATTTGGACGCATTGGT-3′
GAPDH	Reverse	5′-TTTGCACTGGTACGTGTTGAT-3′

Table 3. Cox regression analysis for relapse-free survival in glioma patients
	Univariate analysis		Multivariate analysis
	Hazard ratio (95% CI)	p value	Hazard ratio (95% CI)	p value
SETDB1 expression (high vs. low)	2.234 (0.981-4.128)	0.023	2.459 (1.232-5.483)	0.018
Age	1.321 (0.548-2.325)	0.563	1.431 (0.674-2.543)	0.554
Male vs. Female	1.453 (0.674-3.092)	0.345	1.543 (0.783-3.482)	0.323
Stage (III, IV vs. I. II)	1.982 (1.128-3.763)	0.007	1.889 (1.093-3.542)	0.011

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published 15 Oct, 2020

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Review #1 received at journal
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Editorial decision: Major revision
05 Jul, 2020
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Reviewer #1 agreed at journal
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SETDB1 Promotes the Growth of Glioma via CSF-1-dependent Macrophage Recruitment by Activating the AKT/mTOR Signaling Pathway

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