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. 2023 Mar 23;14(13):1135–1144. doi: 10.1111/1759-7714.14834

Sauchinone inhibits breast cancer cell proliferation through regulating microRNA‐148a‐3p/HER‐2 axis

Xiaolei Hu 1,✉, Jie Wang 2, Pei Shang 1, Shan Wang 1, Lujia Chen 1, Changsheng Ye 1, Guangyu Yao 1,✉
PMCID: PMC10151132  PMID: 36959089

Abstract

Background

Sauchinone is extracted from the root of Saururus chinensis and exhibits potent antitumor effects in various human cancers. However, how sauchinone is involved in breast cancer has not been well studied.

Methods

Cells apoptosis, cell proliferation, and cycle distribution were evaluated. Xenograft tumor mouse model was constructed to investigate the roles of sauchinone. The relevant protein expression was detected by western blot.

Results

We found that sauchinone significantly reduced proliferation and survival, also induced apoptosis of MCF‐7 and Bcap‐37 cells in vitro. Sauchinone significantly increased miR‐148a‐3p expression, and human epidermal growth factor receptor (HER)‐2 targeted on miR‐148a‐3p. Sauchinone exposure downregulated HER‐2 expression whose overexpression partly eliminated the inhibitory effect of sauchinone. Further, sauchinone efficiently inhibited breast cancer progression through downregulating HER‐2 expression in vivo.

Conclusion

Our results indicate that sauchinone efficiently inhibits breast cancer progression through regulating miR‐148a‐3p/HER‐2 axis, suggesting that sauchinone could be an effective anticancer agent for breast cancer.

Keywords: apoptosis, breast cancer, miR‐148a‐3p, proliferation, sauchinone

Sauchinone efficiently inhibits breast cancer progression through regulating miR‐148a‐3p/HER‐2 axis.

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INTRODUCTION

Breast cancer is a common type of cancer and accounts for 16% of cancer deaths among adult women. 1 For breast cancer, current therapies are multimodal majorly including drug therapy, surgery, and radiation. 2 Despite dramatic advances in breast cancer diagnosis, it is still a major health challenge. 3 Therefore, a better understanding of the complex mechanism in this disease contributes to develop efficient anticancer agents for breast cancer.

Sauchinone is isolated from the roots of Saururus chinensis (SC), and is reported to have hepatoprotective, antioxidant, and antitumor properties. 4 However, for breast cancer, sauchinone can downregulate the expression of vascular endothelial growth factor (VEGF), proliferation‐related gene including cyclin D1, and anti‐apoptotic Bcl‐2, which are responsible for apoptosis through the caspase‐3 activation. 5 However, the specific molecular mechanism of sauchinone in breast cancer remains unclear.

Human epidermal growth factor receptor (HER)‐2 is a proto‐oncogene 6 and highly expressed HER‐2 has been observed in prostate, lung, bladder, pancreatic cancer, and osteosarcoma. 7 In breast cancer, ~15% to 20% of patients exhibit obvious overexpression of HER2, which results in a poorer prognosis and survival. 8 Therefore, the identification and development of antitumor agent targeting HER‐2 could be used for HER2‐positive breast cancer patients. MicroRNAs (miRNAs) are reported to be involved in breast cancer. MicroRNA‐223 can promote breast cancer cell proliferation by targeting FOXO 1 and function as a potential tumor marker. 9 In addition, a series of studies have indicated that abnormal miRNA expression involved in invasion, metastasis, and resistance to chemotherapy, and also can act as biomarkers in breast cancer. 10 , 11 MicroRNA‐21 and MicroRNA‐210 are closely involved in breast cancer invasion and poor prognosis. 12 MiR‐148a‐3p inhibits breast cancer progression through downregulating WNT1. 13 Furthermore, the miR‐148a‐3p/HER‐2 axis has been identified as predictive signature to evaluate the breast cancer patients respond to single agent trastuzumab‐based neoadjuvant therapy. 14 However, whether miR‐148a‐3p is involved in sauchinone treatment and the interaction with HER‐2 is still unknown. In this study, the antitumor effect of sauchinone for breast cancer was investigated. In mechanism aspect, the relationship between it and existing miR‐148a‐3p/HER‐2 signature would also be discussed.

