Mechanism of action of miR-330-5p targeting ITGA5 in the regulation of proliferation, migration, and invasion of gastric cancer

Mechanism of action of miR-330-5p targeting ITGA5 in the regulation of proliferation, migration, and invasion 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 Mechanism of action of miR-330-5p targeting ITGA5 in the regulation of proliferation, migration, and invasion of gastric cancer Jun-fu Wang, Jian-ming Wei, Ting He, Jun-wen Hu, Jiang-nan Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4747650/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2026 Read the published version in BMC Cancer → Version 1 posted 4 You are reading this latest preprint version Abstract BACKGROUND: ITGA5 is an oncogene that performs its biological function by integrating the intracellular structure and extracellular matrix. Our research group found that ITGA5 is a gastric cancer-related gene highly expressed in this tumor and is closely related to its proliferation and metastasis. The ITGA5 gene is regulated by multiple miRNAs during the occurrence and development of tumors. This study aimed to explore the role of targeting miRNAs upstream of ITGA5 in the regulation of the proliferation, invasion, and migration of gastric cancer cells. METHODS: The target miRNA molecules regulating the ITGA5 gene were predicted by four bioinformatics databases (TargetScan、miRDB、miRTarBase and mirDIP), and the unreported miRNAs with high correlation were selected and their expression in gastric cancer was assessed by qRT-PCR and western blot. The miRNAs with potential targeting abilities were further verified by dual luciferase reporter gene experiment. The effects of miR-330-5p and ITGA5 on the proliferation, invasion, and migration of gastric cancer cells were evaluated by CCK8, clonogenic assay, and Transwell chamber assay, respectively. RESULTS: Six miRNAs (miR-26a-5p、miR-92a-3p、miR-148a-3p、miR-148b-3p、miR-330-5p and miR-152-3p) with high stability and conservation were found, and miR-330-5p was the one targeting and regulating ITGA5. In vitro experiments demonstrated that miR-330-5p mimic significantly inhibited the proliferation, invasion, and migration of gastric cancer cells compared with the control group ( p < 0.05). The transfection of miR-330-5p mimic into gastric cancer cells overexpressing ITGA5 (OE-ITGA5) resulted in a significant reversion of the promoting effect of OE-ITGA5 on the proliferation, invasion, and migration of gastric cancer cells ( P < 0.05). In addition, miR-330-5p mimic reduced ITGA5 expression in gastric cancer cells and partially reversed the FAK/AKT signaling pathway activated by the ITGA5 gene. miR-330-5p inhibitor increased ITGA5 expression in gastric cancer cells, and they partially reversed the FAK/AKT signaling pathway blocked by sh-ITGA5. CONCLUSIONS: ITGA5 was promotive for GC tumor growth and cell biological behaviors, and miR-330-5p targeted 3'-UTR of ITGA5 and inhibited its expression. ITGA5 was expected to become a new molecular marker, with miR-330-5p representing a novel therapeutic target for GC. This discovery provides a theoretical basis to further understand the mechanism related to the occurrence and development of gastric cancer, improving the diagnosis and prognosis while discovering a new therapeutic target. miR-330-5p ITGA5 gastric cancer tumor progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Gastric cancer is one of the most common malignancies, diagnosed in more than 1 million people worldwide every year, with an incidence and mortality ranking third [1]. Although its incidence is decreasing worldwide, it is still high in East Asian countries. According to the latest cancer data statistics in 2020, the annual incidence of gastric cancer in China is approximately 479000 cases, and the number of annual deaths is approximately 374000 [1]. The risk factors associated with gastric cancer include Helicobacter pylori infection, environmental factors, low intake of fruits and vegetables, smoking, and alcohol consumption [2,3]. Surgery and chemotherapy are still the main treatment for gastric cancer. Although the early diagnosis and treatment of gastric cancer are improved, the prognosis of gastric cancer is still poor due to lymphatic metastasis and invasion [4, 5]. Therefore, it is of the utmost importance to discover new molecular mechanism involved in the occurrence and development of gastric cancer to ultimately contribute to the discovery of new tumor markers and take effective measures for early diagnosis and targeted treatment of gastric cancer metastasis to improve the survival rate and quality of life of patients. In other words, it is necessary to provide a theoretical basis for the early clinical diagnosis, the occurrence and development mechanism, and consequent targeted therapy of gastric cancer. MicroRNAs are a class of endogenous non-coding microRNAs of 18-24 nucleotides. They are a single stranded small molecule RNAs that bind to the 3 'untranslated region (3' UTR) of the target mRNA through base complementary pairing, thereby inhibiting the transcription or degradation of the target gene [6]. miRNAs are involved in the biological process of a variety of tumors, such as tumor cell proliferation, apoptosis, angiogenesis, invasion, and migration [7, 8]. miRNAs are the main regulators of gene expression and are involved in cancer metastasis. The abnormal expression of miRNAs in tumors causes changes in the biological functions of tumor cells. Some miRNAs play an oncogenic role and some a tumor suppressive role [9, 10]. Some aberrantly expressed miRNAs directly affect the occurrence and development of tumors by targeting genes or signaling pathways related to tumor progression [11]. More and more studies confirmed that miRNAs may become potential biomarkers for the diagnosis, treatment, and prognosis of tumor patients [12]. miR-330-5p is closely related to the occurrence and development of a variety of tumors [13, 14]. Previous studies of our research group found that ITGA5 expression is up-regulated in gastric cancer cell lines and tissues, and its expression is closely related to tumor size, lymph node metastasis, and TNM stage. ITGA5 induces proliferation, invasion, and migration of gastric cancer cells by activating the FAK/AKT signaling pathway [15]. ITGA5 is a member of the integrin family, with a molecular weight of approximately 130 kd. More and more reports on the role of ITGA5 in tumors are available, and the regulation of its expression in tumors has attracted extensive research. ITGA5 is a fibronectin receptor working as a proto-oncogene affecting the intracellular and extracellular signal transduction and is closely related to the occurrence and development of a variety of tumors. Other scholars found that ITGA5 is regulated by miRNAs, affecting its expression in tumor cells, thereby causing functional changes. Hara et al. [16] found that ITGA5 mRNA is a direct target of mir-92a. Transfection of mir-92a mimic reduces the expression of ITGA5 in ovarian cancer cells and inhibits their adhesion, invasion, and proliferation. Gong et al. [17] found that miR-17 directly binds ITGA5 and ITGβ1 in the 3' untranslated region (3' UTR) and inhibits their expression, thereby inhibiting the peritoneal seeding of ovarian cancer cells. Xue et al. [13] found that ITGA5 is the target of mir-30b-5p, thus inhibiting the invasion and migration of esophageal cancer cells. Yoo et al. [14] found that miR-330-5p affects the development of colorectal cancer by regulating the expression of ITGA5. Similarly, Feng et al. [18] found that miR-330-5p targets and inhibits the expression of ITGA5 gene in glioma cells, thereby inhibiting their proliferation, invasion, and migration and promoting their apoptosis. Despite all these pieces of evidence, the relationship between miR-330-5p and ITGA5 in gastric cancer is still unclear, without demonstration that targeting ITGA5 results in the regulation of gastric cancer cell proliferation, invasion, and migration. In this study, the miRNAs regulating the target gene ITGA5 were predicted by bioinformatics, finally revealing that miR-330-5p was negatively correlated with ITGA5 expression. The targeting relationship between miR-330-5p and ITGA5 gene was confirmed by dual luciferase reporter experiment. Moreover, miR-330-5p affected the proliferation, invasion, and migration of gastric cancer cells by mediating ITGA5 and affecting the regulation of FAK/AKT signaling pathway. Hence, this study provides a theoretical basis to further understand the mechanism related to the occurrence and development of gastric cancer, improving the diagnosis and prognosis, and discovering a new therapeutic target. 2. Materials and methods 2.1 Bioinformatics database data acquisition Four online websites, TargetScan (http://www.targetscan.org/vert_72/), miRDB (http://mirdb.org/cgi-bin/search.cgi), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/search.php) and mirDIP (http://ophid.utoronto.ca/mirDIP/index.jsp#r) were used to predict the miRNAs that target and regulate the human ITGA5 gene, and the parameters such as target score and PCT were analyzed to discover miRNAs with high correlation scores and intersection in the four databases. 2.2 Cell culture Human gastric cancer cell lines (AGS, MKN28, and HGC27) and normal gastric mucosal epithelial cells GES-1 were purchased from the cell bank of the Chinese Academy of Sciences and stored in liquid nitrogen. AGS and MKN28 cells stably overexpressing ITGA5 (OE-ITGA5) and with silenced ITGA5 (sh-ITGA5), as well as the negative control, were successfully constructed and stored in liquid nitrogen. Cells were incubated at 37 ℃ in a humidified environment with 5% CO 2 . The medium group consisted of RPMI-1640 medium with 10% fetal bovine serum (GIBCO, USA) and 1% double antibody (Dalian Meilun biological Co., Ltd., China). Fresh medium was changed every other day. 2.3 Cell transfection The cell state and activity were optimized, and they were transfected when the confluence reached 60-70%. miRNA mimic, inhibitor, or their negative controls (Genechem, Shanghai, China) were transiently transfected using lipofectamine3000 (Invitrogen, Carlsbad, CA, USA) for further experiments, according to the above protocol. 