miR‐205‐3p promotes proliferation and reduces apoptosis of breast cancer MCF‐7 cells and is associated with poor prognosis of breast cancer patients

Abstract Background To study the expression of microribonucleic acid (miR)‐205 in breast cancer and its effects on the proliferation and apoptosis of breast cancer cells. Methods Breast cancer cell line MCF‐7 cells with stable expression of miR‐205‐3p were constructed. Cell proliferation, invasion, and apoptosis were detected via MTT assay, transwell assay, and flow cytometry, respectively. The expressions of Ezrin, LaminA/C, cleaved caspase‐3, Bcl‐2, and Bax were detected via Western blotting. The expressions of miR‐205‐3p in breast cancer tissues and para‐carcinoma tissues were detected via quantitative PCR (qPCR). Results In transfection group, cell proliferation and invasion capacities were increased significantly (P < 0.01), but apoptotic cells were significantly reduced (P < 0.01). In addition, the expressions of Ezrin, LaminA/C, and cleaved caspase‐3 in the transfection group were significantly decreased (P < 0.01), but the Bcl‐2/Bax ratio was significantly increased (P < 0.01). The miR‐205‐3p expression in tumor tissues of breast cancer patients was significantly higher than that in para‐carcinoma tissue, but Ezrin, LaminA/C, and cleaved caspase‐3 expressions in tumor tissues were remarkably declined (P < 0.01), while the Bcl‐2/Bax ratio was remarkably increased (P < 0.01). Moreover, the 5‐year survival of patients with high expression of miR‐205‐3p was significantly shorter than patients with normal or low expression (P < 0.01). Conclusion Highly expressed miR‐205‐3p can promote the proliferation and invasion and reduce the apoptosis of breast cancer cells, and the high expression of miR‐205‐3p can significantly reduce the survival time of patients.

cities 2 A variety of stress responses in cells will be produced when various external stimuli (various carcinogenic factors) act on breast epithelial cells, resulting in cell proliferation, thus leading to breast cancer 3 At present, surgical operation is dominated in the clinical treatment approaches of breast cancer, but the range of surgical resection in clinic is wide, the trauma is large, and a lot of related complications often occur in patients after clinical surgical treatment, causing serious physiological and psychological damage to patients 4

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A large number of studies in recent years have revealed that various specific gene mutations were identified in tumor patients and several microribonucleic acids (miRNAs) are closely related to the tumor metastasis and invasion 5, 6 Osipov et al 7 found high expression of miR-205 in epithelial ovarian cancer cells, indicating that miR-205 is possibly associated with the incidence of ovarian cancer. There is a great amount of research evidence proving that the miR-205 expressions in a variety of tumors are lower than that in normal tissues [8][9][10][11] miRNAs are therefore applied in the diagnosis of cancer 12,13 For instance, miR-205 presents as the potential biomarkers for early prognosis or diagnosis while its level is proposed in predicting lymph node metastasis in triple-negative breast cancer patients 14,15 Moreover, it has been demonstrated that miR-205/ASPP2 axis promoted cell migration and also increased cell proliferation, which may be potential diagnostic and therapeutic biomarkers in cervical and lung cancers 16

| Patients and grouping
A total of 58 patients who were pathologically diagnosed with breast cancer, treated, and underwent mastectomy in Guangdong Provincial People's Hospital from June 2011 to June 2013 were collected. None of the patients received chemotherapy and radiotherapy before operation. The para-carcinoma tissues of breast cancer (more than 5 cm away from the breast cancer tissues) were taken during operation as the control. All enrolled patients were females aged 42-69 years with a median age of 56.2 years, and they all suffered from invasive ductal carcinoma. According to the tumor-nodemetastasis (TNM) staging criteria issued by the International Union Against Cancer, patients were staged, and there were 8 cases in stage I, 41 cases in stage II, 6 cases in stage III, and 3 cases in stage IV. Inclusion criteria were as follows: (1) patients without severe hepatic and renal function impairment, (2) patients who agreed to be followed up, (3) patients without a history of serious cardiovascular and immune system diseases, and (4) patients without chronic or acute infectious diseases. All enrolled patients personally signed the informed consent and had complete clinical and pathological data and complete therapeutic regimens. The experimental scheme was reviewed and approved ethically by our hospital.

