METTL14-dependent m 6 A modification of circUGGT2 drives gastric cancer progression by sponging miR-186-3p and upregulating MAP3K9

doi:10.21203/rs.3.rs-2737082/v1

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METTL14-dependent m 6 A modification of circUGGT2 drives gastric cancer progression by sponging miR-186-3p and upregulating MAP3K9

https://doi.org/10.21203/rs.3.rs-2737082/v1

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Background: N6-methyladenosine (m⁶A) RNA modification has been shown to act fundamental roles in cancer progression. However, the underlying mechanisms by which m⁶A modification of circRNAs contributes to gastric cancer (GC) remain unknown.

Methods: METTL14-mediated m⁶A modification of circRNAs was identified by an m⁶A-circRNA epi-transcriptomic microarray and verified by methylated RNA immunoprecipitation (MeRIP) and m⁶A dot blot. Cell bio-behaviors were measured by molecular oncology experiments in vitro and in vivo. The mechanisms underlying gastric tumorigenesis were conducted by RIP, luciferase gene report, RT-qPCR. and Western blot analyses. The prognostic significance was analyzed by FISH and TCGA dataset.

Results: We found that hsa_circ_0030632 (circUGGT2) was a predominant m⁶A target of METTL14, and METTL14 knockdown (KD) reduced circUGGT2 m⁶A levels but increased its mRNA levels. circUGGT2 KD impaired cell growth and metastasis in vitro and in vivo, but circUGGT2 overexpression prompted these effects. Furthermore, circUGGT2 was validated to sponge miR-186-3p and upregulate MAP3K9 and could abolish METTL14-caused miR-186-3p upregulation and MAP3K9 downregulation in GC cells. circUGGT2 negatively correlated with miR-186-3p expression and harbored a poor prognosis in patients with GC.

Conclusion: Our findings unveil that METTL14-dependent m⁶A modification of circUGGT2 promotes GC progression by sponging miR-186-3p and upregulating MAP3K9.

m6A

METTL14

circUGGT2

miR-186-3p

gastric cancer

growth

Gastric cancer (GC) is one of most prevalent gastrointestinal cancers and presents the fourth incidence and the third leading cause of cancer-related deaths [1, 2]. Tumor metastasis constitutes the fundamental cause of discouraging outcomes for advanced patients [3]. The drivers leading to premetastatic microenvironment of GC remain unclear and further observations of the molecular mechanisms underlying GC progression are indispensable for early detection and treatment of GC.

N6-methyladenosine (m⁶A), one of most common modifications in eukaryotic mRNAs is launched by m⁶A methyltransferases (METTL3/14, WTAP, ZC3H13, etc) and dynamically abolished by m⁶A demethylases (FTO and ALKBH5) [4]. Dysregulated METTL3/14 or FTO/ALKBH5 is implicated in a wide body of malignancies including GC [4-11]. METTL3-mediated m⁶A modification of HDGF mRNA advances GC metastasis [12] and ALKBH5 represses GC invasion via PKMYT1 m⁶A modification [13]. Our previous studies uncovered that YTHDF1 as an m⁶A reader promotes gastric tumorigenesis by enhancing USP14 translation [14] and METTL14 restrains GC progression in an m⁶A-dependent manner [15].

It is acknowledged that circular RNAs (circRNAs), a subgroup of noncoding RNAs (ncRNAs) characterized by covalently closed continuous loops and resistance to RNase R exert emerging roles in cancer [16]. circRNAs can act as diagnostic biomarkers [17, 18], oncogenes [19-21] or anti-oncogenes [22-24] in multiple malignancies including GC. It has been reported that circRNAs influence GC progression depending on sponging miRNAs [21, 25-27], interacting with RNA-binding proteins [28-30] and encoding proteins [31-33]. Our previous studies corroborated that circDLST, circLARP4 and circYAP1 participate in gastric carcinogenesis and may offer the potential therapeutic targets for GC [34-36].

