Study on miR-144-3p targeting CDH11 to suppress malignant biological behaviors of gastric cancer

Study on miR-144-3p targeting CDH11 to suppress malignant biological behaviors of gastric cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Study on miR-144-3p targeting CDH11 to suppress malignant biological behaviors of gastric cancer Xinyuan Chen, Chengting Wu, Ting Wang, Ruidi Li, Yinhang Cui, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8700555/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective : miR-144-3p is a tumor suppressor in gastric cancer, however, the mechanisms by which it inhibits gastric cancer invasion and metastasis have not been fully elucidated. This study aimed to determine whether miR-144-3p suppresses epithelial–mesenchymal transition (EMT) and tumor metastasis by targeting the cell adhesion molecule CDH11, thereby providing a novel molecular target for the intervention of gastric cancer metastasis. Methods : Colony formation, cell cycle analysis, wound-healing, and Transwell assays were performed to evaluate the effects of miR-144-3p and CDH11 on gastric cancer cell proliferation, cell cycle progression, migration, and invasion. Subcutaneous xenograft and lung metastasis models in nude mice were established to assess tumorigenicity and distant colonization in vivo. Dual-luciferase reporter assays and quantitative PCR were used to validate the targeting relationship between miR-144-3p and CDH11. Rescue experiments using wound-healing and Transwell assays were conducted to further confirm the reversal effects of CDH11 overexpression on miR-144-3p function, and Western blotting was performed to analyze changes in EMT-related protein expression. Results : Overexpression of miR-144-3p significantly inhibited gastric cancer cell proliferation, migration, invasion, and the growth of subcutaneous xenografts and lung metastases both in vitro and in vivo, accompanied by suppression of EMT. Dual-luciferase reporter assays demonstrated that miR-144-3p directly targets and negatively regulates CDH11. Silencing of CDH11 similarly suppressed gastric cancer cell proliferation, migration, invasion, and tumor growth in both subcutaneous and pulmonary metastasis models, while inhibiting EMT. Rescue experiments confirmed that CDH11 overexpression partially reversed the inhibitory effects of miR-144-3p on gastric cancer cell migration and invasion and restored the miR-144-3p–induced EMT phenotype. Conclusion : The miR-144-3p/CDH11 axis may serve as a potential diagnostic biomarker and therapeutic target for gastric cancer. miR-144-3p gastric cancer CDH11 epithelial–mesenchymal transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Gastric cancer (GC) is one of the most common malignant tumors worldwide, ranking sixth in incidence and fourth in cancer-related mortality among all cancer types [1]. The prognosis of patients with GC remains poor and is closely associated with clinical stage, with distant metastasis representing the principal cause of the sharp decline in survival among patients with advanced disease. Epidemiological data indicate that the 5-year survival rates for patients with stage I, II, and III GC are approximately 65%, 35%, and 25%, respectively, whereas once distant metastasis occurs and the disease progresses to stage IV, only about 20% of patients survive for more than one year [2]. These clinical realities highlight that suppression of GC metastasis is critical for improving patient prognosis and overcoming current therapeutic limitations. Therefore, identifying novel molecular targets capable of effectively blocking GC metastasis is of substantial clinical importance. MicroRNAs (miRNAs) have attracted considerable attention due to their extensive post-transcriptional regulation of gene expression and their involvement in key steps of tumor metastasis, including epithelial–mesenchymal transition (EMT) [3]. Among them, miR-144-3p has been identified as an important tumor-suppressive miRNA and is downregulated in multiple types of cancer, with low expression levels correlating with enhanced metastatic potential and poor clinical outcomes [4–8]. Downregulation of miR-144-3p has been shown to promote the EMT process, suggesting that it plays a central regulatory role in suppressing GC invasion and metastasis [9]. However, the critical downstream targets and molecular networks through which miR-144-3p precisely regulates EMT and metastasis remain incompletely defined. EMT is a fundamental mechanism driving tumor metastasis and is characterized by alterations in the expression of classical molecules such as CDH1 (E-cadherin) and CDH2 (N-cadherin). CDH11 (cadherin-11) is a type II classical cadherin that not only participates in the regulation of cell proliferation and differentiation [10–13] but is also closely associated with the EMT process [14]. CDH11 has been reported to suppress malignant progression in several cancers, including breast, prostate, and pancreatic cancers [15–17]. In gastric cancer tissues, elevated CDH11 expression is significantly associated with poor prognosis and aggressive behaviors such as bone and liver metastasis [18–22]. Bioinformatics analyses further support this association at the data level, suggesting that CDH11 may serve as a potential molecular marker for predicting GC prognosis [18,19]. CDH11 can function as a direct target of microRNAs and participate in the progression of GC [23]. Previous studies have shown that the long noncoding RNA AP000695.2 can competitively bind miR-144-3p, thereby regulating the expression of multiple downstream genes, including CDH11, and promoting GC development [24]. However, whether miR-144-3p directly targets CDH11 and modulates its biological functions in GC remains to be experimentally validated. Therefore, the present study aimed to verify the targeting regulatory relationship between miR-144-3p and CDH11 through both in vitro and in vivo experiments and to further elucidate the specific role of this regulatory axis in EMT, with the ultimate goal of providing new theoretical evidence for therapeutic strategies targeting GC metastasis. Methods Cell culture The GC-derived cell line HGC-27-LUC was purchased from Servicebio (STCC00080P), and the human gastric cancer cell line AGS was obtained from Procell (CL-0022; lot no. 230911041801). Cells were cultured in RPMI-1640 or Ham’s F-12K medium (Gibco; Procell), respectively, with all media supplemented with 10% fetal bovine serum (FBS; Servicebio, G8002) and 1% penicillin–streptomycin (Solarbio, T1300). All cells were maintained at 37 °C in a humidified incubator containing 5% CO₂. Bioinformatics analysis The association between CDH11 expression and gastric cancer prognosis was obtained from the Human Protein Atlas database. CDH11 mRNA expression data were retrieved from the GEPIA database. The potential targeting relationship between miR-144-3p and CDH11 was predicted using the TargetScan, miRCODE, and starBase databases. Lentiviral infection The miR-144-3p mimic and negative control (miR-NC) lentiviral vectors were synthesized by Shanghai HanHeng Biotechnology Co., Ltd. Logarithmically growing HGC-27 cells were seeded into 6-well plates at a density of 5 × 10⁴ cells per well. When cell confluence reached approximately 30% on the following day, 1 mL of lentiviral suspension at a multiplicity of infection (MOI) of 30 was added to each well. Cells in the miR-144-3p mimic group were transduced with the miR-144-3p mimic lentivirus, whereas the NC group was transduced with the negative control lentivirus. After 4 h, 1 mL of complete culture medium was added, and the medium was replaced after 24 h. Successfully transduced cells were selected using puromycin at a concentration of 2 μg/mL and subsequently passaged for further experiments. The expression levels of miR-144-3p in each group were determined by quantitative PCR (qPCR). The lentiviral constructs LV–miR-144-3p mimic and the corresponding control LV–NC were successfully generated. The sequences of the miR-144-3p mimic and miR-144-3p negative control are listed in Table 1. siRNA and vector transfection GenePharma provided the CDH11 siRNA、CDH11 mimic.and negative control RNA. The siRNA-mate Plus transfection reagent、GP-transfect-Mate transfection reagent which was made by Suzhou GenePharma Co., Ltd., was used to transfect cells for 24 hours at 37°C in accordance with the manufacturer's instructions. Western blot and RT-qPCR were used to confirm the effectiveness of the transfection. Table 1 contains a list of the siRNA sequences. Colony formation assay After transfection, 500 cells were seeded into six-well plates containing complete medium and allowed to grow for approximately 2 weeks until visible colonies formed. Cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.05% crystal violet (Solarbio, 240009007) for 30 min. After washing with PBS, the plates were air-dried at room temperature. Colonies were photographed and counted using Image J software. All assays were performed in triplicate. Migration assay An assay for wound healing was used to evaluate the ability of cells to migrate. 