ALKBH5-dependent upregulation of circARHGEF12 by Oridonin impairs gastric cancer progression and cisplatin resistance via regulating miR-130b-5p/LATS2 signaling

doi:10.21203/rs.3.rs-5240498/v1

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Research Article

ALKBH5-dependent upregulation of circARHGEF12 by Oridonin impairs gastric cancer progression and cisplatin resistance via regulating miR-130b-5p/LATS2 signaling

https://doi.org/10.21203/rs.3.rs-5240498/v1

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Background

Accumulating evidence unveils that N6-methyladenosine (m⁶A) methylation modifications and deregulated circular RNAs (circRNAs) play critical roles in diverse malignancies including gastric cancer (GC). However, the underlying mechanisms by which ALKBH5 mediates m⁶A modification of circRNAs in GC progression and chemoresistance remain unknown.

Methods

The m⁶A-circRNA epi-transcriptomic microarray was applied to screen ALKBH5-mediated m⁶A demethylation of hsa_circ_0002089 (circARHGEF12), which was verified by m⁶A dot blot, RT-qPCR, methylated RNA immunoprecipitation (Me-RIP) and RIP assays. The cellular localization and mRNA expression levels of circARHGEF12 in GC tissue samples were detected by fluorescence in situ hybridization. Gain- or loss-of-function assays as well as in vivo tumorigenesis and lung metastasis models were executed to assess the role of circARHGEF12 in GC cells. The specific binding of circARHGEF12 with miR-130b-5p was validated by RT-qPCR, RIP, and luciferase gene report assays. The effects of Oridonin-mediated ALKBH5 and (or) circARHGEF12 on miR-130b-5p/LATS2/YAP signaling were determined by RT-qPCR, Western blot and functional assays.

Results

We found that circARHGEF12 was identified as an m⁶A-modified target of ALKBH5 in GC cells. Overexpression of circARHGEF12 impaired cell proliferation and cisplatin resistance in vitro as well as repressed gastric tumorigenesis and lung metastasis in vivo, but circARHGEF12 knockdown (KD) drove these effects. Furthermore, circARHGEF12 could act as the sponge of miR-130b-5p to attenuate ALKBH5 KD-induced miR-130b-5p upregulation and LATS2/YAP inactivation in GC cells. Oridonin activates ALKBH5 to enhances GC suppression via miR-130b-5p/LATS2 signaling.

Conclusion

Our findings reveal that ALKBH5-dependent upregulation of circARHGEF12 by oridonin impairs GC progression and cisplatin resistance via regulating miR-130b-5p/LATS2 signaling and may provide a novel therapeutic target for GC.

m6A

ALKBH5

circARHGEF12

miR-130b-5p

gastric cancer

cisplatin

Gastric cancer (GC), one of most common gastrointestinal malignancies ranks the fourth incidence and the third leading cause of cancer-related deaths worldwide, especially in China ^1,2. Despite the widespread application of Helicobacter pylori eradication and endoscopic resection of early gastric tumors, the outcome of advanced patients with GC remains discouraging ascribed to the malignant dissemination ³. Therefore, further exploration of potential mechanisms underlying GC progression is essential for early diagnosis and treatment of GC.

N6-methyladenosine (m⁶A), one of the most prevalent modifications in mammal mRNAs has been reported to be of supreme significance in cancer progression ⁴. m⁶A methylation modifications can be initiated by methyltransferases, named as “writers” including (METTL3/14, WTAP, RBM15/15B and KIAA1429), eliminated by demethylases FTO and ALKBH5 (termed as “erasers”), and discriminated by RNA-binding proteins (“readers”) such as YTHDF1-3, YTHDC1-2 and IGF2BP1-3 ^4,5. The deregulation of m⁶A “writers”, “erasers” or “readers” is involved in the pathogenesis and progression of GC ^6–10. ALKBH5 as an m⁶A “eraser” acts a dual role in GC. It can repress GC cell invasion ¹¹, but also promote bile acid-induced gastric intestinal metaplasia ¹² and GC metastasis ¹³. However, the role and molecular mechanism of ALKBH5 in GC need to be investigated.

