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Abstract

BACKGROUND:

ARHGDIB, a Rho GDP dissociation inhibitor protein, has been reported playing critical roles in regulation of multiple biological responses. However, whether ARHGDIB serves as a valuable biomarker in cancer is little known so far, especially in breast cancer.

OBJECTIVE:

In this study, we aimed to investigate the importance of ARHGDIB in breast cancer, including but not limited to biomarker-like role, as well as potential mechanisms.

METHODS:

Total 100 breast cancer samples and 100 benign breast disease samples were enrolled and underwent detailed pathological assessment and IHC analysis. Human breast cancer cell lines and epithelial cell line were subjected to siRNA-mediated knock-down, RT-qPCR, western blot, MTT staining, cell cycle assay, transwell analysis respectively.

RESULTS:

We observed the expression of ARHGDIB is significantly higher in human breast cancer tissues compared with the benign tissues. ARHGDIB expression was positively correlated with tumor size, lymph node metastasis and TNM stage in breast cancer patients. Moreover, ARHGDIB depletion decreased proliferation, migration and invasion of breast cancer cells. Furthermore, we found ARHGDIB mediated epithelial-mesenchymal transition, and MMP2 is the key downstream effector of ARHGDIB.

CONCLUSIONS:

Hence, our results suggested the significance and predictive role of ARHGDIB in breast cancer. High expression of ARHGDIB indicated the poor prognosis for breast cancer patients.

Keywords

ARHGDIB biomarker breast cancer EMT prognosis

1. Introduction

Breast cancer, one of the most common malignant diseases, is one of the leading causes of cancer death for woman worldwide. According to the report, from 2005 to 2014, overall breast cancer incidence rates increased among Asian/Pacific Islander (1.7% per year) women [1]. As a result of the increasing longevity, increased urbanization and the adoption of western lifestyles, the incidence of breast cancer is increasing in the developing world [2]. Metastatic dissemination of cancer cells from the primary tumor and their spread to distant sites in the body is the leading cause of mortality in breast cancer patients, which is often associated with epithelial-mesenchymal transition (EMT) mediated metastasis [3, 4]. It has been shown that EMT is regulated by transcription factor Snai1 [5]. Despite kinds of therapies for the clinical treatment of breast cancer, such as radiation, chemotherapy and surgical resection, 41070 women with breast cancer died in the United States in 2017 [6]. However, the molecular mechanisms underlying the recurrence and metastasis of breast cancer remain to be elucidated. Thus, new biomarkers for the diagnosis and prognosis of breast cancer are required.

Rho GDP dissociation inhibitors (RhoGDIs), the pivotal regulators of Rho GTPase function, with the character of forming a complex with Rho GTPase, regulates their nucleotide exchange and membrane association [7, 8]. ARHGDIB is one of the most critical members in RhoGDIs. It is mainly located in hematopoietic, endothelial, and epithelial cells [9] and involved in a variety of cellular functions, including signaling, secretion, cytoskeletal organization and proliferation [10]. Previous studies have reported the dysregulation of ARHGDIB in a variety of human tumors, while the role of ARHGDIB appears as controversial in tumor development and progression. For example, low expression of ARHGDIB was reported in bladder cancer and Hodgkin’s lymphoma and was identified as a presumptive metastatic suppressor [11, 12]. On the other side, overexpression of ARHGDIB was observed in pancreatic cancer and gastric cancer [13, 14]. Furthermore, few studies conducted in the role of ARHGDIB in breast cancer [15]. The clinical significance of the expression of in breast cancer patients and the underlying mechanism remain to be elucidated. In this study, we investigated the role of ARHGDIB in cell proliferation and metastasis in breast cancer and the clinical significance in breast cancer patients. Our study suggest ARHGDIB might play an important role in breast cancer progression.