| Detection of miR-205-3p expression level in breast cancer tissues
After breast cancer tissues and para-carcinoma tissues were obtained, they were added into the tube (100 mg/mL) together with TRIzol, and smashed using an ultrasonic disruptor followed by being incubated on ice for 10 minutes, followed by centrifugation at 12 000 g and 4°C for 10 minutes. 700 μL supernatant was taken, added with chloroform, and manually vibrated for 10 times, followed by incubation on ice for 10 minutes and centrifugation at 12 000 g and 4°C for 10 minutes. After the supernatant was discarded, 1 mL freshly prepared 75% ethanol was added into each tube, followed by centrifugation at 12 000 g and 4°C for 15 minutes. The ethanol was discarded, the cap of the centrifuge tube was removed for natural air-drying, and 30 μL diethyl pyrocarbonate (DEPC)-treated water was added to dissolve the RNA. The optical density (OD) value and purity of RNA were measured. The reaction system was prepared strictly according to instructions of the reverse transcription kit (Invitrogen): 5 μg total RNA, 1 μL Oligo(dT) 20 , 1 μL 10 mmol/L dNTP, and 3 μL 0.1mol/L DTT were added, and DEPC-treated water was also added until the total volume was 20 μL. After reaction at 60°C for 15 minutes and incubation at 37°C for 2 minutes, the reverse transcription was performed. After RT-qPCR system was prepared, the amplification was performed on the RT-qPCR instrument.
Amplification conditions are as follows: pre-denaturation at 92°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, extension at 72°C for 2 minutes, and a total of 35 cycles. Primers were synthesized by Invitrogen, and RNU6B was used

| Survival curve analysis
The miR-205-3p expression in cancer tissues of each patient with breast cancer was detected and compared with that in para-carcinoma tissue, and divided into normal or low expression group and high expression group. Data of patients were recorded in detail, and patients were followed up for 5 years. The 5-year survival rate of patients was recorded, and the survival curve was drawn. Influencing factors of patient's prognosis in both groups were analyzed via Cox multivariate regression analysis.

| Cell culture and construction of cell lines with miR-205-3p overexpression
Human breast cancer MCF-7 cell lines were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences. After resuscitation, cells were cultured using Dulbecco's modified Eagle medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) in an incubator with 5% CO 2 at 37°C till the logarithmic growth phase.
Then, cells were digested with trypsin and continued to be cultured, followed by passage till the 3rd generation used for the experiment. was connected to the vector using T4 ligase (Takara), followed by plate clone using LB solid medium at 37°C and sequencing identification using positive colonies. The miR-205-3p lentivirus was packaged with the plasmid (TransGen), the titer was detected, and the miR-205-3p lentivirus was used to infect MCF-7 cells after meeting requirements: After digestion, MCF-7 cells were inoculated into a 24-well plate at a density of 5 × 10 4 /well. After culturing for 24 hours, 440 μL DMEM, 10 μL hsa-miR-205-3p, and 50 μL polybrene were added into the centrifuge tube, mixed evenly, and inoculated into the plate. After culturing for 12 hours, the original medium was replaced with DMEM containing 10% FBS for culture for another 72 hours. Then, the fluorescence intensity of cells was observed under a microscope. After cells were screened using puromycin (Sigma) and cloned, the MCF-7 cell lines with stable infection were constructed. Overexpression of miR-205-3p was then confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) using RNU6B as an internal control.

| Detection of cell proliferation and invasion capacities
Cell proliferation capacity was detected via methyl thiazolyl tetrazolium (MTT) assay: After cells in the logarithmic growth phase were digested, the cell density was adjusted to 1 × 10 4 /well, and cells were inoculated into a 96-well plate and divided into blank control group (MCF-7 cells without any treatment), negative control group (MCF-7cells inoculated with empty vector), and hsa-miR-205-3p group (MCF-7 cells inoculated with hsa-miR-205-3p), followed by culture in an incubator for 24 hours. Then, 100 μL MTT was added to culture cells for another 4 hours, the supernatant was discarded, and 150 μL dimethyl sulfoxide (DMSO) was added into each well and vibrated on a shaking table for 1 minute. Finally, cell proliferation in each group was detected using a microplate reader.
Cell invasion capacity was detected via transwell assay: After cells in the logarithmic growth phase were digested, the cell density was adjusted to 1.5 × 10 6 /well, and the cell suspension was pre-  Then, the cell suspension was inoculated into a 6-well plate and added with serum-free DMEM, followed by culture in the incubator for 24 hours. After the culture solution was discarded, cells were washed with phosphate-buffered saline (PBS), scraped off, centrifuged, and resuspended with PBS for 3 times strictly according to instructions of the apoptosis kit (BD Bioscience). The fluorescence maker was added and cells were incubated in a dark place at room temperature for 15 minutes, followed by sample loading and detection via flow cytometry. All samples should be detected within 1 hour to ensure the effect.