It is revealed that m⁶A modification of circALG1 or circMDK facilitates tumorigenesis and metastasis in colorectal cancer (CRC) [37] and hepatocellular carcinoma (HCC) [38]. We previously found that METTL14-mediated m⁶A modification of circORC5 impedes GC tumorigenesis [15]. Herein, we discovered a novel circRNA circUGGT2, which could be modified by METTL14 in an m⁶A-dependent manner, promoted growth and metastasis of GC cells by sponging miR-186-3p and upregulating MAP3K9, providing a prognostic and therapeutic target for GC.

Clinical data

The clinical and prognostic data as well as miRNA expression levels were downloaded from The Cancer Genome Atlas database (http://xena.ucsc.edu/getting-started/). 10 pair-matched GC tissue samples used for assessment of circUGGT2 and miR-186-3p expression levels were frozen at −80°C in our Digestive Diseases Research Lab. A tissue microarray (TMA, Cat No. HStmA180Su16; Lot No.XT18-003) consisting of 90 cases of GC tissue samples was provided by Shanghai Outdo Biotech (Shanghai, China). Our study protocols were approved by the Ethics Committee of Shanghai Sixth People’s Hospital.

RNA fluorescence in situ hybridization (FISH)

FISH analysis was utilized to pinpoint the subcellular localization of hsa_circ_0030632 (circUGGT2) and miR-186-3p in GC tissues from TMA. Biotin-labeled probe sequence for circUGGT2 (Green fluorescence, 5’-GAGCTTGGTTTCTTTTGATTTTCTTAAA A-3') and Digoxin-labeled probe sequence for miR-186-3p (Red fluorescence, 5’- CCCAAAAAATTCACCTTTGGGCAAAAAA-3’) were used for FISH analysis of the expression levels of circUGGT2 and miR-186-3p in GC tissues. The detailed experimental process was performed as previously reported [15].

RNA extraction and Real-Time fluorescence Quantitative PCR (RT-qPCR)

The RNA extraction in our study was executed by Trizol method (Lot No. 15596-026, Ambion) and then was reversely transcribed into cDNA for further qPCR analysis by HiScript® II Q Select RT SuperMix (Lot No. R233, VAZYME) and SYBR Green Master Mix (Lot No. Q111-02, VAZYME) according to the manufacturer’s instructions. The primer sequences used for qPCR analysis were included in Supplementary Table S1. Relative expression levels for circUGGT2, miR-186-3p and MAP3K9 were quantified using the 2^−ΔΔCt.

Western blot

GC tissues and cells were lysed with RIPA Pyrolysis liquid (P0013B, Beyotime). The supernatants were resolved in SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes, which were probed with anti-MAP3K9 (121KD, Affinity, 1:2000, DF8808) and anti-GAPDH (37KD, 1:1000, AB-P-R001) overnight at 4°C. The protein bands were developed by enhanced chemiluminescence (ECL).

Plasmid, shRNA, miRNA mimic and inhibitor

Lentivirus-mediated sh-METTL14 [15] or sh-circUGGT2 (5’- AATCAAAAGAAA CCAAGCTCA-3’) as well as miR-186-3p mimic or inhibitor was offered by GenePharma (Shanghai, China). The negative control (NC or CON) and miR-NC were considered as the control groups. MKN-28 and BGC-823 cells were planted in 6-well plates 48 h prior to sh-METTL14, sh-circUGGT2, miR-186-3p mimic or inhibitor transfection with 50-60% confluence, and then mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture instructions.

m⁶A-circRNA epi-transcriptomic microarray

Total RNA was extracted from sh-METTL14 or sh-NC stably-transfected MKN-28 cells and m⁶A-circRNA epi-transcriptomic microarray was utilized to screen METTL14-dependent m⁶A modification of circRNAs in GC cells as previously reported [15].

RNA immunoprecipitation (RIP) and m⁶A-RIP (MeRIP)

RIP assay was performed using RNA-Binding Protein Immunoprecipitation Kit (17-700, Millipore) and HiScript II qRT SuperMix II kit (R223, VAZYME). The antibodies against m⁶A (68055-1-Ig, Proteintech, Wuhan, China), METTL14 (26158-1-AP, Proteintech, Wuhan, China) and Ago2 (67934-1-Ig, Proteintech, Wuhan, China) were used for MeRIP or RIP analysis.

