αO-Conotoxin GeXIVA Inhibits the Growth of Breast Cancer Cells via Interaction with α9 Nicotine Acetylcholine Receptors.

The α9-containing nicotinic acetylcholine receptor (nAChR) is increasingly emerging as a new tumor target owing to its high expression specificity in breast cancer. αO-Conotoxin GeXIVA is a potent antagonist of α9α10 nAChR. Nevertheless, the anti-tumor effect of GeXIVA on breast cancer cells remains unclear. Cell Counting Kit-8 assay was used to study the cell viability of breast cancer MDA-MD-157 cells and human normal breast epithelial cells, which were exposed to different doses of GeXIVA. Flow cytometry was adopted to detect the cell cycle arrest and apoptosis of GeXIVA in breast cancer cells. Migration ability was analyzed by wound healing assay. Western blot (WB), quantitative real-time PCR (QRT-PCR) and flow cytometry were used to determine expression of α9-nAChR. Stable MDA-MB-157 breast cancer cell line, with the α9-nAChR subunit knocked out (KO), was established using the CRISPR/Cas9 technique. GeXIVA was able to significantly inhibit the proliferation and promote apoptosis of breast cancer MDA-MB-157 cells. Furthermore, the proliferation of breast cancer MDA-MB-157 cells was inhibited by GeXIVA, which caused cell cycle arrest through downregulating α9-nAChR. GeXIVA could suppress MDA-MB-157 cell migration as well. This demonstrates that GeXIVA induced a downregulation of α9-nAChR expression, and the growth of MDA-MB-157 α9-nAChR KO cell line was inhibited as well, due to α9-nAChR deletion. GeXIVA inhibits the growth of breast cancer cell MDA-MB-157 cells in vitro and may occur in a mechanism abolishing α9-nAChR.


Introduction
Cancer is the second leading cause of death worldwide, and it was estimated to account for 9.6 million deaths in 2018 [1]. According to the statistics, breast cancer is the second most common carcinoma in the world after lung cancer, and is also the highest-incidence cancer among women. In a global context, breast cancer remains the leading cause of cancer incidence and mortality, with 2.1 million newly diagnosed cases and 630 thousand deaths in 2018 [1,2]. Recurrence and metastasis are the major cause of these deaths [2]. Additionally, genetic and reproductive risk factors play important roles in susceptibility to breast cancer [3]. Conventional therapeutic strategies usually provide limited specificity, resulting in severe side effects and toxicity to normal organisms. In contrast, targeted cancer therapy could improve the therapeutic potential of anti-tumor agents and reduce adverse side effects [4].

A Detection of the Stability of GeXIVA in Cell Culture Medium by RP-UPLC
The stability of GeXIVA was tested in cell culture medium (Figure 1). RP-UPLC (Reverse phase high-performance liquid chromatography) was used to determine the extent of the degradation of the peptides, then, a time-changing trend diagram of GeXIVA in culture media with different concentrations of serum was drawn to judge the stability of the GeXIVA in cell cultures. After 24 h incubation in different concentrations of serum (H 2 0 Control, 0% FBS, 5% FBS, 10% FBS), the remaining amounts of GeXIVA were~93%,~73%,~66% and~54% respectively. The results showed that the content of GeXIVA decreased while the serum concentration increased, and as time went on the trend continued. To assure maximum efficacy of drugs, we selected DMEM medium without serum in the process of GeXIVA-treated cells.
Mar. Drugs 2020, 18, x 3 of 14 remaining amounts of GeXIVA were ~93%, ~73%, ~66% and ~54% respectively. The results showed that the content of GeXIVA decreased while the serum concentration increased, and as time went on the trend continued. To assure maximum efficacy of drugs, we selected DMEM medium without serum in the process of GeXIVA-treated cells.

GeXIVA Affected the Growth of Breast Cancer Cells MDA-MB-157
We compared the effects of GeXIVA on human epithelial cancer cell line MDA-MB-157 and human normal mammary gland epithelial cell line HS578BST (Figure 2). Microscopic examination revealed that cell density and cell morphology were changed significantly after 24 h treatment of GeXIVA at different concentrations, and cancer cells were more sensitive to GeXIVA than normal cells. In the MDA-MB-157 breast cancer cell line, cell proliferation was remarkably decreased after treatment of GeXIVA in a concentration-and time-dependent manner ( Figure 2A). In MDA-MB-157 cells, the IC50 of 24 h for GeXIVA was approximately 78.31 µM. These results indicated that GeXIVA could effectively inhibit the proliferation of breast cancer cells MDA-MB-157, but had no toxic effects on normal cells HS578BST.

