Skip to main content
NCBI home page
Oncology Research logoLink to Oncology Research
. 2018 Dec 27;27(1):19–27. doi: 10.3727/096504018X15193469240508

MicroRNA-623 Targets Cyclin D1 to Inhibit Cell Proliferation and Enhance the Chemosensitivity of Cells to 5-Fluorouracil in Gastric Cancer

Lihua Jiang *,1, Wenchuan Yang *,1, Weishi Bian †, Hailin Yang *, Xia Wu *, Yuhua Li *, Wen Feng *, Xuejian Liu *
PMCID: PMC7848397  PMID: 29495973

Abstract

The dysregulation of microRNAs (miRNAs) plays an important function in the onset and progression of gastric cancer (GC). In addition, aberrantly expressed miRNAs affect the chemosensitivity of GC cells to chemotherapeutic drugs. Hence, miRNA-based targeted therapy might be applied to treat patients with GC exhibiting chemotherapeutic resistance. In this study, miRNA-623 (miR-623) expression was downregulated in GC tissues and cell lines. Functional analysis showed that the restored miR-623 expression could inhibit the proliferation of GC cells and enhance their chemosensitivity to 5-FU via the cell apoptosis pathway. Cyclin D1 (CCND1) was identified as a direct target gene of miR-623 in GC. The overexpressed CCND1 in GC tissues was negatively correlated with miR-623 level. The recovered CCND1 expression counteracted the effects of miR-623 on GC cell proliferation, chemosensitivity, and 5-FU-induced apoptosis. Thus, our results suggest that miR-623 might function as a tumor suppressor in GC and could be a promising therapeutic target for patients with GC, especially those with chemotherapeutic resistance.

Key words: MicroRNA-623, Gastric cancer (GC), Chemosensitivity, 5-Fluorouracil (5-FU), Cyclin D1 (CCND1)

INTRODUCTION

Gastric cancer (GC), the most common cancer affecting the digestive tract, ranks as the fifth most frequently occurring cancer and the third leading cause of cancer-associated mortalities globally1. Approximately 1,000,000 novel GC cases and over 700,000 GC-related deaths are annually recorded worldwide2. Several risk factors, such as dietary habits, Helicobacter pylori infection, pernicious anemia, and chronic atrophic gastritis, have been validated to be involved in the occurrence and development of GC3,4. Despite advancements in treatments for GC, the prognosis of patients with advanced GC remains unsatisfactory5. The 5-year overall survival rate of patients with GC diagnosed in advanced stages is 4%6. Clinical studies have emphasized that the poor therapeutic outcomes of patients with GC is associated with late diagnosis, metastasis, and recurrence7,8. Moreover, chemoresistance impedes successful cancer treatment9. Therefore, the molecular mechanism associated with GC pathogenesis should be investigated to develop novel treatment options and improve the clinical outcomes of patients with this disease.

A previous study on microRNAs (miRNAs) has helped enhance our understanding of chemotherapy resistance10. miRNAs are a large family of short noncoding single-stranded RNA molecules that can play major regulatory roles11. miRNAs participate in gene regulation by directly interacting with the 3′-untranslated regions (3′-UTRs) of their target genes in a base-pairing manner, resulting in the degradation of mRNA or inhibition of translation12. Aberrant miRNA expression has been widely reported in almost all types of human malignancies, such as GC13, renal cell carcinoma14, colorectal cancer15, and ovarian cancer16. Such expression is also implicated in tumorigenesis and tumor development through the regulation of cell proliferation, cell cycle, apoptosis, and metastasis17,18. miRNAs are also involved in the chemosensitivity and chemoresistance of human cancer types19. For example, miR-939 upregulation increases the chemosensitivity of drug-resistant GC cells to vincristine and adriamycin by directly targeting EZH220. Therefore, targeting miRNAs may be a new therapeutic strategy for the treatment of chemoresistant GC.

miR-623 plays important roles in lung adenocarcinoma21. However, its expression pattern, biological functions, and biological mechanism in GC remain unclear. In this study, we confirmed that miR-623 expression was reduced in GC tissues and cell lines. Upregulation of miR-623 suppressed the proliferation of GC cells and increased their chemosensitivity to 5-fluorouracil (5-FU). Cyclin D1 (CCND1) was also identified as a direct target of miR-623 in GC.