MATERIALS AND METHODS

Materials

Sauchinone was obtained from Nature‐standard (purity >98%) and used for subsequent experiments in this study. The structural formula of sauchinone is shown in Figure 1.

FIGURE 1.

FIGURE 1

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Sauchinone significantly inhibited MCF‐7 and Bcap‐37 cell function. (a) The structural formula of sauchinone. (b) MCF‐7 and Bcap‐37 cell viability was detected by ent‐sauchinone (20, 40, 80, 160, and 320 μM) or dimethyl sulfoxide vehicle (control) for 48 h, or treated with sauchinone (0, 50, and 100 μg/mL) for indicated times. (c), (d) Cell proliferation of MCF‐7 (c) and Bcap‐37 cells (d) was measured. (e), (f) The numbers of colony formation of MCF‐7 (e) and Bcap‐37 (f) cells were evaluated and counted by a microscope.

Cell culture

From the American Type Culture Collection, MCF‐7, and Bcap‐37 (human breast cancer cell lines) were obtained and cultured with Roswell Park Memorial Institute (RPMI)‐1640 medium containing 10% fetal bovine serum (FBS) with 5% CO2 at 37°C.

Cell transfection

MCF‐7 and Bcap‐37 cells were transfected with miR‐148a‐3p mimics and negative control miR (miR‐NC) (Sigma Aldrich) by Lipofectamine 2000. MiR‐148a‐3p antagomir was transfected with Lipofectamine RNAi MAX Kit. After 48 h for transfection, the efficiency of transfection was determined by quantitative reverse transcription (qRT)‐polymerase chain reaction (PCR).

Luciferase reporter assay

The fragments of 3′untranslated regions (3′UTR) HER‐2 containing mutant type (MUT) and wild‐type (WT) of binding site with miR‐148a‐3p were inserted into pRL (pRL vectors are wild‐type Renilla luciferase control vectors) reporter plasmid to construct the luciferase reporter plasmids. The luciferase reporter plasmids were co‐transfected with miR‐NC or miR‐148a‐3p mimic into cell lines. Luciferase activity was detected after transfection for 48 h.

Cell viability assay

In 96‐well plates, Bcap‐37 and MCF‐7 Cells (7 × 103 cells/well) were seeded and incubated for 24 h at 37°C. Next, various concentrations of sauchinone were added to cells for 48 h. After incubation, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide staining was permed to evaluate the cell viability.

Cell counting kit‐8 assay

In 96‐well plates, approximately 1 × 103 cells were plated, then different concentrations of sauchinone (0, 50, and 100 μg/mL) was incubated cells for different times. Finally, cell viability was detected.

Colony formation assay

As previously described, 15 ~1000 cells were plated in 6‐well plates for 2 weeks. The numbers of colonies were counted after cells were stained with 1% crystal violet dye.

Transwell assay

As previously described, 16 using 24‐well Transwell plates, cell invasiveness was performed. Briefly, approximately 1 × 105 cells were plated into the upper chambers contained medium without serum. However, the medium in the lower chambers were filled with 20% FBS. Crystal violet was used to stain cells after 24 h culture. Without coating the membranes with Matrigel, the migration assay was performed similarly with invasion assay.

Cell‐cycle and apoptosis analysis

In 6‐well plates, approximately 1 × 105 cells/mL MCF‐7 and Bcap‐37 cells were plated and treated with various concentrations of sauchinone. Cells were stained for 30 min with 50 μg/mL propidium iodide (PI) solution and cell percentage at specific phases was detected by a FACSCalibur cytometry. Cells were treated with 100 μg/mL sauchinone, and apoptosis rate was analyzed with a Annexin V‐FITC/PI apoptosis detection kit as previously described. 17

