Huaier Extract Inhibits Breast Cancer Progression Through a LncRNA-H19/MiR-675-5p Pathway

Breast cancer is the most common cancer worldwide and the leading cause of cancer-related mortality among women [1]. Among the various therapies available, TCM (Traditional Chinese Medicine) has an important role [2, 3]. Huaier extract, which has been used in China for approximately 1600 years [4], has attracted increasing attention as a result of its anti-tumor activity [5, 6]. Studies show that Huaier extract acts as a tumor suppressor by inhibiting tumor growth [7] and metastasis [8]; and preventing resistance to chemotherapy [9]. Our previous studies indicate that Huaier extract inhibits breast cancer progression by inducing apoptosis and autophagy, inhibiting angiogenesis, modulating the ERα pathway, targeting cancer stem cells, and regulating tumor-associated macrophages [4, 10-14]. However, the mechanisms that underlie these effects are unclear.

Noncoding RNAs (ncRNAs) are RNA molecules that do not encode proteins. Recent reports suggest that ncRNAs play critical roles in cancer development and progression [15, 16]. Long noncoding RNAs (lncRNAs) are ncRNAs longer than 200 nucleotides [17]. Accumulating evidence indicates that lncRNAs regulate breast cancer development and progression via multiple pathways [18-20]. LncRNA H19 is encoded by a paternally imprinted gene, H19, whose oncogenic functions have been investigated in the context of pancreatic ductal adenocarcinoma, hepatocarcinoma, bladder cancer, glioma and gastric cancer [21-25]. Recent data and studies of miR-675-5p derived from lncRNA H19 indicate that the lncRNA H19/miR-675-5p axis plays a pro-oncologic role by regulating the expression of several target genes [26-30].

Here, we examined the roles of H19 and miR-675-5p in the anti-tumor effects of Huaier extract. The data show that Huaier extract inhibits the expression of H19 and miR-675-5p. The over-expression of H19 and miR-675-5p reversed the inhibitory effects of Huaier extract, whereas reduced levels of H19 and miR-675-5p sensitized breast cancer cells to Huaier extract. Therefore, this study shows that Huaier extract inhibits breast cancer progression via the H19/miR-675-5p axis. Thus, Huaier extract is a promising therapeutic agent for the clinical treatment of breast cancer.

The breast cancer cell lines MDA-MB-231, MDA-MB-468 and MCF7 were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM/high glucose medium (Gibco-BRL, Rockville, IN, USA) supplemented with 10% fetal bovine serum (CLARK), 100 U/ml penicillin and 100 μg/ml streptomycin in 5% CO₂ at 37°C. Huaier extract was kindly provided by Gaitianli Medicine Co., Ltd. (Jiangsu, China) and was prepared as previously described [14]. MicroRNA mimics, microRNA inhibitors, and siRNA were obtained from GenePharma (Shanghai, China).

Total RNA was extracted from MDA-MB-231 and MCF7 cells and Huaier-treated cells (MDA-MB-231_H and MCF7_H), followed by analysis using GeneChip® Human Transcriptome Array 2.0 (GMINIX, Shanghai, China), according to the manufacturer’s instructions. The microarray covered 40, 000 human noncoding transcripts, 245, 000 coding transcripts, and 339, 000 probe sets that covered exon-exon junctions. Each RNA was detected by corresponding probes and analysis was repeated three times.

Cell viability was measured in an MTT assay. Breast cancer cells were initially transfected with the indicated vectors, microRNAs, or siRNAs. At 48 h post transfection, cells (2 × 10³ cells/well) were cultured overnight in 96-well plates. The medium was subsequently replaced with medium that contained various concentrations of Huaier extract (0, 2, 4, or 8 mg/mL). After incubation for the indicated time, 20 μL of MTT (5 mg/mL in PBS) was added to each well and the cells were incubated for 4-6 h at 37°C. The supernatants were carefully discarded, and 100 μL dimethyl sulfoxide (DMSO) was added to each well. Absorbance values were read in a Microplate Reader (Bio-Rad, Hercules, CA, USA) at 490 nm and 570 nm.

