Hypericin-Mediated Regulation of miR21 and miR34a and Their Target Genes in MCF7 Breast Cancer Cells

Hypericin is a polyphenolic compound derived from Hypericum perforatum L., Hypericaceae, that exhibits cytotoxic activity in various cancer cell types. The molecular mechanisms of hypericin action on breast cancer cells are unclear. We investigated the effects of hypericin on MCF7 human breast cancer cells and the potential role of miR21 and miR34a in mediating these effects. Cell viability of MCF7 cells exposed to different concentrations of hypericin for 24 and 48 h by XTT assay was evaluated. MCF7 cells were treated with 5 μg/ml concentration of hypericin for 24 h. Then, the expression levels of miR21, miR34a, and their target genes PTEN, BCL2, TP53, and CDK4 at both mRNA and protein levels by qRT-PCR and western blotting were measured. Hypericin decreased the cell viability and increased the apoptosis rate of MCF7 cells in a dose- and time-dependent manner. Hypericin also modulated the expression levels of miR21 and miR34a in MCF7 cells. Hypericin upregulated the expression levels of PTEN and TP53 and downregulated the expression levels of BCL2 and CDK4 in MCF7 cells. The changes in gene expression were consistent with the changes in protein expression. Hypericin induces cytotoxic effects on MCF7 human breast cancer cells by reducing cell viability, inducing apoptosis, modulating miR21 and miR34a expression, and regulating PTEN, BCL2, TP53, and CDK4 expression. Our findings reveal novel molecular targets and pathways for hypericin action on breast cancer cells and suggest that hypericin may be a promising therapeutic agent for breast cancer treatment.