Evidence unveils that circular RNAs (circRNAs) act a crucial part in GC ¹⁴. For one thing, circNRIP1, circLMTK2 or circHECTD1 drives GC tumorigenesis and metastasis by sponging miR-149-5p, miR-150-5p or miR-1256 ^15–17, but circCCDC9, circPSMC3 or hsa_circ_0004872 blocks GC progression by sponging miR-6792-3p, miR-296-5p or miR-224 ^18–20. For another thing, circHuR, circURI1 or circGSK3B impairs GC progression by interacting with CNBP, hnRNPM or EZH2 ^21–23, but circAGO2 binds with AGO2 to facilitate gastric carcinogenesis ²⁴. Moreover, circDIDO1 or circMAPK1 can code MAPK1 or DIDO1-529aa to suppress GC metastasis ^25,26. Our previous findings indicated that circLARP4 or circDLST modifies GC by sponging miR-424 or miR-502-5p ^27,28. Currently, it has been demonstrated that m⁶A modification of circALG1, circMDK or circSORE promotes metastasis and sorafenib resistance in colorectal cancer (CRC) and hepatocellular carcinoma (HCC) ^29–31. We also found that METTL14-mediated m⁶A modification of circORC5 or circUGGT2 downregulation impedes GC progression and cisplatin resistance ^7,32.

Historically, naturally derived compounds have been predominant sources of anticancer agents. Oridonin (Ori), a tetracyclic diterpenoid, is naturally found in the leaves of Rabdosia rubescens ³³. Existing research has uncovered the anticancer activity of Ori ^34,35. However, the potential mechanisms by which Ori mediates m⁶A-circRNA axis to resist GC remain unclear. Herein, we found that ALKBH5-dependent upregulation of circARHGEF12 by Ori impaired GC progression and cisplatin resistance via regulating the miR-130b-5p/LATS2 signaling, and might offer a potential therapeutic target for GC.

Clinical data

The clinical data including miRNAs and LATS2 expression levels were downloaded from The Cancer Genome Atlas database (TCGA, http://xena.ucsc.edu/getting-started/). 10 pair-matched GC tissue samples used for detection of circARHGEF12 and miR-130b-5p expression levels were frozen at − 80°C in Central Laboratory of Shanghai Sixth People's Hospital. The tissue microarray (TMA, Lot No. XT18-003) containing 90 pairs 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 used to confirm the subcellular localization and expression levels of hsa_circ_0002089 (circARHGEF12) and miR-130b-5p in GC tissue samples from TMA. Biotin-labeled probe sequence for circARHGEF12 (Green fluorescence, 5’- AAGCTTTGTTATCTGGACTCTGCTTAAAA-3') and Digoxin-labeled probe sequence for Homo sapiens miR-130b-5p (Red fluorescence, 5’- GTAGTGCAACAGGGAAAGAGTAAAAAA-3’) were used for FISH analysis as previously reported ⁷.

Cell culture and Drug treatment

Oridonin (C20H28O6, CAS No. 28957-04-2, purity: 99.89%) was purchased from MedChemExpress (Shanghai, China). Oridonin was dissolved in dimethyl sulfoxide (DMSO) as a stock solution and stored at − 20 ℃. MKN-28 and HGC-27 cells were treated for 48h with the specified concentrations of oridonin (0, 5, 10 and 20 µM). The human GC cell lines (MKN-28, HGC-27) and the human embryonic kidney cell HEK293T were obtained from stocks at Digestive Disease Laboratory of Shanghai Sixth People’s Hospital. These cell lines were maintained in RPMI 1640 medium (Hyclone, Logan, Utah) supplemented with 10% of fetal bovine serum (Gibco, United States), 100 U/mL of penicillin and 100 µg/mL of streptomycin (PenicillinStreptomycin-Glutamine, Gibco, United States). The cells were grown at 37°C in a humidified incubator containing 5% CO₂. GC cell (MKN-28) were exposed to gradually increasing doses of Cisplatin (DDP) (Selleck, USA) over a 6-month period to establish DDP-resistant cells (MKN-28/DDP) until the cells acquired resistance to 1µg/ml. Prior to each experiment, MKN-28/DDP cells were cultured in drug-free RPMI 1640 medium for 2 weeks.

Molecular docking model

The crystal structure of ALKBH5 (code: 4o7x) was obtained from the Protein Data Bank (https://www.rcsb.org/). The compound Oridonin was retrieved through the TCMSP database (https://old.tcmsp-e.com/tcmsp.php). Subsequently, AutoDock Vina1.5.6 software was employed for hydrogenation, dehydration and molecular docking analysis. PyMOL2.1.0 was utilized for 3-D structure visualization.

