miR-654-5p promotes gastric cancer progression via the GPRIN1/NF-κB pathway.

BACKGROUND: Gastric carcinoma (GC) ranks the fifth most common cancer worldwide, with high incidence and mortality rates. Numerous microRNAs (miRNAs), including miR-654-5p, have been implicated in the pathophysiological processes of tumorigenesis. Nevertheless, the mechanism of miR-654-5p in GC is unclear.
OBJECTIVES: Our study is devoted to exploring the function and molecular mechanism of miR-654-5p on the malignant cell behaviors of GC.
METHODS: The gene expression was detected by reverse transcription quantitative polymerase chain reaction. GC cell proliferation and motion were assessed by colony formation assay and transwell assay. The binding capacity between miR-654-5p and G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) was explored by luciferase reporter and RNA pulldown assays. The protein levels were detected by Western blotting.
RESULTS: miR-654-5p expression was higher in GC cells and tissues than control cells and tissues. miR-654-5p promoted GC cell growth and motion. Moreover, our findings showed that miR-654-5p was bound with GPRIN1. Importantly, downregulation of GPRIN1 rescued the inhibitory influence of miR-654-5p knockdown on GC cell malignant behaviors. Additionally, miR-654-5p activated the nuclear factor kappa-B (NF-κB) pathway by regulation of GPRIN1.
CONCLUSIONS: miR-654-5p facilitated cell proliferation, migration, and invasion in GC via targeting the GPRIN1 to activate the NF-κB pathway.

Introduction

Gastric carcinoma (GC) is one of the most common malignant tumors worldwide due to its high incidence and mortality rates [1]. Every year about one million people are diagnosed with gastric cancer all over the world, of whom about three quarters die from this horrible disease [2]. The incidence rates of GC vary significantly between different genders and across different countries [3]. Despite greatly improved GC treatments, the prognosis is still unsatisfactory. Additionally, the overall 5-year survival rate and disease-free rate are very low in most areas of the world [4]. Thus, exploring the molecular basis of GC is essential for developing novel therapeutic treatments to improve the current poor survival rate.

MicroRNAs (miRNAs) are short noncoding RNAs, which are 22 nucleotides long and modulate gene expression in animals, plants, and protozoa [5,6]. miRNAs often bind to specific mRNAs in the 3′ untranslated region (3′ UTR) to repress protein synthesis, indicating its function after transcription [7]. Furthermore, miRNAs have been proposed to participate in not only biological processes, but also the development of numerous human diseases including cancers [8]. In almost all kinds of cancers, miRNAs are dysregulated and serve as either tumor promoters or suppressors [9,10]. Additionally, these aberrantly expressed miRNAs have significant clinical use along with diagnostic and prognostic values in human cancers [11]. Therefore, miRNAs can be recognized as biomarkers in cancers, providing novel therapeutic approaches for cancer treatment [12,13]. Increasing studies clarified that miR-654-5p is abnormally expressed in lots of cancers such as colorectal cancer, colon cancer, breast cancer, and ovarian cancer, and thus, regulates the development of cancers by targeting specific genes [14,15]. All the findings suggested that miR-654-5p may promote tumorigenesis. Previously, it has been reported that miR-654-5p downregulation influenced transmembrane protein 52B (TMEM52B, also abbreviated as C12orf59) expression in GC [16]. Nevertheless, the detailed molecular mechanism of miR-654-5p in GC requires to be elucidated.

Our previous study revealed that G protein-regulated inducer of neurite outgrowth 3 (GPRIN3) was modulated by miR-6838-5p to affect cell proliferation and motion in GC [17]. As another member of GPRIN family, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) has prognostic value and exerts biological functions in lung cancer, regulating cell epithelial–mesenchymal transition to promote lung cancer growth and metastasis, which indicates that GPRIN1 may become a useful target for lung cancer treatments [18]. Nevertheless, the function of GPRIN1 in gastric cancer is still unclear.

