MicroRNA-495 Confers Increased Sensitivity to Chemotherapeutic Agents in Gastric Cancer via the Mammalian Target of Rapamycin (mTOR) Signaling Pathway by Interacting with Human Epidermal Growth Factor Receptor 2 (ERBB2).

BACKGROUND In recent years, the incidence of gastric cancer (GC) has been increasing worldwide. Emerging evidence shows that microRNAs (miRs) may be involved in the pathogenesis of GC. Thus, this study explored the mediatory role of miR-495 in GC chemosensitivity, and investigated the mechanism by which it affects the biological behaviors of GC cells via the mTOR signaling pathway. MATERIAL AND METHODS After GC and paracancerous tissue collection, the positive rate of ERBB2 and mTOR was evaluated by immunohistochemistry. Subsequently, the expression of miR-495, ERBB2, and mTOR was determined by RT-qPCR and Western blot analysis. Next, the targeting relationship between miR-495 and ERBB2 was confirmed by dual-luciferase reporter gene assay. In addition, chemosensitivity and proliferation were detected by MTT assay and apoptosis was assessed by flow cytometry. RESULTS We found higher positive rates of ERBB2 and mTOR and decreased expression of miR-495 in GC tissues and showed that ERBB2 is the target gene of miR-495. Furthermore, we determined that overexpression of miR-495 and silencing of ERBB2 enhanced GC cell chemosensitivity and apoptosis, but inhibited GC cell proliferation. We also found that the effect of miR-495 inhibition was lost when ERBB2 was suppressed. CONCLUSIONS The key findings of our study demonstrate that the miR-495 exerts promotive effects on GC chemosensitivity via inactivation of the mTOR signaling pathway by suppressing ERBB2. The study provides reliable evidence supporting the use of miR-495 as a novel potential target in the chemotherapy of GC.

Background

Gastric cancer (GC) is the fifth most common malignancy and the second most frequent cause of cancer-related mortality around the world [1]. Annually, 989 600 cases were diagnosed and East Asia accounts for more than 50% of these [2]. Helicobacter pylori infection (the predominant risk factor), dietary factors, tobacco use, and obesity are the most significant factors associated with GC infection [3]. Despite the significant improvement of survival due to early detection and radical surgery, many patients are diagnosed with advanced GC and thus cannot receive the best therapy [4]. Current treatments for GC are mainly endoscopic detection with gastrectomy and chemotherapy (CT) or chemo-radiotherapy (CRT) in a neoadjuvant or adjuvant setting [2]. Chemotherapy is responsible for the reduction of tumor size by stress-induced apoptosis after direct and irreparable physical or chemical damage to DNA, but cancer cells may show resistance to chemotherapy drugs [5]. In treatment of GC, chemotherapy has been widely applied and treatments based on platinum and 5-fluorouracil (5-FU) have been shown to prolong GC patient survival [6,7].

miRNAs are abnormally expressed in a variety of hematological and solid cancers and altered miRNA expression is involved in malignancy progression and prognosis of GC [8]. It has been revealed that miR-495 suppresses GC progression through the inhibition of cell migration and invasion [3]. In the field of cancer chemotherapy, miRNA is regarded as a prognostic biomarker and a potential candidate for relieving multidrug resistance [9]. miR-495 regulates human epidermal growth factor receptor 2 (ERBB2) by binding to the 3′-untranslated regions (3′UTR) in connection with the target prediction program, microRNA.org. ERBB2, an oncogenic agent located in chromosome 17 (17q12–q21), encodes a 185-kDa transmembrane tyrosine kinase receptor (p185) [10]. ERBB2 has been identified to be implicated in cancerous initiation and progression, and its overexpression or amplification served as an independent prognostic factor in GC [11]. Furthermore, Tornillo et al. revealed that ERBB2 overexpression activated the mammalian target of rapamycin (mTOR)/p70S6K signaling [12]. mTOR is considered to be a potential prognostic biomarker of GC and significantly functions in cellular processes [13]. A previous study has identified that GC can activate the mTOR signaling pathway, while the inhibited mTOR signaling pathway is a contributor to trichostatin A (TSA) ability to affect GC cell proliferation and cell cycle [14]. In esophageal cancer, the mTOR pathway is involved in chemotherapy by the interaction with metformin [15]. In the present study we investigated the hypothesis that miR-495 can act as a significant biomarker of GC and promotes GC chemosensitivity via inactivation of the mTOR signaling pathway by binding to ERBB2. The results of our study could form an experimental basis for improvement of GC chemotherapy.

Material and Methods

Ethical statement

The study was approved by the Institutional Review Board of the Cancer Hospital Affiliated to Xinjiang Medical University. Written informed consent was obtained from each participant in accordance with the Declaration of Helsinki.

Microarray analysis

The UALCAN website () was used to analyze gene expression data in The Cancer Genome Atlas (TCGA) () by RAN-seq and clinical data on 31 kinds of different cancers with TCGA. Box and whisker plots showed gene expression level in different cancers and their subtypes/sub-stages. Level 3 TCGA RNA-seq data corresponding to the primary tumor and normal (if available) samples for each gene are represented as box and whisker plots in every TCGA cancer type. ERBB2 was input in UALCAN, and then GC was chosen among cancer types for obtaining corresponding data.

