# MicroRNA-139-5p Suppresses Cell Malignant Behaviors in Breast Cancer through Targeting MEX3A

Authors: Jian Chu; Tangya Li; Lei Li; Huiwen Fan
Journal: Computational and Mathematical Methods in Medicine
 (2021)

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## Abstract

Objective. The study was designed to evaluate the underlying mechanism of microRNA-139-5p in breast cancer (BC). Methods. Expression statuses of microRNA-139-5p and MEX3A were measured by qRT-PCR and western blotting. The anticancer effect of microRNA-139-5p in vitro was tested by a set of assays. Interaction between microRNA-139-5p and MEX3A was validated by dual-luciferase detection. Results. MicroRNA-139-5p expression in BC cells was obviously low, while MEX3A was significantly overexpressed. MicroRNA-139-5p restrained proliferative, invasive, and migratory abilities of BC cells and increased apoptosis level of BC cells, while MEX3A exerted a promoting effect on BC cell growth. Dual-luciferase reporter detection confirmed that microRNA-139-5p bound to MEX3A 3′-UTR. Conclusions. MicroRNA-139-5p inhibited the development of BC by targeting MEX3A. MicroRNA-139-5p/MEX3A may be a target for BC therapy.


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## Body

## 1. Introduction

Breast cancer (BC), which is commonly diagnosed in women, gives rise to the second frequent cancer-related death [1]. In 2018, up to 2.1 million women were diagnosed with BC [2]. The 5-year survival rate of BC patients has been improved, but the 5-year survival rate of those patients with metastatic BC is just about 26% [3]. Because of the heterogeneity of BC metastasis, it is difficult to determine therapies and assess metastasis risk [4]. Therefore, focusing on molecular mechanism and therapies of occurrence and metastasis of BC may help to improve the survival rate of patients and offer new ideas for targeted therapy of BC.MicroRNAs take part in physiological and pathological processes in the human body, like cell apoptosis, cell differentiation, and organ development, thus inhibiting or facilitating the occurrence of human cancer [5]. Many studies have demonstrated that various microRNAs regulate the occurrence and development of BC, such as microRNA-193a [4], microRNA-140-5p [6], and microRNA-1228 [7]. MicroRNA-139-5p has been shown to modulate the growth of cancer cells. Yang et al. [8] discovered that microRNA-139-5p facilitates prostate cancer progress by modulating SOX5 and significantly affects the prognosis of prostate cancer. In addition, Chen et al. [9] have reported that microRNA-139-5p is significantly downregulated in uterine leiomyoma tissues, and microRNA-139-5p blocks proliferation of uterine leiomyoma cells, as well as induces cell apoptosis via degenerating TPD52. Li et al. [10] demonstrated that microRNA-139-5p targeting Notch1 restrains metastasis and EMT in glioma. However, how microRNA-139-5p functions in BC needs further in-depth investigation.MEX3 family participates in physiological processes such as immune response, metabolism, and cancer [11]. MEX3A is a new component in mammals containing GW-182 or Dcp that functions as a cellular site for mRNA degradation [12]. MEX3A has been confirmed to be significantly related to the occurrence of cancers, such as bladder cancer [13] and Wilms tumor [14]. However, the role of MEX3A in BC has less been studied.In the current study, microRNA-139-5p expression and its biological functions in BC were analyzed. MicroRNA-139-5p and MEX3A interaction was also evaluated to reveal the effects of microRNA-139-5p on BC development. We indicated an underlying role of microRNA-139-5p/MEX3A in BC process, which could be a theoretical support for novel therapeutical targets or biomarkers.


## 2. Materials and Methods

### 2.1. Bioinformatics Approaches

Expression data information of mature microRNAs (normal: 104, tumor: 1,103) and mRNAs (normal: 113, tumor: 1,109) of TCGA-BRCA was accessed from The Cancer Genome Atlas (TCGA) database. MicroRNA-139-5p expression was analyzed on basis of the downloaded data. R package “edgeR” was used to carry out differential expression analysis on mRNAs in TCGA-BRCA dataset with the normal group as the control and∣logFC∣>2, padj<0.01 as thresholds to acquire differentially expressed mRNAs (DEmRNAs). MicroRNA-139-5p target binding prediction was conducted by using targetScan, miRDB, and mirDIP databases, and the predicted mRNAs were intersected with upregulated DEmRNAs to screen the potential miR-139-5p targets. Correlation analysis was conducted to select miR-139-5p target mRNA presenting the highist negative correlation efficiency with miR-139-5p at expression level. Survival analysis of 11 target genes was processed by the GEPIA database.


