Overexpression of miR-195-5p suppresses gastric cancer progression by regulating LAMP2-mediated autophagy

Overexpression of miR-195-5p suppresses gastric cancer progression by regulating LAMP2-mediated autophagy | 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 Overexpression of miR-195-5p suppresses gastric cancer progression by regulating LAMP2-mediated autophagy Chenglong Shen, Fang Zhou, Xiaolei Zhang, Jiazhe Shao, Yuchao Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6189299/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 May, 2025 Read the published version in Biochemical Genetics → Version 1 posted 4 You are reading this latest preprint version Abstract MiR-195-5p has been confirmed to be intimately linked to many cancers development, whereas its role in gastric cancer (GC) remains controversial. The objective of this study was to explore the potential function and mechanism of miR-195-5p in GC. First, the miR-195-5p level in GC was analyzed based on qRT-PCR and TCGA STAD. Then, the function of miR-195-5p in GC cells was detected via CCK-8, clone formation assay, flow cytometry, transwell, and wound healing assay. Subsequently, we explored the possible mechanism of miR-195-5p using KEGG analysis, database analysis, Western blot, and rescue assays. In this research, miR-195-5p was declined in GC and associated to malignant progression of GC. The introduction of miR-195-5p weakened cell growth, metastasis, and facilitated cell apoptosis. KEGG enrichment analysis indicated that miR-195-5p influenced the malignant progression of GC through mechanisms related to autophagy. MiR-195-5p overexpression leads to a diminish in cellular autophagy. Further studies identified that miR-195-5p bound to LAMP2. Mechanism studies confirmed that miR-195-5p introduction attenuated cell growth, and this process was rescued by LAMP2 overexpression. MiR-195-5p affects the malignant progression of GC by regulating LAMP2-mediated autophagy. Gastric cancer miR-195-5p LAMP2 autophagy malignant progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Gastric cancer (GC) is recognized as a frequent form of cancer in the digestive system. Baded on the analysis of statistics in 2022, saw an estimated 970,000 new cases of GC and 660,000 fatalities worldwide (Bray et al. 2024 ). Several factors contribute to the occurrence of GC, including late-night habits, an unbalanced diet, alcohol consumption, and Helicobacter pylori infection (Machlowska et al. 2020 ). In recent years, human beings have made remarkable achievements in the fields of chemotherapy, surgical treatment and molecular targeted therapy for GC. With the progress of technology, the incidence rate and mortality of GC have reduced (Song et al. 2017 ). However, GC often has an insidious onset, which means most patients are diagnosed at advanced stages. This increases their likelihood of recurrence and metastasis after surgery, resulting in a poor prognosis and a 5-year survival rate of around 20% (Díaz Del Arco et al. 2024). Therefore, exploring the mechanisms of GC development and searching for specific markers have become important issues that need to be addressed urgently. The miRNAs constitute a category of endogenous, non-coding RNAs, characterized by their high degree of conservation and an average length of around 22 nucleotides. miRNAs act as negative regulators of gene expression through binding to the 3′ UTR of messenger RNAs (mRNAs), hindering the translation process or enhancing the degradation of these mRNAs (Bartel 2009 ). Research have reported that miRNAs act a vital role in regulating tumorigenesis and development. For instance, miR-339-5p weakens the migration and proliferation of GC cells via ALKBH1 (Wang et al. 2020 ). Jiang et al. found that miRNA-15a-3p expression was lower in hepatocellular carcinoma tissues. Patients with reduced levels of miRNA-15a-3p had higher rates of metastasis and poorer prognoses, and overexpression of miRNA-15a-3p decreased cancer cell proliferation and metastatic potential (Jiang et al. 2020 ). miRNAs also participate in the regulation of autophagy process via mediating target genes (Shan et al. 2021 ). Therefore, miRNAs serve as ideal candidate biomarkers for almost all human diseases, including tumors. miR-195-5p, which originates from the 5p arm of the miR-195 precursor, has been found to be associated to a wide range of cancers in humans, including lung cancer, esophageal cancer, and GC (Xu et al. 2022 ). Nevertheless, the specific mechanism of miR-195-5p in GC still to be elucidated and need to be explored in depth. In the cells of eukaryotes, autophagy is a highly conserved process whose main function is the effective degradation and reuse of intracellularly damaged and destroyed organelles and biomolecules (Fang et al. 2016 ). A multitude of research have reported that differential changes in autophagy levels have been found in several types of tumors, and that changes in autophagy may serve a position in facilitating or hindering tumor cells in tumorigenesis and development. Autophagy plays a crucial role in maintaining tissue homeostasis and physiological functions (Ding et al. 2024 ). Therefore, autophagy-related interventions are a promising new strategy for tumor therapy. The Lysosome-associated membrane protein 2 (LAMP2) protein, a singular transmembrane constituent of the lysosomal membrane, serves to preserve the integrity of the lysosomal membrane against the action of lysosomal hydrolases and modulates the process of membrane coalescence between lysosomes and other organelles during autophagic activities (Pajares et al. 2018 ). LAMP2 exerts a rate-limiting effect during the lysosomal degradation phase by affecting immunity, macroautophagy (MA) and molecular chaperone-mediated autophagy (CMA) (Eskelinen 2006 ). During neuroendocrine differentiation of prostate cancer cells, LAMP2 is upregulated and autophagic activity is increased (Morell et al. 2016 ). In addition, TSTA3 overexpression accelerates lung squamous cell carcinoma cell growth and metastasis through regulating LAMP2-mediated autophagy and the tumor microenvironment (Guo et al. 2023 ). However, the mechanism by which LAMP2-mediated autophagy affects GC progression is still imperfect and requires in-depth study. The main objective of the present study was to explore the mechanism of aberrantly expressed miR-195-5p in the development of GC. MiR-195-5p was depressed in GC in this research. MiR-195-5p overexpression weakened cell growth and metastasis and facilitated cell apoptosis. Furthermore, this process was discovered to be linked with autophagy by KEGG enrichment analysis. Online database analysis disclosed that miR-195-5p bound to LAMP2. This study reveals that miR-195-5p regulates GC development through LAMP2-mediated autophagy, offering new insights for treating patients with GC. Material and Methods Clinical tissue sample acquisition Human GC tumor tissues and paracancerous normal tissues were obtained from patients who underwent surgery at our institution. The tumor tissues were rapidly frozen in liquid nitrogen and subsequently maintained at -80°C for preservation. Written informed consent was secured from each patient, the research was sanctioned by the hospital Medical Ethics Committee, with all protocols adhering to the principles outlined in the Declaration of Helsinki. Cell culture and transfection GC cell lines (AGS, MKN45, SGC-7901, BGC-823) and human normal gastric mucosal epithelial cell line (GSE-1) were purchased from China Center for Type Culture Collection (Wuhan, China). Cells were cultured in DMEM medium containing 10% (v/v) FBS, 100 U/mL penicillin (Life Technologies), and streptomycin (Life Technologies) at 5% CO 2 at 37°C. Specific LAMP2 siRNA and miR-195-5p mimics were designed and synthesized from GenePharma (Shanghai, China). Recombinant overexpression vectors were constructed by inserting LAMP2 sequences into pLCDH-ciR, respectively. The pLCDH-LAMP2 or miR-195-5p mimics were transfected into GC cells using Lipofectamine 3000 (Invitrogen) based on the manufacturer's instructions. qRT-PCR RNA was isolated from treated cells and tissues by TRizol reagent. The extracted RNA was reverse transcribed to cDNA via the Prime Script™ RT kit (Takara, China). qRT-PCR was performed on a quantitative PCR system(Applied Biosystems 7500, USA). Relative expression was calculated through the 2 -ΔΔCt method. Cell proliferation The CCK-8 kit (MilliporeSigma, USA) was used to assess cell proliferation. The density of cells was adjusted to 1x10 4 cells/mL, and 100 μL/well was taken and inoculated in 96-well plates, with 5 replicates set up per well. At 0, 24, 48, and 72 hours into the incubation period, 10 μL of CCK-8 reagent was introduced, respectively, and the incubation was continued for 2 h. Later, the microplate reader was utilized to quantify the absorbance at 450 nm, which corresponds to the optical density (OD). Clone formation assay Post-transfection, cells were adjusted to a concentration of 500 cells per well and then plated into 6-well plates for incubation at 37°C for a fortnight. After a fixation process with methanol for 30 min, the cells were subjected to staining using a 0.1% crystal violet solution. The subsequent procedures included capturing images and conducting a cell count of the stained cells. Apoptosis assay Flow cytometry was employed to assess cellular apoptosis. Cells were plated in culture dishes and allowed to grow for 24 h before the transfection process. At 48 h post-transfection, the cells were collected, washed with PBS, and then processed for dual staining with the Annexin V FITC/PI Apoptosis Detection Kit procured from Solarbio. Wound healing assay