Research Communication MiRNA Expression in Breast Cancer Varies with Lymph Node Metastasis and Other Clinicopathologic Features
Bin Wang1 Jindong Li1 Mei Sun2 Lihua Sun3 Xingyi Zhang1*
1
Department of Thoracic Surgery, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China 2 Department of Pathology, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China 3 Department of Anesthesiology, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China
Abstract Breast carcinoma is the most common malignant tumor in females, and lymph node (LN) status is one of the most important prognostic factors in patients with breast cancer. MiRNAs have been shown to have important role in oncogenesis, invasion, and metastasis via epigenetic posttranscriptional gene regulation. However, lymphatic metastasis-related miRNAs of breast cancer has not been well documented. The aim of this study was to identify and evaluate miRNAs related to breast cancer LN metastasis and to explore the clinical significance of the differentially expressed miRNAs in patients with breast cancer. The expression of miRNAs in patients with primary breast cancer with LN metastasis and that without LN metastases was compared by miRNA microarray. We further validated the miRNAs (miR-185-5p, miR-339-5p, miR-542-5p, and miR-3923) between LN (n 5 31) and nonlymph node (NLN; n 5 42) group using real-time reverse transcriptase polymerase chain reaction. Furthermore,
the relationship between miRNA expression and clinical pathological features was analyzed. The miRNA microarray initially identified that eight miRNAs (miR-206, miR-3923, miR-181a, miR-92a, miR-421, miR-339-5p, miR-3196, and miR-29b) were downregulated in LN metastasis group, whereas five miRNAs (miR-542-5p, miR-200a, miR-564, miR-451, miR-30c, miR-200b, miR-191-3p, miR-142-5p, and miR-185-5p) were upregulated in LN group when compared with those in NLN group. In the validation cohort, the expression levels of miR-185-5p and miR542-5p were significantly expressed higher in LN group (P 5 0.002 and P 5 0.001, respectively), and the expression levels of miR-339-5p and miR-3923 were significantly expressed lower in LN group (P 5 0.001 and P 5 0.001, respectively). Our results indicate the potential role of miR-185-5p, miR-542-5p, miR-339-5p, and miR-3923 in predicting metastasis to the LN C and prognosis in breast cancers. V 2014 IUBMB Life, 66(5):371–377, 2014
Keywords: breast cancer; microRNAs; microarray; lymph node metastasis; prognosis
Introduction C 2014 International Union of Biochemistry and Molecular Biology V
Volume 66, Number 5, May 2014, Pages 371–377 *Address correspondence to: Xingyi Zhang, Department of Thoracic Surgery, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China. E-mail: [email protected] Received 12 April 2014; Accepted 1 May 2014 DOI 10.1002/iub.1273 Published online 20 May 2014 in Wiley Online Library (wileyonlinelibrary.com)
IUBMB Life
Breast cancer is the most common malignant tumor in females, and the incidence of breast cancer is increasing in the developing world (1). Metastasis is one of the basic characteristics of malignant tumors, and lymphatic metastasis is the most common phenomenon for carcinoma, which is closely related to the poor prognosis because effective treatments are still lacking (2). Axillary lymph node (LN) status is one of the most important prognostic indicators in breast cancer, and the detection of nodal metastases is a key factor in recommending adjuvant chemotherapy after surgery (3). Widespread use of mammography has resulted in marked increase in early
371
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detection of breast cancer, improvement in therapy, and declining mortality (4). The positive yield of axillary LN dissection also decreases, and LN negative patients do not benefit from axillary dissection but may suffer from its complications (5). Axillary LN dissection is now no longer considered to be the standard treatment in all patients with invasive breast cancer (6). Therefore, it is essential to find predictive factors for axillary LN involvement in early breast cancer. MiRNAs are endogenous, small noncoding single-stranded RNAs of 18–25 nucleotides in length, found in both plants and animals, which function at post-transcriptional and transcriptional levels as negative regulators of gene expression. The binding of miRNAs to complementary sites in the 30 untranslated regions and other regions of protein-coding mRNA sequences cause either degradation of the mRNA or inhibition of translation (7, 8). It is estimated that up to 60% of genes may be regulated by more than 1,900 human miRNAs thus far identified (9, 10). The involvement of miRNAs in cancer pathogenesis is now well established, and miRNA expression profiles accurately classify tumors at different stages and distinguish among subsets of patients with different molecular pathologies (11–13). There are some reports on the association of the clinicopathogenetic features of breast cancer with miRNA expression (12–15). However, lymphatic metastasisrelated miRNAs of breast cancer has not been well documented. The aim of this study was to identify and evaluate miRNAs related with breast cancer LN metastasis. In this study, we investigated the differences in miRNA profiling between primary breast cancers with LN metastasis and that without LN metastasis, and then selected several miRNAs and detected the expression of them in 73 breast cancer specimens in an effort to identify the miRNAs involved in breast cancer LN metastasis. Furthermore, association between miRNA expression and clinicopathologic characteristics as well as Her-2/neu, ER, and PR status in breast carcinoma was determined.
