Contribution of DNA methylation and EZH2 in SRBC down‑regulation in gastric cancer
Shiva Rezaei1 · Mohammad Ali Hosseinpourfeizi1 · Yaghoub Moaddab2 · Reza Safaralizadeh1

Received: 26 April 2020 / Accepted: 22 June 2020
© Springer Nature B.V. 2020

Gastric cancer (GC), a high mortality malignancy, is induced by genetic and epigenetic factors. DNA and histone meth- ylation play critical roles in tumor suppressor genes inactivation. SRBC (serum deprivation response factor-related gene product that binds to the c-kinase), suggested as a tumor suppressor gene, participates in apoptosis, tumor chemoresistance and DNA damage response and is repressed in various cancers. Inspecting the mechanisms underlying SRBC suppression is important for cancer treatments. We investigated SRBC promoter DNA methylation status and expression of SRBC and EZH2 histone methyltrasferase in gastric cancer. Also, we surveyed SRBC expression after 5-azacitidine and UNC1999 treatments of AGS cell line. In current work, we used gastric adenocarcinoma tissues, marginal samples and normal gastric biopsies. DNA methylation was detected by Methylation- Specific PCR and mRNA expression was measured by Real-Time PCR. SRBC promoter methylation analysis, showed fully and partial methylated versions that were associated with patient’s age (p = 0.001). SRBC expression significantly decreased in GC compare with marginal and normal samples (p-value < 0.001). EZH2 showed remarkable up-regulation in GC than controls and demonstrated a strong inverse correlation with SRBC expression (r = − 0.69). Restoration of SRBC expression was observed after 5-azacitidine and UNC1999 applications with a remarkable increase by combinational treatment. We showed that EZH2 plays role in SRBC silencing in addition to DNA methylation. Our study, suggests that DNA methylation and EZH2 are involved in SRBC silencing and their inhibitors can be considered in cancer treatment investigations to overcome chemoresistance induced by SRBC inactivation.
Keywords Gastric cancer · DNA methylation · Histone methylation · SRBC · EZH2

Gastric cancer (GC) is the fourth most common cancer and second leading cause of cancer related mortalities in the world [1]. The incidence of gastric cancer in Asian countries including Iran, especially in the north, northwest and west of Iran, is increasing [2–4]. Cancer as a complicated multi- factorial disease is an interplay between genetics and epige- netics pathways [5]. DNA methylation in the 5th carbon of cytosine in the regions containing CpG (C-phosphate-G),

and as a result formation of 5-methyl cytosine is one of the most common epigenetic changes [6]. DNA methylation cat- alyzed by the enzymes generally called DNA methyltrans- ferases (DNMTs) [7, 8]. DNA methylation status alternation, histone modifications and nucleosome rearrangements are implications of epigenetic changes in the cells [9, 10]. Epi- genetic investigations suggested that DNA methylation have pivotal interactions with histone modifications resulting in alternation of gene expression [11].
SRBC gene (serum deprivation response factor-related

gene product that binds to the c-kinase) suggested as a puta-

 Mohammad Ali Hosseinpourfeizi [email protected]
 Reza Safaralizadeh [email protected]
1 Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
2 Liver and Gastroenterology Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
tive tumor suppressor gene, was shown to be down regulated in different malignancies like breast, gastric and lung can- cers [12, 13]. Evidence has demonstrated that inactivation of SRBC (as a BRCA1 interactor protein) involves in tumor resistance against chemotherapeutic agents such as oxalo- platin treatments [14]. There is also findings highlighting the critical role of SRBC in apoptosis induced by TNFα [15]. Therefore, the study of mechanisms undying SRBC