5×10 ^5 cells were seeded into 6-well plates following a 48-hour treatment period. A 200 μL pipette tip was used to make a scratch after the cells had fully confluenced. The medium used to cultivate the cells contained 10% FBS. Picture times were 0, 24, and 48 hours. For each well, three typical fields were chosen, and Image J was used to determine the cell movement distance.Migration rate = (distance at 0 h—distance at 48 h) / distance at 0 h*100%. Matrigel invasion assay In the upper chamber, which contained serum-free medium, cells were seeded at a density of 2.5×10 ^4 after being pre-coated with Matrigel (Biosharp, 54234， Matrigel (Corning) diluted at a 1:8 ratio with serum-free medium.) for four hours. The lower chamber was filled with medium that contained 10% FBS. Membranes were preserved with 4% paraformaldehyde for 30 minutes following a 48-hour incubation period. After 30 minutes of staining with 0.05% crystal violet, the membranes were imaged, twice washed, and ImageJ was used to count the cells. Cell cycle analysis Cells were seeded into six-well plates and cultured at 37 °C for 48 h. The culture medium was then discarded, and cells were collected and centrifuged to remove the supernatant. Each tube was resuspended in 1 mL of DNA staining solution and 10 μL of permeabilization solution, followed by vortexing for 10 s to ensure thorough mixing. Samples were incubated at room temperature for 30 min in the dark and subsequently analyzed by flow cytometry. Cell cycle distribution was assessed using FlowJo software. Dual-luciferase assay CDH11 3′UTR wild-type (WT) and mutant-type (MUT) luciferase reporter constructs were synthesized by Shanghai HanHeng Biotechnology Co., Ltd. These reporters were co-transfected with either miR-144 mimic or miR-144 mimic negative control (NC) using the transfection reagent Lipofiter 3.0 (HB-LF3-1000, Hanbio Biotechnology Co., Ltd., Shanghai, China). After 48 h, cells were collected, and luciferase activity was measured. All procedures were performed following the manufacturer’s instructions for the Dual-Luciferase Assay Kit (HB-DLR-100, Hanbio Biotechnology Co., Ltd., Shanghai, China).。 Animal study Purchased from Hunan Silek Jingda Experimental Animal Co., Ltd., male BALB/c nude mice weighing 12–14 g at 4 weeks of age were kept in a pathogen-free, temperature-controlled environment. Food and water were freely available to mice. Mice (5 mice per group) received subcutaneous injections of HGC-27 Luc cells (1×10 ^6 ) overexpressing miR-144-3p, miR-NC, or transfected with si-CDH11、si-NC via the right axilla and tail vein. Every two days, the tumor's size was measured, and its volume was computed using the formula volume = (length × width ^2 )/2. Mice were put to death by cervical dislocation after 21 days. Tumors were removed, weighed, and captured on camera. The Guangxi University of Chinese Medicine Ethics Committee gave its approval to all animal study protocols.The study is reported in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ).Anesthesia in mice was induced and maintained using isoflurane inhalation.mice were initially induced with 5% isoflurane in an induction chamber and maintained on 1.5–2% isoflurane via a nose cone, with anesthetic depth monitored by the absence of pedal withdrawal reflex. At the experimental endpoint, mice were humanely euthanized by inhalation anesthetic overdose with isoflurane, a method compliant with the AVMA Guidelines for the Euthanasia of Animals. Specifically, animals were exposed to 5% isoflurane in an induction chamber until cessation of vital signs (respiratory and cardiac arrest), and death was confirmed by the absence of breathing, heartbeat, and corneal reflex. Small-animal in vivo CT multimodal fusion imaging system A small-animal in vivo CT multimodal fusion imaging system was used to monitor the development of lung metastases. Fluorescein potassium salt (Aladdin, L120798) was prepared in PBS at a concentration of 15 mg/mL and administered intraperitoneally to nude mice at a dose of 10 mL/kg. Thirty seconds after injection, mice were anesthetized by inhalation of isoflurane, and imaging was performed using the SkyView small-animal in vivo CT multimodal fusion system (Guangzhou BoluTeng Biotechnology Co., Ltd.) for 120 seconds. Data acquisition, processing, and three-dimensional reconstruction were carried out on the system workstation. Immunohistochemistry The following day, sections were counterstained with hematoxylin, detected using a DAB detection kit, and incubated with secondary antibodies for an hour at room temperature. The sections were then cleared, mounted, and dehydrated. Immunohistochemical pictures were subjected to optical density analysis using Image J software. To measure optical density, 400× magnification photographs were taken for each slice. For additional investigation, the mean optical density was employed. Western blot analysis RIPA lysis buffer (Epizyme, P0013B) was used to extract proteins from cells and tissues, and the BCA assay (Epizyme, P0012S) was used to quantify the proteins. SDS-PAGE (Servicebio, G2184-50T) was used to separate the proteins, which were then transferred to 0.2μm PVDF membranes and blocked for two hours with skim milk. After around 14 hours of incubation with particular primary antibodies at 4°C, the membranes were treated for an additional hour at room temperature with secondary antibodies. Vimentin (1:1000, Servicebio, GB11192-100), N-cadherin (1:1000, Servicebio, GB12135-100), OB-cadherin (1:1000, Servicebio, GB111008-100), E-cadherin (1:1000, Servicebio, GB12083-100), GAPDH (1:5000, Servicebio, GB15002-100) were the particular primary antibodies that were used. RT-qPCR analysis Following the manufacturer's instructions, RNA was extracted from cells and tissues using an RNA extraction kit (Beyotime, R0018S). Using a cDNA synthesis kit and a SYBR Green PCR kit (TOLOBIO Biotech, 22107, 22204), RNA was then reverse transcribed into cDNA in accordance with the procedure. 2^−ΔΔCt was used to quantify the results. SinoGene Biotech, located in Shanghai, China, synthesized the miR-144-3p and U6 primers. Table 1 lists the primer sequences that were employed. Statistical analysis To conduct statistical studies, GraphPad Prism 10 was utilized. The mean±standard deviation (SD) is used to display the data. If the data were normally distributed, a t-test was used for comparisons between two groups; if not, a nonparametric Mann-Whitney test was used. Data that were normally distributed were analyzed using ANOVA for multiple group comparisons, whereas data that were not normally distributed were analyzed using one-way analysis. P-values< 0.05 were regarded as statistically significant. Results miR-144 suppresses gastric cancer progression in vitro and in vivo In our study, a series of experiments were conducted to investigate the role of miR-144 in gastric cancer (GC). Colony formation assays demonstrated that both GC cell lines formed fewer colonies in the miR-144 overexpression group compared with the miR-NC group (Fig. 1A). Moreover, wound-healing and Transwell assays indicated that upregulation of miR-144 inhibited GC cell migration (Fig. 1B) and invasion (Fig. 1C). Compared with the miR-NC group, cells in the miR-144 overexpression group exhibited G0/G1 phase cell cycle arrest (Fig. 1D). Western blot analysis of EMT-related proteins revealed that miR-144 overexpression significantly increased E-cadherin levels while reducing N-cadherin and Vimentin expression in both HGC-27 and AGS cells (Fig. 1F). In vivo, equal numbers of HGC-27-Luc cells transduced with either miR-144 overexpression or control lentiviral vectors were injected subcutaneously into the flanks of male nude mice, and tumor sizes were measured every three days (Fig. 2). Mice were sacrificed 21 days later, and tumor weights were recorded (Fig. 2). In the lung metastasis model, equal numbers of HGC-27-Luc cells with miR-144 overexpression or control vectors were injected via the tail vein, and mice were sacrificed 21 days post-injection. Photographs of the subcutaneous xenografts showed a pronounced inhibitory effect of miR-144 on tumor growth (Fig. 2). Immunohistochemistry (IHC) analysis revealed that, compared with the NC group, tumors from the miR-144 overexpression group exhibited significantly increased E-cadherin and decreased N-cadherin and Vimentin expression (Fig. 2). In the lung metastasis