Materials and Methods
Of all cases, fresh frozen tissue from 12 patients with breast cancer were used to undertake miRNA array (LN metastasis 5 6 cases; LN negative group 5 6 cases), which further validated the miRNAs between primary breast cancer with LN metastasis (n 5 31) and nonlymph node (NLN) metastases (n 5 42) group using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and analyzed the relationship between the expression and the clinicopathological characteristics of the specimens. Furthermore, miRNA expression was correlated with Her-2/neu, ER, and PR expression, and their expression in breast carcinoma was determined.
RNA Isolation Total RNA was extracted and purified using mirVanaTM miRNA Isolation Kit (Cat. No. AM1560; Ambion, Austin, TX, USA) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
MiRNA Expression Array MiRNA microarray was executed by Agilent human miRNA array (8*60K, version 16.0; Agilent Technologies, Santa Clara, CA, USA), which contains capture probes targeting 1,205 human miRNA sequences registered in the miRBASE database (available from: http:\\www.mirbase.org). MiRNA molecule in total RNA was labeled by miRNA Complete Labeling and Hyb Kit (Cat. No. 5190-0456; Agilent Technologies) following the manufacturer’s instructions (labeling section). Each slide was hybridized with 100 ng of Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit (Cat. No. 5190-0456; Agilent Technologies) in hybridization oven (Cat. No. G2545A; Agilent Technologies) at 55 C, 20 rpm, and for 20 h according to the manufacturer’s instructions (hybridization section). After hybridization, the slides were washed in staining dishes (Cat. No. 121; Thermo Shandon, Waltham, MA, USA) with Gene Expression Wash Buffer Kit (Cat. No. 5188–5327; Agilent Technologies). The slides were scanned by Agilent Microarray Scanner (Cat. No. G2565BA; Agilent Technologies) and Feature Extraction software 10.7 (Agilent Technologies) with default settings. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent Technologies).
Patient Material A total of 73 invasive ductal breast cancer tissue samples with (i.e., 31) or without (i.e., 42) LN metastasis were obtained from the Department of Oncology, Jilin Medical University, Jilin, China, between December 2011 and March 2012. The median age of the patients at surgery was 49 years (ranging from 38 to 65 years), and all patients were female. Both tissue samples used in the experiments were carefully snap-frozen in liquid nitrogen at the time of resection and stored at 280 C until total RNA was extracted. None of the patients of our study received any neoadjuvant chemotherapy or radiotherapy. We obtained written informed consent from each patient, and the Institute Research Medical Ethics Committee of Jilin Medical University granted approval for this study.
372
MiRNA Quantitative RT-PCR MiRNAs expression in cancer and noncancer tissues of each patient was detected using SYBR-green real-time RT-PCR on a Light Cycler 480 system according to the manufacturer’s instructions. Briefly, 50 ng of total RNA was reverse transcribed using miRNA cDNA Synthesis Kit (Takara Life Technologies, Japan). The measurement of the expression levels of individual miRNAs was performed using miRNA sequencespecific primers (Takara Life Technologies). The RT-PCR conditions used were as follows: 95 C for 30 sec, followed by 35 cycles of 95 C for 10 sec and 60 C for 25 sec. MiRNA expression levels were accessed by relative quantification and the fold expression changes determined by 22䉭䉭CT method (16), and U6 RNA was used as endogenous control.
Mirna Related to Lymph Node Metastasis
for 48 h. Invading cells on the lower surface that passed through the filter were fixed and stained using crystal violet in gluteraldehyde and photographed.