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

The RNA extraction 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 mRNA levels of circARHGEF12, miR-130b-5p, ALKBH5, LATS2 and YAP 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-ALKBH5 (44KD, Affinity, 1:1000, DF2585), anti-LATS2 (150KD, Affinity, 1:1000, AF7940), anti-p-LATS2 (150KD, Affinity, 1:1000, AF7440), anti-YAP (78KD, CST, 1:1000, 14074) 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 ALKBH5 ¹⁵ or circARHGEF12 and si-circARHGEF12 (5’-GAGTCCAGATAACAAAG CTTT-3’) as well as miR-130b-5p 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 HGC-27 cells were planted in 6-well plates 48 h prior to ALKBH5, circARHGEF12, miR-130b-5p mimic or inhibitor transfection with 50–60% confluence, and then mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture instructions.

mA-circRNA epi-transcriptomic microarray

Total RNA was extracted from ALKBH5 or NC stably-transfected MKN-28 cells and m⁶A-circRNA epi-transcriptomic microarray was utilized to screen ALKBH5-dependent m⁶A modification of circRNAs in GC cells as previously reported ⁷.

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), ALKBH5 (44KD, Affinity, 1:1000, DF2585) and Ago2 (67934-1-Ig, Proteintech, Wuhan, China) were used for MeRIP or RIP analysis.

Bioinformatic analysis

The specific binding between circARHGEF12 and miRNAs (miR-103a-2-5p, miR-335-5p, miR-130b-5p, miR-339-3p and miR-605-5p) 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 cells were seeded into 96-well plates and co-transfected with PRL-TK-pMIR-circARHGEF12 or PRL-TK-pMIR-LATS2 3’UTR, miR-130b-5p inhibitor, and miR-130b-5p 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 and caudal vein pulmonary metastasis model

Female BALB/c nude mice (6-8-week, SPF) were purchased from the Shanghai Laboratory Animal Central (SLAC, Shanghai, China). MKN-28 cells (1×10⁷) transfected with circARHGEF12 or NC lentiviruses were resuspended in 200µL of sterile PBS and injected subcutaneously under the right forearm armpit of mice. Based on recent studies, the mice were treated with 5 ~ 6 mg/kg DDP three times per week^36–38. Therefore, the mice were intraperitoneally injected with 0, 3 or 6 mg/kg DDP at days 10, 13, 16, 19, 22, and 25 after inoculation with HGC-27 cells. After 30 days, the mice were anaesthetized with isoflurane and sacrificed, then the xenografted tumors were collected. Tumor volume = (length * width2)/2.

MKN-28 cells stably transfected with circARHGEF12 or 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 mice were sacrificed after 28 days, and the lung tissues were removed, photographed, and fixed. All animal studies were conducted in accordance with institutional ethical guidelines for animal experiments and approved by the Institutional Animal Care and Use Committee or the Ethics Committee for Animal Experiments of Shanghai Sixth People’s Hospital (No: DWLL2024-966).

Cell Counting Kit-8 (CCK8), Colony formation, Transwell assays, m ⁶ A dot blot, RNase R treatment and Nuclear and cytoplasmic fractionation, hematoxylin-eosin (HE) staining and Immunohistochemical (IHC) analysis

These experiments were performed as previously reported ^7,32.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 9 (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. Survival curves were generated using the Kaplan-Meier method and log-rank testing. Pearson Correlation Analysis was used to analyze the correlation of circARHGEF12 with miR-130b-5p. The half-maximal inhibitory concentration (IC₅₀) values were determined using a curve fitting model with a four-parameter logistic equation model in GraphPad Prism software. P < 0.05 was considered statistically significant.

Upregulation of CircARGEF12 by ALKBH5-Mediated m⁶A Modification and Its Characteristics in GC

ALKBH5, an m⁶A demethylase, modulates m⁶A methylation levels on mRNAs and its downregulation has been observed in GC tissues ³⁹ (Our data not shown). The m⁶A dot blot assay demonstrated that overexpression of ALKBH5 (ALKBH5-OE) led to a reduction in total m⁶A levels in MKN-28 and HGC-27 cells (Fig. S1A). To identify the circRNAs potentially regulated by ALKBH5, we performed m⁶A-circRNA epi-transcriptomic analysis of ALKBH5-OE and NC-transfected MKN-28 cells. Differential expression analysis, with thresholds set at |log2(FC)| ≥ 2 and p < 0.05, revealed obvious alterations in circRNA m⁶A levels, among which has_circ_0002089 exhibited the most pronounced downregulation in m⁶A levels (log2(FC) = 2.54, p = 0.002) (Fig. 1A).