2. Results

2.1 Expression of ARHGDIB in clinical breast tissue specimens and cell lines

To address the role of ARHGDIB on the development and progression of breast cancer, we first detected the expression of ARHGDIB in clinical breast specimens by IHC. ARHGDIB was predominantly expressed at the cytoplasm of breast epithelium, and the expression of ARHGDIB protein was frequently observed stronger in breast cancer tissues, in comparison to benign breast disease tissues (Fig. 1A). Moreover, ARHGDIB protein expression was observed in 74 of the 100 (74%) breast cancer specimens which was significantly higher than benign breast disease tissue ( $P<$ 0.01, Table 1). Furthermore, we analyzed the expression levels of ARHGDIB in multiple breast cell lines by western blot. The results showed ARHGDIB was poorly expressed or undetectable in human breast benign derived epithelial cells (MCF-10A), ER ( $+$ ) breast cancer cell lines (MCF-7 and T47D), as well as HER2 ( $+$ ) breast cancer cell lines (BT474). In contrast, it was strongly expressed in the triple negative breast cancer cell lines (MDA-MB-231 and SUM159). Of note, MDA-MB-231, a highly invasive breast cancer cell line, exhibited the most abundant ARHGDIB expression. These observations suggested that ARHGDIB expression may be associated with high aggressive behavior in breast cancer cells (Fig. 1B).

Table 1
Expression of ARHGDIB in breast cancer samples and benign tissues

Group	$n$	ARHGDIB expression		$P$ value
		Negative, $n$ (%)	Positive, $n$ (%)
Breast cancer tissue	100	26 (26%)	74 (74%)	$<$ 0.001
Benign breast disease	100	61 (61%)	39 (39%)

Figure 1.

Differential expression of ARHGDIB in breast clinical samples and cell lines. A. Expression of ARHGDIB protein in breast cancer tissues and benign breast disease tissues was detected using IHC. B. Cell lysates, as indicated, were subjected to western blot analysis using antibodies specific for ARHGDIB. $\beta$ -actin was used as loading control for cell lysates.

Table 2

Correlation of ARHGDIB expression and clinicopathological parameters in breast cancer patients

Parameter	$n$	ARHGDIB expression	$P$ value
		Positive, $n$ (%)
Age (years)
$\leqslant$ 50	58	44 (76%)	0.618
$>$ 50	42	30 (71%)
Tumor size
$\leqslant$ 2	17	6 (35%)
$>$ 2, $\leqslant$ 5	64	51 (80%)	0.001
$>$ 5	19	15 (80%)
Lymph node metastasis
No	54	33 (61%)	0.002
Yes	46	41 (89%)
Grade
I $+$ II	73	55 (75%)	0.615
III	27	19 (70%)
Stage
I $+$ II	83	58 (70%)	0.038
III $+$ IV	17	16 (94%)
ER
Positive	47	32 (68%)	0.204
Negative	53	42 (79%)
PR
Positive	58	35 (60%)	0.371
Negative	42	29 (69%)
HER-2
0, 1 $+$	71	54 (76%)	0.463
2 $+$ , 3 $+$	29	20 (69%)

2.2 Association of ARHGDIB expression with clinicopathological parameter from breast cancer patients

To explore the clinical significance, we next investigated the association of ARHGDIB expression with clinicopathological parameters in breast cancer patients. As shown in the Table 2, the expression of ARHGDIB protein was significantly associated with large tumor size ( $P=$ 0.001), lymph node metastasis ( $P=$ 0.002) and advanced stage ( $P=$ 0.038), while no significant difference was observed in patients’ age, grade, or ER, PR and HER-2 expression (all $P>$ 0.05).

2.3 Impact of ARHGDIB expression on breast cancer cell proliferation

To assess the functional role of ARHGDIB in breast cancer, we transfected either ARHGDIB specific siRNA (siARHGDIB) or negative control (NC) into MDA-MB-231 cells. The results of western blot and RT-qPCR showed that ARHGDIB was remarkably decreased in the siARHGDIB groups compared to the NC group (Fig. 2A and B). In colony formation assays, the cells with transiently transfected into siARHGDIB formed smaller and fewer colonies per plate than that in the NC cells (Fig. 3A). In MTT assay, we observed that cell viability of the cells with transfection of siARHGDIB was significantly decreased compared with transfection with NC (Fig. 3B). Similarly, the cell numbers were decreased significantly in the group of siARHGDIB transfection compared with cells transfected with NC (Fig. 3C).

Figure 2.

Construction of ARHGDIB knockdown system in MDA-MB-231 cells. A. Western blot analysis showed that siARHGDIB markedly reduced the protein expression of ARHGDIB. B. RT-qPCR analysis showed that siARHGDIB markedly reduced the mRNA level of ARHGDIB in MDA-MB-231 cells. ${}^{**}$ , $P<$ 0.01.