MATERIALS AND METHODS

Acquisition of Tissue Specimens

This research was approved by the Ethics Committee of Linyi Third People’s Hospital (Shandong, P.R. China). Full written informed consent was provided by all GC patients who participated in the current study. A total of 31 paired GC tissues and corresponding adjacent normal tissues were obtained from patients diagnosed with GC at the Linyi Third People’s Hospital. All the patients were treated with surgical resection between August 2010 and January 2016. No patients had been treated with chemotherapy or radiotherapy before surgery. Both GC tissues and corresponding adjacent normal tissues were quickly frozen in liquid nitrogen and kept in the cryopreservation refrigerator at −80°C.

Cell Culture and Transfection Condition

Four human GC cell lines (MKN-45, SGC-7901, BGC-823, and MGC-803) and the human normal gastric epithelial cell line GES-1 were ordered from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, P.R. China). All these cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin and streptomycin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and grown at 37°C in a humidified chamber containing 5% CO2 and 95% air.

miR-623 mimic and negative control miRNA mimic (miR-NC) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, P.R. China). Empty pcDNA3.1 vector and constructed CCND1 overexpression vector pcDNA3.1-CCND1 were designed and synthesized by Integrated Biotech Solutions (Shanghai, P.R. China). For cell transfection, cells were inoculated into six-well plates with a density of 5 × 105 cells/well. Cell transfection was conducted using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer’s protocol, when the cells reached about 60% confluence. Eight hours posttransfection, cell culture medium was replaced with fresh DMEM containing 10% FBS. Transfection efficiency was evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) or Western blot analysis.

RNA Isolation and RT-qPCR

Total RNA was prepared from tissue samples or cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. For the quantification of miRNA expression, total RNA was reverse transcribed into cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). Subsequently, qPCR was carried out on the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.) using a TaqMan MicroRNA PCR Kit (Applied Biosystems). For the detection of mRNA expression, reverse transcription was performed with M-MLV Reverse Transcription system (Promega Corporation, Madison, WI, USA). Afterward, SYBR Green I Mix (Takara Biotechnology Co., Ltd., Dalian, P.R. China) was utilized to carry out qPCR. U6 snRNA and GAPDH were applied as internal control for miR-623 and CCND1 mRNA, respectively. Data were analyzed using the 2−ΔΔCt method22.

Cell Counting Kit-8 (CCK-8) and Cell Chemosensitivity Assays

Transfected cells were collected and inoculated into 96-well plates at a density of 3 × 103 cells/well in triplicate. The extent of cell proliferation was evaluated at 0, 24, 48, and 72 h after incubation at 37°C in a humidified chamber containing 5% CO2 and 95% air. CCK-8 solution (10 μl; Beyotime Institute of Biotechnology, Haimen, P.R. China) was added to each well. After the cells were incubated at 37°C for another 2 h, absorbance was determined at a wavelength of 450 nm with a SpectraMax M5 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

Cell chemosensitivity was determined using the CCK-8 method. At 24 h after transfection, the cells were seeded into 96-well plates with a density of 3 × 103 cells/well. Afterward, the cells were incubated overnight and treated with 5-FU (Sigma-Aldrich, St. Louis, MO, USA) at different concentrations (0–32 μM). Cell chemosensitivity assay was conducted at 48 h after 5-FU was administered. The dose–response curve was plotted at various concentrations.

Flow Cytometry Cell Apoptosis Analysis

A total of 5 × 105 cells were plated into six-well plates and cultured at 37°C with 5% CO2. At 48 h after transfection, the cells were probed with 8 μM 5-FU and further incubated at 37°C for another 48 h. The cells were harvested, washed with ice-cold phosphate-buffered saline, and collected into a 1.5-ml tube. Cell apoptosis was then detected using an Annexin-V-FITC apoptosis detection kit (Invitrogen Corporation). In brief, 300 μl of 1× binding buffer was added to a 1.5-ml tube, gently mixed, and added with 5 μl of annexin V and 5 μl of PI. The cells were further incubated at room temperature in the dark for 15 min, and cell apoptosis was analyzed using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). Each assay was performed in triplicate.

Bioinformatics Analysis

TargetScan (www.targetscan.org/ ) and miRanda (www.microrna.org ) were utilized to predict the potential targets of miR-623. Among these candidates, CCND1 was selected for further confirmation, as it has previously been demonstrated to contribute to the chemosensitivity of GC cells.

Dual-Luciferase Reporter Assay

A wild-type (WT) CCND1 3′-UTR containing the predicted binding sequences of miR-623 and a mutant (MUT)-type CCND1 3′-UTR lacking the binding sequences of miR-623 were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, P.R. China), cloned into a psiCHECK-2 vector (Promega Corporation), and designated as psiCHECK-WT-CCND1-3′-UTR and psiCHECK-MUT-CCND1-3′-UTR, respectively. The cells were seeded into 24-well plates (2.0 × 103/well) 24 h prior to transfection and then transfected with miR-623 mimic or miR-NC and psiCHECK-WT-CCND1-3′-UTR or psiCHECK-MUT-CCND1-3′-UTR using Lipofectamine™ 2000 according to the manufacturer’s protocol. Luciferase activities were detected using a Dual-Luciferase Reporter Assay System (Promega Corporation) at 48 h posttransfection. Renilla luciferase activity was used as an internal reference.

