ELK4-mediated lncRNA SNHG22 promotes gastric cancer progression through interacting with EZH2 and regulating miR-200c-3p/Notch1 axis

doi:10.21203/rs.3.rs-494915/v1

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ELK4-mediated lncRNA SNHG22 promotes gastric cancer progression through interacting with EZH2 and regulating miR-200c-3p/Notch1 axis

https://doi.org/10.21203/rs.3.rs-494915/v1

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Background

Long non-coding RNAs (lncRNAs) play important regulatory roles in the initiation and progression of various cancers. However, the biological roles and the potential mechanisms of lncRNAs in gastric cancers remain unclear.

Methods

The expression of SNHG22 in gastric cancer was analyzed in public databases (TCGA) and validated via qRT-PCR. SNHG22 knockdown cell lines were construced, and cell proliferation and invasion were analyzed. CHIP and luciferase reporter assays were performed to clarify the transcriptional role of ELK4. RNA pull-down followed MS and RIP assays were employed to identify the interaction between SNHG22 and EZH2. Luciferase reporter assays and RIP assays were used to confirm the regulation of SNHG22 on Notch1 by sponging miR-2003-3p.

Results

Knockdown of SNHG22 inhibited the proliferation and invasion ability of GC cells. Moreover, we identified that the transcriptional factor, ELK4, could promote SNHG22 expression in GC cells. In addition, using RNA pull-down followed MS assay, we found that SNHG22 directly bound to EZH2 to suppress the expression of tumor suppressor genes. At the same time, SNHG22 sponged miR-200c-3p to increase Notch1 expression.

Conclusions

Taken together, our findings demonstrated the role of SNHG22 on promoting proliferation and invasion of GC cells. And we revealed a new regulatory mechanism of SNHG22 in GC cells. SNHG22 is a promising lncRNA biomarker for diagnosis and prognosis and a potential target for GC treatment.

Cancer Biology

Clinical Pharmacology

Gastroenterology & Hepatology

Critical Care & Emergency Medicine

SNHG22

gastric cancer

EZH2

Notch1

Gastric cancer (GC) is one of the most frequently and malignant cancer and the third cause of death of cancer patients¹. Despite of the aggressive treatment including surgical resection and chemotherapy, the five-year net survival rate of GC patients is still poor, less than 40%². The aggressive phenotypes of GC cells, like enforced proliferation and invasion, is the main reason for the poor prognosis of GC cells^{3, 4}. Therefor, revealing the potential molecular mechanism of malignancy of GC cells is necessary. Recent studies have demonstrated the important role of lncRNAs as critical regulators in a lot of cancers including GC^{5, 6}. Long noncoding RNAs (lncRNAs) are a class of RNAs molecules that comprise more than 200 nucleotides in length^{5, 7}. LncRNAs have been reported to be dysregulated in cancer tissue compared with adjacent normal tissues, indicating the special function of lncRNAs on regulating tumorigenesis and progression^{8, 9}.

Many lncRNAs have been reported to be involved in mediating GC progression. For example, lncRNA HCP5 is down-regulated in GC tissues and HCP5 expression and overall survival of GC patients is significantly corrected. Moreover, decreased HCP5 had a facilitating effect on proliferation and invasion of GC cells¹⁰. Similarly, lncRNA BX357664 was proved lowered expressed in GC cells than adjacent tissues. And BX357664 could sponging miR-183-5p to regulating PTEN and PI3K/Akt pathway to inhibit the proliferation and metastasis of GC¹¹. However, the mechanism of lncRNAs remains unclear. And more lncRNAs needs to be explored in GC cells. SNHG22 is a newly discovered lncRNA, which is translated from SNHG22 genes located on chromosome 18q21.1. SNHG22 was proved to be overexpressed in epithelial ovarian carcinoma and acts as an oncogene promoting tumor progression¹². However, the molecular mechanism of SNHG22 in GC cells is not clearly discovered.