qRT‐PCR

Total RNA samples of cultured cells were extracted. PCR reactions were performed with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and U6 as the internal reference. The target gene expression was calculated by 2−ΔΔCt method. 18 MiR‐148a‐3p: 5′‐ CCCGGATTCGCAAACTCAGCTTTAC ‐3′; 5′‐ CGGAATTCGTTGCGACCGTGATACC ‐3′; HER‐2: 5′‐ AGAAAGTGGTGCCATTTTTG ‐3′, 5′‐ AGCAGATGCCCATGCTTTCT ‐3′; U6 forward: 5′‐GACCGATTCGTTCTGTGGCAC‐3′, 5′‐GATTACCCGTCGGCCATCGATC‐3′; GAPDH: 5′‐ GAAGGTGAAGGTCGGAGTC ‐3′, 5′‐ GAAGATGGTGATGGGATTTC ‐3′.

Western blotting

Total protein from cultured cells or tumors was extracted. Approximately 50 μg samples were separated and transferred to polyvinylidene fluoride membranes. Primary antibodies including HER‐2 (1:1000, MA5‐13675, Abcam) and β‐actin (1:1000, Ab8227, Abcam) incubated the membrane at 4°C overnight. The membranes were incubated with horseradish peroxidase ‐labeled immunoglobulin G and visualized with Bio‐Rad imaging system.

Xenograft model

As previously described with minor modulation, 19 a total of 1 × 106 Bcap‐37 cells were subcutaneously inoculated into nude BALB/c mice (4‐week‐old and female). Mice were treated for 3 days with or without miR‐148a‐3p antagomir 80 mg/kg/day via intravenous tail injections, phosphate‐buffered saline (PBS) as the control. In each group, mice were treated with or without 5 mg/kg sauchinone every day for 5 days (PBS was used as control). Tumor volume was calculated: V = 0.524 × L × W2. Finally, mice were euthanized, and then the size and weight of tumors was evaluated.

Ki‐67 staining assay

Xenograft tumors was isolated from mice and embedded in paraffin. With anti‐Ki67 antibody as described previously, 20 these 4 μM‐thick sections were immunohistochemically stained.

Statistical analysis

All data are presented as the means ± standard error (SE) method. Statistical analysis was performed using the SPSS software (version 18.0), and Student's t‐test was conducted to compare the difference between two groups. The value p < 0.05 was considered as the significant threshold.

RESULTS

Sauchinone inhibits breast cancer cell function

The molecular structure of sauchinone was showed in Figure 1(a). The cell viability treated by sauchinone was evaluated in a dose dependent manner (Figure 1(b)). IC50 values of sauchinone against MCF‐7 and Bcap‐37 were 97.8 ± 0.58 μM and 102.1 ± 2.11 L μM. Sauchinone significantly inhibited the MCF‐7 (Figure 1(c)) (p < 0.01) and Bcap‐37 (Figure 1(d)) (p < 0.01) cell viability. Furthermore, the colony formation of MCF‐7 (Figure 1(e)) (p < 0.01) and Bcap‐37 cells (Figure 1f)) (p < 0.01) was significantly decreased by sauchinone treatment. The results indicated that sauchinone can inhibit breast cancer cell viability, cell colony formation, and proliferation via a low dosage.

Sauchinone significantly inhibits breast cancer cell migration and invasion

A total of 100 μM sauchinone significantly inhibited MCF‐7 and Bcap‐37 cell migration and invasion (Figure 2(a),(b)) (p < 0.01), indicating that sauchinone reduced the breast cancer cell migration and invasion. The results indicated that sauchinone may contribute to inhibit breast cancer cell metastasis via a low dosage.

FIGURE 2.

FIGURE 2

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Sauchinone significantly inhibited breast cancer migration and invasion. The MCF‐7 and Bcap‐37 cells were treated with sauchinone (0, 50, and 100 μg/mL). The migration (a) and invasion (b) capacity was detected by transwell assay.