Bioinformatic analysis

The specific binding between circUGGT2 and miRNAs (miR-30d-3p, miR-579-3p, miR-664b-3p and miR-186-3p) was identified using the miRbase database (http://www.mirbase.org /index.shtml). The target genes of miR-186-3p were identified byTargetScanHuman7.1 (http://www.targetscan.org/vert_71/).

Luciferase gene reporter assay

Luciferase gene reporter assay was performed by double luciferase gene reporter kit (RG027, Beyotime). HEK293T were seeded into 96-well plates and co-transfected with PRL-TK-pMIR-circUGGT2 or PRL-TK-pMIR-MAP3K9 3’UTR, and miR-186-3p mimics or miR-NC. After 48 h of incubation, the firefly and Renilla luciferase activities were examined with a dual-luciferase reporter assay.

In vivo tumorigenesis assay

Female BALB/c nude mouse (6-8 week, SPF) were provided by the Shanghai Laboratory Animal Central (SLAC, Shanghai, China). MKN-28 cells (1 × 10⁷) transfected with sh-circUGGT2 or sh-NC lentiviruses were resuspended in 200μL of sterile PBS and injected subcutaneously under the right forearm armpit of mice. After 30 days, the mice were sacrificed, and the xenografted tumors were collected. Tumor volume = (length x width²)/2. The animal experiments were approved by the Ethics Committee of Shanghai Sixth People’s Hospital.

Caudal vein pulmonary metastasis model

MKN-28 cells stably transfected with sh-circUGGT2 or sh-NC lentiviruses were cultured in complete medium. When the cells were 70% confluent, the medium was replaced with fresh medium to remove dead and detached cells. 1 × 10⁷ MKN-28 cells were injected into the mice tail vein. The progression of pulmonary metastasis was investigated for 30 days.

Cell culture, m⁶A dot blot, MTT, Colony formation, Transwell assays, RNase R treatment and Nuclear and cytoplasmic fractionation, hematoxylin-eosin (HE) staining and Immunohistochemical analysis

These experiments were performed as previously reported [12, 15].

Statistical analysis

Statistical analysis was performed with GraphPad Prism 7 (La Jolla, CA, USA). In brief, the values are expressed as the mean ± standard deviation (SD). Student’s test and analysis of variance were used for comparisons between groups. Kaplan-Meier analysis was used to assess the association of circUGGT2/miR-186-3p with GC prognosis. Pearson Correlation Analysis was used to analyze the correlation of circUGGT2 with miR-186-3p. P <0.05 was considered statistically significant.

METTL14 mediated m⁶A modification of circUGGT2 in GC cells

According to our previous report, METTL14 has been indicated as an oncogenic factor in GC [15]. The m⁶A dot blot assay showed METTL14 knockdown (KD) decreased the total m⁶A levels whereas METTL14 overexpression (OE) increased the total m⁶A levels in MKN-28 and BGC-823 cells (Figure 1A). Then, m⁶A-circRNA epi-transcriptomic microarray was used to screen m⁶A-modified circRNAs between sh-METTL14 and sh-NC-transfected MKN-28 cells, among which top 20 circRNAs with low m⁶A levels and 7 circRNAs with high m⁶A levels were identified in METTL14 KD group as compared with the sh-NC group based on the P < 0.05 and fold change (FC) > 2 (Figure 1B). Moreover, we found that hsa_circ_0030632 (circUGGT2) harbored the lowest m⁶A levels (FC=3.66, P = 0.0026) when METTL14 was knocked down in MKN-28 cells. Both of m⁶A-circRNA microarray and MeRIP analyses verified that METTL14 KD diminished the m⁶A levels of circUGGT2 in MKN-28 and BGC-823 cells (Figure 1C). qRT-PCR analysis showed that METTL14 KD raised circUGGT2 mRNA levels (Figure 1D) while METTL14 OE inhibited its expression in MKN-28 and BGC-823 cells (Figure 1E). RIP assay further confirmed that circUGGT2 could be enriched in METTL14-interacting mRNAs and METTL14 OE decreased circUGGT2 enrichment levels as compared with the IgG group (Figure 1F).