Western Blot Analysis

In this study, mouse anti-human monoclonal CCND1 (1:1,000 dilution; Catalog No. sc-8396) and mouse anti-human monoclonal β-actin (1:1,000 dilution; Catalog No. sc-81178) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). β-Actin was utilized as a loading control for protein level normalization. Total protein was isolated from tissues or cells using a total protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, P.R. China). The total protein concentration was assessed with a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal quantities of protein sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% fat-free milk in TBS containing 0.1% Tween 20 (TBST), incubated with primary antibodies at 4°C overnight, washed with TBST, and probed with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; Catalog No. sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. The blots were visualized using an enhanced chemiluminescence solution (ECL; Pierce; Thermo Fisher Scientific, Inc.).

Statistical Analysis

Data were expressed as mean ± standard deviation and statistically analyzed using SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). Differences between two groups were examined using Student’s t-test, and one-way ANOVA was performed when there were more than two groups. Student–Newman–Keuls test was conducted as a post hoc test following ANOVA. A two-tailed value of p < 0.05 was considered significant.

RESULTS

miR-623 Expression Is Downregulated in GC Tissues and Cell Lines

To explore the expression pattern of miR-623 in GC, we initially detected its expression in 31 paired GC tissues and the corresponding adjacent normal tissues. RT-qPCR analysis revealed that miR-623 expression was downregulated in GC tissues compared with those in adjacent normal tissues (p < 0.05) (Fig. 1A). We then measured the miR-623 expression levels in four human GC cell lines and a human normal gastric epithelial cell line GES-1. miR-623 was underexpressed in all of the tested GC cell lines compared with that of GES-1 (p < 0.05) (Fig. 1B). SGC-7901 and BGC-823 cell lines, which exhibited a relatively lower miR-623 expression out of the four examined cell lines, were selected for subsequent experiments. These results suggested that the downregulation of miR-623 might be associated with GC progression.

Figure 1.

Figure 1

Open in a new tab

MicroRNA-623 (miR-623) is downregulated in gastric cancer (GC) tissues and cell lines. (A) Relative miR-623 expression levels in 31 paired GC tissues and their corresponding adjacent normal tissues were detected through RT-qPCR. *p < 0.05 compared with normal tissues. (B) Relative miR-623 expression was determined in four human GC cell lines (MKN-45, SGC-7901, BGC-823, and MGC-803) and a human normal gastric epithelial cell line (GES-1). *p < 0.05 compared with GES-1.

miR-623 Overexpression Represses Cell Proliferation and Increases the Chemosensitivity of Cells to 5-FU in GC

miR-623 was markedly overexpressed in SGC-7901 and BGC-823 cells transfected with miR-623 mimic to explore the biological function of miR-623 in GC (p < 0.05) (Fig. 2A). CCK-8 assay was then performed to investigate the effect of miR-623 overexpression on GC cell proliferation. The results revealed that the restored miR-623 expression significantly repressed the proliferation of SGC-7901 and BGC-823 cells (p < 0.05) (Fig. 2B). Therefore, decreased miR-623 expression might promote GC progression by inducing the proliferation of GC cells.

Figure 2.

Figure 2

Open in a new tab

miR-623 inhibits the proliferation and enhances the chemosensitivity of SGC-7901 and BGC-823 cells to 5-fluorouracil (5-FU). (A) SGC-7901 and BGC-823 cells were transfected with miR-623 mimic or miR-negative control (NC). At 48 h posttransfection, relative miR-623 expression was evaluated through reverse transcription quantitative polymerase chain reaction (RT-qPCR). *p < 0.05 compared with miR-NC. (B) The proliferation of SGC-7901 and BGC-823 cells was determined through cell counting kit-8 (CCK-8) assay following transfection with miR-623 mimic or miR-NC. *p < 0.05 compared with miR-NC. (C) miR-623 mimic or miR-NC was introduced to SGC-7901 and BGC-823 cells. After 24 h of transfection, different concentrations of 5-FU were added and incubated for another 48 h. Cell chemosensitivity assay was performed to detect the chemosensitivity of SGC-7901 and BGC-823 cells to 5-FU. *p < 0.05 compared with miR-NC. (D) SGC-7901 and BGC-823 cells were transfected with miR-623 mimic or miR-NC and incubated with 8 μM 5-FU 48 h after transfection. Cell apoptosis was analyzed through flow cytometry to examine the cell apoptosis rate. *p < 0.05 compared with miR-NC + 5-FU.

