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Abstract
Introduction: miR-125a-3p could have a role in gastric cancer by targeting HER2. This study aimed to investigate the expression pattern of miR-125a-3p, identify the expression level of its target gene in gastric carcinoma, and test its effect in HER-2 positive gastric carcinoma cells. Materials and Methods: The levels of miR-125a-3p in both cancer and noncancer tissues were measured by using Quantitative real-time polymerase chain in 70 gastric carcinomas. Immunohistochemical study was used to measure the expression of HER2 protein in these carcinomas. In addition, the level of expression of this miRNA is correlated to different pathological and clinical parameters. The effects of miR-125a-3p alone and in combination with 5-FU (fluorouracil) on the growth of HER2 positive (NUGC4) and HER2 negative (ECC10) gastric carcinoma cells were also analyzed by in vitro studies. Results: Most gastric cancer tissues samples showed downregulation of miR-125a-3p (84%) when compared to their noncancer tissues. Significant correlations of downregulation of miR-125a-3p with cancer recurrence and pathological staging of gastric carcinoma (P = 0. 02 and 0.02, respectively) were noted. HER2 protein expression correlated significantly and inversely with miR-125a-3p expression (P < 0.05). A reduction in cell growth rate was noted significantly in miR-125a-3p transfected gastric carcinoma cells when 5-FU was added to them in comparison to other control cells (P < 0.01). When both gastric carcinoma cell lines were transfected with miR-125a-3p, a significantly higher growth inhibition percentage in HER2 positive (NUGC4) cell line was seen in comparison to the HER2 negative (ECC10) cells (P < 0.01). Conclusion: miR-125a-3p plays a significant role in the pathogenesis of gastric carcinoma. Therapeutic transfection of miR-125a-3p in HER2 positive gastric cancer cells resulted in reduced cell proliferation and potentiate the effect of 5-FU.
Keywords: Carcinoma, gastric, HER2, immunohistochemistry, miR-125a-3p
How to cite this article: Introduction 
Gastric cancer is the 3rd most common deadly cancer and the 5th most common neoplasm with about 783,000 deaths in 2018 according to GLOBOCAN 2018 data. The major histology type of gastric cancer is adenocarcinoma.[1] MicroRNAs (miRNAs) are noncoding small RNAs (their length is about 21–23 nucleotides), which could regulate gene expression by mRNA degradation or translational repression.[2] Aberrant miRNAs expression has been found to be related to a diversity of human diseases like tumors, viral infections, neurodegenerative diseases, cardiovascular diseases, diabetes, and other diseases.[3] The miRNAs expression is tissue-specific and can be used to detect tumor origin and its type. The miRNA-expression profiles could be novel biomarkers for diagnosis and predicting prognosis and have potential therapeutic strategies by relevant miRNA expression or function manipulation of many human diseases.[4]
Up to now, the first approach in clinical studies using miRNA is by either miRNA mimics or miR-inhibitors. In addition, pharmacological approaches with miRNA could use miRNA sponges or anti-miRs. An important example of an effective therapeutic anti-miRNA method is miravirsen, “a locked nucleic acid (LNA)-modified anti-miR-122” that successfully fights a hepatitis C virus infection.[5] Another element that entered clinical phase 1 evaluation very recently is MRG-106, “an LNA anti-miR of miRNA-155” that could have roles in hematological malignancies.[6] A modified RNA nucleotide LNA or called bridged nucleic acid, which has a modified ribose moiety with an extra bridge that connects the 4' carbon and 2' oxygen, “locks” the ribose in a conformation of 3'-endo, to resist enzymatic degradation.[7]
miR-125a-3p was shown to control main cellular processes in some types of cancer. For instance, miR-125a-3p over-expression could impair migration and invasion of breast cancer cells.[8] Ectopic expression of miR-125a-3p in hepatocellular carcinoma cells resulted in the proliferation of cancer cells and inhibition of metastases, which are the result from miR-125a-3p ability to target vascular endothelial growth factor A and matrix metalloproteinase 11 (MMP11).[9] In addition, the roles of miR-125a-3p in the invasion and migration processes have also been implied in gastric and lung cancer.[10],[11]
Human epidermal growth factor receptor 2 (HER2 or ErbB2) (HER2) is one member of the human epidermal growth factor receptor (EGFR) family that comprises four members (ErbB 1–4) and has been amplified at the genomic level and/or overexpressed at the protein level in many cancers. ErbB signalling modules downstream pathways include the phosphatidylinositol 3-kinase/Akt (PKB) pathway and the Ras/Raf/MEK/ERK1/2 pathway, which have implications in survival, proliferation, and growth pathways of cells. Besides, members of ErbB family induce the focal adhesion kinase (FAK) activation playing an important role in cell attachment, migration, and invasion as it regulates the cell membrane protrusions formation and extension.[12]
In gastric carcinoma, HER2-overexpression depends on the primary location of carcinoma and ranges between 6%–30%.[13] HER-2 target therapy is a major therapy for treating metastatic or recurrent gastric and gastroesophageal carcinoma.[14] As both miR-125a-3p and HER-2 are important in the carcinogenesis of gastric carcinoma, this study aimed to investigate the expression pattern of miR-125a-3p, identify the expression level of its target gene in gastric cancer tissues, and test its anticancer role in HER2 positive gastric cancer.
