miR-223-3p promotes cell proliferation and invasion by targeting Arid1a in gastric cancer

Abstract

Accumulating evidence has indicated that microRNAs can regulate downstream signaling pathways and play an important role in various tumors. In this study, we found that miR-223-3p was differentially expressed in 40 paired gastric cancer tissues and adjacent tissues and that miR-223-3p was positively correlated with tumor invasion depth and lymph node metastasis. Luciferase reporter assay confirmed that Arid1a was the target gene of miR-223-3p. Functional assays showed that miR-223-3p promoted the proliferation and invasion of gastric cancer cells by regulating the expression of Arid1a. We also confirmed that miR-223-3p regulated the growth of gastric cancer cells in vivo, while an antagomir against miR-223-3p significantly inhibited tumor growth. In conclusion, our results demonstrated that miR-223-3p inhibits gastric cancer cell progression by decreasing the expression of Arid1a. Therefore, miR-223-3p may act as a potential therapeutic target for patients with gastric cancer.

Introduction

Gastric cancer is one of the most common malignant tumors in the world. Approximately 1,033,701 patients were diagnosed with gastric cancer, ranking it the fifth among all new cancer cases in 2018; moreover, approximately 782,682 people died of gastric cancer, ranking it the third in cancer-related deaths [1]. According to data reported by Chen et al. [2], there were 679,100 new cases of gastric cancer and 498,000 deaths in China in 2015, the morbidity was ranked the second and the mortality was ranked the third. Although the diagnosis and treatment of early gastric cancer have been greatly improved, nearly 80% of patients are in an advanced stage when it is diagnosed. These patients have no opportunity to undergo surgery and have few options except for chemotherapy. They all have a short survival time, with a 5-year survival rate of less than 20% [3]. Traditional chemicals have a limited effect on prolonging the survival time of patients with gastric cancer. In recent years, with the development of molecular biology, targeted treatments such as anti-HER2 [4] and anti-VEGFR-2 [5] therapies have achieved good results. However, because of the heterogeneity of gastric cancer patients and highly complex epigenetic inheritance, there are no clear predictors of efficacy and prognosis.

MicroRNAs (miRNAs), a class of endogenous small RNAs of approximately 20–24 nucleotides in length, play a variety of important regulatory roles in cells [6]. More than 1500 miRNAs have been discovered in the human genome, and each miRNA can target multiple mRNAs; moreover, each mRNA can also be regulated by several miRNAs [7,8]. miRNAs are involved in regulating cell growth, proliferation, differentiation, and apoptosis by participating in several signaling pathways, such as the MAPK [9] and PI3K-AKT [10] signaling pathways; moreover, they also modulate cell invasion and metastasis through microvesicles and exosomes [11,12]. Therefore, using miRNAs as a target of tumor therapy has become a hot topic in the field of gene regulation research. Guo et al. [13] reported that miR-371a-3p was upregulated in gastric cancer tissues, and its expression level was closely related to the prognosis of patients. The silencing of miR-371a-3p was confirmed to inhibit the invasion and migration of gastric cancer in vivo and in vitro. This suggests that miRNA may be a potential target for the treatment of gastric cancer.

miR-223-3p was found to be upregulated in a variety of tumors, such as non-small cell lung cancer [14], clear cell renal carcinoma [15], and bladder cancer [16]. Yang et al. [17] reported that miR-223-3p might play a crucial role in the progression of Helicobacter pylori-related chronic gastritis developing into gastric cancer. However, the mechanism is still unclear.

In our previous study [18], we demonstrated that the expression level of Arid1a is closely related to the prognosis of patients with gastric cancer. We found that there was a complementary sequence between miR-223-3p and the 3'UTR of Arid1a mRNA, which was predicted by TargetScan and miRDB. Therefore, we speculated that there was a close relationship between Arid1a and mir-223-3p.

In this study, we investigated the expression of miR-223-3p and Arid1a in gastric cancer cell lines and 40 paired gastric samples. We identified that Arid1a is the target gene of miR-223-3p and revealed that miR-223-3p promotes tumor cell proliferation and invasion by modulating the expression of Arid1a.