Cell chemosensitivity assays were conducted to evaluate whether miR-623 is involved in the development of chemoresistance in GC cells. In Figure 2C, SGC-7901 and BGC-823 cells transfected with miR-623 mimic exhibited a significantly decreased survival rate after they were treated with 5-FU (p < 0.05), suggesting that miR-623 overexpression might reverse the chemoresistance of GC cells to 5-FU. The antitumor activity of 5-FU was mainly caused by promoting cell apoptosis. Hence, cell apoptosis was analyzed through flow cytometry to elucidate whether miR-623 mediated the chemosensitivity of GC cells to 5-FU by promoting 5-FU-induced cell apoptosis. In Figure 2D, enforced expression of miR-623 expedited the 5-FU-stimulated apoptosis of SGC-7901 and BGC-823 cells (p < 0.05). These results implied that miR-623 was involved in the regulation of the chemosensitivity of GC cells to 5-FU via the cell apoptosis pathway.

CCND1 Is a Direct Target Gene of miR-623 in GC

Bioinformatic analysis was conducted to search for the potential targets of miR-623 and to explore the mechanisms that might be responsible for the action of miR-623 in GC. Among these candidates, CCND1 (Fig. 3A) was selected for further confirmation because it contributes to the chemosensitivity of GC cells23–26. RT-qPCR and Western blot analysis were applied to detect the mRNA and protein expression of CCND1 in SGC-7901 and BGC-823 cells transfected with miR-623 mimic or miR-NC. Our data showed that the ectopic miR-623 expression decreased the CCND1 expression in SGC-7901 and BGC-823 cells at the mRNA (p < 0.05) (Fig. 3B) and protein (p < 0.05) (Fig. 3C) levels, suggesting that miR-623 negatively regulated the endogenous CCND1 expression in GC. To further confirm that CCND1 is a direct target of miR-623 and to determine whether miR-623 can directly target the 3′-UTR of CCND1, we performed a dual-luciferase reporter assay. SGC-7901 and BGC-823 cells were transfected with miR-623 mimic or miR-NC in combination with psiCHECK-WT-CCND1-3′-UTR or psiCHECK-MUT-CCND1-3′-UTR. The transfection of SGC-7901 and BGC-823 cells with miR-623 mimic reduced the luciferase activities of the reporter containing the WT 3′-UTR of CCND1 (p < 0.05) but not the activities of the reporter with the respective MUT 3′-UTR (Fig. 3D and E). These findings suggested that CCND1 is a direct target of miR-623 in GC.

Figure 3.

Figure 3

Open in a new tab

miR-623 directly targets cyclin D1 (CCND1) in GC. (A) Wild-type (WT) and mutated (MUT) miR-623 binding sequences in the 3′-untranslated region (3′-UTR) of CCND1. (B) RT-qPCR and (C) Western blot were performed to determine the mRNA and protein levels of CCND1 in SGC-7901 and BGC-823 cells transfected with miR-623 mimic or miR-NC. *p < 0.05 compared with miR-NC. miR-623 or miR-NC was transfected in (D) SGC-7901 and (E) BGC-823 cells with psiCHECK-WT-CCND1-3′-UTR or psiCHECK-MUT-CCND1-3′-UTR. Relative luciferase activity levels were measured at 48 h posttransfection. *p < 0.05 compared with miR-NC.

CCND1 Expression Increases in GC Tissues and Shows a Negative Correlation With miR-623 Levels

The expression level of CCND1 in GC tissues and adjacent normal tissues was measured through RT-qPCR and Western blot analysis. The results indicated that the mRNA expression level of CCND1 increased in GC tissues compared with that in adjacent normal tissues (p < 0.05) (Fig. 4A). In addition, CCND1 protein expression was upregulated in GC tissues compared with that in adjacent normal tissues (Fig. 4B). Spearman’s correlation analysis revealed that miR-623 was remarkably associated with the mRNA of CCND1 in these GC tissues (r = −0.5849, p = 0.0005) (Fig. 4C).

Figure 4.

Figure 4

Open in a new tab

CCND1 overexpression in GC tissues is inversely correlated with miR-623 level. (A) RT-qPCR and (B) Western blot were applied to measure the mRNA and protein expression levels of CCND1 in GC tissues and adjacent normal tissues, respectively. *p < 0.05 compared with normal tissues. (C) The association between CCND1 mRNA and miR-623 levels in GC tissues was assessed through Spearman’s correlation analysis. r = −0.5849, p = 0.0005.