In this study, we showed that SNHG22 was overexpressed in tumors of GC patients and it was correlated with advanced pathological phenotype and poor survival. ELK4 transcriptionally elevating SNHG22 expression. SNHG22 promoted GC progression in vitro and in vivo by recruiting EZH2 and sponging miR-200c-3p. Our study provided a potential new target for GC treatment.

Patients samples

A total of 60 GC cases who had surgically proven primary GC were obtained from the Affiliated Hospital of Qingdao University. This study was approved by the Ethics Committee on Human Research of the Affiliated Hospital of Qingdao University, and written informed consent was received from all patients. All GC and paired normal tissue samples were collected during surgery and frozen immediately in liquid nitrogen for further total RNA or protein extraction.

Cell culture

The GSE-1, BGC-823, SGC-7901, MGC-803, AGS, MKN-45, NUGC-3 and HEK 293T cell lines were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplementary with 10% FBS, 100μg/ml streptomycin and 100 U/ml penicillin. All cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.

RNA preparation and quantitative real-time PCR (qRTP-CR)

Total RNA extraction from GC cells or clinical samples was performed using the TRIzol reagent (Invitrogen, USA) as previously described¹³. For SNHG22 distribution analysis, PARISTM Kit (Thermo Fisher, USA) was used to extract the nuclear and cytoplasmic RNA separately as previously described. Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix (Vazyme) with an ABI Prism 7900 Sequence detection system (Applied Biosystems, Canada). U6 and beta-actin were used as endogenous controls. The primers are shown in Supplementary Table 1.

Protein extracton and western blot

Protein extraction from cells and tumor tissues and western blot analysis were performed as described as previously described¹³. Antibodies against ELK4 (abcam, ab86002), EZH2 (abcam, ab191250), H3K9me1 (abcam, ab16989), H3K9me2 (abcam, ab1220), H3K9me3 (abcam, ab8898), H3K27me2 (abcam, ab24684), H3K27me3 (abcam, ab6002), H3 (abcam, ab1791), E-cadherin (CST, 14472), EAF2 (CST, 14159), ADRB2 (abcam, ab182136), rap1GAP (abcam, ab32373), RUNX3 (abcam, ab224641), GAPDH (CST, 5174) and Notch1 (CST, 3608) were used.

Plasmids and siRNA transfection and lentiviral transduction

Lentivirus carrying sh-SNHG22 or sh-ctrl was packaged in human embryonic kidney 293T cells using the lentiviral packaging kit purchased from Genechem (Shanghai, China). Stable cell lines were established by infecting BGC-823 and MGC-803 cells with lentivirus followed by puromycin selection. siRNA targeting ELK4, miR-200c-3p mimics and anti-miR-200c-3p were designed and synthesized by RiboBio (Guangzhou, China).

RNA fluorescence in situ hybridization (FISH)

Cy5-labeled specific probe to SNHG22 was purchased from RiboBio and a FISH kit (RiboBio) was used to detect the signals according to the manufacturer’s instruction.

Cell counting kit-8 (CCK-8) assay

To measure the viability of GC cells, 3 x 10³cells were seeded into 96-well plates, and the CCK-8 solution (Dojindo, KMJ, Japan) was added to each well at the indicated time. After a 1 h incubation at 37 °C, absorbance was detected (OD value) at a wavelength of 450 nm.

Colony formation assay

To measure the viability of GC cells, 1000 cells were seeded into 6-well plates and cultured for approximately 2 weeks until colony formation was observed.

Transwell assay

Transwell invasion assay was performed using a 6.5-mm diameter Transwell chamber with 8-μm pore polycarbonate membrane insert (Corning). Matrigel (Corning) was used to cover the upper chambers. Transfected or control GC cells were plated on the upper chambers. 12 h later, cells were fixed, stained and counted.

In vitro 3D migration assay

For 3D migration assays, 20 μl GC cell suspension (containing 2000 cells) was placed onto the lid of a 6-cm dish. The lids were inverted for 2 days to obtain a cellular aggregate. The cellular aggregates were then implanted into three-dimensional collagen I gels (PureCol, Inamed, Fremont, CA, USA). The migration of GC cells was monitored using a Leica DMI3000B microscope system at indicated time.

