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MiR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3
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MiR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3 Qian Wu1, Huiyu Zhong1, Lin Jiao, Yang Wen, Yi Zhou, Juan Zhou, Xiaojun Lu, Xingbo Song*, Binwu Ying* Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: MicroRNA Integrin Gastric cancer Tumor migration and invasion Mechanism
BACKGROUND: Gastric cancer is one of the major malignant tumors in the world. Integrins expressed in cancer cells can promote tumor progression and migration. MiRNAs can inhibit the expression of target genes by directly binding to their mRNAs and can affect various important biological processes. The aim of this study was to investigate the role of miR-124- 3p and ITGB3 in gastric cancer. METHODS: RT-PCR and western blot are used to detect the expression of miR-124-3p, ITGB3 and integrin β3 in gastric cancer tissues and cells. The wound healing, CCK-8 assay, transwell migration and invasion assay were performed to determine the cell proliferation, migration and invasion. What’s more, bioinformatics prediction and luciferase assay was conducted to demonstrated the binding efficiency between miR-124-3p and ITGB3. RESULTS: We verified that ITGB3 and miR-124-3p changes the migration and invasion of gastric cancer cells in vitro. The overexpression or silencing of miR-124-3p inhibited or promoted the proliferation, migration and invasion of both selected gastric cancer cells, and ITGB3 is just the reverse. Meanwhile, we validated that ITGB3 is the target of miR-124-3p by bioinformatics prediction and luciferase assay. Lastly, the expression of ITGB3 in 40 pairs of gastric cancer tissues were significantly higher than that in the adjacent normal tissues, while the expression level of miR-124-3p was significantly decreased in cancer tissues. CONCLUSIONS: miR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3 in gastric cancer cells. Our results suggested that miR-124-3p and ITGB3 may reasonably serve as a promising therapeutic target.
1. Introduction Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer death worldwide [1].Although 80 to 90% of tumors occurs sporadically, genetic factors contribute to gastric carcinogenesis [2]. Research on gastric cancer at the molecular and genetic levels is in urgent need of updating diagnostic methods and therapeutic strategies. In recent years, numerous studies have been demonstrating the relationship between protein-coding genes and tumorigenesis. However, the latest evidence suggests an emerging role of non-coding RNA in the occurrence and development of cancer, especially microRNAs (miRNAs) [3]. MicroRNAs (miRNAs) are single-stranded RNAs that are transcribed from independent non-coding region RNAs or introns of protein-coding genes. MicroRNAs can inhibit the expression of target genes by directly binding to their mRNAs and can affect various important biological
processes including cell cycle regulation, differentiation, proliferation and apoptosis [4]. There is sufficient evidence to confirm that miRNAs are closely related to gastric cancer and can act as potential biomarkers for the disease [5,6]. Single nucleotide polymorphism (SNP) is the DNA sequence polymorphism caused by single nucleotide variation at the genome level. It has reported that some SNPs located at the 3'UTR of the mRNA binding site are not only associated with cancer susceptibility [7,8], but also affect the binding ability between miRNAs and messenger RNA (mRNA). The ability of miRNAs binding to messenger (mRNA) is critical for regulating mRNA level and protein expression, SNPs in the 3′UTR region of mRNA can affect the binding ability. Integrins expressed in cancer cells can promote tumor progression and migration by increasing proliferation, invasion, and migration of cancer cells. Integrins are formed by non-covalent bond of α and β subunits in a 1:1 ratio. There are 18 alpha subunits and 8 beta subunits, which together form at least 24 integrins [9]. Studies have found that
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Corresponding authors. E-mail addresses: [email protected] (X. Song), [email protected] (B. Ying). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.prp.2019.152762 Received 23 August 2019; Received in revised form 11 November 2019; Accepted 25 November 2019 0344-0338/ © 2019 Elsevier GmbH. All rights reserved.
Please cite this article as: Qian Wu, et al., Pathology - Research and Practice, https://doi.org/10.1016/j.prp.2019.152762
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124-3p) were obtained from Genechem Co., Ltd, (Shanghai, China). Transfection was performed in six-well plates using lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.
tumor-suppressive miRNAs can regulate the expression of integrins [10,11].Previous research by our study found that rs3809865 in the 3′ UTR region of ITGB3 in microRNA-binding sites was associated with gastric cancer stage in Chinese Han population [12]. In this work, we verified that ITGB3 rs3809865 SNP alteration of miR-124-3p binding on ITGB3 and miR-124-3p/ITGB3 was involved in the proliferation, migration, and invasion of gastric cancer. MiR-124-3p exerts tumor suppressor function in gastric cancer by regulating integrin β3 expression. Our study revealed a novel mechanism of miR-124-3p on regulation of ITGB3 in the proliferation and migration of gastric cancer and the importance of the single nucleotide acid rs3809866 on the miR-1243p/ITGB3 binding regulation, providing promising molecular target for gastric cancer treatment.
2.3. RNA extraction and Real-time PCR (RT-PCR) analysis
2. Materials and Methods
The total RNA was extracted from tumor tissues and cells by using TRIzol (Thermo Fisher Scientific) in accordance with the manufacturer’s instruction. The PrimeScript™ RT Master Mix (TaKaRa, Japan) was reverse-transcribed into cDNA. Then gene expression was assessed by RT-PCR using SYBR® PrimeScript™ miRNA RT-PCR Kit (Takara, Japan) using U6 small RNA or ACTIN as internal controls. The relative expression levels were calculated by 2−ΔΔCt method. The primer sequences used are listed in Supplementary Table 1.
2.1. Patients and tissue samples
2.4. Bioinformatics predictions
40 pairs of gastric cancer tissues and adjacent normal tissues were obtained from West China Hospital of Sichuan University. The enrolled patients had primary gastric cancer tissue that had been surgically removed without adjuvant therapy. Tissues were immediately frozen in liquid nitrogen after isolation from clinical surgery, and part of tissues were separated and placed in 4% paraformaldehyde and DEPC-treated paraformaldehyde at the same time. The collection of all tissue and clinical information were approved by the Ethics Committee of West China Hospital of Sichuan University and informed consent was obtained from patients and their families. The clinical characteristics of patients are collected as shown in Table 1, The TNM stage, Lymph node status and Metastasis status were referenced The eighth edition of gastric cancer TNM staging system [13].