2.4 Real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted and isolated from tissues and cells using Trizol Kit (Macherey Nagel, Germany), and the concentration and purity of RNA were detected using nanodrop ND-2000 spectrophotometer. RNA and miRNA were reverse transcribed using a reverse transcription kit (Takara Biotechnology Co, Ltd.). The expression of mRNAs and miRNAs was detected using SYBR Green I fluorescence detection kit (Takara, China), and ABI 7500 real time PCR detection system, with GAPDH and U6 as internal controls, ITGA5, miR-26a-5p, miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-330-5p and miR-152-3p were detected. Finally, their expression was calculated using the 2 − ∆∆ CT method. Primers were purchased from Shanghai Bioengineering Technology Co., Ltd., and the sequences were the following:Table 1 and Table 2. 2.5. Western blotting The RIPA lysis buffer (Thermo Fisher Scientific) containing the protease inhibitor PMSF was used to lyse the cells and extract the protein. Cells were lysed on ice for 30 min, centrifuged at 11000 × g at 4 ℃, and total protein concentration in the supernatant was detected by the protein concentration BCA (Thermo Fisher Scientific) method. The loading buffer (Beijing solabo science and Technology Co., Ltd.) was added, and the proteins were heated at 100 ℃ for 10 min. Total proteins were separated by 10% polyacrylamide gel electrophoresis, transferred to the membrane, blocked with 5% skimmed milk powder at room temperature for 30 min, and incubated with primary antibody overnight at 4 ℃. The antibodies and concentrations used were the following: ITGA5 (Proteintech, 1:1000 dilution), AKT (Cell Signaling Technology, 1:1000 dilution), p-AKT (Cell Signaling Technology, 1:1000 dilution), GAPDH (Cell Signaling Technology, 1:10000 dilution), FAK (Cell Signaling Technology, 1:1000 dilution), and p-FAK (Cell Signaling Technology, 1:1000 dilution). The membrane was washed and incubated with the secondary antibody (Cell Signaling Technology 1:5000 dilution) for 60 min at room temperature, and then the color was developed using ECL developer. GAPDH was used as the loading control to normalize protein expression. 2.6. Luciferase reporter assay 293T cells were seeded into 48-well plates and cultured in a 5% CO 2 incubator at 37 ℃ until reaching 70% - 80% confluence. The wild-type and mutant dual luciferase reporter vectors psicheck-ITGA5-wt and psicheck-ITGA5-mut in the seed region of ITGA5 gene were designed and constructed. The miR-330-5p mimic was co-transfected with the mimic of 30utr type or mutant ITGA5 of wild-type or mutant ITGA5 using lipo3000 transfection agent (Life Technologies，Gaithersburg，MD，USA). Cells were washed with PBS after 48 h. Luciferase activity was determined using a dual luciferase reporter assay system (Promega) and a micro plate fluorescence reader (BioTek, winooski, Vermont, USA). 2.7 CCK8 The gastric cancer cells grown in logarithmic phase were collected and seeded into 96-well plates at a density of approximately 1000 cells / well. CCK8 reagent (10 µL) was added to each well at 0, 24, 48, 72, and 96 hours, and incubated at 37 ℃ for 2 h. The absorbance of each well was read at 450 nm using a microplate reader (BIA RAD) and the cell growth curve was plotted. 2.8. Clonogenic assay Approximately 400 cells in good growth condition and the logarithmic phase were seeded into a Petri dish, and incubated at 37 ℃ under 5% CO 2 . Cells were fixed when clumps of clonal cells were visible to the naked eye in the Petri dish after 10-14 days by adding 2 ml 4% paraformaldehyde working solution for 20-30 minutes, and 0.1% crystal violet staining solution was used for 20 minutes. The number of clones of the different cell lines with a diameter greater than 2 mm was counted under the microscope, and compared to evaluate the proliferation ability of each cell line according to the number of cell clones. 2.9. Cell invasion and migration assay Transwell chambers (BD, USA) were placed into 24-well plates. Gastric cancer cells (4 × 10 5 ) treated with a fully mix were suspended in 800 µL serum-free medium and added to the upper chamber, while the medium containing 20% FBS was added to the lower chamber. The cells in the lower side of the membrane were fixed with 4% formaldehyde and stained with crystal violet. Subsequently, five fields of view were randomly selected, the cells were counted under the microscope, and the average value was calculated. Those with more crossing cells were considered to have stronger invasion ability. 2.10. Statistical analysis Statistical analysis was performed using SPSS 20.0 and GraphPad prism 7.0 was used for the graphical processing of the results. Results were expressed as mean ± standard deviation (c±s). T-test was used to evaluate the difference between two groups, and one-way ANOVA was used to compare multiple groups. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Prediction and screening of miRNAs targeting the 3'UTR of ITGA5 gene The results shown in Table 3 revealed that 27 miRNAs targeting ITGA5 were found in TargetScan v2.0 database, 120 in miRDB, 30 in miRTarBase, and 51 in mirDIP. The 3'UTR of ITGA5 mRNA has multiple potential miRNA targeting sequences. The intersection of data from the four databases was performed, and six miRNAs with high stability and conservation were found, such as miR-152-3p, miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-26a-5p and miR-330-5p. All of them were potentially targeting ITGA5, as shown in Figure 1A. According to the prediction analysis of the public database of the Sun Yat sen University on the ENCORI pan-cancer analysis platform, ITGA5 mRNA was negatively correlated with the expression of miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-26a-5p and miR-330-5p in 372 gastric cancer tissue samples, while it was positively correlated with miR-152-3p, as shown in Figure 1b-g. After the transfection of miRNA mimic NC, miR-26a-5p mimic, miR-92a-3p mimic, miR-148a-3p mimic, miR-148b-3p mimic, miR-330-5p mimic, and miR-152-3p mimic, the ITGA5 mRNA and protein expression in 293T cells after transfection of miR-330-5p mimic alone was significantly reduced compared with the control group miRNA mimic NC ( P < 0.05, Figure 1 h-j). Thus, the upstream miRNA miR-330-5p targeting ITGA5 was chosen for further evaluation. 3.2 Targeting relationship between miR-330-5p and ITGA5 Sequencing identification confirmed that the vector was successfully constructed, as shown in Figure 2 a-c. The upregulation of miR-330-5p inhibited the luciferase activity of psicheck-ITGA5 wt group, but had no effect on the vector of NC group. The relative fluorescence intensity of psicheck-ITGA5 wt group and blank control group was 5.103±0.075 and 9.547±0.035, respectively. The luciferase expression of miR-330-5p in the psicheck-ITGA5 wt group was significantly higher than that in the blank control group ( P < 0.001). In addition, miR-330-5p significantly inhibited the fluorescence expression of psicheck-ITGA5-wt ( P 0.05, Table 2 and Figure 2D). ITGA5 protein in gastric cancer cells was significantly increased after the transfection of miR-330-5p inhibitor compared with the control group ( P < 0.05). However, ITGA5 protein in gastric cancer cells was significantly decreased After transfection of miR-330-5p mimic ( P < 0.05) (Figure. 2 E-H). The above experimental results demonstrated that miR-330-5p targeted the 3'UTR of human ITGA5 gene and exerted a targeted regulatory effect on this gene. 3.3 miR-330-5p significantly attenuated the proliferative effect of ITGA5 on gastric cancer cells The proliferation ability of the miR-330-5p-mimic group was significantly lower than that of the control group ( P < 0.05), the cell proliferation ability of the miR-mimic-NC+OE-ITGA5 group was significantly higher than that of the control group ( P < 0.05), and the cell proliferation ability of the miR-330-5p-mimic+OE-ITGA5 group was reduced than that of miR-mimic-NC+OE-ITGA5 group ( P < 0.05) (Figure. 3 a-b). The number of gastric cancer cell clones in the miR-330-5p-mimic group was significantly less than that in the control group ( P < 0.05), the number of gastric cancer cell clones in the miR mimic NC+OE-ITGA5 group was significantly higher than that in the control group ( P < 0.05), and the number of gastric cancer cell clones in the miR-330-5p-mimic+OE-ITGA5 group was less compared with that in the miR mimic NC+OE-ITGA5 group ( P < 0.05) (Figure. 3 C-F). These results indicated that miR-330-5p-mimic significantly inhibited the proliferation of gastric cancer cells, and significantly inhibited the proliferation promoting effect of ITGA5. 3.4 miR-330-5p significantly inhibited the migration and invasion of gastric cancer cells induced by ITGA5 The migration ability of gastric cancer cells transfected with miR-330-5p mimic was significantly reduced compared with that of the control group miR mimic NC ( P < 0.05), the migration ability of miR mimic NC + OE-ITGA5 group was significantly increased compared with that of the miR mimic NC group ( P < 0.05), while the migration ability of gastric cancer cells in the miR-330-5p-mimic + OE-ITGA5 group was significantly decreased compared with that of the miR mimic NC + OE-ITGA5 group ( P < 0.05) (Figure. 4 A-F). The invasion of gastric cancer cells transfected with miR-330-5p mimic was significantly reduced compared with that of the control group miR mimic NC ( P < 0.05), the invasion of cells in the miR mimic NC+OE-ITGA5 group was significantly increased than that in the miR mimic NC group ( P < 0.05), while the invasion of the miR-330-5p-mimic+OE-ITGA5 cells was significantly decreased than that in the miR mimic NC+OE-ITGA5 group ( P < 0.05) (Figure. 4 A-F). These results indicated that miR-330-5p-mimic significantly inhibited the invasion and migration of gastric cancer cells. 3.5 miR-330-5p reversed the activation of FAK/AKT pathway by ITGA5 p-FAK and p-AKT protein expression in the miR-330-5p mimic group of gastric cancer cells MKN-28 and AGS was significantly inhibited compared with the miR mimic NC group ( P < 0.05), while FAK and AKT protein expression was not significantly changed. p-FAK and p-AKT protein expression in the miR mimic NC+OE-ITGA5 group was significantly increased ( P < 0.05), but the expression of FAK and AKT protein was not significantly changed. p-FAK and p-AKT protein expression was inhibited in the OE-ITGA5+miR-330-5p mimic group compared with that in the OE-ITGA5+miR-330-5p mimic NC group ( P < 0.05). However, FAK and AKT protein expression was not significantly changed (Figure. 