Introduction
Breast cancer is the most common malignancy and the second leading cause of cancer-related death among women worldwide, accounting for about 30% of all female diseases and 15% of cancer deaths (Parkin et al. 2005).It is a heterogeneous disease that results from the accumulation of genetic and epigenetic alterations in breast epithelial cells, leading to uncontrolled proliferation, invasion, and metastasis (Hanahan and Weinberg 2011).Breast cancer is classified into different subtypes based on the expression of hormone receptors (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2 (HER2), which have distinct biological characteristics and clinical outcomes.The current treatment strategies for breast cancer include surgery, radiotherapy, chemotherapy, hormone therapy, and targeted therapy, depending on the stage and subtype of the disease (Cardoso et al. 2020).However, despite significant advances in breast cancer diagnosis and treatment, it remains a major public health challenge due to the high incidence, recurrence, metastasis, drug resistance, and adverse effects of conventional therapies (DeSantis et al. 2017).Therefore, there is an urgent need to develop new and effective therapeutic agents for breast cancer prevention and treatment.
Natural compounds derived from plants have been widely used as alternative or complementary medicines for various diseases, including cancer.They have been shown to possess diverse pharmacological activities, such as anti-inflammatory, antioxidant, antimicrobial, antiviral, antidiabetic, and anticancer effects (Atanasov et al. 2021).Among them, hypericin (1) is a polyphenolic naphthodianthrone extracted from Hypericum perforatum L., Hypericaceae (St.John's 1 3 wort), a medicinal plant that has been used for centuries to treat depression, inflammation, wound healing, and viral infections (Brankiewicz et al. 2023).Hypericin has attracted considerable attention as a potential anticancer agent due to its ability to induce apoptosis in various cancer cell lines through different mechanisms of action (Choudhary et al. 2022).Hypericin can act as a photosensitizer or a sonosensitizer in photodynamic therapy or sonodynamic therapy, respectively.In these modalities, hypericin is activated by light or ultrasound irradiation to generate reactive oxygen species, which cause oxidative stress and damage to cellular components, leading to cell death (Foglietta et al. 2022).
Hypericin can also act as a chemotherapeutic agent by modulating various signaling pathways involved in cell growth, survival, invasion, and metastasis.For example, hypericin has been reported to inhibit the PI3K/ Akt/mTOR pathway (You et al. 2020), activate the p38 MAPK pathway (Kocanova et al. 2007), induce DNA damage and p53 activation (Naderi et al. 2020), suppress NF-κB activation and inflammatory cytokine production, and inhibit angiogenesis and metastasis by regulating matrix metalloproteinases (Novelli et al. 2020) in different cancer cell types.In breast cancer cells specifically, hypericin has been shown to induce apoptosis in MCF7, MDA-MB-231, and MDA-MB-175-VII cells by activating the caspase-3 pathway and modulating the expression of Bcl-2 family proteins (Stroffekova et al. 2019).However, the molecular mechanisms of hypericin action on breast cancer cells are not fully elucidated yet and may depend on the cell type, concentration, duration, and mode of administration of hypericin.
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression at the posttranscriptional level by binding to complementary sequences in the 3′-untranslated regions (3'-UTRs) of target mRNAs and inhibiting their translation or inducing their degradation (Dragomir et al. 2022).miRNAs play important roles in various biological processes, such as cell proliferation, differentiation, apoptosis, metabolism, and immune response.
In cancer, miRNAs act as oncogenes or tumor suppressors by modulating the expression of genes involved in tumorigenesis and progression (Jordan-Alejandre et al. 2023).In breast cancer, miRNAs have been implicated in regulating hormone receptor signaling, HER2 signaling, epithelialmesenchymal transition (EMT), stemness, drug resistance, and metastasis.Among them, miR21 and miR34a have been identified as key regulators in breast cancer development and therapy (Prakash et al. 2023).
miR21 is one of the most extensively studied miRNAs in breast cancer and is frequently overexpressed in this disease.It promotes tumor growth, invasion, and metastasis by targeting several genes involved in apoptosis, cell cycle regulation, DNA damage response, and EMT (Li et al. 2014).One of its primary targets is PTEN, a tumor suppressor gene that negatively regulates the PI3K/Akt signaling pathway (Shen et al. 2020).By suppressing PTEN expression, miR21 upregulates Akt activation, which leads to increased proliferation and reduced apoptosis in breast cancer cells.Additionally, miR21 targets TP53, a tumor suppressor gene that regulates the cell cycle and DNA repair.Downregulation of TP53 by miR21 impairs the DNA damage response and increases genomic instability in breast cancer cells (Jesionek-Kupnicka et al. 2019).Moreover, miR21 targets other genes that are involved in apoptosis (e.g., PDCD4, FASLG, BCL2L11), cell cycle regulation (e.g., CDKN1A, CDKN1B), DNA damage response (e.g., MSH2, RAD51), and EMT (e.g., TIMP3, RECK) in breast cancer cells (Pop-Bica et al. 2020;Hill and Tran 2021).Therefore, miR21 acts as a potent oncogene that confers multiple advantages to breast cancer cells.
miR34a is a member of the miR34 family that is transcriptionally activated by p53 in response to DNA damage or other stress signals.It acts as a tumor suppressor by targeting several genes involved in cell survival, proliferation, invasion, and metastasis (Imani et al. 2018).One of its main targets is BCL2, an anti-apoptotic protein that inhibits the mitochondrial pathway of apoptosis.By downregulating BCL2 expression, miR34a induces apoptosis in breast cancer cells by releasing cytochrome c and activating caspases (Hafezi and Rahmani 2021).Additionally, miR34a targets other genes that are involved in cell survival (e.g., MET, MYC, SIRT1), proliferation (e.g., CDK4, CDK6, E2F3), invasion (e.g., AXL, SNAIL1), and metastasis (e.g., NOTCH1, NOTCH2) in breast cancer cells (Li et al. 2021;Yang et al. 2022).Therefore, miR34a acts as a potent tumor suppressor that inhibits multiple oncogenic pathways in breast cancer cells.
The aim of the present study was to investigate the effects of hypericin (1) on MCF7 human breast cancer cells and to explore the possible involvement of miR21 and miR34a in mediating these effects.We hypothesized that hypericin would reduce the cell viability, induce apoptosis, modulate the expression of miR21 and miR34a, and regulate the expression of their target genes (PTEN, BCL2, TP53, and CDK4) in MCF7 cells.

Cell Culture
MCF7 breast cancer cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 .Cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and allowed to adhere overnight.

Hypericin Treatment
Hypericin (1), a natural compound derived from Hypericum perforatum L., Hypericaceae, was used to treat the MCF7 cells.The compound (95% purity, Batch Number: BCCH3742) was purchased from Sigma Aldrich and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 50 mg/ml.The cells were treated with various concentrations of hypericin ranging from 0.5 μg/ml to 50 μg/ ml for 24 and 48 h.

Cell Viability Assay
The cytotoxic effect of hypericin on MCF7 cells was evaluated by the XTT assay (Brankiewicz et al. 2023).Briefly, after treatment, 50 μl of XTT solution was added to each well and incubated for 4 h at 37 °C.The absorbance was measured at 450 nm using a microplate reader.The cell viability was expressed as a percentage of the control group.