ERBB2 function analysis

The Kyoto Encyclopedia of Genes and Genome (KEGG) database () was used to analyze target gene functions. By inputting ERBB2 and selecting GC among the search results, the metabolic pathway involved in the target gene and regulatory association with the metabolic pathway were discovered in a majority of metabolic pathways in the KEGG database.

Study subjects

A total of 359 GC patients with the mean age of 59.06±10.61 years were obtained from the Cancer Hospital Affiliated to Xinjiang Medical University from December 2015 to December 2017. Among them, 219 patients were qualified for receiving the surgery and 177 patients had complete information. In the Department of Gastrointestinal Surgery, 132 patients were selected to undergo experiments under the conditions of safety. The GC tissues and paracancerous tissues at 3 cm were placed into cryopreservation tubes, and then immediately stored in liquid nitrogen tanks at −196°C within 10 min after the tissues were obtained in vitro. Among enrolled patients, 22 were at tumor stage I, 30 were at stage II, 65 were at stage III, and 15 were at stage IV according to node metastasis (TNM) classification [16]; 73 cases had intestinal type of histological classification [16], 20 had diffuse type, and 39 had mixed type; 76 cases had highly differentiated tumor and 56 had lowly differentiated tumor; 93 cases had lymph node metastasis (LNM) and 39 cases were without LNM. The inclusion criteria were: patients confirmed by pathology and/or cytology with primary GC; patients had no obvious abnormal medullary hematopoiesis, liver function, and kidney function; and patients were never treated with chemotherapy. The exclusion criteria were: patients did not receive treatment in our hospital after diagnosis; patients had history of treatment in other hospitals; patients had other malignancies [17].

Evaluation of chemotherapy effect

From December 2015 to December 2017, GC patients selected were treated with common chemotherapy and according to the Revised Response Evaluation Criteria in Solid Tumors (RECIST) issued by the World Health Organization (WHO) [18], the chemotherapeutic effects were classified into complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD), in which CR + PR was total efficacy. Among 132 patients, 20 cases were CR, 30 cases were PR, 32 cases were SD, and 50 cases PD. In accordance with curative effect, the experiment subjects were assigned into the sensitive group (CR + PR; n=50) and the resistant group (SD + PD; n=82).

Immunohistochemistry (IHC)

The samples were fixed with formaldehyde, embedded in paraffin, and sliced into 4-μm serial sections. Then, the sections were incubated at 60°C for 1 h, dewaxed by xylene, and dehydrated with gradient ethanol of 80%, 90%, and 100% and N-butanol. Next, the sections were immersed in 3% H2O2 for 10 min, washed with distilled water 3 times (3 min each time), repaired with high-pressure antigen for 1~3 min and cooled to room temperature in an ice bath, and washed twice with 0.01M phosphate-buffered saline (PBS, pH7.4) (3 min each time). After being incubated with 10% normal goat serum blocking solution (Cwbiotech, Beijing, China) at room temperature for 20 min, superfluous liquid was removed and we added an appropriate amount of rabbit anti-human ERBB2 monoclonal antibody (ab134182, 1: 100, Abcam Inc., MA, USA) to the sections to incubate with rabbit anti-human mTOR polyclonal antibody (1: 2000, ab2732, Abcam Inc., Cambridge, MA, USA) at 4°C overnight. Next, PBS washing was repeated 3 times (3 min each time), and then we added biotin labeled goat anti-rabbit immunoglobulin G (IgG) (ab6789, 1: 1000, Abcam Inc., MA, USA) secondary antibody for incubation at 37°C for 30 min. After washing 3 times in PBS (3 min each time), we added streptomycin anti-biotin protein-peroxidase (SP, Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) to the sections in an incubator at 37°C for 20 min. Then, the PBS washing for 3 times was repeated again (5 min each time) and sections were developed with 3,3-diaminobenzidine tetrahydrochloride (DAB, DA1010-3mL, Solarbio Life Sciences, Beijing, China) for 5~10 min. The degree of staining was observed under a microscope. The sections were washed with distilled water for 10 min, immersed in hematoxylin for 4 min and 1% hydrochloric acid alcohol for 10 s, washed, and immersed in 1% ammonia for 10 s. Furthermore, the sections were dehydrated with gradient alcohol, cleared with xylene, and sealed with natural gum. In order to reflect the expression levels of ERBB2 and mTOR, the percentage of positive cells was counted in 5 randomly chosen high-power fields for each section.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The Hyperpure RNA extraction kit (D203-01, GenStar BioSolutions Co., Ltd., Beijing, China) was used to extract total RNA. Premiers of miR-495, ERBB2, mTOR, CyclinD1, ataxia-telangiectasia mutated (ATM), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X (Bax), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Takara Biotechnology Ltd. (Dalian, Liaoning, China) (Table 1). RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFi-MMLV, and RNA enzyme without water were used for further experiments. According to the instructions of the TaqMan MicroRNA Assays Reverse Transcription Primer kit (4366596, Thermo Scientific, Waltham, MA, USA), reverse transcription was performed with 20 μL of reaction system. The reaction conditions were as follows: reverse transcription reaction at 42°C for 30~50 min and inactivation of reverse transcriptase enzyme at 85°C for 5 s. Fluorescence quantitative PCR was conducted with reacting liquid according to the instructions of the SYBR® Premix Ex Taq™ II kit (RR820A, Xingzhi Biological Technology Co. Ltd., Guangzhou, Guangdong, China). The 50-μL reaction system included 25 μL SYBR® Premix Ex Taq™ II (2×), 2 μL PCR forward premier, 2 μL PCR reverse premier, 1 μL ROX Reference Dye (50×), 4 μL DNA template, and 16 μL ddH2O. Fluorescent quantitative PCR was carried out using the ABI PRISM® 7300 system (Shanghai Kun Ke Instrument and Equipment Co., Ltd., Shanghai, China) with reaction conditions as follow: pre-denaturation at 95°C for 10 min, a total of 40 cycles of denaturation at 95°C for 15 s, and anneal at 60°C for 30 s, followed by extension at 72°C for 1 min. The U6 gene served as the internal control of miR-495 and GAPDH was the internal control of ERBB2, mTOR, CyclinD1, ATM, and Bcl-2. 2−ΔΔCt indicated the multiple proportions of target genes in the experiment and control groups. The formula was ΔΔCT=ΔCt (experiment group) – ΔCt (control group), in which ΔCt=Ct (target gene) – Ct (reference gene). Ct was the number of amplifying cycles when the real-time fluorescence intensity reached threshold, and the amplification was at logarithmic growth. The expressions of miR-495, ERBB2, mTOR, CyclinD1, ATM, Bcl-2, and Bax were calculated. Parallel experiments were conducted in triplicate.

