Regular paper

MiR-96-5p is involved in permethrin-promoted proliferation and migration of breast cancer cells

Yi Yan, Tian Wen Long, Xi Niu, Jia Fu Wang and Sheng Li✉

Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology & Agro-Bioengineering, CICMEAB, College of Life Sciences/Institute of Agro-Bioengineering, Guizhou University, Guiyang 550025, Guizhou Province, China

MicroRNAs (miRNAs) are major players in cellular responses to xenobiotic compounds and toxins. However, the role of miRNAs in pyrethroid pesticide-induced cancer progression remains unclear. This study aimed to investigate the function of miR-96-5p in permethrin-induced proliferation and migration in breast cancer cells. In our study, the expression of miR-96-5p was upregulated in permethrin-treated MCF-7 cells. MiR-96-5p promoted MCF-7 cell proliferation and migration, accompanied bychanges in the expression of proteins involved in cell proliferation, migration, and apoptosis. Homeobox A5 (HOXA5) was identified as a direct target of miR-96-5p. HOXA5 silencing had the opposite effects with miR-96-5p inhibition. In conclusion, these results suggest that miR-96-5p is involved in permethrin-promoted proliferation and migration of breast cancer cells by targeting HOXA5.

Keywords: miR-96-5p, permethrin, proliferation and migration, MCF-7 cells, HOXA5

Received: 23 October, 2022; revised: 16 November, 2022; accepted: 22 March, 2023; available on-line: 17 August, 2023

✉e-mail: sli1@gzu.edu.cn

Acknowledgments of Financial Support: This study was supported by the National Natural Science Foundation of China (No. 22166010 and No. 31401091).

Abbreviations: miRs, MicroRNAs; HOXA5, homeobox A5; CCK-8, Cell counting kit-8; 3’-UTR, 3’-untranslated regions; WT, Wild-type

Introduction

Permethrin is a synthetic pyrethroid pesticide widely used in agriculture. Permethrin typically disturbs the nervous system of insects through slackening sensitiveness toward voltage on the intramembrane and extramembrane of axons in the nervous system of insects (Park et al., 2021; Wang et al., 2016). Due to the efficient insecticidal properties of permethrin, it is not only applied to control crop diseases and pests but also used in mosquito killing and malaria treatment (Richards et al., 2017; Richards et al., 2018; Thievent et al., 2019). However, the residual toxicity of permethrin has attracted great attention from the public. In vitro studies indicated that permethrin competitively combined with estrogen receptor (ER) against estradiol (E2) (McCarthy et al., 2006) and promoted the proliferation of breast cancer cells, which could be blocked by estrogen antagonists (Kim et al., 2004; Sun et al., 2014). In addition, the wet weight of the uterus of female rats increased after being injected subcutaneously with permethrin, which was similar to the effect caused by E2, and permethrin-induced weight increase he the uterus could be inhibited by antiestrogens. When male mice were given permethrin per os, their luteinizing hormone (LG) significantly increased, while testosterone (T) level and sperm vitality dramatically decreased (Kim et al., 2005). These studies indicate that pyrethroid pesticides such as permethrin have estrogen effects and belong to environmental hormones. According to epidemiological observation, workers exposed to low-dose permethrin for a long time have higher risks of colorectal cancer, breast cancer, lung cancer, and leukemia (Rusiecki et al., 2009). However, the underlying mechanisms remain elusive.

MicroRNA (miRNA) is a kind of non-coding small RNA and regulates protein expression by binding to the 3’-untranslated region (3-UTR) of target genes (Burnett et al., 2021; Cao et al., 2021). miRNAs play an essential role in regulating the organismic response to toxic effects caused by xenobiotic compounds, including pesticides. For example, miR-22 restrained endothelial cell dysfunction caused by the pesticide endosulfan (Xu et al., 2017). MiR-513 protected renal cells from DDVP-induced apoptosis by targeting Bcl-2 (Li et al., 2018). MiR-96-5p was initially identified as a 23-nucleotide miRNA and abundant evidence has shown that miR-96 is related to cell proliferation and apoptosis. For example, overexpression of miR-96-5p was shown to induce gastric adenocarcinoma cell apoptosis through targeting FOXO3 (He & Zou, 2020), and it also inhibited hepatocellular carcinoma cell apoptosis (Iwai et al., 2018). MiR-96-5p inhibited the expression of the target gene MTSS1 and enhanced the growth of glioma cells (Zhang & Guo, 2019). These findings indicate that miR-96-5p is involved in tumor cell proliferation, migration, and apoptosis. However, the role of miR-96 in the cellular response to exogenous toxicants remains unclear.