Statistical Analysis The delta Ct (DCt) in each sample represents the relative expression amount of miRNA: DCt 5 Ct (miRNA) 2 Ct (U6). Comparison of mean DCt values between the independent groups was calculated using Mann-Whitney U test. For determining the association between miRNAs expression and clinical features, the miRNAs expression was represented as 22DCT. The Mann-Whitney U test was used to analyze the relationship between the levels of miR-185-5p, miR-339-5p, miR542-5p, and miR-3923 and the clinical features of the patients. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 13.0 software.
FIG 1
Hierarchical clustering analysis of microRNAs expression data from microarray analysis. Each row indicates a miRNA, and each column indicates a sample. The miRNA subset tree is on the left, and the sample subset is at the top.
Results Results of the miRNA Array
Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection. Adherent cells were maintained in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 C.
Microarray analysis in breast cancer resulted in the detection of 17 differentially expressed miRNAs (LN metastasis vs. NLN metastasis). Figure 1 shows two main gene clusters (clusters I and II) of altogether 17 miRNAs. A markedly (more than 1.5-fold) decreased level when compared with NLN group was found in LN group for miR-206, miR-3923, miR-181a, miR-92a, miR-421, miR-339-5p, miR-3196, and miR-29b that belongs to the cluster I. Cluster II grouped upregulated miRNAs in LN group; more than 1.5-fold change was detected for miR-542-5p, miR-200a, miR-564, miR-451, miR30c, miR-200b, miR-191-3p, miR-142-5p, and miR-185-5p.
Cell Transfection
Real-Time RT-PCR Validation of Selected miRNAs
The following items were purchased from Guangzhou Ribo-Bio (Guangzhou, China): miRNA mimics; miRNA mimic NC, a negative control for miRNA mimic; miRNA inhibitor with a sequence complementary to mature miRNA; and miRNA inhibitor NC, a negative control for miRNA inhibitor. Breast cancer cell line MDA-MB-231 (3 3 105) was seeded onto six-well plates and cultured overnight. Cells were then transfected with 20 nM mimics (for miR-339-5p and miR3923) or 40 nM inhibitors (for miR-185-5p and miR-542-5p) and the corresponding doses of miR NC using Lipofectamine 2000 according to the manufacturer’s instruction. After 48 h, cells were used for further qRT-PCR and transwell analysis.
Two upregulated (miR-185-5p and miR-542-5p) and two downregulated miRNAs (miR-339-5p and miR-3923) with more than 1.5-fold change in their expression over the control were selected for array data validation and for the evaluation of LN metastasis precision by the RT-qPCR. MiR-185-5p and miR-542-5p were significantly expressed higher in LN group (P 5 0.002 and P 5 0.001, respectively), while miR-339-5p and miR-3923 were significantly expressed lower in LN group (P 5 0.001 and P 5 0.001, respectively; Fig. 1).
Cell Culture
Invasion Assay Invasion of cells was measured using a Matrigel invasion assay (Becton Dickinson, Bedford, MA, USA). Transwell inserts of 8mm pore size were coated with a final concentration of 1 mg/mL of Matrigel in cold serum-free DMEM. Cells were trypsinized, and 1 3 105 cells were added in triplicate wells. The lower chamber of the transwell was filled with 750 mL of culture media containing 0.5% serum as a chemoattractant, along with the treatment of nestin shRNA and allowed to incubate at 37 C
Wang et al.