GeXIVA Affected the Growth of Breast Cancer Cells MDA-MB-157
We compared the effects of GeXIVA on human epithelial cancer cell line MDA-MB-157 and human normal mammary gland epithelial cell line HS578BST (Figure 2). Microscopic examination revealed that cell density and cell morphology were changed significantly after 24 h treatment of GeXIVA at different concentrations, and cancer cells were more sensitive to GeXIVA than normal cells. In the MDA-MB-157 breast cancer cell line, cell proliferation was remarkably decreased after treatment of GeXIVA in a concentration-and time-dependent manner ( Figure 2A). In MDA-MB-157 cells, the IC 50 of 24 h for GeXIVA was approximately 78.31 µM. These results indicated that GeXIVA could effectively inhibit the proliferation of breast cancer cells MDA-MB-157, but had no toxic effects on normal cells HS578BST.
Mar. Drugs 2020, 18, x 3 of 14 remaining amounts of GeXIVA were ~93%, ~73%, ~66% and ~54% respectively. The results showed that the content of GeXIVA decreased while the serum concentration increased, and as time went on the trend continued. To assure maximum efficacy of drugs, we selected DMEM medium without serum in the process of GeXIVA-treated cells.

GeXIVA Affected the Growth of Breast Cancer Cells MDA-MB-157
We compared the effects of GeXIVA on human epithelial cancer cell line MDA-MB-157 and human normal mammary gland epithelial cell line HS578BST (Figure 2). Microscopic examination revealed that cell density and cell morphology were changed significantly after 24 h treatment of GeXIVA at different concentrations, and cancer cells were more sensitive to GeXIVA than normal cells. In the MDA-MB-157 breast cancer cell line, cell proliferation was remarkably decreased after treatment of GeXIVA in a concentration-and time-dependent manner ( Figure 2A). In MDA-MB-157 cells, the IC50 of 24 h for GeXIVA was approximately 78.31 µM. These results indicated that GeXIVA could effectively inhibit the proliferation of breast cancer cells MDA-MB-157, but had no toxic effects on normal cells HS578BST. Values were expressed as mean ± SD of three independent assays. Statistical analysis was performed with one-way ANOVA and Tukey's test. ** p < 0.01, *** p < 0.001 indicating a significant difference between the treatments compared to medium control. (A) MDA-MB-157; (B) Hs578BST. Values were expressed as mean ± SD of three independent assays. Statistical analysis was performed with one-way ANOVA and Tukey's test. ** p < 0.01, *** p < 0.001 indicating a significant difference between the treatments compared to medium control. (A) MDA-MB-157; (B) Hs578BST.

GeXIVA Induced Apoptosis in MDA-MB-157 Cells
Apoptosis is a major cause of cancer cell growth inhibition, and previous studies have confirmed that α9-nAChR affects cell proliferation in MDA-MB-157 breast cancer cells. Herein, two-color flow cytometry with Annexin V-FITC and Propidium iodide (PI) labeling showed necrosis to be the predominant mode of cell death in MDA-MB-157 cells treated with various concentrations of GeXIVA (11.25, 22.5, 45 and 90 µM) for 24 h. The significant difference is shown in the representative scatter plots of cells treated by a series of concentrations of GeXIVA ( Figure 3A-E). The percentage of early/late apoptosis cells was summarized in Figure 3F. In control group, the proportion of early and late apoptotic cells was 0.73%. After 24 h treatment with 11.25-90 µM GeXIVA, the ratio of early and late apoptotic cells was significantly increased by up to 27.05%. These results showed that GeXIVA inhibits the growth of MDA-MB-157, probably by inducing cell apoptosis.
Apoptosis is a major cause of cancer cell growth inhibition, and previous studies have confirmed that α9-nAChR affects cell proliferation in MDA-MB-157 breast cancer cells. Herein, two-color flow cytometry with Annexin V-FITC and Propidium iodide (PI) labeling showed necrosis to be the predominant mode of cell death in MDA-MB-157 cells treated with various concentrations of GeXIVA (11.25, 22.5, 45 and 90 µM) for 24 h. The significant difference is shown in the representative scatter plots of cells treated by a series of concentrations of GeXIVA ( Figure 3A-E). The percentage of early/late apoptosis cells was summarized in Figure 3F. In control group, the proportion of early and late apoptotic cells was 0.73%. After 24 h treatment with 11.25-90 µM GeXIVA, the ratio of early and late apoptotic cells was significantly increased by up to 27.05%. These results showed that GeXIVA inhibits the growth of MDA-MB-157, probably by inducing cell apoptosis.