Reintroduction of CCND1 Counteracts the Effects of miR-623 on GC Cells

Considering that CCND1 served as a direct functional target of miR-623, we hypothesized that the recovered CCND1 expression in the miR-623-expressing cells might rescue the effects of miR-623 overexpression on GC. To confirm this hypothesis, we performed rescue experiments and introduced miR-623 mimic to SGC-7901 and BGC-823 cells in combination with pcDNA3.1 or pcDNA3.1-CCND1. We evaluated the transfection efficiency through Western blot analysis and observed that the protein level of CCND1 was restored in SGC-7901 and BGC-823 cells after they were cotransfected with pcDNA3.1-CCND1 (p < 0.05) (Fig. 5A). Similarly, the subsequent experiments confirmed that the restored CCND1 expression counteracted the effects on SGC-7901 and BGC-823 cell proliferation (p < 0.05) (Fig. 5B), chemosensitivity (p < 0.05) (Fig. 5C), and 5-FU-induced apoptosis (p < 0.05) (Fig. 5D) caused by miR-623 overexpression. These results demonstrated that miR-623 might play tumor-suppressive roles in GC partly by downregulating CCND1.

Figure 5.

Figure 5

Open in a new tab

CCND1 overexpression reverses the effects of miR-623 on GC cells. (A) CCND1 protein expression was detected in SGC-7901 and BGC-823 cells cotransfected with miR-623 mimic and pcDNA3.1 or pcDNA3.1-CCND1 through Western blot. *p < 0.05 compared with miR-NC. #p < 0.05 compared with miR-623 mimics + pDNA3.1-CCND1. CCK-8 assay (B), cell chemosensitivity assay (C), and flow cytometry analysis of cell apoptosis (D) were performed to determine cell proliferation, chemosensitivity to 5-FU, and apoptosis induced by 5-FU in differently treated SGC-7901 and BGC-823 cells, respectively. *p < 0.05 compared with miR-NC. #p < 0.05 compared with miR-623 mimics + pcDNA3.1-CCND1.

DISCUSSION

The dysregulation of miRNAs plays an important function in the onset and progression of GC27–29. Aberrantly expressed miRNAs affect the chemosensitivity of cells to chemotherapeutic drugs in multiple types of human cancer. For example, the restored miR-16 expression enhances the chemosensitivity of breast cancer cells to paclitaxel by directly targeting inhibitor of nuclear factor κ B kinase subunit β (IKBKB)30. Xiao et al.31 revealed that the chemosensitivity of acute myeloid leukemia cells to doxorubicin is increased by miR-217 overexpression. Cheng et al. found that ectopic expression of miR-451 sensitizes lung cancer cells to cisplatin by blocking Mcl-119. Zhang et al.32 demonstrated that resumption expression of miR-625 increases the chemosensitivity of glioma cells to temozolomide through regulation of AKT2. Xu et al.33 reported that enforced expression of miR-330 targets thymidylate synthase (TYMS) to enhance the sensitivity of colorectal cancer cells to 5-FU by regulating TYMS. In GC, miR-14734, miR-320a35, miR-12620, and miR-21836 are implicated in the regulation of cell chemosensitivity. These findings suggested that miRNA-based targeted therapy might be applied to treat patients with tumors exhibiting chemotherapeutic resistance.

miR-623 is downregulated in lung adenocarcinoma tissues and cell lines. Decreased miR-623 level is significantly associated with tumor differentiation, lymphatic metastasis, and increased serum carcinoembryonic antigen level. The median overall survival of the patients with lung adenocarcinoma and low miR-623 expression is shorter than that of the patients with high miR-623 expression. Furthermore, miR-623 reexpression prevents the in vitro and in vivo growth and metastasis of lung adenocarcinoma cells by directly targeting Ku8021. In our study, miR-623 was underexpressed in GC tissues and cell lines. In the subsequent in vitro experiments, miR-623 overexpression attenuated the proliferation of GC cells and increased their chemosensitivity to 5-FU. CCND1 was validated as a direct target of miR-623 in GC. The upregulation of CCND1 in GC tissues was also associated with miR-623 level. Rescue experiments revealed that the recovered CCND1 expression counteracted the effects of miR-623 on GC cells. These results demonstrated that miR-623 directly targeted CCND1 to play tumor-suppressive roles in GC growth and chemoresistance, implying that miR-623 might be investigated as a therapeutic target to rapidly block the tumor growth and chemoresistance of patients with this malignancy.