Bioinformatics information software NIH (https://snpinfo.niehs.nih. gov/), PolymiRTs Database 3.0 (http://compbio.uthsc.edu/miRSNP/ home.php), miRNASNP (http://www.bioguo.org/miRNASNP/) and miRdSNP (http://mirdsnp.ccr.buffalo.edu/index.php) were used to predict miRNAs that bind to rs3809865 in the 3'UTR region of ITGB3. The potential targets intersection was demonstrated by the Venn diagram. 2.5. Luciferase reporter assay Luciferase reporter assay was performed to verify if ITGB3 was a direct target of miR-124-3p. Construction of psiCEHCK-2 dual luciferase vector (Promega Corporation, Fitchburg, WI, USA) with rs3809865 A / T was named as wild type of ITGB or the mutant type of ITGB3. The constructs and miR-124-3p mimics or mimics-NC (Genechem Co., Ltd, Shanghai, China) were transiently co-transfected with the luciferase reporter plasmid in HEK-293 T cells. Transfected cells were collected after 48 hours of incubation at 37 °C. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA) in accordance with the manufacturer’s instructions.
2.2. Cell culture and transfection Human Gastric Cancer Cell Line (MKN-45, MGC803) and HEK-293 T were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, Hubei, China). Cells were cultured with DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C and 5% CO2. pEGFP-ITGB3, ITGB3 siRNA, pEGFP-NC, control siRNA (the control of ITGB3), miR-124-3p mimics, miR-124-3p inhibitor, miR-NC and inhibitor-NC (the control of miR-
2.6. Wound healing assay Table 1 Pathological information of 40 gastric cancer tissues. Variable
GC Patients (n = 40)
Age, mean ± SD (years) Male/female Tumor size (maximum diameter) ≥5 cm ＜5 cm Lymph node status N0 N1 N2 N3 Metastasis status 0 1 TNM stage Ⅰ Ⅱ Ⅲ Ⅳ Differentiation low differentiation Medium-high differentiation
59.65 ± 10.21 23/17
The migration of cells was evaluated by wound healing assay. In brief, Cells were incubated n 6-well plates with a density of 5 × 105 cells. Micropipette tip was used to make a scratch When the cells reached 90% confluence. Photos were captured from ten randomly selected fields in each group. The width (mm) of the scratch on each photo was measured using Image-Pro Plus 6.0 software (Media Cybernetics, USA).
16(40.0%) 24(60.0%)
2.7. CCK-8 assay
14(35.0%) 5(12.5%) 10(25.0%) 11(27.5%)
Cell Counting Kit-8 (CCK-8 assay kit, Dojindo, Japan) was conducted to measure cell proliferation capacity at a density of 5 × 103 cells in each well of 96-well plates. After incubation for 0, 12, 24, 48 h, 10 μl of CCK-8 solution was added into each well. Absorbance was examined utilizing MRX II microplate reader (Dynex Technologies, Chantilly, VA, USA) at 450 nm.
36(90.0%) 4(10.0%) 12(30.0%) 10(25.0%) 14(35.0%) 4(10.0%)
2.8. Transwell migration and invasion assays
22(55.0%) 18(45.0%)
Transwell migration and invasion assay were carried out to evaluate cells migration and invasion capacity. For transwell migration assay, A total of 1 × 105 cells were plated into the upper chamber of transwell chamber (Corning incorporated, USA) while the bottom chamber was
N: the regional lymph node involvement. M: the presence or otherwise of distant metastatic spread 2
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databases predicted that rs3809865 was located in the target sequence of miR-124-3p and miR-506-3p, as shown in Fig. 2A. Further prediction was made by the software of miRanda and RNA22v2.0, miR-124-3p performed better than miR-506-3p. To evaluate whether the change from A to T at rs3809865 affects the targeted binding of miR-124-3p and ITGB3, luciferase reporter assays were performed. ITGB3 3’UTRs containing the T-allele and A-allele of rs3809865 and miR-124-3p mimics or miR-NC were co-transfected into HEK-293 T cell, relative firefly and Renilla luciferase activity was measured. As shown in Fig. 2C, both of miR-124-3p co-transfected with A-allele or T-allele of ITGB3 can inhibited luciferase activity compared to controls. But the luciferase activity of the T allele have a higher inhibition rate compared with A allele(25.0% ± 1.7% VS 10.9% ± 2.0%, p < 0.001, Fig. 2D)
supplied with 10% FBS-contained medium. After 24 h, the migrated cells were washed with PBS and dyed with 0.5% crystal violet in 20% methanol. Take pictures at least ten randomly selected fields in each chamber and count the number of cells. The transwell invasion assay was the same as migration assay except the upper chamber was precoated with Matrigel medium at a 1:8 ratio. 2.9. Western blotting Total proteins were extracted from the collected tissues and cells with RIPA (Servicebio Biotechnology, China). The proteins were separated on SDS-PAGE and then transferred to PVDF membrane. The membranes were separately incubated with monoclonal rabbit antiITGB3 (Abcam, USA), mouse anti-GAPDH (Abcam, USA), or HRP-coupled secondary antibodies (Servicebio, China). The gray value calculation of Western blots was analyzed using Image J software (National Institutes of Health, Bethesda, MD).
3.3. miR-124-3p plays a role in gastric cancer by repressing ITGB3 expression To further confirm the relationship between miR-124-3p and ITGB3 in the regulation of gastric cancer, the miR-124-3p mimics, miR-NC, miR-124-3p inhibitor and inhibitor-NC were transfected to MGC-803 and MKN45 cells, respectively. RT-PCR analysis confirmed that miR124-3p levels were markedly increased or decreased in MGC-803 and MKN45 cells after transfection. (Figs. 3A and S2A). RT-PCR analysis and Western blotting were used to verify the changes in the expression of ITGB3 and integrin β3. The date showed that the expression of ITGB3 and integrin β3 was lower in miR-124-3p mimics group than in miR-NC group in both MGC-803 and MKN45 cells. On the contrary, downregulation of miR-124-3p increased the expression level of ITGB3 and integrin β3. (Figs. 3B, C and S2B, C). Next, we examined the role of miR-124-3p in proliferation and migration of gastric cancer. As the result of CCK-8 assay shows, the capacity of cell proliferation was inhibited after miR-124-3p overexpression. Meanwhile, the capacity of cell proliferation was increased after transfection with mir-124-3p inhibitor (Figs. 3D and S2D). Similar results were observed in wound healing assay when investigating the migration ability cells (Figs. 3E and S2E). Cell migration and invasion ability was examined by transwell migration and invasion assays. The result showed that the migration capacities of MGC-803 and MKN45 cells transfected with miR-124-3p mimics were significantly decreased compared to the miR-NC group while transfected with miR-124-3p inhibitor were increased compared with the inhibitor-NC group (Figs. 3F and S2F). These results are in good consistent with the prediction that miR-124-3p plays a role in gastric cancer by repressing ITGB3 expression.