5 a-b). The above results suggested that miR-330-5p mimic significantly reduced p-FAK and p-AKT protein expression ( P < 0.05), and ITGA5 overexpression significantly increased p-FAK and p-AKT protein expression ( P < 0.05). miR-330-5p mimic reversed the effect of ITGA5 overexpression on gastric cancer cells, indicating that it inhibited the activation effect of ITGA5 on FAK/AKT signaling pathway. p-FAK and p-AKT protein expression in the miR-330-5p inhibitor group of gastric cancer cells MKN-28 and AGS was significantly increased compared with that in the miR inhibitor NC group ( P < 0.05), while FAK and Akt protein expression was not significantly changed. p-FAK and p-AKT protein expression in the sh-ITGA5+miR inhibitor NC group was significantly decreased ( P < 0.05), but the expression of FAK and AKT protein was not significantly changed. p-FAK and p-AKT protein expression was increased in the sh-ITGA5+miR-330-5p inhibitor group compared with that in the sh-ITGA5+miR-330-5p inhibitor NC group ( P < 0.05). However, FAK and AKT protein expression was not significantly changed (Figure. 5 C-D). The above results suggested that miR-330-5p inhibitor significantly increased p-FAK and p-AKT protein expression ( P < 0.05), and ITGA5 silencing significantly reduced p-FAK and p-AKT protein expression ( P < 0.05). After co-transfection of miR-330-5p inhibitor and sh-ITGA5, miR-330-5p inhibitor reversed the effect of ITGA5 silencing on gastric cancer cells, indicating that it reversed the effect of ITGA5 gene silencing and inhibited the inhibitory effect of ITGA5 silencing on the FAK/AKT signaling pathway. 4. Discussion The occurrence and development of tumor is a very complex and multi-step process, such as a combination of internal and external factors, participation of oncogenes and tumor suppressor genes, and mutual regulation and dynamic changes of coding and non-coding genes. A large number of studies showed that the ITGA5 gene is up-regulated in a variety of tumors and affects the initiation and development of tumors, thus being a very promising molecule [ 19 , 20 ]. The occurrence and development of tumors are not simply caused by the change of a certain molecule, but by the change of a certain molecule or some molecules causing a series of reactions, which eventually lead to the occurrence and progression of tumors [ 21 ]. Previous studies of our and other research groups found that the ITGA5 gene mainly acts as an oncogene in gastric cancer. Therefore, the discovery of upstream miRNAs that are negatively correlated with ITGA5 expression and target and regulate it, was the topic of our present study. miRNAs possess regulatory functions, which are estimated to regulate 30% of genes in the human genome, and each miRNA regulates hundreds of genes [ 22 ]. They also regulate cancer-related pathways, and some dysregulated miRNAs cause changes in the biological behavior of tumors, indicating that they can be used as potential biomarkers for human tumor diagnosis and prognostic therapeutic targets [ 23 ]. Generally, miRNAs form RNA induced silencing complex (RISC) with related proteins and inhibit their transcription and translation by binding to the 3'UTR region of the target RNA, thereby regulating the biological behavior of the body [ 24 ]. So far, 800–1000 miRNAs have been experimentally proved, and most of them are located in the tumor genome region [ 25 ]. miRNAs are involved in various types of tumors, such as gastric cancer, intestinal cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer, thyroid cancer, and ovarian cancer [ 26 , 27 ]. More and more studies proved that miRNA expression may be used to diagnose tumors and as prognostic markers [ 28 , 29 ]. The miRNAs targeting ITGA5 gene were found through the analysis of a large number of articles, and multiple miRNAs are able to target the ITGA5 gene, including miR-330-5p, miR-26a-5p, miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-205-5p, miR-152-3p, miR − 128-3p and miR-31-3p. For example, in a study of liver cancer, miR-128 inhibits the metastasis and stem cell like properties of liver cancer cells by targeting the expression of itga2 and ITGA5. miR-128 plays a tumor suppressive role in hepatocellular carcinogenesis [ 30 ]. miR-26a inhibits the proliferation, invasion, and apoptosis of liver cancer cells by targeting the ITGA5 gene protein [ 31 ]. miR-92a-3p reduces the expression of ITGA5 in ovarian cancer cells, and inhibits the proliferation, adhesion, and invasion of cancer cells [ 16 ]. In vitro and in vivo experiments of cardiovascular disease revealed that miR-92a targets ITGA5 modifying its gene expression in endothelial cells to block angiogenesis [ 32 ]. miR-148b-3p inhibits the proliferation, invasion, and migration of malignant vascular endothelial cells by inhibiting the expression of ITGA5 and ALCAM in melanoma and breast cancer [ 33 ]. A miRNA can target and regulate multiple target genes, and multiple miRNAs can jointly regulate a target gene and participate in the regulation of multiple signaling pathways [ 34 ]. At present, hundreds of predicted target gene websites are available. The predicted target genes must be comprehensively analyzed and experimentally verified in order to clarify their targeting relationship. Once a specific miRNA is selected, all target mRNAs can be predicted. For example, once it is known a specific mRNA3 '-utr, the sequences of all miRNAs and binding sites can be predicted. The upstream miRNAs targeting the ITGA5 gene were predicted in this work, which were reported in the reviewed literature, indicating that these miRNAs do have a targeting relationship with the ITGA5 gene. Only after the transfection of miR-330-5p mimic, ITGA5 mRNA and protein expression was reduced, and other miRs showed no evident changes. Thus, miR-330-5p was discovered as an upstream molecule targeting the ITGA5 gene. miR-330-5p also plays different roles in a variety of tumors. Some scholars found that the expression of miR-330 is decreased in prostate cancer [ 35 ] and malignant melanoma [ 36 ]; it indeed mediates cell proliferation, apoptosis, invasion, and migration. Further in-depth research revealed that miR-330-5p is closely related to the progression of nasopharyngeal carcinoma [ 37 ], lung cancer [ 38 ], cervical cancer [ 39 ], ovarian cancer [ 40 ], and is closely related to the biological characteristics of tumors. The role of miRNAs in gastric cancer has gradually received attention. It is worth noting that miRNAs and their target gene mRNAs play a synergistic network role in the process of tumor progression. Thus, it is also necessary to explore the relationship between miRNAs and target genes. Yoo et al. [ 14 ] found that miR-330-5p targeted the ITGA5 gene in colorectal cancer, affecting its occurrence and development by regulating the expression of this gene. Although miR-330-5p and ITGA5 have been discussed in many tumors, the relationship between them and their functional roles in gastric cancer remain unknown. Our results revealed that miR-330-5p directly acted on the binding site on the 3 'UTR of ITGA5 revealing the targeting relationship between miR-330-5p and ITGA5; thus, ITGA5 was the target gene of miR-330-5p. The results of our study were consistent with those in the literature. ITGA5 is closely related to the biological behavior of gastric cancer cells. Can the regulation of miR-330-5p targeting ITGA5 gene cause changes in its biological behavior? The effect of miR-330-5p and ITGA5 on cell biological behavior and their regulatory mechanism should be further explored to provide a reliable theoretical basis for the role of miR-330-5p and ITGA5 in the diagnosis and treatment of gastric cancer. Previous studies found that ITGA5 is a proto-oncogene, which significantly promotes the proliferation, invasion, and migration of MKN28 and AGS cells. This study found that the high miR-330-5p expression inhibited the proliferation, migration, and invasion of gastric cancer cells. Further co-transfection of miR-330-5p silencing and overexpression mimic as well as ITGA5 silencing and overexpression plasmids revealed that miR-330-5p inhibited the proliferation, invasion, and migration effect induced by ITGA5. Our study showed that miR-330-5p targeted ITGA5 modifying its expression in gastric cancer and regulated the migration and invasion ability of gastric cancer cells. miR-330-5p inhibits ITGA5 protein expression in colorectal cancer by directly binding to the 3 'UTR untranslated region of ITGA5 mRNA [ 14 ]. Similar to this study, Feng et al. [ 18 ] found that miR-330-5p targeted the ITGA5 gene to inhibit its expression in glioma, thereby inhibiting the proliferation, invasion, and migration of glioma cells, but did not further study the mechanism. The above findings are consistent with the results of this experimental study. Thus, miR-330-5p reversed the promoting effect of ITGA5 on the proliferation, invasion, and migration of gastric cancer cells. Previous studies found that ITGA5 may activate the FAK/AKT signaling pathway. However, the relationship and mechanism between miR-330-5p, ITGA5 gene, and FAK/AKT have not been reported in gastric cancer, thus needing further elucidation. ITGA5 is closely related to the expression of PI3K and AKT. After overexpression of ITGA5 at the gene and protein levels, the expression levels of p-PI3K/PI3K, p-AKT/AKT and their downstream proteins are correspondingly upregulated [ 41 ]. Studies have shown that upregulating LBH gene can significantly increase the expression of ITGA5, p-FAK and p-AKT, activate the ITGA5 /FAK/AKT pathway, and promote the proliferation and invasion of gastric cancer cells [ 42 ]. In the study of colon cancer and glioblastoma, the down-regulation of miR-330-5p expression may affect the occurrence and development of tumors by regulating the expression of ITGA5 [ 14 , 18 ]. In oral squamous cell carcinoma,ITGA5 can promote the progression of oral squamous cell carcinoma by activating PI3K/AKT signaling pathway [ 43 ]. ITGA5 is aberrantly expressed in a variety of malignant tumors, and regulates the occurrence and development of tumors by participating in the proliferation, migration and invasion of tumor cells. The main mechanism of ITGA5 regulating tumorigenesis and development is through mirna-ITGA5 and ITGA5-PI3K/AKT/FAK pathways, but the specific biological mechanism of its role and its upstream and downstream events are still not fully understood. Our study found that miR-330-5p mimics could significantly reduce the protein expression levels of p-FAK and p-AKT, and ITGA5 overexpression could significantly increase the protein expression levels of p-FAK and p-AKT. After CO transfection, miR-330-5p mimics could reverse the effect of ITGA5 overexpression on gastric cancer cells, indicating that miR-330-5p mimics may reverse the overexpression of ITGA5 gene and inhibit the activation of FAK/AKT signaling pathway by ITGA5. This is similar to the results of this study. Our study also found that miR-330-5p can promote the expression of ITGA5 and activate FAK/AKT pathway to affect the occurrence and development of gastric cancer. 