Wound healing assay was used to detect the migration ability of cells. Marker pen was used to draw a line on the back side of the 6-well plate, and BGC-823 and SGC-7901 cells with 80% density were digested with trypsin, resuspended in complete medium, counted on cell counting plate, and then inoculated into the 6-well plate according to the amount of 3x10 5 cells in each well, and cultured at constant temperature for 24 h. The liquid was changed regularly, when the confluence of cells reaches 100%, insert a pipette gun with a 100 μL tip perpendicular to the bottom of the plate and make a scratches at equal distances in each well, aspirate and discard the medium, wash three times by carefully adding 1 mL PBS along the side wall, and add 2% FBS to the complete medium and put it in the incubator for incubation. The healing status of the same scratch at 0 h and 48 h was photographed under a microscope, and the migration area of each time period was calculated and analyzed in Image J software. Transwell assay Extracellular matrix gel was uniformly spread across the upper face of the polycarbonate membranes in the Transwell inserts. After a preliminary cultured with serum-free medium for 24 h, the cellular concentration was adjusted to 5x10 5 /ml. 100 μL this suspension was introduced into the upper chamber of the chambers, 600 μL of serum-containing medium was added to the lower chamber, and the chambers were placed in a cell culture incubator for 48 h. Then, the chambers were removed. The cells on the lower surface of the upper chamber were carefully wiped off with a cotton swab, fixed with methanol and stained with crystal violet. The number of cells was observed under a microscope with a 400x field of view, and 5 fields of view were photographed and calculated, and the average value was taken. Dual luciferase reporter assay The downstream target mRNAs of miRNAs were predicted based on the online software. Luciferase reporter vectors including LAMP2-WT, LAMP2-MUT. The SGC-7901, BGC-823 cells were inoculated into 96-well plates and cultured for 24 h until a confluence of 50%-70%. The cells were then co-transfected with the indicated reporter vectors and miR-195-5p mimics or miR-NC via Lipofectamine 3000. 48 h later, luciferase activity was measured using the Dual Luciferase Reporter Assay System. Western blot (WB) Total proteins were extracted using RIPA lysate (Solarbio, China). Protein concentration was measured by BCA kit (Solarbio, China). Then, proteins were separated by 10% SDS/PAGE, and proteins were transferred to nitrocellulose membranes (Biosharp, China). The membranes were sealed in 5% skimmed milk powder and incubated with primary antibody at 4°C overnight. After that, the membrane was incubated with secondary antibody for 60 min. At last, protein bands were visualized via an ECL kit (Beyotime Biotech, China). Immunofluorescence analysis Cells were attached and grown on slides. Cell fixation was performed using a 4% solution of paraformaldehyde for 30 min. Then, cells were subjected to permeabilization using a 3% BSA solution for an equivalent duration. Cells were incubated with LC3B primary antibody overnight in a humidified chamber. After labeling with primary antibody overnight, cells were incubated with fluorescent secondary antibody (Alexa Fluor 488, USA) for 1 h. They were then stained with DAPI (Thermo Fisher Scientific, USA). Slides were evaluated on a microscope. Analysis was performed with Image J software. Statistical analysis Statistical data were analyzed and expressed as mean ± standard deviation using GraphPad Prism 8.0 software. Each experiment was repeated 3 times and the average value. Differences between two or more groups were assessed by Student's t-test or one-way ANOVA. p<0.05 was considered significantly different. Results miR-195-5p is reduced in GC To understand the role of miR-195-5p in GC, we examined miR-195-5p expression in GC tissues. miR-195-5p was identified to be clearly diminished in GC tissues contrasted to normal tissues adjacent to the cancer (Fig. 1 A). According to the TCGA database, miR-195-5p was also found to be downregulated in GC tissues (Fig. 1 B). Furthermore, we examined the miR-195-5p level in GC cells. Compared to GSE-1, miR-195-5p was declined in AGS, MKN45, SGC-7901, and BGC-823 cells. Among them, miR-195-5p had the lowest expression in SGC-7901 and BGC-823 cells, so this two cells were used for subsequent experiments (Fig. 1 C). The above findings indicated miR-195-5p might be a novel target for GC. miR-195-5p overexpression weakens cell proliferation and induces apoptosis MiR-195-5p overexpression vector was constructed to explore the function of miR-195-5p in GC. The outcome displayed miR-195-5p was obviously enhanced in cells after of miR-195-5p overexpression compared with control, indicating that miR-195-5p was successfully overexpression in BGC-823 and SGC-7901 cells (Fig. 2 A). Subsequently, cells growth, colony formation and apoptotic ability were performed. The findings illustrated that miR-195-5p introduction evidently suppressed the growth and proliferation ability of cells, and markedly heightened the apoptotic ability of the cells (Fig. 2 B-D). The above results indicated that miR-195-5p introduction induced apoptosis and restrained cell proliferation. Overexpression of miR-195-5p suppresses GC cell metastasis through autophagy Next, we investigated the impact of miR-195-5p overexpression cell migration and invasion ability. Wound healing assay was applied to determine the changes in cell migration ability. It was observed that the migration ability of SGC-7901 and BGC-823 cells was reduced after miR-195-5p introduction (Fig. 3 A). The effect of overexpression of miR-195-5p on cell invasion ability was measured via transwell assay. Introduction of miR-195-5p was discovered to clearly decline the invasion ability of cells (Fig. 3 B). To elucidate the mechanism by which miR-195-5p affects GC progression, miR-195-5p differentially expressed genes were found to be enriched in autophagy-related pathways by KEGG enrichment analysis (Fig. 1 D). Subsequently, LC3 aggregation during autophagosome formation was identified by immunofluorescence analysis (Fig. 3 C). LC3 II/I was obviously depressed after overexpression of miR-195-5p, while p62 level was raised, demonstrating that miR-195-5p overexpression repressed cellular autophagy (Fig. 3 D). The above outcomes indicated that introduction of miR-195-5p restrained the metastasis of GC cells in vitro through autophagy. miR-195-5p acts as a functional regulator of LAMP2 To investigate the mechanism by which miR-195-5p affects GC progression, downstream targets of miR-195-5p were screened using autophagy related genes in TCGA database and miRWalk, mirDIP, starBase databases. It was discovered that there were two overlapping genes, LAMP2 and HSPA8 (Fig. 4 A). Moreover, miR-195-5p was discovered to have a binding site with LAMP2 3'-UTR through the database. Therefore, we hypothesized that LAMP2 is a downstream target of miR-195-5p (Fig. 4 B). Next, we assessed the binding relationship between miR-195-5p and the 3'-UTR of LAMP2 using a dual luciferase assay. The outcomes declared that the luciferase activity of LAMP2-WT was considerably reduced in cells transfected with miR-195-5p mimics, whereas the luciferase activity of LAMP2-MUT did not change evidently, indicating a binding relationship between miR-195-5p and LAMP2 (Fig. 4 C). Subsequently, we examined the level of LAMP2 in GC tissues. LAMP2 was heightened in GC tissues contrasted to normal tissues adjacent to the cancer (Fig. 4 D). TCGA database findings also showed high expression of LAMP2 (Fig. 4 E). Pearson correlation analysis was applied on GC tissues to measure the association between miR-195-5p and LAMP2. A significant inverse association was disclosed between miR-195-5p and LAMP2 expression (Fig. 4 F). Moreover, LAMP2 level in cell lines was detected via qRT-PCR, and LAMP2 was raised in GC cells (Fig. 4 G). Next, to investigate the function of LAMP2 in GC, LAMP2 expression was knockdown in the cells, and the knockdown efficiency of LAMP2 was tested via WB. The findings displayed that LAMP2 silencing clearly diminished LAMP2 protein expression in GC cells (Fig. 4 H). CCK-8 results discovered that knockdown of LAMP2 restrained the viability of GC cells (Fig. 4 I). Furthermore, we found that LAMP2 introduction returned the repression impact of miR-195-5p overexpression on cell viability (Fig. 4 J). The above outcomes confirmed that overexpression of miR-195-5p weakened the GC development through LAMP2. Based on the above findings, we conclude that overexpression of miR-195-5p attenuated the malignant progression of GC cells through LAMP2 mediated autophagy. Moreover, according to these outcomes, we have drawn a hypothesis diagram of the mechanism by which miR-195-5p affects the progression of GC (Fig. 5 ). Discussion The development of GC is closely related to a variety of elements, with its etiology being multifaceted. It is subject to a complex interplay of various regulatory systems that influence and modulate its development. The propensity for GC to recur and spread to other parts of the body is a primary obstacle in achieving a cure for the disease (Yang et al. 2023 ). Therefore, elucidating the molecular underpinnings of GC metastasis process is crucial for developing precision treatments for patients with advanced gastric cancer. Here, our study analyzed the impact of miR-195-5p dysregulation in GC on the malignant biology of GC cells. Moreover, our study delineates the regulatory interplay between miR-195-5p and LAMP2 in GC, demonstrating that overexpression of miR-195-5p suppresses the malignant