Dual luciferase reporter assay

To confirm that SNHG22 could sponge miR-200C-3p, HEK-293T cells were co-transfected with the mixture of luciferase reporter vectors (pmirGLO) containing SNHG22-miR-200c-3p binding sequences or mutant sequences and miRNA mimics (20 nM) to examine the interaction between SNHG22 and miR-200c-3p. A dual luciferase reporter assay system (Promega, Madison, WI, USA) was employed to measure the luciferase activity according to the manufacturer’s protocol.

To explore the transcriptional regulation of ELK4 on SNHG22, HEK293T cells were co-transfected with luciferase reporter comprising wild type or mutant SNHG22 promoter region and empty vector or ELK4 plasmid (Genechem). A dual luciferase reporter assay system (Promega, Madison, WI, USA) was employed to measure the luciferase activity according to the manufacturer’s protocol.

Chromatin immunoprecipitation (ChIP)

The Simple Chip Enzymatic Chromatin IP kit (CST, USA) was used to identify the regulation of ELK4 on SNHG22. Briefly, BGC-823 and MGC-803 cells were incubated with formaldehyde for 10 min to obtain the DNA-protein crosslinks. Then, cell lysates were subjected to sonication to get the chromatin fragments and specific antibody or IgG as a control were used for immunoprecipitation. Later, qPCR analysis was employed to analyze the precipitated chromatin DNA.

RNA pull-down and mass spectrometry

SNHG22 pull-down was performed by Magnetic RNA-protein Pull-down Kit (Thermo) according to the manufacturer’s protocol. Firstly, 5′Biotin-labeled oligonucleotide probe targeting SNHG22 was synthesized and purchased from KeyGEN (Jiangsu, China). The RNA was then bound to the beads to orient the RNA for protein binding. RNA-bound beads were incubated with nuclear extraction at 70 °C for 5 min. After RNA slowly cooled down to room temperature, Streptavidin Magnetic Beads were added and incubated at room temperature for 30 min. After washing away the unbound RNA, the beads were incubated with Elution buffer and the Supernatant was obtained for further mass spectrum and western blot. The identified proteins were listed in Supplementary Table 2.

RNA-protein immunoprecipitation (RIP)

RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used for detecting the binding between SNHG22 and EZH2 or SNHG22 and Ago2. Briefly, GC cell lysates were incubated with magnetic beads conjugated with indicated antibody or IgG as a negative control. Followed qRT-PCR analysis was performed to detect the enrichment of SNHG22.

Animal studies

All animal experiments were proved by xxxxxxx. Briefly, BGC823 cells that stably transfected with sh-ctrl or sh-SNHG22 were collected and suspended in PBS on ice. Twelve female BALB/c nude mice were divided into 2 groups randomly. 5 x 106 cells were subcutaneously injected in the thigh root of each mouse. Tumor volumes were monitored every day after injection. At end, the mice were executed and tumors were harvested for further qRT-PCR, western blot and IHC experiments.

Statistical analysis

Data are presented as mean ± SEM and were analyzed using GraphPad 8.01. Student’s t-test, two-way ANOVA, and chi-square tests were used for comparison. Overall survival (OS) was calculated using the Kaplan-Meier method. Statistical significance was set at p < 0.05.

SNHG22 is overexpressed in gastric cancer

The expression levels of SNHG22 were analyzed using TCGA dataset by GEPIA¹⁴ and found that SNHG22 was upregulated in gastric cancers compared with non-tumor tissues (Figure 1A). In addition, we observed that the expression of SNHG22 was distinctly upregulated in all human GC cell lines compared with the GSE-1 cell line (Figure 1B). Then, we analyzed the expression of SNHG22 in 60 paired GC tissues and normal tissues. qRT-PCR analysis showed that SNHG22 was upregulated in 44 GC tissues compared with the matched normal tissues (Figure 1C and D). Furthermore, The overall survival (OS) data showed that patients with high level of SNHG22 developed poor survival rate (Figure 1E). Together, the up-regulated SNHG22 might contribute to the GC development.