5. Conclusion In Conclusion, miR-330-5p inhibition promoted the expression of ITGA5 and activated FAK/AKT pathway, and promoted the proliferation, invasion, and migration of gastric cancer cells. The overexpression of miR-330-5p inhibited the expression of ITGA5 and the activation of FAK/AKT pathway, thereby reducing the proliferation, invasion, and migration of gastric cancer cells. This study explored the inhibitory and regulatory mechanism of miR-330-5p on the function of the target gene ITGA5, providing a reliable theoretical basis for the role of miR-330-5p and ITGA5 in the diagnosis, treatment, and prognosis of gastric cancer. Declarations Authors’ Contributions All authors consulted the final manuscript. Jun-fu Wang and Long-zi Liu conceived the research, analyzed the data and drafted the manuscript; Jun-wen Hu was responsible for collecting the data; Jian-ming Wei and Ting He participated in the interpretation and statistical analysis of the data; Jiang-nan Zhang was responsible for checking and revising the manuscript. Funding This work was supported bythe National Natural Science Foundation of China (grant no.82260465, 82260560 and 82002603); the Science and technology plan of Jiangxi Provincial Health Commission (grant no.202310237); the Science and technology plan of Jiangxi Provincial Administration of traditional Chinese Medicine (grant no.2023B1021) . Availability of data and materials The data used to support the findings of this study are included within the article. Competing interests The authors declare no conflict of interest. Ethics approval and consent to participate Not application. Consent for publication Not application. Acknowledgments Not application. Author details 1 Department of General Surgery, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang 330031, China. 2 Department of General Surgery, Second Affiliated Hospital of Nanchang University, No.1, Minde Road, Nanchang330006, China. References Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021. Plummer M, Franceschi S, Vignat J, et al. Global burden of gastric cancer attributable to Helicobacter pylori[J]. Int J Cancer, 2015, 136(2): 487-490. Ramos M, Ribeiro Junior U, Viscondi JKY, et al. Risk factors associated with the development of gastric cancer - case-control study[J]. Rev Assoc Med Bras, 2018, 64(7): 611-619. Li Z, Guo Q, Lu Y, et al. Increased expression of miR-181d is associated with poor prognosis and tumor progression of gastric cancer[J]. Cancer Biomark, 2019, 26(3): 353-360. Bu Z, Zheng Z, Li Z, et al. 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The circulating level of miR-122 is a potential risk factor for endothelial dysfunction in young patients with essential hypertension[J]. Hypertens Res, 2020, 43(6): 511-517. Liu DC, Song LL, Liang Q, et al. Long noncoding RNA LEF1-AS1 silencing suppresses the initiation and development of prostate cancer by acting as a molecular sponge of miR-330-5p via LEF1 repression[J]. J Cell Physiol, 2019, 234(8): 12727-12744. Su BB, Zhou SW, Gan CB, et al. MiR-330-5p regulates tyrosinase and PDIA3 expression and suppresses cell proliferation and invasion in cutaneous malignant melanoma[J]. J Surg Res, 2016, 203(2): 434-440. Peng Song S-CY. Long non‐coding RNA EWSAT1 promotes human nasopharyngeal carcinoma cell growth in vitro by targeting miR‐326/‐330‐5p[J]. AGING, 2016, 8: 2948-2960. Shen L, Yi S, Huang L, et al. miR-330-3p promotes lung cancer cells invasion, migration, and metastasis by directly targeting hSOD2b[J]. Biotechnol Appl Biochem, 2019, 66(1): 21-32. Cui L, Nai M, Zhang K, et al. lncRNA WT1-AS inhibits the aggressiveness of cervical cancer cell via regulating p53 expression via sponging miR-330-5p[J]. Cancer Manag Res, 2019, 11: 651-667. Lin M, Xia B, Qin L, et al. S100A7 Regulates Ovarian Cancer Cell Metastasis and Chemoresistance Through MAPK Signaling and Is Targeted by miR-330-5p[J]. DNA Cell Biol, 2018, 37(5): 491-500. Zhang CZ, Wang XD, Wang HW, et al, Sorafenib inhibits liver cancer growth by decreasing mTOR, AKT, and PI3K expression[J]. J BUON, 2015, 20( 1) : 218-222． Yu R, Li Z, Zhang C, et al, Elevated limb-bud and heart development ( LBH) expression indicates poor prognosis and promotes gastric cancer cell proliferation and invasion via upregulating Integrin /FAK/ Akt pathway[J]. Peer J, 2019, 7: e6885． Fan QC, Tian H, Wang Y, et al, Integrin-alpha5 promoted the progression of oral squamous cell carcinoma and modulated PI3K/ AKT signaling pathway[J]. Arch Oral Biol, 2019, 101: 85-91． Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 11 Feb, 2026 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 26 Jul, 2024 Editor assigned by journal 22 Jul, 2024 Submission checks completed at journal 22 Jul, 2024 First submitted to journal 16 Jul, 2024 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-4747650","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":332057701,"identity":"1f9eb131-397e-4d8d-9f15-01e49323ee0b","order_by":0,"name":"Jun-fu Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jun-fu","middleName":"","lastName":"Wang","suffix":""},{"id":332057702,"identity":"e7aeea89-bc38-42ba-b02a-e504b3a901ed","order_by":1,"name":"Jian-ming Wei","email":"","orcid":"","institution":"First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jian-ming","middleName":"","lastName":"Wei","suffix":""},{"id":332057703,"identity":"c296dc55-9f10-49a9-94b2-a3a34ae6a8ad","order_by":2,"name":"Ting He","email":"","orcid":"","institution":"First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"He","suffix":""},{"id":332057704,"identity":"26cd95ca-4a6f-4c0a-a5e3-bcf9fa6ece61","order_by":3,"name":"Jun-wen Hu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jun-wen","middleName":"","lastName":"Hu","suffix":""},{"id":332057705,"identity":"ca2abd56-cd7a-463a-9592-89604fbf7dab","order_by":4,"name":"Jiang-nan Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jiang-nan","middleName":"","lastName":"Zhang","suffix":""},{"id":332057706,"identity":"eec284f2-e5ca-415d-a36c-842f675fe879","order_by":5,"name":"Long-zi Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACPmYGxgMgBht7Y+ODD8RoYWNmYIBo4TncbDiDKC0MUC0MEult0hxEaWHnMTjwcUetPZ/kwwZpBgY7Od0Ggg7jMTg488xxZjbpxAbjAoZkY7MDRGg5zNt2jA2kJXkGw4HEbcRq4WGTPNhwmIcELTUSbBKMjc1EamErODiz7YABG09iM+MMAyL8ws9/eOODj2119vLtx5//+FBhJ0dQCxQchtIGxCkHgTrilY6CUTAKRsHIAwAPLDvkXWhJDgAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Long-zi","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-07-16 07:00:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4747650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4747650/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-026-15676-1","type":"published","date":"2026-02-11T15:59:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62426411,"identity":"066ecc15-a0fb-488e-9c78-a16bb07a89e4","added_by":"auto","created_at":"2024-08-14 05:18:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242421,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction and screening of miRNAs targeting the 3'UTR of ITGA5 gene. A. Four databases predict miRNAs targeting ITGA5. B-G. Correlation between ITGA5 and target related miRNA in gastric cancer tissue samples by the public database of Sun Yat sen University on the ENCORI pan-cancer analysis platform, *P \u0026lt; 0.05. H. Targeted screening of the 3'UTR of ITGA5 mRNA and miR-26a-5p, miR-92a-3p, miR-148a-3p, miR-148b-3p, and miR-330-5p in 293T cells by qRT-PCR. I-J Experimental verification and statistical results of the targeting relationship in 293T cells by western blot (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001). K-L. Validation of the targeting relationship between the 3'UTR and miR-330-5p of ITGA5 mRNA in 293T cells, by qRT PCR and western blot.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/a0e0b921de3f33e5bd6d4954.png"},{"id":62426918,"identity":"9ca17b69-b7b8-4433-a0ff-95d86dd67c81","added_by":"auto","created_at":"2024-08-14 05:26:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162323,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting relationship between miR-330-5p and ITGA5. A. ITGA5 wt sequence. B. ITGA5 mut sequence. C. Schematic diagram of 3'UTR and miR-330-5p binding sequence of ITGA5 mRNA. D. Relative luciferase activity in 293 T cells co-transfected with miR-330-5p mimic and ITGA5 wt/mut by dual luciferase reporter assay. E-H. Validation of ITGA5 protein changes targeted by miR-330-5p in AGS and MKN-28 cells.