To elucidate the regulatory effects of ALKBH5 in GC cells, we synthesized specific siRNAs and constructed lentiviral vectors to modulate ALKBH5 expression. The efficiency of ALKBH5 knockdown (KD) or overexpression (OE) was confirmed by Western blot and RT-qPCR (Fig. S1B, C). Based on circRNA annotation, we identified has_circ_0002089 originating from the linear gene Rho guanine nucleotide exchange factor 12 (ARHGEF12), hereafter referred to as circARHGEF12 (Fig. S1D). MeRIP analysis confirmed that ALKBH5-OE reduced the m⁶A levels of circARHGEF12 in MKN-28 and HGC-27 cells (Fig. 1B), while ALKBH5-KD increased its m⁶A levels (Fig. S1E). RT-qPCR analysis indicated a marked upregulation of circARHGEF12 in ALKBH5-OE cells (Fig. 1C). RIP assays further validated the association of ALKBH5 with circARHGEF12, indicating that ALKBH5 KD decreased circARHGEF12 enrichment compared to IgG (Fig. 1D, E), whereas ALKBH5-OE enhanced its enrichment (Fig. S1F).

RNase R treatment confirmed the high stability of circARHGEF12 in MKN-28 and HGC-27 cells relative to its linear counterpart ARHGEF12, as circARHGEF12 remained resistant to RNase R degradation (Fig. S1G). Subcellular localization analyses using RT-qPCR and FISH revealed that circARHGEF12 predominantly resided in the cytoplasm of GC cells (Fig. 1F and Fig. S1H). RT-qPCR analysis of 10 paired GC and adjacent non-tumor tissues authenticated an obvious downregulation of circARHGEF12 in GC samples (Fig. 1G), which was corroborated by FISH analysis of 90 paired GC tissue samples using tissue microarray (TMA) (Fig. 1H).

CircARHGEF12 Mitigates Cisplatin Resistance in GC Cells

To explore the molecular mechanisms underlying cisplatin (DDP) resistance in GC, we established a DDP-resistant GC cell line (MKN-28/DDP). Compared to its parental cell line, MKN-28/DDP cells exhibited a significant downregulation of circARHGEF12 (Fig. 2A), suggesting its potential role in DDP resistance. RT-qPCR analysis of 10 freshly collected cisplatin-resistant GC tissues and 10 cisplatin-sensitive tissues revealed a similar finding (Fig. 2B). To investigate the functional relevance of circARHGEF12 with DDP resistance, we generated GC/DDP cell lines with circARHGEF12-OE. The overexpression efficiency was validated via RT-qPCR (Fig. 2C). Cell viability assay using CCK-8 revealed that circARHGEF12 OE reduced the viability of DDP-resistant cells (Fig. 2D). Colony formation and Transwell assays displayed that circARHGEF12 OE markedly decreased colony numbers and cell invasiveness in DDP-resistant GC cells (Fig. 2E and Fig. S2A).

We established subcutaneous xenograft models in nude mice using MKN-28 cells stably transfected with circARHGEF12-OE or control (NC) lentiviruses. Once tumors were established, mice were treated with intraperitoneal injections of DDP (0, 3 or 6 mg/kg) every three days. Following DDP treatment, tumor volumes and weight were remarkably reduced, Ki-67 staining displayed decreased cell proliferation in tumors overexpressing circARHGEF12, and tumors derived from circARHGEF12-OE cells exhibited an enhanced response to DDP treatment (Fig. 2F and Fig S2B), highlighting the role of circARHGEF12 in suppression of DDP resistance in GC cells.

CircARHGEF12 Restrains GC Progression and Abolishes ALKBH5 KD-Induced Tumor-promoting Effects

To explore the role of circARHGEF12 in GC cells, we transfected MKN-28 and HGC-27 cells with circARHGEF12-OE lentiviruses or siRNA targeting circARHGEF12 (si-circARHGEF12), and RT-qPCR confirmed the significant modulation of circARHGEF12 mRNA expression (Fig. 3A, B and Fig. S2C, D). Functional assays unveiled that circARHGEF12 OE decreased GC cell viability, colony formation, and invasiveness, and counteracted si-ALKBH5-caused tumor-promoting effects (Fig. 3C, E and Fig. S2E). In contrast, circARHGEF12-KD drove tumorigenic traits and reversed ALKBH5-caused tumor-suppressive effects (Fig. 3D, F and Fig. S2F). These results indicated that ALKBH5 mediated circARHGEF12 to inhibit GC progression.

