Identification of miR-758-3p as Potential Modulator of CBX5 Expression in Gastric Cancer.

Gastric cancer is one of the most frequently diagnosed cancer types in China and also the leading causes of cancer-related death. Previous study showed chromobox 5 expression was elevated in gastric cancer, but little is known regarding the precise molecular mechanisms by which chromobox 5 expression was modulated. In this study, we revealed that chromobox 5 could promote gastric cancer cell proliferation, migration, and invasion in vitro. We screened and identified microRNA-758-3p, whose expression was downregulated in gastric cancer tissues and cell lines, which was a potential upstream molecule of chromobox 5. Upregulation of microRNA-758-3p could markedly downregulate the expression of chromobox 5. Additionally, expression of microRNA-758-3p and chromobox 5 was inversely correlated in gastric cancer tissues. Moreover, microRNA-758-3p overexpression suppressed gastric cancer cell proliferation, migration, and invasion, but these effects can be partially reversed by chromobox 5 overexpression. Collectively, our results indicate that microRNA-758-3p serves as a tumor suppressor and plays a crucial role in inhibiting the proliferation, migration, and invasion of gastric cancer via targeting chromobox 5 and implicate its potential application in cancer therapy.

Introduction

Gastric cancer (GC) ranks as the second most commonly diagnosed cancer types in China and also the leading causes of cancer-related deaths among both males and females in China.[1] It was estimated that there were approximately 679 100 new cases with 498 000 deaths in 2015, even though both the incidence and mortality of GC declined from 2000 to 2011 in China.[1] However, these data made a significant contribution to the global burden of cancer owing to the large population size.[1,2]

The development of GC is accompanied with the abnormal expression of a series of oncogenes and tumor suppressor genes.[3] Mounting evidence has demonstrated that microRNAs (miRNAs) were capable to regulate the expression of both oncogenes and tumor suppressor genes mainly through targeting the 3′-untranslated region (3′-UTR) of messenger RNAs.[4,5] MicroRNAs are characterized as a class of noncoding RNAs with approximately 19 to 25 nucleotides in length which have been reported to regulate all the hallmarks of cancer.[6,7] Moreover, many miRNAs have been identified to have a connection with the initiation and progression of GC.[8-11] For example, miR-100 expression was strongly correlated with expression of chemokine receptor 7, which in turn promotes the progression of GC.[8] Abnormal overexpression of miR-377 downregulates the tumor suppressors, including p53, PTEN, and TIMP1 expression, and serves as a novel predictive biomarker for GC tumorigenesis and prognosis, which would be useful for the therapy of GC.[9] Although abnormal expression of miRNAs has been frequently described in GC, imperfection is known regarding the precise underlying mechanisms of how miRNAs modulate the behavior of cancer cells.

MicroRNA-758-3p (miR-758-3p) has been reported to be abnormally expressed in various diseases including cancers.[12-15] For instance, miR-758 regulated cervical cancer infiltration and invasion through regulating matrix extracellular phosphoglycoprotein.[12] In addition, miR-758-3p functions as a tumor suppressor to inhibit proliferation, migration, and invasion of hepatocellular carcinoma via targeting mouse double minute 2 homolog (MDM2) and mechanistic target of rapamycin kinase (mTOR).[13] A recent study indicated miR-758-3p also serves as tumor suppressor in non-small cell lung cancer and was regulated by long noncoding RNA-DANCR to regulate the malignancy of non-small cell lung cancer.[15]

In the present study, we measured the expression pattern and biological function of miR-758-3p in GC and found it functions as a tumor suppressor gene in GC. In addition, we revealed that miR-758-3p regulates GC cell proliferation and migration by downregulating chromobox 5 (CBX5).

Materials and Methods

Clinical Samples

Seventy-three paired cancerous tissues and surrounding noncancerous tissues were obtained from patients with GC who underwent treatment at Renhe Hospital between 2013 and 2016. None of these patients have ever received anticancer treatment before surgery. Samples were immediately frozen using liquid nitrogen. All patients have provided written informed consents. This study was carried out following the Declaration of Helsinki, and study protocol was approved and monitored by ethics committee of Renhe Hospital (KJ2017-03). The clinicopathological features of enrolled patients are summarized in Table 1.

Table 1.

Correlations of miR-758-3p Expression and the Clinicopathological Features of GC.

Variables	N	miR-758-3p Expression		P Valuea
		Low (n = 42)	High (n = 31)
Age, years
≥50	39	23	16	.340
<50	34	19	15
Gender
Male	38	21	17	.423
Female	35	21	14
Lymph node metastasis
Yes	43	25	18	.017
No	30	17	13
Tumor stage
I-II	31	15	16	.002
III	42	27	15
Lauren typing
Intestinal	46	29	17	<.001
Diffuse	14	9	5
Mixed	13	8	5

Abbreviations: GC, gastric cancer; miR-758-3p, microRNA-758-3p.

a Chi-square test.