Material and Methods

Patient and tissue specimens

The gastric cancer tissues and matched paracancerous tissues in this study were obtained from 40 patients with gastric cancer who were admitted to the First Affiliated Hospital of Wannan Medical College from January 1, 2017 to April 30, 2017. All samples were confirmed by two independent pathologists. The patients enrolled in our study did not receive chemotherapy or radiotherapy before surgery. Clinicopathological features were collected, including tumor infiltration depth, lymph node metastasis, vascular invasion, and disease recurrence time (Table 1). The gastric cancer tissues and adjacent tissues were stored in liquid nitrogen. Informed consent was obtained from patients. The study was performed in accordance with the Helsinki Declaration and approved by the Ethics Committee of the First Affiliated Hospital of Wannan Medical College (approval No. 2018-34).

Cell culture

Gastric cancer cell lines (MKN-45, SGC-7901, and AGS) and the normal human gastric cell line GES-1 were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI 1640 medium (Media, Durham, USA) containing 10% calf serum and incubated in an atmosphere with 5% CO₂ at a temperature of 37°C.

RNA extraction and quantitative reverse transcriptase–polymerase chain reaction

Trizol (Invitrogen, Carlsbad, USA) was used to extract RNA from all tissues and cells according to standard protocols. The concentration and purity of RNA were measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, USA). Reverse transcription was performed to produce cDNA by using the Reverse Transcription kit (Ribo, Guangzhou, China). Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) was performed on the 7500 PCR operating system using the Bulge-Loop miRNA qRT-PCR Starter kit (Ribo). The primer sequences used were as follows: U6, 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse); miR-223-3p, 5′-AGCTGGTGTTGTGAATCAGGCCG-3′ (forward) and 5′-TGGTGTCGTGGAGTCG-3′ (reverse); GAPDH, 5′-GAACGGGAAGCTCACTGG-3′ (forward) and 5′-GCCTGCTTCACCACCTTCT-3′ (reverse); and Arid1a, 5′-CTTCAACCTCAGTCAGCTCCCA-3′ (forward) and 5′-GGTCACCCACCTCATACTCCTTT-3′ (reverse). U6 was considered an internal control for miR-223-3p, and GAPDH was considered an internal control for Arid1a. The relative expression levels of miR-223-3p and Arid1a were normalized using the 2^-ΔΔCT calculation method [19].

Cell transfection

Mimics and inhibitors of miR-223-3p and their negative controls were chemically synthesized by Ribo. The sequences were as follows: miR-223-3p mimic, 5′-UGUCAGUUUGUCAAAUACCCCA-3′; miR-223-3p mimic negative control, 5′-UAGCUUAUCAGACUGAUGUUGA-3′ (forward) and 5′-UCAACAUCAGUCUGAUAAGCUA-3′ (reverse); miR-223-3p inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′; and miR-223-3p inhibitor negative control, 5′-UCAACAUCAGUCUGAUAAGCUA-3′. The transfection was performed according to the manufacturer’s protocol using Lipofectamine 3000 (Invitrogen). We added 500 μl of Opti-MEM, with a final concentration of 100 nM mimics/inhibitor or their negative control, and 2 μl of Lipofectamine 3000 into a 24-well plate seeded with cells. The overexpression plasmid of Arid1a and the empty pcDNA3.1 vector were purchased from Addgene (Watertown, USA). A total of 1 μg pcDNA-Arid1a or pcDNA3.1, 2 μl of Lipofectamine 3000, and 500 μl of Opti-MEM were added into a 24-well plate. Forty-eight hours after transfection, cells were harvested for further experiments.

Methyl thiazolyl tetrazolium assay

Cell proliferation was measured by MTT assay. Cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and incubated with 180 μl of the culture medium for 24, 48, and 72 hours. Subsequently, 20 μl of MTT solution was added per well, and incubated for 10 minutes. Then, 150 μl of Dimethyl Sulfoxide (DMSO) was added into each well, and a microplate reader was used to measure the optical density of each well at the wavelength of 490 nm.

Cell apoptosis and cell cycle analysis by flow cytometry

Forty-eight hours after transfection, floating cells and adherent cells cultured in 6-well plates were collected, centrifuged, and resuspended in 5 × binding buffer. Then, the cells were double-stained with Annexin V-FITC and propidium iodide. Cell apoptosis was analyzed by flow cytometry. For the detection of cell cycle, a total of 2 × 10⁵ cells were collected and centrifuged. Then, the cells were treated with a DNA staining solution and permeabilization solution for 30 minutes in the dark. The percentage of cells at various stages was detected and analyzed by flow cytometry.