model, miR-144 overexpression markedly inhibited tumor development, as evidenced by reduced lung nodule counts, with IHC results consistent with those observed in subcutaneous xenografts. Collectively, these findings indicate that miR-144 significantly suppresses both subcutaneous tumor growth and pulmonary metastasis in nude mice. In summary, our results are consistent with previous studies, supporting an antitumor role for miR-144 in GC. MicroRNAs (miRNAs) suppress cancer progression by targeting and inhibiting specific mRNAs. We used online databases to predict potential post-transcriptional targets of miR-144. As shown in Figures 3A–D, the results indicated that miR-144 may bind to the 3′-UTR of CDH11. Data from the Human Protein Atlas database revealed that CDH11 is overexpressed in GC, and high CDH11 levels are associated with significantly reduced survival in GC patients (Figs. 3E, F). Transfection of the miR-144 overexpression vector into HGC-27 cells led to a marked reduction in CDH11 mRNA levels (Fig. 3G). Furthermore, dual-luciferase reporter assays demonstrated that miR-144 overexpression suppressed the luciferase activity of CDH11-WT, confirming that CDH11 is a direct target of miR-144.As shown in the HPA database, CDH11 expression is upregulated in gastric cancer tissues relative to normal gastric tissues (Fig. 3H). CDH11 promotes gastric cancer growth and metastasis in vitro To investigate the role of CDH11 in gastric cancer (GC), we conducted a series of in vitro experiments to assess its biological functions in GC cells. Colony formation assays were performed to evaluate the effect of CDH11 on GC cell proliferation. The results showed that the number of colonies in the si-CDH11 group was significantly lower than that in the si-NC group (Fig. 4A). In addition, wound-healing assays demonstrated that the scratch width in the si-CDH11 group was narrower than in the si-NC group (Fig. 4B), indicating reduced cell migration. Transwell invasion assays further revealed that the number of invading cells in the si-CDH11 group was lower than in the si-NC group (Fig. 4C). Compared with the si-NC group, HGC-27 cells in the si-CDH11 group exhibited G0/G1 phase cell cycle arrest (Fig. 4D).Western blot analysis showed that in both HGC-27 and AGS cells, silencing CDH11 significantly increased E-cadherin expression while reducing N-cadherin and Vimentin levels (Fig. 4F). In vivo, HGC-27-Luc cells transfected with si-CDH11 or control vectors were injected subcutaneously into male nude mice. After 21 days, mice were sacrificed, and tumors were excised (Fig. 5A). Photographs of the subcutaneous xenografts demonstrated that si-CDH11 markedly inhibited tumor growth (Figs. 5B, C). IHC staining revealed that, compared with the si-NC group, tumors in the si-CDH11 group exhibited significantly increased E-cadherin and decreased N-cadherin and Vimentin expression (Fig. 5). Consistent results were observed in the lung metastasis model. Collectively, these findings indicate that silencing CDH11 significantly suppresses GC tumor growth in nude mice. CDH11 function in gastric cancer is regulated by miR-144 Furthermore, we found that overexpression of CDH11 could partially reverse the inhibitory effects of miR-144. HGC-27 and AGS cells were transfected with miR-NC, miR-144, miR-144 plus control vector, or miR-144 plus CDH11 overexpression vector. As shown, CDH11 overexpression counteracted the suppressive effects of miR-144 on gastric cancer cell growth. Wound-healing and Transwell assays demonstrated that restoring CDH11 significantly reversed the inhibitory effects of miR-144 on GC cell migration and invasion (Figs. 7B–C). Mechanistically, CDH11 overexpression led to downregulation of E-cadherin and upregulation of N-cadherin, Vimentin, and CDH11 itself. These results confirm that miR-144 acts as an upstream regulator of CDH11 and inhibits gastric cancer progression by targeting CDH11. Discussion Gastric cancer (GC) is a severe health threat. Despite substantial advances in therapy over the past decades, including surgery, chemotherapy, radiotherapy, immunotherapy, and combination treatments, GC patients still experience rapid disease progression and poor prognosis due to tumor metastasis [25,26]. Epithelial–mesenchymal transition (EMT) is a central initiating step in tumor metastasis. Under specific signaling stimuli, gastric epithelial-derived cancer cells undergo programmed changes in their biological characteristics, transitioning from a differentiated epithelial phenotype to a more invasive mesenchymal phenotype. This process is characterized by downregulation of the epithelial marker E-cadherin and upregulation of mesenchymal markers such as N-cadherin and Vimentin, thereby promoting tumor metastasis. Cell cycle regulation is a core process governing cell growth and division, and dysregulation of this process is a key factor leading to uncontrolled proliferation and tumorigenesis. Cell cycle arrest can therefore inhibit gastric cancer progression. Although miR-144 is recognized as a tumor suppressor, its precise mechanisms in GC metastasis remain unclear. Hence, we explored the effects of miR-144-3p on CDH11-mediated inhibition of EMT in GC using both in vitro and in vivo experiments. Our study first demonstrated that overexpression of miR-144 suppresses malignant behaviors in GC. Specifically, miR-144 inhibited proliferation, migration, and invasion of HGC-27 and AGS cells, inducing G0/G1 cell cycle arrest. miR-144 significantly upregulated the epithelial protein E-cadherin while downregulating mesenchymal proteins, including N-cadherin and Vimentin. In xenograft and lung metastasis models, miR-144 markedly inhibited tumor growth. IHC results indicated that miR-144 increased E-cadherin expression and decreased N-cadherin and Vimentin levels in tumor tissues. These findings confirm the tumor-suppressive role of miR-144 in GC, consistent with previous reports [27]. To better understand the molecular mechanism by which miR-144 suppresses proliferation, migration, invasion, and EMT, CDH11 was identified as a potential target of miR-144 via bioinformatics analysis. Dual-luciferase reporter assays combined with qPCR confirmed that miR-144 directly binds to the 3′-UTR of CDH11, reducing CDH11 expression. We found that silencing CDH11 inhibited malignant behaviors of GC cells, induced G0/G1 cell cycle arrest, and increased E-cadherin while decreasing N-cadherin, Vimentin, and CDH11 protein levels. In vivo, tumors from the si-CDH11 xenograft group were smaller and grew more slowly, and in the lung metastasis model, si-CDH11 significantly reduced the number of lung nodules. IHC results indicated that si-CDH11 increased E-cadherin expression and decreased N-cadherin, Vimentin, and CDH11 levels in both subcutaneous and pulmonary tumors. To further validate that CDH11 is a functional target of miR-144, rescue experiments were conducted. Co-transfection of miR-144 with a CDH11 overexpression vector partially reversed the inhibitory effects of miR-144 on GC cell proliferation, migration, and invasion, confirming that CDH11 is a functionally relevant downstream target of miR-144 in GC cells. Although this study establishes the miR-144/CDH11 signaling axis as a key regulator of GC progression and metastasis, several limitations exist. First, the study primarily focused on a lung metastasis model, without assessing other CDH11-related metastatic sites such as bone. Additionally, simultaneous evaluation of miR-144 and CDH11 protein expression in clinical samples would further strengthen the translational relevance of these findings. Future studies should address these issues to more comprehensively elucidate the regulatory network and therapeutic potential of the miR-144/CDH11 axis in gastric cancer. Declarations Ethics approval consent to participate: All experimental procedures in this study strictly adhered to the ethical principles of laboratory animal welfare and were approved by the Experimental Animal Ethics Committee of Guangxi University of Chinese Medicine (Ethics Approval No. DW20240507-091). Consent for publication ： Written informed consent for participation and publication was obtained from all participants. Availability of data and material: The datasets generated and analysed during the current study are not publicly available as the data are still under active investigation by the research team to prevent preemption and ensure accurate interpretation but are available from the corresponding author on reasonable request. Competing interests: The authors declare no conflict of interest. Funding: This research was funded by the National Natural Science Foundation of China grant number [No. 82360959], Guangxi Graduate Education Innovation Plan Project (Grant No.YCBZ2025193). Authors' contributions: Data curation, Ting Wang, Ruidi Li, and Yinhang Cui; Software, Jiacheng Xie and Changzhou Xiong; Validation, Peibin Wu and Xinyuan Chen; Visualization, Xinyuan Chen and yuanqin du; Methodology, investigation, Writing – original draft, Xinyuan Chen and Chengting Wu; Writing – review & editing, Meiwen Tang and Caizhi Lin. All the authors revised the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgments We sincerely thank the Meiwen Tang research group members for providing resources and support during the study. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Oncol Lett. 2020 Jun;19(6):4011-4023. doi: 10.3892/ol.2020.11531. Epub 2020 Apr 10. PMID: 32391104; PMCID: PMC7204628. Mita H, Katoh H, Komura D, Kakiuchi M, Abe H, Rokutan H, Yagi K, Nomura S, Ushiku T, Seto Y, Ishikawa S. Aberrant Cadherin11 expression predicts distant metastasis of gastric cancer. Pathol Res Pract. 2023 Feb;242:154294. doi: 10.1016/j.prp.2022.154294. Epub 2022 Dec 28. PMID: 36610328. Huang CF, Lira C, Chu K, Bilen MA, Lee YC, Ye X, Kim SM, Ortiz A, Wu FL, Logothetis CJ, Yu-Lee LY, Lin SH. Cadherin-11 increases migration and invasion of prostate cancer cells and enhances their interaction with osteoblasts. Cancer Res. 2010 Jun 1;70(11):4580-9. doi: 10.1158/0008-5472.CAN-09-3016. Epub 2010 May 18. PMID: 20484040; PMCID: PMC2923552. Sandoval-Bórquez A, Polakovicova I, Carrasco-Véliz N, Lobos-González L, Riquelme I, Carrasco-Avino G, Bizama C, Norero E, Owen GI, Roa JC, Corvalán AH. MicroRNA-335-5p is a potential suppressor of metastasis and invasion in gastric cancer. Clin Epigenetics. 2017 Oct 17;9:114. doi: 10.1186/s13148-017-0413-8. Erratum in: Clin Epigenetics. 2021 Mar 8;13(1):50. doi: 10.1186/s13148-021-01036-2. PMID: 29075357; PMCID: PMC5645854. An Y, Liu X, Liu J, Wang D, Yan W, Hu G, Xu L, Li W. Identification of a LncRNA based CeRNA network signature to establish a prognostic model and explore potential therapeutic targets in gastric cancer. Sci Rep. 2025 Jul 1;15(1):20891. doi: 10.1038/s41598-025-05105-x. PMID: 40595924; PMCID: PMC12219596. Zhang Z, Pi J, Zou D, Wang X, Xu J, Yu S, Zhang T, Li F, Zhang X, Zhao H, Wang F, Wang D, Ma Y, Yu J. microRNA arm-imbalance in part from complementary targets mediated decay promotes gastric cancer progression. Nat Commun. 2019 Sep 27;10(1):4397. doi: 10.1038/s41467-019-12292-5. PMID: 31562301; PMCID: PMC6764945. Smyth EC, Moehler M. Late-line treatment in metastatic gastric cancer: today and tomorrow. Ther Adv Med Oncol. 2019 Aug 28;11:1758835919867522. doi: 10.1177/1758835919867522. PMID: 31489035; PMCID: PMC6713955. Liu J, Xue H, Zhang J, Suo T, Xiang Y, Zhang W, Ma J, Cai D, Gu X. MicroRNA-144 inhibits the metastasis of gastric cancer by targeting MET expression. J Exp Clin Cancer Res. 2015 Apr 17;34(1):35. doi: 10.1186/s13046-015-0154-5. PMID: 25927670; PMCID: PMC4417226. Table 1 Table 1 is not available with this version. Additional Declarations No competing interests reported. Supplementary Files OriginalblotimagesincludedintheSupplementaryInformation.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8700555","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588977543,"identity":"ae93a5aa-3005-4023-8f53-95eafc652d85","order_by":0,"name":"Xinyuan Chen","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinyuan","middleName":"","lastName":"Chen","suffix":""},{"id":588977544,"identity":"6cc93873-4bcd-49b1-8b5a-a455b2beea2b","order_by":1,"name":"Chengting Wu","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chengting","middleName":"","lastName":"Wu","suffix":""},{"id":588977548,"identity":"39a12382-9781-4b9f-b1f3-24fa4c340b9e","order_by":2,"name":"Ting Wang","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Wang","suffix":""},{"id":588977549,"identity":"688f29ce-ef48-4d3b-908d-a8d09392134e","order_by":3,"name":"Ruidi Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ruidi","middleName":"","lastName":"Li","suffix":""},{"id":588977552,"identity":"7d59eb84-c90e-4567-8dae-b2ba0f5ac382","order_by":4,"name":"Yinhang Cui","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yinhang","middleName":"","lastName":"Cui","suffix":""},{"id":588977553,"identity":"02374723-0bf9-4239-a43e-77ed674a8ac6","order_by":5,"name":"Jiacheng Xie","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiacheng","middleName":"","lastName":"Xie","suffix":""},{"id":588977555,"identity":"a2bb01b4-af9c-4a3c-9622-f3fab040e798","order_by":6,"name":"Changzhou Xiong","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Changzhou","middleName":"","lastName":"Xiong","suffix":""},{"id":588977558,"identity":"08f1576d-0307-449b-8580-9a501fad6962","order_by":7,"name":"Yuanqin Du","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuanqin","middleName":"","lastName":"Du","suffix":""},{"id":588977560,"identity":"467a25de-e9a7-43fc-90ae-0d77eeb25770","order_by":8,"name":"Peibin WU","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Peibin","middleName":"","lastName":"WU","suffix":""},{"id":588977562,"identity":"98a62f8b-273b-47dc-b529-5a4764f8deca","order_by":9,"name":"Caizhi Lin","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Caizhi","middleName":"","lastName":"Lin","suffix":""},{"id":588977563,"identity":"7f72047b-22c6-4f0a-9f5f-6f7b100a1eeb","order_by":10,"name":"Meiwen Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYPACGziLsYGQWh4IlcbAwEailsMkaLFnP3v41c2283Yb7jc/3czDYCO74QDzswd4beHJS7PObbudPLONzew2D0Oa8YYDbOYG+B2WY2acu+12Mj8bDxtQy+HEDQd42CTwauF/A9JyLpkNouU/EVokcowf5247YAe15QARWm68MWPO/ZecINmWZnZzjkGy8czDbGZ4tbD35xh/zjljZ29w+PCzG28q7GT7jjc/w6sFCMDOSGwAs0FBxUxAPUjJByBhT1jdKBgFo2AUjFgAAO8BRZnaeIUEAAAAAElFTkSuQmCC","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Meiwen","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2026-01-26 13:08:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8700555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8700555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102811035,"identity":"0388278f-c745-4009-9329-e36521bd1d22","added_by":"auto","created_at":"2026-02-17 03:34:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1146928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-144 suppresses GC progression in vitro.\u003c/strong\u003e (A) Colony formation assay showing the effect of miR-144 overexpression in HGC-27 and AGS cells.(B) Wound healing assay assessing cell migration ability. (C) Transwell invasion assay measuring cell invasive capacity.(D) Flow cytometry analysis of the cell cycle.（E）Western blot analysis of E-cadherin, N-cadherin, and Vimentin expression.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/7da22caf494404d6b40507a9.png"},{"id":102962916,"identity":"d83b82d1-5654-4bf9-98c5-3d0b6c6de9c1","added_by":"auto","created_at":"2026-02-19 04:12:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1095535,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-144 suppresses GC progression in vivo.\u003c/strong\u003e(A)Images of GC subcutaneous xenograft tumors in the miR-NC and miR-144 overexpression groups. (B) Subcutaneous xenograft tumor weight. (C) Subcutaneous xenograft tumor growth curve. (D )Representative images of E-cadherin, N-cadherin, and vimentin IHC staining in tumor sections of subcutaneous xenografts from the NC and miR-144 groups. (E) In vivo imaging and multimodal fusion schematic of the gastric cancer lung metastasis mouse model. (F)Images of gastric cancer lung metastasis. (G) Statistical analysis of fluorescence values from in vivo imaging of the gastric cancer lung metastasis mouse model. (h) Representative images of E-cadherin, N-cadherin, and vimentin IHC staining in lung metastasis tumor sections from the NC and miR-144 groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/a925760a2b4c799d1bbd2df3.png"},{"id":102811037,"identity":"d4c4392c-eb7e-46b5-a659-8fde27bad79a","added_by":"auto","created_at":"2026-02-17 03:34:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1292931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatic prediction and experimental validation of miR-144-3p targeting CDH11\u003c/strong\u003e. (A) Volcano plot of differentially expressed genes in gastric cancer. (B)Overlapping target genes of gastric cancer and miR-144-3p. (C )Prediction of target interaction between miR-144-3p and CDH11. (D )Prediction of the binding energy between miR-144-3p and CDH11. (E) schematic diagram of the binding site between miR-144-3p and the CDH11 3'UTR. (F) dual-luciferase reporter assay detecting the interaction between miR-144-3p and the CDH11 3'UTR. (G) q-PCR analysis of CDH11 levels following miR-144-3p overexpression.(H) Immunohistochemical staining of CDH11 in human gastric tissues and gastric cancer.