Correlation Between miRNAs Expression and Clinicopathological Parameters To investigate whether the change in the four miRNAs expression of breast cancer was associated with any of the available clinical characteristics, we studied the association of miRNA expression levels with the clinical and pathological parameters of breast cancer. The correlation analysis of miR-185-5p, miR339-5p, miR-542-5p, and miR-3923 expression with clinicopathologic factors in breast carcinoma is shown in Table 1. The expression of miR-185-5p in patients with stages I and II was 0.13 6 0.14, which was higher than, but not significantly different from, that in patients with stages III and IV
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TABLE 1
Association between miRNAs and clinicopathological characteristics in patients with breast cancer
n
miR-185-5p (22DCt)
miR-339-5p (22DCt)
miR-542-5p (22DCt)
miR-3923 (22DCt)
Bin Wang1 Jindong Li1 Mei Sun2 Lihua Sun3 Xingyi Zhang1*
1
Department of Thoracic Surgery, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China 2 Department of Pathology, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China 3 Department of Anesthesiology, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China
Abstract Breast carcinoma is the most common malignant tumor in females, and lymph node (LN) status is one of the most important prognostic factors in patients with breast cancer. MiRNAs have been shown to have important role in oncogenesis, invasion, and metastasis via epigenetic posttranscriptional gene regulation. However, lymphatic metastasis-related miRNAs of breast cancer has not been well documented. The aim of this study was to identify and evaluate miRNAs related to breast cancer LN metastasis and to explore the clinical significance of the differentially expressed miRNAs in patients with breast cancer. The expression of miRNAs in patients with primary breast cancer with LN metastasis and that without LN metastases was compared by miRNA microarray. We further validated the miRNAs (miR-185-5p, miR-339-5p, miR-542-5p, and miR-3923) between LN (n 5 31) and nonlymph node (NLN; n 5 42) group using real-time reverse transcriptase polymerase chain reaction. Furthermore,
the relationship between miRNA expression and clinical pathological features was analyzed. The miRNA microarray initially identified that eight miRNAs (miR-206, miR-3923, miR-181a, miR-92a, miR-421, miR-339-5p, miR-3196, and miR-29b) were downregulated in LN metastasis group, whereas five miRNAs (miR-542-5p, miR-200a, miR-564, miR-451, miR-30c, miR-200b, miR-191-3p, miR-142-5p, and miR-185-5p) were upregulated in LN group when compared with those in NLN group. In the validation cohort, the expression levels of miR-185-5p and miR542-5p were significantly expressed higher in LN group (P 5 0.002 and P 5 0.001, respectively), and the expression levels of miR-339-5p and miR-3923 were significantly expressed lower in LN group (P 5 0.001 and P 5 0.001, respectively). Our results indicate the potential role of miR-185-5p, miR-542-5p, miR-339-5p, and miR-3923 in predicting metastasis to the LN C and prognosis in breast cancers. V 2014 IUBMB Life, 66(5):371–377, 2014
Keywords: breast cancer; microRNAs; microarray; lymph node metastasis; prognosis
Introduction C 2014 International Union of Biochemistry and Molecular Biology V
Volume 66, Number 5, May 2014, Pages 371–377 *Address correspondence to: Xingyi Zhang, Department of Thoracic Surgery, Second Hospital of Jilin University, Chang Chun, Jilin 130000, China. E-mail: [email protected] Received 12 April 2014; Accepted 1 May 2014 DOI 10.1002/iub.1273 Published online 20 May 2014 in Wiley Online Library (wileyonlinelibrary.com)
IUBMB Life
Breast cancer is the most common malignant tumor in females, and the incidence of breast cancer is increasing in the developing world (1). Metastasis is one of the basic characteristics of malignant tumors, and lymphatic metastasis is the most common phenomenon for carcinoma, which is closely related to the poor prognosis because effective treatments are still lacking (2). Axillary lymph node (LN) status is one of the most important prognostic indicators in breast cancer, and the detection of nodal metastases is a key factor in recommending adjuvant chemotherapy after surgery (3). Widespread use of mammography has resulted in marked increase in early
371
IUBMB LIFE
detection of breast cancer, improvement in therapy, and declining mortality (4). The positive yield of axillary LN dissection also decreases, and LN negative patients do not benefit from axillary dissection but may suffer from its complications (5). Axillary LN dissection is now no longer considered to be the standard treatment in all patients with invasive breast cancer (6). Therefore, it is essential to find predictive factors for axillary LN involvement in early breast cancer. MiRNAs are endogenous, small noncoding single-stranded RNAs of 18–25 nucleotides in length, found in both plants and animals, which function at post-transcriptional and transcriptional levels as negative regulators of gene expression. The binding of miRNAs to complementary sites in the 30 untranslated regions and other regions of protein-coding mRNA sequences cause either degradation of the mRNA or inhibition of translation (7, 8). It is estimated that up to 60% of genes may be regulated by more than 1,900 human miRNAs thus far identified (9, 10). The involvement of miRNAs in cancer pathogenesis is now well established, and miRNA expression profiles accurately classify tumors at different stages and distinguish among subsets of patients with different molecular pathologies (11–13). There are some reports on the association of the clinicopathogenetic features of breast cancer with miRNA expression (12–15). However, lymphatic metastasisrelated miRNAs of breast cancer has not been well documented. The aim of this study was to identify and evaluate miRNAs related with breast cancer LN metastasis. In this study, we investigated the differences in miRNA profiling between primary breast cancers with LN metastasis and that without LN metastasis, and then selected several miRNAs and detected the expression of them in 73 breast cancer specimens in an effort to identify the miRNAs involved in breast cancer LN metastasis. Furthermore, association between miRNA expression and clinicopathologic characteristics as well as Her-2/neu, ER, and PR status in breast carcinoma was determined.
