RESEARCH ARTICLE Jianjun Han, et al.: mir-361-5p in breast cancer

The Bosnian Journal of Basic Medical Sciences publishes an Advanced online manuscript format as a free service to authors in order to expedite the dissemination of scientific findings to the research community as soon as possible after acceptance following peer review and corresponding modification (where appropriate). An Advanced online manuscript is published online prior to copyediting, formatting for publication and author proofing, but is nonetheless, fully citable through its Digital Object Identifier (doi ). Nevertheless, this Advanced online version is NOT the final version of the manuscript. When the final version of this paper is published within a definitive issue of the journal with copyediting, full pagination, etc. the new final version will be accessible through the same doi and this "Advanced online" version of the paper will disappear. RESEARCH ARTICLE Jianjun Han, et al.: mir-361-5p in breast cancer Overexpression of mir-361-5p in triple-negative breast cancer (TNBC) inhibits migration and invasion by targeting RQCD1 and inhibiting the EGFR/ PI3K/Akt pathway Jianjun Han 1, Jingjing Yu 2, Yuna Dai 1, Jumei Li 1, Meiyan Guo 1, Jingzhen Song 1, Xuefeng Zhou 3 * 1 Breast Surgery, Affiliated Hospital of Hebei University of Engineering, Handan, Hebei, China 2 Glandular Surgery, Xingtai Third Hospital, Xingtai, Hebei, China 3 Department of Oncology, Dongtai People s Hospital, Dongtai, Jiangsu, China *Corresponding author: Xuefeng Zhou, Department of Oncology, Dongtai People s Hospital, No. 2 Kangfu West Road, Dongtai 224200, Jiangsu, China. E-mail: dr_xuefengzhou@sina.com Submitted: 23 March 2018 /Accepted: 15 May 2018 1

DOI: http://dx.doi.org/10.17305/bjbms.2018.3399 Licence: 2018 ABMSFBIH. 2

ABSTRACT Triple-negative breast cancer (TNBC) is the leading cause of cancer-related death in women. Previous studies indicated that mir-361-5p was downregulated in breast cancer, however, the exact effect of mir-361-5p on TNBC requires further investigation. In the present study, we investigated whether mir-361-5p can act as a tumor suppressor by targeting required for cell differentiation 1 homolog (RQCD1) and inhibiting epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway in TNBC. The expression of mir-361-5p and RQCD1 was determined by quantitative reverse transcription PCR (qrt-pcr) and/or western blot in TNBC and the adjacent tissues. mir-361-5p mimics were constructed and transfected to TNBC cell line MDA-MB-231. Expression of mir-361-5p and mrna and protein expression of RQCD1, PI3K, Akt, EGFR and matrix metallopeptidase 9 (MMP-9) in MDA-MB-231 were measured by qrt-pcr and/or western blot after transfection. Cell viability was determined by CCK-8 assay. Cell migration and invasion ability were evaluated by scratch and transwell assay respectively. Confirmation of mir-361-5p target was determined by bioinformatics analysis and luciferase reporter assay. Downregulated mir-361-5p and upregulated RQCD1 were observed in tumor tissues. Expression of EGFR, PI3K, Akt and MMP-9 was inhibited in cells treated with mir-361-5p mimics. Transfection of mir-361-5p mimics also inhibited the phosphorylation of EGFR, PI3K, and Akt. Suppressed cell viability, migration, and invasion was found in mir-361-5p mimics groups. Luciferase reporter assay showed that RQCD1 was the target of mir-361-5p. These results indicated that overexpression of mir-361-5p might act as a suppressor in TNBC by targeting RQCD1 to inhibit the EGFR/PI3K/Akt signaling pathway. 3

KEY WORDS: mir-361-5p; RQCD1; EGFR/PI3K/Akt signaling pathway; cell migration and invasion; triple-negative breast cancer; TNBC 4