SNHG22 silencing inhibits GC cell proliferation and invasion

To explore the biologic function of SNHG22 in GC cells, we designed two shRNAs targeting the sequence of SNHG22 and qRT-PCR confirmed the silencing efficacy (Figure 2A). CCK-8 and colony formation assays demonstrated that shRNAs transfection but not scrambled oligonucleotides significantly suppressed the growth and proliferation of GC cells (Figure 2B and C). We also tested the effect of SNHG22 on the invasion of GC cells. Transwell assays indicated that SNHG22 silencing markedly suppressed normally strong invasive capacity of BGC-823 and MGC-803 cells (Figure 2D). Meanwhile, SNHG22 silencing inhibited GC cells infiltration in a 3D collagen matrix (Figure 2E). Taken together, these data indicated that SNHG22 depletion inhibited the GC progression in vitro.

ELK4 binds to the promoter region of SNHG22 and upregulates its expression in GC cells

We next sought to uncover the molecular mechanisms, which contributed to the upregulation of SNHG22 in GC cells. Accumulating researches have revealed that some lncRNAs transcription could be modulated by transcriptional factors (TFs) via directly binding^{15, 16}. Therefore, we tend to explore if TFs can regulate SNHG22 expression. Using the JASPAR database (http://jaspar.binf.ku.dk/)¹⁷, we found that ELK4 is a potential TF which promoting SNHG22 expression (Figure 2A). In accordance with SNHG22, ELK4 expression levels were significantly higher in GCs compared with non-tumor tissues in TCGA dataset (Supplementary Figure 1A). Next, we knocked down or overexpressed ELK4 in GC cells (Supplementary Figure 1B and C). qRT-PCR results showed that SNHG22 level was positively correlated with ELK4 level (Figure 2B and C). Furthermore, we found two possible binding sites (site 1 and site 2) of ELK4 in SNHG22 promoter regions (Figure 2D). Then, we generated luciferase reporter constructs containing fragments of nucleotides -2000 to +1 of the human SNHG22 promoter region and examined their responsiveness to ELK4 (Figure 2D). We found that ELK4 overexpression could not induce the activation of the reporter constructs containing the site2 fragment, while the activity of reporter constructs containing both site 1 and site 2 fragments was significantly increased induced by ELK4 (Figure 2D). In addition, we mutated site 1 and the reporter construct containing a mutant site 1 coul not be activated by ELK4 overexpression(Figure 2D). Meanwhile, we performed a chromatin immunoprecipitation (ChIP) assay using BGC-823 and MGC-803 cells. We found that site 1-containing DNA fragment, but not site 2 DNA fragment, of the SNHG22 gene was coprecipitated with ELK4, supporting the notion that ELK4 directly activates the SNHG22 promoter through binding the site 1 (Figure 2F). Finally, we explored the impact of ELK4 on the malignancy development of GC cells. As shown in Supplementary Figure 1D-G, ELK4 knockdown led to significant decrease of cell viability and invasion of GC cells. Together, our findings proved that ELK4 activated the transcription of SNHG22 in GC.

SNHG22 interacts with EZH2 inducing accumulation of H3K27 trimethylation to multiple tumor suppressive genes

To further evaluate the molecular mechanism by which SNHG22 promotes GC progress, we firstly used FISH and subcellular fractionation to analyze its distribution. The results showed that SNHG22 was primarily observed in both nuclear and cytoplasm (Supplementary Figure 2A and B). Hence, we tend to figure out the role of SNHG22 in different distribution separately.