phenotype of GC cells through LAMP2-mediated autophagy. Existing literature underscores the pivotal role of miRNAs in the etiology and progression of gastric malignancies. The miR-195-5p, belonging to the miR-15 family, is mapped to the 17th chromosome (Nie et al. 2018 ). miR-195-5p exerts tumor-suppressive activity in a spectrum of cancers such as colorectal, lung adenocarcinoma, gallbladder, and cervical cancers (Liu et al. 2023 ). By targeting FOSL1, miR-195-5p effectively downregulates the Wnt/β-catenin pathway, thereby inhibiting the uncontrolled proliferation and invasive behavior characteristic of gallbladder cancer cells (Zhu et al. 2022 ). In non-small cell lung cancer, miR-195-5p overexpression restrains cancer cell growth and induces apoptosis via targeting CEP55 (Luo et al. 2019 ). Furthermore, insertion of miR-195-5p attenuated the growth, metastasis and EMT of laryngeal cancer cells through E2F3 (Zhou et al. 2021b ). The present investigation displayed a marked diminish in miR-195-5p expression within gastric cancerous tissues and cells compared to their non-cancerous counterparts, echoing a similar pattern of downregulation noted across a spectrum of malignancies. The upregulation of miR-195-5p exerts a suppressive effect on cell growth and metastatic potential, concurrently facilitating apoptotic activity. This underscores the miRNA function as a tumor suppressor, demonstrating its efficacy in controlling the proliferative and invasive behaviors of GC cells. Autophagy exerts a complex and context-dependent part in different types of tumors. The process of autophagy exhibits a biphasic influence on oncogenesis, initially acting to restrain tumor development in the early stages. Conversely, in advanced stages of the disease, it may foster the neoplastic process, thereby accelerating tumorigenesis (Debnath et al. 2023 ). A substantial array of miRNAs has been demonstrated to play a role in the modulation of autophagic processes (Li et al. 2020 ). For instance, miR-519a enhances apoptosis and sensitizes glioblastoma to temozolomide treatment via facilitating autophagy and targeting STAT3/Bcl-2/Beclin-1 axis (Li et al. 2018 ). Research has demonstrated that miR-138-5p weakened autophagy and blocked serum starvation-induced autophagic fluxes in pancreatic cancer cells, and further studies revealed that miR-138-5p hampered autophagy in these cells via SIRT1 (Tian et al. 2017 ). Moreover, the autophagic flux is typified by fluctuations in specific marker proteins. The p62 protein, integral to cellular homeostatic mechanisms through its involvement in autophagy modulation and intracellular signaling (Zhou et al. 2021a ). In parallel, the LC3 protein complex stands as a quintessential biomarker of autophagic activity. The abundance of LC3II is positively correlated with the degree of autophagy occurring within the cell (Tanida et al. 2004 ). Our research demonstrated that increased expression of miR-195-5p led to a decrease in the LC3 II/I ratio and an increase in p62 levels, indicating that autophagy was suppressed in the cells. This reveals miR-195-5p introduction restrained cell growth and metastasis through mediating autophagy. Despite identification of various candidate targets for miR-195-5p, the intricate molecular mechanisms mediating its impact on GC development are not yet comprehensively understood. In this research, we screened LAMP2 as a target gene of miR-195-5p by bioinformatics analysis. LAMP2 has three splice isoforms: LAMP2A, LAMP2B, and LAMP2C. Emerging evidence highlighting the pivotal role of lysosomes in multiple cancer types has intensified the scientific community interest in exploring the function of LAMP2 in the progression of cancer (Zheng et al. 2018 ). There is evidence that enhanced LAMP2 levels are linked to tumor progression. Huang et al. found that lncRNA FAM215A stimulates LAMP2 expression to confer drug-resistant and progression in HCC (Huang et al. 2020 ). Another study discovered miR-1-3p attenuated the malignant phenotype of enamel cell tumors via downregulating LAMP2-mediated autophagy (Niu et al. 2021 ). In this study, LAMP2 was markedly elevated in GC and inversely related to the expression of miR-195-5p. Knockdown of LAMP2 weakened cell growth. Additionally, overexpression of LAMP2 reversed the suppressive effect of miR-195-5p introduction on cell growth. The above findings suggested miR-195-5p affects the malignant progression of GC through LAMP2-mediated autophagy. Although this study offers fresh perspectives on the role of miR-195-5p in GC, it also has certain inherent limitations. First, this study was mainly conducted in an in vitro cellular model, and the relevance between miR-195-5p and LAMP2 needs to be validated in more clinical samples in the future. Furthermore, the upstream regulatory mechanisms and downstream impact of miR-195-5p in GC need to be further investigated. In summary, miR-195-5p affects the advancement of GC through the LAMP2-mediated autophagy process. This finding facilitates our understanding of the molecular mechanisms of GC, also provides novel potential targets for the diagnosis and treatment of GC. Declarations Acknowledgments We thank the donors whose names were not included in the author list, but who participated in this program. Author Contribution Chenlong Shen, Xiaolei Zhang and Fang Zhou analyzed data and wrote the paper. Yichen Li and Yuchao Wang performed experiments. Jiazhe Shao and Haichen Liu guided the experiments and the analysis. Guoqiang Zhou and Zhiliang Shi initiated the study, designed experiments. All authors read and approved the final manuscript. Funding Changshu Key Laboratory of Digestive System Tumor Innovation and Diagnosis and Treatment (CS202313), Suzhou City clinical key disease diagnosis and treatment technology special project (LCZX202224), Suzhou Medical and Health science and technology Innovation Project (SKY2022022) Data Availability The data are available from the corresponding author on reasonable request. Competing interests The authors have no conflict of interest, financial or otherwise. Ethical approval Approval of Changshu NO.2 People's Hospital Ethics Committee. All participants were provided with written informed consent at the time of recruitment, and all experiments involving human tissue specimens comply with the Declaration of Helsinki. Consent for Publication Not applicable. References Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215-33. https://doi.org/10.1016/j.cell.2009.01.002 Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I , Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229-263. https://doi.org/10.3322/caac.21834 Debnath J, Gammoh N , Ryan KM (2023) Autophagy and autophagy-related pathways in cancer. Nat Rev Mol Cell Biol 24(8):560-575. https://doi.org/10.1038/s41580-023-00585-z Díaz Del Arco C, Fernández Aceñero MJ , Ortega Medina L (2024) Molecular Classifications in Gastric Cancer: A Call for Interdisciplinary Collaboration. Int J Mol Sci 25(5). https://doi.org/10.3390/ijms25052649 Ding Y, Huang X, Ji T, Qi C, Gao X , Wei R (2024) The emerging roles of miRNA-mediated autophagy in ovarian cancer. Cell Death Dis 15(5):314. https://doi.org/10.1038/s41419-024-06677-8 Eskelinen EL (2006) Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol Aspects Med 27(5-6):495-502. https://doi.org/10.1016/j.mam.2006.08.005 Fang W, Shu S, Yongmei L, Endong Z, Lirong Y , Bei S (2016) miR-224-3p inhibits autophagy in cervical cancer cells by targeting FIP200. Sci Rep 6:33229. https://doi.org/10.1038/srep33229 Guo Y, Hao Y, Shen L, Du Y, Wang X, Gao L, Feng X, Zhai Y, Liu Z, Xu E, Yang Y, Xi Y, Yang B , Zhang L (2023) TSTA3 overexpression promotes malignant characteristics in LUSC by regulating LAMP2-mediated autophagy and tumor microenvironment. Cancer Cell Int 23(1):285. https://doi.org/10.1186/s12935-023-03109-z Huang PS, Lin YH, Chi HC, Tseng YH, Chen CY, Lin TK, Yeh CT , Lin KH (2020) Dysregulated FAM215A Stimulates LAMP2 Expression to Confer Drug-Resistant and Malignant in Human Liver Cancer. Cells 9(4). https://doi.org/10.3390/cells9040961 Jiang C, He ZL, Hu XH , Ma PY (2020) MiRNA-15a-3p inhibits the metastasis of hepatocellular carcinoma by interacting with HMOX1. Eur Rev Med Pharmacol Sci 24(24):12694-12700. https://doi.org/10.26355/eurrev_202012_24167 Li H, Chen L, Li JJ, Zhou Q, Huang A, Liu WW, Wang K, Gao L, Qi ST , Lu YT (2018) miR-519a enhances chemosensitivity and promotes autophagy in glioblastoma by targeting STAT3/Bcl2 signaling pathway. J Hematol Oncol 11(1):70. https://doi.org/10.1186/s13045-018-0618-0 Li X, He S , Ma B (2020) Autophagy and autophagy-related proteins in cancer. Mol Cancer 19(1):12. https://doi.org/10.1186/s12943-020-1138-4 Liu X, Liu Z, Liu Y , Wang N (2023) ATG9A modulated by miR-195-5p can boost the malignant progression of cervical cancer cells. Epigenetics 18(1):2257538. https://doi.org/10.1080/15592294.2023.2257538 Luo J, Pan J, Jin Y, Li M , Chen M (2019) MiR-195-5p Inhibits Proliferation and Induces Apoptosis of Non-Small Cell Lung Cancer Cells by Targeting CEP55. Onco Targets Ther 12:11465-11474. https://doi.org/10.2147/ott.S226921 Machlowska J, Baj J, Sitarz M, Maciejewski R , Sitarz R (2020) Gastric Cancer: Epidemiology, Risk Factors, Classification, Genomic Characteristics and Treatment Strategies. Int J Mol Sci 21(11). https://doi.org/10.3390/ijms21114012 Morell C, Bort A, Vara-Ciruelos D, Ramos-Torres Á, Altamirano-Dimas M, Díaz-Laviada I , Rodríguez-Henche N (2016) Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells. PLoS One 11(9):e0162977. https://doi.org/10.1371/journal.pone.0162977 Nie H, Mu J, Wang J , Li Y (2018) miR‑195‑5p regulates multi‑drug resistance of gastric cancer