Introduction
Globally, gastric cancer (GC) is the fifth leading cause of cancer and the third leading cause of death from cancer making up 7% of cases and 9% of deaths. In 2012 GC occurred in 950,000 people and caused 723,000 deaths [
1]. GC occurs most commonly in East Asia and Eastern Europe and it occurs twice as often in males as in females. The most common cause is infection by the bacterium
Helicobacter pylori (
Hp), which accounts for more than 60% of cases. The current diagnostic system was proved to be relatively poor in early-stage diagnosis GC, and accumulating evidence revealed the great potential of microRNAs (miRNAs) as biomarkers in GC diagnosis [
2].
Receptor tyrosine-protein kinase ErbB-3, is encoded by the ErbB3 gene and a member of the epidermal growth factor receptor (EGFR/ERBB) family of receptor tyrosine kinases [
3]. The kinase-impaired ErbB3 is known to form active heterodimers with other members of the ErbB family, most notably the ligand binding-impaired ErbB2 [
4]. ErbB3 was reported to be overexpressed in human gastric cancer, it acts as a tumor promoter by activation of a serial of complicated signaling including PI3K/AKT, RAS/RAF/MAPK and etc. The diversity of expression of ErbB3 was related to its upstream regulation, among all this mechanism miRNA regulation was one of the important reasons. miR-125, miR-199a, miR-205, and miR-450 etc. were all reported to potentially capable of regulating ErbB3, generally by targeting its three prime untranslated regions (3′UTR) [
5‐
9].
MicroRNAs (miRNAs) are a class of small, noncoding, approximately 22-nucleotides-long RNAs, which may function as a post-transcriptional regulator of gene expression. Single nucleotide polymorphism (SNP) is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population. It commonly occurs in the human genome, some SNP were functional associating with the structure of functional proteins. However, SNP also occurred in 3′UTR of certain genes which might effect on the binding force by various miRNA [
10,
11].
Thus, in this study, we focused on the SNPs in the 3′UTR of ERBB3. By using the bioinformatics software (
http://bioinfo.life.hust.edu.cn/miRNASNP/), we obtained all the SNPs which could regulate by miRNAs (Table
2). Via the bioinformatics prediction and statistical analysis, we found that the rs3202538 (G/T) in ErbB3 3′-UTR might potential effect on the regulation by miR-204 and miR-211. Moreover, a previous study has revealed that rs3202538 (G/T) in ErbB3 3′-UTR was seriously related to type I diabetes just via regulation of miR-204 and miR-211 [
12], we further investigated the allele distribution in a case–control study.
Materials and methods
Study subjects
A total of 851 GC cases and sex plus age-matched 799 controls obtained from Zhenjiang Hospital of Chinese Traditional Medicine, were included in this study. Patients were consecutively recruited between February 2010 and January 2015. All cases are incident ones during enrollment of the current case–control study. The diagnosis of all patients was histological confirmed. A face-to-face questionnaire was administered to collect demographic data and environmental exposure information, including alcohol use and cigarette consumption status as well as family cancer history. The normal tissues sampled was obtained at least 2 cm away from the margin of the tumor. All participants have provided their written informed consents to participate in this study. This study was approved by the Institutional Review Board of Zhenjiang Hospital of Chinese Traditional Medicine.
Genotype
The polymorphism was genotyped through the PCR-restriction fragment length polymorphism (RFLP) method as described previously [
13]. The PCR reactions were carried out in a total volume of 5 μL containing TaqMan Universal Master Mix, 80X SNP Genotyping AssayMix, DNase-free water and 10 ng genomic DNA. The PCR conditions were 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min by the 384-well ABI 7900HT Real Time PCR System. A 10% random sample was reciprocally examined by different persons, and the reproducibility was 100%.
Real-time PCR assay
Real-time polymerase chain reaction (RT-PCR) was performed to determine whether the mutation changed the expression level of ERBB3. The primers used for amplification were forward primer: CAGCAGCTTGACACACGGTA, and reverse primer: AAACACCAAAGTGGCATGTGA for ErbB3 and forward primer: TGTGGGCATCAATGGATTTGG, reverse primer: ACACCATGTATTCCGGGTCAAT for GAPDH. The amplification conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 40 s, and 72 °C for 30 s, and finally 4 °C for 30 min for cooling by the 384-well ABI 7900HT Real Time PCR System.