Colony formation assay

To further investigate the proliferative capacity of gastric cancer cells, 48 hours after transfection, cells were seeded into 12-well plates at a density of 500 cells per well. The cells were incubated with medium containing 10% calf serum for 2 weeks. The medium was changed every 4 days until visible colonies were observed. After washing and fixation, colonies were stained with crystal violet. Colonies containing more than 50 cells were counted, and the results were analyzed with ImageJ software (Bethesda, USA).

Wound healing assay

The migration capacity of gastric cancer cells was assessed by wound healing assay. Forty-eight hours after transfection, cells were seeded into 6-well plates and incubated for 24 hours until cells were confluent. Then a 20-μl pipette tip was used to scratch the surface of the cell layer. Floating cells were washed away with phosphate-buffered saline (PBS), and the culture medium was replaced by serum-free medium. At 0 and 24 hours, the wound healing ability of the cells was observed under fluorescence microscope (EVOS FL Auto; Invitrogen, Carlsbad, USA). Images were acquired, and the migration ability of gastric cancer cells was analyzed by ImageJ software.

Cell invasion assay

Matrigel and RPMI 1640 were mixed at a ratio of 1:9. Then, 50 μl of the mixture was added to the upper chamber of a transwell system and incubated at 37°C for 5 hours to develop the Matrigel solution. Forty-eight hours after transfection, 200 μl of cell suspension, at a density of 10⁵ cells/ml in RPMI 1640, was added into the upper chamber, and 500 μl of medium containing 50% calf serum was added into the lower chamber. After incubation for 24 hours at 37°C, the cells that invaded the Matrigel were fixed and stained and then examined by fluorescence microscopy. Cells were counted and analyzed by ImageJ software.

Western blot analysis

Cells and tissues were harvested and lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) on ice. After centrifugation at 4°C for 30 minutes, the protein concentration of the lysate was measured using a NanoDrop 2000 Spectrophotometer. Next, the total protein was separated by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. After being blocked with 5% bovine serum albumin for 1 hour, the membrane was incubated with primary antibody overnight at 4°C. The next day, after extensive wash with PBS, the membrane was incubated for 2 hours at 37°C with the horseradish peroxidase (HRP)-conjugated secondary antibody. Finally, protein bands were visualized by using the Novex ECL Chemiluminescent Substrate Kit (Invitrogen) and analyzed by ImageJ software. The primary antibodies used were as follows: anti-Arid1a antibody (1:1000, ab242377; Abcam, Cambridge, UK) and anti-β-actin antibody (1:1000, ab8226; Abcam). β-actin was used as an internal control.

Luciferase reporter assay

A luciferase reporter assay was used to verify whether Arid1a directly targets miR-223-3p in gastric cancer cells. First, by using TargetScan [20] and miRDB [21], we predicted that the 3'UTR of Arid1a mRNA contains a site that binds to miR-223-3p. The wild-type and mutant-type 3' UTRs of Arid1a were amplified by PCR, and the products were cloned into a pMIR-REPORT vector (Addgene). Then, miR-223-3p mimic or its negative control was co-transfected into MKN-45 cells together with pMIR-REPORT vector containing Arid1a-wild-type 3' UTR or Arid1a-mutant 3' UTR. Forty-eight hours after transfection, luciferase activity was detected using a dual luciferase reporter assay system (Promega, Madison, USA). Renilla luciferase was used as an internal control.

Mouse xenograft model

A total of 2 × 10⁶ MKN-45 cells were subcutaneously inoculated into the left axilla of 4- to 6-week-old nude mice. All nude mice were purchased from the Experimental Animal Center of Nanjing Medical University (Nanjing, China). After 3 days of subcutaneous inoculation, tumor formation was observed. On the 8th day of subcutaneous injection, the mice were randomly divided into two groups (six in each group). AntagomiR-223-3p (5'-UsGsGGGUAUUUGACAAACUGsAsCsAs-Chol-3′) and antagomiR-NC (5'-CsAsGUACUUUUGUGUAGUAsCsAsAs-Chol-3′) (GenePharma, Shanghai, China) were injected intratumorally at a dose of 15 μg every 3 days. The length and width of all transplanted tumors were measured every 3 days using a vernier caliper. Sixteen days after the intratumoral injection, the nude mice were sacrificed, and the transplanted tumors were excised. The size and weight of the tumors were measured. The expression of miR-223-3p and Arid1a in tumor tissues was detected by RT-PCR, and the expression of Arid1a protein was detected by western blot analysis.