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/548136dee22b30a411a955cc.png"},{"id":62426409,"identity":"da3a0ccd-e068-48fd-a9b4-dc2a30b858c0","added_by":"auto","created_at":"2024-08-14 05:18:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":288498,"visible":true,"origin":"","legend":"\u003cp\u003emiR-330-5p targeted inhibition of ITGA5 on gastric cancer cell proliferation. A-B Effect of CO transfection of miR-330-mimic and OE-ITGA5 on the proliferation of AGS and MKN-28 gastric cancer cells detected by CCK8 cell proliferation assay. C-D Effect of co-transfection of miR-330-mimic and OE-ITGA5 on the proliferation of AGS and MKN-28 gastric cancer cells (100x magnification) detected by clonogenic assay. E-F Corresponding statistical data results of invasion and migration experiments; the results are expressed as mean ± SD of three independent experiments (*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/0c15a470022848db4a677e32.png"},{"id":62426413,"identity":"125d4cfe-e2f5-4659-befb-fc7be3fbad49","added_by":"auto","created_at":"2024-08-14 05:18:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":534992,"visible":true,"origin":"","legend":"\u003cp\u003emiR-330-5p targeted and inhibited the effect of ITGA5 on the invasion and migration of gastric cancer cells. A-B. Invasion and migration of AGS gastric cancer cells after CO transfection of miR-330-mimic and OE-ITGA5 and the corresponding statistical data detected by Transwell chamber experiment. C-D. Invasion and migration of MKN-28 gastric cancer cells after CO transfection of miR-330-mimic and OE-ITGA5 and the corresponding statistical data detected by Transwell chamber experiment. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/af5b800708db2f8f1a2263d0.png"},{"id":62426412,"identity":"02a05e5e-e386-4d6f-8129-3997a11072f4","added_by":"auto","created_at":"2024-08-14 05:18:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135476,"visible":true,"origin":"","legend":"\u003cp\u003emiR-330-5p reversed the effect of ITGA5 on the FAK/AKT signaling pathway. A-B. Expression of FAK / p-FAK and AKT / p-AKT proteins in AGS and MKN-28 gastric cancer cells after CO transfection of miR-330-mimic and OE-ITGA5 analyzed by western blot. C-D. Expression of FAK / p-FAK and AKT / p-AKT proteins in AGS and MKN-28 gastric cancer cells after CO transfection of miR-330-5p inhibitor and sh-ITGA5 analyzed by western blot.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/affd6729ab39f03b50a48bd1.png"},{"id":102785807,"identity":"d4993d97-3149-4ae9-a629-f35b3ba7b064","added_by":"auto","created_at":"2026-02-16 16:10:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2060926,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/c0d7525e-d54c-4999-ad75-bcea94cd5943.pdf"},{"id":62426917,"identity":"489e2db3-0088-4ae4-a24c-9568aae5c6be","added_by":"auto","created_at":"2024-08-14 05:26:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":38118,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4747650/v1/f4c67c63b0f7b6748b40040f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism of action of miR-330-5p targeting ITGA5 in the regulation of proliferation, migration, and invasion of gastric cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGastric cancer is one of the most common malignancies, diagnosed in more than 1 million people worldwide every year, with an incidence and mortality ranking third [1]. Although its incidence is decreasing worldwide, it is still high in East Asian countries. According to the latest cancer data statistics in 2020, the annual incidence of gastric cancer in China is approximately 479000 cases, and the number of annual deaths is approximately 374000 [1]. The risk factors associated with gastric cancer include Helicobacter pylori infection, environmental factors, low intake of fruits and vegetables, smoking, and alcohol consumption [2,3]. Surgery and chemotherapy are still the main treatment for gastric cancer. Although the early diagnosis and treatment of gastric cancer are improved, the prognosis of gastric cancer is still poor due to lymphatic metastasis and invasion [4, 5]. Therefore, it is of the utmost importance to discover new molecular mechanism involved in the occurrence and development of gastric cancer to ultimately contribute to the discovery of new tumor markers and take effective measures for early diagnosis and targeted treatment of gastric cancer metastasis to improve the survival rate and quality of life of patients. In other words, it is necessary to provide a theoretical basis for the early clinical diagnosis, the occurrence and development mechanism, and consequent targeted therapy of gastric cancer.\u003c/p\u003e\n\u003cp\u003eMicroRNAs are a class of endogenous non-coding microRNAs of 18-24 nucleotides. They are a single stranded small molecule RNAs that bind to the 3 \u0026apos;untranslated region (3\u0026apos; UTR) of the target mRNA through base complementary pairing, thereby inhibiting the transcription or degradation of the target gene [6]. miRNAs are involved in the biological process of a variety of tumors, such as tumor cell proliferation, apoptosis, angiogenesis, invasion, and migration [7, 8]. miRNAs are the main regulators of gene expression and are involved in cancer metastasis. The abnormal expression of miRNAs in tumors causes changes in the biological functions of tumor cells. Some miRNAs play an oncogenic role and some a tumor suppressive role [9, 10]. Some aberrantly expressed miRNAs directly affect the occurrence and development of tumors by targeting genes or signaling pathways related to tumor progression [11]. More and more studies confirmed that miRNAs may become potential biomarkers for the diagnosis, treatment, and prognosis of tumor patients [12]. miR-330-5p is closely related to the occurrence and development of a variety of tumors\u0026nbsp;[13, 14].\u003c/p\u003e\n\u003cp\u003ePrevious studies of our research group found that ITGA5 expression is up-regulated in gastric cancer cell lines and tissues, and its expression is closely related to tumor size, lymph node metastasis, and TNM stage. ITGA5 induces proliferation, invasion, and migration of gastric cancer cells by activating the FAK/AKT signaling pathway [15]. ITGA5 is a member of the integrin family, with a molecular weight of approximately 130 kd. More and more reports on the role of ITGA5 in tumors are available, and the regulation of its expression in tumors has attracted extensive research. ITGA5 is a fibronectin receptor working as a proto-oncogene affecting the intracellular and extracellular signal transduction and is closely related to the occurrence and development of a variety of tumors. Other scholars found that ITGA5 is regulated by miRNAs, affecting its expression in tumor cells, thereby causing functional changes. Hara et al. [16] found that ITGA5 mRNA is a direct target of mir-92a. Transfection of mir-92a mimic reduces the expression of ITGA5 in ovarian cancer cells and inhibits their adhesion, invasion, and proliferation. Gong et al. [17] found that miR-17 directly binds ITGA5 and ITG\u0026beta;1 in the 3\u0026apos; untranslated region (3\u0026apos; UTR) and inhibits their expression, thereby inhibiting the peritoneal seeding of ovarian cancer cells. Xue et al. [13] found that ITGA5 is the target of mir-30b-5p, thus inhibiting the invasion and migration of esophageal cancer cells. Yoo et al. [14] found that miR-330-5p affects the development of colorectal cancer by regulating the expression of ITGA5. Similarly, Feng et al. [18] found that miR-330-5p targets and inhibits the expression of ITGA5 gene in glioma cells, thereby inhibiting their proliferation, invasion, and migration and promoting their apoptosis. Despite all these pieces of evidence, the relationship between miR-330-5p and ITGA5 in gastric cancer is still unclear, without demonstration that targeting ITGA5 results in the regulation of gastric cancer cell proliferation, invasion, and migration.\u003c/p\u003e\n\u003cp\u003eIn this study, the miRNAs regulating the target gene ITGA5 were predicted by bioinformatics, finally revealing that miR-330-5p was negatively correlated with ITGA5 expression. The targeting relationship between miR-330-5p and ITGA5 gene was confirmed by dual luciferase reporter experiment. Moreover, miR-330-5p affected the proliferation, invasion, and migration of gastric cancer cells by mediating ITGA5 and affecting the regulation of FAK/AKT signaling pathway. Hence, this study provides a theoretical basis to further understand the mechanism related to the occurrence and development of gastric cancer, improving the diagnosis and prognosis, and discovering a new therapeutic target.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003ch2\u003e2.1 Bioinformatics database data acquisition\u003c/h2\u003e\n\u003cp\u003eFour online websites, TargetScan (http://www.targetscan.org/vert_72/), miRDB (http://mirdb.org/cgi-bin/search.cgi), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/search.php) and mirDIP (http://ophid.utoronto.ca/mirDIP/index.jsp#r) were used to predict the miRNAs that target and regulate the human ITGA5 gene, and the parameters such as target score and PCT were analyzed to discover miRNAs with high correlation scores and intersection in the four databases.\u003c/p\u003e\n\u003ch2\u003e2.2 Cell culture\u003c/h2\u003e\n\u003cp\u003eHuman gastric cancer cell lines (AGS, MKN28, and HGC27) and normal gastric mucosal epithelial cells GES-1 were purchased from the cell bank of the Chinese Academy of Sciences and stored in liquid nitrogen. AGS and MKN28 cells stably overexpressing ITGA5 (OE-ITGA5) and with silenced ITGA5 (sh-ITGA5), as well as the negative control, were successfully constructed and stored in liquid nitrogen. Cells were incubated at 37 ℃ in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium group consisted of RPMI-1640 medium with 10% fetal bovine serum (GIBCO, USA) and 1% double antibody (Dalian Meilun biological Co., Ltd., China). Fresh medium was changed every other day.\u003c/p\u003e\n\u003ch2\u003e2.3 Cell transfection\u003c/h2\u003e\n\u003cp\u003eThe cell state and activity were optimized, and they were transfected when the confluence reached 60-70%. miRNA mimic, inhibitor, or their negative controls (Genechem, Shanghai, China) were transiently transfected using lipofectamine3000 (Invitrogen, Carlsbad, CA, USA) for further experiments, according to the above protocol.\u003c/p\u003e\n\n\u003ch2\u003e2.4 Real-time quantitative polymerase chain reaction (qRT-PCR)\u003c/h2\u003e\n\u003cp\u003eTotal RNA was extracted and isolated from tissues and cells using Trizol Kit (Macherey Nagel, Germany), and the concentration and purity of RNA were detected using nanodrop ND-2000 spectrophotometer. RNA and miRNA were reverse transcribed using a reverse transcription kit (Takara Biotechnology Co, Ltd.). The expression of mRNAs and miRNAs was detected using SYBR Green I fluorescence detection kit (Takara, China), and ABI 7500 real time PCR detection system, with GAPDH and U6 as internal controls, ITGA5, miR-26a-5p, miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-330-5p and miR-152-3p were detected. Finally, their expression was calculated using the 2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u003csup\u003e∆∆\u003c/sup\u003e\u003csup\u003e CT\u003c/sup\u003e method. Primers were purchased from Shanghai Bioengineering Technology Co., Ltd., and the sequences were the following:Table 1 and Table 2.