Berberine Inhibits the Expression of SCT through miR-214-3p Stimulation in Breast Cancer Cells

In this study, we aimed to evaluate the suppressive abilities of berberine (BBR) on MCF-7 and MDA-MB-231 cells and confirm its underlying mechanisms on miR-214-3p. We first built a panel of 18 miRNAs and 9 lncRNAs that were reported to participate in the mechanism of breast cancer. The RT-qPCR results suggested that BBR illustrated a dosage-dependent pattern in the stimulation to miR-214-3p in both MCF-7 and MDA-MB-231 cells. Then, we performed gain-and-lose function tests to validate the role of miR-214-3p contributing to the anticancer effects of BBR. Both BBR and miR-214-3p mimic reduced the cell viability, repressed migration and invasion capacities, increased rates of total apoptotic cells and ratio of Bax/Bcl-2, and increased the percentage of G2/M cells of MCF-7 and MDA-MB-231 cells by colony formation and CKK8 assay, scratch wound healing and gelatin-based 3D conformation assay, transwell invasion assay, and cell cycle analysis, respectively. However, miR-214-3p inhibitor counteracted all these effects of BBR. Based on the bioinformatics analysis and dual-luciferase reporter test, we identified binding sites between SCT and miR-214-3p. We further confirmed that BBR massively and dose-dependently reduced the mRNA expression and protein levels of SCT in both MCF-7 and MDA-231 cells. We testified that both miR-214-3p mimic and BBR could decrease the mRNA expression and protein levels of SCT, while miR-214-3p inhibitor weakened these reductions. In conclusion, BBR suppressed MCF-7 and MDA-MB-231 breast cancer cells by upregulating miR-214-3p and increasing its inhibition to SCT.


Introduction
Based on the results of epidemiological investigation, it has been reported that breast cancer is becoming one of the major types of cancer and contributes to the highest mortality rate in gynecologic malignancies [1]. Currently, the mainstream treatments of breast cancer are regional avenues, including radiation and surgery and general tools like chemotherapy and biologic therapies [2]. In order to improve the management of breast cancer, it is necessary to exploit novel therapeutic target.
Noncoding RNA, including microRNA (miRNA) and long noncoding RNA (lncRNA), participate in various biological processed [3]. Both miRNAs and lncRNAs related to human cancers are referred to as "oncomirs" [4], which are identified as two types: (i) miRNAs or lncRNAs called oncogenes are upregulated or amplified in cancer; (ii) miRNAs or lncRNAs called suppressors are downregulated or deleted in cancer [5]. A handful of miRNAs and lncRNAs, such as miR-101 [6], miR-21 [7], miR-155 [8], LncRNA-H19 [9], lncRNA-SNHG6 [10], and lncRNA-TALNEC2 [11], are considered to participate in the mechanism of proliferation, invasion, apoptosis, and molecular signaling in breast cancer. erefore, these small regulatory miRNAs work as novel targets for anticancer therapeutic strategies. e chemical name of berberine (BBR) is 2,3-methylenedioxy-9,10-dimenthoxyprotoberberine chloride. BBR is a kind of isoquinoline alkaloid that is extracted from Coptidis Rhizoma or Huanglian. BBR has been proved to possess numerous protective properties, like antimicrobial, cardioprotective, and antidiabetic activities [12,13]. Among these properties, anticancer activity of BBR has been widely accepted. BBR shows anticancer activity in plenty of cancers, including breast cancers [14]. It is believed that BBR moderates mitochondria and pathway of caspase to induce the apoptosis of breast cancer cells [15]. However, how miRNA regulation plays a role in the inhibition of breast cancer cells by BBR to is still under ambiguous.

Cell Culture.
Two human breast cancer cell lines (MCF-7 and MDA-MB-231) were purchased from Chinese Academy of Sciences cell bank (Shanghai, China). e cells were cultured in RPMI-1640 medium (Gibco, ermo) supplemented with 10% fetal bovine serum (Gibco, ermo) and incubated in an atmosphere of 37 o C humidified and 5% CO 2 .