inactivation can enhance knowledge of tumor therapeutic resistances.
During last years, a number of literatures highlighted the role of CpG island DNA hypermethylation in SRBC inacti- vation [13]. Additionally, other findings showed that SRBC loss of activation caused by allelic deletion or gene somatic mutations are infrequent and there might be other involved mechanisms independently works or depends on DNA meth- ylation [16].
Investigation proved that EZH2 (enhancer of zeste homolog 2) as a histone methyltransferase, directs tri- methylation of histone H3 at Lys 27 (H3K27me3) and par- ticipates in the expression status of the genes [17]. Reports authenticated EZH2 as a critical catalytic subunit of PRC2 which plays substantial role in silencing of its target genes [17]. To the best of our knowledge, the role of EZH2 in SRBC transcript regulation has not yet been investigated.
Unlike genetic changes, epigenetic modifications have possibility to reverse by using specific enzyme inhibitors. Therefore, it is worth to investigate the role of epigenetic factors leading to SRBC gene silencing in different cancers including gastric cancer which is significantly important in designing cancer epigenetic therapies [18]. Furthermore, investigations underlying epigenetic mechanisms in SRBC inactivation may introduce predictive biomarkers helping in overcoming chemotherapy resistance.
In current study, we examined DNA methylation status of SRBC promoter among patients with gastric adenocarci- noma cancer originated from northwest of Iran (East Azer- baijan province, Tabriz, Iran) as a high risk papulation for GC. Also, we wondered whether histone methylation by EZH2 is contributed in the SRBC transcript down-regula- tion. For this purpose, we explored SRBC and EZH2 mRNA expression levels among our patient tissues. Additionally, we applied DNA methyltransferases 1 (DNMT1) and EZH2 inhibitors as individual and combinational treatments to evaluate the possible re-expression of SRBC in AGS gas- tric cancer cell line.

Materials and methods
Clinical samples

In current work we used 40 gastric adenocarcinoma tis- sues and 40 corresponding non-cancerous marginal tissues (with no obvious tumor cells confirmed, pathologically), as well as 20 normal gastric biopsies from healthy individuals (matched on gender and age) which were taken by endos- copy procedure or surgical resection in Tabriz hospitals (East Azerbaijan province, Tabriz, Iran). All the tissues were frozen in liquid nitrogen, immediately and conveyed to − 80 °C freezer for future experiments. All the specimens
were evaluated and confirmed pathologically, before using in the experiments. Normal samples free of any diseases were used in the study. All the patients confirmed their vol- untarily participations and signed a written consent before sample collection. The study was conducted according to the research ethics approved by the committee of Tabriz Univer- sity of Medical Sciences (IR.TBZMED.REC.1397.772). We selected the patients without any type of prior radiotherapy or chemotherapy treatments.
DNA extraction and bisulfite conversion

DNA from tissue samples were extracted using Qiagen DNeasy blood & tissue kit (kat.no: 69504) according to manufacturer’s protocol. In order to analysis the quality and quantity of the extracted DNAs, A260/230 and A260/280 ratios were measured using a Nano drop instrument. After that, DNA samples went into bisulfite treatment reaction by using Qiagen EpiTect Bisulfite Kit (kat.no: 59104) and fol- lowing the kit’s instruction.
Methylation‐specific PCR (MSP)
Methylation and unmethylation-specific primers of SRBC promoter region were ordered as described in previous study [19]. PCR reactions were done with bisulfite-treated DNAs to amplify the SRBC promoter region using HotStart Plati- num master mix (Invitrogen, Carlsbad, CA, USA) by follow- ing its manufacture’s instruction and the specific primers. In vitro Sss1 Methylase treated DNA sample was used as positive methylated control. Additionally, normal lympho- cyte DNA and normal gastric biopsies were considered as positive unmethylated controls. PCR program was directed as: 94 °C (2 min), 40 cycles of [94 °C (30 s), 58 °C (30 s), 72 °C (30 s)] followed by an extension step of 72 °C (3 min). PCR products were then run on 2% agarose gels and cap- tured with the Gel Doc XR with Quantity One Version 4.6.1. Software (Bio-Rad, Hercules, CA, USA).
RNA extraction, cDNA synthesis and real‑time PCR

Tissue specimens were subjected to RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) accord- ing to manufacturer’s instruction. RNA samples were then evaluated by Nanodrop instrument in aspects of the qual- ity and quantity. RevertAid RT Reverse Transcription Kit (Thermo Scientific) was used to convert 1 µg of RNA to cDNA by following the kit’s protocol. DNase treatment was also applied to avoid genomic DNA contamination. After synthesis, cDNA samples were kept at − 20 °C freezer for later experiments. qPCR was performed with the specific primers which were described before for FP_SRBC 5′-GTTCTGCTCTTCAAGGAGGA-3′,

using bio-rad SYBR Green supermix (kat.no: 1725270) in bio rad Real-Time detection system. GAPDH were used as internal control to normalize the gene expression data.