\u003c/p\u003e\n\u003ch2\u003e2.5. Western blotting\u003c/h2\u003e\n\u003cp\u003eThe RIPA lysis buffer (Thermo Fisher Scientific) containing the protease inhibitor PMSF was used to lyse the cells and extract the protein. Cells were lysed on ice for 30 min, centrifuged at 11000 \u0026times; g at 4 ℃, and total protein concentration in the supernatant was detected by the protein concentration BCA (Thermo Fisher Scientific) method. The loading buffer (Beijing solabo science and Technology Co., Ltd.) was added, and the proteins were heated at 100 ℃ for 10 min. Total proteins were separated by 10% polyacrylamide gel electrophoresis, transferred to the membrane, blocked with 5% skimmed milk powder at room temperature for 30 min, and incubated with primary antibody overnight at 4 ℃. The antibodies and concentrations used were the following: ITGA5 (Proteintech, 1:1000 dilution), AKT (Cell Signaling Technology, 1:1000 dilution), p-AKT (Cell Signaling Technology, 1:1000 dilution), GAPDH (Cell Signaling Technology, 1:10000 dilution), FAK (Cell Signaling Technology, 1:1000 dilution), and p-FAK (Cell Signaling Technology, 1:1000 dilution). The membrane was washed and incubated with the secondary antibody (Cell Signaling Technology 1:5000 dilution) for 60 min at room temperature, and then the color was developed using ECL developer. GAPDH was used as the loading control to normalize protein expression.\u003c/p\u003e\n\u003ch2\u003e2.6. Luciferase reporter assay\u003c/h2\u003e\n\u003cp\u003e293T cells were seeded into 48-well plates and cultured in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37 ℃ until reaching 70% - 80% confluence. The wild-type and mutant dual luciferase reporter vectors psicheck-ITGA5-wt and psicheck-ITGA5-mut in the seed region of ITGA5 gene were designed and constructed. The miR-330-5p mimic was co-transfected with the mimic of 30utr type or mutant ITGA5 of wild-type or mutant ITGA5 using lipo3000 transfection agent (Life Technologies，Gaithersburg，MD，USA). Cells were washed with PBS after 48 h. Luciferase activity was determined using a dual luciferase reporter assay system (Promega) and a micro plate fluorescence reader (BioTek, winooski, Vermont, USA).\u003c/p\u003e\n\u003ch2\u003e2.7 CCK8\u003c/h2\u003e\n\u003cp\u003eThe gastric cancer cells grown in logarithmic phase were collected and seeded into 96-well plates at a density of approximately 1000 cells / well. CCK8 reagent (10 \u0026micro;L) was added to each well at 0, 24, 48, 72, and 96 hours, and incubated at 37 ℃ for 2 h. The absorbance of each well was read at 450 nm using a microplate reader (BIA RAD) and the cell growth curve was plotted. \u003c/p\u003e\n\u003ch2\u003e2.8. Clonogenic assay\u003c/h2\u003e\n\u003cp\u003eApproximately 400 cells in good growth condition and the logarithmic phase were seeded into a Petri dish, and incubated at 37 ℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were fixed when clumps of clonal cells were visible to the naked eye in the Petri dish after 10-14 days by adding 2 ml 4% paraformaldehyde working solution for 20-30 minutes, and 0.1% crystal violet staining solution was used for 20 minutes. The number of clones of the different cell lines with a diameter greater than 2 mm was counted under the microscope, and compared to evaluate the proliferation ability of each cell line according to the number of cell clones.\u003c/p\u003e\n\u003ch2\u003e2.9. Cell invasion and migration assay\u003c/h2\u003e\n\u003cp\u003eTranswell chambers (BD, USA) were placed into 24-well plates. Gastric cancer cells (4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) treated with a fully mix were suspended in 800 \u0026micro;L serum-free medium and added to the upper chamber, while the medium containing 20% FBS was added to the lower chamber. The cells in the lower side of the membrane were fixed with 4% formaldehyde and stained with crystal violet. Subsequently, five fields of view were randomly selected, the cells were counted under the microscope, and the average value was calculated. Those with more crossing cells were considered to have stronger invasion ability.\u003c/p\u003e\n\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis was performed using SPSS 20.0 and GraphPad prism 7.0 was used for the graphical processing of the results. Results were expressed as mean \u0026plusmn; standard deviation (c\u0026plusmn;s). T-test was used to evaluate the difference between two groups, and one-way ANOVA was used to compare multiple groups. A value of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u003c/strong\u003e Prediction and screening of miRNAs targeting the 3\u0026apos;UTR of ITGA5 gene\u003c/p\u003e\n\u003cp\u003eThe results shown in Table 3 revealed that 27 miRNAs targeting ITGA5 were found in TargetScan v2.0 database, 120 in miRDB, 30 in miRTarBase, and 51 in mirDIP. The 3\u0026apos;UTR of ITGA5 mRNA has multiple potential miRNA targeting sequences. The intersection of data from the four databases was performed, and six miRNAs with high stability and conservation were found, such as\u0026nbsp;miR-152-3p, miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-26a-5p and miR-330-5p. All of them were potentially targeting ITGA5, as shown in Figure 1A. According to the prediction analysis of the public database of the Sun Yat sen University on the ENCORI pan-cancer analysis platform, ITGA5 mRNA was negatively correlated with the expression of miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-26a-5p and miR-330-5p in 372 gastric cancer tissue samples, while it was positively correlated with miR-152-3p, as shown in Figure 1b-g. After the transfection of miRNA mimic NC, miR-26a-5p mimic, miR-92a-3p mimic, miR-148a-3p mimic, miR-148b-3p mimic, miR-330-5p mimic, and miR-152-3p mimic, the ITGA5 mRNA and protein expression in 293T cells after transfection of miR-330-5p mimic alone was significantly reduced compared with the control group miRNA mimic NC (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 1 h-j). Thus, the upstream miRNA miR-330-5p targeting ITGA5 was chosen for further evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;\u003c/strong\u003eTargeting relationship between miR-330-5p and ITGA5\u003c/p\u003e\n\u003cp\u003eSequencing identification confirmed that the vector was successfully constructed, as shown in Figure 2 a-c. The upregulation of miR-330-5p inhibited the luciferase activity of psicheck-ITGA5 wt group, but had no effect on the vector of NC group. The relative fluorescence intensity of psicheck-ITGA5 wt group and blank control group was 5.103\u0026plusmn;0.075 and 9.547\u0026plusmn;0.035, respectively. The luciferase expression of miR-330-5p in the psicheck-ITGA5 wt group was significantly higher than that in the blank control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). In addition, miR-330-5p significantly inhibited the fluorescence expression of psicheck-ITGA5-wt (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05); miR-330-5p did not significantly inhibit the fluorescence expression of psicheck-ITGA5-mut (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, Table 2 and Figure 2D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eITGA5 protein in gastric cancer cells was significantly increased after the transfection of miR-330-5p inhibitor compared with the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). However, ITGA5 protein in gastric cancer cells was significantly decreased After transfection of miR-330-5p mimic (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure. 2 E-H). The above experimental results demonstrated that miR-330-5p targeted the 3\u0026apos;UTR of human ITGA5 gene and exerted a targeted regulatory effect on this gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u003c/strong\u003e miR-330-5p significantly attenuated the proliferative effect of ITGA5 on gastric cancer cells\u003c/p\u003e\n\u003cp\u003eThe proliferation ability of the miR-330-5p-mimic group was significantly lower than that of the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), the cell proliferation ability of the miR-mimic-NC+OE-ITGA5 group was significantly higher than that of the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and the cell proliferation ability of the miR-330-5p-mimic+OE-ITGA5 group was reduced than that of miR-mimic-NC+OE-ITGA5 group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure. 3 a-b). The number of gastric cancer cell clones in the miR-330-5p-mimic group was significantly less than that in the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), the number of gastric cancer cell clones in the miR mimic NC+OE-ITGA5 group was significantly higher than that in the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and the number of gastric cancer cell clones in the miR-330-5p-mimic+OE-ITGA5 group was less compared with that in the miR mimic NC+OE-ITGA5 group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure. 3 C-F). These results indicated that miR-330-5p-mimic significantly inhibited the proliferation of gastric cancer cells, and significantly inhibited the proliferation promoting effect of ITGA5.\u003c/p\u003e\n\u003cp\u003e3.4 miR-330-5p significantly inhibited the migration and invasion of gastric cancer cells induced by ITGA5\u003c/p\u003e\n\u003cp\u003eThe migration ability of gastric cancer cells transfected with miR-330-5p mimic was significantly reduced compared with that of the control group miR mimic NC (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), the migration ability of miR mimic NC + OE-ITGA5 group was significantly increased compared with that of the miR mimic NC group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while the migration ability of gastric cancer cells in the miR-330-5p-mimic + OE-ITGA5 group was significantly decreased compared with that of the miR mimic NC + OE-ITGA5 group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure. 4 A-F). The invasion of gastric cancer cells transfected with miR-330-5p mimic was significantly reduced compared with that of the control group miR mimic NC (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), the invasion of cells in the miR mimic NC+OE-ITGA5 group was significantly increased than that in the miR mimic NC group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while the invasion of the miR-330-5p-mimic+OE-ITGA5 cells was significantly decreased than that in the miR mimic NC+OE-ITGA5 group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure. 4 A-F). These results indicated that miR-330-5p-mimic significantly inhibited the invasion and migration of gastric cancer cells.