2.10. Statistical analysis All statistics are completed with GraphPad Prism 6.0(GraphPad Software, La Jolla, CA, USA) and SPSS 22.0 (Chicago, IL, USA). Continuous variables are described by mean ± standard deviation, and categorical variables are described by frequency and percentage. P values are calculated by Student’s t-test or ANOVA analysis. And P < 0.05 represent statistical differences. Each experiment was replicated at least three times. 3. Results 3.1. ITGB3 changes the migration and invasion of gastric cancer cells in vitro To investigate the role of ITGB3 in gastric cancer, a pEGFP-ITGB3 vector and ITGB3 siRNA was transfected into MGC-803 and MKN45 cells which were human gastric cancer cell lines independently. The transfection efficiency at 48 h after transfection was shown in Figs. 1A, B and S1A, B. CCK-8 assays revealed the enhanced cell proliferation after transfection with pEGFP-ITGB3 compared to pEGFP-NC, while ITGB3 siRNA reduced cell proliferation compared to control siRNA in MGC-803 and MKN45 cells. (p < 0.05, Figs. 1C and S1C,). Furthermore, wound healing assay was performed to investigate the roles of ITGB3 on gastric cancer cell migration ability. The results showed that ITGB3 overexpression promoted gastric cancer cell migration in MGC-803 and MKN45 cells, while siRNA of ITGB3 dramatically decreased gastric cancer cell migration in MKN45 cell. (p < 0.05, Figs. 1D and S1D). SiRNA of ITGB3 in MGC803 cell showed a tendency to reduce cell migration but did not reach statistical difference. Similarly, the transwell migration and invasion assays illustrated that upregulation of ITGB3 promoted MGC-803 and MKN45 cells migration and invasive ability compared with controls, while knockdown of ITGB3 expression impeded the cell migration and invasion. (p < 0.05, Figs. 1E and S1E). In summary, our results demonstrated ITGB3 plays an important role in gastric cancer cells migration and invasion.
3.4. ITGB3 is up-regulated and miR-124-3p is down-regulated in gastric cancer tissues Lastly, we evaluated the relative expression levels of ITGB3 and miR-124-3p in 40 gastric cancer tissues and adjacent normal tissues, and their clinical characteristics are summarized in Table 1. The results of RT-PCR showed that ITGB3 in gastric cancer tissues was significantly up-regulated compared with the adjacent non-cancerous tissue samples (Fig. 4A, tumor tissue vs. normal tissue: 1.657 ± 0.134:1.080 ± 0.076; P < 0.001). Receiver operating characteristic (ROC) curve analysis demonstrated the area of ITGB3 under the ROC curve for gastric cancer diagnosis were 0.721(Fig. 4B). To further understand the correlation between the mRNA levels of ITGB3 and clinicopathological features, we performed a stratified analysis based on Tumor size, Lymph node status, Metastasis status, TNM stage and differentiation. The results showed that ITGB3 was correlated with tumor size. The group with the maximum diameter of ≥5 cm was significantly higher than the group which the diameter of < 5 cm (≥5 cm vs. < 5 cm:1.99 ± 0.198 VS 1.434 ± 0.168, P = 0.039) (Fig. 4C). Statistical differences were also available between the four groups of Lymph node status (Fig. 4D). No significance was discovered
3.2. miR-124-3p differentially regulates ITGB3 expression depending on SNP rs3809865 It was reported that SNP in the 3’UTR of an mRNA can effect the combination of mRNA and miRNA which results in decreased expression of the targeted gene [14]. We had verified that rs3809865 in the 3’UTR of ITGB3 was associated with gastric cancer stage in Chinese Han population [12]. Then we employed the online prediction software NIH, PolymiRTs Database 3.0, miRNASNP and miRdSNP to predict miRNAs that bind to rs3809865 in the 3’UTR region of ITGB3. Three 3
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Fig. 1. ITGB3 changes the migration and invasion of gastric cancer cells in vitro. (A,B) The Expression level of ITGB3 and integrin β3 was detected to verify the transfection efficiency at 48 h after transfection. (C) CCK8 showed the proliferation rates of MGC803 cells transfected with control siRNA, ITGB3 siRNA, pEGFP-NC and pEGFP-ITGB3. (D) Representative images of Wound healing assay in different groups. (E) Representative images of the transwell migration an invasion assays in different groups. (Considering for layout of the document, the results of MKN45 are placed in supplemental materials Fig. 1).
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Fig. 2. miR-124-3p differentially regulates ITGB3 expression. (A) NIH, PolymiRTs Database 3.0, miRNASNP and miRdSNP were used to predict that rs3809865 was located in the target sequence of miR-124-3p and miR-506-3p. (B) miRanda and RNA22v2.0 were predicted that miR-124-3p performed better than miR-506-3p with binding to ITGB3. (C,D) Luciferase reporter assays of miR-124-3p co-transfected with wildtype (A) or variant (T) of ITGB3.
4. Discussion
in TNM stage, Differentiation and Metastasis status (Fig. 4E–G). What's more, Western blotting also confirmed the expression trend of integrin β3 which is the protein of ITGB3 between gastric cancer tissues and normal tissues (Fig. 5A). The area of integrin β3 under the ROC curve for gastric cancer diagnosis was 0.810 (Fig. 5B). As for the integrinβ3 expression in gastric cancer, the same results as ITGB3 were observed, however, no significant difference but similar effect occurred between the expression level of integrin β3 and clinicopathologic feature of gastric cancer (Fig. 5C–G). At the same time, the relative expression of miR-124-3p in gastric cancer tissues was also measured. The results showed that the miR-1243p expression in gastric cancer tissues was significantly lower than that in adjacent normal tissues (0.757 ± 0.075 VS 1.112 ± 0.076, P = 0.001, Fig. 6A). The AUC value of ROC curve was 0.712 (Fig. 6B). Similarly, we performed stratified analysis of miR-124-3p based on clinical characteristics. However, no correlation was found between miR-124-3p and tumor size, lymph node status, metastasis status, TNM stage and differentiation of gastric cancer (Fig. 6C–G).
Gastric cancer is one of the major malignant tumors in the world and the second most common cancer in China. There are nearly 700,000 newly diagnosed gastric cancer patients in China every year [15]. It is urgent to study on the pathogenesis of gastric cancer and find early molecular markers. Integrin has been found to play an important role in the migration and invasion of gastric cancer [16,17]. miRNAs regulates the expression of integrin gene or inhibiting its translation at the transcriptional or post-transcriptional level by binding to the 3’UTR of target gene, thereby the invasion and metastasis of gastric cancer was affected [18,19]. Considerable research efforts have been devoted to demonstrated that miR-124-3p play important roles in tumorigenesis. For example, Zhang et al [20] reported that miR-124-3p inhibited glioblastoma multiforme cell development and growth act as the upstream suppressor of NRP-1 by PI3K/Akt/NFκB pathway. Long et al [21]demonstrated that lower miR-124-3p expression was shown to be correlated with a shorter overall survival and poor prognosis in hepatocellular carcinoma(HCC) by 155 biopsy tissues. Furthermore, Liu et al [22] found that miR-124-3p repressed the carcinogenesis of gastric cancer 5
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Fig. 3. miR-124-3p plays a role in gastric cancer by repressing ITGB3 expression. (A) The Expression level of miR124-3p was detected to verify the transfection efficiency. (B,C) RT-PCR analysis and Western blotting were used to verify that the expression of ITGB3 and integrin β3 could be upregulation or downregulation by miR-124-3p decreased or increased. (D) CCK8 showed the proliferation rates of MGC803 cells transfected with miR-NC, miR mimics, inhibitor-NC and miR inhibitor (E) Representative images of Wound healing assay in different groups. (F) Representative images of the transwell migration an invasion assays in different groups. (Considering for layout of the document, the results of MKN45 are placed in supplemental materials Fig. 2).