HOXA5 is a member of homeobox gene clusters, which is involved in the regulation of cell differentiation. The role of HOXA5 in tumorigenesis is controversial. HOXA5 is generally regarded as a tumor suppressor in many cancers, such as cervical cancer and breast cancer. In these cancers, HOXA5 is normally the target of various miRNAs. On the other hand, HOXA5 is reported to be capable of promoting tumor progression, which needs further investigation.

Notably, the mechanism by which permethrin promotes breast cancer is not fully understood, and the role of miR-96-5p in breast cancer remains elusive. Therefore, in this study, we aimed to determine the regulatory role of miR-96-5p in mediating the estrogen effect of permethrin. Our study confirmed that miR-96-5p targeted HOXA5 to promote breast cancer induced by permethrin.

Materials and methods

Cell line and main reagents

MCF-7 cells were obtained from China Center for Type Culture Collection (CCTCC, China). Cell lines were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 1% penicillin and 1% streptomycin (both from Solarbio) under 5% CO2 at 37°C. Permethrin standard (purity 99.1%) and 17β-estradiol standard (purity 99.2%) were purchased from China Center for Standard Substance; Trypsin was purchased from Sigma (USA); CCK-8 kit, Apoptosis-HOECHST staining kit, RNA Extracting Kit, PBS buffer, enhanced RIPA lysis buffer, BCA protein assay kit, and Lipo8000TM were purchased from Beyotime Technology (China), The primary and secondary antibodies were obtained from Beijing Quailityard Biotechnology (China); U6 snRNA Real-time PCR Normalization Kit was purchased from Shanghai GenePharma (China).

Cell proliferation assay

MCF-7 cells were seeded into 96-well plates at a density of 3×103 cells/well. After overnight incubation, the cells reached about 70% confluency. Different concentrations of permethrin (10–4, 10–5, 10–6, 10–7 mol/L) and estradiol (10–6, 10–7, 10–8, 10–9 mol/L) were used to treat MCF-7 cells for 48h or 72 h. Each treatment has five repeats and the medium with different drugs was refreshed daily. Cells untreated with drugs were set as the control group, and wells added only with the medium were set as the blank group. CCK-8 reagent allows the sensitive colorimetric assay to determine the number of viable cells in the cell proliferation assay. Therefore, 20 μL CCK-8 reagent was added to each well after different periods. After 30 min of incubation, the OD values of the solution in each well at 450 nm were measured to calculate cell viability according to the formula:

Cell transfection

MCF-7 cells were seeded into 6-well plates. After 70% confluency was reached, the cells in the control group were treated with 100 pmol scramble miRNA (marked as miR-NC group), while the cells in the experimental group were treated with 100 pmol miR-96-5p inhibitor or miR-96-5p mimic (marked as miR-96-5p inhibitor group or miR-96-5p mimic group) (GenePharma, China) using Lipofectamine 2000 (Invitrogen, USA). The medium was refreshed after 4 h, followed by 24 h incubation.

Quantitative Real-time PCR

Total RNA was extracted with RNAeasy™ Animal RNA Isolation Kit and was reverse transcribed into cDNA using GenePharma RT Reagent Kit. The expression of miR-96-5p was measured with GenePharma qRT-PCR Kit. U6 snRNA was used as an internal reference for miRNA analysis. QRT-PCR was conducted using the CFX96 system. Primers were listed as follows: miR-96-5p, forward: 5’-CGAACTTTGGCACTAGGCACATT-3’, reverse: 5’-TATGGTTTTGACGACTGTGTGAT-3’; U6, forward: 5’-CAGCACATATACTAAAATTGGAACG-3’, reverse: 5’-ACGAATTTGCGTGTCATCC-3’.

The relative expression of target genes was quantified using the 2–ΔΔCt method.

Luciferase reporter assay

The targets of miR-96 were predicted by Target Scan Human (http://www.targetscan.org), miRDB (http://mirdb.org), and miRTarbase (http://mirtarbase.mbc.nctu.edu.tw/php/index. php). For the luciferase reporter assay, the wild-type (WT-HOXA5) or mutant 3’-untranslated regions (MUT-HOXA5) of HOXA5 were subcloned into the luciferase reporter gene vector (pMIR-REPORTER Luciferase, cat. # QYV0423, Qualityard, China). MCF-7 cells were cultured in 24-well plates, and co-transfected with miR-96-5p or miR-NC and WT-HOXA5 or MUT-HOXA5 plasmids using Lipofectamine 2000. The luciferase activity was calculated through the ratio of firefly and renilla luciferase activity following the manufacturer’s instructions.