MiR-130b-5p Upregulation Negatively Correlates with CircARHGEF12 Expression and Clinical Prognosis in GC

The miRbase predicts five potential miRNA binding sites with circARHGEF12 including miR-103a-2-5p, miR-335-5p, miR-130b-5p, miR-339-3p, and miR-605-5p (Fig. 4A), among which miR-130b-5p exhibited the most significant reduction in luciferase activity when co-transfected with miRNA mimics and circARHGEF12 3’UTR in HEK293T cells (Fig. 4B). TCGA analysis revealed that miR-130b-5p was markedly upregulated in GC tissues compared to adjacent normal tissues (Fig. 4C and Fig. S3A), which was confirmed by RT-qPCR in 10 paired GC tissues (Fig. 4D). FISH analysis revealed the co-localization of circARHGEF12 (green) with miR-130b-5p (red) in the cytoplasm of GC cells (Fig. 4E), suggesting a direct interaction between circARHGEF12 and miR-130b-5p. A negative correlation between circARHGEF12 and miR-130b-5p expression was observed in 10 GC tissue samples (Fig. 4F). Clinical relevance analysis indicated that high miR-130b-5p expression was associated with age and lymph node infiltration in patients with GC (Supplementary Table S2). Kaplan-Meier survival analysis unveiled that high miR-130b-5p expression was associated with poor overall survival in GC patients (Fig. 4G), acting as an independent prognostic factor for poor survival from the TCGA cohort (Supplementary Table S3).

CircARHGEF12 Sponges miR-130b-5p to Inhibit GC Cell Proliferation and Invasion

To explore the regulatory relationship between circARHGEF12 and miR-130b-5p in GC cells, we confirmed miR-130b-5p upregulation or downregulation after transfection of miR-130b-5p mimic or inhibitor in MKN-28 and HGC-27 cells using RT-qPCR (Fig. 5A). Dual-luciferase reporter assays showed that miR-130b-5p mimic decreased the luciferase activity of wild-type (WT) circARHGEF12 3'-untranslated region (3'-UTR), while miR-130b-5p inhibitor increased its luciferase activity, indicating a direct interaction between circARHGEF12 and miR-130b-5p (Fig. 5B). RT-qPCR showed that circARHGEF12 OE reduced miR-130b-5p mRNA levels in both MKN-28 and HGC-27 cells (Fig. 5C, left), while circARHGEF12 KD increased miR-130b-5p expression (Fig. 5C, right). Notably, transfection of miR-130b-5p mimic or inhibitor harbored no effect on circARHGEF12 mRNA levels (Fig. S3B, C). Furthermore, RIP assays confirmed that both circARHGEF12 and miR-130b-5p were enriched in Ago2- associated complexes (Fig. 5D). Functional assays highlighted the interplay between circARHGEF12 and miR-130b-5p, showing that miR-130b-5p mimics propelled cell proliferation and invasion in MKN-28 and HGC-27 cells, and rescued circARHGEF12 OE-induced tumor suppressive effects (Fig. 5E and Fig. S3D). Conversely, miR-130b-5p inhibitor repressed GC cell proliferation and invasion, and reversed circARHGEF12 KD-caused tumor-promoting effects (Fig. 5F and Fig. S3E). These results demonstrated that circARHGEF12 acted as the sponge of miR-130b-5p to inhibit cell proliferation and invasion.

CircARHGEF12 Mediates miR-130b-5p to Regulate LATS2/YAP Signaling in GC Cells

Enrichment analysis highlighted the involvement of miR-130b-5p target genes in Hippo signaling pathway (Fig. 6A). Hippo signaling serves as a crucial tumor suppressor pathway, where the dysregulation of its downstream effector, Yes-associated protein (YAP) contributes to cancer ⁴⁰. Large tumor suppressor kinase 2 (LATS2), a core kinase of the Hippo pathway, directly phosphorylates YAP to expedite tis degradation, thus preventing tumorigenesis ⁴¹. Using TargetScanHuman7.1, we identified LATS2 as a direct target of miR-130b-5p and predicted the binding sites between miR-130b-5p and LATS2 3’UTR (Fig. S4A). TCGA cohort confirmed the negative correlation of miR-130b-5p with LATS2 expression in GC tissues (Fig. S4B, C).