Sauchinone induces breast cancer cell apoptosis

Next, results demonstrate that sauchinone significantly decreased S phase and G2/M phase cells and increased G0/G1 phase cells both in MCF‐7 and Bcap‐37 cells (p < 0.05) (Figure 3(a)). Meanwhile, sauchinone remarkably induced apoptosis of two cell lines (p < 0.01) (Figure 3(b)), suggesting that sauchinone‐induced breast cancer cell apoptosis is followed by arresting cells at the G0/G1 phase.

FIGURE 3.

FIGURE 3

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Sauchinone induced breast cancer cell apoptosis. The MCF‐7 and Bcap‐37 cells were treated with sauchinone (0, 50, and 100 μg/mL). Effect of sauchinone on cell cycle (a) and apoptosis rates (b) was detected.

Sauchinone inhibits breast cancer progression through upregulating microRNA‐148a‐3p

For investigating the specific molecular mechanism of sauchinone in breast cancer progression, MCF‐7 and Bcap‐37 cells were treated with 100 μg/mL sauchinone, and results showed that sauchinone significantly enhanced miR‐148a‐3p expression (p < 0.01) (Figure 4(a)). After MCF‐7 and Bcap‐37 cells were transfected with miR‐148a‐3p antagomir, the efficiency of transfection was determined by qRT‐PCR (Figure 4(b)) (p < 0.01). Moreover, combining miR‐148a‐3p antagomir with sauchinone significantly attenuated sauchinone‐induced cell apoptosis (p < 0.01) (Figure 4(c)). These indicate that sauchinone inhibits breast cancer cell growth through upregulating miR‐148a‐3p.

FIGURE 4.

FIGURE 4

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Sauchinone downregulated human epidermal growth factor receptor (HER)‐2 expression through inducing miR‐148a‐3p. (a) After treated with or without 100 μg/mL sauchinone, HER‐2 mRNA in MCF‐7 and Bcap‐37 cells were detected. (b) miR‐148a‐3p mRNA level was measured. (c) The apoptosis rate was detected by flow cytometry. (d) The predicted binding site of miR‐148a‐3p with the 3′ untranslated region of HER‐2 based on Targetscan. (e) Luciferase reporter assay. (f), (g) The mRNA (f) and protein (g) expression of HER‐2 was detected. *p < 0. 05, **p < 0.01 versus control group.

HER‐2 was a direct target of microRNA‐148a‐3p

To explore the molecular targets of miR‐148a‐3p, prediction results based on the TargetScan database revealed that there was a putative binding site of miR‐148a‐3p on 3′UTR of HER‐2 (Figure 4(d)). To test this, we found that luciferase activity of HER‐2‐WT in two cell lines was reduced after transfection with miR‐148a‐3p mimic, whereas no obvious change was observed in HER‐2‐MUT (Figure 4(e)) (p < 0.01). Meanwhile, miR‐148a‐3p mimic significantly inhibited HER‐2 expression (Figure 4(f),(g)) (p < 0.01), we also found that HER‐2 expression could significantly inhibited miR‐148a‐3p expression (Figure S1). These suggest that HER‐2 is a target of miR‐148a‐3p.

Sauchinone inhibited breast cancer cell growth through downregulating HER‐2

Next, we explored whether sauchinone inhibits breast cancer progression through HER‐2 and found that sauchinone significantly decreased the mRNA (Figure 5(a)) (p < 0.01) and protein level (Figure 5(b)) (p < 0.01) of HER‐2 compared with control both in two cell lines. Meanwhile, sauchinone significantly decreased HER‐2 expression, and miR‐148a‐3p antagomir could rescue sauchinone‐induced inhibition in HER‐2 expression both in Bcap‐37 and MCF‐7 cells (p < 0.01) (Figure 5(c) and Figure S2). Next, rescue experiments were performed, and qRT‐PCR results showed that HA‐HER‐2 efficiently increased the expression of HER‐2 compared with HA‐vector (negative control) both in two cell lines (Figure 5(d)) (p < 0.01). Moreover, HER‐2 overexpression significantly attenuated the inhibition of sauchinone on the growth of two cell lines (p < 0.01) (Figure 5(e),(f)). These indicate that sauchinone inhibits breast cancer cell growth through miR‐148a‐3p‐mediated downregulation of HER‐2. In addition, we also demonstrated miR‐148a‐3p mediated response to HER2 monoclonal antibody of trastuzumab (Figure S3) (p < 0.01).