INTRODUCTION Breast cancer is frequently diagnosed in female over the world. Many factors including lifestyle and family history are associated with the increased risk of breast cancer (1, 2). While familial breast cancer accounts for a minor percentage of all breast cancer cases and occurs in patients whose related family members are diagnosed with breast or ovarian cancer (3). It is well known that germline mutations in BRCA1 and BRCA2 and genes mutation in germline confer a 60 80% and 40 85% lifetime risk of developing breast cancer, respectively (4). Furthermore, in clinical, breast cancer were divided into several subtypes based on the expression of a number of receptors such as estrogen receptor, progesterone receptor and human growth factor receptor (4), among which the triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer with poor prognosis. Although the treatment and the 5-year survival rate of TNBC are dramatically improved, the therapy with more efficiency and less side effects need to be further investigated (5). MicroRNAs (mirnas) are endogenous small non-coding RNAs containing 22-24 nt that could bind to the 3 - untranslated region (UTR) of target mrnas resulting in the promoted mrna degradation or repressed mrna translation (6). mirnas have been found to function as oncogenes or tumor suppressors which differently express in tumor tissues and normal tissues (6). Over the past 10 years, many studies suggested that mirnas play key roles in breast cancer development (7). For example, the members of mir-200 family target ZEB1/ZEB2 which are important for cell proliferation, invasion, and migration (8). It was also reported that the expression of mir-342 is associated with ERα expression and introduction of mir-342 enhanced sensitivity to tamoxifen-induced apoptosis (9). mir-361-5p, located on chromosome X, plays critical roles in several human tumors. In colorectal and gastric cancer, mir-361-5p acts as a 5

tumor suppressor (11). In the case of breast cancer, according to the study by Ma et al, mir-361-5p was downregulated in breast cancer and overexpression of mir-361-5p could inhibit cell invasion by suppressing the expression of FGFR1 and MMP-1 (12). However, the exact impact of mir-361-5p on TNBC by targeting RQCD1 has not been fully explained. It is well known that mirna exert its function through inhibiting the expression of its target mrna. Using online bioinformatic methods, differentiation1 homolog (RQCD1) has been predicted as a target gene of mir-361-5p. Previous studies indicated that RQCD1 can activate EGFR/PI3K/Akt signaling pathway via binding to GIGYF1/2 (13). Moreover, decreased expression of RQCD1 is capable of receding the EGF-induced Akt activation followed by inhibiting cell migration and invasion (14). It has been proved that the epidermal growth factor receptor (EGFR) gene plays vital roles in the development of human cancer including cell proliferation, metastasis, apoptosis and angiogenesis (15). Furthermore, phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) pathway is closely related to cell proliferation and invasion in several cancers such as NSCLC, breast cancer and nasopharyngeal carcinoma (16-18). Therefore, based on the previous study, we investigated the effects of mir- 361-5p on EGFR/PI3K/Akt pathway in TNBC by targeting QRCD1. 6

MATERIALS AND METHODS Patients and tissues Thirty patients who have been pathologically diagnosed as TNBC were recruited in our study. Tumor tissues and their adjacent normal tissues were obtained and stored in liquid nitrogen immediately after surgery. Meanwhile, all patients have signed the informed consent prior to the experiments. The clinical information of the patients are provided in Table 1. Cells MDA-MB-231 TNBC cell line was obtained from the BNCC and maintained in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma) at 37 C in an atmosphere of 5% CO2. Plasmids and cell transfection Cells were seeded into the 24-well plates and incubated in RPMI-1640 at 37 C with 5% CO2 for 24 h to reach the 60-70% confluence. Then, the mir-361-5p mimics plasmids and negative control plasmids were constructed by Addgene and transfected to cells with 1μl Lipo6000 TM Transfection Reagent (Thermo Fisher Scientific). The transfection was performed at 37 with 5% CO2 for 6 h. Subsequently, the transfected cells were further incubated for 72 h. qrt-pcr qrt-pcr was performed to investigate the expression of mir-361-5p and proteins. Under the condition of cells with 60% confluence, the RNAs were extracted using total RNA Plant Mini Kit (Trizol). Subsequently, the concentration of RNAs were measured by Nanodrop 2000 (Thermo Fisher Scientific). Additionally, DNA Polymerase (Pyrobest) and M-MLV RT (Thermo Fisher Scientific) was used for cdna synthesis and PCR reaction. The synthesis of cdna was set as 95 C for 15 min and the PCR reaction was set as 96 C for 5 min (initiation), 94 C for 30 7