Nuclear factor kappa B (NF-κB) consists of a series of transcription factors which play an indispensable role during processes mediating the response to diverse external stimuli, and thus, is a pivotal element in many physiological and pathological processes [19]. The NF-κB pathway promotes the growth and survival of many solid and hematological malignancies, affecting the development of human cancers and then providing numerous targets for novel anticancer therapies [20,21]. For example, the NF-κB pathway is activated to influence nasopharynx cancer recrudescence [22]. The NF-κB pathway is blocked by Morin to suppress ovarian cancer cell proliferation, inhibit the inflammatory response, and reduce tumor size [23]. miR-10b facilitated GC cell proliferation and migration by inhibiting CUB (complement protein C1r/C1s, Uegf, and Bmp1) and Sushi multiple domains 1 expression, which caused the activating of the NF-κB pathway [24]. miR-3664-5p suppressed the growth and motion of GC cells through inhibiting the NF-κB pathway via regulating metadherin [25]. Nevertheless, whether miR-654-5p influences the NF-κB pathway via regulating GPRIN1 is unknown.

Our study was designed to reveal the effects of GPRIN1 in proliferation, migration, invasion of GC cells, and the downstream pathway of GPRIN1. The results provide great possibility for the targeted treatment of GC, driving the development of its therapeutic strategies.

Materials and methods

Clinical samples

Totally, 36 pairs of GC and normal tissue samples were collected from Ningbo First Hospital (Zhejiang, China). All samples were stored at −80°C for further use. This study gained approval of the Ethics Committees of Ningbo First Hospital (Zhejiang, China). Every participant had been informed of the purpose of tissue sample collection and signed an informed consent form.

Cell culture

GC cell lines (KATO III and HGC-27) and a normal gastric epithelial cell line (GES-1) were bought from Gaining Biological (Shanghai, China). The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere at 37°C. In some experiments, GC cells were treated with Ammonium pyrrolidinedithiocarbamate (PDTC) at the concentration of 50 μmol/L for 24 h to inhibit the NF-κB pathway [26].

Cell transfection

miR-654-5p inhibitor was employed to knockdown miR-654-5p expression with negative control (NC) inhibitor as a control. The coding sequence of GPRIN1 was cloned and inserted into the pcDNA3.1 plasmid. The above plasmids and oligonucleotides were purchased from GenePharma (Shanghai, China). Next, we used Lipofectamine 2000 (Beyotime, Shanghai, China) for transfection of the plasmids and oligonucleotides into HGC-27 and KATO III cells following the recommended instructions. Forty-eight hours later, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to assess the transfection efficiency.

RT-qPCR

We used TRIzol reagent (Beyotime) to extract total RNA from GC cells and tissues following the protocols of manufacturers. SuperScript Reverse Transcriptase Ⅲ (Takara, Dalian, China) was used for reverse transcription of RNAs into complementary DNA (cDNA). RT-qPCR was performed with SYBR Premix Ex Taq II (Takara, Dalian, China) on an Mx3000P instrument (Strata-gene, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and RNU6 (U6) were considered as the corresponding internal controls. The 2−ΔΔCt method was used for calculation of the fold changes. All primer sequences are available under requirement.

Cell counting kit-8 (CCK-8) assay

pcDNA3.1/GPRIN1 or empty pcDNA3.1 was transfected into HGC-27 and KATO III cells, and then cells were incubated in 96-well plates (2 × 104 cells/well) for 24 h, 48 h, and 72 h. Each well was added with 10 μL of CCK-8 reagent (Dojindo, Tokyo, Honshu, Japan) for another 2 h of cultivation. Finally, the microplate reader (Thermo-Fisher Scientific) was used to determine the absorbance at 450 nm.