The hallmarks of cancer comprise six biological capabilities which include unlimited proliferation, escape of growth inhibitors, resistance to cell death, promotion of tumor angiogenesis, invasion of tumor cells and maintain proliferative signal transduction [23]. Integrins trigger
with a shorter overall survival and disease-free survival rates. These findings are consistent with our study partially. These findings suggest that miR-124-3p could function as tumor suppressors and may an independent indicator of survival for patients. 6
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Fig. 4. Expression level of ITGB3 in gastric cancer tissues and cells. (A) Relative expression levels of ITGB3 in gastric cancer tissues and normal tissue(n = 40). (B) ROC curve analysis for diagnostic value of ITGB3. (C-G) The expression level of ITGB3 in gastric cancer tissues from patients at different Tumor size, Lymph node status, TNM stage, Differentiation and Metastasis status.
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Fig. 5. Expression level of integrin β3 in gastric cancer tissues. (A) Relative expression levels of integrin β3 in gastric cancer tissues and normal tissue by western blot (n = 40). (B) ROC curve analysis for diagnostic value of integrin β3. (C-G) The expression level of integrin β3 in gastric cancer tissues from patients at different Tumor size, Lymph node status, TNM stage, Differentiation and Metastasis status.
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Fig. 6. Expression level of miR-124-3p in gastric cancer tissues. (A) Relative expression levels of miR-124-3p in gastric cancer tissues and normal tissue(n = 40). (B) ROC curve analysis for diagnostic value of miR-124-3p. (C-G) The expression level of miR-124-3p in gastric cancer tissues from patients at different Tumor size, Lymph node status, Metastasis status, TNM stage and Differentiation.
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Appendix A. Supplementary data
and play key roles in the six hallmarks of cancer [24]. For instance, ITGAX stimulates angiogenesis through overexpression of VEGFR2/ VEGF‐A mediated by PI3K/Akt signaling during cancer development, and overexpression of ITGAX can stimulate the globule formation of ovarian cancer cells in human umbilical vein endothelial cells [25]. In Glioblastoma Stem Cells, Integrin α6 enhanced the transcription of FGFR1 and its downstream targets through ZEB1 and YAP1, thereby enhancing the expression of stem cell-related factors and increasing the ability of neurosphere formation and sphere growth. In addition, When specific siRNA disturbed Integrin α6, stem cell-related factors and the ability of neurosphere formation decreased [26]. It has also been reported that miRNAs can play a role in cancer inhibition by regulating the content of integrin in cancer. Previous studied showed mir-3653 inhibits the growth and migration of hepatocellular cancer by reducing the expression level of ITGB1 [27]. Mir-143-3p ACTS as an anticancer gene by downregulating the expression of ITGA6/ASAP3 protein in the development of colorectal cancer [28]. Besides, our study confirmed that miR-124-3p could be a potential prognostic marker in gastric cancer and functions as a tumor suppressor by regulating ITGB3. In this study, we first demonstrate the ITGB3 is the target of miR124-3p by bioinformatics prediction and luciferase assay, then we investigated 40 pairs of gastric cancer tissues and normal control tissues. The expression levels of ITGB3 in tumor tissues were significantly higher than that in the normal group, while the expression level of miR124-3p was significantly lower than that in the normal group. Although it did not reach the statistically significant threshold, integrin β3 and miR-124-3pshowed correlated expression and related to the clinicopathological features of gastric cancer. This result could be validated in a larger sample size. Collectively, the present study demonstrated that miR-124-3p expression is significantly decreased while ITGB3 expression is significantly increases in gastric cancer tissues. Moreover, we demonstrated the miR-124-3p -mediated mechanism for ITGB3 to affect the gastric cancer cell proliferation migration and invasion for the first time. These results suggested that miR-124-3p and ITGB3 may reasonably be a prognostic and diagnostic biomarker for gastric cancer patients and may serve as a promising therapeutic target.
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Yan, Z. Zhu, B. Liu, L. Su, Cancer-associated fibroblast-derived Lumican promotes gastric cancer progression via the integrin beta1-FAK signaling pathway, Int. J. Cancer. 141 (2017) 998–1010, https://doi.org/10.1002/ijc.30801. [17] L. Gan, J. Meng, M. Xu, M. Liu, Y. Qi, C. Tan, Y. Wang, P. Zhang, W. Weng, W. Sheng, M. Huang, Z. Wang, Extracellular matrix protein 1 promotes cell metastasis and glucose metabolism by inducing integrin beta4/FAK/SOX2/HIF-1alpha signaling pathway in gastric cancer, Oncogene 37 (2018) 744–755, https://doi.org/ 10.1038/onc.2017.363. [18] X.-R. Tang, X. Wen, Q.-M. He, Y.-Q. Li, X.-Y. Ren, X.-J. Yang, J. Zhang, Y.-Q. Wang, J. Ma, N. Liu, MicroRNA-101 inhibits invasion and angiogenesis through targeting ITGA3 and its systemic delivery inhibits lung metastasis in nasopharyngeal carcinoma, Cell Death Dis. 8 (2017) e2566, https://doi.org/10.1038/cddis.2016.486. [19] Y. Xu, L. Shen, F. Li, J. Yang, X. Wan, M. Ouyang, microRNA-16-5p-containing exosomes derived from bone marrow-derived mesenchymal stem cells inhibit proliferation, migration, and invasion, while promoting apoptosis of colorectal cancer cells by downregulating ITGA2, J. Cell. Physiol. (2019), https://doi.org/10.1002/ jcp.28747. [20] G. Zhang, L. Chen, A.A. Khan, B. Li, B. Gu, F. Lin, X. Su, J. Yan, miRNA-124-3p/ neuropilin-1(NRP-1) axis plays an important role in mediating glioblastoma growth and angiogenesis, Int. J. Cancer. 143 (2018) 635–644, https://doi.org/10.1002/ijc. 31329. [21] H.-D. Long, Y.-S. Ma, H.-Q. Yang, S.-B. Xue, J.-B. Liu, F. Yu, Z.-W. Lv, J.-Y. Li, R.T. Xie, Z.-Y. Chang, G.-X. Lu, W.-T. Xie, D. Fu, L.-J. Pang, Reduced hsa-miR-124-3p levels are associated with the poor survival of patients with hepatocellular
Ethical approval This study was approved by the Ethics Committee of West China Hospital of Sichuan University. Informed consent Informed consent was obtained from all individual participants included in the study. Author contributions Qian Wu and Huiyu Zhong wrote the main manuscript and fully participated in all experiments. Lin Jiao and Yi Zhou participated in the acquisition of data. Juan Zhou, Yang Wen and Xiaojun Lu participated in the analysis and interpretation of data. Xingbo Song and Binwu Ying designed the study. All authors made substantial contributions to writing and revising the manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China [81672096] and the Projects in the Science and Technology Department of Sichuan Province pillar program [2017FZ0065] 10