sec (denaturation), 65 C for 30 sec (annealing) and 75 C for 1 min (extension) for 33 cycles. Furthermore, the expression of mir-361-5p was normalized to U6 and the GAPDH was regarded as the internal reference of mrnas. At the same time, the 2 ΔΔCq method was used for mrna relative quantification (19). Western blot The western blot assay was performed to assess the expression levels of proteins. The proteins were isolated from cells employing RIPA lysis buffer (Thermo Fisher Scientific) and the concentrations of proteins were measured by BCA Kit (Thermo Fisher Scientific). Subsequently, the protein samples (2 μl/per lane) were separated via 15% SDS-PAGE. The proteins were transferred to PVDF membrane (Membrane Solutions) blocked with 5% skimmed milk for 2 h. After that, the reaction with primary antibodies anti-rqcd1, EGFR, PI3K, Akt, MMP-9, GAPDH was at room temperature for 1h. Additionally, the incubation with HRP-conjugated secondary antibodies was at room temperature for 45 min. ECL kit (Thermo Fisher Scientific) was used for visualization and the image was analyzed by ImageJ. CCK-8 assay The cell viability was assessed by CCK-8 kit (Dojindo) after transfection. The cells were placed into the 96-well plates. Then, the 10 µl CCK-8 reagent was added to wells and cultured for 24 h in RPMI-1640 media with 5% CO2 at 37. Subsequently, the optical density (OD) was measured by microplate reader under 450 nm. The CCK-8 assays were carried out after 12, 24 and 48 h incubation respectively. Scratch and transwell assay The invasion ability of MDA-MB-231 cells was measured by transwell assay after 6 h transfection. The transwell culture inserts (8-mm pore size; Falcon; BD Biosciences) were placed 8

into the wells of 96-well plates to create the separated upper and lower chambers. Furthermore, the upper side of the membrane was pre-coated with Matrigel (BD Biosciences) and incubated at 37 C for 1 h for gel formation. FBS was utilized to hydrate the membrane 2 h prior to use. Then, RPMI-1640 (600 µl) containing 10% FBS and 1 10 5 cells/well was added to the lower and upper chamber respectively. After 48 h incubation, number of invading cells were counted in chambers. Bioinformatics research and luciferase reporter assay According to the bioinformatics research of TargetScan (http://www.targetscan.org/vert_71/), we found that RQCD1 was the target of mir-361-5p and luciferase reporter assay was conducted to verify the hypothesis. The cells were placed into 96-well plates and incubated in RPMI-1640 media to reach the 60% confluence. Subsequently, the co-transfection of mir-361-5p mimics (50 ng), RQCD1-3 UTR-WT (50 ng) or RQCD1-3 UTR-MUT (50 ng) plasmids was performed using Lipo3000 TM Transfection Reagent (0.2 μl). Furthermore, the luciferase activity was normalized to renilla luciferase. The luciferase assay kit (Thermo Fisher Scientific) was used to evaluate the luciferase activity according to the manufacturer s protocol. Statistical methods Each experiment was performed in triplicate and all data were presented as mean ± standard deviation ( x ± s). Additionally, two groups comparisons were conducted by T-test and one-way ANOVA was performed for multiple groups comparison using SPSS 14.0. P<0.05 was defined as the statistical significance. 9