GeXIVA Decreased the Breast Cancer Cells Abilities of Migration
To test the potential changes in some carcinoma-associated characteristics of the MDA-MB-157 cells after GeXIVA treatment, cell migration ability was measured. The results indicated that cells migrated more slowly to close the scratched wounds after treatment with GeXIVA for 24 h compared with control group ( Figure 5). Then, the effects of different concentration on the migration distance of MDA-MB-157 cells were analyzed statistically. This indicated that GeXIVA had inhibition effects on the migration of MDA-MB-157 cells; following the increasing concentrations, cells migration was retarded (Table 1).

GeXIVA Decreased the Breast Cancer Cells Abilities of Migration
To test the potential changes in some carcinoma-associated characteristics of the MDA-MB-157 cells after GeXIVA treatment, cell migration ability was measured. The results indicated that cells migrated more slowly to close the scratched wounds after treatment with GeXIVA for 24 h compared with control group ( Figure 5). Then, the effects of different concentration on the migration distance of MDA-MB-157 cells were analyzed statistically. This indicated that GeXIVA had inhibition effects on the migration of MDA-MB-157 cells; following the increasing concentrations, cells migration was retarded (Table 1).

GeXIVA Affected Breast Cancer Cell Line MDA-MB-157 Proliferation Through The Inhibition of α9-nAChR-mediated Signals
To test the effect of α9-nAChR while GeXIVA inhibiting breast cancer cells, a stable cell line with α9-nAChR gene knock-out was established. The α9-nAChR CRISPR/Cas9 KO plasmid and HDR plasmid were transfected into MDA-MB-157 cells and the transfected cells were selected under the suppression of puromycin. The positive cells were confirmed by detection of the red fluorescent protein (RFP) via fluorescent microscopy ( Figure S1). Real-time PCR showed that transfection of MDA-MB-157 cells with α9-nAChR CRISPR/Cas9 KO and HDR plasmid led to more than a 10-fold decrease in α9-nAChR mRNA expression, while the expression of α3 and β4 nAChR subunits mRNA(as control) had no differences ( Figure 6A). Flow cytometry showed that knockout of the α9-nAChR gene greatly reduced the expression of functional α9-nAChRs on the cell surface ( Figure S2 and Figure 6B). We also applied Western blotting analysis to confirm the expression of α9-nAChR  Note: The capital letters A, B and C represent the 95% confidence intervals. One-way ANOVA was performed on the same row (same times) and different columns (different concentrations) in the table. The lowercase letters a, b and c represent the 95% confidence intervals. One-way ANOVA was performed on the same columns (same concentrations) and different columns (different times) in the table. Values with same superscript letters in the same line showed no significant differences (p > 0.05), those with different letters showed significant or extreme differences (p < 0.05).