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/5924abf57543e83297c1c09a.png"},{"id":102811036,"identity":"838448d1-08a2-4a07-b7d8-a94405182125","added_by":"auto","created_at":"2026-02-17 03:34:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":939845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003esi-CDH11 suppresses GC progression in vitro. \u003c/strong\u003e(A) Colony formation assay showing the effect of si-CDH11 in HGC-27 and AGS cells.(B) Wound healing assay assessing cell migration ability. (C) Transwell invasion assay measuring cell invasive capacity.(D) Flow cytometry analysis of the cell cycle.Western blot analysis of E-cadherin, N-cadherin, and Vimentin expression.*p \u0026lt;0.05; \u003csup\u003e#\u003c/sup\u003ep \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/9151e35b804abcfbc35a1132.png"},{"id":103049417,"identity":"c29b0f49-1226-4872-b41b-b5cffb48c36f","added_by":"auto","created_at":"2026-02-20 07:41:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2085743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003esi-CDH11 suppresses GC progression in vivo. \u003c/strong\u003e(A)Images of GC subcutaneous xenograft tumors in the si-NC and si-CDH11 groups.(B)Subcutaneous xenograft tumor weight.(C)Subcutaneous xenograft tumor growth curve.(D)Representative images of E-cadherin, N-cadherin, and vimentin IHC staining in tumor sections of subcutaneous xenografts from the si-NC and si-CDH11 groups.(E)In vivo imaging and multimodal fusion schematic of the gastric cancer lung metastasis mouse model from the si-NC and si-CDH11 groups.(F)Images of gastric cancer lung metastasis.(G)Statistical analysis of fluorescence values from in vivo imaging of the gastric cancer lung metastasis mouse model.(H)Representative images of E-cadherin, N-cadherin, and vimentin IHC staining in lung metastasis tumor sections from the si-NC and si-CDH11 groups. \u003csup\u003e*\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/7a636e6b03acb7f005663afa.png"},{"id":102962475,"identity":"c6cb58d5-afc6-4103-9337-cdcf9f874d96","added_by":"auto","created_at":"2026-02-19 04:09:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1408738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe function of CDH11 in GC is regulated by miR-144.\u003c/strong\u003e (A)Cell migration ability was assessed using a wound healing assay. (B)Cell invasion capability was evaluated by a Transwell invasion assay. (C)Western blot analysis of CDH11, E-cadherin, N-cadherin, and Vimentin expression. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/602eddc2bb6af89d1f3ce974.png"},{"id":104401284,"identity":"41946bb3-a278-4b8b-adf0-f5fe92c45b0d","added_by":"auto","created_at":"2026-03-11 12:12:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8613648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/b76876ae-f977-44c1-9dc5-b3e942c2b3fb.pdf"},{"id":102811041,"identity":"6d031941-ee3d-4e14-9f16-7b3b39867cc8","added_by":"auto","created_at":"2026-02-17 03:34:26","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":280498411,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalblotimagesincludedintheSupplementaryInformation.zip","url":"https://assets-eu.researchsquare.com/files/rs-8700555/v1/79dec55268d1354d5aa7765b.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on miR-144-3p targeting CDH11 to suppress malignant biological behaviors of gastric cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is one of the most common malignant tumors worldwide, ranking sixth in incidence and fourth in cancer-related mortality among all cancer types [1]. The prognosis of patients with GC remains poor and is closely associated with clinical stage, with distant metastasis representing the principal cause of the sharp decline in survival among patients with advanced disease. Epidemiological data indicate that the 5-year survival rates for patients with stage I, II, and III GC are approximately 65%, 35%, and 25%, respectively, whereas once distant metastasis occurs and the disease progresses to stage IV, only about 20% of patients survive for more than one year [2]. These clinical realities highlight that suppression of GC metastasis is critical for improving patient prognosis and overcoming current therapeutic limitations. Therefore, identifying novel molecular targets capable of effectively blocking GC metastasis is of substantial clinical importance.\u003c/p\u003e\n\u003cp\u003eMicroRNAs (miRNAs) have attracted considerable attention due to their extensive post-transcriptional regulation of gene expression and their involvement in key steps of tumor metastasis, including epithelial\u0026ndash;mesenchymal transition (EMT) [3]. Among them, miR-144-3p has been identified as an important tumor-suppressive miRNA and is downregulated in multiple types of cancer, with low expression levels correlating with enhanced metastatic potential and poor clinical outcomes [4\u0026ndash;8]. Downregulation of miR-144-3p has been shown to promote the EMT process, suggesting that it plays a central regulatory role in suppressing GC invasion and metastasis [9]. However, the critical downstream targets and molecular networks through which miR-144-3p precisely regulates EMT and metastasis remain incompletely defined.\u003c/p\u003e\n\u003cp\u003eEMT is a fundamental mechanism driving tumor metastasis and is characterized by alterations in the expression of classical molecules such as CDH1 (E-cadherin) and CDH2 (N-cadherin). CDH11 (cadherin-11) is a type II classical cadherin that not only participates in the regulation of cell proliferation and differentiation [10\u0026ndash;13] but is also closely associated with the EMT process [14]. CDH11 has been reported to suppress malignant progression in several cancers, including breast, prostate, and pancreatic cancers [15\u0026ndash;17]. In gastric cancer tissues, elevated CDH11 expression is significantly associated with poor prognosis and aggressive behaviors such as bone and liver metastasis [18\u0026ndash;22]. Bioinformatics analyses further support this association at the data level, suggesting that CDH11 may serve as a potential molecular marker for predicting GC prognosis [18,19].\u003c/p\u003e\n\u003cp\u003eCDH11 can function as a direct target of microRNAs and participate in the progression of GC [23]. Previous studies have shown that the long noncoding RNA AP000695.2 can competitively bind miR-144-3p, thereby regulating the expression of multiple downstream genes, including CDH11, and promoting GC development [24]. However, whether miR-144-3p directly targets CDH11 and modulates its biological functions in GC remains to be experimentally validated. Therefore, the present study aimed to verify the targeting regulatory relationship between miR-144-3p and CDH11 through both in vitro and in vivo experiments and to further elucidate the specific role of this regulatory axis in EMT, with the ultimate goal of providing new theoretical evidence for therapeutic strategies targeting GC metastasis.\u003c/p\u003e"},{"header":"Methods","content":"\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GC-derived cell line HGC-27-LUC was purchased from Servicebio (STCC00080P), and the human gastric cancer cell line AGS was obtained from Procell (CL-0022; lot no. 230911041801). Cells were cultured in RPMI-1640 or Ham\u0026rsquo;s F-12K medium (Gibco; Procell), respectively, with all media supplemented with 10% fetal bovine serum (FBS; Servicebio, G8002) and 1% penicillin\u0026ndash;streptomycin (Solarbio, T1300). All cells were maintained at 37 \u0026deg;C in a humidified incubator containing 5% CO₂.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe association between CDH11 expression and gastric cancer prognosis was obtained from the Human Protein Atlas database. CDH11 mRNA expression data were retrieved from the GEPIA database. The potential targeting relationship between miR-144-3p and CDH11 was predicted using the TargetScan, miRCODE, and starBase databases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentiviral infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe miR-144-3p mimic and negative control (miR-NC) lentiviral vectors were synthesized by Shanghai HanHeng Biotechnology Co., Ltd. Logarithmically growing HGC-27 cells were seeded into 6-well plates at a density of 5 \u0026times; 10⁴ cells per well. When cell confluence reached approximately 30% on the following day, 1 mL of lentiviral suspension at a multiplicity of infection (MOI) of 30 was added to each well. Cells in the miR-144-3p mimic group were transduced with the miR-144-3p mimic lentivirus, whereas the NC group was transduced with the negative control lentivirus. After 4 h, 1 mL of complete culture medium was added, and the medium was replaced after 24 h. Successfully transduced cells were selected using puromycin at a concentration of 2 \u0026mu;g/mL and subsequently passaged for further experiments. The expression levels of miR-144-3p in each group were determined by quantitative PCR (qPCR). The lentiviral constructs LV\u0026ndash;miR-144-3p mimic and the corresponding control LV\u0026ndash;NC were successfully generated. The sequences of the miR-144-3p mimic and miR-144-3p negative control are listed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA and vector transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenePharma provided the CDH11 siRNA、CDH11 mimic.and negative control RNA. The siRNA-mate Plus transfection reagent、GP-transfect-Mate transfection reagent which was made by Suzhou GenePharma Co., Ltd., was used to transfect cells for 24 hours at 37\u0026deg;C in accordance with the manufacturer\u0026apos;s instructions. Western blot and RT-qPCR were used to confirm the effectiveness of the transfection. Table 1 contains a list of the siRNA sequences.