cells via targeting ZNF139. Oncol Rep 40(3):1370-1378. https://doi.org/10.3892/or.2018.6524 Niu X, Huang B, Qiao X, Liu J, Chen L , Zhong M (2021) MicroRNA-1-3p Suppresses Malignant Phenotypes of Ameloblastoma Through Down-Regulating Lysosomal Associated Membrane Protein 2-Mediated Autophagy. Front Med (Lausanne) 8:670188. https://doi.org/10.3389/fmed.2021.670188 Pajares M, Rojo AI, Arias E, Díaz-Carretero A, Cuervo AM , Cuadrado A (2018) Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A. Autophagy 14(8):1310-1322. https://doi.org/10.1080/15548627.2018.1474992 Shan C, Chen X, Cai H, Hao X, Li J, Zhang Y, Gao J, Zhou Z, Li X, Liu C, Li P , Wang K (2021) The Emerging Roles of Autophagy-Related MicroRNAs in Cancer. Int J Biol Sci 17(1):134-150. https://doi.org/10.7150/ijbs.50773 Song Z, Wu Y, Yang J, Yang D , Fang X (2017) Progress in the treatment of advanced gastric cancer. Tumour Biol 39(7):1010428317714626. https://doi.org/10.1177/1010428317714626 Tanida I, Ueno T , Kominami E (2004) LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 36(12):2503-18. https://doi.org/10.1016/j.biocel.2004.05.009 Tian S, Guo X, Yu C, Sun C , Jiang J (2017) miR-138-5p suppresses autophagy in pancreatic cancer by targeting SIRT1. Oncotarget 8(7):11071-11082. https://doi.org/10.18632/oncotarget.14360 Wang C, Huang Y, Zhang J , Fang Y (2020) MiRNA-339-5p suppresses the malignant development of gastric cancer via targeting ALKBH1. Exp Mol Pathol 115:104449. https://doi.org/10.1016/j.yexmp.2020.104449 Xu Q, Xu JL, Chen WQ, Xu WX, Song YX, Tang WJ, Xu D, Jiang MP , Tang J (2022) Roles and mechanisms of miR-195-5p in human solid cancers. Biomed Pharmacother 150:112885. https://doi.org/10.1016/j.biopha.2022.112885 Yang WJ, Zhao HP, Yu Y, Wang JH, Guo L, Liu JY, Pu J , Lv J (2023) Updates on global epidemiology, risk and prognostic factors of gastric cancer. World J Gastroenterol 29(16):2452-2468. https://doi.org/10.3748/wjg.v29.i16.2452 Zheng H, Yang Y, Ye C, Li PP, Wang ZG, Xing H, Ren H , Zhou WP (2018) Lamp2 inhibits epithelial-mesenchymal transition by suppressing Snail expression in HCC. Oncotarget 9(54):30240-30252. https://doi.org/10.18632/oncotarget.25367 Zhou C, Liang Y, Zhou L, Yan Y, Liu N, Zhang R, Huang Y, Wang M, Tang Y, Ali DW, Wang Y, Michalak M, Chen XZ , Tang J (2021a) TSPAN1 promotes autophagy flux and mediates cooperation between WNT-CTNNB1 signaling and autophagy via the MIR454-FAM83A-TSPAN1 axis in pancreatic cancer. Autophagy 17(10):3175-3195. https://doi.org/10.1080/15548627.2020.1826689 Zhou M, Wang Y, Zhang C, Qi M, Yao M, Sun L , Xu X (2021b) MicroRNA-195-5p suppresses the proliferation, migration, invasion and epithelial-mesenchymal transition of laryngeal cancer cells in vitro by targeting E2F3. Exp Ther Med 22(4):1078. https://doi.org/10.3892/etm.2021.10512 Zhu H, Chen Z, Yu J, Wu J, Zhuo X, Chen Q, Liang Y, Li G , Wan Y (2022) MiR-195-5p suppresses the proliferation, migration, and invasion of gallbladder cancer cells by targeting FOSL1 and regulating the Wnt/β-catenin pathway. Ann Transl Med 10(16):893. https://doi.org/10.21037/atm-22-3685 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 05 May, 2025 Read the published version in Biochemical Genetics → Version 1 posted Editorial decision: Revision requested 11 Mar, 2025 Editor assigned by journal 11 Mar, 2025 Submission checks completed at journal 11 Mar, 2025 First submitted to journal 09 Mar, 2025 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. 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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-6189299","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427408887,"identity":"158d571f-0dd9-485c-aa81-652bc72291b6","order_by":0,"name":"Chenglong Shen","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Chenglong","middleName":"","lastName":"Shen","suffix":""},{"id":427408891,"identity":"4cd68541-58db-4467-8047-43033842d5dd","order_by":1,"name":"Fang Zhou","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Zhou","suffix":""},{"id":427408893,"identity":"30cfa25f-e57f-4848-ac63-00176c8230eb","order_by":2,"name":"Xiaolei Zhang","email":"","orcid":"","institution":"Meili People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaolei","middleName":"","lastName":"Zhang","suffix":""},{"id":427408896,"identity":"72d0e96a-2522-4db2-b04c-488e557e9255","order_by":3,"name":"Jiazhe Shao","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Jiazhe","middleName":"","lastName":"Shao","suffix":""},{"id":427408898,"identity":"6e518296-e876-41a8-af0a-1d2bb2fee94d","order_by":4,"name":"Yuchao Wang","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yuchao","middleName":"","lastName":"Wang","suffix":""},{"id":427408901,"identity":"2caa103c-9dda-4c5a-9134-9c281f27bf58","order_by":5,"name":"Haichen Liu","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Haichen","middleName":"","lastName":"Liu","suffix":""},{"id":427408903,"identity":"73168cf0-af24-4dec-a2b2-30852938e1ce","order_by":6,"name":"Yichen Li","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yichen","middleName":"","lastName":"Li","suffix":""},{"id":427408906,"identity":"8e9bdbf3-39cc-4f65-8c78-42b74e174aab","order_by":7,"name":"Guoqiang Zhou","email":"","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Guoqiang","middleName":"","lastName":"Zhou","suffix":""},{"id":427408909,"identity":"752fee21-cd27-47fb-8f29-2bf27647ea0e","order_by":8,"name":"Zhiliang Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDCCA1Caj70BTDM2EKEFooiN5wCYQYoWiQQitfAdb37+4OOeOns2ybfHH/Mw2MhuOMD87AE+LZJnjhk2znh2mJlNOi+xmYchzXjDATZzA3xaDG7kMDbzHDjAxiadYwjUcjhxwwEeNgkitNTxsEmeAWn5T7QWZgk2CR6QlgOEtYD8MnPGgcMGbDw5hjPnGCQbzzzMZoZXCzDEHnz4cKDOnp/9jMGHNxV2sn3Hm5/h1YLuTiBmJkH9KBgFo2AUjALsAABNrUelb8JF7AAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Changshu Hospital of Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Zhiliang","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-03-09 14:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6189299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6189299/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10528-025-11120-4","type":"published","date":"2025-05-05T15:57:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78438669,"identity":"b29d6f06-5310-4a77-bb35-609ff84019b6","added_by":"auto","created_at":"2025-03-13 08:31:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":365849,"visible":true,"origin":"","legend":"\u003cp\u003emiR-195-5p was decreased in gastric cancer. (A) qRT-PCR was used to analyzed the relative expression of miR-195-5p in gastric cancer and normal adjacent tissues. (B) TCGA database was used to analyzed the relative expression of miR-195-5p in gastric cancer cell lines. (C) qRT-PCR was used to analyzed the relative expression of miR-195-5p in gastric cancer cell lines. (D) KEGG enrichment analysis was used to find related signaling pathways. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6189299/v1/11f10a38ac77fce7142d6ca2.png"},{"id":78438668,"identity":"519c1cad-eefb-4f15-a909-d0b7a6ba4afc","added_by":"auto","created_at":"2025-03-13 08:31:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1253474,"visible":true,"origin":"","legend":"\u003cp\u003emiR-195-5p overexpression inhibited cell proliferation and induced apoptosis. (A) Overexpression efficiency of miR-195-5p was confirmed by qRT-PCR. (B) Viability analysis at various intervals of miR-195-5p mimics and miR-NC transfection in gastric cancer cells was measured by CCK-8 assay. (C) Colony formation assay of gastric cancer cells transfected with miR-196-5p mimics or miR-NC. (D) Cell apoptotic rate after miR-195-5p overexpression was detected by flow cytometry. ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6189299/v1/32f416dd6bfb88f54275c90d.png"},{"id":78438672,"identity":"20cc4d32-e2f4-40db-9680-cdda584b56f9","added_by":"auto","created_at":"2025-03-13 08:31:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15353883,"visible":true,"origin":"","legend":"\u003cp\u003emiR-195-5p suppresses GC cell metastasis through autophagy. (A) Wound healing assay was used to investigate cell migration ability of gastric cancer cells transfected with miR-195-5p mimics or miR-NC. (B) Transwell assay was used to measured cell invasion ability of gastric cancer cells transfected with miR-195-5p mimics or miR-NC. (C) Immunofluorescence analysis of the LC3-II puncta in gastric cancer cells. Representative images with quantification of LC3 intensity were shown. (D) Western blot was used to analyze the LC3 and p62 levels in different treated cells.* P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6189299/v1/f0d5bc93b3ebc040f5cba7af.png"},{"id":78439943,"identity":"c40dfba8-8845-494f-a65b-0f7cd3103940","added_by":"auto","created_at":"2025-03-13 08:39:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1290348,"visible":true,"origin":"","legend":"\u003cp\u003eLAMP2 is functionally targeted by miR-195-5p in gastric cancer. (A) Screening of genes associated with miR-195-5p by database. (B) The analysis of binding site between miR-195-5p with LAMP2 3’-UTR. (C) The binding relationship between miR-195-5p and LAMP2 3’-UTR was assessed using dual luciferase assay. (D) qRT-PCR analysis of LAMP2 from normal human and gastric cancer. (E) TCGA database was used to analyzed the relative expression of LAMP2 in gastric cancer cell lines. (F) A Pearson correlation analysis was performed on gastric cancer tissues to determine the correlation between miR-195-5p and LAMP2. (G) qRT-PCR analysis of LAMP2 from normal human and gastric cancer cell lines. (H) Western blot analysis of LAMP2 expression in gastric cancer cells transfected with si-NC or si-LAMP2. (I) CCK-8 assay was used to detect cell viability of gastric cancer cells transfected with si-LAMP2 or si-NC at different intervals. (J) CCK-8 assay was used to detect cell viability of gastric cancer cells transfected with miR-NC, miR-NC+oe-NC, miR-195-5p mimics or miR-195-5p mimics+oe-NC or miR-195-5p mimics+oe-LAMP2 at different intervals. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6189299/v1/0cf877db862e5591dd016a19.png"},{"id":78440334,"identity":"db15e668-398a-45a8-a364-6ac947b3b613","added_by":"auto","created_at":"2025-03-13 08:47:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":397328,"visible":true,"origin":"","legend":"\u003cp\u003eModel showing the molecular mechanism of miR-195-5p through LAMP2-mediated autophagy in gastric cancer.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6189299/v1/3f0e9ccaf6eee0af5134560d.png"},{"id":82537543,"identity":"205aa69a-213d-4019-8021-19b6f2fe1235","added_by":"auto","created_at":"2025-05-12 16:08:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21595286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6189299/v1/58a90ef6-7708-4138-a706-f41aedcef855.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOverexpression of miR-195-5p suppresses gastric cancer progression by regulating LAMP2-mediated autophagy\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is recognized as a frequent form of cancer in the digestive system. Baded on the analysis of statistics in 2022, saw an estimated 970,000 new cases of GC and 660,000 fatalities worldwide (Bray et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several factors contribute to the occurrence of GC, including late-night habits, an unbalanced diet, alcohol consumption, and Helicobacter pylori infection (Machlowska et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In recent years, human beings have made remarkable achievements in the fields of chemotherapy, surgical treatment and molecular targeted therapy for GC. With the progress of technology, the incidence rate and mortality of GC have reduced (Song et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, GC often has an insidious onset, which means most patients are diagnosed at advanced stages. This increases their likelihood of recurrence and metastasis after surgery, resulting in a poor prognosis and a 5-year survival rate of around 20% (D\u0026iacute;az Del Arco et al. 2024). Therefore, exploring the mechanisms of GC development and searching for specific markers have become important issues that need to be addressed urgently.\u003c/p\u003e \u003cp\u003eThe miRNAs constitute a category of endogenous, non-coding RNAs, characterized by their high degree of conservation and an average length of around 22 nucleotides. miRNAs act as negative regulators of gene expression through binding to the 3\u0026prime; UTR of messenger RNAs (mRNAs), hindering the translation process or enhancing the degradation of these mRNAs (Bartel \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Research have reported that miRNAs act a vital role in regulating tumorigenesis and development. For instance, miR-339-5p weakens the migration and proliferation of GC cells via ALKBH1 (Wang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Jiang et al. found that miRNA-15a-3p expression was lower in hepatocellular carcinoma tissues. Patients with reduced levels of miRNA-15a-3p had higher rates of metastasis and poorer prognoses, and overexpression of miRNA-15a-3p decreased cancer cell proliferation and metastatic potential (Jiang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). miRNAs also participate in the regulation of autophagy process via mediating target genes (Shan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, miRNAs serve as ideal candidate biomarkers for almost all human diseases, including tumors. miR-195-5p, which originates from the 5p arm of the miR-195 precursor, has been found to be associated to a wide range of cancers in humans, including lung cancer, esophageal cancer, and GC (Xu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nevertheless, the specific mechanism of miR-195-5p in GC still to be elucidated and need to be explored in depth.\u003c/p\u003e \u003cp\u003eIn the cells of eukaryotes, autophagy is a highly conserved process whose main function is the effective degradation and reuse of intracellularly damaged and destroyed organelles and biomolecules (Fang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A multitude of research have reported that differential changes in autophagy levels have been found in several types of tumors, and that changes in autophagy may serve a position in facilitating or hindering tumor cells in tumorigenesis and development. Autophagy plays a crucial role in maintaining tissue homeostasis and physiological functions (Ding et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, autophagy-related interventions are a promising new strategy for tumor therapy. The Lysosome-associated membrane protein 2 (LAMP2) protein, a singular transmembrane constituent of the lysosomal membrane, serves to preserve the integrity of the lysosomal membrane against the action of lysosomal hydrolases and modulates the process of membrane coalescence between lysosomes and other organelles during autophagic activities (Pajares et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). LAMP2 exerts a rate-limiting effect during the lysosomal degradation phase by affecting immunity, macroautophagy (MA) and molecular chaperone-mediated autophagy (CMA) (Eskelinen \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). During neuroendocrine differentiation of prostate cancer cells, LAMP2 is upregulated and autophagic activity is increased (Morell et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, TSTA3 overexpression accelerates lung squamous cell carcinoma cell growth and metastasis through regulating LAMP2-mediated autophagy and the tumor microenvironment (Guo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the mechanism by which LAMP2-mediated autophagy affects GC progression is still imperfect and requires in-depth study.\u003c/p\u003e \u003cp\u003eThe main objective of the present study was to explore the mechanism of aberrantly expressed miR-195-5p in the development of GC. MiR-195-5p was depressed in GC in this research. MiR-195-5p overexpression weakened cell growth and metastasis and facilitated cell apoptosis. Furthermore, this process was discovered to be linked with autophagy by KEGG enrichment analysis. Online database analysis disclosed that miR-195-5p bound to LAMP2. This study reveals that miR-195-5p regulates GC development through LAMP2-mediated autophagy, offering new insights for treating patients with GC.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical tissue sample acquisition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman GC tumor tissues and paracancerous normal tissues were obtained from patients who underwent surgery at our institution. The tumor tissues were rapidly frozen in liquid nitrogen and subsequently maintained at -80\u0026deg;C for preservation. Written informed consent was secured from each patient, the research was sanctioned by the hospital Medical Ethics Committee, with all protocols adhering to the principles outlined in the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGC cell lines (AGS, MKN45, SGC-7901, BGC-823) and human normal gastric mucosal epithelial cell line (GSE-1) were purchased from China Center for Type Culture Collection (Wuhan, China). Cells were cultured in DMEM medium containing 10% (v/v) FBS, 100 U/mL penicillin (Life Technologies), and streptomycin (Life Technologies) at 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eSpecific LAMP2 siRNA and miR-195-5p mimics were designed and synthesized from GenePharma (Shanghai, China). Recombinant overexpression vectors were constructed by inserting LAMP2 sequences into pLCDH-ciR, respectively. The pLCDH-LAMP2 or miR-195-5p mimics were transfected into GC cells using Lipofectamine 3000 (Invitrogen) based on the manufacturer\u0026apos;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated from treated cells and tissues by TRizol reagent. The extracted RNA was reverse transcribed to cDNA via the Prime Script\u0026trade; RT kit (Takara, China). qRT-PCR was performed on a quantitative PCR system(Applied Biosystems 7500, USA). Relative expression was calculated through the 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CCK-8 kit (MilliporeSigma, USA) was used to assess cell proliferation. The density of cells was adjusted to 1x10\u003csup\u003e4\u003c/sup\u003e cells/mL, and 100 \u0026mu;L/well was taken and inoculated in 96-well plates, with 5 replicates set up per well. At 0, 24, 48, and 72 hours into the incubation period, 10 \u0026mu;L of CCK-8 reagent was introduced, respectively, and the incubation was continued for 2 h. Later, the microplate reader was utilized to quantify the absorbance at 450 nm, which corresponds to the optical density (OD).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClone formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePost-transfection, cells were adjusted to a concentration of 500 cells per well and then plated into 6-well plates for incubation at 37\u0026deg;C for a fortnight. After a fixation process with methanol for 30 min, the cells were subjected to staining using a 0.1% crystal violet solution. The subsequent procedures included capturing images and conducting a cell count of the stained cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry was employed to assess cellular apoptosis. Cells were plated in culture dishes and allowed to grow for 24 h before the transfection process. At 48 h post-transfection, the cells were collected, washed with PBS, and then processed for dual staining with the Annexin V FITC/PI Apoptosis Detection Kit procured from Solarbio.