FIGURE 5.

FIGURE 5

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Sauchinone inhibited breast cancer cell growth through inhibiting the expression of human epidermal growth factor receptor (HER)‐2. (a), (b) The mRNA (a) and protein (b) expression of HER‐2 was detected. (c) The HER‐2 protein was detected. (d) mRNA level of HER‐2 was detected. (e), (f) Cell viability was measured.

Sauchinone inhibited breast cancer progression through downregulating HER‐2 in vivo

Finally, on a xenograft model in vivo, we evaluated the antitumor effect of sauchinone based. We found that sauchinone could significantly decrease tumor size tumor weight and tumor volume, but these effects were rescued after miR‐148a‐3p transfection (Figure 6(a)–(c)) (p < 0.01). The similar trend was also observed in Ki‐67 staining results (Figure 6(d)). Meanwhile, the expression of HER‐2 was dramatically downregulated in sauchinone treatment group, but it was also rescued by miR‐148a‐3p overexpression (Figure 6(e)) (p < 0.01). All these results further confirm that sauchinone plays a potential antitumor role through miR‐148a‐3p mediated downregulation of HER‐2 in breast cancer.

FIGURE 6.

FIGURE 6

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Sauchinone inhibited breast cancer progression via miR‐148a‐3p in vivo. (a) Representative xenograft tumor images. (b), (c) Tumor weight and volume of xenograft tumors. (d) Ki‐67 staining of xenograft tumors. (e) The human epidermal growth factor receptor‐2 protein expression in xenograft tumors was detected.

DISCUSSION

In the last decades, several natural products, including sauchinone, curcumin, lycopene, genipin, denbinobin, capsaicin, and ursolic acid, have been identified to show potent antitumor effect in breast cancer. 21 Lycopene exerts antitumor effects by inactivating of AKT and ERKs signaling pathways. 22 Genipin was reported to induce apoptosis through upregulating Bax expression and downregulating Bcl‐2 expression. 23 Through regulating Src‐mediated signaling pathways, denbinobin can inhibit breast cancer metastasis. 24 Capsaicin could induce apoptosis through regulating epidermal growth factor receptor/HER‐2 pathway. 25 Ursolic acid has been reported to inhibit progression of breast cancer through affecting apoptosis related protein expression. 26 , 27 Therefore, the identification of potential anticancer agents for breast cancer is still urgent.

In various human cancers, sauchinone has also been shown to act a crucial antitumor role. Sauchinone has been identified to prevent gastric cancer progression through inhibiting transforming growth factor‐β‐induced metastasis. 28 Sauchinone exerts a potential anticancer effect in hepatocellular carcinoma through modulating AMPK signaling pathway. 29 Whereas in breast cancer, sauchinone has also been reported to exhibit antitumor effect and the specific mechanism is not well known. Only one previous study has demonstrated that sauchinone exerts anticancer effect through regulating proliferation and apoptosis‐related gene expression. 5 In this study, we revealed a new mechanism of sauchinone involved in breast cancer and found it efficiently inhibits breast cancer progression through downregulating HER‐2 by inducing miR‐148a‐3p.

MiR‐148a‐3p has been reported to participate in many biological processes in various human cancers. For example, miR‐148a‐3p can inhibit progression of epithelial ovarian cancer through directly inhibiting the expression of c‐Met, 30 inhibit esophageal cancer invasion and proliferation by targeting DNMT1, 31 and repress proliferation and Epithelial‐Mesenchymal Transition (EMT) through regulating ERBB3/c‐Myc pathway in bladder cancer. 32 In addition, the abnormal miR‐148a‐3p expression suggests it as a biomarker in multiple human cancers, including prostate cancer, colorectal cancer, and osteosarcoma cell. 33 , 34 , 35 Here, in breast cancer cell lines, our results demonstrate that sauchinone significantly induces miR‐148a‐3p and miR‐148a‐3p antagomir efficiently rescues sauchinone‐induced inhibition.