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfection, 500 cells were seeded into six-well plates containing complete medium and allowed to grow for approximately 2 weeks until visible colonies formed. Cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.05% crystal violet (Solarbio, 240009007) for 30 min. After washing with PBS, the plates were air-dried at room temperature. Colonies were photographed and counted using Image J software. All assays were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn assay for wound healing was used to evaluate the ability of cells to migrate. 5\u0026times;10\u003csup\u003e^5\u0026nbsp;\u003c/sup\u003ecells were seeded into 6-well plates following a 48-hour treatment period. A 200 \u0026mu;L pipette tip was used to make a scratch after the cells had fully confluenced. The medium used to cultivate the cells contained 10% FBS. Picture times were 0, 24, and 48 hours. For each well, three typical fields were chosen, and Image J was used to determine the cell movement distance.Migration rate = (distance at 0 h\u0026mdash;distance at 48 h) / distance at 0 h*100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMatrigel invasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the upper chamber, which contained serum-free medium, cells were seeded at a density of 2.5\u0026times;10\u003csup\u003e^4\u0026nbsp;\u003c/sup\u003eafter being pre-coated with Matrigel (Biosharp, 54234，\u0026nbsp;Matrigel (Corning) diluted at a 1:8 ratio with serum-free medium.) for four hours. The lower chamber was filled with medium that contained 10% FBS. Membranes were preserved with 4% paraformaldehyde for 30 minutes following a 48-hour incubation period. After 30 minutes of staining with 0.05% crystal violet, the membranes were imaged, twice washed, and ImageJ was used to count the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded into six-well plates and cultured at 37 \u0026deg;C for 48 h. The culture medium was then discarded, and cells were collected and centrifuged to remove the supernatant. Each tube was resuspended in 1 mL of DNA staining solution and 10 \u0026mu;L of permeabilization solution, followed by vortexing for 10 s to ensure thorough mixing. Samples were incubated at room temperature for 30 min in the dark and subsequently analyzed by flow cytometry. Cell cycle distribution was assessed using FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCDH11 3\u0026prime;UTR wild-type (WT) and mutant-type (MUT) luciferase reporter constructs were synthesized by Shanghai HanHeng Biotechnology Co., Ltd. These reporters were co-transfected with either miR-144 mimic or miR-144 mimic negative control (NC) using the transfection reagent Lipofiter 3.0 (HB-LF3-1000, Hanbio Biotechnology Co., Ltd., Shanghai, China). After 48 h, cells were collected, and luciferase activity was measured. All procedures were performed following the manufacturer\u0026rsquo;s instructions for the Dual-Luciferase Assay Kit (HB-DLR-100, Hanbio Biotechnology Co., Ltd., Shanghai, China).。\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurchased from Hunan\u0026nbsp;Silek\u0026nbsp;Jingda\u0026nbsp;Experimental\u0026nbsp;Animal\u0026nbsp;Co.,\u0026nbsp;Ltd., male BALB/c nude mice weighing 12\u0026ndash;14 g at 4 weeks of age were kept in a pathogen-free, temperature-controlled environment. Food and water were freely available to mice. Mice (5 mice per group) received subcutaneous injections of HGC-27 Luc cells (1\u0026times;10\u003csup\u003e^6\u003c/sup\u003e) overexpressing miR-144-3p, miR-NC, or transfected with si-CDH11、si-NC via the right axilla and tail vein. Every two days, the tumor\u0026apos;s size was measured, and its volume was computed using the formula volume = (length \u0026times; width\u003csup\u003e^2\u003c/sup\u003e)/2. Mice were put to death by cervical dislocation after 21 days. Tumors were removed, weighed, and captured on camera. The Guangxi University of Chinese Medicine Ethics Committee gave its approval to all animal study protocols.The study is reported in accordance with the ARRIVE guidelines (\u003ca href=\"https://arriveguidelines.org/\" target=\"/Users/chenxinyuan/Documents\\x/_blank\"\u003ehttps://arriveguidelines.org\u003c/a\u003e).Anesthesia in mice was induced and maintained using isoflurane inhalation.mice were initially induced with 5% isoflurane in an induction chamber and maintained on 1.5\u0026ndash;2% isoflurane via a nose cone, with anesthetic depth monitored by the absence of pedal withdrawal reflex. At the experimental endpoint, mice were humanely euthanized by inhalation anesthetic overdose with isoflurane, a method compliant with the AVMA Guidelines for the Euthanasia of Animals. Specifically, animals were exposed to 5% isoflurane in an induction chamber until cessation of vital signs (respiratory and cardiac arrest), and death was confirmed by the absence of breathing, heartbeat, and corneal reflex.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall-animal in vivo CT multimodal fusion imaging system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA small-animal in vivo CT multimodal fusion imaging system was used to monitor the development of lung metastases. Fluorescein potassium salt (Aladdin, L120798) was prepared in PBS at a concentration of 15 mg/mL and administered intraperitoneally to nude mice at a dose of 10 mL/kg. Thirty seconds after injection, mice were anesthetized by inhalation of isoflurane, and imaging was performed using the SkyView small-animal in vivo CT multimodal fusion system (Guangzhou BoluTeng Biotechnology Co., Ltd.) for 120 seconds. Data acquisition, processing, and three-dimensional reconstruction were carried out on the system workstation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following day, sections were counterstained with hematoxylin, detected using a DAB detection kit, and incubated with secondary antibodies for an hour at room temperature. The sections were then cleared, mounted, and dehydrated. Immunohistochemical pictures were subjected to optical density analysis using Image J software. To measure optical density, 400\u0026times; magnification photographs were taken for each slice. For additional investigation, the mean optical density was employed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIPA lysis buffer (Epizyme, P0013B) was used to extract proteins from cells and tissues, and the BCA assay (Epizyme, P0012S) was used to quantify the proteins. SDS-PAGE (Servicebio, G2184-50T) was used to separate the proteins, which were then transferred to 0.2\u0026mu;m PVDF membranes and blocked for two hours with skim milk. After around 14 hours of incubation with particular primary antibodies at 4\u0026deg;C, the membranes were treated for an additional hour at room temperature with secondary antibodies. Vimentin (1:1000, Servicebio, GB11192-100), N-cadherin (1:1000, Servicebio, GB12135-100), OB-cadherin (1:1000, Servicebio, GB111008-100), E-cadherin (1:1000, Servicebio, GB12083-100), GAPDH (1:5000, Servicebio, GB15002-100) were the particular primary antibodies that were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the manufacturer\u0026apos;s instructions, RNA was extracted from cells and tissues using an RNA extraction kit (Beyotime, R0018S). Using a cDNA synthesis kit and a SYBR Green PCR kit (TOLOBIO Biotech, 22107, 22204), RNA was then reverse transcribed into cDNA in accordance with the procedure. 