Key Points
• This is the first study investigating the curcumin/miR-638-5p/potential target genes in triple negative breast cancer (TNBC) cell line MDA-MB-231.
• The relationship between TNBC and miRNAs/genes was studied using bioinformatics tools and in vitro experiments, and many important miRNAs and genes have been identified.
• MiR-638-5p may play an important role in the cancer process through its potential target genes CFL1, SIX4, and MAZ.
Introduction
The molecular tumor complexity of breast cancer (BRCA) is an important obstacle to the treatment. Even though there are many useful therapies (surgery, radiation therapy, or hormone therapy), BRCA metastases, drug resistance, and relapse result in poor patient survival. Curcumin is the most prominent polyphenol component extracted from the turmeric (rhizomes of Curcuma longa). Vogel and Pelletier of the Harvard College Laboratory first identified curcumin in 1815 (1). Much subsequent research has demonstrated that curcumin is extremely beneficial to health (2). Its cytotoxic efficacy in several cancer cell lines, including BRCA, has been demonstrated. The pleiotropic action of curcumin in cancer cell inhibition is due to its numerous targets, which include signaling pathways, proteins/enzymes, and microRNAs (miRNAs) (3). miRNAs are single-stranded RNA molecules with around 18-22 nucleotides that act as master regulators of gene expression by binding to their target mRNAs in the cells (4, 5). In 271 species, 38,589 mature miRNAs have been identified, including 2654 mature human miRNAs (6). By recognizing matching sequences at the 3’ UTR region of the target mRNA, a single miRNA may affect thousands of genes (7, 8). Many studies, especially in the last 10 years, have demonstrated that dysregulation of miRNA expression is associated with almost every kind of cancer, including BRCA (9, 10). Cancer hallmarks, such as maintaining cell proliferative signaling, apoptosis avoidance, stimulating invasion and metastasis, and triggering angiogenesis have been linked to altered miRNAs (11). Studies show that many natural dietary supplements, including curcumin, have important roles in various cellular processes (12, 13). The results suggest that these may make important contributions to the fight against cancer in the future (14).
This study was conducted to investigate the relationship between BRCA, miRNAs, mRNA and curcumin using in silico and in vitro methods. Briefly, geo datasets were used to identify miRNAs and genes that may be linked to BRCA. In silico tools were used to match the detected miRNAs and target genes. Enrichment analyses of selected miRNAs and genes were performed using various bioinformatics tools. The relationship between the selected miRNA and the target genes were then confirmed in in vitro evaluation, and the expression levels of the relevant miRNA and genes were investigated in curcumin-treated cells.
Materials and Methods
Identification of Triple Negative Breast Cancer (TNBC)-Associated miRNAs
Overlapping miRNAs between GSE154255 and GSE40525 datasets, which met the criteria of logFC >2 and logFC >1, respectively, and p<0.05 for both datasets, were identified. This was carried out because the GSE154255 dataset contains very few miRNAs with a logFC >2 value, the logFC >1 value was used for this dataset.
Identification of the Effect of Overlapping miRNAs on Overall Survival in BRCA and TNBC
Whether overlapping miRNAs were effective on overall survival (OS) in both BRCA and TNBC was investigated in METABRIC data using kmplot (https://kmplot.com/analysis/) and a significant miRNA was selected for further in silico and in vitro analysis.
The Detection of Overexpressed Genes in BRCA
To identify significant genes in BRCA, The Cancer Genome Atlas Program (TCGA) BRCA data were searched through the GEPIA2 (http://gepia2.cancer-pku.cn/) web tool. Among the overexpressed genes in TCGA BRCA data, genes that met LogFC >1 and p<0.05 criteria were identified.
In silico Investigation of Potential Target Genes of the Selected miRNA
In silico potential target genes of the selected miRNA were identified using the databases miRNet (https://www.mirnet.ca/), miRTarBase v8.0 (https://mirtarbase.cuhk.edu.cn/) and TarBase v.8 (https://dianalab.e-ce.uth.gr/) tools.
Detection of Overlapping Genes Between in silico Target Genes of the Selected miRNA and Significant Genes in TCGA BRCA Data
The overlapping genes between the in silico potential targets of the selected miRNA and the genes overexpressed in TCGA BRCA and meeting the LogFC >1 and p<0.05 criteria were determined.
Enrichment Analysis of Overlapping Genes
The diseases, hub proteins, and pathways most associated with overlapping genes were identified using the Enrichr (https://maayanlab.cloud/Enrichr/) and ShinyGO 0.77 (http://bioinformatics.sdstate.edu/go/) tools.
Identification of Genes Associated With BRCA Overall Survival
Employing the kmplot tool, it was determined whether overlapping genes were associated with BRCA OS.