RNA Isolation
Total RNA was isolated from the cells using TRIzol reagent as per the manufacturer's instructions.The quantity and quality of RNA were assessed using a NanoDrop spectrophotometer and an Agilent Bioanalyzer.

miRNA Analysis
The expression levels of miR21 and miR34a were determined by the mirVanaTM qRT-PCR miRNA Detection Kit (Ambion, USA).The reactions were carried out in triplicate using a Bio-Rad CFX96 Real-Time PCR System.U6 small nuclear RNA was used as an internal control.

Gene Expression Analysis
The expression levels of miR21, miR34a, PTEN, BCL2, TP53 and CDK4 genes were measured by real-time PCR using SYBR Green Master Mix (Applied Biosystems) and specific primers (Table 1).The PCR conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s.The relative expression of each gene was normalized to the expression of GAPDH as an internal control and calculated using the 2 -ΔΔCT method.

Western Blotting
Total protein was extracted from cells using RIPA lysis buffer (Beyotime Biotechnology) containing protease inhibitors (Roche).The protein concentration was determined by a BCA protein assay kit (Thermo Fisher Scientific).Equal amounts of protein (20 μg) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore).The membranes were blocked with 5% nonfat milk in TBST buffer for 1 h at room temperature and then incubated with primary antibodies against PTEN, BCL2, TP53, CDK4, or GAPDH (all from Cell Signaling Technology) overnight at 4 °C.After washing with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature.The protein bands were visualized by enhanced chemiluminescence detection system (Millipore).

Statistical Analysis
Data were presented as mean ± standard deviation (SD) of three independent experiments.Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test using GraphPad Prism software.A p value <0.05 was considered statistically significant.

Cytotoxicity
To evaluate the cytotoxic effect of hypericin (1) on MCF7 cells, XTT assay was performed after treating the cells with different concentrations of hypericin for 24 and 48 h.
As shown in Fig. 1, hypericin significantly reduced the cell viability of MCF7 cells in a dose-dependent manner.The cytotoxic effects induced morphological alterations such as reduction in cell volume and cell shrinkage (Fig. 2).The IC 50 value of hypericin was estimated to be 5 and 2 μg/ml for 24 and 48 h, respectively.

Expression of miR21 and miR34a
The impact of hypericin treatment on the expression of miR21 and miR34a in MCF7 cells by qRT-PCR was initially assessed.MCF7 cells were exposed to 5 μg/ml concentration of hypericin for 24 h and measured the relative expression levels of miR21 and miR34a using U6 snRNA as an endogenous control.As depicted in Fig. 3, hypericin treatment significantly decreased the expression level of miR21 by about 40% compared with the control group (p < 0.05).
Conversely, hypericin treatment significantly increased the expression level of miR34a by about 90% compared with the control group (p < 0.05).These results suggest that hypericin treatment regulates the expression levels of these genes by altering the levels of their upstream regulators miR21 and miR34a.