Cell Lines and Cell Transfection

Three human GC cell lines, SGC-7901, MGC-803, MKN-45, and a normal human gastric cell line, GES-1, were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). These cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, California) supplemented with 10% fetal bovine serum (Gibco, Invitrogen) at a 37°C containing 5% of CO2 in a humidified incubator.

Overexpression or downregulation of miR-758-3p was established using miR-758-3p mimic (5′-UUUGUGACCUGGUCCACUAACC-3′) or miR-758-3p inhibitor (5′-GGUUAGUGGACCAGGUCACAAA-3′) synthesized by GenePharma (Shanghai, China). The corresponding negative control (NC; 5′-UUCUCCGAACGUGUCACGUTT-3′) was also synthesized by GenePharma. The open reading frame construct for CBX5 and NC were purchased from GenScript (Nanjing, China). Cell transfection was carried out using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction of miR-758-3p

Total miRNA was extracted using a miRNA prep pure FFPE kit (TIANGEN, Beijing, China), according to the recommended protocols. Complementary DNA was synthesized using a QuantScript RT Kit (TIANGEN). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, California), according to the manufacturer’s instructions. The relative gene expression was calculated using the 2−ΔΔCt method. The PCR primers were synthesized by GenePharma and were listed as follows: miR-758-3p: forward: 5′-ACACTCCAGCTGGGTTTGTGACCTGGTCCA-3′, reverse: 5′-TGGTGTCGTGGAGTCG-3′; U6 (snRNA): forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′. U6 snRNA was used as an internal control.

Protein Isolation and Western Blot of CBX5

Radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) was used to isolate total proteins according to the manufacturer’s instruction. Protein samples of 40 μg were loaded onto the sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinyl fluoride membrane. After blocking with 5% non-fat milk for 1.5 hours at room temperature, the membranes were incubated with primary antibodies for overnight at 4°C. The primary antibodies used were as follows: anti-CBX5 (#2616; Cell Signaling Technology, Danvers, Massachusetts) and anti-GAPDH (#5174; Cell Signaling Technology). After washing with Tris-buffered saline Tween, membranes were incubated with the horseradish peroxidase–linked goat antirabbit secondary antibody (#7074; Cell Signaling Technology) for 2 hours at room temperature. Band signals were developed by a chemiluminescence detection system (Beyotime).

Cell Proliferation Assay

Cells proliferation was measured using Cell Counting Kit-8 (CCK-8; Dojinodo, Shanghai, China) assay according to the manufacturer’s protocol. The cells to be measured were incubated in 96-well plate, and the cell proliferation rate was detected at 0, 24, 48, and 72 hours. The CCK-8 solution was added to each well at the indicated points and further incubated for 4 hours. Then, a microplate reader (Bio-Rad Laboratories, Richmond, California) was used to detect the absorbance at 450 nm.

Wound Healing Assay

Cell migration was measured using wound healing assays. Cells were plated in 6-well plates and cultured using about 90% to 100% confluence. A scratch was created using a 200-µL pipette tip. After washing with phosphate-buffered saline, the image of the wound was captured at 0 and 48 hours using an inverted microscope.

Cell Invasion Assay

Cell invasion was measured by transwell invasion assay using BD Matrigel invasion chambers (BD Biosciences, Shanghai, China), according to the manufacturer’s instructions. Cells were seeded in the upper well of the chamber containing serum-free RPMI-1640. After 24-hour incubation, invasion cells in the bottom well were fixed with 3% paraformaldehyde and stained with 0.1% crystal violet. Invasion cell numbers were counted in 3 independent fields.

MicroRNA-758-3p Target Genes Prediction

We predicted potential target genes of miR-758-3p using miRNA databases TargetScan. The CBX5 gene was selected for further investigation based on the prediction results.

Luciferase Reporter Assays

The wild-type (wt) of 3′-UTR of CBX5 was cloned from the genomic DNA of GES-1 cells and cloned into the psiCHECK-2 vector (Promega, Madison, Wisconsin). The mutant (mut) type of 3′-UTR of CBX5 was generated using QuickMutation gene mutagenesis kit (Beyotime) with the following mutagenesis primers (sense: 5′-gatcattaatcagtgtttagatcaaaacctaattatcacagcctaggtaag-3′ and antisense: 5′-aggttttgatctattgtgacattaatgatcacaatcagttgactttgaaat-3′). For luciferase reporter assays, miR-758-3p mimic or NC and wt or mut 3′-UTR of CBX5 were cotransfected into cells. Dual-Luciferase Reporter Assay System (Promega) was used to measure luciferase activity. Renilla luciferase was used for normalization.