2^\u0026minus;\u0026Delta;\u0026Delta;Ct was used to quantify the results. SinoGene Biotech, located in Shanghai, China, synthesized the miR-144-3p and U6 primers. Table 1 lists the primer sequences that were employed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo conduct statistical studies, GraphPad Prism 10 was utilized. The mean\u0026plusmn;standard deviation (SD) is used to display the data. If the data were normally distributed, a t-test was used for comparisons between two groups; if not, a nonparametric Mann-Whitney test was used. Data that were normally distributed were analyzed using ANOVA for multiple group comparisons, whereas data that were not normally distributed were analyzed using one-way analysis. P-values\u0026lt; 0.05 were regarded as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003emiR-144 suppresses gastric cancer progression in vitro and in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our study, a series of experiments were conducted to investigate the role of miR-144 in gastric cancer (GC). Colony formation assays demonstrated that both GC cell lines formed fewer colonies in the miR-144 overexpression group compared with the miR-NC group (Fig. 1A). Moreover, wound-healing and Transwell assays indicated that upregulation of miR-144 inhibited GC cell migration (Fig. 1B) and invasion (Fig. 1C). Compared with the miR-NC group, cells in the miR-144 overexpression group exhibited G0/G1 phase cell cycle arrest (Fig. 1D). Western blot analysis of EMT-related proteins revealed that miR-144 overexpression significantly increased E-cadherin levels while reducing N-cadherin and Vimentin expression in both HGC-27 and AGS cells (Fig. 1F).\u003c/p\u003e\n\u003cp\u003eIn vivo, equal numbers of HGC-27-Luc cells transduced with either miR-144 overexpression or control lentiviral vectors were injected subcutaneously into the flanks of male nude mice, and tumor sizes were measured every three days (Fig. 2). Mice were sacrificed 21 days later, and tumor weights were recorded (Fig. 2). In the lung metastasis model, equal numbers of HGC-27-Luc cells with miR-144 overexpression or control vectors were injected via the tail vein, and mice were sacrificed 21 days post-injection. Photographs of the subcutaneous xenografts showed a pronounced inhibitory effect of miR-144 on tumor growth (Fig. 2). Immunohistochemistry (IHC) analysis revealed that, compared with the NC group, tumors from the miR-144 overexpression group exhibited significantly increased E-cadherin and decreased N-cadherin and Vimentin expression (Fig. 2).\u003c/p\u003e\n\u003cp\u003eIn the lung metastasis model, miR-144 overexpression markedly inhibited tumor development, as evidenced by reduced lung nodule counts, with IHC results consistent with those observed in subcutaneous xenografts. Collectively, these findings indicate that miR-144 significantly suppresses both subcutaneous tumor growth and pulmonary metastasis in nude mice. In summary, our results are consistent with previous studies, supporting an antitumor role for miR-144 in GC.\u003c/p\u003e\n\u003cp\u003eMicroRNAs (miRNAs) suppress cancer progression by targeting and inhibiting specific mRNAs. We used online databases to predict potential post-transcriptional targets of miR-144. As shown in Figures 3A\u0026ndash;D, the results indicated that miR-144 may bind to the 3\u0026prime;-UTR of CDH11. Data from the Human Protein Atlas database revealed that CDH11 is overexpressed in GC, and high CDH11 levels are associated with significantly reduced survival in GC patients (Figs. 3E, F). Transfection of the miR-144 overexpression vector into HGC-27 cells led to a marked reduction in CDH11 mRNA levels (Fig. 3G). Furthermore, dual-luciferase reporter assays demonstrated that miR-144 overexpression suppressed the luciferase activity of CDH11-WT, confirming that CDH11 is a direct target of miR-144.As shown in the HPA database, CDH11 expression is upregulated in gastric cancer tissues relative to normal gastric tissues (Fig. 3H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDH11 promotes gastric cancer growth and metastasis in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of CDH11 in gastric cancer (GC), we conducted a series of in vitro experiments to assess its biological functions in GC cells. Colony formation assays were performed to evaluate the effect of CDH11 on GC cell proliferation. The results showed that the number of colonies in the si-CDH11 group was significantly lower than that in the si-NC group (Fig. 4A). In addition, wound-healing assays demonstrated that the scratch width in the si-CDH11 group was narrower than in the si-NC group (Fig. 4B), indicating reduced cell migration. Transwell invasion assays further revealed that the number of invading cells in the si-CDH11 group was lower than in the si-NC group (Fig. 4C). Compared with the si-NC group, HGC-27 cells in the si-CDH11 group exhibited G0/G1 phase cell cycle arrest (Fig. 4D).Western blot analysis showed that in both HGC-27 and AGS cells, silencing CDH11 significantly increased E-cadherin expression while reducing N-cadherin and Vimentin levels (Fig. 4F).\u003c/p\u003e\n\u003cp\u003eIn vivo, HGC-27-Luc cells transfected with si-CDH11 or control vectors were injected subcutaneously into male nude mice. After 21 days, mice were sacrificed, and tumors were excised (Fig. 5A). Photographs of the subcutaneous xenografts demonstrated that si-CDH11 markedly inhibited tumor growth (Figs. 5B, C). \u0026nbsp; IHC staining revealed that, compared with the si-NC group, tumors in the si-CDH11 group exhibited significantly increased E-cadherin and decreased N-cadherin and Vimentin expression (Fig. 5). Consistent results were observed in the lung metastasis model. Collectively, these findings indicate that silencing CDH11 significantly suppresses GC tumor growth in nude mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDH11 function in gastric cancer is regulated by miR-144\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, we found that overexpression of CDH11 could partially reverse the inhibitory effects of miR-144. HGC-27 and AGS cells were transfected with miR-NC, miR-144, miR-144 plus control vector, or miR-144 plus CDH11 overexpression vector. As shown, CDH11 overexpression counteracted the suppressive effects of miR-144 on gastric cancer cell growth. Wound-healing and Transwell assays demonstrated that restoring CDH11 significantly reversed the inhibitory effects of miR-144 on GC cell migration and invasion (Figs. 7B\u0026ndash;C). Mechanistically, CDH11 overexpression led to downregulation of E-cadherin and upregulation of N-cadherin, Vimentin, and CDH11 itself. These results confirm that miR-144 acts as an upstream regulator of CDH11 and inhibits gastric cancer progression by targeting CDH11.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGastric cancer (GC) is a severe health threat. Despite substantial advances in therapy over the past decades, including surgery, chemotherapy, radiotherapy, immunotherapy, and combination treatments, GC patients still experience rapid disease progression and poor prognosis due to tumor metastasis [25,26]. Epithelial\u0026ndash;mesenchymal transition (EMT) is a central initiating step in tumor metastasis. Under specific signaling stimuli, gastric epithelial-derived cancer cells undergo programmed changes in their biological characteristics, transitioning from a differentiated epithelial phenotype to a more invasive mesenchymal phenotype. This process is characterized by downregulation of the epithelial marker E-cadherin and upregulation of mesenchymal markers such as N-cadherin and Vimentin, thereby promoting tumor metastasis. Cell cycle regulation is a core process governing cell growth and division, and dysregulation of this process is a key factor leading to uncontrolled proliferation and tumorigenesis. Cell cycle arrest can therefore inhibit gastric cancer progression. Although miR-144 is recognized as a tumor suppressor, its precise mechanisms in GC metastasis remain unclear. Hence, we explored the effects of miR-144-3p on CDH11-mediated inhibition of EMT in GC using both in vitro and in vivo experiments.\u003c/p\u003e\n\u003cp\u003eOur study first demonstrated that overexpression of miR-144 suppresses malignant behaviors in GC. Specifically, miR-144 inhibited proliferation, migration, and invasion of HGC-27 and AGS cells, inducing G0/G1 cell cycle arrest. miR-144 significantly upregulated the epithelial protein E-cadherin while downregulating mesenchymal proteins, including N-cadherin and Vimentin. In xenograft and lung metastasis models, miR-144 markedly inhibited tumor growth. IHC results indicated that miR-144 increased E-cadherin expression and decreased N-cadherin and Vimentin levels in tumor tissues. These findings confirm the tumor-suppressive role of miR-144 in GC, consistent with previous reports [27].