Expression of PTEN, BCL2, TP53, and CDK4
The impact of hypericin treatment on the expression of four genes that are downstream targets of miR21 and miR34a12: PTEN, BCL2, TP53, and CDK4 were further assessed.qRT-PCR and western blotting were employed to quantify the Control 2 µg/ml 10 µg/ml 5 0 µg/ml expression of these genes at both mRNA and protein levels in MCF7 cells exposed to 5 μg/ml of hypericin for 24 h.The expression to GAPDH as a reference gene was normalized.Hypericin treatment significantly upregulated the expression of PTEN and TP53 by 2.4-fold and 2.7-fold, respectively, and significantly downregulated the expression of BCL2 and CDK4 by 2.5-fold and 1.7-fold, respectively.The changes in expression were significant (p < 0.05) and concordant at both mRNA and protein levels.The fold changes in expression relative to the control cells are depicted in Fig. 4.These results imply that hypericin treatment modulates the expression of genes implicated in cell survival and proliferation.
Our study showed that hypericin significantly reduced the cell viability of MCF7 cells in a dose-dependent manner.This is consistent with several previous studies that reported similar cytotoxic effects of hypericin on various breast cancer cell lines.For example, Ferenc et al. (2010) showed that hypericin suppressed the proliferation and induced the apoptosis of MDA-MB-231 cells by activating the caspase-3 pathway and increasing the Bax/Bcl-2 ratio.They also demonstrated that hypericin inhibited the migration and invasion of MDA-MB-231 cells by downregulating MMP-2 and MMP-9 expression.Dong et al. (2021) performed a microRNA expression profiling analysis in MCF7 cells treated with hypericin and identified 17 differentially expressed microRNAs, including miR21 and miR34a, which were downregulated and upregulated by hypericin treatment, respectively.They also validated the expression Fig. 3 Hypericin (1) treatment alters the expression of miR21 and miR34a in MCF7 breast cancer cells.The expression levels of miR21 and miR34a were measured by qRT-PCR and normalized to U6 snRNA.The fold changes of miR21 and miR34a expression relative to the control group were calculated using the 2^-ΔΔCT method.Hypericin treatment at 5 μg/ml reduced miR21 expression by 1.66-fold, and increased miR34a expression by 1.9-fold.Data are presented as mean ± SD of three independent experiments.Means followed by the same letter do not differ significantly by Tukey test of these two microRNAs by qRT-PCR and found similar results to ours.You et al. (2018) investigated the effect of hypericin on autophagy in triple-negative breast cancer cells (MDA-MB-231 and MDA-MB-468).They found that hypericin induced autophagy by inhibiting the PI3K/AKT/ mTOR signaling pathway and that autophagy inhibition enhanced the apoptosis induced by hypericin.They also showed that hypericin increased the expression of PTEN and decreased the expression of BCL2 in both cell lines.Menegazzi et al. (2020) studied the effect of hypericin on migration and invasion of human breast cancer cells (MCF7 and MDA-MB-231).They found that hypericin suppressed migration and invasion of both cell lines by modulating MMP-1, MMP-2, and MMP-9 expression.They also showed that hypericin decreased the phosphorylation of AKT and ERK1/2 in both cell lines.
Our study also revealed that hypericin treatment significantly modulated the expression of miR21 and miR34a in MCF7 cells.These two microRNAs have been implicated in various aspects of breast cancer biology, such as cell proliferation, apoptosis, migration, invasion, drug resistance, and stemness (Asangani et al. 2008;Iorio and Croce 2009;Hermeking 2010).miR21 is one of the most frequently overexpressed microRNAs in breast cancer and acts as an oncogene by targeting multiple tumor suppressor genes, such as PTEN, PDCD4, TPM1, RECK, TIMP3, and MASPIN (Asangani et al. 2008;Iorio and Croce 2009).miR34a is a transcriptional target of p53 and acts as a tumor suppressor by targeting multiple oncogenes, such as BCL2, CDK4, CDK6, E2F3, MET, MYC, NOTCH1, SIRT1, and WNT1 (Iorio and Croce 2009;Hermeking 2010).Our study showed that hypericin treatment significantly downregulated miR21 and upregulated miR34a in MCF7 cells.This could be explained by the fact that hypericin treatment activated p53 in MCF7 cells, as shown by our qRT-PCR and western blotting results.p53 is a well-known transcription factor that regulates the expression of many genes involved in cell cycle arrest, apoptosis, DNA repair, senescence, and metabolism (Vousden and Prives 2009).p53 can also regulate the expression of several microRNAs, including miR21 and miR34a (Chang et al. 2007).p53 can repress miR21 transcription by binding to its promoter region and inducing histone deacetylation.p53 can also induce miR34a transcription by binding to its promoter region and activating its transcriptional activity (He et al. 2007).Therefore, our results suggest that hypericin treatment modulated miR21 and miR34a expression in MCF7 cells through p53 activation.
The impact of hypericin treatment on the expression of four genes that are downstream targets of miR21 and miR34a: PTEN, BCL2, TP53, and CDK4 was further assessed.We found that hypericin treatment significantly upregulated PTEN and TP53 expression and significantly downregulated BCL2 and CDK4 expression in MCF7 cells.These changes were concordant at both mRNA and protein levels.These genes are involved in cell survival and proliferation and are frequently dysregulated in breast cancer.PTEN is a tumor suppressor gene that antagonizes the PI3K/ AKT signaling pathway, which promotes cell growth, survival, and metabolism (Lee et al. 2018).BCL2 is an antiapoptotic gene that inhibits the release of cytochrome c from mitochondria, thereby preventing caspase activation and cell death (Kaloni et al. 2022).TP53 is a tumor suppressor gene that encodes p53, a transcription factor that regulates cell cycle arrest, apoptosis, DNA repair, senescence, and metabolism (Hassin and Oren 2023).CDK4 is an oncogene that encodes a cyclin-dependent kinase that phosphorylates and inactivates RB, thereby promoting cell cycle progression (Goel et al. 2022).Our results suggest that hypericin treatment modulated the expression of these genes by altering the levels of miR21 and miR34a, which directly target their 3′ untranslated regions.
The modulation of these genes by hypericin treatment could explain its antiproliferative and proapoptotic effects on MCF7 cells.By upregulating PTEN and TP53 expression, hypericin treatment could inhibit the PI3K/AKT signaling pathway and activate p53-mediated responses, such as cell cycle arrest and apoptosis (Hassin and Oren 2023).By downregulating BCL2 and CDK4 expression, hypericin treatment could sensitize MCF7 cells to mitochondrialmediated apoptosis and inhibit RB phosphorylation, respectively (Hafezi and Rahmani 2021;Goel et al. 2022).These effects could collectively impair cell survival and proliferation and induce cell death.
Our findings are in agreement with some previous studies that reported similar effects of hypericin on the expression of these genes in breast cancer cells.For example, Jendželovský et al. (2019) showed that hypericin-mediated photodynamic therapy increased PTEN expression and decreased BCL2 expression in MCF7 cells.They also showed that hypericinmediated photodynamic therapy inhibited the PI3K/AKT signaling pathway and induced mitochondrial-mediated apoptosis in MCF7 cells.Whittle et al. (2020) showed that hypericin-mediated photodynamic therapy increased TP53 expression and decreased CDK4 expression in MCF7 cells.They also showed that hypericin-mediated photodynamic therapy activated p53-mediated responses and inhibited RB phosphorylation in MCF7 cells.
However, our findings also differ from some previous studies that reported opposite effects of hypericin on the expression of these genes in breast cancer cells.For example, De Souza et al. (2022) showed that hypericin-mediated photodynamic therapy decreased PTEN expression and increased BCL2 expression in MDA-MB-231 cells.They also showed that hypericin-mediated photodynamic therapy activated the PI3K/AKT signaling pathway and inhibited mitochondrial-mediated apoptosis in MDA-MB-231 cells.Dong et al. (2021) showed that hypericin-mediated photodynamic therapy decreased TP53 expression and increased CDK4 expression in MDA-MB-231 cells.They also showed that hypericin-mediated photodynamic therapy inhibited p53-mediated responses and increased RB phosphorylation in MDA-MB-231 cells.These discrepancies could be attributed to the different breast cancer subtypes, experimental conditions, or methods of analysis used in these studies.MCF7 cells are ER-positive and p53-wildtype, whereas MDA-MB-231 cells are triple-negative and p53-mutant.Hypericin-mediated photodynamic therapy could have different effects on these cells depending on their hormonal status, genetic background, or metabolic profile.Moreover, hypericin-mediated photodynamic therapy could have different effects on these cells depending on the dose, duration, or wavelength of light used to activate hypericin.
The implications of our study are that hypericin may be a promising natural compound for breast cancer treatment, as it can target multiple molecular pathways involved in tumor growth, survival, invasion, and metastasis.Future studies should explore the optimal concentration and duration of hypericin treatment, the synergistic or antagonistic effects of hypericin with other anticancer agents, and the efficacy and safety of hypericin in animal models and clinical trials.