In vitro wound healing assay

Cells were cultured in 12-well plates at a density of 3×105 cells per well. After transfection with miR-NC, miR-96-5p mimic, and miR-96-5p inhibitor, cells were inoculated until 95% confluency was reached. A sterile 200-μL pipette tip was used to scratch the cell layer manually, and the time point was set as 0 h. Next, the cells were rinsed with PBS 3 times to remove all cellular debris. Fresh complete medium was then added. The subsequent colonization of the denuded surface was quantified with Image J, and the relative migration distance was calculated by subtracting the width at 0 h from the width at each migration time point.

Western blot analysis

Cells were collected after 48 hours of transfection. Radio immuno-precipitation assay (RIPA) lysis buffer was used to extract total proteins and Bicinchoninic acid (BCA) protein assay kit was used to measure the concentrations of total proteins. Proteins of different molecular weights were separated by SDS-PAGE and transferred onto the membranes. After blocking with 5% skim milk for 1 h, proteins were incubated with primary antibodies, rabbit anti-HOXA5, and rabbit anti-β-actin at 4°C overnight. Subsequently, the membranes were washed with TBST solution, incubated with the secondary antibody goat anti-rabbit IgG coupled with HRP at room temperature for 2 h, and washed with TBST solution. The membranes were exposed using an ECL kit and Gel Imaging System. Each experiment was repeated 3 times.

Statistical analysis

Data analysis, graph generation, and statistical analysis were performed with the software GraphPad Prism 8.3 (GraphPad Software Inc., Philadelphia, USA). Data were represented as mean ± standard deviation (S.D.). The comparison between the two groups was analyzed by t-test and the comparison of multiple groups such as different concentrations were analyzed by One-way analysis of variance (ANOVA). P values less than 0.05 were considered significant.

Results

Low-dose permethrin promoted the proliferation of MCF-7 cells

To determine the effect of permethrin on MCF-7 cell viability, the cells were treated with different concentrations of permethrin for 48 h or 72 h. The results showed that permethrin promoted MCF-7 cell proliferation in a dosage range from 10-7M to 10-4M (Fig. 1A). The maximum viability in MCF-7 cells was seen when cells were treated with 10–4 M permethrin for 72 h. Hoechst staining showed that cells in treatment groups had higher viability. The nuclei were evenly stained, compared with the control group (Fig. 1B). IWH assay showed that MCF-7 cell migration was significantly promoted after treatment with 10–4 M permethrin for 72 h (Fig. 1C). These results indicate that low-dose permethrin promotes MCF-7 cell proliferation and migration.

Permethrin promoted ER-α and STAT3 expression and upregulated miR-96

Next, we investigated whether permethrin regulated the expression of genes involved in cell proliferation, employing estradiol as the positive control. As is shown in Fig. 2A, after treatment with low-dose estradiol and permethrin for 72 h, the expression of estrogen receptor-α (ER-α) and Signal Transducer and Activator of Transcription (STAT3) in MCF-7 cells increased.

We performed a qRT-PCR assay to detect miR-96 expression in MCF-7 cells treated with different concentrations of permethrin. The results showed that miR-96 expression in MCF-7 cells in the permethrin-treated group increased in a dose-dependent manner (Fig. 2B).

HOXA5 was a target of miR-96-5p in MCF-7 cells

HOXA5 gene was predicted to be the target of miR-96-5p by the TargetScan program (Fig. 3A). To verify the prediction, we constructed a luciferase reporter containing either the wild-type HOXA5 3’ UTR or a mutated HOXA5 3’ UTR and then co-transfected into MCF-7 cells with miR-96-5p mimic or negative control (NC) miRNAs. A significant decrease of luciferase activity was detected in cells co-transfected with HOXA5 3’ UTR construct and miR-96-5p mimics, compared with cells co-transfected with miR-NC or mutant HOXA5 3’ UTR (Fig. 3B). Compared with the miR-NC group, a significant decrease of HOXA5 expression in miR-96-5p mimic group was observed (Fig. 3C). These results indicate that HOXA5 is a direct target of miR-96-5p.

miR-96-5p promoted permethrin-induced proliferation and migration of MCF-7 cells by targeting HOXA5

To further investigate the mechanism of miR-96-5p in permethrin-induced MCF-7 cell proliferation and migration, the miR-96 inhibitor was transfected into permethrin-treated MCF-7 cells. IWH assay showed that permethrin-induced migration of MCF-7 cells was increased and HOXA5 expression was upregulated after miR-96 was inhibited, compared with cells treated with permethrin only. In rescue experiments, permethrin-induced proliferation and migration of MCF-7 cells were decreased by silencing HOXA5 expression (Fig. 4A, B, C). These results suggest that miR-96 regulates the proliferation and migration of MCF-7 cells by targeting HOXA5.