\u003c/p\u003e\n\u003cp\u003eTo better understand the molecular mechanism by which miR-144 suppresses proliferation, migration, invasion, and EMT, CDH11 was identified as a potential target of miR-144 via bioinformatics analysis. Dual-luciferase reporter assays combined with qPCR confirmed that miR-144 directly binds to the 3\u0026prime;-UTR of CDH11, reducing CDH11 expression. We found that silencing CDH11 inhibited malignant behaviors of GC cells, induced G0/G1 cell cycle arrest, and increased E-cadherin while decreasing N-cadherin, Vimentin, and CDH11 protein levels. In vivo, tumors from the si-CDH11 xenograft group were smaller and grew more slowly, and in the lung metastasis model, si-CDH11 significantly reduced the number of lung nodules. IHC results indicated that si-CDH11 increased E-cadherin expression and decreased N-cadherin, Vimentin, and CDH11 levels in both subcutaneous and pulmonary tumors.\u003c/p\u003e\n\u003cp\u003eTo further validate that CDH11 is a functional target of miR-144, rescue experiments were conducted. Co-transfection of miR-144 with a CDH11 overexpression vector partially reversed the inhibitory effects of miR-144 on GC cell proliferation, migration, and invasion, confirming that CDH11 is a functionally relevant downstream target of miR-144 in GC cells.\u003c/p\u003e\n\u003cp\u003eAlthough this study establishes the miR-144/CDH11 signaling axis as a key regulator of GC progression and metastasis, several limitations exist. First, the study primarily focused on a lung metastasis model, without assessing other CDH11-related metastatic sites such as bone. Additionally, simultaneous evaluation of miR-144 and CDH11 protein expression in clinical samples would further strengthen the translational relevance of these findings. Future studies should address these issues to more comprehensively elucidate the regulatory network and therapeutic potential of the miR-144/CDH11 axis in gastric cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval consent to participate:\u0026nbsp;\u003c/strong\u003eAll experimental procedures in this study strictly adhered to the ethical principles of laboratory animal welfare and were approved by the Experimental Animal Ethics Committee of Guangxi University of Chinese Medicine (Ethics Approval No. DW20240507-091). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eWritten informed consent for participation and publication was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eThe datasets generated and analysed during the current study are not publicly available\u0026nbsp;as the data are still under active investigation by the research team to prevent preemption and ensure accurate interpretation\u0026nbsp;but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis\u003cstrong\u003e\u0026nbsp;research\u0026nbsp;\u003c/strong\u003ewas funded by the National Natural Science Foundation of China grant number [No. 82360959], Guangxi Graduate Education Innovation Plan Project (Grant No.YCBZ2025193).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u0026nbsp;\u003c/strong\u003eData curation, Ting Wang, Ruidi Li, and Yinhang Cui; Software, Jiacheng Xie and Changzhou Xiong; Validation, Peibin Wu and Xinyuan Chen; Visualization, Xinyuan Chen and yuanqin du; Methodology, investigation, Writing \u0026ndash; original draft, Xinyuan Chen and Chengting Wu; Writing \u0026ndash; review \u0026amp; editing, Meiwen Tang and Caizhi Lin. All the authors revised the manuscript. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the Meiwen Tang research group members for providing resources and support during the study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021 May;71(3):209-249. doi: 10.3322/caac.21660. Epub 2021 Feb 4. PMID: 33538338.\u003c/li\u003e\n\u003cli\u003eThrift AP, Wenker TN, El-Serag HB. Global burden of gastric cancer: epidemiological trends, risk factors, screening and prevention. Nat Rev Clin Oncol. 2023 May;20(5):338-349. doi: 10.1038/s41571-023-00747-0. 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PMID: 23913163; PMCID: PMC3834228.\u003c/li\u003e\n\u003cli\u003ePeran I, Dakshanamurthy S, McCoy MD, Mavropoulos A, Allo B, Sebastian A, Hum NR, Sprague SC, Martin KA, Pishvaian MJ, Vietsch EE, Wellstein A, Atkins MB, Weiner LM, Quong AA, Loots GG, Yoo SS, Assefnia S, Byers SW. Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice. Gastroenterology. 2021 Mar;160(4):1359-1372.e13. doi: 10.1053/j.gastro.2020.11.044. Epub 2020 Dec 9. PMID: 33307028; PMCID: PMC7956114.\u003c/li\u003e\n\u003cli\u003eWang H, Zhang B. The Impact of Transcriptional Profiling Cadherin Family and Therapeutic Approaches of Gastric Cancer: A Translational Outlook on Multi-omics Data Analysis. Appl Biochem Biotechnol. 2024 Nov;196(11):7657-7674. doi: 10.1007/s12010-024-04926-2. Epub 2024 Mar 26. PMID: 38530538.\u003c/li\u003e\n\u003cli\u003eChen PF, Wang F, Nie JY, Feng JR, Liu J, Zhou R, Wang HL, Zhao Q. Co-expression network analysis identified CDH11 in association with progression and prognosis in gastric cancer. Onco Targets Ther. 2018 Oct 2;11:6425-6436. doi: 10.2147/OTT.S176511. PMID: 30323620; PMCID: PMC6174304.\u003c/li\u003e\n\u003cli\u003eWang Q, Jia Y, Peng X, Li C. Clinical and prognostic association of oncogene cadherin 11 in gastric cancer. Oncol Lett. 2020 Jun;19(6):4011-4023. doi: 10.3892/ol.2020.11531. Epub 2020 Apr 10. PMID: 32391104; PMCID: PMC7204628.\u003c/li\u003e\n\u003cli\u003eMita H, Katoh H, Komura D, Kakiuchi M, Abe H, Rokutan H, Yagi K, Nomura S, Ushiku T, Seto Y, Ishikawa S. Aberrant Cadherin11 expression predicts distant metastasis of gastric cancer. Pathol Res Pract. 2023 Feb;242:154294. doi: 10.1016/j.prp.2022.154294. Epub 2022 Dec 28. PMID: 36610328.\u003c/li\u003e\n\u003cli\u003eHuang CF, Lira C, Chu K, Bilen MA, Lee YC, Ye X, Kim SM, Ortiz A, Wu FL, Logothetis CJ, Yu-Lee LY, Lin SH. 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PMID: 25927670; PMCID: PMC4417226.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1 ","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-144-3p, gastric cancer, CDH11, epithelial–mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-8700555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8700555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: miR-144-3p is a tumor suppressor in gastric cancer, however, the mechanisms by which it inhibits gastric cancer invasion and metastasis have not been fully elucidated. This study aimed to determine whether miR-144-3p suppresses epithelial–mesenchymal transition (EMT) and tumor metastasis by targeting the cell adhesion molecule CDH11, thereby providing a novel molecular target for the intervention of gastric cancer metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Colony formation, cell cycle analysis, wound-healing, and Transwell assays were performed to evaluate the effects of miR-144-3p and CDH11 on gastric cancer cell proliferation, cell cycle progression, migration, and invasion. Subcutaneous xenograft and lung metastasis models in nude mice were established to assess tumorigenicity and distant colonization in vivo. Dual-luciferase reporter assays and quantitative PCR were used to validate the targeting relationship between miR-144-3p and CDH11. Rescue experiments using wound-healing and Transwell assays were conducted to further confirm the reversal effects of CDH11 overexpression on miR-144-3p function, and Western blotting was performed to analyze changes in EMT-related protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Overexpression of miR-144-3p significantly inhibited gastric cancer cell proliferation, migration, invasion, and the growth of subcutaneous xenografts and lung metastases both in vitro and in vivo, accompanied by suppression of EMT. Dual-luciferase reporter assays demonstrated that miR-144-3p directly targets and negatively regulates CDH11. Silencing of CDH11 similarly suppressed gastric cancer cell proliferation, migration, invasion, and tumor growth in both subcutaneous and pulmonary metastasis models, while inhibiting EMT. Rescue experiments confirmed that CDH11 overexpression partially reversed the inhibitory effects of miR-144-3p on gastric cancer cell migration and invasion and restored the miR-144-3p–induced EMT phenotype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The miR-144-3p/CDH11 axis may serve as a potential diagnostic biomarker and therapeutic target for gastric cancer.\u003c/p\u003e","manuscriptTitle":"Study on miR-144-3p targeting CDH11 to suppress malignant biological behaviors of gastric cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 03:34:14","doi":"10.21203/rs.3.rs-8700555/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b6807ac6-dabb-47a8-add2-f8611adfd272","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-04T11:43:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 03:34:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8700555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8700555","identity":"rs-8700555","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}