RESULTS Downregulated mir-361-5p and upregulated RQCD1 in TNBC tissues The expression of mir-361-5p and RQCD1 mrna were determined by qrt-pcr and the expression of protein RQCD1 was measured by western blot assay. As shown in Figure 1A and B, the expression of mir-361-5p was significantly downregulated in tumor tissues while the dramatically increased RQCD1 mrna expression was observed in tumor tissues showed by dot plot. Furthermore, the expression levels of RQCD1 protein were significantly increased in tumor tissues in Figure 1C (representative sample). Therefore, the abnormal expressions of mir-361-5p and RQCD1 were observed in TNBC tissues. Inhibited EGFR/PI3K/Akt signaling pathway after mir-361-5p mimics transfection After 6 h transfection and 48 h incubation, the expression of proteins in EGFR/PI3K/Akt signaling pathway were measured by qrt-pcr and western blot. As shown in Figure 2A, B and C, the expressions of mrnas of EGFR/PI3K/Akt were significantly decreased in mimics groups compared with control groups, while there is no significance between NC group and control group. Furthermore, the dramatically inhibited expressions of RQCD1, PI3K, Akt, EGFR and MMP-9 at protein level were found in mimics groups compared with control groups and there is apparent difference between NC group and control group. As shown in Figure 2D and E. Hence, the overexpression of mir-361-5p could restrain the EGFR/PI3K/Akt signaling pathway due to the controlled expression of PI3K, Akt, EGFR and MMP-9. Suppressed cell proliferation after mir-361-5p mimics transfection After mir-361-5p mimics transfection, the evaluation of cell proliferation was conducted at 12, 24 and 48 h time point. The results indicated that the suppressed cell proliferation was observed in mimics groups after 24 and 48 h incubation compared with control groups. Yet the difference 10

between NC group and control group is not that obvious (Figure 3). Thus, upregulation of mir- 361-5p might inhibit cell proliferation Inhibited cell migration and invasion of MDA-MB-231 after mir-361-5p mimics transfection The cell migration and transwell ability of MDA-MB-231 cells were determined by transwell assay after transfection and 48 h incubation. The results demonstrated that both of the cell migration and invasion ability were suppressed in mir-361-5p mimics groups than that of control groups, while there is no significant difference between NC group and control group, as shown in Figure 4. Therefore, overexpression of mir-361-5p could inhibiting cell migration and invasion. RQCD1 was the target of mir-361-5p by luciferase reporter assay The research of TargetScan showed that RQCD1 was the target of mir-361-5p, as shown in Figure 5A. Furthermore, the luciferase reporter assay was performed to verify the target of mir- 361-5p using RQCD1-3 UTR-WT and RQCD1-3 UTR-MUT plasmids. The lower luciferase activity in RQCD1-3 UTR-WT and mir-361 co-transfected groups suggested that RQCD1 was the target of mir-361-5p, as shown in Figure 5B. 11

DISCUSSION The purpose of the present study focused on the impacts of mir-361-5p on EGFR/PI3K/Akt signaling pathway by targeting RQCD1 in TNBC. According to the present study, mir-361-5p was down-regulated in breast cancer tissue, which is consistent with the previous study by Ma F et al (12). Furthermore, the TargetScan research showed that RQCD1 was the target of mir-361-5p and we verified that RQCD1 was target of mir-361-5p by luciferase reporter assay. At the same time, we found that overexpression of mir-361-5p induced the suppressed expression of PI3K, Akt, EGFR and MMP-9 to inhibit EGFR/PI3K/Akt signaling pathway in TNBC cell line MDA-MB-231. Furthermore, the decreased cell proliferation, migration and invasion suggested that mir-361-5p was a tumor suppressor of breast cancer, which was also found by Ma F et al (12). Breast cancer is the significant public health problem and the incidence of breast cancer is growing due to the breast cancer risk factor such as menarche at younger age and first pregnancy at late age (20). `Additionally, mirnas have different effects on breast cancer (21). For example, mir-21, mir-10b, mir-125 and mir-155 are overexpressed in breast and function as the oncogenes, while mir-206, mir-31, mir-146 and mir-91 are downregulated in breast cancer and serve as the tumor suppressor genes (21). Moreover, mir-361-5p can not only inhibit cell proliferation and invasion by targeting FGFR1/MMP-1 breast cancer but also suppress cancer progress by targeting STAT6 in NSCLC (22). Additionally, each kind of mirna can target one or more mrna and each kind of mrna can also be targeted by one or more mirna (23). Therefore, the impacts between different mirnas and proteins could be further investigated for a better understanding of its mechanisms. 12