The BD Pharmingen^TM PE Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect apoptotic cells. Briefly, after transfection with the indicated vectors, microRNAs, and siRNAs, breast cancer cells were treated with various concentrations of Huaier extract for the indicated times. Cells were subsequently harvested and washed twice with PBS. Next, cells (1 x 10⁵) were resuspended in 100 μl binding buffer, followed by the addition of 5 μl PE Annexin V and 5 μl 7-AAD. After incubation for 15 min in the dark, an additional 400 μl of binding buffer was added prior to analysis by flow cytometry using a FACScan flow cytometer (BD Biosciences).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNAs or miRNAs were synthesized using the PrimeScript reverse transcriptase reagent kit (TaKaRa, Shiga, Japan) or the Mir-X^TM miRNA first-strand Synthesis Kit (TaKaRa, Shiga, Japan) respectively. Real-time PCR was performed using an Applied Biosystems StepOne Plus Real-Time PCR System. The PCR products were amplified with the following primers: miR-675-5p forward, 5’- GCGGAGAGGGCCCACAG-3’; H19 forward, 5’- AGCCACCACATCATCCCAGA-3’ and H19 reverse, 5’- GAATGCTTGAAGGCTGCTCC-3’; GAPDH forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’ and GAPDH reverse, 5’- GGCTGTTGTCATACTTCTCATGG-3’. GAPDH or U6 was used to determine equal RNA loading. All experiments were repeated at least three times.

To overexpress H19, H19 cDNA was cloned into the multiple cloning site of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). The expression plasmid vector and the empty vector were subsequently transfected into breast cancer cells using lipofectamine 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

MiR-675-5p mimic, miR mimic control, miR-675-5p inhibitor (miR-675-5pI), miR inhibitor control (miR-NCI), specific H19 siRNAs and a scrambled locus siRNA (NC) were purchased from GenePharma (Shanghai, China). Cells were seeded in 60 mm dishes (4 × 10⁵ cells/dish) and transfected according to the manufacturer’s protocol (Invitrogen, CA, USA). The siRNA sequences for H19 were as follows: H19 siRNA 1, 5’- UAAGUCAUUUGCACUGGUU-3’; H19 siRNA 2, 5’- CCCACAACAUGAAAGAAAC-3’.

Indicated cells were lysed with lysis buffer (1 × PBS, 1% NP40, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate) that contained protease inhibitors. Proteins were separated via SDS-PAGE gels and analyzed by Western blotting. An anti-CBL antibody was purchased from Abcam (Cambridge, MA, USA) and a mouse monoclonal antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The expression of β-actin was used as a loading control.

All experiments were performed in triplicate and data are presented as the mean ± S.D.. SPSS V18.0 software was used for statistical analysis. The data were analyzed with one-way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.

We have previously demonstrated that Huaier extract has anti-tumor effects against breast cancer cells [4, 11, 12, 31]. However, the underlying mechanisms were not clear. To identify candidate ncRNAs involved in Huaier-mediated tumor suppression, we examined the ncRNA expression profiles in MDA-MB-231 and MCF7 cells. As shown in Fig. 1A, 1981 human ncRNAs showed a significant change in expression after treatment with Huaier extract. Among these human ncRNAs, 228 were upregulated (including LOC643401, MIR548X, CGO30, RPS15AP10, and LINC00240) and 925 were downregulated (including SNORA38, MIR4536-1, RNU1-23P, ATG10-AS1, and LINC00529). Combining data from the ncRNA microarray analysis and literature searches identified lncRNA H19, the expression of which decreased in both cell lines after exposure to Huaier extract. qPCR confirmed that Huaier extract reduced H19 expression in a time-dependent manner (Fig. 1B). Taken together, these results show that Huaier extract reduces the H19 levels in breast cancer cells.

To assess the potential functional role of H19 on Huaier-mediated tumor suppression, we knocked down the expression of H19 using siRNA1 and siRNA2 (Fig. 2A). Previous studies have demonstrated that H19 siRNA inhibited the cell viability of cancer cells [32, 33], and we obtained the consistent results (Fig. 2B). In our study, the transfection of H19 siRNAs led to a significant increase in the tumor-inhibitory effects of Huaier extract (2 mg/ml) (Fig. 2C). In MDA-MB-231 cells, H19 siRNA1 reduced the cell viability (51.3±1.3%) compared with the control group (67.0±5.2%) after 48 h of exposure to Huaier extract. H19 siRNA2 had the same effect.