Statistical Analyses

GraphPad Prism 5.0 software (GraphPad, Inc, La Jolla, California) was used to for all statistical analysis in this study. Data were presented as the mean (SD). For the comparison between 2 groups, Student t test was used. For the comparison among 3 or more groups, analysis of variance and Tukey test were used. Chi-square test was used to measure the correlations between miR-758-3p expression and clinicopathological features. P < .05 was considered statistically significant.

Results

MicroRNA-758-3p Expression Was Significantly Downregulated in GC Tissues and Cell Lines

In this present study, we measured the miR-758-3p expression in 73 pairs of GC tissues and surrounding noncancerous tissues. The results in Figure 1A revealed that miR-758-3p expression in GC tissues was significantly lower compared to the surrounding noncancerous tissues. Moreover, miR-758-3p expression in GC cell lines, SGC-7901, MGC-803, and MKN-45, was found significantly lower than in normal human gastric cell line GES-1 (Figure 1B). The levels of miR-758-3p expression in these GC cell lines in descending order were MKN-45, MGC-803, and SGC-7901. Therefore, MGC-803 and SGC-7901 cell lines were selected for further studies.

Figure 1.

The miR-758-3p expression level in GC tissues and cells lines. A, Quantitative real-time polymerase chain reaction to analyze miR-758-3p expression in GC tissues and surrounding noncancerous tissues. B, Quantitative real-time polymerase chain reaction to analyze miR-758-3p expression in human GC cell lines (SGC-7901, MGC-803, and MKN-45) and normal cell line GSE-1. **P < .01, ***P < .001. GC indicates gastric cancer; miR-758-3p, microRNA-758-3p.

Clinical Significance of miR-758-3p Expression in GC

To identify the clinical significance of miR-758-3p expression in GC, we analyzed the associations between miR-758-3p expression and clinicopathological features. These patients were divided into 2 groups using the miR-758-3p expression level. Patients with higher miR-758 expression level in GC tissues than in surrounding noncancerous tissues were classified into high miR-758-3p expression group (n = 31); otherwise, the patients were classified into low miR-758-3p expression group (n = 42). These correlation analysis results revealed that low miR-758-3p expression was correlated with lymph node metastasis, tumor stage, and Lauren typing, but not correlated with age and gender (Table 1).

Chromobox 5 Was a Target of miR-758-3p

To explore the molecular target by which miR-758-3p regulates GC cell behaviors, TargetScan was used to predict the potential targets of miR-758-3p. It was predicted that CBX5 was a possible target of miR-758-3p (Figure 2A). To measure whether miR-758-3p impacted CBX5 expression, we examined CBX5 expression in GC cells transfected with miRNAs. We found CBX5 protein expression level was decreased by miR-758-3p mimic (Figure 2B). Next, luciferase activity reporter assay was conducted to verify whether CBX5 was a direct target of miR-758-3p. The results revealed that miR-758-3p mimic inhibited luciferase activity of wt 3′-UTR CBX5 but did not change the luciferase activity of mut 3′-UTR CBX5 (Figure 2C). Moreover, we examined the correlation between miR-758-3p and CBX5 expression in GC, and we found their expression was inversely correlated in GC (Figure 2D). Notably, we found CBX5 expression in GC tissues was significantly higher than in surrounding noncancerous tissues (Figure 2E).

Figure 2.

The miR-758-3p inhibits CBX5 expression by targeting 3′-UTR. A, Predicted miR-758-3p binding site in CBX5 3′-UTR. B, Western blot to detect CBX5 expression after transfection. C, Relative luciferase activity of CBX5 in wt or mut construct. D, Correlation analysis of miR-758-3p and CBX5 in GC tissues. E, Western blot to detect CBX5 expression in GC tissues and surrounding noncancerous tissues (ns: not significant, ***P < .001). CBX5 indicates chromobox 5; GC, gastric cancer; miR-758-3p, microRNA-758-3p; mut, mutant; NC, negative control; UTR, untranslated region; wt, wild type.