Immunohistochemical staining

The transplanted tumors were stripped from the nude mice, fixed with neutral formaldehyde, embedded in paraffin, and cut into 4-μm sections. The sections were heated in an oven at 60°C for 30 minutes and dewaxed with xylene. Then, 3% H₂O₂ was added for 10 minutes to remove endogenous catalase. After being blocked for 30 minutes, the sections were incubated with primary antibodies against Ki-67 (1:500, ab92742; Abcam) overnight at 4°C. The next day, the sections were washed with PBS and incubated with a HRP-conjugated secondary antibody for 30 minutes at 37°C. The signal was visualized with diaminobenzidine. The staining results were scored by two independent pathologists. All sections were examined by fluorescence microscopy. Staining was confirmed by counting the percentage of positive cells and the intensity of positive staining in five random high-power fields.

Statistical analysis

All data analysis in this study was performed with GraphPad Prism 7.0 (GraphPad Software, San Diego, USA). All experiments were repeated three times. Student’s t-test was used to test the significant differences between groups, and one-way ANOVA was used for comparisons among more than two groups. P < 0.05 was considered to be statistically significant. The Pearson coefficient was used to evaluate the correlation between miR-223-3p and Arid1a. Survival curves were estimated using the Kaplan-Meier method, and recurrence-free survival was assessed with the log-rank test.

Results

Expression of miR-223-3p and Arid1a in gastric cancer tissues and matched adjacent tissues

The expression level of miR-223-3p in 40 paired gastric cancer tissues and adjacent tissues was detected by RT-PCR. The results showed that the expression of miR-223-3p in gastric cancer tissues was significantly higher than that in adjacent tissues (Fig. 1A). The deeper the tumor invasion, the higher the miR-223-3p expression (Fig. 1B). Higher miR-223-3p expression was observed in patients with lymph node metastasis (Fig. 1C). After 2 years of follow-up of the 40 patients, we found that the risk of recurrence was significantly higher in the high-miR-223-3p group (P < 0.001; Fig. 1D). miR-223-3p expressions in three gastric cancer cell lines and normal gastric mucosal epithelial cell lines were also detected by RT-PCR. Results showed that the expression level was significantly higher in MKN-45 and AGS cells than that in GES-1 cells, whereas in SGC-7901 cells, the results were not significantly different (Fig. 1E).

To investigate the function of miR-223-3p, MKN-45 and SGC-7901 cells were selected for further research. Mimics and inhibitors of miR-223-3p were used to overexpress and reduce miR-223-3p expressions, respectively, and the efficiency was detected by PCR. After transfection of the mimics or inhibitor, the expression of miR-223-3p was significantly increased (P < 0.01) or decreased (P < 0.05), respectively, when compared to the control group (Fig. 1F).

miR-223-3p promoted gastric cancer cell proliferation, migration, and invasion

To further investigate the function of miR-223-3p in gastric cancer cell lines, we examined different cell phenotypes by using mimics and inhibitors to regulate the expression of miR-223-3p. The MTT assay showed that the proliferation activity of gastric cancer cells transfected with mimics was significantly higher than that of cells transfected with the negative control (P < 0.001; Fig. 2A); in contrast, the proliferation activity was significantly decreased (P < 0.001) in cells transfected with the inhibitor. Colony formation assay revealed that the number of cell colonies was significantly higher in the mimic group than in the negative control group (P < 0.01) and was markedly reduced in the inhibitor group (P < 0.01; Fig. 2B). These results suggested that miR-223-3p promoted cell proliferation in gastric cancer cells.

Wound healing assays and invasion assays were used to evaluate the effect of miR-223-3p on cell migration and invasion, respectively. The results demonstrated that the migration and invasion abilities were significantly enhanced in gastric cancer cells transfected with mimics (P < 0.001) and significantly attenuated in gastric cancer cells transfected with the inhibitor (P < 0.05; Fig. 2C,D). All these results indicated that miR-223-3p could promote gastric cancer cell proliferation, migration, and invasion.