Introduction of Plasmids and siRNA into Cells.
Lipofectamine ® 3000 ( ermo Fisher Scientific, Inc.) was used to transfect the plasmids into cells in accordance with the protocols. Reagent of miR-214-3p mimic or miR-214-3p inhibitor (Guangzhou RiboBio Co., Ltd.) was employed to up-or downregulate the levels of miR-214-3p in 6-well plates with a density of 2 × 10 5 cells in each well. e negative control (NC) was manipulated by a scrambled miRNA.

Cell Proliferation Assay.
We used the Cell Counting Kit 8 (CCK8, Beyotime, China) assay and colony formation assay to estimate the effect of BBR on cell proliferation. Briefly, 7 × 10 3 cells/well were seeded in 96-well plates and treated with BBR (HPLC ≥ 99%, purchased from Meilun Biologics, Dalian China). After 72 h of incubation, a microplate reader was used at a 450 nm optical density to test the viability of each group cells. In colony formation assay, cells were placed into 6-well plates and maintained in media for two weeks and fixed by methanol and stained by 0.1% crystal violet (Sigma, USA) for 20 min.

Cell Migration and Invasion Assay.
e activity of cell migration was evaluated by scratch wound healing assay and gelatin-based 3D conformation assay. In scratch wound healing assay, a sterile tip was used to scratch each well to form a thin "wound." Before adding the serum-free medium, PBS was manipulated to wash the floating cells. At 0 and 12 h after cell recovery, Image-Pro Plus software was manipulated for measuring cell migration distance, and the data were averaged. In gelatin-based 3D conformation assay, fully-formed 3D structures were transferred to 0.1% gelatincoated plates and treated with BBR or miR-214-3p mimic or miR-214-3p inhibitor. e migration levels were evaluated after 24 and 48 h. e medium containing 2% FBS was employed to reduce influence of cell proliferation in this test. 3D structures were imaged by microscope Nikon Eclipse TS 110 and quantified by ImageJ software. e migration index was calculated by the area of cells migrating outwards the 3D structure.
Invasion activities of MCF-7 and MDA-MB-231 cells were analyzed by using transwell invasion assay. On the upper surface of the membrane, the chambers were coated with Matrigel (1 : 5; 80 μl/well, BD Biosciences). DMEM with 10% FBS was added to the lower chamber. After 24 h incubation, 4% paraformaldehyde was used to fix the upper chambers for 10 min. en, it was stained by crystal violet. e microscope (Olympus) was used to count the numbers of passed cells.

Apoptosis Assay and Cell Cycle Analysis.
e levels of apoptosis were measured by annexin-V and propidium iodide (PI) staining as previously described [35]. Cells were seeded to 6-well plate and treated with DMSO, BBR, miR-214-3p mimic, or BBR with miR-214-3p inhibitor. After 24 h, cells were harvested and stained with annexin-V and propidium iodide for 20 min and then run on a BD FACSCanto II cell analyser (BD Biosciences, USA). At least 10000 single cell events were acquired per sample and analyzed by FlowJo software v10.5.0 (FlowJo, USA). e methods of cell cycle analysis were performed according to previous report [36]. Cells were seeded to 6-well plate and treated with DMSO, BBR, miR-214-3p mimic, or BBR with miR-214-3p inhibitor. After 24 h, cells were resuspended in ice-cold Dulbecco's phosphate buffered saline (DPBS) with 70% ethanol. Cells were centrifuged at 300 xg for 5 min and resuspended in DPBS. After 2 h staining of propidium iodide and bovine pancreas ribonuclease, cells were run in BD FACSCanto II cell analyser (BD Biosciences, USA). 50000 single cell events were captured per sample and analysed by FlowJo software v10.5.0 (FlowJo, USA).

Detection of miRNAs and lncRNAs.
We used TRIzol reagent (Invitrogen, CA) to extract total RNA of MCF-7 and MDA-MB-231 cells in each group. SYBR Green PCR kit ( ermo) was used for PCR amplification. Each sample was provided with three repeated holes. Internal reference of GAPDH was manipulated to adjusting and the data of mRNA expression were calculated by the 2 −ΔΔ Ct method. Primer sequences were shown in Table 1.