Materials and Methods
Of all cases, fresh frozen tissue from 12 patients with breast cancer were used to undertake miRNA array (LN metastasis 5 6 cases; LN negative group 5 6 cases), which further validated the miRNAs between primary breast cancer with LN metastasis (n 5 31) and nonlymph node (NLN) metastases (n 5 42) group using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and analyzed the relationship between the expression and the clinicopathological characteristics of the specimens. Furthermore, miRNA expression was correlated with Her-2/neu, ER, and PR expression, and their expression in breast carcinoma was determined.
RNA Isolation Total RNA was extracted and purified using mirVanaTM miRNA Isolation Kit (Cat. No. AM1560; Ambion, Austin, TX, USA) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
MiRNA Expression Array MiRNA microarray was executed by Agilent human miRNA array (8*60K, version 16.0; Agilent Technologies, Santa Clara, CA, USA), which contains capture probes targeting 1,205 human miRNA sequences registered in the miRBASE database (available from: http:\\www.mirbase.org). MiRNA molecule in total RNA was labeled by miRNA Complete Labeling and Hyb Kit (Cat. No. 5190-0456; Agilent Technologies) following the manufacturer’s instructions (labeling section). Each slide was hybridized with 100 ng of Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit (Cat. No. 5190-0456; Agilent Technologies) in hybridization oven (Cat. No. G2545A; Agilent Technologies) at 55 C, 20 rpm, and for 20 h according to the manufacturer’s instructions (hybridization section). After hybridization, the slides were washed in staining dishes (Cat. No. 121; Thermo Shandon, Waltham, MA, USA) with Gene Expression Wash Buffer Kit (Cat. No. 5188–5327; Agilent Technologies). The slides were scanned by Agilent Microarray Scanner (Cat. No. G2565BA; Agilent Technologies) and Feature Extraction software 10.7 (Agilent Technologies) with default settings. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent Technologies).
Patient Material A total of 73 invasive ductal breast cancer tissue samples with (i.e., 31) or without (i.e., 42) LN metastasis were obtained from the Department of Oncology, Jilin Medical University, Jilin, China, between December 2011 and March 2012. The median age of the patients at surgery was 49 years (ranging from 38 to 65 years), and all patients were female. Both tissue samples used in the experiments were carefully snap-frozen in liquid nitrogen at the time of resection and stored at 280 C until total RNA was extracted. None of the patients of our study received any neoadjuvant chemotherapy or radiotherapy. We obtained written informed consent from each patient, and the Institute Research Medical Ethics Committee of Jilin Medical University granted approval for this study.
372
MiRNA Quantitative RT-PCR MiRNAs expression in cancer and noncancer tissues of each patient was detected using SYBR-green real-time RT-PCR on a Light Cycler 480 system according to the manufacturer’s instructions. Briefly, 50 ng of total RNA was reverse transcribed using miRNA cDNA Synthesis Kit (Takara Life Technologies, Japan). The measurement of the expression levels of individual miRNAs was performed using miRNA sequencespecific primers (Takara Life Technologies). The RT-PCR conditions used were as follows: 95 C for 30 sec, followed by 35 cycles of 95 C for 10 sec and 60 C for 25 sec. MiRNA expression levels were accessed by relative quantification and the fold expression changes determined by 22䉭䉭CT method (16), and U6 RNA was used as endogenous control.
Mirna Related to Lymph Node Metastasis
for 48 h. Invading cells on the lower surface that passed through the filter were fixed and stained using crystal violet in gluteraldehyde and photographed.