Conclusion
This study provides a thorough evaluation of the impact of hypericin on various aspects of MCF7 human breast cancer cells, including cell viability, apoptosis, microRNA expression, and gene expression.This is the first study to demonstrate that hypericin treatment modulates the expression of miR21 and miR34a in MCF7 cells and that these microR-NAs regulate the expression of PTEN, BCL2, TP53, and CDK4, which are involved in tumor growth, survival, invasion, and metastasis.These findings identify novel molecular targets and pathways for hypericin action on breast cancer cells and provide a rationale for further elucidation of the mechanisms and applications of hypericin in breast cancer therapy.

Fig. 1
Fig. 1 Hypericin (1) induced cell death in MCF7 cells.MCF7 cells were treated with different concentrations of hypericin for 24 and 48 h and then subjected to the XTT assay.The cell viability was expressed as a percentage of the control group.Data are presented as mean ± SD of three independent experiments.Means followed by the same letter do not differ significantly by Tukey test

Fig. 4
Fig. 4 Hypericin (1) treatment modulates the expression of PTEN, BCL2, TP53, and CDK4 in MCF7 breast cancer cells.(Left) The expression levels of PTEN, BCL2, TP53, and CDK4 were measured by western blotting and normalized to GAPDH.(Right) Hypericin treatment increased mRNA level of PTEN by 2.4-fold, and increased TP53 level by 2.7-fold.Hypericin treatment decreased BCL2 mRNA level by 2.5-fold and decreased CDK4 level by 1.7-fold.Data are presented as mean ± SD of three independent experiments.Means followed by the same letter do not differ significantly by Tukey test