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWound healing assay was used to detect the migration ability of cells. Marker pen was used to draw a line on the back side of the 6-well plate, and BGC-823 and SGC-7901 cells with 80% density were digested with trypsin, resuspended in complete medium, counted on cell counting plate, and then inoculated into the 6-well plate according to the amount of 3x10\u003csup\u003e5\u003c/sup\u003e cells in each well, and cultured at constant temperature for 24 h. The liquid was changed regularly, when the confluence of cells reaches 100%, insert a pipette gun with a 100 \u0026mu;L tip perpendicular to the bottom of the plate and make a scratches at equal distances in each well, aspirate and discard the medium, wash three times by carefully adding 1 mL PBS along the side wall, and add 2% FBS to the complete medium and put it in the incubator for incubation. The healing status of the same scratch at 0 h and 48 h was photographed under a microscope, and the migration area of each time period was calculated and analyzed in Image J software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular matrix gel was uniformly spread across the upper face of the polycarbonate membranes in the Transwell inserts. After a preliminary cultured with serum-free medium for 24 h, the cellular concentration was adjusted to 5x10\u003csup\u003e5\u003c/sup\u003e/ml. 100 \u0026mu;L this suspension was introduced into the upper chamber of the chambers, 600 \u0026mu;L of serum-containing medium was added to the lower chamber, and the chambers were placed in a cell culture incubator for 48 h. Then, the chambers were removed. The cells on the lower surface of the upper chamber were carefully wiped off with a cotton swab, fixed with methanol and stained with crystal violet. The number of cells was observed under a microscope with a 400x field of view, and 5 fields of view were photographed and calculated, and the average value was taken.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe downstream target mRNAs of miRNAs were predicted based on the online software. Luciferase reporter vectors including LAMP2-WT, LAMP2-MUT. The SGC-7901, BGC-823 cells were inoculated into 96-well plates and cultured for 24 h until a confluence of 50%-70%. The cells were then co-transfected with the indicated reporter vectors and miR-195-5p mimics or miR-NC via Lipofectamine 3000. 48 h later, luciferase activity was measured using the Dual Luciferase Reporter Assay System.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot (WB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted using RIPA lysate (Solarbio, China). Protein concentration was measured by BCA kit (Solarbio, China). Then, proteins were separated by 10% SDS/PAGE, and proteins were transferred to nitrocellulose membranes (Biosharp, China). The membranes were sealed in 5% skimmed milk powder and incubated with primary antibody at 4\u0026deg;C overnight. After that, the membrane was incubated with secondary antibody for 60 min. At last, protein bands were visualized via an ECL kit (Beyotime Biotech, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were attached and grown on slides. Cell fixation was performed using a 4% solution of paraformaldehyde for 30 min. Then, cells were subjected to permeabilization using a 3% BSA solution for an equivalent duration. Cells were incubated with LC3B primary antibody overnight in a humidified chamber. After labeling with primary antibody overnight, cells were incubated with fluorescent secondary antibody (Alexa Fluor 488, USA) for 1 h. They were then stained with DAPI (Thermo Fisher Scientific, USA). Slides were evaluated on a microscope. Analysis was performed with Image J software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical data were analyzed and expressed as mean \u0026plusmn; standard deviation using GraphPad Prism 8.0 software. Each experiment was repeated 3 times and the average value. Differences between two or more groups were assessed by Student\u0026apos;s t-test or one-way ANOVA. p\u0026lt;0.05 was considered significantly different.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003emiR-195-5p is reduced in GC\u003c/h2\u003e \u003cp\u003eTo understand the role of miR-195-5p in GC, we examined miR-195-5p expression in GC tissues. miR-195-5p was identified to be clearly diminished in GC tissues contrasted to normal tissues adjacent to the cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). According to the TCGA database, miR-195-5p was also found to be downregulated in GC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, we examined the miR-195-5p level in GC cells. Compared to GSE-1, miR-195-5p was declined in AGS, MKN45, SGC-7901, and BGC-823 cells. Among them, miR-195-5p had the lowest expression in SGC-7901 and BGC-823 cells, so this two cells were used for subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The above findings indicated miR-195-5p might be a novel target for GC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003emiR-195-5p overexpression weakens cell proliferation and induces apoptosis\u003c/h2\u003e \u003cp\u003eMiR-195-5p overexpression vector was constructed to explore the function of miR-195-5p in GC. The outcome displayed miR-195-5p was obviously enhanced in cells after of miR-195-5p overexpression compared with control, indicating that miR-195-5p was successfully overexpression in BGC-823 and SGC-7901 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, cells growth, colony formation and apoptotic ability were performed. The findings illustrated that miR-195-5p introduction evidently suppressed the growth and proliferation ability of cells, and markedly heightened the apoptotic ability of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). The above results indicated that miR-195-5p introduction induced apoptosis and restrained cell proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of miR-195-5p suppresses GC cell metastasis through autophagy\u003c/h2\u003e \u003cp\u003eNext, we investigated the impact of miR-195-5p overexpression cell migration and invasion ability. Wound healing assay was applied to determine the changes in cell migration ability. It was observed that the migration ability of SGC-7901 and BGC-823 cells was reduced after miR-195-5p introduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The effect of overexpression of miR-195-5p on cell invasion ability was measured via transwell assay. Introduction of miR-195-5p was discovered to clearly decline the invasion ability of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To elucidate the mechanism by which miR-195-5p affects GC progression, miR-195-5p differentially expressed genes were found to be enriched in autophagy-related pathways by KEGG enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Subsequently, LC3 aggregation during autophagosome formation was identified by immunofluorescence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). LC3 II/I was obviously depressed after overexpression of miR-195-5p, while p62 level was raised, demonstrating that miR-195-5p overexpression repressed cellular autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The above outcomes indicated that introduction of miR-195-5p restrained the metastasis of GC cells in vitro through autophagy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003emiR-195-5p acts as a functional regulator of LAMP2\u003c/h2\u003e \u003cp\u003eTo investigate the mechanism by which miR-195-5p affects GC progression, downstream targets of miR-195-5p were screened using autophagy related genes in TCGA database and miRWalk, mirDIP, starBase databases. It was discovered that there were two overlapping genes, LAMP2 and HSPA8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Moreover, miR-195-5p was discovered to have a binding site with LAMP2 3'-UTR through the database. Therefore, we hypothesized that LAMP2 is a downstream target of miR-195-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Next, we assessed the binding relationship between miR-195-5p and the 3'-UTR of LAMP2 using a dual luciferase assay. The outcomes declared that the luciferase activity of LAMP2-WT was considerably reduced in cells transfected with miR-195-5p mimics, whereas the luciferase activity of LAMP2-MUT did not change evidently, indicating a binding relationship between miR-195-5p and LAMP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Subsequently, we examined the level of LAMP2 in GC tissues. LAMP2 was heightened in GC tissues contrasted to normal tissues adjacent to the cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). TCGA database findings also showed high expression of LAMP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Pearson correlation analysis was applied on GC tissues to measure the association between miR-195-5p and LAMP2. A significant inverse association was disclosed between miR-195-5p and LAMP2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Moreover, LAMP2 level in cell lines was detected via qRT-PCR, and LAMP2 was raised in GC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Next, to investigate the function of LAMP2 in GC, LAMP2 expression was knockdown in the cells, and the knockdown efficiency of LAMP2 was tested via WB. The findings displayed that LAMP2 silencing clearly diminished LAMP2 protein expression in GC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). CCK-8 results discovered that knockdown of LAMP2 restrained the viability of