Furthermore, we used flow cytometry analysis to detect apoptotic cells after exposure to Huaier extract and H19 siRNAs. As shown in Fig. 2D, the transfection of H19 siRNA1 led to a significant increase (1.57-fold) in the percentage of apoptotic MDA-MB-231 cells compared with the control group (p=0.035). These results suggest that knocking down H19 expression increased the anti-tumor effects of Huaier extract.

In the present study, the expression of H19 increased after the transfection of cells with pcDNA3.1-H19. qPCR was used to examine the transfection efficiency (Fig. 3A; p＜0.01). A previous study showed that the overexpression of H19 promoted the proliferation of breast cancer cells [34], and we demonstrated the same effect (Fig. 3B). The role of H19 was further examined by assessing cell proliferation and apoptosis. In MDA-MB-231 cells, after treatment with 4 mg/ml Huaier extract for 24 h, the overexpression of H19 led to significant inhibition of Huaier-induced tumor suppression (Fig. 3C; p=0.028). The same results were obtained in MDA-MB-468 cells. In addition, the data shown in Fig. 3D demonstrate that the increased expression of H19 reduced the percentage of apoptotic cells by 0.65-fold (p=0.025). Taken together, these findings indicate that H19 plays a major role in Huaier-mediated tumor suppression.

Previous studies demonstrate that H19 exon 1 encodes the precursor for miR-675, which generates two conserved miRNAs (miR-675-5p and miR-675-3p) [27, 29, 35, 36]. Therefore, we explored the possibility that miR-675-5p was associated with the function of H19 in Huaier-mediated tumor suppression.

As shown in Fig. 4A, treatment with Huaier extract led to a significant and dose-dependent reduction in the miR-675-5p expression by MDA-MB-468 and MDA-MB-231 cells. The miR-675-5p level was significantly upregulated after transfection with miR-675-5p mimics (Fig. 4B). Fig. 4C shows that the over-expression of miR-675-5p led to a slight suppression of Huaier extract-mediated suppression of breast cancer proliferation. Therefore, miR-675-5p is involved in the anti-tumor function of Huaier extract.

The expression of miR-675-5p in MDA-MB-231 cells was reduced by a miR-675-5p inhibitor (Fig. 5A). Downregulation of miR-675-5p increased the anti-tumor effects of Huaier extract. In MDA-MB-231 cells, treatment with Huaier extract for 48 h led to a significant reduction in the viability of breast cancer cells (from 74.9±6.8% in the NCI group to 59.1±8.0% in the miR-675-5pI group; p=0.030; Fig. 5B). Similar results were obtained when MDA-MB-468 cells were treated with Huaier extract. Flow cytometry analysis was further applied to detect the effect of miR-675-5p on apoptosis. As shown in Fig. 5C, inhibition of miR-675-5p significantly increased the percentage of apoptotic cells induced by Huaier extract (p=0.019). These results showed that the downregulation of miR-675-5p promoted the cytotoxicity effect of Huaier extract.

Vennin et al. previously demonstrated that CBL was a direct target of miR-675-5p [29]. H19/miR-675-5p increases cell proliferation by regulating CBL. In addition, we previously showed that Huaier increased the CBL expression via microarray profiling on protein-coding genes [37]. Therefore, we supposed that CBL may be linked to the H19/miR-675-5p axis and may play a role in mediating the cytotoxic effects of Huaier extract. Fig. 6A shows that the CBL expression in breast cancer cells increased in a dose-dependent manner after exposure to Huaier extract, which was consistent with our previous microarray data [37]. As shown in Fig. 6B, the overexpression miR-675-5p decreased the expression of CBL. However, the inhibition of miR-675-5p exerted the opposite effect. Treatment with Huaier extract rescued the suppression effect of miR-675-5p on the CBL expression, which was consistent with the previous study [29]. Thus, CBL plays a critical role in Huaier extract-mediated tumor suppression. In summary, Huaier extract reduced the expression of H19 and miR-675-5p, thereby inhibiting breast cancer progression through increasing CBL (Fig. 7).