Cell lines and cell culture
GC cell lines SGC-7901 and MKN-45 were purchased from the Chinese Academy of Sciences Cell Bank. All cells were cultured in Dulbecco’s Minimum Essential Medium (DMEM) purchased from Gibco (CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, USA) and grown in humidified 5% CO2 at 37 °C. MiR-214 and miR-1225a mimics and a normal control were obtained from Genepharma (Shanghai, China). The transfection was conducted by using Lipofectamine 2000 (Invitrogen Corp, CA, USA).
Cell proliferation assays
Cell proliferation was determined by using CCK-8 (Dojin Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, the control and infected cells were seeded at a density of 1 × 103 cells/well in 96-well plates. 10 μL of CCK-8 was added to each well containing 100 µL of the culture medium, and the plate was incubated for 2 h at 37 °C. The viability of cells was evaluated by measuring the absorbance at 450 nm, using a microplate reader (Thermo Labsystems, CA).
Western blot
For western blotting, proteins were extracted from tissues or cultured cells using RIPA buffer containing Protease Inhibitor Cocktails (P8340) (Sigma-Aldrich). An equal amount of proteins (100 μg) were separated with 7.5%/12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. Primary polyclonal antibodies targeting ErbB3 (ab20161), p-AKT (ab38449), AKT (ab8933), β-actin (ab3280) were purchased from Abcam (Cambridge, MA). The secondary antibodies were anti-rabbit or anti-mouse HRP-linked were purchased from Santa Cruz Biotechnology (CA, USA). The blots were developed using ECL reagent (Millipore, MASS, USA). An equal amount of protein loading in each lane was confirmed using β-actin antibody. ImageJ software quantified the integrated density of the band.
Prediction of miRNAs binding to the SNP
Based on our bioinformatics analysis by using the bioinformatics software (
http://www.bioguo.org/miRNASNP/) to predict the related SNPs in the 3′UTR of ERBB3 which could interact with miRNAs.
Construction of luciferase-based reporter plasmids
A full-length fragment of the 3′UTR containing rs3202538 (G/T) (wild type/mutant) were amplified. The PCR product was cloned into the pGL3-promoter luciferase-based plasmid (Promega) at the cloning site between KpnI and XhoI. The amplified fragment was verified by DNA sequencing. For cell proliferation and invasion assay, the full-length cDNA of ErbB3 was sub-cloned into the pGL3-ErbB3 3′UTR plasmid.
Immunohistochemistry
Sections were de-paraffinized and followed by rehydration steps through a graded ethanol series and distilled water, and then were treated with 3% H2O2 in methanol for 30 min to block the endogenous peroxidase activity. The sections were rinsed in phosphate-buffered saline (PBS) twice, 5 min each time and incubated with 10% normal goat serum for 30 min to block non-specific antibody binding. After washing, the samples were incubated with primary anti-rabbit antibody ErbB3 (ab16901) purchased from Abcam (Cambridge, MA) at 4 °C overnight, and then washed with PBS for three times and then incubated with secondary antibodies. After that, the sections were stained with DAB according to manufacturer’s protocols and mounted and photographed using a digitalized microscope camera (Nikon, Tokyo, Japan).
Dual-luciferase reporter assay
For luciferase activity analysis, SGC-7901 and MKN-45 cells were cotransfected with 100 ng of luciferase reporter constructs 5 ng of the β-gal control plasmid and 10 pmol of miRNAs with 1 µL Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, NY, USA). After incubation for 48 h, we carried out the luciferase assay using the luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s protocol. Measurements of luminescence and absorbance of β-gal were performed on a luminometer (Glomax 20/20; Promega). Three independent experiments were performed in triplicate.
Cell invasion assay
For trans-well assay, the chamber was treated with Matrigel before cells were inoculated, 100 μL cell suspension with serum-free medium was seeded to the upper chamber, cells were stained with crystal violet staining solution (Beyotime, Nantong, China). Migrated cells were counted by using Image-pro Plus 6.0 while cell numbers of the normal control group were normalized to 1.