GeXIVA Affected Breast Cancer Cell Line MDA-MB-157 Proliferation through the Inhibition of α9-nAChR-mediated Signals
To test the effect of α9-nAChR while GeXIVA inhibiting breast cancer cells, a stable cell line with α9-nAChR gene knock-out was established. The α9-nAChR CRISPR/Cas9 KO plasmid and HDR plasmid were transfected into MDA-MB-157 cells and the transfected cells were selected under the suppression of puromycin. The positive cells were confirmed by detection of the red fluorescent protein (RFP) via fluorescent microscopy ( Figure S1). Real-time PCR showed that transfection of MDA-MB-157 cells with α9-nAChR CRISPR/Cas9 KO and HDR plasmid led to more than a 10-fold decrease in α9-nAChR mRNA expression, while the expression of α3 and β4 nAChR subunits mRNA(as control) had no differences ( Figure 6A). Flow cytometry showed that knockout of the α9-nAChR gene greatly reduced the expression of functional α9-nAChRs on the cell surface ( Figure S2 and Figure 6B). We also applied Western blotting analysis to confirm the expression of α9-nAChR protein was significantly silenced in knocked out cell lines (MDA-MB-157 α9-nAChR KO) ( Figure 6C). protein was significantly silenced in knocked out cell lines (MDA-MB-157 α9-nAChR KO) ( Figure  6C).      To further test the possible influence of GeXIVA on the expression of α9-nAChR, breast cancer cell line MDA-MB-157 was treated with GeXIVA for 24 h. Cells were harvested and subjected to total RNA extraction. qRT-PCR assays for the α9-nAChR showed significantly reduced transcriptions after cells treated with GeXIVA; however, β4-nAChR was almost unchanged before and after the treatment ( Figure 8A). The expression of α9-nAChR was determined by indirect fluorescence staining and flow cytometry analysis, the results revealed a significant decrease in the level of α9-nAChR, after 24 h exposure of cells to GeXIVA at concentrations of 22.5 µM and higher ( Figure 8B). mean ± SD of three independent assays. * p < 0.001 for a significant difference in the MDA-MB-157 α9-nAChR KO cell line in comparison to the MDA-MB-157 cell line.
To further test the possible influence of GeXIVA on the expression of α9-nAChR, breast cancer cell line MDA-MB-157 was treated with GeXIVA for 24 h. Cells were harvested and subjected to total RNA extraction. qRT-PCR assays for the α9-nAChR showed significantly reduced transcriptions after cells treated with GeXIVA; however, β4-nAChR was almost unchanged before and after the treatment ( Figure 8A). The expression of α9-nAChR was determined by indirect fluorescence staining and flow cytometry analysis, the results revealed a significant decrease in the level of α9-nAChR, after 24 h exposure of cells to GeXIVA at concentrations of 22.5 µM and higher ( Figure 8B).

Discussion
The α9-nAChR subunit, initially discovered in hair cells of the inner ear, can form a homopentamer or assemble into a hetero-pentamer with α10 or α5-nAChR subunits [25,26]. Including α9-nAChR, almost all of nAChRs are expressed not only in neuronal systems, but also in numerous nonneuronal tissues cells such as skin, pancreas and lung, suggesting that nAChRs may have roles in other biological processes in addition to synaptic transmission [8]. It is reported that α9-nAChR is involved in promoting cancer cell proliferation, angiogenesis, cancer metastasis and apoptosis suppression during carcinogenesis in response to tumor microenvironments [7,11,13,27,28]. In a previous study, we determined the expression profile of the α9-nAChR subunit in human breast cancer cell lines and found that α9-nAChR was expressed differently in breast cancer cells. This suggests that α9-nAChR may become an effective target for breast cancer treatment [29].
GeXIVA, a Conus peptide, contains 28 amino acids and four Cys residues which have three disulfide bonds [16]. The original study of GeXIVA on α9α10 nAChR expressed in Xenopus oocytes revealed that GeXIVA by itself is able to block the α9α10 nAChR current of ACh-evoked currents completely at a 100 nM concentration, and this block was rapidly reversible [16]. GeXIVA is a potent and selective antagonist of α9α10 nAChR subtype [16,30]. In vivo, it also displayed potent alleviation of neuropathic pain in several rat models [18,20]. In vitro, GeXIVA showed an antitumor effect [22]. Therefore, it is worth further investigating GeXIVA as a potential therapeutic agent against breast cancer, etc.
Previous research has indicated that GeXIVA is unstable in serum [30]. In this study, we decided to evaluate the stability of GeXIVA in cell culture medium, and the ability of the GeXIVA to control