Figure 3.

Inhibitory effects of ARHGDIB knockdown on breast cancer cell proliferation and cell cycle distribution. (A) Colony formation assay, (B) MTT assay and (C) cell growth assay indicated that knockdown of ARHGDIB significantly suppressed the proliferation of MDA-MB-231 cells. (D) The cell cycle distribution was determined with flow cytometric analysis. The representative FACS analysis is shown. The percentage of MDA-MB-231 cells in the G0/G1, S, and G2/M phases. The data represent one of three separate experiments. ${}^{**}$ , $P<$ 0.01.

2.4 Effect of ARHGDIB on cell cycle of breast cancer cells

In order to determine whether ARHGDIB knockdown affects cell cycle regulation of breast cancer cells, flow cytometric analysis was performed at 48 h after cell transfection with either siARHGDIB or NC control. As shown in Fig. 3D, the population of both siARHGDIB-transfected MDA-MB-231 cells in the G0/G1 phase increased significantly. Moreover, the increase in G0/G1 phase cell population was accompanied with a concomitant decrease of cell number in S phase and G2/M phase.

2.5 Impact of ARHGDIB expression on migration and invasion of breast cancer cells

In order to determine the role of ARHGDIB in migration and invasion of breast cancer, cell migration and invasion assays were individually performed in MDA-MB-231 cells at 48 h after cell transfection with either siARHGDIB or NC control. Compared with NC group, both cell migration (Fig. 4A) and invasion (Fig. 4B) decreased significantly in MDA-MB-231 cells with siARHGDIB, which suggested that ARHGDIB might be involved in breast cancer metastasis.

Figure 4.

Knockdown of ARHGDIB inhibits migration and invasion of breast cancer cells. (A) Migration assay and (B) Invasion assay indicated that the knockdown of ARHGDIB by siARHGDIB significantly suppressed the migration and invasion of MDA-MB-231 cells. ${}^{**}$ , $P<$ 0.01.

2.6 Influence of ARHGDIB on EMT of breast cancer cells

Considered the close connection between cancer migration, invasion and EMT, we further examined the protein levels of several key EMT biomarkers in breast cancer cells transfected with either siARHGDIB or NC control [16, 17]. As expected, protein levels of epithelium marker (Occludin) was increased, while mesenchymal markers (MMP2, Vimentin and Snai1) were decreased significantly in the group of siARHGDIB compared with NC control (Fig. 5).

Figure 5.

ARHGDIB depletion suppresses EMT in breast cancers. Representative western blot for epithelial marker Occludin, as well as mesenchymal markers MMP2, Vimentin and Snai1 in siARHGDIB cells and NC control. ${}^{**}$ , $P<$ 0.01.

3. Discussion

Rho GTPase proteins are involved in cytoskeleton component remodeling, thus influencing cell mobility [18]. Therefore, the regulation of Rho family proteins might have a profound impact on metastasis, a process that involves in cancer cell migration from one location to another to invade the destination tissue/organ [19]. ARHGDIB is a Rho GTPase regulatory protein that binds and holds GDP bound Rho proteins in an inactive non-membrane localized, cytoplasmic compartment [20]. Alterations of ARHGDIB expression levels have been shown in a variety of human tumor types [21]. Yi et al. showed that ARHGDIB promoted pancreatic carcinoma cell invasion and metastasis in vitro [13]. Yang et al. reported that high ARHGDIB expression level promotes the aggressive behavior of hepatocellular carcinoma [22]. These studies suggest an oncogene role of ARHGDIB in cancer progression. However, Gildea et al. reported that ARHGDIB is an invasion and metastasis suppressor gene in human bladder cancer [23]. Herein we systematically observed the role of ARHGDIB in human breast cancer both in vitro and in clinical tissues. The expression of ARHGDIB is frequently increased in breast cancer cell lines and clinical breast tissue specimens. Both the capacity of proliferation and metastasis were dramatically decreased after the knockdown of ARHGDIB in breast cancer cells. This is consistent with previous reports in the literature [15, 24]. In addition, the G0/G1 phase increased while S and G2/M phase decreased after transiently transfected into siARHGDIB in breast cancer cells. It is known that uncontrolled proliferation is a major feature of cancer cells, and these cells have typically acquired a complex defect in cell cycle progression. However, a previous report contradicts our existing conclusions. This study shows Rho-GDI $\beta$ has two opposite activities, one inhibiting tumor progression by supressing migration and the other promoting tumor by co-expressing with the transcription factor ETS1 (a tumor-promoting protein) [25]. We hypothesized that ARHGDIB may be involved in the induction of EMT in breast cancer, resulting in one of the functions predominating. Therefore, the significance of ARHGDIB for breast cancer needs to be further verified through more in-depth experiments and studies on a large number of patients.