Transwell assay

Cell migratory and invasive capabilities were evaluated by transwell assays. Transwell chamber (Corning Inc., Corning, NY) was applied to measure cell motion. In brief, cells in medium containing 1% FBS were incubated in the upper well at 37°C for 24 h. Cells failing to migrate were removed by cotton swabs and 100% methanol was utilized to fix migratory cells followed by staining with 0.5% crystal violet. The inverted microscope (Olympus Corporation, Tokyo, Japan) was utilized to image the cells. Cell invasive ability was detected by Transwell invasion assay, which is like the migration assay except that the chambers contained 1 mg/mL matrigel (Corning Inc.). Number of migratory and invaded cells was counted to measure cell migratory and invasive abilities, respectively.

Colony formation assay

Transfected GC cells were diluted and seeded onto 6-well plates (1,000 cells/well). HGC-27 and KATO III cells were cultured for 14 days with 5% CO2 and 95% air at 37°C. After fixation and staining, the colony numbers were observed and counted under a microscope.

RNA pulldown assay

RNA pulldown assay was conducted to examine the binding of GPRIN1 and miR-654-5p. After lysing, GC cells were treated with RNase-free DNase I (Beyotime). Afterwards, the biotin-labeled wild-type miR-654-5p (bio-miR-654-5p WT), biotin-labeled mutant miR-654-5p (bio-miR-654-5p MUT) or bio-NC, and streptavidin-coated magnetic beads (Sigma-Aldrich, St Louis, MO, USA) were incubated with cell lysate at 4°C for 3 h. Finally, RNA samples were isolated by TRIzol and subjected to RT-qPCR analysis.

Dual luciferase reporter assay

The 3′ UTR of GPRIN1 was amplified from the full-length cDNA by PCR. After digestion with XhoI and NotI, the 3′-UTR fragment was cloned into the XhoI/NotI sites of the psiCHECK-2 vector (Promega) to obtain a wild-type (Wt) GPRIN1 3′ UTR-luciferase reporter plasmid. A mutant (Mut) GPRIN1 3′ UTR-luciferase reporter plasmid was constructed using a QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime). Luciferase reporter assay was performed by cotransfection with 250 ng of luciferase reporter vectors and miR-654-5p inhibitor or NC inhibitor (10 nM) using Lipofectamine 2000 (Invitrogen). After 48 h, the GC cells were assayed for luciferase activities on a Dual Luciferase Reporter Assay System (Promega). The firefly luciferase activity values were normalized to Renilla luciferase values to exclude the effects of difference in transfection efficiency.

Western blotting

The protein contents in GC cells and tissues were isolated by radio-immunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride. The concentration of protein was determined using a bicinchoninic acid kit (Beyotime). Afterwards, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, USA) after being separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. After being blocked with 5% nonfat milk powder, the membranes were cultured with primary antibodies at 4°C overnight, followed by cultivation with secondary antibodies (TransGen Biotech, Beijing, China) for 1 h. The signals of protein bands were observed, and the images were analyzed using ImageJ software (National Institutes of Health, MD, USA).

NF-κB activity assay

KATO III and HGC-27 cells were cultured for 24 h in 6-well plates (5 × 104 cells/well). Next, GC cells were transfected with the NF-κB reporter luciferase construct. After 6 h, cells were washed and co-transfected with miR-654-5p inhibitor and sh-GPRIN1 for another 24 h. Afterwards, transfected cells were washed in PBS and harvested in lysis buffer. A Promega luciferase assay kit was then used to examine luciferase activity. Finally, the experimental values were recorded in comparison with the positive and negative control samples.

Animal studies

Twenty-four-week-old female BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and raised in the specific pathogen-free animal room. All mice experiments were performed according to the guideline of the Ethics Committee of the Institutional Animal Care and Use Subcommittee of the Ningbo First Hospital (Zhejiang, China). Mice were randomly divided into four groups: (i) NC inhibitor (n = 5), (ii) miR-654-5p inhibitor (n = 5), (iii) miR-654-5p inhibitor + sh-GPRIN1 (n = 5), and (iv) miR-654-5p inhibitor + sh-GPRIN1 + PDTC (n = 5). 2 × 106 HGC-27 cells stably transfected by lentivirus conveying NC inhibitor, miR-654-5p inhibitor, or sh-GPRIN1 were injected into the left subcutaneous armpit areas of mice to develop a tumor-bearing mice model. After tumor inoculation for 7 days, PDTC (50 mg/kg/day) was intraperitoneally daily injected into mice in the miR-654-5p inhibitor + sh-GPRIN1 + PDTC group. The tumor volume was calculated (V = 0.5 × A × B 2, V refers to volume, A refers to longest diameter, B refers to the shortest diameter) every 3 days. On day 21, mice were euthanized, and the tumors were collected for weighing.

