Ginsenoside Rh 2 differentially Mediates microRNA Expression to Prevent Chemoresistance of Breast Cancer

Breast cancer is one of the most common cancer in women, drug-resistance and toxicity are the leading causes that limit the success of aggressive breast cancer therapy (Deng et al., 2013; Liu et al., 2013; Gong et al., 2014; Wang et al., 2014). Ginsenoside Rh2 has been used for more than 2, 000 years in oriental countries. It is the major pharmacological active component of ginseng that includes a similar basic structure, which consists of a gonane steroid nucleus that holds 17 carbon atoms settled in a four-trans-ring (Zhang et al., 2012) (Figure 1). As a potent novel class phytoestrogen that exhibits a multi-target, multisystem and a significant low toxicity, Studies shows that G-Rh2 treatment can effect human


Introduction
Breast cancer is one of the most common cancer in women, drug-resistance and toxicity are the leading causes that limit the success of aggressive breast cancer therapy (Deng et al., 2013;Liu et al., 2013;Gong et al., 2014;Wang et al., 2014).Ginsenoside Rh2 has been used for more than 2, 000 years in oriental countries.It is the major pharmacological active component of ginseng that includes a similar basic structure, which consists of a gonane steroid nucleus that holds 17 carbon atoms settled in a four-trans-ring (Zhang et al., 2012) (Figure 1).As a potent novel class phytoestrogen that exhibits a multi-target, multisystem and a significant low toxicity, Studies shows that G-Rh2 treatment can effect human RESEARCH ARTICLE
MicroRNAs (miRNAs) are involved in the regulation of various biological processes: tumorigenesis, development, cellular migration, apoptosis, signal transduction and carcinogenesis etc.Recently, increasing evidence revealed that miRNAs act an important role in anticancer drug resistance and miRNAs expression profiling can be related with the development of anticancer drug-resistance, such as miR-181a, miR-34a, miR-222, etc.There are several mechanisms have been shown to be targeted by miRNAs in drug-resistant breast cancer such as DNA repair (Chen et al., 2012;Ratert et al., 2013;Chan et al., 2013;Kutanzi et al., 2011).
Some studies showed G-Rh2 mediates changes in the miRNA expression profile of human lung cancer A549 cells.It also inhibits glioma cell proliferation by targeting miRNA-128 (Wu et al., 2011;Jiao et al., 2013).Adriacin (Adr) and Docetaxel (Doc) are two of the most common chemotherapeutic drugs in BCA.There has been no report to demonstrate the mechanisms of G-Rh2 to reverse drug-resistance in BCA from miRNA levels.Our team conducted the profiling of miRNAs expression Figure 1.The Structure of G-Rh2 in human BCA Adr-resistant MCF-7 cells (MCF-7/Adr) and Doc-resistant MCF-7 cells (MCF-7/Doc), we are the first to report the findings and testify that 5 miRNA is related to drug-resistance (An et al., 2013;Zhong et al., 2013).We propose a logical hypothesis: G-Rh2 could mediate the drug-resistance specific miRNA, which could then regulate corresponding target genes through the gene regulatory network --cutting-off the process and development of drug-resistant tumor cells and improving the treatment effect.

Cell culture
Human breast cancer cell line MCF-7 was purchased from ATCC (Rockville, MD).The resistant sublines, selected at 100nm Docetaxel (MCF-7/Doc) or at 500nm Adriamycin (MCF-7/Adr), were successfully established from human breast cancer parental cell line MCF-7 by exposing MCF-7 to gradually increasing concentrations of Doc or Adr in vitro in our laboratory.All cell lines were cultured in DMEM high glucose (HyClone), supplemented with 10% fetal bovine serum (Gibco) in a humidified atmosphere containing 5% CO 2 at 37°C.

Cell proliferation assay
Cells were seeded into 96-well plates (6×10 3 cells/ well), treated with different concentrations of G-Rh2 and incubated for 24 hours.Then 20µl of MTT solution (5mg/ ml) was added to each well and the cells were maintained in a humidified atmosphere for 3-4 hours at 37°C.The MTT-containing medium was removed and 150lL of DMSO (AMRESCO, America) was added to each well; each experiment was performed in quAdruplicate.The absorbance was measured at 570 nm using CliniBio128 (ASYS-Hitech, Austria)

Real-time quantitative PCR
Total RNA was extracted using TRIzol ® Reagent (Invitrogen, Carlsbad, CA); afterwards a reverse transcription was done using TaqMan ® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA); mature miRNA was spotted using TaqMan ® MicroRNA Assay (Applied Biosystems, Foster City, CA); all procedures were done according to manufacturer's instructions.Relative expression levers were calculated using the ΔΔCt method, normalized with endogenous control and was presented along with negative control.Clustal X software was used to analyze measured miRNA sequences; the sequences were similarity not high in a reverse transcription system.All reverse transcriptions and PCR assays were presented in triplicate.