EMT is an important cellular mechanism during tumor invasion and metastasis development and results in enhanced cell motility [26]. However, it has not been determined whether ARHGDIB expression result in EMT in breast cancer. In this study, the knockdown of ARHGDIB was observed to inhibit EMT by down-regulating mesenchymal-associated markers MMP2, Vimentin and transcription factor Snai1 while up-regulating the epithelial-associated marker Occludin. To our knowledge, this is the first report suggesting that ARHGDIB can induce EMT in breast cancer.

In summary, our study demonstrated that the altered expression of ARHGDIB was exist in breast cancer and ARHGDIB play an important role in breast cancer cell proliferation and metastasis. Our finding suggests a role of ARHGDIB in the molecular etiology of breast cancer and provides a possible target for therapeutic strategies.

4. Materials and methods

4.1 Clinical sample collection

In total, 100 breast cancer samples and 100 benign breast disease samples were enrolled in the present study. These tissue specimens were collected from the patients treated with surgery between January 2013 and December 2014 at the First Affiliated Hospital of Anhui Medical University (Hefei, China). All resected tissues underwent detailed pathological assessment, and all patients had not treated with chemotherapy of radiation therapy before surgery, and no evidence of rheumatic disease, acute infection, human immunodeficiency virus or other type of cancer. The clinicopathological parameters of these patients were collected which were determined based on the 2012 World Health Organization (WHO) classification system [27]. The protocol to use these tissues was approved by the Biomedical Ethics Committee of Anhui Medical University with informed consent of all the involved patients.

4.2 Immunohistochemical analysis

For immunohistochemistry (IHC) of ARHGDIB expression in tissue samples, a rabbit anti-ARHGDIB polyclonal antibody was obtained from Proteintech Group (Proteintech Group, Chicago, IL, USA) and was used at a dilution of 1:50 according to our previous studies [28, 29]. Expression of ARHGDIB protein in breast cancer and benign breast disease tissues were observed by a pathologist who was blinded to the patients. The staining intensity was scored as: 0, negative; 1, weak; 2, medium and 3, strong. The extent of staining was scored as: 0, 0%; 1, 1–25%; 2, 26–50%; 3, 51–75% and 4 $>$ 76%. The final score was obtained by the sum of the intensity score and the quantity score. A score of $\geqslant$ 3 was considered as positive expression, while a score of $<$ 3 was considered as negative expression [30].

4.3 Cell culture

All human breast cancer cell lines (MCF-7, T47D, BT474, MDA-MB-231 and SUM159) and human breast epithelial cell line (MCF-10A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). MCF-7, T47D and BT474 cells were maintained in the RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone). MDA-MB-231 cells were cultured in L15 medium supplemented with 10% FBS. SUM159 cells were cultured in the Ham’s F12 medium supplemented with 5% FBS, 10 $\mu$ g/ml insulin and 2.5 $\mu$ g/ml dexamethasone. MCF-10A were cultured in DMEM/F12 medium supplemented with 5% super horse serum (SHS, Sangon biotech). In addition, MDA-MB-231 were cultured in an incubator under CO ${}_{2}$ -free at 37 ${{}^{\circ}}$ C. All cell lines except MDA-MB-231 were cultured in the atmosphere of 5% CO ${}_{2}$ at 37 ${{}^{\circ}}$ C.

4.4 siRNA transfection and knock-down

Small interfering RNAs (siRNA) for targeting ARHGDIB and negative control (NC) RNA were purchased from GenePharma (Shanghai, China). The sequences of the siRNAs and NC used for transfection are shown in Table 3. The MDA-MB-231 cells were seed in 6-well plates overnight and then transiently transfected with siARHGDIB or NC using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturers’ protocol. Real-time PCR analysis was used to confirm the efficiency of siRNA transfection.