| Detection of expression levels of related proteins
Cells in each group were cultured in the 6-well plate, collected, followed by addition of radioimmunoprecipitation assay (RIPA) lysis solution (Wuhan Servicebio) and 1% protease inhibitor (Wuhan Servicebio) into each well (500 μL/well). After breast cancer tissues and para-carcinoma tissues were obtained, they were added with RIPA lysis solution and 1% protease inhibitor (100 mg/mL) and smashed using the ultrasonic disruptor. The above cell suspension and tissue homogenate were incubated on ice, followed by centrifugation at 1000 g and 4°C for 10 minutes to obtain the total protein solution. The total protein concentration in each sample was detected using the BCA protein assay kit (PIERCE). After the protein loading buffer in an equal concentration was prepared, 10% gel was prepared and added with an equal amount of loading buffer, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under constant pressure. Then, the protein was transferred onto a PVDF membrane (Millipore), and the target band was cut and sealed with freshly prepared 5% skim milk powder for 2 hours. Ezrin, LaminA/C, cleaved caspase-3, Bcl-2, Bax, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (purchased from CST, USA, and prepared at a volume ratio of 1:1000) were incubated at 4°C over-

| Statistical analysis
In this study, three independent experiments were performed for in vitro assay. Data were presented as mean ± SD and processed using  Compared with those in paracarcinoma tissues, Ezrin, LaminA/C, and cleaved caspase-3 expressions are remarkably lower in breast cancer tissues, but the Bcl-2/Bax ratio is higher. Data were presented as a fold change relative to GAPDH (mean ± SD). ** P < 0.01

| miR-205-3p expression in breast cancer tissues
The miR-205-3p expressions in tumor tissues and para-carcinoma tissues of breast cancer patients were detected via RT-qPCR.
According to results, the miR-205-3p expression in tumor tissues of breast cancer patients was increased significantly compared with that in para-carcinoma tissues (1.87 ± 0.63 vs 1, P < 0.01) (Figure 1).

| Related protein expressions in breast cancer tissues
Ezrin, LaminA/C, cleaved caspase-3, Bcl-2, and Bax protein expressions in tumor tissues and para-carcinoma tissues of breast cancer patients were detected via Western blotting. Results manifested that compared with those in para-carcinoma tissues, Ezrin, LaminA/C, and cleaved caspase-3 expressions were significantly declined in tumor tissues (P < 0.01), but the Bcl-2/Bax ratio was increased significantly (P < 0.01) ( Figure 2).

| Correlations of miR-205-3p with clinicopathological features of breast cancer patients
The miR-205-3p level was classified as high or low in relation to the median value (cutoff value = 1.704). The above patients were accordingly divided into low or normal expression group and high expression group based on the miR-205-3p expression level in breast cancer tissues, with para-carcinoma tissues as the control. The clinicopathological data of patients in each group were recorded in details, and correlations of miR-205-3p expression level with clinicopathological data of patients were analyzed. Results showed that miR-205-3p was not related to the age and menstrual condition of breast cancer patients, but statistically closely related to the size, TNM staging, lymph node metastasis, and recurrence of breast cancer (P < 0.05) ( Table 1).
Further univariate and multivariate analysis showed that miR-205-3p expression was an independent factor for the survival (recurrence-free survival) of breast cancer patients (Table 2).

| Correlation of miR-205-3p with prognosis of breast cancer patients
All breast cancer patients enrolled were followed up for 5 years, the survival rate of patients was recorded, and the survival curve was drawn. According to results, the 5-year survival rate of breast cancer patients with low or normal expression of miR-205-3p was significantly higher than that in patients with high expression of miR-205-3p (P < 0.01) (Figure 3).

| Construction of cell lines with miR-205-3p overexpression
After human breast cancer MCF-7 cells were transfected with miR-205-3p lentivirus, the miR-205-3p expression in each group was TA B L E 1 Clinicopathological data of breast cancer patients detected via RT-qPCR. Results showed that miR-205-3p expression levels in cells in blank control group and negative control group were significantly lower than that in hsa-miR-205-3p group (P < 0.01), indicating successful construction of MCF-7 cell lines with miR-205-3p overexpression (Figure 4).