Statistical analysis

The SPSS 20.0 software was employed to analyze relative statistical data. The comparison between different groups was analyzed by the Student’s t-test or one-way ANOVA. Pearson’s correlation analysis was conducted to evaluate gene expression correlation. p values less than 0.05 were significant.

Results

GPRIN1 is downregulated in GC cells and tissues

Although GPRIN1 was proved to be a vital regulator in numerous human cancers, its role in GC remains unclear yet. To clarify this, we first compared GPRIN1 expression between GC and NC. RT-qPCR showed that GPRIN1 expression was lower in GC tissues than in non-tumor tissues (Figure 1a). According to starBase website (http://starbase.sysu.edu.cn/), low expression level of GPRIN1 is closely associated with the low survival rates of GC patients (Figure 1b). Furthermore, GPRIN1 expression was decreased at the mRNA and protein levels in GC cells (HGC-27 and KATO III) compared to normal gastric epithelial cell line GES-1 (Figure 1c and d). In conclusion, GPRIN1 expresses at a low level in GC.

Figure 1

GPRIN1 is downregulated in GC cells and tissues. (a) Relative GPRIN1 mRNA expression level in 36 GC tissues compared to that in 36 normal gastric tissues was analyzed by RT-qPCR. (b) The relationship between GPRIN1 expression and overall survival of GC patients was predicted by starBase website (http://starbase.sysu.edu.cn/). (c) Relative GPRIN1 mRNA expression level in GC cell lines (KATO III and HGC-27) compared to that in a normal gastric epithelial cell line (GES-1) was evaluated through RT-qPCR. (d) GPRIN1 protein levels in GC cell lines (KATO III and HGC-27) and in a normal gastric epithelial cell line (GES-1) were detected by Western blotting, and the quantitative analysis was performed using ImageJ software. ** p < 0.01, *** p < 0.001.

GPRIN1 inhibits growth and motion of GC cells

To explore the underlying function of GPRIN1 in GC, GPRIN1 was overexpressed in HGC-27 and KATO III cells to examine potential gain-of-functions. The RT-qPCR results revealed that the GPRIN1 overexpression vector significantly elevated GPRIN1 expression in HGC-27 and KATO III cells (Figure 2a). Next, we detected the regulatory effects of GPRIN1 on GC cell proliferation and motion. CCK-8 assay showed that GPRIN1 overexpression suppressed GC cell viability compared with the NC group (Figure 2b). Colony formation assay demonstrated that GC cell proliferation was inhibited by GPRIN1 (Figure 2c). The results of Transwell assays demonstrated that GPRIN1 attenuated the migratory and invasive ability of GC cells (Figure 2d and e). Collectively, GPRIN1 inhibited GC cell proliferation, migration, and invasion.

Figure 2

GPRIN1 inhibits proliferation, migration, and invasion of GC cells. (a) HGC-27 and KATO III cells were transfected with pcDNA3.1/GPRIN1, and the overexpression efficiency was examined by RT-qPCR. (b) GC cell viability after GPRIN1 overexpression was detected by a CCK-8 assay. (c) Influences of GPRIN1 on GC cell proliferation were explored by a colony formation assay. (d and e) Transwell assays were performed to reveal the migration and invasion of GC cells transfected with pcDNA3.1/GPRIN1. ** p < 0.01, *** p < 0.001.