Table 1

Primer sequences of related genes for reverse transcription quantitative polymerase chain reaction.

Genes	Premier sequences
miR-495	F: 5′-TCCGATTCTTCACGTGGTAC-3′
	R: 5′-GTGCAGGGTCCGAGGT-3′
U6	F: 5′-CTCGCTTCGGCAGCACATATACT-3′
	R: 5′-ACGCTTCACGAATTTGCGTGTC-3′
ERBB2	F: 5′-TCTGACGTCCATCGTCTCTG-3′
	R: 5′-AGGGCATAAGCTGTGTCACC-3′′
mTOR	F: 5′-GCCCCACCCCCATAGCTTCTCTC-3′
	R: 5′-CAGGACTCAGGACACAACTAGCCC-3′
CyclinD1	F: 5′-CCCACTCCTACGATACGC-3′
	R: 5′-AGCCTCCCAAACACCC-3′
ATM	F: 5′-TGGATCCAGCTATTTGGTTTGA-3′
	R: 5′-CCAAGTATGTAACCAACAATAGAAGAAG-3′
Bcl-2	F: 5′-GGAGGATTGTGGCCTTCTTTG-3′
	R: 5′-GGTGCCGGTTCAGGTACTCA-3′
Bax	F: 5′-CCTTTTGCTTCAGGGTTTCAT-3′
	R: 5′-CTCCATGTTACTGTCCAGTTCGT-3′
GAPDH	F: 5′-GGTGTGAACCACGAGAAATATGAC-3′
	R: 5′-TCATGAGCCCTTCCACAATG-3′

miR-495 – miRNA-495; ERBB2 – human epidermal growth factor receptor 2; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; Bcl-2 – B-cell lymphoma-2; mTOR – mammalian target of rapamycin; Bax – BCL2 associated X; ATM – ataxia-telangiectasia mutated; F – forward; R – reverse.

Western blot analysis

GC cells were collected after 48-h transfection, washed with cold PBS (Beyotime Institute of Biotechnology, Jiangsu, China) 3 times, and then lysed with total protein lysate. Next, the cells were centrifuged at 8000 rpm at 4°C for 30 min and the supernatant was collected to measure protein concentration in each sample with bicinchoninic acid (BCA) kit (20201ES76, Yeasen Company, Shanghai, China). A total of 50 μg protein in each group was subjected to electrophoresis with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After that, the protein was transferred to nitrocellulose (NC) membranes (ZY-160FP, Shanghai Ze Ye Biological Technology Co. Ltd., Shanghai, China), blocked in 5% non-fat milk powder for 2 h at room temperature, and then washed with Tris-buffered saline (TBS) 3 times (10 min each). After that, the membrane was placed into diluted primary antibody as follows: anti-rabbit HER2 monoclonal antibody (ab134182, 1: 10000), anti-rabbit mTOR polyclonal antibody (ab2732, 1: 2000), anti-rabbit CyclinD1 monoclonal antibody (ab134175, 1: 10000), anti-rabbit ATM monoclonal antibody (ab81292, 1: 50000), anti-mouse Bcl-2 monoclonal antibody (ab692, 1: 500), anti-rabbit Bax monoclonal antibody (ab53154, 1: 500), and anti-mouse GAPDH monoclonal antibody (ab8245, 1: 5000) at 4°C overnight. All of the above antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Next, the membranes were washed with TBS 3 times (10 min each), diluted secondary antibody labeled IgG goat anti-rabbit polyclonal antibody (ab20272, 1: 500, Abcam Inc., Cambridge, MA, USA) was added, followed by shaking for 1 h at room temperature, washing with TBS 3 times (10 min each time) again, and reacting with electro-chemiluminescence (ECL) for 1 min. With the liquid absorbed, the membrane was covered with preservative film and observed using an X-ray machine (36209ES01, Shanghai QC Bio Science & Technologies Co., Ltd., Shanghai, China). GAPDH served as the internal control, and gray value ratio of target band to internal control band indicated the relative expression of protein. The parallel experiment was conducted in triplicate.