Table 3
Sequence of the oligonucleotides used for knockdown experiments

Gene	Sense strand	Antisense strand
Negative control	5’-UUCUCCGAACGUGUCACGUTT-3’	5’-ACGUGACACGUUCGGAGAATT-3’
siARHGDIB#1	5’-CCAUUGUGUUAAAGGAAGGTT-3’	5’-CCUUCCUUUAACACAAUGGTT-3’
siARHGDIB#2	5’-GCUCAAUUAUAAGCCUCCATT-3’	5’-UGGAGGCUUAUAAUUGAGCTT-3’

4.5 RNA isolation and RT-qPCR

Total RNA was extracted from MDA-MB-231 cells using Trizol reagent (Invitrogen). The sequences of the primers used for RT-qPCR are shown in Table 4. The reverse transcription reaction was performed for total RNA using the all-in one first-strand cDNA Synthesis Supermix for qPCR (TransGen Biotech, Beijing) and Light Cycler Fast Start DNA Master Plus SYBR Green I kit (TransGen Biotech, Beijing) was used for RT-qPCR according to the manufacturers’ instructions protocol to analyze the mRNA expression of ARHGDIB. The expression level of the target gene was detected using the 2 ${}^{-\Delta\Delta\text{Ct}}$ method.

Table 4
Specific primers used for RT-qPCR experiments

Gene	Forward sequence	Reverse sequence
ARHGDIB	5’-GGATGACGATGATGAGCTGGACAG-3’	5’- CAGTCCTGTAGGTGTGCTGAACG-3’
GAPDH	5’-AGCAAGAGCACAAGAGGAAG-3’	5’-GGTTGAGCACAGGGTACTTT-3’

4.6 Protein extraction and western blot

Total proteins were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) from each group of MDA-MB-231 cells, and total proteins were extracted to measure the concentration by a BCA protein quantification kit (Thermo, USA). Then about 15 $\mu$ g total proteins was fractionated by electrophoresis through 10% SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Germany) following the manufacturers’ instructions. Then the membranes were blocked with 5% defatted milk at the room temperature for one hour. After that, the membranes were washed with phosphate buffer saline with Tween-20 (PBST). Afterwards, the membranes were incubated with specific primary antibodies and the secondary antibodies (goat anti-rabbit or anti-mouse) at the room temperature for two hours and one hour respectively. Finally, the protein bands were identified using the ECL system (Millipore, Bedford, MA, USA) and $\beta$ -actin was examined as an endogenous control. The antibodies used were as follows: ARHGDIB (Proteintech Group, Chicago, IL, USA), MMP2 (Proteintech Group, Chicago, IL, USA), $\beta$ -actin (Proteintech Group, Chicago, IL, USA), Occludin (Proteintech Group, Chicago, IL, USA), Vimentin (BD Biosciences, San Jose, CA, USA), Snai1 (Proteintech Group, Chicago, IL, USA).

4.7 Cell proliferation assays

For cell proliferation, methyl-thiazolyl-tetrazolium (MTT) assay, total number assay and colony formation assay were used. In total number assay, 1 $\times$ 10 ${}^{5}$ original cells were seeded into six-well plates and cultured in normal conditions, and then these cells were counted every day for 5 days [31]. The MTT assay is based on conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells and provides a quantitative determination of viable cells. In MTT assay, each group of cells were seeded into 96-well plates with an original cell number of 1500 on the same day. After 24 h of culture, the number of viable cells was measured by adding 0.5 mg/ml MTT reagent at 100 $\mu$ l. Two hours later, the medium was replaced by 100 $\mu$ l dimethyl sulfoxide (DMSO) and the cell viability was tested at OD 570 nm with an enzyme-linked immunosorbent assay reader (Bio-Rad, CA, USA) every day for 5 days. In colony formation assay, cells were seeded into 6-well plates with an original cell number of 1000, and each group contained three wells. 7–12 days later, the cells were washed twice with PBS, and then fixed it with 90% alcohol for 40 min. After that, the cells were stained with 0.1% crystal violet for 10 min. Finally, four individual fields were randomly selected under the light microscope, and subsequently clone numbers were manually calculated with the standard of more than 100 cells per clone. All the cell proliferation-associated assays were technically in triplicate and biologically repeated thrice.