MiRNA microarray
Total RNA including miRNAs was extracted using MirVana miRNA Isolation Kit (Ambion, AM1560).The concentration and quality of the RNA were measured by the UV absorbance at 260 and 280 nm (260/280 nm) on Nanodrop 2000 spectrophotometry (Thermo Scientific) and by formaldehyde denaturing gel electrophoresis.
The RNA was labeled using the FlashTag RNA Labeling Kit (Genishere), according to Affymetrix manufacturer's recommendations.Hybridization and washing were performed using the Affymetrix Fluidics Station 450 and Hybridization Oven 640 under standard conditions.Image processing was conducted using the Affymetrix GeneArray 3000 scanner.The Affymetrix GeneChip miRNA 2.0 Array contains 15, 644 probe sets including 1105 human mature miRNAs.The raw data was treated using miRNA QC tool software (Affymetrix).The data output was received in Excel spreadsheets containing the normalized micro-RNA expression profiles.Differentially expressed miRNAs were filtered to exclude those changes less than 2.0-fold compared with MCF-7/S.

Establish stable expression of green fluorescent protein in breast cancer cell lines.
In order to facilitate observation, a recombinant lentiviral vector stable expression of green fluorescent protein was used in breast cancer cell lines.MCF-7/Doc or Adr and MCF-7 cells in logarithmic growth phase were seeded in 24-well plates ( 3×10 4 cells/well) after digestion until cell fusion becomes 50% to 60%; which was carried out in accordance with reagent instruction lentivirus infections.After 72 hours, the collected fluorescence was stronger in each well, which resurfaced after digestion was covered with 50% to 60%; added 2μg/ml puromycin to screen.After one week, 1μg/ml puromycin was added to maintain the pressure; three generations were continued to be cultured to observe the expression of the green fluorescence.Then, the green fluorescence MCF-7 cells, MCF-7/Adr cells and MCF-7/Doc cells were inoculated for 24 hours in equal amounts with G-Rh2 intervention; then afterwards intervened with Doc and Adr for 24 hours.

Western blot
Total protein was extracted and lysed in the RIPA buffer (Beyotime, Jiangsu, China).Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to the polyvinylidene difluoride membranes (Sigma, Germany).After blocking with 5% skim milk, the membranes were incubated with primary antibodies against human Bax (1:500, Santa Cruz, CA) overnight at 4°C; after washing with TBS, the horseradish peroxidase-conjugated secondary antibody (Kangwei Ltd., Beijing, China) was further incubated; the protein band was visualized by Chemiluminescence with pierce ECL kits (Millipore, Billerica, MA).β-actin (1:4000, Bioworld, MN) was used as an internal load to normalize the expression patterns of each sample.Three separate experiments were performed to show the protein expression.

Statistical analysis
All experiments were performed in triplicate and a representative data was shown from three separate experiments.A statistical analysis was performed using a t-test or One-way ANOVA and Spearman rank test with a SPSS 16.0 statistic.All experiments were performed in triplicate;a level of p<0.05 was considered statistically significant.

G-Rh2 treatment decreases the viability of breast cancer cells
We utilized different ginsenoside Rh2 concentrations to determine its antiproliferative effects.As shown in Figure 2 ABC, the viability of MCF-7, MCF-7/Doc and MCF-7/Adr cells gradually decreased with the increase of G-Rh2 concentration in a dose-dependent manner.MCF-7 cells with G-Rh2 (40 μM/l) treatment; MCF-7/Adr cells with G-Rh2 (80 μM/l) treatment and MCF-7/Doc cells with G-Rh2 (80 μM/l) treatment for 24 hours compared to MCF-7, MCF-7/Adr and MCF-7/Doc dramatically decreased cell viability with statistical significance; In particular, MCF-7/Doc and MCF-7/Adr cells exhibited a relatively high sensitivity to G-Rh2; indicating that G-Rh2 has a strong anti-proliferative activity in MCF-7 cell lines.
As shown in Figure 3, The three cells with G-Rh2 (80μM/l) treatment for 0, 12, 24, 48 hours dramatically decreased cell viability, In a word, it shows G-Rh2 treatment decreases the viability of breast cancer cells in a dose and time-dependent manner.