| Effects of miR-205-3p on proliferation, invasion, and apoptosis of breast cancer cells
Effects of miR-205-3p on proliferation, invasion, and apoptosis of MCF-7 cells were detected via MTT assay, transwell assay, and flow cytometry, respectively. Results showed that both cell proliferation and invasion capacities in hsa-miR-205-3p group were significantly higher than those in blank control group and negative control group (P < 0.01, P < 0.01). After transfection with miR-205-3p, the number of apoptotic cells in hsa-miR-205-3p group was significantly decreased compared with those in the blank control group and negative control group (P < 0.01) ( Figure 5).

| Effects of miR-205-3p on related protein expressions
Ezrin, LaminA/C, cleaved caspase-3, Bcl-2, and Bax protein expressions in each group were detected via Western blotting. Results demonstrated that compared with those in the blank control group and negative control group, Ezrin, LaminA/C, and cleaved caspase-3 expressions were significantly declined in hsa-miR-205-3p group (P < 0.01), but the Bcl-2/Bax ratio was increased significantly (P < 0.01) ( Figure 6). F I G U R E 4 Detection of miR-205-3p expression after transfection by RT-qPCR. After MCF-7 cells were transfected with has-miR-205-3p, total RNA was isolated for analysis of the expression of miR-205-3p by RT-qPCR. The miR-205-3p expression level in cells in hsa-miR-205-3p group is significantly higher than those in blank control group and negative control group, ** P < 0.01

| D ISCUSS I ON
Breast cancer ranks first in the common malignant tumors in women, the current incidence of which accounts for more than 10% of the systemic tumors and seriously affects the physical health of women 19 Breast cancer occurs frequently in postmenopausal women mainly the study revealed that patients who had negative or low Ezrin expression had a significantly shorter survival than those whose tumors had moderate or high Ezrin expression, 30 which is consistent with our finding that Ezrin expressions were significantly declined in breast cancer tissues. Also, it has been shown that radixin, as a group are significantly higher than those in blank control group and negative control group, but the apoptosis level is obviously decreased, ** P < 0.01 methylation and histone deacetylation as a means to downregulate caveolin expression 32 The similar mechanisms may reduce ezrin expression, in addition to genetic alterations associated with breast carcinogenesis.
Expression of A-type lamins increases the risk of death from colorectal cancer because its presence gives rise to increased invasiveness but not affect cell proliferation 33 By contrast, previous data indicate that low expression of lamin A/C is associated with an increased disease recurrence in stage II and III colon cancer patients 34 Moreover, evidence unraveled that cultured breast cancer cell lines expressed less lamin A/C compared with noncancerous mammary gland cells, which is in line with our result that LaminA/C was significantly decreased in breast cancer tissues 35 The loss of lamin A/C expression is associated with variables of poor prognosis and shorter outcome in patients with breast cancer 36 The loss of A-type lamins was shown to induce changes in nuclear distribution of telomeres and to affect telomere function, thereby inducing chromosomal instability 37 The malfunction of A-type lamins will increase chromosomal instability and stimulate carcinogenesis.
Increasing genomic instability may enhance tumor progression, Compared with those in the blank control group and negative control group, Ezrin, LaminA/C, and cleaved caspase-3 expressions are remarkably lower in hsa-miR-205-3p group, but the Bcl-2/Bax ratio is higher. Data were presented as a fold change relative to GAPDH (mean ± SD). ** P < 0.01 Our data demonstrate that correlation of miR-205-3p with prognosis of breast cancer patients was established in those patients that died because the breast cancer but no other causes and miR-205-3p may serve as an important gene for prognosis evaluation of breast cancer. The limitation in our study still exists that the controversial result regarding the changes in Ezrin, LaminA/C in tumor tissues, and the potential mechanisms remain to be further investigated. We propose that cancer cells exhibit a variety of features indicative of atypical nuclei, while the changes in these indicators may be further related to tumor's pathological classification and cancer progression.
In conclusion, significantly higher miR-205-3p expression is found in breast cancer tissues and associated with shorter survival of patients. Highly expressed miR-205-3p promotes the proliferation and invasion and reduces the apoptosis of breast cancer cells in vitro, suggesting therapeutic targeting miR-205-3p might be a novel approach for the treatment of breast cancer.