Upregulation of circUGGT2 was associated with poor survival in patients with GC

On the basis of the annotation of circRNAs, hsa_circ_0030632, originated from the linear gene UDP-glucose glycoprotein glucosyltransferase 2 (UGGT2) was named as circUGGT2 (Figure 2A). After MKN-28 and BGC-823 cells were exposed to RNase R treatment for 48h, we found that circUGGT2 produced a resistance to RNase R relative to UGGT2 (Figure 2B). Subcellular location analysis revealed that circUGGT2 was primarily located in the cytoplasm in MKN-28 and BGC-823 cells (Figure 4C, D). qRT-PCR displayed that circUGGT2 expression was markedly elevated in GC tissue samples as compared with the adjacent normal tissues (Figure 4E), and this result was validated by FISH analysis (Figure 4F). Kaplan-Meier uncovered that GC patients with circUGGT2-high levels possessed worse overall survival as compared with those with circORC5-low levels (Figure 4G).

circUGGT2 facilitated GC cell growth and invasion and abolished METTL14-induced antitumor effects.

To clarify the function of circUGGT2 in GC cells, we constructed circUGGT2 OE cell model in MKN-28 and BGC-823 cells with plasmids and validated its expression levels by qRT-PCR analysis (Figure 3A). We found that METTL14 OE repressed cell viability (Figure 3B), colony formation (Figure 3C) and cell invasion capabilities (Figure 3D), while circUGGT2 OE encouraged these effects and deteriorated METTL14-induced antitumor effects in MKN-28 and BGC-823 cells (Figure 3B-D). These results indicated that METTL14 mediated circUGGT2 to regulate GC progression.

miR-186-3p was downregulated and negatively correlated with circUGGT2 expression in GC

In the light of the miRbase, circUGGT2 has the greatest potential to interact with 4 miRNAs (miR-30d-3p, miR-579-3p, miR-664b-3p and miR-186-3p ) (Figure 4A). We estimated their expression levels in GC and found that only miR-186-3p indicated decreased expression in GC as compared with normal tissues (Figure 4B). FISH analysis showed that circUGGT2 was co-located with miR-186-3p in the cytoplasm in GC tissue cells (Figure 4C). qRT-PCR analysis showed that miR-186-3p was remarkably downregulated in 10 pairs of GC tissues (Figure S1) and this result was validated by FISH analysis in 90 pairs of GC samples (Figure 4D, E). Pearson correlation analysis indicated that circUGGT2 had a negative correlation with miR-186-3p expression in 10 cases of GC samples (Figure 4F). However, Kaplan-Meier suggested that miR-186-3p high or low expression had no difference in overall survival and tumor recurrence in patients with GC (Figure S2).

circUGGT2 acted as a sponge of miR-186-3p in GC cells

The binding sites between circUGGT2 and miR-186-3p can be shown in Figure 5A. We then confirmed that miR-186-3p mimic lowered the luciferase activity of the WT circUGGT2 3’-untranslated region (UTR), but exhibited no effect on that of the Mut circUGGT2 3’-UTR compared to the miR-NC group in HEK293T cells (Figure 5B). qRT-PCR analysis revealed that the expression levels of miR-186-3p were substantially increased by circUGGT2 KD but decreased by circUGGT2 OE in MKN-28 and BGC-823 cells (Figure 5C, D). Moreover, we implemented RIP analysis for Ago2 protein in MKN-28 and BGC-823 cells and found that both of circUGGT2 and miR-186-3p were enriched in the Ago2-interacting mRNAs compared to the IgG group (Figure 5E, F). Functional analysis demonstrated that miR-186-3p mimic suppressed cell viability and attenuated circUGGT2-induced proliferation-promoting effects in MKN-28 and BGC-823 cells (Figure 5G).