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carcinoma, Mol. Biol. Rep. 45 (2018) 2615–2623, https://doi.org/10.1007/s11033018-4431-1. F. Liu, H. Hu, J. Zhao, Z. Zhang, X. Ai, L. Tang, L. Xie, miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer, Biomed. Reports. 9 (2018) 147–155, https://doi.org/10.3892/br.2018.1113. D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674, https://doi.org/10.1016/j.cell.2011.02.013. D. Bianconi, M. Unseld, W.G. Prager, Integrins in the Spotlight of Cancer, Int. J. Mol. Sci. 17 (2016), https://doi.org/10.3390/ijms17122037. J. Wang, L. Yang, F. Liang, Y. Chen, G. Yang, Integrin alpha x stimulates cancer angiogenesis through PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression in blood vessel endothelial cells, J. Cell. Biochem. 120 (2019)
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Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp
MiR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3 Qian Wu1, Huiyu Zhong1, Lin Jiao, Yang Wen, Yi Zhou, Juan Zhou, Xiaojun Lu, Xingbo Song*, Binwu Ying* Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
A R T I C LE I N FO
A B S T R A C T
Keywords: MicroRNA Integrin Gastric cancer Tumor migration and invasion Mechanism
BACKGROUND: Gastric cancer is one of the major malignant tumors in the world. Integrins expressed in cancer cells can promote tumor progression and migration. MiRNAs can inhibit the expression of target genes by directly binding to their mRNAs and can affect various important biological processes. The aim of this study was to investigate the role of miR-124- 3p and ITGB3 in gastric cancer. METHODS: RT-PCR and western blot are used to detect the expression of miR-124-3p, ITGB3 and integrin β3 in gastric cancer tissues and cells. The wound healing, CCK-8 assay, transwell migration and invasion assay were performed to determine the cell proliferation, migration and invasion. What’s more, bioinformatics prediction and luciferase assay was conducted to demonstrated the binding efficiency between miR-124-3p and ITGB3. RESULTS: We verified that ITGB3 and miR-124-3p changes the migration and invasion of gastric cancer cells in vitro. The overexpression or silencing of miR-124-3p inhibited or promoted the proliferation, migration and invasion of both selected gastric cancer cells, and ITGB3 is just the reverse. Meanwhile, we validated that ITGB3 is the target of miR-124-3p by bioinformatics prediction and luciferase assay. Lastly, the expression of ITGB3 in 40 pairs of gastric cancer tissues were significantly higher than that in the adjacent normal tissues, while the expression level of miR-124-3p was significantly decreased in cancer tissues. CONCLUSIONS: miR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3 in gastric cancer cells. Our results suggested that miR-124-3p and ITGB3 may reasonably serve as a promising therapeutic target.
1. Introduction Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer death worldwide [1].Although 80 to 90% of tumors occurs sporadically, genetic factors contribute to gastric carcinogenesis [2]. Research on gastric cancer at the molecular and genetic levels is in urgent need of updating diagnostic methods and therapeutic strategies. In recent years, numerous studies have been demonstrating the relationship between protein-coding genes and tumorigenesis. However, the latest evidence suggests an emerging role of non-coding RNA in the occurrence and development of cancer, especially microRNAs (miRNAs) [3]. MicroRNAs (miRNAs) are single-stranded RNAs that are transcribed from independent non-coding region RNAs or introns of protein-coding genes. MicroRNAs can inhibit the expression of target genes by directly binding to their mRNAs and can affect various important biological
processes including cell cycle regulation, differentiation, proliferation and apoptosis [4]. There is sufficient evidence to confirm that miRNAs are closely related to gastric cancer and can act as potential biomarkers for the disease [5,6]. Single nucleotide polymorphism (SNP) is the DNA sequence polymorphism caused by single nucleotide variation at the genome level. It has reported that some SNPs located at the 3'UTR of the mRNA binding site are not only associated with cancer susceptibility [7,8], but also affect the binding ability between miRNAs and messenger RNA (mRNA). The ability of miRNAs binding to messenger (mRNA) is critical for regulating mRNA level and protein expression, SNPs in the 3′UTR region of mRNA can affect the binding ability. Integrins expressed in cancer cells can promote tumor progression and migration by increasing proliferation, invasion, and migration of cancer cells. Integrins are formed by non-covalent bond of α and β subunits in a 1:1 ratio. There are 18 alpha subunits and 8 beta subunits, which together form at least 24 integrins [9]. Studies have found that
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Corresponding authors. E-mail addresses: [email protected] (X. Song), [email protected] (B. Ying). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.prp.2019.152762 Received 23 August 2019; Received in revised form 11 November 2019; Accepted 25 November 2019 0344-0338/ © 2019 Elsevier GmbH. All rights reserved.
Please cite this article as: Qian Wu, et al., Pathology - Research and Practice, https://doi.org/10.1016/j.prp.2019.152762
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124-3p) were obtained from Genechem Co., Ltd, (Shanghai, China). Transfection was performed in six-well plates using lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.
tumor-suppressive miRNAs can regulate the expression of integrins [10,11].Previous research by our study found that rs3809865 in the 3′ UTR region of ITGB3 in microRNA-binding sites was associated with gastric cancer stage in Chinese Han population [12]. In this work, we verified that ITGB3 rs3809865 SNP alteration of miR-124-3p binding on ITGB3 and miR-124-3p/ITGB3 was involved in the proliferation, migration, and invasion of gastric cancer. MiR-124-3p exerts tumor suppressor function in gastric cancer by regulating integrin β3 expression. Our study revealed a novel mechanism of miR-124-3p on regulation of ITGB3 in the proliferation and migration of gastric cancer and the importance of the single nucleotide acid rs3809866 on the miR-1243p/ITGB3 binding regulation, providing promising molecular target for gastric cancer treatment.