Materials and Methods Tissue samples
Formalin-fixed paraffin-embedded (FFPE) human tissue samples were prospectively collected from gastric cancer with corresponding noncancer tissues from patients undergone gastric resection or endoscopic biopsy in private clinics in Hilla city after obtaining the consents from the patients. The study was approved from ethical committee of scientific research in the University of Babylon in Iraq (approval number 4/2020).
Patients clinical and pathologic data
After tissue collection, histopathological confirmation of the diagnosis of the collected samples from the surgical specimens was done. Of these, 70 patients with gastric adenocarcinoma were selected for this study. The information regarding the grading (degree of differentiation) and staging (level of extent) of these samples were recorded according to the World Health Organization (WHO) grading system and the TNM staging system (level of extent) for GCa of the American Joint Committee on Cancer (AJCC) 2018,[1] respectively. In addition, the other clinical and pathologic parameters including age, gender of the patients, and recurrence of the disease were also noted. All the patients were followed-up clinically.
MicroRNA extraction and cDNA conversion of the samples
From the FFPE tissues, total RNA was extracted after sectioning using a microtome. Tissues were sectioned using (5 μm) thickness and then RNA extracted using Qiagen miRNeasy Mini Kit (Qiagen, Germany) following the guidelines from the manufacturer. The quality and purity of purified RNAs including miRNAs were assessed as previously performed.[15] The RNA concentration was noted in ng/μl.
Reverse transcription reactions were done by using a miScript Reverse Transcription Kit (Qiagen) as described.[15] cDNA samples were diluted to the final concentration of 1.5 ng/μl to be used for real-time polymerase chain (PCR) reaction.
Quantitative real-time PCR for miR-125a-3p
miR-125a-3p expression in gastric cancer was done by using an IQ5 Multicolour Real-Time PCR Detection system (Bio-Rad, Hercules, CA, USA) as discussed previously.[16] RNU6B (Hs_RNU6B_2 miScript Primer Assay, Qiagen) was used for normalizing the quantity of target miRNA. Cancer and noncancer samples were run in the machine as described in previous research paper.[17] 2-ΔΔCT (fold change) method was used to obtain the results for each sample.[15] Upregulation and downregulation of this gene were noted and assessed in the same way as described in our previous research.[18]
HER2 staining by Immunohistochemistry in gastric cancer tissues
The immunohistochemical analysis of HER2 protein was performed by using ENVision™ FLEX Mini Kit (Dako, Denmark). Antigen retrieval was done as previously reported.[15] Peroxidase blocking reagent was used to block the tissue samples for 7 min. Rabbit Monoclonal antibody (anti-HER2 antibody), SP3, 1:100 dilutions (Thermo Fisher Scientific, Waltham, MA, USA) was used as a primary antibody. Tissues were incubated for 60 min at room temperature. Then, the samples were proceeded for further steps of staining as suggested by the manufacture company. A gastric adenocarcinoma tissue that displayed strong membranous staining was considered as a positive control in each run of the experiment. With the same procedure, the negative control was prepared, without primary antibody incubation.
By using a light microscope, the scoring of slides was done according to the scoring system of HER2 in gastric cancer, which was suggested by Hofmann et al.[19] in which, a grading scale ranging from 0 to 3 was used in scoring calculation. The 0 scale represented negative staining in less than 10% of tumor cells, 1 represented weak staining in more than 10% of tumor cells, 2 denoted basolateral membranous staining in either nonuniform or weak staining in at least 10% of cells, and 3 was considered when complete or basolateral membranous reactivity of strong intensity in more than 10% of cells. Scoring of slides was done by two different pathologists to confirm the data.