Overexpression of miR-758-3p Inhibits GC Cell Proliferation, Migration, and Invasion Through Targeting CBX5

To investigate the roles of miR-758-3p and the involvement of CBX5 in GC progression, miRNAs or CBX5 construct was transfected into GC cell lines. The expression of miR-758-3p in synthetic miRNA transfection GC cell lines was confirmed by qRT-PCR (Figure 3A). Western blot assay showed CBX5 expression can be elevated by CBX5 construct (Figure 3B). Meanwhile, the inhibitory effect of miR-758-3p mimic on CBX5 expression can be reversed by CBX5 construct (Figure 3B). The CCK-8 assay revealed that cell proliferation of GC cell lines was enhanced by miR-758-3p inhibitor but inhibited by miR-758-3p mimic (Figure 3C). In addition, wound healing assay indicated that miR-758-3p overexpression inhibited GC cell migration, and the downregulation of miR-758-3p has the opposite effects (Figure 3D). Furthermore, decreased cell invasion was also observed in GC cells transfected with miR-758-3p mimic (Figure 3E). Importantly, it was found that overexpression of CBX5 reversed the inhibitory effects of miR-758-3p mimic on cell proliferation, migration, and invasion (Figure 3C-3E). Taken together, these results demonstrated that miR-758-3p could inhibit GC cell proliferation, migration, and invasion through targeting CBX5.

Figure 3.

The miR-758-3p regulates GC cell proliferation, migration, and invasion through regulating CBX5. A, Quantitative real-time polymerase chain reaction to analyze miR-758-3p expression in GC cell lines transfected with miR-758-3p mimic, inhibitor, and NC. B, Western blot to detect CBX5 expression after miR-758-3p mimic or CBX5 construct transfection. C, The CCK-8 assay to detect cell proliferation after synthetic miRNAs or CBX5 construct transfection. D, Wound healing assay to detect cell migration after synthetic miRNAs or CBX5 construct transfection. E, Transwell assay to detect cell invasion after synthetic miRNAs or CBX5 construct transfection (ns, not significant, **P < .01, ***P < .001). CBX5 indicates chromobox 5; CCK-8, Cell Counting Kit-8; GC, gastric cancer; miR-758-3p, microRNA-758-3p; NC, negative control.

Discussions

Treatment of GC nowadays includes chemotherapy, surgery, and radiation therapy, but the therapeutic results are still disappointing.[16,17] Although extensive efforts have been put on the development of novel targeted therapy strategies and showed promising results in animal model, they are still far away from clinical use.[18,19] However, these efforts emphasized the importance to search for biomarkers that can be used for targeted therapy to benefit survival of patients with GC.

In recent decades, it has been demonstrated that miRNAs serve as modulators for GC’s progression.[8-11] In the present study, miR-758-3p expression level was examined in GC tissues, and its biological role and mechanisms in regulating GC progression were investigated. The measurement on the expression level of miR-758-3p indicated that miR-758-3p expression was downregulated in GC tissues compared to the matched noncancerous tissues. Furthermore, the low expression of miR-758-3p was correlated with worse lymph node metastasis and advance tumor stage. Overexpression miR-758-3p using miR-758-3p mimic inhibits GC cell proliferation, migration, and invasion. It has been well-documented that cancer cells exhibited unrestrained growth and increased cell migration and invasion.[20] Hence, these results revealed that miR-758-3p serves as tumor suppressor in GC and might be used as indicators for the malignancy of patients with GC.

MicroRNA-758-3p has been downregulated in hepatocellular carcinoma and cervical cancer.[12,13] Importantly, numerous downstream targets including matrix extracellular phosphoglycoprotein, MDM2, and mTOR have been identified which helped to establish the importance of miR-758-3p in cancers.[12,13] CBX5 was previously reported to elevate expression in GC tissues and can be downregulated by the treatment of vorinostat, indicating the oncogenic role of CBX5 in GC.[21] However, it was rarely understood how CBX5 expression was regulated in the progression and development of GC. In this study, we predicted CBX5 might be a target of miR-758-3p in GC through the online target prediction algorithm TargetScan. Furthermore, we found CBX5 expression was negatively correlated with the expression levels of miR-758-3p by either in vitro miRNAs cell transfection or the expression correlation analysis in GC tissues. The luciferase activity reporter assay validated CBX5 was a direct target of miR-758-3p. Moreover, the in vitro experiments revealed that miR-758-3p regulates GC cell behaviors through targeting CBX5 expression.

Taken together, we established the tumor suppressor role of miR-758-3p in GC, as it was downregulated in GC tissues and can negatively regulate the malignancy behaviors of GC cells. Furthermore, the investigation on the mechanism revealed that miR-758-3p inhibits GC progression through targeting CBX5. On the whole, these findings suggested miR-758-3p might be a possible target for GC treatment. However, we still have to admit that this study is a preliminary investigation on the biological roles of miR-758-3p and CBX5 in the progression of GC. More efforts are needed to deeply investigate the detailed mechanisms in this process.