Dual-luciferase reporter gene assay

Target genes of miR-495 were analyzed using the biological prediction website (microRNA.org), and dual-luciferase reporter was employed to validate that ERBB2 was the target gene of miR-495. Endonuclease sites of SpeI and Hind III were used to introduce target fragments to pMIR-reporter. Mutational site of complementary sequence of seed sequence was designed in ERBB2 wild-type. After restriction endonuclease digestion, target fragments of wild-type and mutant were inserted into the pMIR-reporter report plasmid by T4 DNA ligase. Luciferase reporter plasmids of wild and mutant types with correct sequences were co-transfected into HEK-293T cells (CRL-1415, Shanghai Xin Yu Biotech Co., Ltd., Shanghai, China) with miR-495 mimic. Cells were collected and lysed 28 h later, centrifuged for 3–5 min, and the supernatant was obtained. Luciferase activity was analyzed using the Dual-luciferase Reporter Assay System (Promega corporation, Madison, WI, USA). A total of 100 μL LAR II and 20 μL cell lysate were added into tubes to examine the luciferase activity of the target, and the luciferase activity of the internal control was also assessed with the addition of 100 μL Stop&Glo reagent. The luciferase value was determined with fluorescence detector (Promega Corporation, Madison, WI, USA) and the ratio of target luciferase value to internal control luciferase value served as relative luciferase activity. The experiment was conducted in triplicate.

Cell treatment

SGC-7901 cells purchased from Shanghai Xin Yu Biotech Co., Ltd. (Shanghai, China) were seeded in a culture medium and cultured in the 5% CO2 incubator at 37°C. Changed every 24~48 h, the culture medium was composed of 10% fetal bovine serum (FBS), RPMI-1640 culture medium, and penicillin. After being treated with 0.25% pancreatin for subculturing, cells during logarithmic growth were chosen for the following experiments.

The cells were assigned into blank, negative control (NC) (transfected with miR-495 NC sequence), miR-495 mimic (transfected with miR-495 mimic), miR-495 inhibitor (transfected with miR-495 inhibitor), siRNA-ERBB2 (transfected with siRNA-ERBB2), and miR-495 inhibitor + siRNA-ERBB2 (co-transfected with miR-495 inhibitor + siRNA-ERBB2) groups. The SGC-790 1 cells during logarithmic growth were seeded into a 6-well plate. When cells reached 50% confluence, the cells were transfected according to the instructions of the Lipofectamine 2000 kit (Invitrogen Inc., Carlsbad, CA, USA). A total of 250 μL serum-free culture medium Opti-MEM (Gibco, Gaithersburg, MD, USA) was utilized to dilute 100 pmol miR-495 mimic, miR-495 inhibitor, siRNA-ERBB2, miR-495 inhibitor + siRNA-ERBB2, and NC plasmids (final concentration of 50 nM), and then mixed and incubated at room temperature for 5 min. Next, 250 μL serum-free culture medium Opti-MEM was used to dilute 5 μL lipofectamine 2000, mixed and incubated at room temperature for another 5 min. Finally, the above mixtures were mixed together, incubated at room temperature (20 min), and added into cell culture wells at 37°C and 5% CO2 for 6~8 h. Then, complete medium was replaced and cells were prepared for subsequent experiments after another 24 h~48 h of culture.

MTT assay

In the cell sensitivity testing for chemotherapeutic drugs, transfected cells in the 6 groups were cultured in a 96-well plate with matched enzyme scale. Five repeated wells and NC wells in non-cell culture medium were designed in each group, with 4000 cells per well cultured at 37°C for 24 h. After cell adherence, the cells were treated with PBS, Mitomycin (MMC), Adriamycin (ADR), 5-FU, and cisplatin (DDP) for 72 h. Before testing, we added 20 μL MTT (5 mg/mL, GD-Y1317, Shanghai Guduo Biological Technology Co., Ltd., Shanghai, China) to each well for incubation at 37°C for 4 h with culture fluid carefully absorbed. Then, we added 200 μL dimethyl sulfoxide (DMSO, D5879-100ML, Sigma-Aldrich, St. Louis MO, USA) to each well, followed by mixing. Optical value (OD) at 570 nm was determined by a microplate reader. The experiment was carried out in triplicate. Sensitivity to tumor drugs was regarded as the inhibition rate of drugs. The inhibitory rate was calculated as follows: [1–(OD group with drugs–OD NC group without cell)/OD group without drugs]×100%. Parallel experiments were carried out in triplicate.

Measurement of cell proliferation rate was as follows: when the growth density reached 80%, the transfected cells were washed with PBS 2 times, treated with 0.25% trypsin for single cell suspension. After counting, cells were seeded into a 96-well plate at 3×103~6×103 cells/well, and the volume of each well was 0.2 mL. Six duplicate wells were prepared. After incubation, the culture plate was taken out at 12 h, 24 h, 48 h, and 72 h, and then further cultured in a culture medium maintaining 10% MTT solution (5 g/L, GD-Y1317, Shanghai Guduo Biological Technology Co. Ltd., Shanghai, China) for 4 h with the supernatant removed. To each well we added 100 μL DMSO (Sigma-Aldrich, St. Louis MO, USA), followed by slight shaking for 10 min to fully dissolve formazan crystals produced by living cells. The OD value at 570 nm of each well was determined by a microplate reader (Nanjing Detie Laboratory Equipment Co., Ltd., Jiangsu, China). A cell viability curve was drawn with time as the abscissa and the OD value the vertical axis. The experiment was carried out in triplicate.