G-Rh2 can reverse drug resistance
MCF-7, MCF-7/Doc, MCF-7/Adr, MCF-7/Doc or MCF-7/Adr with G-Rh2 intervention divided into two groups: the Doc or Adr treated and untreated group, by Doc and Adr, remnants of green fluorescent cells had significant differences between MCF-7/Doc, MCF-7/Adr and MCF-7 cells; proving the existence of MCF-7/Doc and MCF-7/Adr drug resistance.However, the residual green fluorescent cells of the MCF-7/Doc and MCF-7/ Adr cells with G-Rh2 intervention coped with the drug was significantly reduced more than the two cells without intervention; suggesting that drug resistance can be reversed by G-Rh2 (Figure 5AB).

G-Rh2 treatment alters the expression of Bax protein in breast cancer cells
As we all know, the gene Bax expression has a role in inducing apoptosis and anti-chemoresistance, study shows some miRNAs related to Bax in it, such as miR-34a, miR-511, miR-365, miR-214, etc. we utilized western blot to detect Bax expresses.As shown in Figure 7AB, the results revealed the Bax expression in MCF-7/Doc and MCF-7/ Adr cells significantly decreased, compared with MCF-7 cells; however, the Bax expression in MCF-7/Doc and MCF-7/Adr cells with G-Rh2 intervention significantly increased, compared with the two cells with intervention.

Discussion
One important factor which limits the application of advanced breast cancer treatment is acquired drugresistance.G-Rh2 has showed a great diversity of biological activities, including immunomodulatory effects, antioxidative, anti-inflammatory, anti-tumor and vasorelaxing activities; In this study, we proved G-Rh2 could mediate the MDR specific miRNA, then regulate the corresponding target genes --reversing the drug-resistant of BCA cells.
We researched the miRNA expression profiles and explored miRNAs that could be associated to drug resistance.There are several mechanisms have recently  doi.org/10.7314/APJCP.2015doi.org/10.7314/APJCP. .16.3.1105Ginsenoside Rh2 Prevents Chemoresistance of Breast Cancer been shown to be targeted by miRNAs in drug-resistant breast cancer, including: decreased intracellular drug concentrations; mediated by drug transporters and metabolic enzymes; impaired cellular responses that affect cell cycle arrest, apoptosis; DNA repair and alterations in the availability of drug targets.
To further verify whether the differential miRNAs expression has a major influence in preventing the process of acquiring drug resistance with G-Rh2 intervention, We tested the five drug-resistance specific miRNAs from both MCF-7/Adr and MCF-7/Doc cells.The results showed that only miR-222, miR-34a and miR-29a changed the characteristics of drug resistant breast cancer cells to Doc and ADR, out of the five selected miRNAs.We therefore conclude that G-Rh2 could mediate the miRNA expression to reduce the drug resistance of breast cancer.
The residual green fluorescent cells of the G-Rh2mediated cells groups was lesser than another groups; suggesting that anticancer effect and reverse the drug resistance of G-Rh2 has a synergistic effect.
MDR is often related with lowerexpression of Bax, leading to chemotherapeutic failure.Study shows miR-34a regulatory ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells.In order to further explore a possible role of Bax in the effect of G-Rh2 on reversing drug resistance, we assessed Bax expression in beast cancer cells.Our results indicate that G-Rh2 effectively activated drug-resistant BCA cells to Doc and Adr through upregulation of Bax.
In summary, G-Rh2 causes a strong antichemoresistance effect in BCA cells, therefore, miRNA plays a major influence in the G-Rh2-mediated effect, particularly, the three specified drug-resistant miRNAs.Moreover, this study provides a novel insight of the molecular mechanisms of G-Rh2 --reversing drugresistance by using the latest technology to detect miRNA communication mechanisms.However, this study still has some shortcomings, if animal models are embraced into our study --through pharmaceutical interventions on the above subjects --we could further explore how Chinese medicine can inverse and inhibit chemotherapy resistance in BCA from a clinical perspective; which could make our conclusions more persuasive.