METTL14 mediated circUGGT2 to modulate miR-186-3p/MAP3K9 axis

According to the cumulative weighted context++ score from the TargetScanHuman7.1,

we identified mitogen-activated protein kinase kinase kinase 9 (MAP3K9) as a direct

target of miR-186-3, and the binding sites between miR-186-3p and MAP3K9 can be

shown in Figure 6A. We then verified that miR-186-3p mimic dwindled the luciferase

activity of the WT MAP3K9 3’-UTR, but displayed no effect on that of the Mut MAP3K9 3’-UTR compared to the miR-NC group in HEK293T cells (Figure 6B). qRT-PCR and Western blot analyses unveiled that the expression levels of MAP3K9 were declined by miR-186-3p mimic (Figure 6C, D) but elevated by miR-186-3p inhibitor in MKN-28 and BGC-823 cells (Figure 6E and Figure S3). Furthermore, we found that METTL14 OE repressed the expression of MAP3K9, whereas circUGGT2 OE promoted its expression and abolished METTL14-induced MAP3K9 downregulation in MKN-28 and BGC-823 cells (Figure 6F, G).

Knockdown of circUGGT2 suppressed in vivo tumorigenesis and lung metastasis

To illuminate the effects of circUGGT2 on tumor growth in vivo, we used sh-circUGGT2 or sh-NC stably transfected MKN-28 cells to construct subcutaneous xenograft tumor models. After investigations for 30 days, we found that, the size of the tumors formed by sh-circUGGT2-transfected MKN-28 cells was smaller than that formed by NC-transfected cells (Figure 7A). The growth curve indicated that, the tumors in the sh-circUGGT2 group displayed a decreasing trend in a time-dependent manner relative to the sh-NC group (Figure 7B). The tumor volume and weight were dwindled in the sh-circUGGT2 group compared to the NC group (Figure 7C and Figure S4). HE and IHC analyses demonstrated that the positive expression of Ki-67 and MAP3K9 was decreased in xenograft tumor tissues in the sh-circUGGT2 group compared to the NC group (Figure 7D). In addition, we constructed a caudal vein lung metastasis model (Figure 7E) and found that the body weight in the sh-circUGGT2 group exhibited a declining trend in a time-dependent manner relative to the sh-NC group (Figure 7F). HE and IHC analyses uncovered that the positive expression of Ki-67 and MAP3K9 was reduced in metastatic lung tumor tissues in the sh-circUGGT2 group compared to the NC group (Figure 7G).

Increasing evidence indicates that METTL14 plays an anti-oncogenic role by m⁶A modification of mRNAs [39, 40], miRNAs [41, 42] or lncRNAs [43, 44], and m⁶A modification of circRNAs also takes a part in cancer progression [37, 38]. Our previous study authenticated that METTL14-mediated m⁶A modification of circORC5 inhibits GC progression [15]. Herein, we identified a novel circRNA circUGGT2, which was subject to METTL14-mediated m⁶A modification in GC cells. METTL14 KD decreased circUGGT2 m⁶A levels but increased its mRNA expression. Further investigations revealed that METTL14-dependeent m⁶A modification of circUGGT2 promoted GC progression by sponging miR-186-3p and upregulating MAP3K9, and provided a potential therapeutic target for GC.

Accumulating data have verified that circRNAs play crucial toles in GC progression by acting as oncogenes [21, 25-28] or tumor suppressors [29-32]. Our previous studies showed that circDLST drives but circLARP4 and circYAP1 hinders GC progression [34-36]. Herein, we found that circUGGT2 OE accelerated but circUGGT2 KD prevented GC growth and metastasis in vitro and in vivo, and circUGGT2 OE abolished METTL14-caused antitumor effects. These findings indicated that m⁶A modification of circUGGT2 by METTL14 promoted gastric tumorigenesis and metastasis.