Discussion
The α9-nAChR subunit, initially discovered in hair cells of the inner ear, can form a homo-pentamer or assemble into a hetero-pentamer with α10 or α5-nAChR subunits [25,26]. Including α9-nAChR, almost all of nAChRs are expressed not only in neuronal systems, but also in numerous non-neuronal tissues cells such as skin, pancreas and lung, suggesting that nAChRs may have roles in other biological processes in addition to synaptic transmission [8]. It is reported that α9-nAChR is involved in promoting cancer cell proliferation, angiogenesis, cancer metastasis and apoptosis suppression during carcinogenesis in response to tumor microenvironments [7,11,13,27,28]. In a previous study, we determined the expression profile of the α9-nAChR subunit in human breast cancer cell lines and found that α9-nAChR was expressed differently in breast cancer cells. This suggests that α9-nAChR may become an effective target for breast cancer treatment [29].
GeXIVA, a Conus peptide, contains 28 amino acids and four Cys residues which have three disulfide bonds [16]. The original study of GeXIVA on α9α10 nAChR expressed in Xenopus oocytes revealed that GeXIVA by itself is able to block the α9α10 nAChR current of ACh-evoked currents completely at a 100 nM concentration, and this block was rapidly reversible [16]. GeXIVA is a potent and selective antagonist of α9α10 nAChR subtype [16,30]. In vivo, it also displayed potent alleviation of neuropathic pain in several rat models [18,20]. In vitro, GeXIVA showed an antitumor effect [22]. Therefore, it is worth further investigating GeXIVA as a potential therapeutic agent against breast cancer, etc.
Previous research has indicated that GeXIVA is unstable in serum [30]. In this study, we decided to evaluate the stability of GeXIVA in cell culture medium, and the ability of the GeXIVA to control the growth of MDA-MB-157 cells. The remaining amount of GeXIVA reached~80% in serum-free DMEM medium after 48 h, so, in the next steps, the cells were co-incubated with GeXIVA in serum-free DMEM medium to assure maximum efficacy. Prolonged 24 h incubation of MDA-MB-157 cells with GeXIVA resulted in the pronounced concentration-dependent inhibition of the cell growth with IC 50 of 78 µM. Moreover, GeXIVA had an obvious inhibitory effect on breast cancer cell line MDA-MB-157 compared with normal breast epithelia cell line HS578BST in vitro. This means that the same doses of GeXIVA could kill more cancer cells than normal cells. In other words, GeXIVA is less toxic to normal cells than cancer cells. Moreover, apoptosis analysis demonstrated that the apoptosis rates in the drug treatment group were significantly higher than in the control group. This result indicated that GeXIVA inhibit the growth of MDA-MB-157, probably by inducing cell apoptosis. We also demonstrated that inhibition of α9-nAChR by GeXIVA could inhibit MDA-MB-157 cancer cell proliferation through the induction of G0/G1 arrest. Metastasis is a major obstacle in clinical cancer therapy [31]. Previous studies have indicated that nicotine can enhance the migratory abilities of cancer cells [31][32][33]. However, some studies reported the antagonists of nAChRs could reduce the progression of cancer cells [34]. Here, we found that GeXIVA could abolish the abilities of cancer migration.
Previous studies have shown that natural polyphenol compounds can reduce nicotine-mediated carcinogenic effects by inhibiting the expression of α9-nAChR [13,15,35,36]. It is reported that α9-containing nAChRs may play an important role in breast cancer [11,13], but we knew little about the antitumor effect of GeXIVA previously. To determine whether the anti-proliferative effect of GeXIVA is associated with α9-nAChRs, we blocked expression of this receptor by CRISPR/Cas9 knockdown technique. The anti-proliferative effects were profoundly abolished by GeXIVA treatment in α9-nAChR knockdown MDA-MB-157 cells compared with wild-type cells. Thus, we can conclude that the GeXIVA antiproliferative effect on MDA-MB-157 cells is a result of the protein interaction of α9-nAChR with GeXIVA. Therefore, the GeXIVA inhibitory effects might be through downregulating of the protein and gene expression of α9-nAChR. More experiments are needed to gain insight into the underlying molecular mechanisms.

Cell Culture
Human breast cancer cell line MDA-MB-157 and human normal mammary gland epithelial cell line HS578BST were purchased from the Kunming Institute of Zoology (Yunnan, China). To make the complete growth medium, all the cells were maintained in Dulbecco's modified eagle medium (DMEM), supplemented with fetal bovine serum (FBS) to a final concentration of 10%, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 • C in an atmosphere of 5% CO 2 .