2.3. RNA extraction and Real-time PCR (RT-PCR) analysis
2. Materials and Methods
The total RNA was extracted from tumor tissues and cells by using TRIzol (Thermo Fisher Scientific) in accordance with the manufacturer’s instruction. The PrimeScript™ RT Master Mix (TaKaRa, Japan) was reverse-transcribed into cDNA. Then gene expression was assessed by RT-PCR using SYBR® PrimeScript™ miRNA RT-PCR Kit (Takara, Japan) using U6 small RNA or ACTIN as internal controls. The relative expression levels were calculated by 2−ΔΔCt method. The primer sequences used are listed in Supplementary Table 1.
2.1. Patients and tissue samples
2.4. Bioinformatics predictions
40 pairs of gastric cancer tissues and adjacent normal tissues were obtained from West China Hospital of Sichuan University. The enrolled patients had primary gastric cancer tissue that had been surgically removed without adjuvant therapy. Tissues were immediately frozen in liquid nitrogen after isolation from clinical surgery, and part of tissues were separated and placed in 4% paraformaldehyde and DEPC-treated paraformaldehyde at the same time. The collection of all tissue and clinical information were approved by the Ethics Committee of West China Hospital of Sichuan University and informed consent was obtained from patients and their families. The clinical characteristics of patients are collected as shown in Table 1, The TNM stage, Lymph node status and Metastasis status were referenced The eighth edition of gastric cancer TNM staging system [13].
Bioinformatics information software NIH (https://snpinfo.niehs.nih. gov/), PolymiRTs Database 3.0 (http://compbio.uthsc.edu/miRSNP/ home.php), miRNASNP (http://www.bioguo.org/miRNASNP/) and miRdSNP (http://mirdsnp.ccr.buffalo.edu/index.php) were used to predict miRNAs that bind to rs3809865 in the 3'UTR region of ITGB3. The potential targets intersection was demonstrated by the Venn diagram. 2.5. Luciferase reporter assay Luciferase reporter assay was performed to verify if ITGB3 was a direct target of miR-124-3p. Construction of psiCEHCK-2 dual luciferase vector (Promega Corporation, Fitchburg, WI, USA) with rs3809865 A / T was named as wild type of ITGB or the mutant type of ITGB3. The constructs and miR-124-3p mimics or mimics-NC (Genechem Co., Ltd, Shanghai, China) were transiently co-transfected with the luciferase reporter plasmid in HEK-293 T cells. Transfected cells were collected after 48 hours of incubation at 37 °C. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA) in accordance with the manufacturer’s instructions.
2.2. Cell culture and transfection Human Gastric Cancer Cell Line (MKN-45, MGC803) and HEK-293 T were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, Hubei, China). Cells were cultured with DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C and 5% CO2. pEGFP-ITGB3, ITGB3 siRNA, pEGFP-NC, control siRNA (the control of ITGB3), miR-124-3p mimics, miR-124-3p inhibitor, miR-NC and inhibitor-NC (the control of miR-
2.6. Wound healing assay Table 1 Pathological information of 40 gastric cancer tissues. Variable
GC Patients (n = 40)
Age, mean ± SD (years) Male/female Tumor size (maximum diameter) ≥5 cm ＜5 cm Lymph node status N0 N1 N2 N3 Metastasis status 0 1 TNM stage Ⅰ Ⅱ Ⅲ Ⅳ Differentiation low differentiation Medium-high differentiation
59.65 ± 10.21 23/17
The migration of cells was evaluated by wound healing assay. In brief, Cells were incubated n 6-well plates with a density of 5 × 105 cells. Micropipette tip was used to make a scratch When the cells reached 90% confluence. Photos were captured from ten randomly selected fields in each group. The width (mm) of the scratch on each photo was measured using Image-Pro Plus 6.0 software (Media Cybernetics, USA).
16(40.0%) 24(60.0%)
2.7. CCK-8 assay
14(35.0%) 5(12.5%) 10(25.0%) 11(27.5%)
Cell Counting Kit-8 (CCK-8 assay kit, Dojindo, Japan) was conducted to measure cell proliferation capacity at a density of 5 × 103 cells in each well of 96-well plates. After incubation for 0, 12, 24, 48 h, 10 μl of CCK-8 solution was added into each well. Absorbance was examined utilizing MRX II microplate reader (Dynex Technologies, Chantilly, VA, USA) at 450 nm.
36(90.0%) 4(10.0%) 12(30.0%) 10(25.0%) 14(35.0%) 4(10.0%)
2.8. Transwell migration and invasion assays
22(55.0%) 18(45.0%)
Transwell migration and invasion assay were carried out to evaluate cells migration and invasion capacity. For transwell migration assay, A total of 1 × 105 cells were plated into the upper chamber of transwell chamber (Corning incorporated, USA) while the bottom chamber was
N: the regional lymph node involvement. M: the presence or otherwise of distant metastatic spread 2
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databases predicted that rs3809865 was located in the target sequence of miR-124-3p and miR-506-3p, as shown in Fig. 2A. Further prediction was made by the software of miRanda and RNA22v2.0, miR-124-3p performed better than miR-506-3p. To evaluate whether the change from A to T at rs3809865 affects the targeted binding of miR-124-3p and ITGB3, luciferase reporter assays were performed. ITGB3 3’UTRs containing the T-allele and A-allele of rs3809865 and miR-124-3p mimics or miR-NC were co-transfected into HEK-293 T cell, relative firefly and Renilla luciferase activity was measured. As shown in Fig. 2C, both of miR-124-3p co-transfected with A-allele or T-allele of ITGB3 can inhibited luciferase activity compared to controls. But the luciferase activity of the T allele have a higher inhibition rate compared with A allele(25.0% ± 1.7% VS 10.9% ± 2.0%, p < 0.001, Fig. 2D)
supplied with 10% FBS-contained medium. After 24 h, the migrated cells were washed with PBS and dyed with 0.5% crystal violet in 20% methanol. Take pictures at least ten randomly selected fields in each chamber and count the number of cells. The transwell invasion assay was the same as migration assay except the upper chamber was precoated with Matrigel medium at a 1:8 ratio. 2.9. Western blotting Total proteins were extracted from the collected tissues and cells with RIPA (Servicebio Biotechnology, China). The proteins were separated on SDS-PAGE and then transferred to PVDF membrane. The membranes were separately incubated with monoclonal rabbit antiITGB3 (Abcam, USA), mouse anti-GAPDH (Abcam, USA), or HRP-coupled secondary antibodies (Servicebio, China). The gray value calculation of Western blots was analyzed using Image J software (National Institutes of Health, Bethesda, MD).