TCM has gained increasing attention because of its role in cancer prevention and treatment [38-40]. Huaier extract has been used in China for approximately 1600 years [4]; however, its anti-tumor effects were confirmed more recently [8, 13, 41]. We have previously demonstrated that Huaier extract induces autophagy and apoptosis [4, 11, 31], regulates tumor-associated macrophages [10], inhibits cancer stem cells [12], and suppresses the ERa signaling pathway [13]. However, its underlying mechanisms were not completely understood. Here, we examined the role of lncRNAs in Huaier-mediated tumor suppression. The data demonstrate that Huaier extract inhibits breast cancer progression by regulating the H19/miR-675-5p axis. To the best of our knowledge, this investigation represents the first report to demonstrate the participation of lncRNA in Huaier-mediated cytotoxicity. LncRNAs are a class of RNAs longer than 200 nucleotides that lack protein-coding potential [42]. An increasing number of lncRNAs have been identified as playing critical roles in cancer development and progression [43-45]. LncRNA H19 is located on the 11p15.5 locus and functions as an oncogene in several cancers [46-48]. Here, we initially analyzed lncRNA microarray profiles to identify candidate lncRNAs associated with the cytotoxic role of Huaier extract. H19 was among the most downregulated lncRNAs. qPCR data confirmed that Huaier extract inhibited the expression of H19 in three breast cancer cell lines. As shown in Fig. 1B, exposure to Huaier extract for 24 h significantly reduced the expression of H19. The detail mechanisms that underlie the suppression will be discovered in our future research. The knockdown of H19 sensitized breast cancer cells to Huaier extract; however, the overexpression of H19 reversed the effect of Huaier extracts. Taken together, these data show that the cytotoxic effects of Huaier extract against breast cancer cells are mediated by H19.

Of all ncRNAs, microRNAs have been the subject of substantial studies, many of which have verified their roles in breast cancer development and progression [49-52]. Increasing evidence suggests cross-regulation between microRNAs and lncRNAs [53]. LncRNAs may function as a molecular sponge or a precursor that regulates both the expression and function of miRNAs [54, 55]. miR-675-5p is derived from the first exon of H19, and its expression in gastric cancer positively correlates with H19 expression [56]. Recent studies have indicated that miR-675-5p regulates several H19-mediated effects, including intestinal epithelial barrier function, osteoblast differentiation and cancer invasion [23, 30, 35]. We previously performed a miRNA microarray profiling that compared breast cancer tissues with normal breast tissues [57]. According to the data, 21 miRNAs were upregulated, and 27 miRNAs were downregulated. MiR-675-5p was among the most upregulated miRNAs, which predicted the oncogenic effects of miR-675-5p. As shown in Fig. 4C, the overexpression of miR-675-5p reduced the inhibitory effect of Huaier extract on cell proliferation; however, the inhibition of miR-675-5p increased the effect of Huaier extract on the cell viability and apoptosis (Fig. 5B and C). These data demonstrated that miR-675-5p promoted the progression of breast cancer cells after exposure to Huaier extract. In addition, we determined that Huaier extract suppressed the expression of miR-675-5p in breast cancer cells. As shown in Fig. 4A, Huaier extract inhibited the levels of miR-675-5p in a dose-dependent manner, which indicated that Huaier extract exerted its anti-tumor effect via miR-675-5p inhibition. CBL is a direct target of miR-675-5p [29]. The current data show that Huaier extract increases the expression of CBL. Therefore, miR-675-5p mediates the function of H19 by targeting CBL. However, in a recent study, Costa et al. demonstrated that miR-675-5p exerted a metastatic phenotype through an H19 independent mechanism [58]. Thus, further research should be performed to investigate whether Huaier extract could directly affect miR-675-5p.

In summary, the expression levels of H19, miR-675-5p and CBL in breast cancer cell lines are affected by Huaier extract. The overexpression of H19 or miR-675-5p reverses the tumor-inhibitory effects of Huaier extract. However, knocking down H19 or miR-675-5p sensitizes breast cancer cells to Huaier extract. These data provide evidence that Huaier extract inhibits breast cancer cell proliferation and induces apoptosis via the lncRNA H19/miR-675-5p/CBL signaling pathway. Thus, we have identified a novel mechanism that underlies the anti-tumor effects of Huaier extract, a TCM that has potential as a clinical treatment for breast cancer.

This work was supported by the National Natural Science Foundation of China (No. 81272903; No. 81672613), Key Research and Development Program of Shandong Province (No. 2016GGE2775), and Special Support Plan for National High Level Talents (“Ten Thousand Talents Program”) to Qifeng Yang.

J.W., L.J. and Q.Y. designed and performed the study. J.W. and X.W. wrote the manuscript. T.C. analyzed the data. X.W.drew the figures. J.W., L.J. and X.W. conducted most of the experiments. All authors reviewed the manuscript.

The authors have no conflict of interest.