Statistical Analysis The delta Ct (DCt) in each sample represents the relative expression amount of miRNA: DCt 5 Ct (miRNA) 2 Ct (U6). Comparison of mean DCt values between the independent groups was calculated using Mann-Whitney U test. For determining the association between miRNAs expression and clinical features, the miRNAs expression was represented as 22DCT. The Mann-Whitney U test was used to analyze the relationship between the levels of miR-185-5p, miR-339-5p, miR542-5p, and miR-3923 and the clinical features of the patients. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 13.0 software.
FIG 1
Hierarchical clustering analysis of microRNAs expression data from microarray analysis. Each row indicates a miRNA, and each column indicates a sample. The miRNA subset tree is on the left, and the sample subset is at the top.
Results Results of the miRNA Array
Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection. Adherent cells were maintained in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 C.
Microarray analysis in breast cancer resulted in the detection of 17 differentially expressed miRNAs (LN metastasis vs. NLN metastasis). Figure 1 shows two main gene clusters (clusters I and II) of altogether 17 miRNAs. A markedly (more than 1.5-fold) decreased level when compared with NLN group was found in LN group for miR-206, miR-3923, miR-181a, miR-92a, miR-421, miR-339-5p, miR-3196, and miR-29b that belongs to the cluster I. Cluster II grouped upregulated miRNAs in LN group; more than 1.5-fold change was detected for miR-542-5p, miR-200a, miR-564, miR-451, miR30c, miR-200b, miR-191-3p, miR-142-5p, and miR-185-5p.
Cell Transfection
Real-Time RT-PCR Validation of Selected miRNAs
The following items were purchased from Guangzhou Ribo-Bio (Guangzhou, China): miRNA mimics; miRNA mimic NC, a negative control for miRNA mimic; miRNA inhibitor with a sequence complementary to mature miRNA; and miRNA inhibitor NC, a negative control for miRNA inhibitor. Breast cancer cell line MDA-MB-231 (3 3 105) was seeded onto six-well plates and cultured overnight. Cells were then transfected with 20 nM mimics (for miR-339-5p and miR3923) or 40 nM inhibitors (for miR-185-5p and miR-542-5p) and the corresponding doses of miR NC using Lipofectamine 2000 according to the manufacturer’s instruction. After 48 h, cells were used for further qRT-PCR and transwell analysis.
Two upregulated (miR-185-5p and miR-542-5p) and two downregulated miRNAs (miR-339-5p and miR-3923) with more than 1.5-fold change in their expression over the control were selected for array data validation and for the evaluation of LN metastasis precision by the RT-qPCR. MiR-185-5p and miR-542-5p were significantly expressed higher in LN group (P 5 0.002 and P 5 0.001, respectively), while miR-339-5p and miR-3923 were significantly expressed lower in LN group (P 5 0.001 and P 5 0.001, respectively; Fig. 1).
Cell Culture
Invasion Assay Invasion of cells was measured using a Matrigel invasion assay (Becton Dickinson, Bedford, MA, USA). Transwell inserts of 8mm pore size were coated with a final concentration of 1 mg/mL of Matrigel in cold serum-free DMEM. Cells were trypsinized, and 1 3 105 cells were added in triplicate wells. The lower chamber of the transwell was filled with 750 mL of culture media containing 0.5% serum as a chemoattractant, along with the treatment of nestin shRNA and allowed to incubate at 37 C
Wang et al.
Correlation Between miRNAs Expression and Clinicopathological Parameters To investigate whether the change in the four miRNAs expression of breast cancer was associated with any of the available clinical characteristics, we studied the association of miRNA expression levels with the clinical and pathological parameters of breast cancer. The correlation analysis of miR-185-5p, miR339-5p, miR-542-5p, and miR-3923 expression with clinicopathologic factors in breast carcinoma is shown in Table 1. The expression of miR-185-5p in patients with stages I and II was 0.13 6 0.14, which was higher than, but not significantly different from, that in patients with stages III and IV
373
IUBMB LIFE
TABLE 1
Association between miRNAs and clinicopathological characteristics in patients with breast cancer
n
miR-185-5p (22DCt)
miR-339-5p (22DCt)
miR-542-5p (22DCt)
miR-3923 (22DCt)