Dual-luciferase reporter assays demonstrated that miR-130b-5p mimics reduced luciferase activity of the WT LATS2 3'-UTR, while miR-130b-5p inhibitor increased this activity (Fig. 6B). RT-qPCR and Western blot analyses revealed that miR-130b-5p mimics repressed LATS2 activities and increased YAP levels and these effects were reversed by circARHGEF12-OE in MKN-28 and HGC-27 cells (Fig. 6C and Fig. S4D, E). Conversely, miR-130b-5p inhibitor elevated LATS2 activities and decreased YAP levels and these effects were also reversed by si-circARHGEF12 (Fig. 6D and Fig. S4F, G).

Overexpression of LATS2 in MKN-28 and HGC-27 cells was validated by RT-qPCR and Western blot (Fig. S5A). Functional assays, including colony formation, and transwell assays, indicated that transfection with miR-130b-5p mimics reversed LATS2 OE-induced impairment of colony formation invasion capabilities of GC cells (Fig. 6E). Additionally, RT-qPCR and Western blot analyses indicated that miR-130b-5p mimics abolished LATS2 OE-induced YAP degradation in MKN-28 and HGC-27 cells (Fig. S5B, C).

Oridonin Activates ALKBH5 to Enhances GC Suppression via miR-130b-5p/LATS2 Pathway

We have verified the antitumor effects of oridonin (Ori) in GC cells in vitro and in vivo (data not shown). To explore whether Ori curbs the malignant phenotypes of GC through the ALKBH5/miR-130b-5p/LATS2 pathway, MKN-28 and HGC-27 cells were treated with Ori at the indicated concentrations (0, 5, 10, 20µM). We selected a concentration of 10 µM oridonin for subsequent experiments. Then, we conducted molecular docking modeling analysis to evaluate the interaction between Ori and ALKBH5. As shown in Fig. 7A, ALKBH5 contains an active binding pocket well-suited for Ori, with a binding affinity of -7.7 kcal/mol. Hydrogen bonds are generated between Ori and ALKBH5 at Lys132, Glu153 and Val202, providing the primary molecular basis for their interaction. Additionally, three hydrophobic interactions involving Pro150, Ile201, and His204 further stabilize the binding. RT-qPCR and Western blot analyses confirmed that Ori increased the expression levels of ALKBH5 (Fig. 7B, E), circARHGEF12 (Fig. 7C), and LATS2 (Fig. 7E), but suppressed the levels of miR-130b-5p (Fig. 7D) and YAP (Fig. 7E) in a dose-dependent manner. Functional assays, including colony formation and transwell assays, demonstrated that Ori effectively inhibited cell invasion and migration, and ALKBH5-OE enhanced Ori-caused tumor-suppressive effects but si-ALKBH5 reversed these effects in MKN-28 and HGC-27 cells (Fig. 7F). The similar results were shown in GC cells with circARHGEF12-OE or si-circARHGEF12 synergized with Ori (Fig. S6A). In DDP-resistant GC cell line MKN-28/DDP, circARHGEF12-OE increased Ori-induced suppressive effects on proliferation and invasion of MKN-28/DDP cells but si-circARHGEF12 reversed these effects (Fig. S6B).

CircARHGEF12 Suppresses Tumor Growth and Lung Metastasis in vivo

To validate our in vitro findings, MKN-28 cells stably overexpressing circARHGEF12 or a control vector were subcutaneously injected into BALB/c nude mice. Tumor volumes were measured every two days for 30 days before the mice were sacrificed.

Tumor volume and weight were reduced in the circARHGEF12 OE group compared to controls (Fig. 8A-C). HE staining and IHC analysis of subcutaneous tumors revealed decreased protein expression of Ki-67 and YAP but increased expression of LATS2 in the circARHGEF12 OE group (Fig. 8D). To assess the effect of circARHGEF12 on tumor metastasis, we utilized lung metastasis model in nude mice with the tail vein injection of MKN-28 cells stably overexpressing circARHGEF12 or the control vector. Increased body weight of mice (Fig. S6C) and LATS2 protein expression but decreased protein expression of Ki-67 and YAP (Fig. 8E) were investigated in metastatic lung tumor tissues in the circARHGEF12 OE group. These findings confirmed that circARHGEF12 OE inhibited in vivo GC growth and metastasis.