miR-654-5p targets GPRIN1 in GC cells

miRNAs were confirmed to relate to GC progression [27,28,29]. Increasing evidence suggested that miR-654-5p had regulatory influence on numerous human cancers [14,15]. To explore the role of miR-654-5p in GC, we first detected miR-654-5p level in GC cells. RT-qPCR indicated that miR-654-5p expression level was increased in GC tissues (Figure 3a). StarBase website also showed that miR-654-5p level was higher in GC samples compared to normal samples (Figure 3b). High expression of miR-654-5p is closely associated with poor overall survival of GC patients according to Kaplan-Meier Plotter website (https://kmplot.com/) (Figure 3c). Furthermore, a negative expression correlation between miR-654-5p and GPRIN1 was observed in 36 collected GC tissues (Figure 3d). miR-654-5p was also overexpressed in GC cells compared to that in normal cells (Figure 3e). To figure out the exact role of miR-654-5p in GC, a series of loss-of-function experiments were taken by transfecting miR-654-5p inhibitor or NC inhibitor into GC cells. RT-qPCR showed that miR-654-5p inhibitor decreased miR-654-5p expression in HGC-27 and KATO III cells (Figure 3f). Importantly, both GPRIN1 mRNA expression and protein level were significantly increased by miR-654-5p inhibition (Figure 3g). Additionally, an RNA pulldown assay revealed that GPRIN1 was highly enriched in bio-miR-654-5p WT in comparison with bio-NC (Figure 3h), suggesting that GPRIN1 was bound to the wild-type miR-654-5p. GPRIN1 was not significantly pulled down by bio-miR-654-5p MUT. Results of the luciferase reporter assay revealed that luciferase activity of psiCHECK-2-GPRIN1 3′UTR Wt was higher in HGC-27 and KATO III cells transfected with miR-654-5p inhibitor than those transfected with NC inhibitor. There was no significant difference between luciferase activity of psiCHECK-2-GPRIN1 3′UTR Mut under transfections of miR-654-5p inhibitor and NC inhibitor (Figure 3i). Figure S1a revealed that GPRIN3 expression was not impacted by miR-654-5p inhibitor. miR-654-5p inhibitor had more stimulating effects on GPRIN1 expression than on C12orf59 expression in GC cells.

Figure 3

miR-654-5p targets GPRIN1 in GC cells. (a) Relative miR-654-5p level in 36 GC tissues compared to that in 36 normal tissues was assessed by RT-qPCR. (b) miR-654-5p expression pattern in 372 GC samples and 32 normal samples was analyzed according to starBase website (http://starbase.sysu.edu.cn/). (c) High level of miR-654-5p was related to poor overall survival of GC patients according to Kaplan-Meier Plotter website. (d) The correlation between miR-654-5p and GPRIN1 expression levels in GC tissues was analyzed by Spearman’s correlation analysis. (e) Relative miR-654-5p level in GC cells compared to that in GES-1 cells was measured by RT-qPCR. (f) HGC-27 and KATO III cells were transfected with miR-654-5p inhibitor, and RT-qPCR assessed the transfection efficiency. (g) RT-qPCR and Western blotting were conducted to reveal relative mRNA and protein levels of GPRIN1 in GC cells transfected with miR-654-5p inhibitor compared to GC cells transfected with NC inhibitor. (h) An RNA pulldown assay showed the relative enrichment of GPRIN1 in bio-miR-654-5p WT and bio-miR-654-5p MUT compared to that in bio-NC. (i) A luciferase reporter assay was conducted to reveal the binding of miR-654-5p and GPRIN1 3′UTR. ** p < 0.01, *** p < 0.001.

miR-654-5p downregulation inhibits proliferation, migration, and invasion of GC cells by GPRIN1

A series of rescue experiments were performed to verify whether GPRIN1 contributes to the miR-654-5p-induced tumor promotion in GC. We transfected sh-GPRIN1 into miR-654-5p-silenced HGC-27 and KATO III cells. The findings indicated that miR-654-5p downregulation inhibited GC cell proliferation and motion, while transfection of sh-GPRIN1 rescued the inhibitory effects of miR-654-5p inhibitor on GC cells (Figure 4a–d). Figure S1b revealed that the rescue effect of sh-GPRIN1 on miR-654-5p inhibitor in GC cell viability was more significant than that of sh-C12orf59, indicating that GPRIN1 plays a more important role than C12orf59 in miR-654-5p-mediated GC cells.