To identify the mechanism of nuclear SNHG22, biotinylated SNHG22 and antisense control were incubated with nuclear extraction. Mass spectrometry (MS) confirmed the specific band of SNHG22 pull-down products was enhancer of zeste homolog 2 (EZH2) (Figure 4A). EZH2 is the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), with a nuclear distribution in GC cells¹⁸. We confirmed the interaction of SNHG22 with EZH2 using RNA pull-down assay followed by western blotting (Figure 4B and Supplementary Figure 2C). Consistently, RNA immunoprecipitation (RIP) showed enrichment of SNHG22 by EZH2 (Figure 4C and Supplementary Figure 2D). These results validated the interaction between SNHG22 and EZH2. Moreover, serial deletion analysis validated that the 538–1077 nt region of SNHG22 was indispensable for direct interaction with EZH2 (Supplementary Figure 2E). Sequence analysis by POSTAR2 (http://lulab.life.tsinghua.edu.cn/postar/)^{19, 20} indicated a sequence motif and structural preference of the RBP binding site for EZH2 (Figure 4E), which was located in the 737-763 nt region of SNHG22 and formed a stem-loop structure (Figure 4F). RIP performed after mutagenesis of this stem-loop region revealed that it was critical to SNHG22 interaction with EZH2 (Figure 4G and Supplemental Figure 2F). These data suggest that SNHG22 interacts with EZH2 in nuclear.

As EZH2 is the enzymatic subunit of polycomb-repressive complex 2(PRC2). And PRC2 functions as a histone H3 lysine 27 (H3K27) methyltransferase to promotes transcriptional silencing via regulating chromatin structure. At the same time, histone methylation is involved in transcriptome reprogramming during GC development. Considering the interaction between SNHG22 and EZH2, we therefore tend to figure bout whether SNHG22 could regulate histone methylation. The results showed that SNHG22 deficiency in GC cells preferably decreased trimethylation at H3K27 instead of the levels of methylation at H3K9 (Figure 5A), indicating a critical control mechanism of H3K27me3 by SNHG22. qPCR was performed to examine the mRNA levels of five common EZH2 target suppressive genes. The results demonstrated that the silence of SNHG22 remarkably increased the mRNA and protein levels in both BGC-823 and MGC-803 cells (Figure 5B and C). Furthermore, ChIP assays revealed that SNHG22 knockdown remarkably decreased the binding of EZH2 to the promoter regions of these tumor suppressive genes (Figure 5D and E).

Taken together, SNHG22 represses multiple suppressive genes by recruiting EZH2 to promote GC progression.

SNHG22 promotes Notch1 expression through sponging miR-200c-3p

Using online prediction tools (SNP2 and starbase)^{21, 22}, we found four potential miRNAs which could interact with SNHG22 (Supplementary Figure 3A). To figure out which one is the target of SNHG22, a dual luciferase reporter assay was performed and the results showed that miR-200c-3p significantly reduced the relative luciferase activity of the SNHG22 3’UTR (Supplementary Figure 3B). Furthermore, we constructed mutant SNHG22 reporter plasmid. The results of dual luciferase reporter assay showed that miR-200c-3p significantly suppressed the activity while the mutation of miR-200c-3p binding sites abolished this effect (Supplementary Figure 3D). In addition, Ago2 based RIP assays confirmed the interaction between SNHG22 and miR-200c-3p mediated by Ago2 (Supplementary Figure 3E). Next, we transfected GC cells with sh-SNHG22 or co-transfected sh-SNHG22 together with miR-200c-3p or anti-miR-200c-3p. In vitro experiments, including colony formation assays and 3D 3D collagen matrix invasion assays, verified that knockdown of SNHG22 mediated inhibition of GC cell proliferation and invasion were partially rescued by co-transfection with anti-miR-200c-3p. Together, these results suggested that carcinogenic effect of SNHG22 was partially mediated by sponging miR-143-3p.

Next, using online prediction tools (DIANA TOOLS, miRDB and Targetscan)^23-25, we found 411 potential targets of miR-200c-3p (Supplementary Figure 4A). GO analysis results indicated that these targets are involved in cell proliferation and invasion process (Supplementary Figure 4B). Among these putative targets, we selected eight common genes as potential components of the SNHG22 and miR-200c-3p ceRNA network. qRT-PCR revealed that overexpression of miR-200c-3p significantly inhibited Notch1 expression in GC cells, while anti-miR-200c-3p exhibited the opposite effect (Supplementary Figure C and D). Then, we constructed wild type and mutant Notch1 reporter plasmids (Supplementary Figure 4E). The results of dual luciferase activity assays demonstrated that miR-200c-3p bound directly to the Notch1 3′-UTR region (Supplementary Figure 4F). Overexpression of miR-200c-3p remarkably increased Notch1 expression, while knockdown of miR-200c-3p significantly reduced Notch1 expression (Supplementary Figure 4G). These results suggested that Notch1 is a direct target of miR-200c-3p.