In vitro Studies
Cell Culture
For cell culture, the TNBC cell line, MDA-MB-231, was cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) (EcoTech Biotechnology, Erzurum, Turkey) with 1% penicillin (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) 10% fetal bovine serum (FBS) (EcoTech Biotechnology, Turkey) in a humidified incubator (Sanyo) with 5% CO2 at 37 °C.
Curcumin Treatment
Highly purified curcumin (Bio Basic Inc., Canada) was dissolved in dimethyl sulfoxide (DMSO) (1 mg/mL). Curcumin was prepared at different concentrations (1 µM, 3 µM, 5 µM, 10 µM, 30 µM and 50 µM). As curcumin was dissolved in DMSO, the control cells were treated with DMSO at the same quantities as the experimental groups. Cells were maintained in 6-well or 96-well plates (Nest Biotechnology Co., China) for 24 hours at 37 °C. The 50% inhibition concentration (IC50) value of curcumin was determined (10 µM). For further investigation, this value was used to treat MDA-MB-231 cells.
miR-638-5p Mimic Transfection
MDA-MB-231 cells were seeded at sixty percent confluency into 96-well or 6-well cultivation plates. Afterward, using the supplier’s protocol for transient overexpression of miR-638-5p, cells were transfected with 30 pM miR-638-5p mimic (5¢-AGGGAUCGCGGGCGGGUGGCGGCCU-3¢) (Thermo Fisher Scientific), or non-targeting (NT) control miRNA using lipofectamine 2000 (Invitrogen). Following 24 hours of culture, transfected cells were used for functional assays.
RNA Isolation, cDNA Synthesis Process, and Quantitative Real-time PCR
Total RNA was extracted from curcumin-treated and miR-638-5p transfected cells and control cultures using TRIzol (Invitrogen). A NanoDrop spectrophotometer (Thermo) was used to assess the quality and quantity of the RNA samples. To examine the expression of selected genes or miRNAs, equal amounts of RNA from the specimens were reverse-transcribed into cDNA using a cDNA Reverse Transcription Kit (Invitrogen, Thermo Fisher Scientific) or TaqMan Kit (Invitrogen, Thermo Fisher Scientific), respectively. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) reactions were carried out via 5x HOT FIRE qPCR Mix Plus (Solis Bio-Dyne Co, Estonia) or TaqMan Advanced Master Mix (Invitrogen, Thermo Fisher Scientific). Table 1 shows the primer sequences used for qRT-PCR experiments. B-actin or RNU43 expression were used to normalize gene or miRNA expression. All reactions were performed at least twice. The 2-ΔΔCt method was employed to calculate the relative expressions of the genes and miRNAs that were investigated.
Detection of Cell Viability Using Cell Viability Detection Kit-8
Cell viability was determined via the cell viability detection kit-8 (CVDK-8) assay (EcoTech Biotechnology) MDA-MB-231 cells (3 x 103 cells per well) were seeded into 96-well plates in five replicates and incubated for 24 hours. Then the cells were transfected with lipofectamine 2000 (Invitrogen) reagent to express miR-638-5p mimic or NT miRNA. After 24 hours, each well was treated with CVDK-8 reagent, and the plates were incubated for three hours. A Multiskan spectrophotometer (Thermo Fisher Scientific) was used to measure absorbance at 450 nm.
Detection of Cell Proliferation Using the Viability Imaging Method
After enzyme-linked immunoabsorbance (ELISA) evaluation for cell viability, the 96 well plate was inverted and the liquid was removed. Then the wells were washed with PBS. After removal of the PBS, a light microscope image was taken at x10 and recorded.
Scratch Assay
5 x105 MDA-MB-231 cells in DMEM with 10% FBS were seeded in 6-well plates. When the cells reached 95% confluency, scratches were made with a 10 µL pipette tip. After removing the medium from the plate and washing with PBS, the attached cells were cultured in DMEM. The cells that migrated to the “wound area” were measured from multiple microscopic areas, and images were captured at 0 and 24 hours with a light microscope at x100 magnification.
Statistical Analysis
Publicly available data were used in part of the bioinformatics the study, and in the miRNA analysis, those with logFC >2 for GSE154255, logFC >1 for GSE40525, and p<0.05 for both datasets were selected. For genes, among the TCGA BRCA data, those with logFC >+1 and p<0.05 values were considered significant. In terms of the in vitro studies, all data is shown as the mean ± standard deviation of a minimum of two independent experiments that yielded comparable results. Student’s t-test was used to analyze significant differences using GraphPad Prism 7.0. A difference that was statistically significant was indicated by p<0.05.