Identifying miRNAs and their targets involved in gastric carcinogenesis and progression may help develop effective therapeutic strategies. In this study, CCND1 was validated to be a direct and functional target of miR-623 in GC. CCND1, located in chromosome 11q13, is a well-described oncogene overexpressed in numerous human cancer types, such as thyroid carcinoma36, breast cancer37, lung cancer38, and bladder cancer39. In GC, CCND1 is also upregulated and strongly correlated with poor differentiation40. Survival analysis indicated that the CCND1 expression level was significantly associated with the disease-free survival and overall survival of patients with GC. The prognosis of these patients who exhibited a high CCND1 level was poorer than that of the patients with low CCND1. Multivariate analysis has identified CCND1 expression as an independent prognostic indicator of disease-free survival and overall survival in GC23. Functional assays have demonstrated that CCND1 contributes to the tumor initiation and progression of GC through the regulation of cell proliferation, cycle, migration, invasion, and apoptosis23–25. The inhibition of CCND1 enhances the chemosensitivity of GC cells to 5-FU26. These findings suggested that targeting CCND1 may provide advantages to treat patients with GC, especially those exhibiting chemotherapeutic resistance.

CCND1 has been determined to be regulated by miRNAs in multiple types of human cancer. For instance, miR-193a-3p reexpression targets CCND1 to prohibit cell growth in prostate cancer41. In T-cell lymphoma, miR-373 overexpression represses cell proliferation by regulating CCND142. In colon cancer, the miR-374a upregulation reduces cell growth, motility, and intrahepatic metastasis by inhibiting CCND143. In non-small cell lung cancer, miR-326 attenuates cell proliferation and colony formation and promotes apoptosis by directly targeting CCND144. In GC, miR-193b24, miR-33a45, and miR-449a25 directly target CCND1 and inhibit GC progression. Combined with the present findings, the miRNA/CCND1 axis shows potential for the therapy of patients with GC.

In conclusion, miR-623 was significantly underexpressed in GC tissues and cell lines. In vitro studies demonstrated that miR-623 inhibited GC cell proliferation and increased cell chemosensitivity to 5-FU. CCND1 was mechanistically identified as a direct target gene of miR-623 in GC. However, further experiments should be conducted to evaluate whether miR-623 can enhance the chemosensitivity of GC cells to other chemotherapeutic drugs.

ACKNOWLEDGMENT

The authors declare no conflicts of interest.

Footnotes

The authors declare no conflicts of interest.