Notch1 expression was decreased upon SNHG22 knockdown but was rescued by anti-miR-200c-3p. Conversely, Notch1 expression was increased upon SNHG22 overexpression but was rescued by miR-200c-3p in GC cells (Figure 6A). SNHG22 knockdown in GC cells elicited a marked increase in the recruitment of Ago2 to Notch1 transcripts, and overexpression of SNHG22 led to the increased enrichment of Ago2 transcripts bound to SNHG22 but decreased enrichment of Notch1 transcripts (Figure 6B). The luciferase activity of Notch1 reporter was decreased upon SNHG22 knockdown and was rescued by miR-200c-3p sponge, while the luciferase activity of mutant reporter was unchanged (Figure 6C). After identifying Notch1 as direct target of SNHG22, we next analyzed the functional role of Notch1 in GC progression. We observed that overexpression of Notch1 restored the proliferation and invasion in GC cells with knocked-down SNHG22 (Figure 6 D and E).

Collectively, these results demonstrated that SNHG22 functions as a molecular sponge for miR-200c-3p to facilitate the expression of Notch1 to promote the proliferation and invasion of GC cells.

Knockdown of SNHG22 inhibits GC progression in vivo

To determine the impact of SNHG22 on tumorigenic capacities in vivo, a xenograft tumor model was constructed. SNHG22-stable-knockdown BGC-823 cells or control cells were subcutaneously injected into nude mice. We found that the tumors derived from the control group were markedly larger than tumors obtained from the SNHG22-stable-knockdown group (Figure 7A and B). Additionally, tumors derived from the control group revealed higher SNHG22 levels (Figure 7C) together with stronger Notch1 and lower E-cadherin, EAF2, ADRB2, rap1GAP and RUNX3 expression in tumors obtained from the SNHG22-stable-knockdown group (Figure 7D and E). Together, these data indicated that SNHG22 promotes GC progression through elevating Notch1 and recruiting EZH2 to suppress tumor suppressive genes in vivo.

We next determined the expression levels of SNHG22 and the indicated genes in GC clinical samples. Consistent with the findings in mice, the higher Notch1 expression was observed in the SNHG22^high subtype GC clinical samples (Figure 7F), and lower expression of E-cadherin, EAF2, ADRB2, rap1GAP and RUNX3 expression were ovserved in the SNHG22^high subtype GC clinical samples (Figure 7G-K). These results confirmed the effect of SNHG22 on GC progression through regulating multiple genes.

Previous studies have revealed considerable roles for lncRNAs in the progression and development of human cancers^{26, 27}. In gastric cancers, a lot of lncRNAs, like CRNDE²⁸, RNA1²⁹, have been characterized and their functional mechanism have been reported as well. Recently, Shen k et. al demonstrated that SNHG22 induces migration, invasion and angiogenesis of gastric cancer cells via miR-361-3p/HMGA1/Wnt axis³⁰. However, the mechanism for high SNHG22 expression remains unknown. Meanwhile, deeper mechanism for SNHG22 regulating GC progression needs to be explored. In this study, we confirmed higher expression of SNHG22 in GC cells, and higher SNHG22 expression confers poor prognosis. In vitro and in vivo experiments supported the promotion of SNHG22 on proliferation and invasion of GC cells. Next, we found that ELK4 transcriptionally promotes SNHG22 expression. SNHG22 could not only sponge miR-200c-3p to elevating Notch1 expression, but also recruit EZH2 to the promoter regions of multiple tumor suppressive genes. These findings suggest that SNHG22 exerts an oncogenic function and play a key role in GC development.