Cell culture experiment

AGS Cell line (ATCC) was grown in DMEM medium con- taining 10% FBS (Gibco, 11,573,397), 4 mM L-glutamine, 4500 mg/L glucose, sodium pyruvate and 1% of penicil- lin/streptomycin under 5% (v/v) CO2 at 37 °C. In the way to investigate the effect of DNMT1 and EZH2 inhibi- tors on SRBC expression level, 5-Azacitidine (5-aza) and UNC1999 treatments were applied, respectively. For this purpose, 1 µM of 5-aza (Sigma-Aldrich) and 1 µM of UNC1999 (Sigma-Aldrich) were used to direct individual treatments as well as a combinational treatment of both inhibitors for 72 h. AGS cells were seeded in 6-well plates 24 h before treating the cells. DMSO was considered as control treatment in all the experiments. The treatments were done in triplicate.
Statistical analysis

Statistical analysis was done by SPSS v.26 software. Stu- dent’s t-test and one-way analysis of variance (ANOVA) using Duncan test were performed to compare the means. P-values of < 0.05 were inferred as statistically significant. Pearson’s correlation coefficient (r) was used to investigate the relation between EZH2 and SRBC expression levels.

Methylation specific PCR was directed to compare SRBC promoter methylation of GC tissues and their matched non-cancerous marginal samples. The analysis determined fully methylated and partially methylated versions of the SRBC promoter methylation in GC (Fig. 1). However, there was no fully non-methylated sample among them. In particular, promoter methylation analysis demonstrated 71.43% full methylation and 28.57% partial methylation in SRBC promoter region of the examined GC tissues, in total. In contrast, among matched marginal tissues, 85.71% showed fully non-methylated version. Additionally, we found 14.29% of marginal tissues as partially methylated in SRBC promoter region. After that, the relative expres- sion level of SRBC was determined in GC and marginal tissues to consider the effect of SRBC promoter methyla- tion status on the SRBC mRNA expression (Fig. 1d and e).

Fig. 1 Methylation-specific PCR and mRNA expression analysis of SRBC. MSP products of SRBC promoter directed in non-cancerous marginal tissues (a) and GC samples (b) as well as the controls (c). MSP with methylated and unmethylated specific primers using a gas- tric specimen of a healthy individual (obtained by endoscopy) was considered as additional positive unmethylated control. H2O was used
as zero control. d qPCR analysis of SRBC mRNA expression in GC samples (T), marginal tissues (M) and normal individuals (N); e real- time PCR products of SRBC and GAPDH on 2% agarose gel. T GC tissue, M non-cancerous marginal tissue, N normal healthy individ- ual, M methylated, UM unmethylated. ***Shows p-value < 0.001; NS non-significant

Normal gastric biopsies from healthy individuals was also evaluated as additional control in expression experiments. SRBC mRNA expression was defined by using real-time PCR. Unlike marginal and normal tissues, level of expres- sion was very low or even undetectable in some of the GC tissues. Transcript expression of SRBC (Fig. 1d) exhib- ited significant down regulation in GC samples compare to marginal samples (p-value < 0.001) and normal tissues (p-value < 0.001). Also, it was not detected any significant differences in relative mRNA expression between GC sam- ples exhibiting fully methylated SRBC promoter with the partial methylated ones. Our data analysis demonstrated a significant correlation of DNA methylation status and age of the patients (full methylated version in patient < 65 was 0.81%, > 65 was 0.14%; p-value = 0.001). However, there was no association with gender, lymph node metastasis and tumor type. Despite the presence of both methylation- and unmethylation specific products in parts of GC tissues, the SRBC mRNA expression was still significantly lower in this group than marginal tissues (p-value < 0.001), as well as the normal samples (p-value < 0.001). According to these results, we then explored EZH2 histone methyl- transferase expression as another possible epigenetic factor participate in regulation of SRBC expression together with
DNA methylation or independently.
As Fig. 2 shows qPCR analysis determined up-regu- lation of EZH2 expression in GC tissues in comparison with marginal samples (p-value < 0.001) and normal con- trols (p-value < 0.001). Additionally, comparison of EZH2 expression levels in partial methylated group of GC and partial methylated set of marginal tissues, exhibited a sig- nificant EZH2 up-regulation (p-value = 0.013) in these cancerous samples (mean = 1.00 ± 0.03) rather than the marginal ones (mean = 0.68 ± 0.01). Pearson correlation coefficient (r) demonstrated a strong inverse relationship between EZH2 and SRBC expression levels in gastric can- cer (p-value = 0.000; r = − 0.69), which is in accordance with previous finding showed a negative correlation of
EZH2 expression with its target genes in breast cancer [22].
Further confirmation of EZH2 and DNA methylation participations in SRBC down regulation, obtained in the analysis of AGS cell line, as a gastric cancer cell line pro- ducing low SRBC mRNA transcript. For this purpose, AGS cells were treated by 5-aza, UNC1999 and their combina- tions. qPCR mRNA expression analysis showed a significant increased expression of SRBC in the cells underwent 5-aza and UNC1999 treatments. Remarkably, this value was much higher in combinational treatment than the single treatments (Fig. 3a). Additionally, methylation analysis showed demeth- ylation effects of these treatments on SRBC promotor region (Fig. 3b). This can suggest an important role of EZH2 as a histone methyltransferase in SRBC silencing in AGS cells. Possible correlation between SRBC and EZH2 expression levels and clinicopathological features of the GC patients were assessed by SPSS v.26. Data analysis results dem- onstrated a significant association between SRBC expres- sion and the age of GC patients (p-value = 0.02). However, it was not found any correlation of SRBC expression with other clinicopathological characteristics like gender, lymph node metastasis and tumor type (Table 1). In case of EZH2 expression, we detected no link regarding all the examined
clinicopathological characteristics (Table 1).