Flow cytometry

After 24-h transfection, the cells were cultured at 5% CO2 incubator at 37°C for 48 h, collected, and washed with PBS 2 times. Then, the cells were centrifuged, re-suspended with 200 μL binding buffer, and mixed with 10 μL Annexin V-FITC and 5 μL propidium iodide (PI) for reaction, avoiding exposure to light at room temperature for 15 min, followed by adding 300 μL binding buffer. Then, flow cytometry (6HT, Cellwar Biotechnology Co., Ltd., Wuhan, Hubei, China) was employed for assessment of cell apoptosis at the excitement wavelength of 488 nm. The experiment was carried out in triplicate. With Annexin V/PI double-parameter method, the normal cell FITC and PI staining (FITC−PI−) was shown as upper right upper zone cell cluster in the flow cytometry, apoptotic cells FITC and PT staining (FITC+PI−) in lower right zone cell cluster, and dead cells FITC and PI staining (FITC+PI+) in lower left zone cell cluster.

Statistical analysis

All data were analyzed by SPSS 21.0 (IBM Corp., New York, USA). Measurement data are indicated as mean ± standard deviation (SD). Comparisons between 2 groups with normal distribution and homogenous variance were performed using the t test. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA). Data normality was checked with Kolmogorov-Smirnov test. Comparisons between multiple groups were done with one-way ANOVA with Tukey post hoc test when data were determined to have Gaussian distribution and with Kruskal-Wallis analysis of variance with Dunn’s post hoc test for non-Gaussian distribution. Cell viability at different time points was analyzed by repeated-measures ANOVA. A value of p<0.05 was considered to be statistically significant.

Results

ERBB2 mediates the mTOR signaling pathway in GC patients

The UALCAN website was utilized to predict ERBB2 gene expression in GC (Figure 1A). ERBB2 gene expression was higher in GC samples than that in normal samples by TCGA database.

Figure 1

ERBB2 is a target gene of miR-495 and mediates the mTOR signaling pathway in GC patients. (A) Expression of ERBB2 was higher in GC samples than that in normal samples in TCGA database; ERBB2 expression in 34 normal samples shown by blue box diagram; ERBB2 expression in 415 GC samples revealed by red box diagram; all these samples were from TCGA database; (B) Research results of ERBB2 in microRNA.org database; Homo sapiens was set; only miR-495 was predicted and the detection of luciferase activity was indicated in the histogram which suggested that miR-495 directly regulate ERBB2; (C) Metabolic signaling pathway participated by ERBB2 was searched by KEGG database; ERBB2 was located in forward of mTOR signaling pathway and affected its expression in GC; * p<0.05, vs. the NC group; these data was measurement data and analyzed by the t test; the experiment was repeated 3 times; ERBB2 – human epidermal growth factor receptor 2; mTOR – mammalian target of rapamycin; GC – gastric cancer; KEGG – Kyoto Encyclopedia of Genes and Genome; TCGA – The Cancer Genome Atlas; NC – negative control.

MiRNA that could mediate ERBB2 gene was searched in microRNA.org. As shown in Figure 1B, only miR-495 targets ERBB2 from this database, which indicated that mediatory role of ERBB2 was potentially influenced by miR-495. Target relationship was further validated with online software and dual-luciferase reporter gene assay. The outcomes revealed that there was a specific binding region between ERBB2-3′UTR and miR-495 sequences, and ERBB2 was the target gene of miR-495. Compared with the NC group, luciferase activity of ERBB2 wild 3′UTR was significantly inhibited by miR-495 mimic, but no difference was found in mutant 3′UTR (p>0.05). Therefore, we concluded that miR-495 can bind with ERBB2-3′UTR and downregulate ERBB2 expression after transfection.

For better understanding of the ERBB2 potential mediatory mechanism, the possible signaling pathway participated by ERBB2 was predicted with KEGG. As shown in Figure 1C, ERBB2 affected the mTOR signaling pathway through P13K and other genes. The above results suggest that ERBB2 has an impact on GC via the mTOR signaling pathway.

ERBB2 and mTOR signaling pathway-related genes are upregulated in GC tissues

The positive rates of ERBB2 and mTOR protein expression were estimated by immunohistochemistry. As shown in Figure 2, ERBB2 was mainly located in cell membrane, presenting as brown particles. Activated positive rate of mTOR protein mainly localized in cytoplasm, presenting as pale brown under the microscope. Positive rates of ERBB2 and mTOR protein expression were higher in the GC tissues than in the paracancerous tissues (p<0.05). The results indicated that ERBB2 was highly expressed and the mTOR signaling pathway was activated in GC tissues.