Discussion

Estrogens are crucially involved in the development of breast cancer (Yi et al., 2009; Chaudhuri et al., 2021). Estrogen activates the ER dimer in the promoter domain of the target gene to regulate gene expression in cells (Krieg et al., 2001). Estrogen also promotes the phosphorylation of related transcription factors (Sengupta et al., 2019). Human breast cancer cell line MCF-7 is capable of expressing estrogen receptor and is sensitive to estrogen. Therefore, in the present study, we employed the MCF-7 cell line as the experiment model and found that low-dose permethrin promoted the proliferation and migration of estrogen-sensitive MCF-7 cells. Moreover, we found that permethrin increased ERα protein expression as E2 did, indicating that permethrin plays a role in enhancing the metabolism of the estrogen receptor.

As a critical downstream transcription factor of ER, phosphorylated STAT3 passes through the nuclear pore to activate the transcription of target genes (Kettner et al., 2019; Wang et al., 2021). In our study, when MCF-7 cells were treated with low-dose permethrin, the expression of STAT3 and ER-α increased, compared with E2 treatment as the positive control. These results suggest that permethrin has an estrogen effect and promotes cell proliferation by activating the estrogen receptor and STAT-related estrogen pathway.

STAT3 is an important transcription factor of the miR-96-182-183 family (Lei et al., 2021; Xiao et al., 2021). MiR-96 was reported to influence the cell cycle (Xu et al., 2018). miR-96 inhibitor arrested the cell cycle at the G1 phase, while fewer cells entered the S phase, suggesting that inhibition of miR-96 induces cell arrest in the G1 phase. Therefore, we speculated that miR-96 might promote cell proliferation and migration induced by permethrin. Our results showed that the miR-96 inhibitor attenuated permethrin-induced proliferation and migration of MCF-7 cells, which confirmed our speculation.

Next, TargetScan, miRDB, and miRTarbase were employed to predict the target genes of miR-96 (Guo et al., 2021). HOXA5 is highly correlated with cell proliferation and tumorigenesis. HOXA5 is a member of the homeobox gene family encoding homologous proteins, and its biological function is to control embryonic development and cell differentiation. HOXA5 is regulated by AKT/mTORC1/S6K1 signal pathway (Feng et al., 2017), and inhibits cell progression by regulating AKT/p27 pathway (Wang et al., 2019). HOXA5 expression was closely related to tumor histological grade (Dziobek et al., 2020; Aubin et al., 2002). Inhibition and overexpression of HOXA5 promoted proliferation and apoptosis of osteosarcoma cells through the p53 and p38α/MAPK pathways (Chen et al., 2019). Inhibition of HOXA5 significantly enhanced viability and proliferation in U2OS and MG63 cells, while overexpression of HOXA5 promoted cell apoptosis and caspase-3 activity. Knockdown of HOXA5 in HCC cells provoked capillary tube formation, while overexpression of HOXA5 inhibited cell proliferation and invasion and promoted cell apoptosis. Based on the luciferase assay, we confirmed that HOXA5 was a target gene of miR-96. When HOXA5 was overexpressed, proliferation and migration in MCF-7 cells induced by permethrin were inhibited. Therefore, overexpression of HOXA5 could reverse the effects of miR-96. These results suggest that miR-96 promotes permethrin-induced proliferation and migration in MCF-7 cells via the downregulation of HOXA5. A recent study reported the potential of miRNAs as biomarkers of breast cancer (Shaaban et al., 2022). It would be interesting to evaluate the application of miR-96 in the early diagnosis of breast cancer.

This study has certain limitations. First, the downstream mechanism of Permethrin, miR-96-5p, and HOXA5 in promoting breast cancer is still obscure. In addition, we need to perform in vivo experiments to confirm our conclusion in future studies. Beyond these limitations, this is the first study to demonstrate that miR-96-5p targeted HOXA5 to promote cancer progression.

In conclusion, our results suggest that permethrin, as a pseudo estrogen, upregulates ER-α and STAT3 expression in MCF-7 cells. STAT3 activates the transcription of miR-96, which specifically inhibits HOXA5 expression and promotes the proliferation and migration of MCF-7 cells.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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