Results
Bioinformatics Analysis
TNBC-Associated miRNAs
The geo dataset analysis revealed 16 downregulated miRNAs to be common in both geo datasets (Table 2).
The Prognostic Importance of Selected miRNA
The KMplot survival evaluation revealed that decreased expressions of miR-638-5p and miR-139-3p had an effect on the OS of BRCA patients in general and also for the TNBC subtype of BRCA (Figure 1). As miR-638-5p was found to be more closely associated with BRCA on literature review, it was chosen for the remaining in silico analyses and the in vitro study.
Overexpressed Genes in TCGA BRCA Data
Analysis of TCGA BRCA data identified 248 genes which met the LogFC >+2 and p<0.05 criteria.
Detection of Potential Target Genes of the Selected miRNA
Using miRNet (miRTarBase v8.0 and TarBase v.8), it was found that miR-638-5p could potentially target 1416 genes (Figure 2).
Detection of Overlapping Genes
Thirteen genes were found to overlap between the TCGA data and potential in silico targets of miR-638-5p (Table 3).
Enrichment Analysis Results
It was found that the thirteen overlapping genes were linked to various cancers, particularly BRCA, and that these genes are associated with cancer-related pathways, such as those involved in cell division and chromosome segregation, as well as being related to hub proteins which are closely associated with BRCA (Figure 3 and Figure 4).
The Prognostic Importance of Selected Genes
The prognostic importance of four hub genes in BRCA patient survival was investigated. It was revealed that the differential expression of SIX4 and CDH1 influenced patient survival (Figure 5).
In vitro Investigations
Cell Viability Assay Results
Both ELISA absorbance measurement results and the viability imaging method results showed that curcumin treatment or miR-638-5p mimic transfection significantly reduced the proliferation of MDA-MB-231 cells at 24 hours (p<0.01) (Figure 6 and Figure 7).
Scratch Assay Results
Scratch assay results showed that curcumin treatment at a concentraton of 10 µM significantly diminished the cell migration of MDA-MB-231 compared to the untreated control group at 24 hours of evaluation. Furthermore, at 24 hours, miR-638-5p mimic transfection reduced cell migration compared to the NT miRNA mimic group (Figure 8).
qRT-PCR Results
The effect of curcumin treatment or miR-145-5p mimic transfection on the expression of the selected genes were investigated using qRT-PCR. The selected CDH1, MAZ, SIX4, CFL1, and SMC1A genes were quantified using the primers shown in Table 1. To normalize gene expression, the b-actin housekeeping gene was used. It was observed that the expression of CFL1, SIX4, and MAZ genes decreased significantly in both MDA-MAB-231 cells administered curcumin and MDA-MB-231 cells transfected with the miR-638-5p mimic (Figure 9).
Discussion and Conclusion
Numerous studies have shown that curcumin inhibits cancer cell proliferation, increases apoptosis, and disrupts migration via its effect on miRNAs. It has been reported that curcumin inhibits the progression of colorectal cancer cells by regulating the CDCA3/CDK1 pathway via miR-134-5p (15). Curcumin has been shown to inhibit cell growth in BRCA through the miR-21/PTEN/Akt pathway. Liang et al. (16) reported that curcumin suppressed the survival, migration, and invasion of papillary thyroid cancer cells by modulating the miR-301a-3p/STAT3 axis.
In the bioinformatics section of the present study, we found that only miR-638 and miR-139-3p had a significant effect on the OS of both BRCA, and specifically TNBC, patients among the overlapping miRNAs in the GSE154255 and GSE40525 datasets (Figure 1). MiR-638-5p was chosen for the in vitro study based on the data obtained from the literature review, as miR-638-5p may be more closely associated with BRCA. However, taking the current study’s bioinformatics data and literature results into account, we would like to emphasize that miR-139-3p may also be closely related to BRCA and that more comprehensive studies on the relationship between this miRNA and BRCA and specifically TNBC, are required.
To the best of our knowledge, there is no previous study into the effects of curcumin on cancer processes in BRCA cells specifically via miR-638 and its target genes. Using TCGA data and in silico tools, 13 potential miR-638-5p target genes were identified. Five of these 13 genes (CFL1, SIX4, MAZ, CDH1, and SMC1A) were chosen for in vitro examination. In the in vitro study, MDA-MB-231 cells were treated with curcumin, and it was found that the expression of miR-638-5p increased in the curcumin-supplemented group compared to the control group, while the expression of CFL1, SIX4, MAZ, and CDH1 genes decreased. Subsequently, MDA-MB-231 cells were transfected with a miR-638-5p mimic. The expression of CFL1, SIX4, and MAZ genes was found to be reduced in the transfected group (Figure 9B).
miR-638-5p is a tumor suppressor miRNA that has been linked to a variety of cancers (17, 18). Zheng et al. (19) showed that miR-638-5p acts as a tumor suppressor in glioma by regulating HOXA9. Another study found that miR-638-5p inhibited cell proliferation in human osteosarcoma by repressing PIM1 expression (20).