Figure 4

miR-654-5p knockdown inhibits proliferation, migration, and invasion of GC cells by GPRIN1. (a–d) CCK-8, colony formation, and Transwell assays revealed that GPRIN1 knockdown rescued the inhibition in the proliferation, migration, and invasion of HGC-27 and KATO III cells caused by miR-654-5p inhibitor. ** p < 0.01, *** p < 0.001.

miR-654-5p regulates the NF-κB pathway by GPRIN1

The NF-κB pathway was proposed many times to participate in GC tumorigenesis [30,31,32,33]. We inferred that miR-654-5p regulates the NF-κB pathway by GPRIN1. To examine the connection between GPRIN1 and the NF-κB pathway, pcDNA3.1/GPRIN1 and the corresponding control were transfected into GC cells. NF-κB activity assay showed that NF-κB transcriptional activity was suppressed by GPRIN1 overexpression compared with the NC group (Figure 5a). Furthermore, Western blotting indicated that GPRIN1 overexpression significantly decreased the protein levels of p-NF-κB p65 and p-NF-κB IκBα. Basal protein levels of NF-κB p65 and NF-κB IκBα are not significantly influenced (Figure 5b). We found that silencing of miR-654-5p suppressed the NF-κB pathway, while GPRIN1 knockdown rescued the inhibitory influence of miR-654-5p inhibitor on NF-κB transcriptional activity (Figure 5c). The decreased protein levels of p-NF-κB p65 and p-NF-κB IκBα caused by miR-654-5p downregulation were reversed by GPRIN1 knockdown (Figure 5d). In summary, miR-654-5p activated the NF-κB pathway by regulation of GPRIN1. Moreover, we detected the influence of PDTC, an inhibitor of the NF-κB pathway, on the miR-654-5p/GPRIN1 axis. Figure 5e revealed that PDTC had no significant effects on miR-654-5p expression. Figure 5f revealed that PDTC influenced neither GPRIN1 expression nor the effect of silenced miR-654-5p on GPRIN1 expression.

Figure 5

miR-654-5p modulates the NF-κB pathway by GPRIN1. (a) NF-κB transcriptional activity was measured in HGC-27 and KATO III cells after GPRIN1 overexpression. (b) HGC-27 and KATO III cells were transfected with pcDNA3.1/GPRIN1 and empty pcDNA3.1. Protein levels of NF-κB p65, p-NF-κB p65, IκBα, and p-NF-κB IκBα in HGC-27 and KATO III cells were examined by Western blotting. (c) NF-κB transcriptional activity in HGC-27 and KATO III cells co-transfected with miR-654-5p inhibitor and sh-GPRIN1 was measured. (d) GC cells were co-transfected with miR-654-5p inhibitor and sh-GPRIN1 and corresponding controls. Protein levels of NF-κB p65, p-NF-κB p65, IκBα, and p-NF-κB IκBα were detected by Western blotting. (e) Influence of PDTC on relative expression of miR-654-5p was detected by RT-qPCR. (f) Relative expression of GPRIN1 in the control, PDTC, miR-654-5p inhibitor, miR-654-5p inhibitor + PDTC, miR-654-5p inhibitor + sh-GPRIN1, and miR-654-5p inhibitor + sh-GPRIN1 + PDTC groups was detected by RT-qPCR. *p < 0.05, ** p < 0.01.