Cell lines and cell culture
Human gastric cancer cell lines (NUGC4 with HER2 overexpression, ECC10 with no HER2 protein detected)[20] were purchased from the American type culture collection (ATCC). They were cultivated and maintained in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal blood serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured as a monolayer in a 37°C incubator and 5% CO2.
Cell transfection
Human miR-125a-3p plasmids were used as permanent transfection of gastric cancer cells. miR-control plasmid was purchased from GenePharma Co., Ltd. (Shanghai, China). The transfection of miR-125a-3p plasmids and miR-control plasmids was performed according to the manufacturer's instructions. By doubling the concentration of miR-125a-3p plasmid, optimization was achieved. Approximately 0.5 μg of DNA was used to optimize the transfection in 96 well plates when 80%–90% confluence was reached.[21]
Cell proliferation assay
By using MTT assay (3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide), the role of miR-125a-3p in gastric cancer cell proliferation was detected. Transfected cells, nontransfected cells (in addition to the 5-fluoruracil 500 μg/mL) (positive control), and RPMI media (Sigma–Aldrich, Castle Hill, NSW, Australia) only treated cells (negative control) were seeded in flat-bottom 96-well plates with 1 × 104 cells/well in triplicate and were incubated at 37°C for 24 h, 48 h, 72 h, and 96 h. Then, to each well, 10 mL cell counting kit-8 (CCK8)/100 mL RPMI media was added.[15] Afterwards, the cells were incubated at 37°C for 1 h. Finally, by using the optical density of each well read with an ELISA plate reader, the absorbance was determined. The cellular growth inhibition was calculated according to the following equation: Growth inhibition% = 100 − mean of the optical density of experimental wells/mean of the optical density of control wells.[15]
Statistical analysis
Statistical analysis was achieved by Statistical Package for the Social Sciences (SPSS), version 26 (IBM, New York, USA). For categorical variables, Chi-square test or likelihood ratio was used. For continuous variables, Pearson correlation test was used. One-way ANOVA and Independent t-test were performed for the analysis of continuous variables in categories. Value < 0.05 for these tests was considered as significant.
Results Correlation of miR-125a-3p expression to the gastric carcinoma patient's parameters [Table 1]
Table 1: Quantitative real-time PCR expression of miR-125a-3p in relation to clinicopathologic parameters of the patients with gastric carcinomaGastric adenocarcinoma tissues showed downregulation of miR-125a-3p in comparison to the adjacent nonmalignant tissues [Table 1]. miR-125a-3p downregulated in 84% (59/70) and overexpressed in 7% (5/70) of Gca. The expression levels of miR-125a-3p showed no change in the remaining carcinomas (6/70; 9%).
In gastric adenocarcinoma with advanced cancer stages (stage III or stage IV), downregulation of miR-125a-3p predominated and a significant correlation between miR125a-3p downregulation and the advanced cancer stages of gastric cancer was noted (P = 0.02). Cancer recurrence after surgery was noted in 29% (20/70) of the patients. Approximately 80% of the patients with recurrent cancer had a low expression level of miR-125a-3p (P = 0.02) when compared to those with no cancer recurrence. miR-125a-3p expression showed no significant correlation with other parameters of the patients as shown in [Table 1] (P > 0.05).
miR-125a-3p expression in correlation with HER2 protein expression in gastric cancer
Membranous and cytoplasmic expression of HER2 protein was found in an immunohistochemical study [Figure 1]. Of the 64 cases that had a high or low level of miR-125a-3p, 22 cases showed score = 0 or 1 (low or no expression) of HER2 protein and 42 cases showed a score = 2 or 3 (HER2 protein high expression). In general, 71.2% of the miR-125a-3P downregulated gastric carcinoma showed score = 2 or 3 (HER2 protein overexpression) and 100% of the miR-125a-3P upregulated gastric cancer tissues showed score = 0 or 1 (HER2 protein downregulation). A significant inverse correlation between miR-125a-3p and its target HER2 was suggested from these data [Table 2].