Cell apoptosis and cell cycle was regulated by the downregulation of miR-223-3p expression

Flow cytometry analysis showed that the overexpression of miR-223-3p significantly reduced cell apoptosis, while cell apoptosis was significantly increased after the knockdown of miR-223-3p. Our results suggested that downregulation of miR-223-3p might promote apoptosis in gastric cancer cell lines (Fig. 2E). Additionally, changes in the cell distribution in the cell cycle were associated with miR-223-3p expression. The proportion of cells in the S phase was markedly increased in cells overexpressing miR-223-3p compared with that in the negative control cells. Furthermore, the proportion of S phase cells was decreased significantly with the knockdown of miR-223-3p. These results revealed that miR-223-3p promoted the cell cycle transition from the G1 to the S phase, cellular DNA synthesis, and cell proliferation (Fig. 2F).

miR-223-3p targets Arid1a in gastric cancer cell lines

miRNAs negatively regulate the translation of mRNA [6,22]. They mainly exert their function by inhibiting the expression of their target genes and then attenuate or eliminate the function of downstream genes. In our previous research, we found that Arid1a expression was closely related to the pathological features and prognosis of patients with gastric cancer [18]. Based on the prediction of TargetScan and miRDB, there is a complementary sequence between miR-223-3p and the 3'UTR of Arid1a. The 3'UTR of Arid1a contains a binding site for miR-223-3p, suggesting that Arid1a may be a direct target gene of miR-223-3p. Therefore, we investigated whether Arid1a is directly regulated by miR-223-3p by luciferase reporter assay. The wild-type or mutant-type Arid1a mRNA 3'UTR was cloned into a luciferase reporter vector to construct a luciferase reporter plasmid. Our results showed that the overexpression of miR-223-3p significantly reduced the luciferase activity of the wild-type Arid1a 3'UTR but could not change the luciferase activity of the mutant-type Arid1a 3'UTR (Fig. 3A). We further detected the expression of Arid1a protein in 40 paired gastric cancer tissues and found that Arid1a expression in gastric cancer tissues was lower than that in adjacent tissues (Fig. 3B,J). Moreover, Arid1a expression was significantly decreased with the depth of tumor invasion and lymph node metastasis (Fig. 3C,D). It was also found that the risk of recurrence was significantly higher in the low-Arid1a-expression group than that in the high-Arid1a-expression (P < 0.05; Fig. 3E). Simultaneously, miR-223-3p and Arid1a showed a significant negative correlation in the 40 gastric cancer patients (r = −0.6875, P < 0.001; Fig. 3F). We also observed that the expressions of Arid1a mRNA and protein were significantly decreased when miR-223-3p was overexpressed; in contrast, the expressions of Arid1a mRNA and protein were significantly increased when miR-223-3p was knocked down in MKN-45 and SGC-7901 cells (Fig. 3G-I).

miR-223-3p exerts its biological function by targeting Arid1a

Rescue experiments were performed to verify whether miR-223-3p exerts its biological function by targeting Arid1a in SGC7901 cells. SGC7901 cells were selected because of the significantly lower expression of miR-223-3p in SGC7901 cells compared with MKN-45 cells. Results showed that the mRNA and protein levels of Arid1a were decreased after the overexpression of miR-223-3p and were restored after co-transfection with either the miR-223-3p mimic or negative control and pcDNA-Arid1a or pcDNA3.1. The MTT assay showed that the overexpression of Arid1a attenuated the effect of miR-223-3p on gastric cancer cells compared with its effect on the negative control (Fig. 4A). Co-transfection with the Arid1a plasmid attenuated the inhibitory effect of miR-223-p on Arid1a (Fig. 4B,C). The colony formation assay and transwell invasion assay also confirmed that the overexpression of miR-223-3p significantly enhanced colony formation and invasion, while Arid1a overexpression attenuated colony formation and invasion (Fig. 4D,E). These results indicated that miR-223-3p promoted cell proliferation and invasion by targeting Arid1a.

miR-223-3p promoted tumor growth in vivo

The effect of miR-223-3p on tumor cells was further explored in vivo by the inoculation of xenograft in nude mice. A total of 2 × 10⁶ cells were subcutaneously inoculated in the left axilla of the nude mice. On day 8, each nude mouse was intratumorally injected with 15 μg of antagomiR-223-3p or antagomiR-NC. The injection was performed every 3 days, and the size of the transplanted tumor was measured to calculate the tumor volume. Results showed that the volume and weight of tumor in the antagomiR-223-3p group were significantly smaller than those in the control group (Fig. 5A,C), and tumor growth was significantly slower in the antagomiR-223-3p group (Fig. 5B). Compared with the control group, the expression of miR-223-3p was significantly decreased, and the mRNA and protein expression of Arid1a was markedly increased in the antagomiR-223-3p group (Fig. 5D,E). Then we examined the expression of Ki-67 in the transplanted tumors by immunohistochemical staining to evaluate tumor growth in vivo. More cell apoptosis was found in the group treated with antagomiR-223-3p; moreover, the antagomiR-223-3p group had a significantly lower intensity staining of Ki-67 than the control group. These results confirmed that the knockdown of miR-223-3p could inhibit tumor proliferation.

