miR, microRNA; NC, negative control; wt, wild-type; mut, mutant. miR-220b expression is downregulated, whereas radixin level is elevated in BC tissue. TNM stage. miR-200b expression levels exhibited the opposite trend. Radixin mRNA expression in breast cancer cells was notably higher, whereas miR-200b expression was lower compared with that in normal breast epithelial MCF-10A cells. The expression of radixin was higher, whereas miR-200b was lower in MDA-MB-231 cells compared with that in MCF-7 cells. miR-200b mimic or siRNA-radixin transfection downregulated the expression of radixin in MDA-MB-231 cells and attenuated the invasive and proliferative abilities of these cells. miR-200b-knockdown and radixin overexpression were associated with enhanced cell invasion in breast cancer. In conclusion, miR-200b regulates breast cancer cell proliferation and invasion by targeting radixin expression. cells (Thermo Fisher Scientific, Inc.) to screen a positive clone. Following sequencing, pMIR-Radixin-wild-type (wt) and pMIR-Radixin-mutant (mut) plasmids were selected. 293T cells (Thermo Fisher Scientific, Inc.) were transfected with 1 g pMIR-Radixin-wt or pMIR-Radixin-mut with the miR-200b mimic, inhibitor or NC using riboFECT? CP transfection reagent. Following incubation for 48 h, luciferase activity was detected using a Dual-Luciferase assay kit according to the manufacturer’s protocol. All luciferase activities were normalized to that of luciferase. Manipulation of miR-200b expression in MDA-MB-231 cells. MDA-MB-231 cells were divided into two groups, inoculated into 10-cm culture dishes and cultured to 50-60% confluency, followed by transfection with miR-NC or miR-200b mimic. A total of 5 nM miR-NC-mimic or miR-200b mimic and miR-NC-inhibitor or miR-200b inhibitor were diluted in 100 l riboFECT? CP Buffer at room temperature for 5 min and incubated with 10 l riboFECT? CP Reagent at room temperature for 0-15 min. The mixture was added to the cell culture medium and incubated for 72 h at 37?C prior to further experiments. Radixin siRNA transfection MDA-MB-231 cells were divided into two groups, inoculated into Licofelone 10-cm culture dishes and cultured to 50-60% confluency, followed by transfection with siRNA-NC or siRNA-radixin. The transfection protocol was the same as that aforementioned. Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was extracted from MDA-MB-231 cells using the miRNeasy FFPE kit Licofelone (Qiagen China Co., Ltd.). TransScript? Green One-Step qRT-PCR SuperMix (Beijing Transgen Biotech Co., Ltd.) was used for one step RT-qPCR detection. The PCR system comprised 1 g template RNA, 0.3 M forward and reverse primers, 10 l 2X TransStart Tip Green qPCR SuperMix, 0.4 l One-Step RT Enzyme mix and 0.4 l Passive Reference Dye II dissolved in RNase-free water. The reaction was performed on an ABI ViiA?7 PCR system at 94?C for 5 min, followed by 40 cycles of 94?C for 5 sec and 60?C for 30 sec. U6 and GAPDH was used as reference genes for Licofelone miRNA and mRNA expression, respectively. Quantitative analysis was performed using the 2-Cq method (23). Primer sequences used for RT-qPCR were as follows: Radixin forward, 5′-CTCGAAAAGCTCTAGAACTGG-3′ and reverse, 5′-GGTTCATTACCCCTTCATTTG-3′; miR-200b forward, 5′-ACAGTAATACTGCCTGGTAATG-3′ and reverse, 5′-GGTCCAGTTTTTTTTTTTTTTTCATC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-ACGCTTCACGAATTTGCGT-3′; GAPDH forward, 5′-CAGCGACACCCACTCCTCCACCTT-3′ and reverse, 5′-CATGAGGTCCACCACCCTGTTGCT-3′. Western blotting. Total protein was extracted from MDA-MB-231 cells by SDS lysis and quantified by the BCA method. A total of 40 g protein/lane was separated by 8-10% SDS-PAGE and transferred to a PVDF membrane at 300 mA for 1.5 h. Following blocking by 5% skimmed milk at room temperature for 6 h, the membrane was incubated with primary antibodies at 4?C overnight (N-cadherin, 1:2,000; E-cadherin, 1:2,000; radixin, 1:1,000; and -actin, 1:10,000). The membrane was washed with PBST and further incubated with the HRP-conjugated secondary antibody (1:10,000) at room temperature for 60 min. Finally, the membrane was treated with ECL chemiluminescence reagent and developed. Image J software 1.52 (National Institutes of Health) was used for densitometric analysis of band intensity. Flow cytometry MDA-MB-231 Cells (1×106/ml) were treated with 10 M 5-ethynyl-2′-deoxyuridine (EdU) solution in the logarithmic phase. Following 48-h RASGRP1 incubation, the cells were digested with trypsin, collected, stained with Alexa Fluor-488 labeled reaction liquid (cat. no. “type”:”entrez-nucleotide”,”attrs”:”text”:”C10337″,”term_id”:”1535408″,”term_text”:”C10337″C10337; Thermo Fisher Scientific, Inc.) at room temperature for 30 min and detected using a Licofelone Beckman Coulter FC 500 MCL/MPL flow cytometer (Becton, Dickinson and Company). FlowJo software (version 7.6.1; FlowJo LLC) was used for the analysis of results. Cell Counting Kit-8 (CCK-8) assay CCK-8 (Shanghai Shenggong Biology Engineering Technology Service) was used to evaluate cell.