Statistical analysis
Differences between cases and controls were evaluated by the Student’s t test for continuous variables and the χ2 test for categorical variables. The association between SNPs and GC risk was estimated by the OR and 95% CI using the general genetic model. The potential gene–environment interaction was evaluated by logistic regression analysis and tested by comparing changes in deviance between the models of main effects with or without the interaction term. The overall survival and relapse free survival rate in different groups were analyzed by using Kaplan–Meier curve. Comparisons between groups were analyzed by the t test (two-sided). All statistical analyses were performed using Prism Graphpad software.
Discussion
Overexpression of ErbB3 was reported in human gastric cancer in several independent types of research, and also a high expression of ErbB3 was regarded as an indicator of poor prognosis in post-surgery patients [
3,
4,
14]. Recent works have implied the role of ErbB3 in gastric cancer as a key signaling hub [
15]. On the one hand, HER3 overexpression may promote tumor progression and invasion by activation of PI3K/AKT signaling pathway [
16]. On the other hand, a lot of direct evidence has emerged the benefit of anti-HER3 agents in combination with EGFR tyrosine kinase inhibitors as well as anti-HER2 agents in gastric cancer [
17]. And moreover, alternative activation of c-MET mediated signaling was also dependent on ErbB3, which reflected as the development of resistance to c-MET inhibitors may result from the overexpression of ErbB3 [
18].
The expression of ErbB3 was diverse in different GC patients, therefore, the signaling activation was also different. So far there was rarely research addressing the diversity of expression of ErbB3 in human GC, we addressed this issue here as SNP in its 3′UTR region and might cause deregulation of certain miRNA. miR-204 and miR-211 were found to have a potential suppression effect on ErbB3 transcription and SNP rs3202538 from G to T might increase ErbB3 expression by actuating such suppressive effect. In the present study, the difference of ErbB3 expression was verified between the G and T allele, the expression of ErbB3 can be attenuated dramatically by both miR-204 and miR-211 even in exogenous ErbB3 overexpression cells regulated by G allele but not the T allele 3′UTR. Also, the cell proliferation, invasion ability as well as downstream pathway activation also decreased corresponding to the expression of ErbB3.
The roles of miR-211 were complicated and controversial. Some of the researchers implied it as a tumor suppressor by targeting oncogenic genes such as IGF2R, TGFBR2 and NFAT5 in melanoma [
19]. And moreover, in human breast cancer, miRNA-211 directly inhibit CDC25B expression in breast cancer cells, alters other related target proteins CCNB1 and FOXM1, and then inhibits breast cancer cells growth, migration, and invasion and lead G2/M arrest [
20]. However, some studies defined miR-211 as a tumor promoter, by inhibiting the expression of SRC kinase signaling inhibitor 1 (SRCIN1), miR-211 was proved to promote human non-small cell lung cancer [
21], and by targeting tumor suppressive gene CHD 5, miR-211 can promote human colon cancer [
22,
23]. However, our study indicated that mIR-211 as a tumor suppressor in human GC by targeting ErbB3.
Compared to miR-211, the role of miR-204 was more explicit. In 2014 Zhou et al. reported that
Hp infection can down-regulate expression of miR-204 and thus to promote human GC by targeting SOX4 [
24], And miR-204 was extensively reported to suppression of EMT by targeting varying genes including snail and sirt1 [
25,
26]. And in the present study, we revealed that miR-204 can also relate to metastasis of human GC by targeting ErbB3.
Conclusion
In the present study, we found miR-211 and miR-204 have a potential transcriptional suppressive effect on ErbB3, and SNP in 3′UTR region of ErbB3 can effectively associate significantly up-regulation of ErbB3 which might occur due to loss regulation of miRNA. These findings are novel and might contribute to explicit the individual differences in GC susceptibility.
Authors’ contributions
Conception and design: SXC and SH. Collection and assembly of data: CX and YXW; Data analysis and interpretation: OYJ and HCX. Contribution of reagents, materials, and analysis tools, wrote the paper: QYC, OYJ and WDF. All authors read and approved the final manuscript.
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