Cell Culture Medium Stability
Medium stability assay was performed in cell culture media with different concentrations of serum for GeXIVA [1,2]. With setting experimental group (DMEM + GeXIVA, 5% FBS + DMEM + GeXIVA, 10% FBS + DMEM + GeXIVA) and comparison group (GeXIVA was incorporated into the aqueous phase), the sample of each group was placed in an incubator at 37 • C with 5% CO 2 atmosphere and 100% relative humidity for 0, 12, 24 and 48 h. 30 µL triplicate aliquots were taken out at different time points. Each serum aliquot was quenched with 30 µL of 6 M urea and incubated for 10 min at 4 • C. Then, each serum aliquot was treated with 30 µL of 20% trichloroacetic acid (TCA) for another 10 min at 4 • C to precipitate serum proteins. Precipitated serum proteins were then spun down at 14,000 g [19]. Qualitative and quantitative analysis were performed by RP-UPLC, through drawing time-changing trend diagrams of GeXIVA in culture media with different concentrations of serum to judge the stability of the GeXIVA in cell cultures.

Cell Counting Kit-8 Assay (CCK-8)
To study effects of GeXIVA on cell growth, 5 × 10 3 cells were seeded in 96-well culture plates. After 24 h, the cells were incubated in a complete medium at 37 • C for adhesion. Thereafter, GeXIVA (dissolved in water) was added to cells at different concentrations (11.25, 22.5, 45, 90 and 180 µM) and grown during 24 h in medium without FBS at 37 • C, 5% CO 2 . The medium was removed and the CCK-8 solution was added to cell wells for 1 h at 37 • C. The absorbance at 450 nm (OD 450) each well was obtained using a microplate reader. Do not add any compounds to cells as control. The blank group was supplemented with culture medium and contained no cells.
Cell viability was measured with a commercial Cell Counting Kit-8. Briefly, 2 × 10 3 cells were seeded onto 96-well plates and the CCK-8 solution was directly added to the cell wells and incubated at 37 • C for 1 h. The absorbance at 450 nm (OD 450 ) for each well was obtained by using a microplate reader with a background control as blank. The cell viability was expressed as the percentage of the untreated control.

Migration Assay
Wound assay was used to evaluate the migration ability of cells. A total of 1 × 10 5 cells were seeded in a 12-well plate and grew to confluence followed by scratching the monolayer cells with a 200 µL pipette tip to create a wound. After the medium and floating cells were removed, the cells were rinsed with phosphate-buffered saline three times. The cells were treated with different concentrations of GeXIVA and incubated for 24 h in medium without FBS at 37 • C, 5% CO 2 . Plates were washed to remove floating cells and debris, then cell migration images were taken at 0, 24 and 48 hours after drug treatment. Cells without addition of any compounds were used as a control. Each testing group contained at least three independent wells.

Flow Cytometry
Apoptosis assay was detected with an Annexin V-FITC/PI Apoptosis Detection Kit (KeyGENBioTECH, Nanjing, China) and flow cytometry analysis. A total of 2 × 10 5 cells were seeded onto 6-well plates. After a 24 h incubation for adhesion, the cells were treated with GeXIVA for 24 h. Cells were collected by trypsinization and dual stained with Annexin V-FITC and PI for 15 min at room temperature in dark. For each sample, data from approximately 1 × 10 4 cells were recorded in list mode on logarithmic scales. Apoptosis and necrosis were analyzed by quadrant statistics on PI-negative, Annexin V-positive cells and cells positive for both, respectively.
Cells were seeded in 6-well culture plates and incubated with different concentrations of GeXIVA for 24 h. Then the cells were detached from the wells by Trypsin Solution (Without EDTA), washed with PBS containing 3% bovine serum albumin (BSA), and fixed in pre-cooling 70% ethanol for 4 h. After fixation, the cells were washed twice by 3% BAS-PBS, resuspended in 10% RNase-PI staining solution, and analyzed by Guava easyCyte TM flow cytometer.
The expression of α9-nAChR in cells was determined by indirect fluorescence staining and flow cytometry analysis. The treated cells were harvested and washed with PBS twice, fixed with 4% PFA for 20 min and washed twice by centrifugation at 1000 rpm for 5 min each time. Cells were suspended in 0.1% Triton X-100 in 1 × PBS buffer for 10 min and washed twice with PBS by centrifugation at 1300 rpm for 5 min each time. The cells were incubated with 3% BSA in 1 × PBS buffer for 30 min. Primary antibody at an appropriate dilution was added and incubated for 2 hr at room temperature, then washed and incubated with secondary antibody (FITC-conjugated Goat anti-rabbit Ig (G+L)) for 1 hr at room temperature. Finally, the cells were washed twice and re-suspended in 300 µL 1 × PBS and analyzed the expression of α9-nAChRs by flow cytometry. The primary antibody used was as follows: CHRNA9 Rabbit Polychonal Antibody (26025-1-AP, proteintech, Chicago, IL, USA).