GC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Furthermore, we found that LAMP2 introduction returned the repression impact of miR-195-5p overexpression on cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). The above outcomes confirmed that overexpression of miR-195-5p weakened the GC development through LAMP2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the above findings, we conclude that overexpression of miR-195-5p attenuated the malignant progression of GC cells through LAMP2 mediated autophagy. Moreover, according to these outcomes, we have drawn a hypothesis diagram of the mechanism by which miR-195-5p affects the progression of GC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of GC is closely related to a variety of elements, with its etiology being multifaceted. It is subject to a complex interplay of various regulatory systems that influence and modulate its development. The propensity for GC to recur and spread to other parts of the body is a primary obstacle in achieving a cure for the disease (Yang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, elucidating the molecular underpinnings of GC metastasis process is crucial for developing precision treatments for patients with advanced gastric cancer. Here, our study analyzed the impact of miR-195-5p dysregulation in GC on the malignant biology of GC cells. Moreover, our study delineates the regulatory interplay between miR-195-5p and LAMP2 in GC, demonstrating that overexpression of miR-195-5p suppresses the malignant phenotype of GC cells through LAMP2-mediated autophagy.\u003c/p\u003e \u003cp\u003eExisting literature underscores the pivotal role of miRNAs in the etiology and progression of gastric malignancies. The miR-195-5p, belonging to the miR-15 family, is mapped to the 17th chromosome (Nie et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). miR-195-5p exerts tumor-suppressive activity in a spectrum of cancers such as colorectal, lung adenocarcinoma, gallbladder, and cervical cancers (Liu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By targeting FOSL1, miR-195-5p effectively downregulates the Wnt/β-catenin pathway, thereby inhibiting the uncontrolled proliferation and invasive behavior characteristic of gallbladder cancer cells (Zhu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In non-small cell lung cancer, miR-195-5p overexpression restrains cancer cell growth and induces apoptosis via targeting CEP55 (Luo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, insertion of miR-195-5p attenuated the growth, metastasis and EMT of laryngeal cancer cells through E2F3 (Zhou et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). The present investigation displayed a marked diminish in miR-195-5p expression within gastric cancerous tissues and cells compared to their non-cancerous counterparts, echoing a similar pattern of downregulation noted across a spectrum of malignancies. The upregulation of miR-195-5p exerts a suppressive effect on cell growth and metastatic potential, concurrently facilitating apoptotic activity. This underscores the miRNA function as a tumor suppressor, demonstrating its efficacy in controlling the proliferative and invasive behaviors of GC cells.\u003c/p\u003e \u003cp\u003eAutophagy exerts a complex and context-dependent part in different types of tumors. The process of autophagy exhibits a biphasic influence on oncogenesis, initially acting to restrain tumor development in the early stages. Conversely, in advanced stages of the disease, it may foster the neoplastic process, thereby accelerating tumorigenesis (Debnath et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A substantial array of miRNAs has been demonstrated to play a role in the modulation of autophagic processes (Li et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, miR-519a enhances apoptosis and sensitizes glioblastoma to temozolomide treatment via facilitating autophagy and targeting STAT3/Bcl-2/Beclin-1 axis (Li et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Research has demonstrated that miR-138-5p weakened autophagy and blocked serum starvation-induced autophagic fluxes in pancreatic cancer cells, and further studies revealed that miR-138-5p hampered autophagy in these cells via SIRT1 (Tian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, the autophagic flux is typified by fluctuations in specific marker proteins. The p62 protein, integral to cellular homeostatic mechanisms through its involvement in autophagy modulation and intracellular signaling (Zhou et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). In parallel, the LC3 protein complex stands as a quintessential biomarker of autophagic activity. The abundance of LC3II is positively correlated with the degree of autophagy occurring within the cell (Tanida et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Our research demonstrated that increased expression of miR-195-5p led to a decrease in the LC3 II/I ratio and an increase in p62 levels, indicating that autophagy was suppressed in the cells. This reveals miR-195-5p introduction restrained cell growth and metastasis through mediating autophagy.\u003c/p\u003e \u003cp\u003eDespite identification of various candidate targets for miR-195-5p, the intricate molecular mechanisms mediating its impact on GC development are not yet comprehensively understood. In this research, we screened LAMP2 as a target gene of miR-195-5p by bioinformatics analysis. LAMP2 has three splice isoforms: LAMP2A, LAMP2B, and LAMP2C. Emerging evidence highlighting the pivotal role of lysosomes in multiple cancer types has intensified the scientific community interest in exploring the function of LAMP2 in the progression of cancer (Zheng et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). There is evidence that enhanced LAMP2 levels are linked to tumor progression. Huang et al. found that lncRNA FAM215A stimulates LAMP2 expression to confer drug-resistant and progression in HCC (Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another study discovered miR-1-3p attenuated the malignant phenotype of enamel cell tumors via downregulating LAMP2-mediated autophagy (Niu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, LAMP2 was markedly elevated in GC and inversely related to the expression of miR-195-5p. Knockdown of LAMP2 weakened cell growth. Additionally, overexpression of LAMP2 reversed the suppressive effect of miR-195-5p introduction on cell growth. The above findings suggested miR-195-5p affects the malignant progression of GC through LAMP2-mediated autophagy. Although this study offers fresh perspectives on the role of miR-195-5p in GC, it also has certain inherent limitations. First, this study was mainly conducted in an in vitro cellular model, and the relevance between miR-195-5p and LAMP2 needs to be validated in more clinical samples in the future. Furthermore, the upstream regulatory mechanisms and downstream impact of miR-195-5p in GC need to be further investigated.\u003c/p\u003e \u003cp\u003eIn summary, miR-195-5p affects the advancement of GC through the LAMP2-mediated autophagy process. This finding facilitates our understanding of the molecular mechanisms of GC, also provides novel potential targets for the diagnosis and treatment of GC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe thank the donors whose names were not included in the author list, but who participated in this program.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e Chenlong Shen, Xiaolei Zhang and Fang Zhou analyzed data and wrote the paper. Yichen Li and Yuchao Wang performed experiments. Jiazhe Shao and Haichen Liu guided the experiments and the analysis. Guoqiang Zhou and Zhiliang Shi initiated the study, designed experiments. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eChangshu Key Laboratory of Digestive System Tumor Innovation and Diagnosis and Treatment (CS202313), Suzhou City clinical key disease diagnosis and treatment technology special project (LCZX202224), Suzhou Medical and Health science and technology Innovation Project (SKY2022022)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe data are available from the corresponding author on reasonable request. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors have no conflict of interest, financial or otherwise.