It has been reported that ALKBH5 acts as an anti-oncogene in several malignancies including GC ^11,42–44, and low expression of ALKBH5 is an independent prognostic factor of poor survival ⁴³. Reversely, ALKBH5 acts as an oncogene in renal cell carcinoma (RCC) ⁴⁵ and GC ¹³. Our findings showed that, in accordance with the report ¹¹, ALKBH5 acts a tumor suppressive role in GC and represents a favorable prognostic in patients with GC (data not shown).

ALKBH5 regulates gastric carcinogenesis by modification of mRNA ¹¹ or lncRNA ¹³ in an m⁶A-dependent manner. The m⁶A-mediated modification of circRNAs participates in cancer progression ^29–31. Our previous report uncovered that m⁶A modification of circORC5 ⁷ or circUGGT2 ³² facilitates GC tumorigenesis and metastasis. Herein, m⁶A-circRNA microarray identified that ALKBH5 exerted m⁶A-dependent modification of circARHGEF12, and ALKBH5-OE reduced the m⁶A level of circARHGEF12 but elevated its mRNA expression. Overexpression of circARHGEF12 impaired colony cell proliferation, metastasis and DDP resistance in vitro and in vivo, and counteracted METTL14 KD-induced tumor-enhancing effects, but circARHGEF12 KD harbored the opposite effects, indicating that ALKBH5-mediated m⁶A modification of circARHGEF12 suppresses GC progression and DDP resistance.

It has been shown that circRNAs function as miRNA sponges involved in GC progression ^15–20. Our previous reports unveiled that circLARP4 or circDLST impacts GC tumorigenesis by sponging miR-424 or miR-502-5p ^27,28. Herein, we found that circARHGEF12 was downregulated in GC tissues and could act as the sponge of miR-130b-5p in GC cells. The upregulation of miR-130b-5p is reported in HCC ⁴⁶, GC ⁴⁷ and breast cancer ⁴⁸ and drives cell proliferation ⁴⁷. We also found that miR-130b-5p was upregulated and correlated with poor survival in GC and abolished circARHGEF12-caused suppressive effects on GC cells. Overexpression of ALKBH5 or circARHGEF12 led to decreased miR-130b-5p expression. circSLC8A1 can act as a sponge of miR-130b to suppress bladder cancer progression ⁴⁹. These findings suggested that ALKBH5-mediated m⁶A modification of circARHGEF12 suppressed GC progression by sponging miR-130b-5p.

The Hippo kinase LATS2 can control cancer cell fate ^50,51 and LATS2/YAP signaling is involved in mediation of carcinogenesis ⁵². LATS2 restrains Helicobacter pylori-caused epithelial-mesenchymal transition and intestinal metaplasia in gastric mucosa ⁵³ and YAP initiates gastric tumorigenesis ⁵⁴. We herein identified LATS2 as a direct target of miR-130b-5p, and inhibition of miR-130b-5p or overexpression of circARHGEF12 increased LATS2 activity and YAP1 degradation, and circARHGEF12 counteracted ALKBH5 KD-induced LATS2 inactivation and YAP increase in GC cells. ALKBH5 can repress cancer progression by inhibition of miR-107/LATS2-/YAP1 signaling ⁵⁵. These findings suggested that ALKBH5-mediated m⁶A modification of circARHGEF12 could act as a miR-130b-5p sponge to activate LATS2 and increease YAP degradation, contributing to suppression of GC progression and DDP resistance (Fig. 9).

Our study highlights the important role of ALKBH5-mediated m⁶A modification of circARHGEF12 in suppressing GC progression and DDP resistance via regulating miR-130b-5p/LATS2 signaling and discovers the utilization of oridonin in regulating the ALKBH5/circARHGEF12 axis in GC. These insights not only advance our understanding of circRNA biology in GC but also position a promising candidate for therapeutic intervention of GC.