Figure 2

Higher positive rates of ERBB2 and mTOR protein expression in GC tissues. (A) Positive rates of ERBB2 (×200) and mTOR (×400) protein expression using IHC; (B) Histogram of positive rates of ERBB2 and mTOR protein expression in GC and paracancerous tissues; * p<0.05 vs. the paracancerous tissues; ERBB2 – human epidermal growth factor receptor 2; mTOR – mammalian target of rapamycin; GC – gastric cancer; the results were enumeration data expressed in percentage form; the chi-square test was performed to analyze data; n=132.

Low expression of miR-495 and overexpression of ERBB2 in GC tissues

RT-qPCR (Figure 3A) and Western blot analysis (Figure 3B) were employed to determine miR-495 and ERBB2 expression in GC tissues and corresponding paracancerous tissues. The results indicated that compared with paracancerous tissues, miR-495 was downregulated in the GC tissues, but mRNA and protein levels of ERBB2 were highly expressed (p<0.05). As shown in Table 2, miR-495 expression and ERBB2 mRNA expression in GC tissues were significantly associated with GC diameter, TNM classification, histological type, pathological differentiation, and LNM (all p<0.05), but not related to patient age or sex (both p>0.05).

Figure 3

RT-qPCR and Western blot analysis show downregulation of miR-495 and upregulation of ERBB2 in GC tissues. (A) RT-qPCR was employed to determine miR-495 level and ERBB2 mRNA level; (B) Gray value of ERBB2 protein level by Western blot analysis; (C) ERBB2 protein level with Western blot analysis; * p<0.05 vs. the paracancerous tissues; ERBB2 – human epidermal growth factor receptor 2; miR-495 – microRNA-495; GC – gastric cancer; LNM – lymph node metastasis; RT-qPCR – reverse transcription quantitative polymerase chain reaction; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; these data was measurement data and analyzed by the t test; n=132.

Table 2

Correlation of miR-495 and ERBB2 expression with clinicopathological characteristics of GC patients.

Clinicopathological characteristic	n	MiR-495	p	ERBB2	p
Age (year)			0.526		0.660
<60	61	0.67±0.09		1.36±0.13
≥60	71	0.66±0.09		1.37±0.13
Gender			0.567		0.414
Male	95	0.67±0.09		1.36±0.12
Female	37	0.66±0.09		1.38±0.14
Max GC diameter(cm)			0.006		0.021
<4	101	0.68±0.08		1.35±0.13
≥4	31	0.63±0.11		1.40±0.12
Histological classification			0.007		0.003
Intestinal type	73	0.69±0.07		1.33±0.11
Diffuse type	20	0.65±0.07		1.40±0.12
Mixed type	39	0.64±0.11		1.41±0.15
Pathological differentiation type			0.013		0.008
High differentiation	76	0.68±0.09		1.34±0.13
Medium and low differentiation	56	0.64±0.09		1.40±0.12
LNM			0.003		0.001
No	39	0.70±0.09		1.31±0.07
Yes	93	0.65±0.10		1.39±0.14
Clinical classification			0.001		<0.001
I	22	0.72±0.04		1.29±0.05
II	30	0.70±0.04		1.32±0.06
III	65	0.65±0.10		1.40±0.14
IV	15	0.63±0.11		1.41±0.17

ERBB2 – human epidermal growth factor receptor 2; miR-495 – microRNA-495; LNM – lymth node metastasis.

MiR-495 participates in GC chemosensitivity by inhibiting ERBB2 via the mTOR signaling pathway

IHC (Figure 4A), RT-qPCR (Figure 4B) and Western blot analysis (Figure 4C) were performed to measure the expression of miR-495, ERBB2, mTOR, CyclinD1, ATM, Bcl-2, and Bax. The resistant group revealed increased mRNA and protein levels of ERBB2, mTOR, and Bcl-2 in GC tissues (all p<0.05), but expression of miR-495, mRNA, and protein levels of CyclinD1, ATM, and Bax were reduced (all p<0.05) in comparison to the sensitivity group. Comparatively higher expression of ERBB2 and lower expression of miR-495 were associated with GC chemosensitivity.

Figure 4

Higher expression of ERBB2 and mTOR and lower expression of miR-495 participated in GC chemosensitivity. (A) ERBB2 (×200) and mTOR (×400) expression levels of GC tissues in the sensitivity and resistant groups determined by IHC; (B) miR-495 expression and mRNA levels of related genes in the sensitivity and resistant groups by RT-qPCR; (C) Protein levels of related genes in sensitivity and resistant groups by Western blot analysis; * p<0.05 vs. the sensitivity group; miR-495, miRNA-495; ERBB2 – human epidermal growth factor receptor 2; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; Bcl-2 – B-cell lymphoma-2; mTOR – mammalian target of rapamycin; ATM – ataxia-telangiectasia mutated; Bax – BCL2 associated X; GC – gastric cancer; IHC – immunohistochemistry; RT-qPCR – reverse transcription quantitative polymerase chain reaction; the data in A was enumeration data expressed in percentage form; the comparison between 2 groups was detected by chi-square test; the data in B, C were measurement data and expressed as mean ± standard deviation; the comparison between 2 groups was detected by t test; n=132.