ELK4 (SRF accessory factor-1, SAP-1) belongs to the TCF subfamily of the ETS domain transcription factors, and was described to have crucial roles in a lot of cancers³¹. ELK4 together with SRF is a significant regulator for many genes, for example c-fos and LAMB3^{32, 33}. In the present study, we found that ELK4 overexpression promotes proliferation and invasion of GC cells. Moreover, ELK4 can bind the promoter region of SNHG22 and regulate the expression level of SNHG22. Above all, this study found the regulatory mechanism of SNHG22 and indicated the important role of ELK4 on progression of GC.

LncRNAs were reported to regulate gene expression at the pre-transcriptional, transcriptional, and post-transcriptional levels³⁴. The distribution of lncRNAs largely determines what types of functions they can perform. At the transcriptional level, lncRNA regulates gene expression by modifying gene, histone and chromatin without changing the DNA sequences³⁵. Recently, many researches have been proved to have the capability of binding the PRC2 core component EZH2³⁶. In this study, we found that a large of SNHG22 was located at nuclear in GC cells, which conferred the potential of SNHG22 remodeling genes expression through EZH2. Using RIP and RNA pull-down assays, we found that SNHG22 directly binds EZH2 in GC cells, suggesting that SNHG22 regulates potential targets at the transcriptional level. Further results of ChIP assays revealed that SNHG22 could recruit EZH2 to the promoter regions of multiple tumor suppressor genes (E-cadherin, EAF2, ADRB2, rap1GAP and RUNX3), and modulating H3K27me3 levels to repress their transcription. These findings indicate that lncRNA SNHG22 plays a crucial role in EZH2-mediated repression of tumor suppressor genes in GC cells.

At the post-transcriptional level, lncRNAs and miRNAs networks control the display of numbers of target genes. This ceRNA mechanisms emerged as imperative regulator for target genes^{35, 37}. By online predictive tools and following RIP or luciferase activity assays, we discovered miR-200c-3p as the sponging miRNA of SNHG22. In addition, we found that Notch1 is the direct target of miR-200c-3p, and further results confirmed the regulation of SNHG22 on Notch1. Overall, our findings revealed the essential role of SNHG22/miR-200c-3p/Notch1 network on promoting GC progression.

In summary, our results establish highly expression of SNHG22 as a regulator of proliferation and invasion of GC cells. We further investigated the mechanism of SNHG22 in different distribution of GC cells on promoting tumor progression. Our results demonstrate that SNHG22 induces epigenetic silencing of E-cadherin, EAF2, ADRB2, rap1GAP and RUNX3 expression following binding with EZH2. Meanwhile, SNHG22 functions as a ceRNA to regulate Notch1 expression. These findings suggested that SNHG22 may serve as a potential therapeutic target and a prognostic biomarker in GC.

GC: Gastric cancer; lncRNA: Long non-coding RNA; SNHG22: small nucleolar RNA host gene 22; ceRNA: Competing endogenous RNA; ELK4: ETS transcription factor ELK4; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; TCGA: The Cancer Genome Atlas; Notch1: notch receptor 1; EAF2: ELL associated factor 2; ADRB2: adrenoceptor beta 2; RUNX3: RUNX family transcription factor 3.

Availability of data and materials

All data used or analyzed during this study are included in this article and are available from the corresponding author upon reasonable request.

ACKNOWLEDGMENTS

Not applicated.

Funding

Not applicated.

Contributions

X.M and Y.W designed this research. X.M, T.J and A.X performed the experiments and data analysis. X.M drafted the manuscript and the other authors revised the manuscript.

Ethics declarations

Ethics approval and consent to participate

The study protocols were approved by the Ethics Committee of The Affiliated Hospital of Qingdao University. Informed consent was obtained from all participants included in this study according to ethical committee regulations.

Consent for publication

All authors agree with the content of the manuscript.

Competing interests

The authors declare that they have no competing interests.

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ELK4-mediated lncRNA SNHG22 promotes gastric cancer progression through interacting with EZH2 and regulating miR-200c-3p/Notch1 axis

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