Protein Detection of Levels of SCT.
e total protein of MCF-7 and MDA-MB-231 cells was extracted with RIPA lysis method. e protein concentration of each well was detected with a BCA method. Protein content was adjusted to 4 μg/μl, 12% SDS-PAGE electrophoresis separation was carried out, and the membrane was transferred to PVDF membranes after ionization. Staining was carried out with Ponceau working solution.
e antibodies of GAPDH (Sigma no. 2275-PC-020) and SCT (Sigma no. AF6387-SP) were diluted to 1 : 5000 and 1 : 1000, respectively. e diluents were added to be sealed overnight at 4°C. Quantity One software was employed to analyze the gray value of scanned protein bands. e relative expression was equal to the ratio of target protein gray value and GAPDH gray value.

Prediction of Target Genes.
Targetscan7.2 and miRwalk were used to predict downstream target gene of miR-214-3p. Reporter gene plasmids containing wild-type and mutant SCT 3′UTR, psiCHECK-2 plasmids (Promega, C8021, USA), miR-214-3p mimic, and their controls were transfected into MCF-7 and MDA-MB-231 cells for 48 hours and then used. Dual-luciferase reporter gene detection kits were used for operation. Finally, collected cells were detected by chemiluminescence. ree repetitions were designed for each group of experiments, and each experiment was repeated for three times.

Statistical Analysis.
e data were analyzed by SPSS 20.0 software and the results were drawn by Graph Prism 8.0. All the data were expressed as mean ± standard deviation (SD ± means). e comparisons of each group were calculated by Student's t-test or one-way ANOVA methods. When p < 0.05, there were statistical differences.

BBR Can Obviously Increase the Expression of miR-214-3p
in Both MCF-7 and MDA-MB-231 Cells. We first built a panel of 18 miRNAs and 9 lncRNAs that were reported to participate in the mechanism of breast cancer. e RT-qPCR results suggested that BBR illustrated a dosage-dependent pattern in the stimulation to miR-214-3p in both MCF-7 and MDA-MB-231 cells ( Figure 1).

BBR Upregulates miR-214-3p Expression to Repress Cell Growth, Invasion, and Migration.
en, we performed gainand-lose function tests to validate the role of miR-214-3p contributing to the anticancer effects of BBR on MCF-7 and MDA-MB-231 cells.
e transfection efficiency was confirmed by RT-qPCR (Figure 2 conformation assay ( Figure 4) suggested that both BBR treatment and miR-214-3p mimic could repress the migration capacities of MCF-7 and MDA-MB-231 cells. It was observed that both BBR and miR-214-3p mimic prevented the invasion capacities of MCF-7 and MDA-MB-231 cells by transwell invasion assay ( Figure 5). However, miR-214-3p inhibitor counteracted all these suppressions of BBR treatment (Figures 2-5).  Figure 7). However, miR-214-3p inhibitor counteracted all these stimulations of BBR treatment (Figures 6 and 7).  sites (Figures 8(a) and 8(b)). So, we first verified it by dualluciferase reporter. Activity of luciferase in miR-214-3p mimic group inpsiCHECK-2-SCT-WT was obviously lower than that of independent sequence group (p < 0.05), while activity of luciferase in miR-214-3p mimic group in psi-CHECK-2-SCT-MUT group was not significantly different from that of independent sequence group (p > 0.05) (Figure 8(c)). en, to ensure the assumption that BBR could promote miR-214-3p expression and suppress the protein expression of its targets SCT, we further confirmed that BBR could massively and dose-dependently reduce the mRNA expression and protein levels of SCT in both MCF-7 and MDA-231 cells (p < 0.05) (Figures 8(d) and 8(e)). Next, we testified that both miR-214-3p mimic and BBR could decrease the mRNA expression and protein levels of SCT, while miR-214-3p inhibitor weakened these reductions (Figures 8(f ) and 8(g)). ese results indicated that BBR promoted miR-214-3p expression and repressed the protein expression of its targets SCT.