REFERENCES

  • 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86. [DOI] [PubMed] [Google Scholar]
  • 2. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–917. [DOI] [PubMed] [Google Scholar]
  • 3. Cheng XJ, Lin JC, Tu SP. Etiology and prevention of gastric cancer. Gastrointest Tumors 2016;3:25–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Compare D, Rocco A, Nardone G. Risk factors in gastric cancer. Eur Rev Med Pharmacol Sci. 2010;14:302–8. [PubMed] [Google Scholar]
  • 5. Kim TH, Shivdasani RA. Stomach development, stem cells and disease. Development 2016;143:554–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Cunningham SC, Kamangar F, Kim MP, Hammoud S, Haque R, Maitra A, Montgomery E, Heitmiller RE, Choti MA, Lillemoe KD, Cameron JL, Yeo CJ, Schulick RD. Survival after gastric adenocarcinoma resection: Eighteen-year experience at a single institution. J Gastrointest Surg. 2005;9:718–25. [DOI] [PubMed] [Google Scholar]
  • 7. Matouk IJ, Halle D, Raveh E, Gilon M, Sorin V, Hochberg A. The role of the oncofetal H19 lncRNA in tumor metastasis: Orchestrating the EMT-MET decision. Oncotarget 2016;7:3748–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Geredeli C, Dogru O, Omeroglu E, Yilmaz F, Cicekci F. Gastric metastasis of triple negative invasive lobular carcinoma. Rare Tumors 2015;7:5764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Sun F, Lu X, Li H, Peng Z, Wu K, Wang G, Tong Q. Special AT-rich sequence binding protein 1 regulates the multidrug resistance and invasion of human gastric cancer cells. Oncol Lett. 2012;4:156–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Dehghanzadeh R, Jadidi-Niaragh F, Gharibi T, Yousefi M. MicroRNA-induced drug resistance in gastric cancer. Biomed Pharmacother. 2015;74:191–9. [DOI] [PubMed] [Google Scholar]
  • 11. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. [DOI] [PubMed] [Google Scholar]
  • 12. Cortes-Sempere M, Ibanez de Caceres I. microRNAs as novel epigenetic biomarkers for human cancer. Clin Transl Oncol. 2011;13:357–62. [DOI] [PubMed] [Google Scholar]
  • 13. Tao Y, Yang S, Wu Y, Fang X, Wang Y, Song Y, Han T. MicroRNA-216a inhibits the metastasis of gastric cancer cells by targeting JAK2/STAT3-mediated EMT process. Oncotarget 2017;8:88870–81. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 14. Yoshino H, Yonezawa T, Yonemori M, Miyamoto K, Sakaguchi T, Sugita S, Osako Y, Tatarano S, Nakagawa M, Enokida H. Downregulation of microRNA-1274a induces cell apoptosis through regulation of BMPR1B in clear cell renal cell carcinoma. Oncol Rep. 2018;39:173–81. [DOI] [PubMed] [Google Scholar]
  • 15. Yang X, Yang L, Ma Y, Zhao X, Wang H. MicroRNA-205 mediates proteinase-activated receptor 2 (PAR2)-promoted cancer cell migration. Cancer Invest. 2017;35:601–9. [DOI] [PubMed] [Google Scholar]
  • 16. Wang Y, Xu C, Wang Y, Zhang X. MicroRNA-365 inhibits ovarian cancer progression by targeting Wnt5a. Am J Cancer Res. 2017;7:1096–106. [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 17. Wang J, Song YX, Wang ZN. Non-coding RNAs in gastric cancer. Gene 2015;560:1–8. [DOI] [PubMed] [Google Scholar]
  • 18. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR. MicroRNA expression profiles classify human cancers. Nature 2005;435:834–8. [DOI] [PubMed] [Google Scholar]
  • 19. Cheng D, Xu Y, Sun C, He Z. MicroRNA-451 sensitizes lung cancer cells to cisplatin through regulation of Mcl-1. Mol Cell Biochem. 2016;423:85–91. [DOI] [PubMed] [Google Scholar]
  • 20. Wang P, Li Z, Liu H, Zhou D, Fu A, Zhang E. MicroRNA-126 increases chemosensitivity in drug-resistant gastric cancer cells by targeting EZH2. Biochem Biophys Res Commun. 2016;479:91–6. [DOI] [PubMed] [Google Scholar]
  • 21. Wei S, Zhang ZY, Fu SL, Xie JG, Liu XS, Xu YJ, Zhao JP, Xiong WN. Hsa-miR-623 suppresses tumor progression in human lung adenocarcinoma. Cell Death Dis. 2016;7:1–13. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25:402–8. [DOI] [PubMed] [Google Scholar]
  • 23. Yuan C, Zhu X, Han Y, Song C, Liu C, Lu S, Zhang M, Yu F, Peng Z, Zhou C. Elevated HOXA1 expression correlates with accelerated tumor cell proliferation and poor prognosis in gastric cancer partly via cyclin D1. J Exp Clin Cancer Res. 2016;35:15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Wang L, Zhang Y, Zhao L, Liu S, Yu S, Ma Y, Sun G. MicroRNA-193b inhibits the proliferation, migration and invasion of gastric cancer cells via targeting cyclin D1. Acta Histochem. 2016;118:323–30. [DOI] [PubMed] [Google Scholar]
  • 25. Hu J, Fang Y, Cao Y, Qin R, Chen Q. miR-449a regulates proliferation and chemosensitivity to cisplatin by targeting cyclin D1 and BCL2 in SGC7901 cells. Dig Dis Sci. 2014;59:336–45. [DOI] [PubMed] [Google Scholar]
  • 26. Seo JH, Jeong ES, Lee KS, Heo SH, Jeong DG, Choi YK. Lentivirus-mediated shRNA targeting of cyclin D1 enhances the chemosensitivity of human gastric cancer to 5-fluorouracil. Int J Oncol. 2013;43:2007–14. [DOI] [PubMed] [Google Scholar]