Human SRBC gene, located in a tumor suppressor region of the genome, is considered to play roles in apoptosis, tumor chemoresistance and DNA damage response by interacting with BRACA1 [14, 15]. Inactivation through DNA meth- ylation of the CpGs especially in the regulatory elements is suggested as a critical cause of tumor suppressor genes inactivation [12].
During last years, studies have reported hypermethylation of SRBC promoter region in various human malignancies

Fig. 2 EZH2 relative mRNA expression in GC samples (T), marginal tissues (M) and
normal individuals (N). a Real- time PCR products of EZH2 on 2% agarose gel; b qPCR analysis of EZH2 expression
in GC tissues, non-cancerous marginal and normal controls.
***Shows p-value < 0.001. NS

Fig. 3 SRBC methylation status and mRNA expression after 5-aza and UNC1999 individual and combinational treatments. Re-expres- sion of SRBC after 5-aza and UNC1999 treatments was remark-

Characteristics No. of patients (%) SRBC relative expression (2−Δct) p- value EZH2 relative expression (2−Δct) p- value
< 65
18 (45%)
0.06 ± 0.02
1.11 ± 0.14
> 65 22 (55%) 0.22 ± 0.06 0.81 ± 0.11
Male 26 (65%) 0.18 ± 0.05 NS 0.95 ± 0.11 NS
Female 14 (35%) 0.09 ± 0.04 0.95 ± 0.15
Lymph node metastasis
Positive 26 (65%) 0.15 ± 0.04 NS 0.9 ± 0.11 NS
Negative 14 (35%) 0.14 ± 0.06 1.03 ± 0.15
Tumor type
Intestinal 23 (57.5%) 0.16 ± 0.05 NS 0.87 ± 0.1 NS
Signet ring type 6 (27.5%) 0.18 ± 0.01 0.85 ± 0.19
Diffuse 11 (15%) 0.11 ± 0.06 1.15 ± 0.22

Table 1 The relationship between SRBC and EZH2 expression levels and
able in AGS cell line (a). The effect of the treatments on the meth- ylation status of SRBC (b). M methylated, UM unmethylated. *Shows p-value < 0.05 and **shows 0.001 < p-value < 0.01

the clinicopathological Characteristics of GC patients
mean ± SEM
mean ± SEM

Bold value with an asterisk shows significant p-value (p < 0.05)
NS non-significant

such as colon, ovarian, lung and breast cancers [13, 14, 19]. Lee et al. demonstrated hypermethylation of CpGs in SRBC promoter in a remarkable part of “gastric cell lines and pri- mary tumors” leading to its transcript down regulation [12]. Our findings determined a higher percentage of SRBC meth- ylation in GC tissues in comparison with matched marginal samples. The presence of partial methylation in part of GC specimens can be explained by intratumoral heterogeneity or possible contamination with normal cells. Additionally,
we detected 14.29% partially methylation in marginal group. This may be as a result of precancerous stage already pre- sents in marginal cells as previous reports [16]. However, our data demonstrated no significant differences in SRBC and EZH2 relative expression levels in marginal tissues and healthy samples. This indicated that marginal tissues can consider as normal control containing the least number of cancerous cells. In total, SRBC expression was suppressed in GC tissues compare to marginal and normal samples.