Discussion
BBR is a natural alkaloid mainly found in the famous Chinese herb Coptidis Rhizoma. In the beginning, BBR was used in treating diarrhea and gastroenteritis [37]. In subsequent studies, BBR was proved to possess other properties, for instance, antibiosis, cardioprotection, glucose regulation, and antineoplastic activity [12,13,35,38]. In this study, it was found that BBR obviously suppressed the abilities of growth and invasiveness in both MCF-7 and MDA-MB-231 cells.
ese results were consistent with previous studies [39,40]. Kim et al. found that berberine could efficiently Evidence-Based Complementary and Alternative Medicine inhibit growth by inducing cell cycle arrest in anoikis-resistant MCF-7 and MDA-MB-231 cells [39]. It was also indicated that the growth inhibitory effects of berberine treatment on MCF-7 cells might be partly due to the effects on side population cells and ABCG2 expression [40]. Further analysis of these phenotypes is essential for understanding the effect of berberine on anoikis-resistant breast cancer cells. Nonetheless, the antitumor mechanism of BBR in breast cancer cells still remains ambiguous.
In our investigation, the focus was to seek the key ncRNA and its target pathway in the antibreast cancer mechanism of BBR. We first listed a panel of 18 miRNAs [6][7][8][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] and 9 lncRNAs [9,10,15,[29][30][31][32][33][34] that were previously reported to be related with breast cancer, and we found that BBR illustrated a dosage-dependent pattern in the stimulation to miR-214-3p in both MCF-7 and MDA-MB-231 cells. miRNA is one of the important regulators that act as a posttranscriptional suppression officer. Abnormal levels of miRNA could result in a diversity of regulation in diverse cellular pathways. ere is a strong proof that some crucial miRNAs are involved in the progress of breast cancer [41]. To validate the role of miR-214-3p in the suppression of BBR to breast cancer cells, we performed a rescue test and observed that both BBR and miR-214-3p mimic could repress the abilities of growth, invasiveness, and migration in MCF-7 and MDA-MB-231 cells, while miR-214-3p inhibitor counteracted these suppression. We also found that BBR upregulated miR-214-3p expression to induce cell apoptosis and G2/M arrest. ese results indicated that BBR presented anticancer effects through miR-214-3p. Another study showed that lncRNATSLNC8 inhibited miR-214-3p/FOXP2 axis to suppress the proliferation and G1/S phase transition of breast cancer cells [42]. It has been reported that the expression of miR-214-3p is correlated with the proliferation and apoptosis of breast cancer cells [21] and acts as a breast tumor suppressor through the regulation of EMT [43]. In the fields of breast cancer, these studies have established that miR-214-3p had a vital role in the progress of breast cancer. Our results indicated that miR-214-3p was also the target of BBR to present anticancer effects. en, after using bioinformatics analysis and searching previous studies, we identified that SCT might be the downstream target of miR-214-3p. To confirm the assumption that BBR could promote miR-214-3p transcription and raise the suppression of its downstream target SCT, we first confirmed that BBR could dose-dependently reduce SCT in both levels of mRNA and protein. After that, we found that both miR-214-3p mimic and BBR repressed SCT mRNA and protein, while miR-214-3p inhibitor weakened these reductions. In physiological conditions, SCT binds to * *       its receptor to mediate the effect of the gastrointestinal hormone on digestion and water homeostasis. e overexpression of SCT in MCF-7 cells led to an increase of the cell proliferation index and cellular migration [44]. e data of this study revealed the fact that miR-214-3p inversely acted on SCT in both MCF-7 and MDA-MB-231 cells. However, whether SCT is an oncogene or a tumor suppressor is still controversial. A number of studies hold the idea that it is an oncogene, as high expression was observed in breast cancer [44], whereas other studies indicated the downregulation of the gene in colorectal cancer [45] and prostate cancer [46]. SCT acts as a gene with double-edge sword activities, which possesses both oncogenic and tumorsuppressive effects. It plays a tumor-suppressive role in normal cells and a proliferation and migration stimulating role in cancer cells. It has been reported that SCT suppresses the proliferation of normal breast cells, while the gene stimulates the proliferation and migration of cancer cells [44].
In conclusion, BBR is indicative of the suppression to MCF-7 and MDA-MB-231 breast cancer cells by upregulating the expression of miR-214-3p and increasing its inhibition to SCT. e miR-214-3p/SCT axis is the therapeutic target in the mechanism of BBR to suppress breast cancer.

Data Availability
e datasets used and/or analyzed in the present study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no competing interests.