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eApproval of Changshu NO.2 People\u0026apos;s Hospital Ethics Committee. All participants were provided with written informed consent at the time of recruitment, and all experiments involving human tissue specimens comply with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215-33. https://doi.org/10.1016/j.cell.2009.01.002\u003c/li\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I , Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229-263. https://doi.org/10.3322/caac.21834\u003c/li\u003e\n\u003cli\u003eDebnath J, Gammoh N , Ryan KM (2023) Autophagy and autophagy-related pathways in cancer. Nat Rev Mol Cell Biol 24(8):560-575. https://doi.org/10.1038/s41580-023-00585-z\u003c/li\u003e\n\u003cli\u003eD\u0026iacute;az Del Arco C, Fern\u0026aacute;ndez Ace\u0026ntilde;ero MJ , Ortega Medina L (2024) Molecular Classifications in Gastric Cancer: A Call for Interdisciplinary Collaboration. Int J Mol Sci 25(5). https://doi.org/10.3390/ijms25052649\u003c/li\u003e\n\u003cli\u003eDing Y, Huang X, Ji T, Qi C, Gao X , Wei R (2024) The emerging roles of miRNA-mediated autophagy in ovarian cancer. Cell Death Dis 15(5):314. https://doi.org/10.1038/s41419-024-06677-8\u003c/li\u003e\n\u003cli\u003eEskelinen EL (2006) Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol Aspects Med 27(5-6):495-502. https://doi.org/10.1016/j.mam.2006.08.005\u003c/li\u003e\n\u003cli\u003eFang W, Shu S, Yongmei L, Endong Z, Lirong Y , Bei S (2016) miR-224-3p inhibits autophagy in cervical cancer cells by targeting FIP200. Sci Rep 6:33229. https://doi.org/10.1038/srep33229\u003c/li\u003e\n\u003cli\u003eGuo Y, Hao Y, Shen L, Du Y, Wang X, Gao L, Feng X, Zhai Y, Liu Z, Xu E, Yang Y, Xi Y, Yang B , Zhang L (2023) TSTA3 overexpression promotes malignant characteristics in LUSC by regulating LAMP2-mediated autophagy and tumor microenvironment. Cancer Cell Int 23(1):285. https://doi.org/10.1186/s12935-023-03109-z\u003c/li\u003e\n\u003cli\u003eHuang PS, Lin YH, Chi HC, Tseng YH, Chen CY, Lin TK, Yeh CT , Lin KH (2020) Dysregulated FAM215A Stimulates LAMP2 Expression to Confer Drug-Resistant and Malignant in Human Liver Cancer. Cells 9(4). https://doi.org/10.3390/cells9040961\u003c/li\u003e\n\u003cli\u003eJiang C, He ZL, Hu XH , Ma PY (2020) MiRNA-15a-3p inhibits the metastasis of hepatocellular carcinoma by interacting with HMOX1. Eur Rev Med Pharmacol Sci 24(24):12694-12700. https://doi.org/10.26355/eurrev_202012_24167\u003c/li\u003e\n\u003cli\u003eLi H, Chen L, Li JJ, Zhou Q, Huang A, Liu WW, Wang K, Gao L, Qi ST , Lu YT (2018) miR-519a enhances chemosensitivity and promotes autophagy in glioblastoma by targeting STAT3/Bcl2 signaling pathway. J Hematol Oncol 11(1):70. https://doi.org/10.1186/s13045-018-0618-0\u003c/li\u003e\n\u003cli\u003eLi X, He S , Ma B (2020) Autophagy and autophagy-related proteins in cancer. Mol Cancer 19(1):12. https://doi.org/10.1186/s12943-020-1138-4\u003c/li\u003e\n\u003cli\u003eLiu X, Liu Z, Liu Y , Wang N (2023) ATG9A modulated by miR-195-5p can boost the malignant progression of cervical cancer cells. Epigenetics 18(1):2257538. https://doi.org/10.1080/15592294.2023.2257538\u003c/li\u003e\n\u003cli\u003eLuo J, Pan J, Jin Y, Li M , Chen M (2019) MiR-195-5p Inhibits Proliferation and Induces Apoptosis of Non-Small Cell Lung Cancer Cells by Targeting CEP55. Onco Targets Ther 12:11465-11474. https://doi.org/10.2147/ott.S226921\u003c/li\u003e\n\u003cli\u003eMachlowska J, Baj J, Sitarz M, Maciejewski R , Sitarz R (2020) Gastric Cancer: Epidemiology, Risk Factors, Classification, Genomic Characteristics and Treatment Strategies. Int J Mol Sci 21(11). https://doi.org/10.3390/ijms21114012\u003c/li\u003e\n\u003cli\u003eMorell C, Bort A, Vara-Ciruelos D, Ramos-Torres \u0026Aacute;, Altamirano-Dimas M, D\u0026iacute;az-Laviada I , Rodr\u0026iacute;guez-Henche N (2016) Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells. PLoS One 11(9):e0162977. https://doi.org/10.1371/journal.pone.0162977\u003c/li\u003e\n\u003cli\u003eNie H, Mu J, Wang J , Li Y (2018) miR‑195‑5p regulates multi‑drug resistance of gastric cancer cells via targeting ZNF139. Oncol Rep 40(3):1370-1378. https://doi.org/10.3892/or.2018.6524\u003c/li\u003e\n\u003cli\u003eNiu X, Huang B, Qiao X, Liu J, Chen L , Zhong M (2021) MicroRNA-1-3p Suppresses Malignant Phenotypes of Ameloblastoma Through Down-Regulating Lysosomal Associated Membrane Protein 2-Mediated Autophagy. Front Med (Lausanne) 8:670188. https://doi.org/10.3389/fmed.2021.670188\u003c/li\u003e\n\u003cli\u003ePajares M, Rojo AI, Arias E, D\u0026iacute;az-Carretero A, Cuervo AM , Cuadrado A (2018) Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A. Autophagy 14(8):1310-1322. https://doi.org/10.1080/15548627.2018.1474992\u003c/li\u003e\n\u003cli\u003eShan C, Chen X, Cai H, Hao X, Li J, Zhang Y, Gao J, Zhou Z, Li X, Liu C, Li P , Wang K (2021) The Emerging Roles of Autophagy-Related MicroRNAs in Cancer. Int J Biol Sci 17(1):134-150. https://doi.org/10.7150/ijbs.50773\u003c/li\u003e\n\u003cli\u003eSong Z, Wu Y, Yang J, Yang D , Fang X (2017) Progress in the treatment of advanced gastric cancer. Tumour Biol 39(7):1010428317714626. https://doi.org/10.1177/1010428317714626\u003c/li\u003e\n\u003cli\u003eTanida I, Ueno T , Kominami E (2004) LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 36(12):2503-18. https://doi.org/10.1016/j.biocel.2004.05.009\u003c/li\u003e\n\u003cli\u003eTian S, Guo X, Yu C, Sun C , Jiang J (2017) miR-138-5p suppresses autophagy in pancreatic cancer by targeting SIRT1. Oncotarget 8(7):11071-11082. https://doi.org/10.18632/oncotarget.14360\u003c/li\u003e\n\u003cli\u003eWang C, Huang Y, Zhang J , Fang Y (2020) MiRNA-339-5p suppresses the malignant development of gastric cancer via targeting ALKBH1. Exp Mol Pathol 115:104449. https://doi.org/10.1016/j.yexmp.2020.104449\u003c/li\u003e\n\u003cli\u003eXu Q, Xu JL, Chen WQ, Xu WX, Song YX, Tang WJ, Xu D, Jiang MP , Tang J (2022) Roles and mechanisms of miR-195-5p in human solid cancers. Biomed Pharmacother 150:112885. https://doi.org/10.1016/j.biopha.2022.112885\u003c/li\u003e\n\u003cli\u003eYang WJ, Zhao HP, Yu Y, Wang JH, Guo L, Liu JY, Pu J , Lv J (2023) Updates on global epidemiology, risk and prognostic factors of gastric cancer. World J Gastroenterol 29(16):2452-2468. https://doi.org/10.3748/wjg.v29.i16.2452\u003c/li\u003e\n\u003cli\u003eZheng H, Yang Y, Ye C, Li PP, Wang ZG, Xing H, Ren H , Zhou WP (2018) Lamp2 inhibits epithelial-mesenchymal transition by suppressing Snail expression in HCC. Oncotarget 9(54):30240-30252. https://doi.org/10.18632/oncotarget.25367\u003c/li\u003e\n\u003cli\u003eZhou C, Liang Y, Zhou L, Yan Y, Liu N, Zhang R, Huang Y, Wang M, Tang Y, Ali DW, Wang Y, Michalak M, Chen XZ , Tang J (2021a) TSPAN1 promotes autophagy flux and mediates cooperation between WNT-CTNNB1 signaling and autophagy via the MIR454-FAM83A-TSPAN1 axis in pancreatic cancer. Autophagy 17(10):3175-3195. https://doi.org/10.1080/15548627.2020.1826689\u003c/li\u003e\n\u003cli\u003eZhou M, Wang Y, Zhang C, Qi M, Yao M, Sun L , Xu X (2021b) MicroRNA-195-5p suppresses the proliferation, migration, invasion and epithelial-mesenchymal transition of laryngeal cancer cells in vitro by targeting E2F3. Exp Ther Med 22(4):1078. https://doi.org/10.3892/etm.2021.10512\u003c/li\u003e\n\u003cli\u003eZhu H, Chen Z, Yu J, Wu J, Zhuo X, Chen Q, Liang Y, Li G , Wan Y (2022) MiR-195-5p suppresses the proliferation, migration, and invasion of gallbladder cancer cells by targeting FOSL1 and regulating the Wnt/\u0026beta;-catenin pathway. Ann Transl Med 10(16):893. https://doi.org/10.21037/atm-22-3685\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biochemical-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bigi","sideBox":"Learn more about [Biochemical Genetics](http://link.springer.com/journal/10528)","snPcode":"10528","submissionUrl":"https://submission.nature.com/new-submission/10528/3","title":"Biochemical Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gastric cancer, miR-195-5p, LAMP2, autophagy, malignant progression","lastPublishedDoi":"10.21203/rs.3.rs-6189299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6189299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMiR-195-5p has been confirmed to be intimately linked to many cancers development, whereas its role in gastric cancer (GC) remains controversial. The objective of this study was to explore the potential function and mechanism of miR-195-5p in GC. First, the miR-195-5p level in GC was analyzed based on qRT-PCR and TCGA STAD. Then, the function of miR-195-5p in GC cells was detected via CCK-8, clone formation assay, flow cytometry, transwell, and wound healing assay. Subsequently, we explored the possible mechanism of miR-195-5p using KEGG analysis, database analysis, Western blot, and rescue assays. In this research, miR-195-5p was declined in GC and associated to malignant progression of GC. The introduction of miR-195-5p weakened cell growth, metastasis, and facilitated cell apoptosis. KEGG enrichment analysis indicated that miR-195-5p influenced the malignant progression of GC through mechanisms related to autophagy. MiR-195-5p overexpression leads to a diminish in cellular autophagy. Further studies identified that miR-195-5p bound to LAMP2. Mechanism studies confirmed that miR-195-5p introduction attenuated cell growth, and this process was rescued by LAMP2 overexpression. MiR-195-5p affects the malignant progression of GC by regulating LAMP2-mediated autophagy.\u003c/p\u003e","manuscriptTitle":"Overexpression of miR-195-5p suppresses gastric cancer progression by regulating LAMP2-mediated autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 08:30:56","doi":"10.21203/rs.3.rs-6189299/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-11T18:04:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-11T11:18:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-11T11:15:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biochemical Genetics","date":"2025-03-09T14:40:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biochemical-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bigi","sideBox":"Learn more about [Biochemical Genetics](http://link.springer.com/journal/10528)","snPcode":"10528","submissionUrl":"https://submission.nature.com/new-submission/10528/3","title":"Biochemical Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e21f5f07-02e4-41b3-86b9-1a4523498ff4","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:02:39+00:00","versionOfRecord":{"articleIdentity":"rs-6189299","link":"https://doi.org/10.1007/s10528-025-11120-4","journal":{"identity":"biochemical-genetics","isVorOnly":false,"title":"Biochemical Genetics"},"publishedOn":"2025-05-05 15:57:41","publishedOnDateReadable":"May 5th, 2025"},"versionCreatedAt":"2025-03-13 08:30:56","video":"","vorDoi":"10.1007/s10528-025-11120-4","vorDoiUrl":"https://doi.org/10.1007/s10528-025-11120-4","workflowStages":[]},"version":"v1","identity":"rs-6189299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6189299","identity":"rs-6189299","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}