It is mechanically observed that circRNAs can act as miRNA sponges involved in GC progression [25-27]. Our previous studies uncovered that circDLST, circLARP4 or circYAP1 regulates GC growth and invasion by sponging miR-502-5p, miR-424 or miR-367-5p [34-36]. Herein, we found that circUGGT2 was upregulated in GC tissues and correlated with poor survival in patients with GC. Moreover, circUGGT2 could act as a sponge of miR-186-3p in GC cells. The downregulation of miR-186-3p has been reported in cervical cancer and CRC and represses tumorigenesis by targeting IGF1 or KRT18 [45-47]. We herein found that miR-186-3p was also downregulated in GC tissues, harbored a negative correlation with circUGGT2 expression and abolished circUGGT2-induced cell proliferation. These findings suggested that m⁶A modification of circUGGT2 by METTL14 facilitated GC progression by sponging miR-186-3p.

Mitogen-activated protein kinase kinase kinase 9 (MAP3K9), a member of serine/threonine protein kinases, acts as an oncoprotein as well as a target of mIR-7, miR-148a-3p or miR-125b-5p in several cancers [48-50]. We herein identified MAP3K9 as a direct target of miR-186-3p, and METTL14 OE decreased MAP3K9 expression levels, but circUGGT2 OE promoted its expression and attenuated METTL14-induced MAP3K9 downregulation in GC cells. These findings suggested that METTL14-dependent m⁶A modification of circUGGT2 could act as a sponge of miR-186-3p to upregulate MAP3K9, contributing to GC tumorigenesis (Figure 8).

Our findings uncover that m⁶A modification of circUGGT2 by METTL14 promotes growth and invasion of GC cells by sponging miR-186-3p and upregulating MAP3K9 and may provide a potential therapeutic target for GC.

GC: Gastric cancer; CRC: colorectal cancer; HCC: hepatocellular carcinoma; circRNA: circular RNA; miRNA: microRNA; lncRNA: long noncoding RNA; m⁶A: N6-methyladenosine; METTL3/14: methyltransferase 3/14; ALKBH5: alkB homolog 5, RNA demethylase; YTHDF1: YTH N6-methyladenosine RNA binding protein 1; HDGF: heparin binding growth factor; IGF1: insulin like growth factor 1; KRT18: keratin 18; USP14: ubiquitin specific peptidase 14; MAP3K9: mitogen-activated protein kinase kinase kinase 9; UGGT2: UDP-glucose glycoprotein glucosyltransferase 2; KD: knockdown; OE: overexpression; Me-RIP: methylated RNA immunoprecipitation; RIP: RNA immunoprecipitation; TCGA: The Cancer Genome Atlas; FISH: Fluorescence in situ hybridization; TMA: Tissue microarray; FBS: Fetal bovine serum; IOD: Immunofluorescence accumulation optical density; RT-qPCR: real-time quantitative PCR.

Acknowledgements

Thanks for the technical support from Aksomics Biotechnology Co., Ltd.

Authors’ contributions

JZ and JSZ designed this study. JZ and XYC drafted the manuscript. XYC and YLY performed the experiments and contributed equally to the article. ZYC, MC, HNF collected the data and YY conducted the statistical analysis and JZ revised this manuscript. All authors read and approved the final manuscript.

Funding

Our work was supported by the grants from Shanghai Science and Technology Innovation Plan Star Cultivation (Sailing Special Project, No. 22YF1432600)., Natural Science Foundation of Shanghai (No. 21ZR1448700), National Natural Science Foundation of China (No. 82074161, 81873143).

Availability of data and materials

All data generated or analysed during this study are included in this published article and its additional files.

Ethics approval and consent to participate

The present study was approved by the Hospital’s Protection of Human Subjects Committee.

Consent for publication

Consent for publication has been obtained from the patients.

Competing interests

The authors declare that they have no competing interests.

Author details

Department of Gastroenterology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China.

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METTL14-dependent m 6 A modification of circUGGT2 drives gastric cancer progression by sponging miR-186-3p and upregulating MAP3K9

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