Although the expression of mir-361-5p in TNBC and its impacts on cell proliferation, migration and invasion ability were evaluated in our present study, the specific factors involved in cell proliferation, migration and invasion have not been fully investigated, for instance, the alteration of proliferation-related factors in cell proliferation process. At the same time, some studies showed that EGFR/PI3K/Akt signaling pathway is associated with cell apoptosis (24, 25). Therefore, the impacts of mir-361-5p on cell apoptosis in TNBC could be determined by flow cytometry. 13

CONCLUSION To sum up, mir-361-5p was down-regulated in TNBC. Furthermore, mir-361-5p might inhibit the proliferation, migration and invasion of TNBC cells by targeting RQCD1 resulting in the repressed EGFR/PI3K/Akt signaling pathway. Therefore, in the present study, we further concluded that mir-361-5p acted as tumor suppressor and could be a potential target for TNBC treatment. DECLARATION OF INTERESTS The authors declare no conflict of interests. 14

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FIGURES AND TABLES FIGURE 1. Downregulated mir-361-5p and upregulated RQCD1 in breast cancer tissues. The expression of mir-361-5p and RQCD1 in tissues were determined by qrt-pcr and western blot. (A) Relative expression of mir-361-5p in normal and tumor tissues. (B) Relative expression of RQCD1 mrna in normal and tumor tissues (C) Relative expression of RCD1 protein in tissues of representative patient. **p<0.01 vs. Normal group. 19

FIGURE 2. Inhibited EGFR/PI3K/Akt signaling pathway after mir-361-5p mimics transfection. After mir-361-5p mimics transfection and 48 h incubation in RPMI-1640 medium, the RT-PCR and western blot were performed for mrnas and protein expression determination. (A) Relative expression of mrna of Akt. (B) Relative expression of mrna of EGFR. (C) Relative expression of mrna of PI3K. (D) 20

Image of western blot for RQCD1, PI3K, p-egfr, p-akt and MMP-9 protein expression. (E) Grey analysis of RQCD1, PI3K, p-egfr, p-akt and MMP-9 expression. Control: blank group. NC group: cells treated with control plasmids. mimics group: cells treated with mir-361-5p mimics plasmids. **p<0.01 vs. control group. 21

FIGURE 3. Suppressed cell proliferation after mir-361-5p mimics transfection. The cell viability was evaluated at 0, 12, 24 and 48 h time point by CCK-8 assay. The inhibited cell proliferation was observed after mir-361-5p mimics transfection. Control: blank group. NC group: cells treated with control plasmids. mimics group: cells treated with mir-361-5p mimics plasmids **p<0.01 vs. Control group. 22

FIGURE 4. Inhibited cell migration and invasion of MCF-7 after mir-361-5p mimics transfection. Transwell assay of MCF-7 and the number of invasion cells. Control: blank group. NC group: cells treated with control plasmids. mimics group: cells treated with mir-361-5p mimics plasmids. **p<0.01 vs. Control group. 23

FIGURE 5. RQCD1 was the target of mir-361-5p by luciferase reporter assay. (A) Bioinformatics research for the target of mir-361-5p. (B) Relative luciferase activity after co-tranfection. Control: blank group. NC group: cells treated with control plasmids. mimics group: cells treated with mir-361-5p mimics plasmids**p<0.01 vs. NC group. 24

TABLE 1. Clinicopathological information of the patients with TNBC Characteristics Cases (n) mir-361 low (n) mir-361 high (n) P-value Age (years) 0.3329 40 6 3 3 >40 24 17 7 Tumor size (cm) 0.4475 <2 4 2 2 2 26 18 8 Differentiation 0.0153* Well 5 1 4 Poor 25 19 6 Tumor stage 0.0410* I+II 8 3 5 III+IV 22 17 5 Lymph node metastases 0.0098* Yes 3 0 3 No 27 20 7 25