  • 27. Chang L, Guo F, Huo B, Lv Y, Wang Y, Liu W. Expression and clinical significance of the microRNA-200 family in gastric cancer. Oncol Lett. 2015;9:2317–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Ma G, Dai W, Sang A, Yang X, Gao C. Upregulation of microRNA-23a/b promotes tumor progression and confers poor prognosis in patients with gastric cancer. Int J Clin Exp Pathol. 2014;7:8833–40. [PMC free article] [PubMed] [Google Scholar]
  • 29. Qiao W, Cao N, Yang L. MicroRNA-154 inhibits the growth and metastasis of gastric cancer cells by directly targeting MTDH. Oncol Lett. 2017;14:3268–74. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 30. Tang X, Jin L, Cao P, Cao K, Huang C, Luo Y, Ma J, Shen S, Tan M, Li X, Zhou M. MicroRNA-16 sensitizes breast cancer cells to paclitaxel through suppression of IKBKB expression. Oncotarget 2016;7:23668–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Xiao Y, Deng T, Su C, Shang Z. MicroRNA 217 inhibits cell proliferation and enhances chemosensitivity to doxorubicin in acute myeloid leukemia by targeting KRAS. Oncol Lett. 2017;13:4986–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Zhang J, Zhang J, Zhang J, Qiu W, Xu S, Yu Q, Liu C, Wang Y, Lu A, Zhang J, Lu X. MicroRNA-625 inhibits the proliferation and increases the chemosensitivity of glioma by directly targeting AKT2. Am J Cancer Res. 2017;7:1835–49. [PMC free article] [PubMed] [Google Scholar]
  • 33. Xu W, Jiang H, Zhang F, Gao J, Hou J. MicroRNA-330 inhibited cell proliferation and enhanced chemosensitivity to 5-fluorouracil in colorectal cancer by directly targeting thymidylate synthase. Oncol Lett. 2017;13:3387–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Shen J, Niu W, Zhang H, Jun M, Zhang H. Downregulation of microRNA-147 inhibits cell proliferation and increases the chemosensitivity of gastric cancer cells to 5-Fluorouracil by directly targeting PTEN. Oncol Res. 2017. [Epub ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 35. Ge X, Cui H, Zhou Y, Yin D, Feng Y, Xin Q, Xu X, Liu W, Liu S, Zhang Q. miR-320a modulates cell growth and chemosensitivity via regulating ADAM10 in gastric cancer. Mol Med Rep. 2017;16:9664–70. [DOI] [PubMed] [Google Scholar]
  • 36. Brzezianska E, Cyniak-Magierska A, Sporny S, Pastuszak-Lewandoska D, Lewinski A. Assessment of cyclin D1 gene expression as a prognostic factor in benign and malignant thyroid lesions. Neuro Endocrinol Lett. 2007;28:341–50. [PubMed] [Google Scholar]
  • 37. Li X, Huo X, Li W, Yang Q, Wang Y, Kang X. Genetic association between cyclin D1 polymorphism and breast cancer susceptibility. Tumour Biol. 2014;35:11959–65. [DOI] [PubMed] [Google Scholar]
  • 38. Betticher DC, Heighway J, Hasleton PS, Altermatt HJ, Ryder WD, Cerny T, Thatcher N. Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer. Br J Cancer 1996;73:294–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Kopparapu PK, Boorjian SA, Robinson BD, Downes M, Gudas LJ, Mongan NP, Persson JL. Expression of cyclin D1 and its association with disease characteristics in bladder cancer. Anticancer Res. 2013;33:5235–42. [PMC free article] [PubMed] [Google Scholar]
  • 40. Shan YS, Hsu HP, Lai MD, Hung YH, Wang CY, Yen MC, Chen YL. Cyclin D1 overexpression correlates with poor tumor differentiation and prognosis in gastric cancer. Oncol Lett. 2017;14:4517–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Liu Y, Xu X, Xu X, Li S, Liang Z, Hu Z, Wu J, Zhu Y, Jin X, Wang X, Lin Y, Chen H, Mao Y, Luo J, Zheng X, Xie L. MicroRNA-193a-3p inhibits cell proliferation in prostate cancer by targeting cyclin D1. Oncol Lett. 2017;14:5121–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Tian YY, Jia CM, Li Y, Wang Y, Jiang L, Liu AC. Restoration of microRNA-373 suppresses growth of human T-cell lymphoma cells by repressing CCND1. Eur Rev Med Pharmacol Sci. 2016;20:4435–44. [PubMed] [Google Scholar]
  • 43. Chen Y, Jiang J, Zhao M, Luo X, Liang Z, Zhen Y, Fu Q, Deng X, Lin X, Li L, Luo R, Liu Z, Fang W. MicroRNA-374a suppresses colon cancer progression by directly reducing CCND1 to inactivate the PI3K/AKT pathway. Oncotarget 2016;7:41306–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Sun C, Huang C, Li S, Yang C, Xi Y, Wang L, Zhang F, Fu Y, Li D. Hsa-miR-326 targets CCND1 and inhibits non-small cell lung cancer development. Oncotarget 2016;7:8341–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Wang Y, Zhou X, Shan B, Han J, Wang F, Fan X, Lv Y, Chang L, Liu W. Downregulation of microRNA33a promotes cyclin-dependent kinase 6, cyclin D1 and PIM1 expression and gastric cancer cell proliferation. Mol Med Rep. 2015;12:6491–500. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Oncology Research are provided here courtesy of Tech Science Press

RESOURCES