Gastric cancer

CRC

colorectal cancer

HCC

hepatocellular carcinoma

m⁶A

N6-methyladenosine

circRNA

circular RNA

miRNA

microRNA

lncRNA

long noncoding RNA

METTL3/14

methyltransferase 3/14

ALKBH5

alkB homolog 5, RNA demethylase

WTAP

WT1 associated protein

RBM15/15B

RNA binding motif protein 15/15B

KIAA1429/VIRMA

vir like m6A methyltransferase associated

YTHDF1/2/3

YTH N6-methyladenosine RNA binding protein 1/2/3

YTHDC1/2

YTH domain containing 1/2

IGF2BP1/2/3

IGF2 mRNA binding protein 1/2/3

knockdown

Overexpression

TCGA

The Cancer Genome Atlas

TMA

Tissue microarray

FISH

RNA fluorescence in situ hybridization

DDP

Cisplatin

ECL

enhanced chemiluminescence

RIP

RNA immunoprecipitation

Me-RIP

methylated RNA immunoprecipitation

CCK8

Cell Counting Kit-8

hematoxylin-eosin

IHC

Immunohistochemical

IC50

The half-maximal inhibitory concentration

ARHGEF12

Rho guanine nucleotide exchange factor 12

qRT-PCR

Quantitative real-time PCR

3'-UTR

3'-untranslated region

wild-type

MUT

mutated

LATS2

large tumor suppressor kinase 2

YAP

Yes associated protein.

Acknowledgements

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

Authors’ contributions

JZ, GPZ and XYC designed this study. YLY and JZ drafted the manuscript. YLY and PT performed the experiments and contributed equally to the article. HXZ, HNF, YY collected the data. YLY and ZYC 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 Natural Science Foundation of Shanghai (No. 21ZR1448700), National Natural Science Foundation of China (No. 82074161, 81873143), Shanghai Science and Technology Innovation Plan Star Cultivation (Sailing Special Project, No. 22YF1432600) and the Key Research Project of Science and Technology Department of Sichuan Province (2022YFS0176).

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 Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.

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ALKBH5-dependent upregulation of circARHGEF12 by Oridonin impairs gastric cancer progression and cisplatin resistance via regulating miR-130b-5p/LATS2 signaling

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Clinical data

RNA fluorescence in situ hybridization (FISH)

Cell culture and Drug treatment

Molecular docking model

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

Western blot

Plasmid, shRNA, miRNA mimic and inhibitor

mA-circRNA epi-transcriptomic microarray

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

Bioinformatic analysis

Luciferase gene reporter assay

Statistical analysis

Results

Upregulation of CircARGEF12 by ALKBH5-Mediated m⁶A Modification and Its Characteristics in GC

CircARHGEF12 Mitigates Cisplatin Resistance in GC Cells

CircARHGEF12 Restrains GC Progression and Abolishes ALKBH5 KD-Induced Tumor-promoting Effects

MiR-130b-5p Upregulation Negatively Correlates with CircARHGEF12 Expression and Clinical Prognosis in GC

CircARHGEF12 Sponges miR-130b-5p to Inhibit GC Cell Proliferation and Invasion

CircARHGEF12 Mediates miR-130b-5p to Regulate LATS2/YAP Signaling in GC Cells

Oridonin Activates ALKBH5 to Enhances GC Suppression via miR-130b-5p/LATS2 Pathway

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

ALKBH5-dependent upregulation of circARHGEF12 by Oridonin impairs gastric cancer progression and cisplatin resistance via regulating miR-130b-5p/LATS2 signaling

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Clinical data

RNA fluorescence in situ hybridization (FISH)

Cell culture and Drug treatment

Molecular docking model

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

Western blot

Plasmid, shRNA, miRNA mimic and inhibitor

mA-circRNA epi-transcriptomic microarray

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

Bioinformatic analysis

Luciferase gene reporter assay

Statistical analysis

Results

Upregulation of CircARGEF12 by ALKBH5-Mediated m6A Modification and Its Characteristics in GC

CircARHGEF12 Mitigates Cisplatin Resistance in GC Cells

CircARHGEF12 Restrains GC Progression and Abolishes ALKBH5 KD-Induced Tumor-promoting Effects

MiR-130b-5p Upregulation Negatively Correlates with CircARHGEF12 Expression and Clinical Prognosis in GC

CircARHGEF12 Sponges miR-130b-5p to Inhibit GC Cell Proliferation and Invasion

CircARHGEF12 Mediates miR-130b-5p to Regulate LATS2/YAP Signaling in GC Cells

Oridonin Activates ALKBH5 to Enhances GC Suppression via miR-130b-5p/LATS2 Pathway

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

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

Upregulation of CircARGEF12 by ALKBH5-Mediated m⁶A Modification and Its Characteristics in GC