\u003c/p\u003e\n\u003cp\u003e3.5 miR-330-5p reversed the activation of FAK/AKT pathway by ITGA5 \u003c/p\u003e\n\u003cp\u003ep-FAK and p-AKT protein expression in the miR-330-5p mimic group of gastric cancer cells MKN-28 and AGS was significantly inhibited compared with the miR mimic NC group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while FAK and AKT protein expression was not significantly changed. p-FAK and p-AKT protein expression in the miR mimic NC+OE-ITGA5 group was significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), but the expression of FAK and AKT protein was not significantly changed. p-FAK and p-AKT protein expression was inhibited in the OE-ITGA5+miR-330-5p mimic group compared with that in the OE-ITGA5+miR-330-5p mimic NC group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). However, FAK and AKT protein expression was not significantly changed (Figure. 5 a-b). The above results suggested that miR-330-5p mimic significantly reduced p-FAK and p-AKT protein expression (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and ITGA5 overexpression significantly increased p-FAK and p-AKT protein expression (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). miR-330-5p mimic reversed the effect of ITGA5 overexpression on gastric cancer cells, indicating that it inhibited the activation effect of ITGA5 on FAK/AKT signaling pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ep-FAK and p-AKT protein expression in the miR-330-5p inhibitor group of gastric cancer cells MKN-28 and AGS was significantly increased compared with that in the miR inhibitor NC group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while FAK and Akt protein expression was not significantly changed. p-FAK and p-AKT protein expression in the sh-ITGA5+miR inhibitor NC group was significantly decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), but the expression of FAK and AKT protein was not significantly changed. p-FAK and p-AKT protein expression was increased in the sh-ITGA5+miR-330-5p inhibitor group compared with that in the sh-ITGA5+miR-330-5p inhibitor NC group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). However, FAK and AKT protein expression was not significantly changed (Figure. 5 C-D). The above results suggested that miR-330-5p inhibitor significantly increased p-FAK and p-AKT protein expression (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and ITGA5 silencing significantly reduced p-FAK and p-AKT protein expression (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). After co-transfection of miR-330-5p inhibitor and sh-ITGA5, miR-330-5p inhibitor reversed the effect of ITGA5 silencing on gastric cancer cells, indicating that it reversed the effect of ITGA5 gene silencing and inhibited the inhibitory effect of ITGA5 silencing on the FAK/AKT signaling pathway.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe occurrence and development of tumor is a very complex and multi-step process, such as a combination of internal and external factors, participation of oncogenes and tumor suppressor genes, and mutual regulation and dynamic changes of coding and non-coding genes. A large number of studies showed that the ITGA5 gene is up-regulated in a variety of tumors and affects the initiation and development of tumors, thus being a very promising molecule [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The occurrence and development of tumors are not simply caused by the change of a certain molecule, but by the change of a certain molecule or some molecules causing a series of reactions, which eventually lead to the occurrence and progression of tumors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Previous studies of our and other research groups found that the ITGA5 gene mainly acts as an oncogene in gastric cancer. Therefore, the discovery of upstream miRNAs that are negatively correlated with ITGA5 expression and target and regulate it, was the topic of our present study.\u003c/p\u003e \u003cp\u003emiRNAs possess regulatory functions, which are estimated to regulate 30% of genes in the human genome, and each miRNA regulates hundreds of genes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. They also regulate cancer-related pathways, and some dysregulated miRNAs cause changes in the biological behavior of tumors, indicating that they can be used as potential biomarkers for human tumor diagnosis and prognostic therapeutic targets [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Generally, miRNAs form RNA induced silencing complex (RISC) with related proteins and inhibit their transcription and translation by binding to the 3'UTR region of the target RNA, thereby regulating the biological behavior of the body [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. So far, 800\u0026ndash;1000 miRNAs have been experimentally proved, and most of them are located in the tumor genome region [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. miRNAs are involved in various types of tumors, such as gastric cancer, intestinal cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer, thyroid cancer, and ovarian cancer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. More and more studies proved that miRNA expression may be used to diagnose tumors and as prognostic markers [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe miRNAs targeting ITGA5 gene were found through the analysis of a large number of articles, and multiple miRNAs are able to target the ITGA5 gene, including miR-330-5p, miR-26a-5p, miR-92a-3p, miR-148a-3p, miR-148b-3p, miR-205-5p, miR-152-3p, miR \u0026minus;\u0026thinsp;128-3p and miR-31-3p. For example, in a study of liver cancer, miR-128 inhibits the metastasis and stem cell like properties of liver cancer cells by targeting the expression of itga2 and ITGA5. miR-128 plays a tumor suppressive role in hepatocellular carcinogenesis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. miR-26a inhibits the proliferation, invasion, and apoptosis of liver cancer cells by targeting the ITGA5 gene protein [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. miR-92a-3p reduces the expression of ITGA5 in ovarian cancer cells, and inhibits the proliferation, adhesion, and invasion of cancer cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments of cardiovascular disease revealed that miR-92a targets ITGA5 modifying its gene expression in endothelial cells to block angiogenesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. miR-148b-3p inhibits the proliferation, invasion, and migration of malignant vascular endothelial cells by inhibiting the expression of ITGA5 and ALCAM in melanoma and breast cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A miRNA can target and regulate multiple target genes, and multiple miRNAs can jointly regulate a target gene and participate in the regulation of multiple signaling pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At present, hundreds of predicted target gene websites are available. The predicted target genes must be comprehensively analyzed and experimentally verified in order to clarify their targeting relationship. Once a specific miRNA is selected, all target mRNAs can be predicted. For example, once it is known a specific mRNA3 '-utr, the sequences of all miRNAs and binding sites can be predicted. The upstream miRNAs targeting the ITGA5 gene were predicted in this work, which were reported in the reviewed literature, indicating that these miRNAs do have a targeting relationship with the ITGA5 gene. Only after the transfection of miR-330-5p mimic, ITGA5 mRNA and protein expression was reduced, and other miRs showed no evident changes. Thus, miR-330-5p was discovered as an upstream molecule targeting the ITGA5 gene.\u003c/p\u003e \u003cp\u003emiR-330-5p also plays different roles in a variety of tumors. Some scholars found that the expression of miR-330 is decreased in prostate cancer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and malignant melanoma [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]; it indeed mediates cell proliferation, apoptosis, invasion, and migration. Further in-depth research revealed that miR-330-5p is closely related to the progression of nasopharyngeal carcinoma [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], lung cancer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], cervical cancer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and is closely related to the biological characteristics of tumors. The role of miRNAs in gastric cancer has gradually received attention. It is worth noting that miRNAs and their target gene mRNAs play a synergistic network role in the process of tumor progression. Thus, it is also necessary to explore the relationship between miRNAs and target genes.\u003c/p\u003e \u003cp\u003eYoo et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] found that miR-330-5p targeted the ITGA5 gene in colorectal cancer, affecting its occurrence and development by regulating the expression of this gene. Although miR-330-5p and ITGA5 have been discussed in many tumors, the relationship between them and their functional roles in gastric cancer remain unknown. Our results revealed that miR-330-5p directly acted on the binding site on the 3 'UTR of ITGA5 revealing the targeting relationship between miR-330-5p and ITGA5; thus, ITGA5 was the target gene of miR-330-5p. The results of our study were consistent with those in the literature. ITGA5 is closely related to the biological behavior of gastric cancer cells. Can the regulation of miR-330-5p targeting ITGA5 gene cause changes in its biological behavior? The effect of miR-330-5p and ITGA5 on cell biological behavior and their regulatory mechanism should be further explored to provide a reliable theoretical basis for the role of miR-330-5p and ITGA5 in the diagnosis and treatment of gastric cancer.