Role of the miR-654-5p/GPRIN1/NF-κB pathway in a tumor-bearing mouse model

miR-654-5p expression was lower, while GPRIN1 expression was higher, in tumors of the miR-654-5p inhibitor group than NC inhibitor group. PDTC had no significant effects on miR-654-5p or GPRIN1 expression (Figure 6a). Mice with miR-654-5p deficiency exhibited smaller tumor size and reduced tumor volume and weight than control mice. Mice in the miR-654-5p inhibitor + sh-GPRIN1 group showed increased tumor size, volume, and weight than mice in the miR-654-5p inhibitor group. Moreover, reduced tumor size, volume, and weight were observed in mice with NF-κB inhibition by PDTC administration (Figure 6b–d). All these findings implied that silenced miR-654-5p suppressed GC tumor growth in mice by the GPRIN1/NF-κB pathway.

Figure 6

Role of the miR-654-5p/GPRIN1/NF-κB pathway in a tumor-bearing mouse model. (a) Expression of miR-654-5p and GPRIN1 in tumors of mice in the NC inhibitor, miR-654-5p inhibitor, miR-654-5p inhibitor + sh-GPRIN1, and miR-654-5p inhibitor + sh-GPRIN1 + PDTC groups was detected by RT-qPCR. (b) Images of tumors. (c) Tumor volume was recorded every three days. (d) Tumor weight was recorded at the end of the animal experiments. ** p < 0.01, *** p < 0.001.

Discussion

As one of the leading cancers worldwide, GC is a great threat to human health due to its high morbidity and mortality [34]. It is crucial to clarify the underlying molecular mechanisms of GC to diagnose tumors and develop effective therapeutic strategies [35]. Although increasing studies reported that many miRNAs participated in biological processes associated with GC progression, including cell proliferation, differentiation, apoptosis, and invasion [36,37,38,39], further investigations are required to ascertain the underlying mechanisms of the miRNA-induced network in GC. Furthermore, accumulating evidence demonstrated that miR-654-5p is a significant regulator involved in numerous cancers. For instance, miR-654-5p is upregulated to regulate colorectal cancer progression through targeting hematopoietic cell-specific Lyn substrate 1-associated protein X-1 [14]. miR-654-5p targets growth factor receptor-bound protein 2-related adaptor protein to drive oral squamous cell carcinoma cell growth and migration [40]. miR-654-5p suppresses ovarian cancer tumorigenesis by targeting CUB domain containing protein 1 and pleiomorphic adenoma gene 1 like zinc finger 2 [41]. Nevertheless, the specific role and detailed mechanism of miR-654-5p in GC require further investigation. Our work was designed to explore the biological function, as well as the molecular mechanisms of miR-654-5p in GC. Bioinformatic analysis showed elevated expression of miR-654-5p in GC cells and tissues. Additionally, miR-654-5p downregulation suppressed the growth and motion of GC cells. Finally, high expression of miR-654-5p is associated with the poor overall survival of GC patients. Overall, these findings illustrated that miR-654-5p promoted the progression of GC.

Previously, GPRIN3, a member of GPRIN family, has been indicated to be modulated by miR-6838-5p to affect cell growth and migration in GC [17]. Herein, we explored the role of another member of GPRIN family, GPRIN1, in the development of GC. Our study found that GPRIN1 expression was downregulated in GC cells and tissues. Furthermore, GPRIN1 inhibited the malignant behaviors of GC cells. GPRIN1 was also confirmed to be a miR-654-5p target. Additionally, GPRIN1 was found to be negatively modulated by miR-654-5p in GC. Rescue assays indicated that miR-654-5p facilitated the development of GC via modulating GPRIN1.

The NF-κB pathway has been widely acknowledged as a significant regulator during biological processes in gastric cancer [42,43,44]. In our current study, miR-654-5p downregulation blocked the NF-κB signaling pathway, while the inhibitory effects were reversed by GPRIN1 knockdown. It could be inferred that the NF-κB pathway was involved in the miR-654-5p-mediated regulation of GC. Importantly, the miR-654-5p/GPRIN1 axis was not impacted by the NF-κB pathway.

In conclusion, our study revealed that miR-654-5p promoted cell proliferation, migration, and invasion of GC via targeting GPRIN1 and activating the NF-κB pathway, which provided novel sights for GC treatment.