Generation of Stable α9-nAChR-KO Cell Lines
MDA-MB-157 cells were plated in a 6-well culture plate at a density of 1.5-2.5 × 10 5 cells in 3 mL antibiotic-free standard growth medium per well. After 24 h, the cells were co-transfected with α9-nAChR CRISPR/Cas9 Knock out (KO) plasmid (Santa Cruz, sc-402753-KO-2, CA, USA) and α9-nAChR homology-directed repair (HDR) plasmid (Santa Cruz, sc-402753-HDR-2, CA, USA). After incubation for 24 h at 37 • C in a CO 2 incubator, the plasmid-containing medium was removed and fresh growth medium containing 10% serum was added in the presence of 6 µg/mL of puromycin. Selected cells, for a minimum of 3-5 days, approximately every 2-3 days, were aspirated and replaced with freshly prepared selective medium. This resulted in the establishment of MDA-MB-157 KO cells in which α9-nAChR gene expression was knocked out.

RNA Isolation and Quantitative Real-time PCR
Total RNA of cells was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

Protein Extraction and Western Blot Analysis
The cultured cells were washed with cold phosphate buffer saline (PBS) three times and harvested using a cell lysis buffer containing protease inhibitors PMSF (Solarbio Life Sciences, Beijing, CHN). Equal amounts of protein from control and treated cell lysates were separated using a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel under reducing conditions and transferred onto polyvinylidene fluoride (PVDF) membranes (Solarbio Life Sciences, Beijing, China), which were subsequently probed with primary antibodies (AChRα9, Santa Cruz, sc-293282, CA, USA). In all Western blots, membranes were additionally probed with an antibody for GAPDH (Santa Cruz, sc-47724, CA, USA) to ensure equal loading of protein between samples. Horseradish peroxidase-conjugated secondary antibodies (m-IgGκ BP-HRP, Santa Cruz, sc-516102, CA, USA) were used with enhanced chemoluminescence reagent (Biosharp, Guangzhou, China) to visualize the protein bands. Images of the films were captured using the Alpha FluorChem E (ProteinSimple, CA, USA).

Statistical Analysis
Three to five independent repeats were conducted for all experiments. Error bars represent these repeats. Statistical comparisons between groups were performed using ANOVA (Prism GraphPad Software and SPSS 17.0). A Student's t-test was used and a p-value < 0.05 was considered significant. Analysis was performed with the SPSS software package (version Version 17.0).

Conclusions
In this study, the anti-proliferative activity of GeXIVA on breast cancer cells was confirmed in vitro, which deserves further study as a potential agent for anti-cancer therapy. Our results demonstrated that α9-nAChR played a key role in breast cancer cells, which was differentially over-expressed in breast cancer cell lines than in normal cells. As an antagonist of α9-nAChR, αO-conotoxin GeXIVA induced cell apoptosis and inhibited the proliferation of breast cancer MDA-MB-157 cells. On the other hand, we found that the proliferation of breast cancer cells was blocked by GeXIVA through S-phase cell cycle arrest. Moreover, GeXIVA induced a downregulation of α9-nAChR expression, and the anti-tumor effect of GeXIVA was abolished by α9-nAChR silencing. These findings provide obvious molecular evidence that GeXIVA is effective drug lead for targeting therapy of breast cancer, which can inhibit the growth of breast cancer cells.

Conflicts of Interest:
The authors declare no conflict of interest.