On November 28, 2023, a Pubmed search with the keywords “miR-638, breast cancer” yielded 19 results. Studies into BRCA, specifically investigating the role of miR-638-5p, have revealed that miR-638-5p expression was reduced in BRCA, and that miR-638-5p may be connected with resistance to various chemotherapeutics, radiotherapy, and ultraviolet (UV) sensitivity. He et al. (21) showed that CircNCOR1 regulates the efficacy of radiotherapy in BRCA through the miR-638-5p/CDK2 axis. Wang et al. (22) reported that miR-638-5p could be used as a biomarker for 5-fluorouracil sensitivity in BRCA treatment. Another study revealed that miR-638-5p/BRCA1 regulation affects DNA repair, as well as sensitivity to UV and cisplatin in TNBC.
The enrichment analysis performed in the present study on the potential target genes of miR-638-5p revealed that the disease was most likely to be associated with the identified genes is mammary neoplasms (Figure 4). Cell cycle and chromosome segregation are two biological events in which these genes may play important roles that are closely related to the cancer process. All of these suggest that further research into the relationship between miR-638-5p and the candidate genes may assist in understanding BRCA, and specifically TNBC, biology.
The decreased expression of all three selected miR-638-5p potential target genes (CFL1, SIX4, and MAZ) in both the curcumin-added and miR-638-5p transfected groups is an important clue about the functioning of the curcumin/miR-638/target gene axis. Despite the fact that no studies have been conducted to explain the relationship between miR-638-5p and CFL1 in BRCA, it has been demonstrated that CFL1 may contribute to the BRCA process by changing its expression via different miRNAs. miR-342 has been shown to inhibit the growth, migration, and invasion of BRCA cells by targeting CFL1 (23). Another study demonstrated that miR-200b-3p and miR-429-5p inhibit the growth and motility of BRCA cells by targeting the LIMK1/CFL1 pathway (24).
Although SIX4 is a gene linked to some cancers, including BRCA, there are fewer details about it compared to other selected miR-638-5p targeted genes. SIX4 promotes metastasis in BRCA via STAT3 induction, according to one of the few studies (25). Wu et al. (26) reported elevated SIX4 expression in BRCA that serves an oncogenic role by reducing the immune response, particularly in luminal subtypes, and is associated with diminished promoter methylation levels.
MAZ represents one of the genes involved in gene expression regulation and development of tumors. MAZ dysregulation has been related to the progression of many tumors, involving BRCA (27). MAZ-regulated SIPL1 has been shown to promote tumor progression in TNBC, and dysregulation of this MAZ expression may be associated with a poor prognosis in TNBC (28).
In the present study, using in silico and in vitro methods, curcumin was shown to affect cancer processes in MDA-MB-231 cells by altering the expressions of miR-638-5p and its potential target genes. Numerous studies have suggested that miRNAs may be potential therapeutic molecules in cancer in the future (29). However, research into the complicated interactions between miRNAs and their target genes is currently incomplete. The findings of the present study will contribute to the existing literature. It should be noted, however, that the expression of the selected genes was determined at the mRNA level. Therefore it is recommended that in future studies, the findings obtained using in silico and in vitro approaches should be validated in BRCA tissue samples and with other in vivo methods.
The findings of this study showed that curcumin appears to inhibit MDA-MB-231 cancer cell proliferation and migration by altering the expression of miR-638-5p and its potential target genes CFL1, SIX4, and MAZ. It is suggested that miR-638-5p and its target gene axis in BRCA, should be investigated further in future studies.
Ethics Committee Approval: Because of this study was prepared using publicly available bioinformatics data and in vitro study utilizing MDA-MB-231 cell line it does not require ethical approval.
Informed Consent: Because of this study was prepared using publicly available bioinformatics data and in vitro study utilizing MDA-MB-231 cell line it does not require informed consent.
Authorship Contributions
Concept: M.K., A.A., S.O.; Design: M.K., A.A., S.P.; Data Collection and/or Processing: M.K., A.A., I.S., S.P., Analysis and/or Interpretation: M.K., S.P., K.C., S.O.; Literature Search: M.K., I.S., S.E., F.A.; Writing: M.K., A.A., S.E., F.A., S.P., S.O.
Conflict of Interest: The authors have no conflicts of interest to declare.
Financial Disclosure: Council of Higher Education Research Universities Support Program project, ADEP-Istanbul University, TSA-2023-39483.