Discussion

miR-223-3p has been found to be abnormally expressed in a variety of diseases and can participate in cell proliferation and apoptosis through various pathways, mediating the progression of the disease. Liu et al. [23] reported that the overexpression of miR-223-3p attenuated abnormal proliferation induced by hypoxia, thereby improving the pathological features of pulmonary hypertension and ameliorating its progression. Another study revealed that miR-223-3p expression predicted a favorable survival outcome in patients with osteosarcoma, and the overexpression of miR-223-3p significantly inhibited osteosarcoma cell invasion, migration, growth, and proliferation [24]. Until now, few studies have reported on miR-223-3p in gastric cancer, and the mechanism is still unclear.

In this study, the expression level of miR-223-3p was found to be significantly upregulated in gastric cancer tissues compared with that in the adjacent tissues, and its expression level was closely related to the clinicopathological features of gastric cancer patients. We also found that patients with a high expression of miR-223-3p had a significantly higher risk of recurrence. Overexpression of miR-223-3p significantly enhanced gastric cancer cell proliferation, migration, and invasion, resulting in less apoptosis, while knockdown of miR-223-3p significantly reduced proliferation, migration, and invasion in gastric cancer. Simultaneously, we observed that miR-223-3p promoted more cells into the S phase. These findings suggest that miR-223-3p may play a role in gastric cancer as an oncogene miRNA.

To further explore the potential mechanism, the target genes of miR-223-3p were predicted by bioinformatics analysis with TargetScan and miRDB, and Arid1a was screened. Arid1a is located on human chromosome 1p35.3, which is rich in A-T sequences and has ATPase activity. Arid1a, which is encoded by Arid1a, is an important constituent subunit of SWI/SNF [25]. It affects the assembly of SWI/SNF complexes, thereby targeting the downstream genes and binding to other transcription factors [26–28]. Arid1a mutations are one of the main mechanisms leading to the loss of Arid1a in tumor tissues [29]. Since 2011, exome sequencing has revealed that Arid1a has different degrees of mutation in a variety of tumors [30–33], mainly involving nonsense mutations and frame shift mutations [34]. It was reported that knockdown of Arid1a promoted the proliferation of gastric cancer cells, while overexpression of Arid1a significantly inhibited the colony formation of gastric cancer cells [35]. Our previous study found that, as a tumor suppressor gene, Arid1a was expressed at low levels in tumor tissues and was downregulated in patients with deep infiltration and lymph node metastasis [18]. In this study, we revealed a negative expression relationship between Arid1a and miR-223-3p in specimens. The luciferase reporter assay confirmed that the Arid1a acted as the target gene of miR-223-3p.

In the present study, we also observed that the expression of Arid1a was significantly decreased after the overexpression of miR-223-3p, and the expression of Arid1a protein was significantly increased after the knockdown of miR-223-3p. Moreover, miR-223-3p binds to the 3'UTR of Arid1a and reduces the mRNA and protein levels of Arid1a. Rescue experiments showed that Arid1a attenuated the proliferation and invasion of miR-223-3p-overexpressed gastric cancer cells, compared with the control group, indicating that miR-223-3p exerts its oncogene miRNA function by targeting Arid1a in gastric cancer. We also found that nude mice treated with antagomiR-223-3p had a significant decrease in tumor volume and cell proliferation compared with the mice in the negative control group, suggesting that miR-223-3p may be a potential therapeutic target for gastric cancer.

In summary, we confirmed that miR-223-3p was highly expressed in gastric cancer tissues and that patients with a high expression of mir-223-3p had a poor prognosis. We also found that miR-223-3p regulated gastric cancer cell proliferation, migration, and invasion by targeting Arid1a in vitro. The antagomir against miR-223-3p significantly inhibited the growth of xenograft. This study suggests that miR-223-3p may be a potential therapeutic target for gastric cancer; however its regulation of downstream pathways remains to be further studied.

Funding

This work was supported by the grants from the Natural Science Research Project of College and Universities in Anhui Province (No. KJ2017A262) and the Funding of the ‘Peak’ Training Program for Scientific Research of Yijishan Hospital, Wannan, Medical College (No. GF2019G11).

References