4.8 Cell cycle assay

MDA-MB-231 cells were seed in 6-well plates overnight and then transiently transfected with siARHGDIB or NC. Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China) was used to explore the cell cycle, when the confluence of each group of cells approximately reach 70%. Briefly, the MDA-MB-231 cells were collected, washed with PBS for three times, and fixed with 70% ice-cold ethanol overnight. The ethanol-suspended cells were then washed and stained with propidium iodide (PI) for 30 min in the dark at 37 ${{}^{\circ}}$ C, and subsequently subjected to Flow cytometry (BD Biosciences, CA, USA) immediately.

4.9 Migration and invasion assays

MDA-MB-231 cells were seed in 6-well plates overnight and then transiently transfected with siARHGDIB or NC and incubated for 48 hours before the migration and invasion experiments. For the cell migration assay, cells were performed using uncoated (8 $\mu$ m pore size, Coring Costar, USA) filters in 24-well plates. In detail, the mixture of cells and L15 medium with no FBS was added into the upper chambers (1 $\times$ 10 ${}^{5}$ cells per well). Below the insert, the chambers of 24-well plates contained medium supplemented with 10% FBS as the chemoattractant. Both of siARHGDIB or NC into MDA-MB-231 cells were cultured in an incubator (CO ${}_{2}$ -free, 37 ${}^{\circ}$ C) and detected after 12 h. For the cell invasion assay, cells were seeded into transwells with 1:7 Matrigel mixture (mixed with 7.5 $\mu$ l Matrigel (BD Biosciences, San Jose, CA, USA) and 52.5 $\mu$ l cell culture medium) and were detected after 24 h. In detail, after cells were counted, the mixture of cells and L15 medium with no FBS was added into the upper chambers coated with the mixture of Matrigel (1 $\times$ 10 ${}^{5}$ cells per well). L15 medium containing 10% FBS was added to the lower chambers. Both of siARHGDIB or NC into MDA-MB-231 cells were cultured in an incubator (CO ${}_{2}$ -free, 37 ${}^{\circ}$ C) and detected after 24 h. After that, the lower chambers were discarded and the upper chambers (for both migration and invasion assay) were put into chambers with PBS and washed for twice. Then the cells in the upper chambers were dyed with 0.1% crystal violet (A100528; Sangon Biotech) for 10 min at room temperature and then washed with PBS three times. The Matrigel was erased with a swab, then randomly select four fields under the light microscope for cell counting.

4.10 Statistical analysis

The association between the expression of ARHGDIB and clinicopathological parameter in clinical samples was analyzed using the chi-square test. In addition, the statistical differences of the experimental data were evaluated by one-way analysis of variance (ANOVA) using GraphPad Prism 7 software package. All experiments were performed at least three times. $P<$ 0.05 was considered as statistically significant respectively.

Footnotes

Acknowledgments

This work was supported by the National Nature Science Foundation of China (81972472, 31970696 and 81502975), Anhui provincial academic and technical leader reserve candidate (#2016H074), and Key Program of Outstanding Young Talents in Higher Education Institutions of Anhui (#gxyqZD2016046). The research was supported in part by the China Postdoctoral Science Foundation (2016T90413 and 2015M581693).

Conflict of interest

The authors declare that they have no conflicts of interest.

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Systematic investigation of biomarker-like role of ARHGDIB in breast cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Results

2.1 Expression of ARHGDIB in clinical breast tissue specimens and cell lines

Table 1 Expression of ARHGDIB in breast cancer samples and benign tissues

2.3 Impact of ARHGDIB expression on breast cancer cell proliferation

2.5 Impact of ARHGDIB expression on migration and invasion of breast cancer cells

4. Materials and methods

4.1 Clinical sample collection

4.2 Immunohistochemical analysis

4.3 Cell culture

4.4 siRNA transfection and knock-down

Table 3 Sequence of the oligonucleotides used for knockdown experiments

Table 4 Specific primers used for RT-qPCR experiments

4.7 Cell proliferation assays

4.8 Cell cycle assay

4.9 Migration and invasion assays

4.10 Statistical analysis

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Expression of ARHGDIB in breast cancer samples and benign tissues

Table 3
Sequence of the oligonucleotides used for knockdown experiments

Table 4
Specific primers used for RT-qPCR experiments