3.3. miR-124-3p plays a role in gastric cancer by repressing ITGB3 expression To further confirm the relationship between miR-124-3p and ITGB3 in the regulation of gastric cancer, the miR-124-3p mimics, miR-NC, miR-124-3p inhibitor and inhibitor-NC were transfected to MGC-803 and MKN45 cells, respectively. RT-PCR analysis confirmed that miR124-3p levels were markedly increased or decreased in MGC-803 and MKN45 cells after transfection. (Figs. 3A and S2A). RT-PCR analysis and Western blotting were used to verify the changes in the expression of ITGB3 and integrin β3. The date showed that the expression of ITGB3 and integrin β3 was lower in miR-124-3p mimics group than in miR-NC group in both MGC-803 and MKN45 cells. On the contrary, downregulation of miR-124-3p increased the expression level of ITGB3 and integrin β3. (Figs. 3B, C and S2B, C). Next, we examined the role of miR-124-3p in proliferation and migration of gastric cancer. As the result of CCK-8 assay shows, the capacity of cell proliferation was inhibited after miR-124-3p overexpression. Meanwhile, the capacity of cell proliferation was increased after transfection with mir-124-3p inhibitor (Figs. 3D and S2D). Similar results were observed in wound healing assay when investigating the migration ability cells (Figs. 3E and S2E). Cell migration and invasion ability was examined by transwell migration and invasion assays. The result showed that the migration capacities of MGC-803 and MKN45 cells transfected with miR-124-3p mimics were significantly decreased compared to the miR-NC group while transfected with miR-124-3p inhibitor were increased compared with the inhibitor-NC group (Figs. 3F and S2F). These results are in good consistent with the prediction that miR-124-3p plays a role in gastric cancer by repressing ITGB3 expression.
2.10. Statistical analysis All statistics are completed with GraphPad Prism 6.0(GraphPad Software, La Jolla, CA, USA) and SPSS 22.0 (Chicago, IL, USA). Continuous variables are described by mean ± standard deviation, and categorical variables are described by frequency and percentage. P values are calculated by Student’s t-test or ANOVA analysis. And P < 0.05 represent statistical differences. Each experiment was replicated at least three times. 3. Results 3.1. ITGB3 changes the migration and invasion of gastric cancer cells in vitro To investigate the role of ITGB3 in gastric cancer, a pEGFP-ITGB3 vector and ITGB3 siRNA was transfected into MGC-803 and MKN45 cells which were human gastric cancer cell lines independently. The transfection efficiency at 48 h after transfection was shown in Figs. 1A, B and S1A, B. CCK-8 assays revealed the enhanced cell proliferation after transfection with pEGFP-ITGB3 compared to pEGFP-NC, while ITGB3 siRNA reduced cell proliferation compared to control siRNA in MGC-803 and MKN45 cells. (p < 0.05, Figs. 1C and S1C,). Furthermore, wound healing assay was performed to investigate the roles of ITGB3 on gastric cancer cell migration ability. The results showed that ITGB3 overexpression promoted gastric cancer cell migration in MGC-803 and MKN45 cells, while siRNA of ITGB3 dramatically decreased gastric cancer cell migration in MKN45 cell. (p < 0.05, Figs. 1D and S1D). SiRNA of ITGB3 in MGC803 cell showed a tendency to reduce cell migration but did not reach statistical difference. Similarly, the transwell migration and invasion assays illustrated that upregulation of ITGB3 promoted MGC-803 and MKN45 cells migration and invasive ability compared with controls, while knockdown of ITGB3 expression impeded the cell migration and invasion. (p < 0.05, Figs. 1E and S1E). In summary, our results demonstrated ITGB3 plays an important role in gastric cancer cells migration and invasion.
3.4. ITGB3 is up-regulated and miR-124-3p is down-regulated in gastric cancer tissues Lastly, we evaluated the relative expression levels of ITGB3 and miR-124-3p in 40 gastric cancer tissues and adjacent normal tissues, and their clinical characteristics are summarized in Table 1. The results of RT-PCR showed that ITGB3 in gastric cancer tissues was significantly up-regulated compared with the adjacent non-cancerous tissue samples (Fig. 4A, tumor tissue vs. normal tissue: 1.657 ± 0.134:1.080 ± 0.076; P < 0.001). Receiver operating characteristic (ROC) curve analysis demonstrated the area of ITGB3 under the ROC curve for gastric cancer diagnosis were 0.721(Fig. 4B). To further understand the correlation between the mRNA levels of ITGB3 and clinicopathological features, we performed a stratified analysis based on Tumor size, Lymph node status, Metastasis status, TNM stage and differentiation. The results showed that ITGB3 was correlated with tumor size. The group with the maximum diameter of ≥5 cm was significantly higher than the group which the diameter of < 5 cm (≥5 cm vs. < 5 cm:1.99 ± 0.198 VS 1.434 ± 0.168, P = 0.039) (Fig. 4C). Statistical differences were also available between the four groups of Lymph node status (Fig. 4D). No significance was discovered
3.2. miR-124-3p differentially regulates ITGB3 expression depending on SNP rs3809865 It was reported that SNP in the 3’UTR of an mRNA can effect the combination of mRNA and miRNA which results in decreased expression of the targeted gene [14]. We had verified that rs3809865 in the 3’UTR of ITGB3 was associated with gastric cancer stage in Chinese Han population [12]. Then we employed the online prediction software NIH, PolymiRTs Database 3.0, miRNASNP and miRdSNP to predict miRNAs that bind to rs3809865 in the 3’UTR region of ITGB3. Three 3
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Fig. 1. ITGB3 changes the migration and invasion of gastric cancer cells in vitro. (A,B) The Expression level of ITGB3 and integrin β3 was detected to verify the transfection efficiency at 48 h after transfection. (C) CCK8 showed the proliferation rates of MGC803 cells transfected with control siRNA, ITGB3 siRNA, pEGFP-NC and pEGFP-ITGB3. (D) Representative images of Wound healing assay in different groups. (E) Representative images of the transwell migration an invasion assays in different groups. (Considering for layout of the document, the results of MKN45 are placed in supplemental materials Fig. 1).
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Fig. 2. miR-124-3p differentially regulates ITGB3 expression. (A) NIH, PolymiRTs Database 3.0, miRNASNP and miRdSNP were used to predict that rs3809865 was located in the target sequence of miR-124-3p and miR-506-3p. (B) miRanda and RNA22v2.0 were predicted that miR-124-3p performed better than miR-506-3p with binding to ITGB3. (C,D) Luciferase reporter assays of miR-124-3p co-transfected with wildtype (A) or variant (T) of ITGB3.
4. Discussion
in TNM stage, Differentiation and Metastasis status (Fig. 4E–G). What's more, Western blotting also confirmed the expression trend of integrin β3 which is the protein of ITGB3 between gastric cancer tissues and normal tissues (Fig. 5A). The area of integrin β3 under the ROC curve for gastric cancer diagnosis was 0.810 (Fig. 5B). As for the integrinβ3 expression in gastric cancer, the same results as ITGB3 were observed, however, no significant difference but similar effect occurred between the expression level of integrin β3 and clinicopathologic feature of gastric cancer (Fig. 5C–G). At the same time, the relative expression of miR-124-3p in gastric cancer tissues was also measured. The results showed that the miR-1243p expression in gastric cancer tissues was significantly lower than that in adjacent normal tissues (0.757 ± 0.075 VS 1.112 ± 0.076, P = 0.001, Fig. 6A). The AUC value of ROC curve was 0.712 (Fig. 6B). Similarly, we performed stratified analysis of miR-124-3p based on clinical characteristics. However, no correlation was found between miR-124-3p and tumor size, lymph node status, metastasis status, TNM stage and differentiation of gastric cancer (Fig. 6C–G).