Data analysis showed a significant correlation of DNA methylation status and age of the patients (p-value = 0.001) and no associations with gender, lymph node metastasis and tumor type which has been also reported in other studies, previously [23, 24]. Although, gene silencing by DNA methylation has been suggested as a possible mechanism of SRBC down-regulation [12, 19], other studies underlined that there may exist alternative mechanisms [16, 25]. Despite of detecting 0.14% fully methylated SRBC promoter among GC patients over than 65, SRBC expression of this group was still lower than marginal as well as normal tissues. Zӧchbauer-Muller et al. showed that 45% of the examined lung cancer sam- ples present SRBC silencing without DNA methylation [16]. Also, lee and coworkers reported that 6% of their investigated tumor specimens have normal SRBC expres- sion in spite of its promoter DNA methylation [12]. These findings indicate the existence of other involved factors in SRBC inactivation.
EZH2 is a methyl transferase which induces tri-methyl-
ation of histone H3 at Lys 27 and regulate gene expression [17]. Dysregulation of EZH2 has been detected in differ- ent cancers including gastric, breast, prostate and ovar- ian cancers [17, 26]. Here, we demonstrated that SRBC is down-regulated in both fully and partially methylated sets of GC tissues without any significant difference between the two groups suggesting the presence of other mechanisms participating in SRBC expression decline, in addition to DNA methylation. In agreement with previous study [27], EZH2 mRNA expression noticeably increased in GCs compare to marginal and normal samples with a significant up regulation in partially methylated group of GCs than the marginal tissues presenting partial methyla- tion. However, we did not determine any links of EZH2 expression with the clinicopathological features of the examined GC patients. On the other hands, our analysis showed an inverse correlation of EZH2 expression with SRBC expression level. These results can be an explana- tion for low expression of SRBC even in partially methyl- ated GC tissues.
Furthermore, re-expression of SRBC by 5-Aza and
UNC1999 as DNA and histone methyltransferase inhibi- tors provided another confirmation for the hypothesis of DNA methylation and EZH2 participations in SRBC silencing. This is in consistent with previous findings confirming the essential role of EZH2 in silencing of the genes independently or in partnership with DNA meth- ylation [18, 28]. In another study, Fujii and colleagues showed the restoration of RUNX3 expression in EZH2 knockdown cells. They also, showed a negative links of EZH2 and RUNX3 protein levels in gastric cancer and indicated RUNX3 as a target which is suppressed by EZH2 [28].
In conclusion, current study suggests EZH2 as another epigenetic factor participating in SRBC gene silencing in GC that could be viewed individually or in partnership with DNA methylation. We showed that marginal tissues have ideal possibility to inspect as normal controls. DNA and histone methyltransferase inhibitors can be considered as epigenetic therapies to re-express SRBC. However, it still needs to be validated in future using EZH2 knockout/ knockdown cells, in vivo experiments, larger populations and using other geographical origins in order to inter to clinical applications.
This work presents a link between SRBC suppression and EZH2 overexpression (in addition to DNA methylation) and opens a window toward future research going into the details of SRBC repression regulation by EZH2 and planning to overcome the tumor chemoresistance.
Acknowledgements We would like to thank to staff in Departments of Pathology and Surgery as well as Endoscopy Section in Imam Reza and Shahid Madani hospitals of Tabriz, Tabriz, Iran that kindly contributed with this investigation.
Author contributions Conceptualization and Study design: MAHF, RS and SR Patient’s sample provision: YM Experiments: SR Data analysis and interpretation: SR, MAHF and RS Writing—original draft preparation: SR Writing—review and editing: SR, MAHF, YM and RS Funding acquisition: MAHF and RS Supervision: MAHF and RS. All authors read and approved the final manuscript.
Funding This work was funded by University of Tabriz, Tabriz, Iran.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All participants received and signed an informed writ- ten consent. The study was conducted according to the research ethics approved by the committee of Tabriz University of Medical Sciences (Reference Number: IR.TBZMED.REC.1397.772).

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