\u003c/p\u003e \u003cp\u003ePrevious studies found that ITGA5 is a proto-oncogene, which significantly promotes the proliferation, invasion, and migration of MKN28 and AGS cells. This study found that the high miR-330-5p expression inhibited the proliferation, migration, and invasion of gastric cancer cells. Further co-transfection of miR-330-5p silencing and overexpression mimic as well as ITGA5 silencing and overexpression plasmids revealed that miR-330-5p inhibited the proliferation, invasion, and migration effect induced by ITGA5. Our study showed that miR-330-5p targeted ITGA5 modifying its expression in gastric cancer and regulated the migration and invasion ability of gastric cancer cells. miR-330-5p inhibits ITGA5 protein expression in colorectal cancer by directly binding to the 3 'UTR untranslated region of ITGA5 mRNA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similar to this study, Feng et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] found that miR-330-5p targeted the ITGA5 gene to inhibit its expression in glioma, thereby inhibiting the proliferation, invasion, and migration of glioma cells, but did not further study the mechanism. The above findings are consistent with the results of this experimental study. Thus, miR-330-5p reversed the promoting effect of ITGA5 on the proliferation, invasion, and migration of gastric cancer cells.\u003c/p\u003e \u003cp\u003ePrevious studies found that ITGA5 may activate the FAK/AKT signaling pathway. However, the relationship and mechanism between miR-330-5p, ITGA5 gene, and FAK/AKT have not been reported in gastric cancer, thus needing further elucidation. ITGA5 is closely related to the expression of PI3K and AKT. After overexpression of ITGA5 at the gene and protein levels, the expression levels of p-PI3K/PI3K, p-AKT/AKT and their downstream proteins are correspondingly upregulated [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Studies have shown that upregulating LBH gene can significantly increase the expression of ITGA5, p-FAK and p-AKT, activate the ITGA5 /FAK/AKT pathway, and promote the proliferation and invasion of gastric cancer cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the study of colon cancer and glioblastoma, the down-regulation of miR-330-5p expression may affect the occurrence and development of tumors by regulating the expression of ITGA5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In oral squamous cell carcinoma,ITGA5 can promote the progression of oral squamous cell carcinoma by activating PI3K/AKT signaling pathway [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. ITGA5 is aberrantly expressed in a variety of malignant tumors, and regulates the occurrence and development of tumors by participating in the proliferation, migration and invasion of tumor cells. The main mechanism of ITGA5 regulating tumorigenesis and development is through mirna-ITGA5 and ITGA5-PI3K/AKT/FAK pathways, but the specific biological mechanism of its role and its upstream and downstream events are still not fully understood. Our study found that miR-330-5p mimics could significantly reduce the protein expression levels of p-FAK and p-AKT, and ITGA5 overexpression could significantly increase the protein expression levels of p-FAK and p-AKT. After CO transfection, miR-330-5p mimics could reverse the effect of ITGA5 overexpression on gastric cancer cells, indicating that miR-330-5p mimics may reverse the overexpression of ITGA5 gene and inhibit the activation of FAK/AKT signaling pathway by ITGA5. This is similar to the results of this study. Our study also found that miR-330-5p can promote the expression of ITGA5 and activate FAK/AKT pathway to affect the occurrence and development of gastric cancer.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn Conclusion, miR-330-5p inhibition promoted the expression of ITGA5 and activated FAK/AKT pathway, and promoted the proliferation, invasion, and migration of gastric cancer cells. The overexpression of miR-330-5p inhibited the expression of ITGA5 and the activation of FAK/AKT pathway, thereby reducing the proliferation, invasion, and migration of gastric cancer cells. This study explored the inhibitory and regulatory mechanism of miR-330-5p on the function of the target gene ITGA5, providing a reliable theoretical basis for the role of miR-330-5p and ITGA5 in the diagnosis, treatment, and prognosis of gastric cancer.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consulted the final manuscript. Jun-fu Wang and Long-zi Liu conceived the research, analyzed the data and drafted the manuscript; Jun-wen Hu was responsible for collecting the data; Jian-ming Wei and Ting He participated in the interpretation and statistical analysis of the data; Jiang-nan Zhang was responsible for checking and revising the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported bythe National Natural Science Foundation of China (grant no.82260465, 82260560 and 82002603); the Science and technology plan of Jiangxi Provincial Health Commission (grant no.202310237); the Science and technology plan of Jiangxi Provincial Administration of traditional Chinese Medicine (grant no.2023B1021) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of General Surgery, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang 330031, China.\u0026nbsp;\u003csup\u003e2\u003c/sup\u003eDepartment of General Surgery, Second Affiliated Hospital of Nanchang University, No.1, Minde Road, Nanchang330006, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, et al. 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Arch Oral Biol, 2019, 101: 85-91．\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miR-330-5p, ITGA5, gastric cancer, tumor progression","lastPublishedDoi":"10.21203/rs.3.rs-4747650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4747650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND:\u003c/strong\u003e ITGA5 is an oncogene that performs its biological function by integrating the intracellular structure and extracellular matrix. Our research group found that ITGA5 is a gastric cancer-related gene highly expressed in this tumor and is closely related to its proliferation and metastasis. The ITGA5 gene is regulated by multiple miRNAs during the occurrence and development of tumors. This study aimed to explore the role of targeting miRNAs upstream of ITGA5 in the regulation of the proliferation, invasion, and migration of gastric cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS: \u003c/strong\u003eThe target miRNA molecules regulating the ITGA5 gene were predicted by four bioinformatics databases (TargetScan、miRDB、miRTarBase and mirDIP), and the unreported miRNAs with high correlation were selected and their expression in gastric cancer was assessed by qRT-PCR and western blot. The miRNAs with potential targeting abilities were further verified by dual luciferase reporter gene experiment. The effects of miR-330-5p and ITGA5 on the proliferation, invasion, and migration of gastric cancer cells were evaluated by CCK8, clonogenic assay, and Transwell chamber assay, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS: \u003c/strong\u003eSix miRNAs (miR-26a-5p、miR-92a-3p、miR-148a-3p、miR-148b-3p、miR-330-5p and miR-152-3p) with high stability and conservation were found, and miR-330-5p was the one targeting and regulating ITGA5. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that miR-330-5p mimic significantly inhibited the proliferation, invasion, and migration of gastric cancer cells compared with the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The transfection of miR-330-5p mimic into gastric cancer cells overexpressing ITGA5 (OE-ITGA5) resulted in a significant reversion of the promoting effect of OE-ITGA5 on the proliferation, invasion, and migration of gastric cancer cells (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). In addition, miR-330-5p mimic reduced ITGA5 expression in gastric cancer cells and partially reversed the FAK/AKT signaling pathway activated by the ITGA5 gene. miR-330-5p inhibitor increased ITGA5 expression in gastric cancer cells, and they partially reversed the FAK/AKT signaling pathway blocked by sh-ITGA5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSIONS: \u003c/strong\u003eITGA5 was promotive for GC tumor growth and cell biological behaviors, and miR-330-5p targeted 3'-UTR of ITGA5 and inhibited its expression. ITGA5 was expected to become a new molecular marker, with miR-330-5p representing a novel therapeutic target for GC. This discovery provides a theoretical basis to further understand the mechanism related to the occurrence and development of gastric cancer, improving the diagnosis and prognosis while discovering a new therapeutic target.\u003c/p\u003e","manuscriptTitle":"Mechanism of action of miR-330-5p targeting ITGA5 in the regulation of proliferation, migration, and invasion of gastric cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-14 05:18:18","doi":"10.21203/rs.3.rs-4747650/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-26T07:59:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-23T02:32:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-23T02:31:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-07-16T06:58:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3337088e-fe7a-47bc-9d52-115611eda22f","owner":[],"postedDate":"August 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:07:59+00:00","versionOfRecord":{"articleIdentity":"rs-4747650","link":"https://doi.org/10.1186/s12885-026-15676-1","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2026-02-11 15:59:17","publishedOnDateReadable":"February 11th, 2026"},"versionCreatedAt":"2024-08-14 05:18:18","video":"","vorDoi":"10.1186/s12885-026-15676-1","vorDoiUrl":"https://doi.org/10.1186/s12885-026-15676-1","workflowStages":[]},"version":"v1","identity":"rs-4747650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4747650","identity":"rs-4747650","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}