MiR-495 inhibits the mTOR signaling pathway via downregulating ERBB2 in GC cells

Expression of miR-495, ERBB2, mTOR, CyclinD1, ATM, Bcl-2, and Bax were determined by RT-qPCR (Figure 5A, 5B) and Western blot analysis (Figure 5C, 5D). No difference was found in these gene expression levels between the blank and NC groups (all p>0.05). When compared with the blank and NC groups, elevated expression of miR-495, CyclinD1, ATM, and Bax and decreased expression of ERBB2, Bcl-2, and mTOR were found in the miR-495 mimic group (all p<0.05). The siRNA-ERBB2 group showed no difference in miR-495 expression when compared with the blank and NC groups, but reduced ERBB2, Bcl-2, and mTOR expression and increased expression of CyclinD1, ATM, and Bax were found (all p<0.05); the miR-495 inhibitor group showed the opposite situation except that miR-495 expression was suppressed (all p<0.05). There was no difference in the mRNA levels of ERBB2, mTOR, Bcl-2, CyclinD1, ATM, and Bax among the blank, NC, and miR-495 inhibitor + siRNA-ERBB2 groups (all p>0.05), but miR-495 expression was reduced in the miR-495 inhibitor + siRNA-ERBB2 group (all p>0.05). The above results suggest that upregulation of miR-495 and ERBB2 gene silencing elevates the mRNA levels of CyclinD1, ATM, and Bax, while inhibiting mRNA levels of ERBB2, Bcl-2, and mTOR in GC cells.

Figure 5

Upregulation of miR-495 or silencing ERBB2 increases expression of CyclinD1, ATM, and Bax but decrease expression of ERBB2, Bcl-2 and mTOR in GC cells. (A) miR-495 expression and mRNA levels of ERBB2, mTOR and Bcl-2 determined by RT-qPCR; (B) mRNA levels of CyclinD1, ATM, and Bax measured through RT-qPCR; (C) Protein levels of ERBB2, mTOR, CyclinD1, ATM, Bcl-2 and Bax by Western blot analysis; (D) Gray values of ERBB2, mTOR, CyclinD1, ATM, Bcl-2 and Bax protein; * p<0.05, vs. the blank and NC groups; miR-495 – microRNA-495; NC – negative control; ERBB2 – human epidermal growth factor receptor 2; Bcl-2 – B-cell lymphoma-2; mTOR – mammalian target of rapamycin; ATM – ataxia-telangiectasia mutated; Bax – BCL2 associated X; RT-qPCR – reverse transcription quantitative polymerase chain reaction; these data was measurement data and analyzed by one-way ANOVA; the experiment was repeated 3 times.

Upregulation of miR-495 or silencing ERBB2 elevates GC cell chemosensitivity

We used the MTT assay to measure the chemosensitivity of SGC-7901 cells to MMC, ADM, 5-FU, and DDP4. As presented in Table 3, compared with the blank and NC groups, cell chemosensitivity to the above chemotherapeutic drugs were increased in the miR-495 mimic and siRNA-ERBB2 groups, but we found the opposite trend in the miR-495 inhibitor group (all p<0.05); there was no difference found in the miR-495 inhibitor + siRNA-ERBB2 group compared with the blank and NC groups (p>0.05). Thus, we conclude that cell chemosensitivity was strengthened by upregulated miR and ERBB2 gene silencing.

Table 3

Sensitivity of SGC-7901 cells to 4 chemotherapeutic drugs after transfection.

	MMC	ADM	5-FU	DDP
Blank	27.46±1.41	35.76±3.38	21.20±2.24	40.88±1.32
NC	23.17±2.01	30.17±2.98	24.38±3.04	41.29±4.01
miR-495 mimic	32.88±2.11*	47.55±3.08*	31.25±2.14*	56.25±5.11*
miR-495 inhibitor	10.55±1.55*	16.11±1.98*	14.17±1.04*	27.11±2.55*
siRNA-ERBB2	35.58±2.17*	52.52±4.08*	36.55±3.14*	53.55±3.25*
miR-495 inhibitor + siRNA-ERBB2	25.06±1.97	27.98±2.94	22.19±2.00	37.10±3.97

p<0.05, compared with the blank and NC groups;

NC – negative control; MMC – mitomycin; ADR – adriamycin; 5-FU – 5- fluorouracil; DDP – cisplatin; ERBB2 – human epidermal growth factor receptor 2; these data was measurement data and analyzed by the one-way ANOVA; the experiment was repeated for three times.

Upregulation of miR-495 or silencing ERBB2 inhibits cell proliferation of GC cells

Cell viability was measured with MTT assay. As revealed in Figure 6, compared with the blank and NC groups, the cell proliferation rate was enhanced in the miR-495 inhibitor group, but we found the opposite trend in the miR-495 mimic and siRNA-ERBB2 groups (all p<0.05); no difference was found in the miR-495 inhibitor + siRNA-ERBB2 group (p>0.05). Therefore, we conclude that cell proliferation was reduced by upregulation of miR-495 or ERBB2 gene silencing.

Figure 6

MTT assay shows that upregulation of miR-495 or silencing ERBB2 inhibits GC cell viability. * p<0.05, vs. the blank and NC groups; NC – negative control; OD – optical density; miR-495 – microRNA-495; MTT – 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ERBB2 – human epidermal growth factor receptor 2; these data was measurement data and analyzed by repeated-measures ANOVA; the experiment was repeated 3 times.