Figure 1: Moderately differentiated adenocarcinoma of stomach stained strongly for HER2 in IHC analysis (high power)
Table 2: Immunohistochemical analysis of HER2 protein in correlation with miR-125a-3p expressionmiR-125a-3p effect on HER2 protein expression in gastric carcinoma cells
miR-125-3p reduces the proliferation and growth of gastric carcinoma cells. Stable transfection data was accomplished [Table 3]. The growth rate of cells was significantly reduced in miR-125a-3p transfected gastric carcinoma cells in both cancer cell lines in comparison to other control cells after 48 h, 72 h, and 96 h (maximum inhibition reached after 72 h) of incubation (P < 0.001). In addition, significantly higher growth inhibition percentages were seen in NUGC4 cells in comparison to the ECC10 cells (P < 0.001). 5-FU treatment together with miR-125a-3p showed a high significance difference from miR-125a-3p alone (P < 0.01) and from other control groups in the same cell line (P < 0.001). The combined treatment of 5-FU with miR-125a-3p showed a significant therapeutic difference in NUGC4 cells in comparison to the ECC10 cells (P < 0.001) [Figure 2] and [Figure 3].
Table 3: Percentages of growth inhibition for cells treated with miR-125a-5p alone or combined to 5-FU (fluorouracil) in comparison to plasmid and RPMI media control groups after 24 h, 48 h, 72 h, and 96 h of exposure in two different gastric cancer cell lines as evidenced by MTT assay represented by Mean±SD
Figure 2: Moderately differentiated adenocarcinoma of stomach stained slightly for HER2 in IHC analysis (high power)
Figure 3: Poorly differentiated adenocarcinoma of stomach stained strongly for HER2 NEU IHC staining (Low power) Discussion Data from several studies demonstrated the downregulation of miR-125a-3p in many cancers and its role in inhibiting tumor cell proliferation, invasion, and migration, as well as enhancing apoptosis.[22],[23] The results of the present study concurred with those of the previous studies. A meta-analysis in 2019 found that miR-125a-3p is a tumor suppressor with prognostic and clinicopathologic values for human gastric and lung cancers. miR-125a-3p overexpression predicted favorable prognosis, early pathological stages, negative lymph node metastatic status, and lower tumor grade in some cancers.[24] In addition, a significant inverse correlation between the levels of miR-125a-3p and its target CDK3 in primary breast cancer samples was reported.[25] Taken together, these findings support that miR-125a-3p has a tumor-suppressive role in cancer development by targeting some downstream genes.
Yan et al.[26] 2019 showed that the roles of interleukin-21 receptor (IL-21R or CD360) in gastric carcinogenesis and dysregulation of long noncoding RNA MALAT1/miR-125a axis are partly responsible for its activation. The regulation of the paracrine of VEGF-A in gastric cancer is attributed partially to miR-125a and thereby controlled the angiogenesis of this cancer.[27] Yet, its roles in the progression of HER2 positive gastric cancer have not been completely reported.
In this study, we report that miR-125a-3p has an important role in HER2 positive gastric cancer, in which the transcriptional activity of HER2 was inhibited with miR-125a-3p that was characterized by a decrease in cell proliferation rate by in vitro study and inverse correlation between miR-125a-3p and HER2. Our study for the first time identified that miR-125a-3p has an inhibitory effect on HER2 overexpressed gastric cancer cells, which is also validated by the clinical sample used in this study.
Wang et al.[28] 2013 showed that Entinostat (a chemotherapeutic agent that stops the growth of tumor cells by blocking some of the enzymes needed for cell growth) encourages miR-205, miR-125a, and miR-125b expression and expression of these miRNAs results in erbB2/erbB3 downregulation in breast cancer cells. Our study strengthens their findings in terms that this miRNA can control the downstream genetic expression in gastric cancer. Their suggestion was that miRNA-dependent mechanisms can produce an epigenetic regulation, which can be considered as a novel line in the development of treatment in HER-2-overexpressing breast cancer.
There are several clinical trials to target HER2 gene as a treatment for HER2 positive gastric cancer. The results of this research suggest that the development of epigenetic target therapy could be a treatment option for gastric carcinoma in the future. Particularly, we propose that miR-125a-3p targeting could be a promising therapy for clinical trials in gastric cancer.
Conclusion The current study confirms the molecular control of miR-125a-3p in gastric cancer and its expression could be a prognostic marker. Moreover, an inverse correlation between miR125a-3p and HER2 suggests the role of this miRNA in the pathogenesis of gastric cancer. Restoration of the miR-125a-3p level in HER2 positive gastric cancer cells can be emerged as a new and effective molecular therapeutic regime to treat this type of cancer. The treatment could overcome 5-FU resistance and potentiate its effect in cancer, which need further molecular investigations.
Acknowledgements
The authors dedicate thanks and gratitude to Professor Alfred Lam (Head of pathology department/School of Medicine/Griffith University/Australia) who help in arranging the manuscript and all persons who help in achieving this work.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_580_21
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