HER‐2 is often highly expressed in human cancers and serves as a direct target gene of miRNAs to affect human cancer progression, including breast cancer. MiR‐9 inhibits tumor engraftment, migration and colony formation of breast cancer cells by directly targeting HER‐2. 36 Long noncoding RNAs SNHG20 can promote breast cancer progression by targeting miR‐495/HER‐2 axis. 37 However, in breast cancer, the role of miR‐148a‐3p/HER‐2 remains unclear. Here, HER‐2 is identified to be a direct target of miR‐148a‐3p. Meanwhile, sauchinone significantly downregulated HER‐2 expression, and miR‐148a‐3p antagomir could reverse sauchinone‐induced inhibition in HER‐2 expression. Overexpression of HER‐2 significantly attenuated the effect of sauchinone on breast cancer. Finally, sauchinone was found to be able to efficiently prevent breast cancer progression with a significant decrease of HER‐2 expression in vivo. Although HER‐2 overexpression significantly attenuates the inhibitory effect of sauchinone in vitro, further experiment is needed to apply HER‐2 overexpression in the xenograft model to further confirm the role of sauchinone in breast cancer.

In this study, we have revealed the potential target for sauchinone. In the future possible clinical application, the expression of HER‐2/miR‐148a‐3p may be a prognostic biomarker. However, there may be more biomarkers for sauchinone. The mechanisms of sauchinone in breast cancer should also be identified in future work. More biomarker molecular about sauchinone should also be identified in future work. These markers together would form a panel to provide a more complete perspective or the pharmacology, toxicology, and drug resistance of sauchinone in future.

CONCLUSION

In summary, our study showed that sauchinone inhibited breast cancer progression through downregulating HER‐2 expression by inducing miR‐148a‐3p. This study suggests that sauchinone acts as an efficient anticancer agent for breast cancer, and miR‐148a‐3p/HER‐2 axis may be a potential target for breast cancer treatment.

AUTHOR CONTRIBUTIONS

Xiaolei Hu, Jie Wang, Pei Shang, Guangyu Yao: Concept, manuscript writing, editing and review. Shan Wang, Lujia Chen, Changsheng Ye: Data collection and analysis, manuscript preparation. All authors have read and approve the submission of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Figure S1. The mRNA expression of miR‐148a‐3p was detected.

Click here for additional data file. (384.6KB, tif)

Figure S2. Sauchinone inhibits breast cancer cell growth through miR‐148a‐3p‐mediated downregulation of HER‐2.

Click here for additional data file. (3MB, tif)

Figure S3. Trastuzumab inhibits breast cancer cell growth through miR‐148a‐3p‐mediated downregulation of HER‐2.

Click here for additional data file. (3MB, tif)

ACKNOWLEDGMENTS

We thank the financial support from Natural Science Foundation of Guangdong Province (2019A1515011331).

Hu X, Wang J, Shang P, Wang S, Chen L, Ye C, et al. Sauchinone inhibits breast cancer cell proliferation through regulating microRNA‐148a‐3p/HER‐2 axis. Thorac Cancer. 2023;14(13):1135–1144. 10.1111/1759-7714.14834

Xiaolei Hu, Jie Wang, and Pei Shang contributed equally to this work.

Contributor Information

Xiaolei Hu, Email: xlhu@smu.edu.cn.

Guangyu Yao, Email: ygy531@hotmail.com.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. The mRNA expression of miR‐148a‐3p was detected.

Click here for additional data file. (384.6KB, tif)

Figure S2. Sauchinone inhibits breast cancer cell growth through miR‐148a‐3p‐mediated downregulation of HER‐2.

Click here for additional data file. (3MB, tif)

Figure S3. Trastuzumab inhibits breast cancer cell growth through miR‐148a‐3p‐mediated downregulation of HER‐2.

Click here for additional data file. (3MB, tif)

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

Articles from Thoracic Cancer are provided here courtesy of Wiley

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