Upregulation of miR-495 or silencing ERBB2 promotes GC cell apoptosis

Annexin-V-FITC/PI double-staining was performed to determine cell apoptosis rates of GC cells in each group (Figure 7). A remarkably lower cell apoptotic rate was observed in the miR-495 inhibitor group, while the miR-495 mimic and siRNA-ERBB2 groups had notably increased cell apoptotic rates (all p<0.05); no difference was found in the miR-495 inhibitor + siRNA-ERBB2 group (p>0.05). We conclude that upregulating miR-495 silencing ERBB2 blocked cell cycle entry, while accelerating apoptosis of GC cells.

Figure 7

Flow cytometry shows that upregulation of miR-495 or silencing ERBB2 promotes GC cell apoptosis. (A) Cell apoptotic rates measured by flow cytometry; (B) cell apoptotic rates in each group; * p<0.05 vs. the blank and NC groups; miR-495 – microRNA-495; ERBB2 – human epidermal growth factor receptor 2; NC – negative control; the one-way ANOVA was performed to analyze the measurement data; the experiment was repeated 3 times.

Discussion

GC is thought to be a biologically and genetically heterogeneous malignancy [19]. In order to search for a potential candidate in potent treatment, we performed our experiments and concluded that miR-495 enhanced chemosensitivity in GC cells via inactivation of the mTOR signaling pathway by downregulating ERBB2 expression.

Firstly, miR-495 was revealed to downregulated in GC tissues in our experiments. Increasing evidence has focused on the inhibitory role of miR-495 on GC, and miR-495 was revealed to be downregulated in many cancers, including GC [20]. Other studies further proved the underling mechanism of inhibited miR-495-3p leads to malignancy and progression of gastric epithelial cells by contributing to expression of multiple oncogenic epigenetic modifiers (Ems) [21]. In addition, Xu et al. reported that miR-495 is a tumor suppressor attenuating GC cell migration and invasion via the regulation of PRL-3 [22]. In the present study, the online website combined with dual-luciferase reporter gene assay determined that ERBB2 is the target gene of miR-495. Additionally, it has been reported that miR-7 and miR-331-3p are direct mediators of ERBB2 [23]. It was also reported that ERBB2 is highly expressed in GC tissues and is an independent negative prognostic factor for cancers [10], which is consistent with our study.

Secondly, we found that mTOR was activated in GC tissues. Activated mTOR is frequently found in many human cancers [24]. In addition, blockade of mTOR was proved to be a new potential therapy for cancer treatment [25]. According to our results, ERBB2 exerted great influence on GC via the mTOR signaling pathway, and mTOR expression level was suppressed by miR-495. Moreover, a previous study has proved that miR-199a-3p inhibits cell proliferation via downregulation of mTOR expression in endometrial cancer cells [26]. In the present study, the mTOR signaling pathway was verified to have a correlation with GC chemosensitivity by the interaction with miR-495 and ERBB2. Several previous reports found a correlation between the mTOR signaling pathway and chemosensitivity in that mTOR inhibitors have the potential to improve the effects of current cisplatin-based chemotherapy regimens in urothelial carcinoma [27] and the suppression of mTOR by rapamycin enhanced chemosensitivity of CaSki cells [28].

Additionally, our results suggest that miR-495 overexpression reduces expression levels of ATM in GC tissues by targeting ERBB2. ATM expression combined with MSI (microsatellite instability) is a potentially significant biomarker of GC, and it is known that inhibited ATM expression induces cancer development and progression [29]. Furthermore, it has been revealed that miR-147 can promote chemosensitivity of GC cell to 5-FU by targeting PTEN [30], which is in agreement with our study.

Lastly, we demonstrated that overexpression of miR-495 and ERBB2 gene silencing elevated Bax expression level while reducing expression levels of Bcl-2 and CyclinD1, which suggests that upregulation of miR-495 and ERBB2 gene silencing can promote GC apoptosis. Cyclin D1 is significantly involved in progression of various tumors, including GC [31]. In addition, it has been identified to be highly expressed in GC related to cell proliferation [32,33]. Bax and Bcl-2 are common members of the Bcl-2 family; but the former is a pro-apoptotic protein but the latter is anti-apoptotic [34]. Consistent with our finding, Liu et al. revealed that miR-495 overexpression promotes cell apoptosis and inhibits cell proliferation in human umbilical vein endothelial cells [20]. Similarly, miR-495 exerts potent anti-tumor activity by suppressing tumor growth and repressing GC cell migration and invasion [35]. In addition, recent data have shown that the downregulation of ERBB2 suppresses human cancer cell proliferation but promotes cancer cell apoptosis by small interfering RNA [36]. All of the above evidence demonstrates that upregulation of miR-495 and ERBB2 gene silencing can inhibit GC apoptosis.

Conclusions

In conclusion, miR-495 functioned as a tumor suppressor in our study; its effects on inhibiting ERBB2 gene and mTOR signaling pathway to promote GC chemosensitivity was demonstrated. Thus, miR-495 is a potent therapeutic target for GC treatment. Unfortunately, the sample size was small and further large-scale clinic studies are required to validate our findings. We only focused on the effect of miR-495 on GC, so ongoing investigations are needed to confirm the association between miR-495 expression, HER2, and chemosensitivity in other cancer subtypes.