Gastric cancer is one of the major malignant tumors in the world and the second most common cancer in China. There are nearly 700,000 newly diagnosed gastric cancer patients in China every year [15]. It is urgent to study on the pathogenesis of gastric cancer and find early molecular markers. Integrin has been found to play an important role in the migration and invasion of gastric cancer [16,17]. miRNAs regulates the expression of integrin gene or inhibiting its translation at the transcriptional or post-transcriptional level by binding to the 3’UTR of target gene, thereby the invasion and metastasis of gastric cancer was affected [18,19]. Considerable research efforts have been devoted to demonstrated that miR-124-3p play important roles in tumorigenesis. For example, Zhang et al [20] reported that miR-124-3p inhibited glioblastoma multiforme cell development and growth act as the upstream suppressor of NRP-1 by PI3K/Akt/NFκB pathway. Long et al [21]demonstrated that lower miR-124-3p expression was shown to be correlated with a shorter overall survival and poor prognosis in hepatocellular carcinoma(HCC) by 155 biopsy tissues. Furthermore, Liu et al [22] found that miR-124-3p repressed the carcinogenesis of gastric cancer 5
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Fig. 3. miR-124-3p plays a role in gastric cancer by repressing ITGB3 expression. (A) The Expression level of miR124-3p was detected to verify the transfection efficiency. (B,C) RT-PCR analysis and Western blotting were used to verify that the expression of ITGB3 and integrin β3 could be upregulation or downregulation by miR-124-3p decreased or increased. (D) CCK8 showed the proliferation rates of MGC803 cells transfected with miR-NC, miR mimics, inhibitor-NC and miR inhibitor (E) Representative images of Wound healing assay in different groups. (F) Representative images of the transwell migration an invasion assays in different groups. (Considering for layout of the document, the results of MKN45 are placed in supplemental materials Fig. 2).
The hallmarks of cancer comprise six biological capabilities which include unlimited proliferation, escape of growth inhibitors, resistance to cell death, promotion of tumor angiogenesis, invasion of tumor cells and maintain proliferative signal transduction [23]. Integrins trigger
with a shorter overall survival and disease-free survival rates. These findings are consistent with our study partially. These findings suggest that miR-124-3p could function as tumor suppressors and may an independent indicator of survival for patients. 6
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Fig. 4. Expression level of ITGB3 in gastric cancer tissues and cells. (A) Relative expression levels of ITGB3 in gastric cancer tissues and normal tissue(n = 40). (B) ROC curve analysis for diagnostic value of ITGB3. (C-G) The expression level of ITGB3 in gastric cancer tissues from patients at different Tumor size, Lymph node status, TNM stage, Differentiation and Metastasis status.
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Fig. 5. Expression level of integrin β3 in gastric cancer tissues. (A) Relative expression levels of integrin β3 in gastric cancer tissues and normal tissue by western blot (n = 40). (B) ROC curve analysis for diagnostic value of integrin β3. (C-G) The expression level of integrin β3 in gastric cancer tissues from patients at different Tumor size, Lymph node status, TNM stage, Differentiation and Metastasis status.
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Fig. 6. Expression level of miR-124-3p in gastric cancer tissues. (A) Relative expression levels of miR-124-3p in gastric cancer tissues and normal tissue(n = 40). (B) ROC curve analysis for diagnostic value of miR-124-3p. (C-G) The expression level of miR-124-3p in gastric cancer tissues from patients at different Tumor size, Lymph node status, Metastasis status, TNM stage and Differentiation.
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Appendix A. Supplementary data
and play key roles in the six hallmarks of cancer [24]. For instance, ITGAX stimulates angiogenesis through overexpression of VEGFR2/ VEGF‐A mediated by PI3K/Akt signaling during cancer development, and overexpression of ITGAX can stimulate the globule formation of ovarian cancer cells in human umbilical vein endothelial cells [25]. In Glioblastoma Stem Cells, Integrin α6 enhanced the transcription of FGFR1 and its downstream targets through ZEB1 and YAP1, thereby enhancing the expression of stem cell-related factors and increasing the ability of neurosphere formation and sphere growth. In addition, When specific siRNA disturbed Integrin α6, stem cell-related factors and the ability of neurosphere formation decreased [26]. It has also been reported that miRNAs can play a role in cancer inhibition by regulating the content of integrin in cancer. Previous studied showed mir-3653 inhibits the growth and migration of hepatocellular cancer by reducing the expression level of ITGB1 [27]. Mir-143-3p ACTS as an anticancer gene by downregulating the expression of ITGA6/ASAP3 protein in the development of colorectal cancer [28]. Besides, our study confirmed that miR-124-3p could be a potential prognostic marker in gastric cancer and functions as a tumor suppressor by regulating ITGB3. In this study, we first demonstrate the ITGB3 is the target of miR124-3p by bioinformatics prediction and luciferase assay, then we investigated 40 pairs of gastric cancer tissues and normal control tissues. The expression levels of ITGB3 in tumor tissues were significantly higher than that in the normal group, while the expression level of miR124-3p was significantly lower than that in the normal group. Although it did not reach the statistically significant threshold, integrin β3 and miR-124-3pshowed correlated expression and related to the clinicopathological features of gastric cancer. This result could be validated in a larger sample size. Collectively, the present study demonstrated that miR-124-3p expression is significantly decreased while ITGB3 expression is significantly increases in gastric cancer tissues. Moreover, we demonstrated the miR-124-3p -mediated mechanism for ITGB3 to affect the gastric cancer cell proliferation migration and invasion for the first time. These results suggested that miR-124-3p and ITGB3 may reasonably be a prognostic and diagnostic biomarker for gastric cancer patients and may serve as a promising therapeutic target.
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Ethical approval This study was approved by the Ethics Committee of West China Hospital of Sichuan University. Informed consent Informed consent was obtained from all individual participants included in the study. Author contributions Qian Wu and Huiyu Zhong wrote the main manuscript and fully participated in all experiments. Lin Jiao and Yi Zhou participated in the acquisition of data. Juan Zhou, Yang Wen and Xiaojun Lu participated in the analysis and interpretation of data. Xingbo Song and Binwu Ying designed the study. All authors made substantial contributions to writing and revising the manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China [81672096] and the Projects in the Science and Technology Department of Sichuan Province pillar program [2017FZ0065] 10
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