### 2.2. Cell Cultivation

Human normal breast epithelial cell line MCF-10A (BNCC337734) and human BC cell lines MDA-MB-231 (BNCC337893), MCF7 (BNCC337656), HCC1937 (BNCC338509), and MX-1 (BNCC100280) were offered by BeNa Culture Collection (Beijing, China). MCF-10A, HCC1937, and MX-1 were grown in 90% RPMI-1640HyClone,USA+10% fetal bovine serum (FBS; Thermo Scientific HyClone, Beijing, China). MDA-MB-231 and MCF7 were cultivated in DMEM (Thermo, USA) with 10% FBS.


### 2.3. Transfection of Cells

MicroRNA-139-5p mimics, microRNA-139-5p inhibitor, oe-MEX3A, and corresponding negative controls were offered by GenePharma Company (Shanghai, P.R. China). Nontransfected control was presented to identify the transfection efficiency (FigureS1). 1.5 μL/mL Lipofectamine 2000 transfection reagent (ThermoFisher, USA) was utilized to get 50 nM gene segment or 1 μg plasmid transfected into BC cells. Experiments were carried out 24 h after cell culture, and transfection lasted for 48 h.


### 2.4. qRT-PCR

After total RNA was isolated by TRIzol reagent (ThemorFisher, USA), it was quantified using NanoDrop (ThermoFisher, USA). Reverse transcription of mRNAs and microRNA was performed by using the SuperScript III First-Strand Synthesis System kit (Invitrogen, USA) and NCode™ VILO™ microRNA cDNA Synthesis kit (Life Technologies). qRT-PCR analysis of mRNAs and microRNAs was conducted by using the SoFast™ EvaGreenH Supermix (Bio-Rad) and EXPRESS SYBR Green ER microRNA qRT-PCR kit (Life Technologies), respectively. U6 and GAPDH were used as internal control genes. The following were primer sequences: microRNA-139-5p forward: 5′-TCTACAGTGCACGTGTC-3′, reverse: 5′-GAATACCTCGGACCCTGC-3′; U6 forward: 5′-CTCGCTTCGGCAGCACATA-3′, reverse: 5′-AACGATTCACGAATTTGCGT-3′; MEX3A forward: 5′-CGGAGTGCACTCTGGCTTTGAG-3′, reverse: 5′-CAGAGGAGAAGAGCACGGAGGT-3′; GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′; reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′. MicroRNA or mRNA relative expression was analyzed by the 2-ΔΔCt method.


### 2.5. Western Blotting

RIPA buffer solution (Solarbio, Beijing, China) was used to isolate total proteins in each group. The concentration of extracted samples was determined by the BCA assay kit (Abcam, Cambridge, UK). Separated by 10% SDS-PAGE, samples were transferred onto PVDF membrane (Invitrogen, USA), followed by incubation with primary antibodies. Then, proteins were treated with a secondary antibody (goat anti-rabbit IgG (1 : 2000, ab205718, Abcam, UK)) for 1 h. Protein levels were detected by the BeyoECL Chemiluminescence Kit (Beyotime Institute of Biotechnology). In this study, primary antibiotics were rabbit anti-MEX3A (1 : 500, ab79046, Abcam, UK) and rabbit anti-GAPDH (1 : 2500, ab9485, Abcam, UK).


### 2.6. CCK-8 Assay

Transfected cells were inoculated into a 96-well plate (2×103 cells/well). By using the CCK-8 kit, cell proliferation was assessed (Dojindo, Kumamoto, Japan) after cell cultivation. Optical density at 450 nm presented cell proliferative ability.


### 2.7. Cell Migration and Invasion Assays

Cells were placed in a 6-well plate (1×105 cells/well). When cells reached 100% confluence, a 10 μL pipette was used to create a scratch area in the central region of the monolayer cells. Then, cells were placed into a medium containing 1% FBS. At 0 h and 30 h, images of cell migration were taken and analyzed.After cell transfection, MDA-MB-231 and MCF7 cells were suspended in a serum-free medium. Next, suspended cells were seeded to the upper side of the chamber (Corning Incorporated, Corning, NY, USA). The bottom side was injected the medium with 10% FBS. After cell incubation, invasive cells were treated with 4% formaldehyde solution and 0.25% crystal violet. Afterwards, cells were washed twice with PBS and air-dried. Number of invading cells was observed by using an inverted microscope (Olympus Corporation, Tokyo, Japan).


### 2.8. Cell Apoptosis Detection

Transfected cells were prepared and double stained through using the PI-Annexin-V apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Apoptosis status was analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).


### 2.9. Dual-Luciferase Reporter Gene Detection

Wild-type (wt) and mutant-type (mut) sequences of MEX3A-3′UTR were subcloned into PGL3-M (Promega, Madison, WI, USA). MicroRNA-139-5p mimics or NC mimics were cotransfected into MDA-MB-231 cells with PGL3-3′UTR-wt or PGL3-MEX3A-3′UTR-mut. Luciferase activity was quantified using a dual-luciferase reporter gene system (Promega, USA).


### 2.10. Data Analysis

SPSS 22.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. Data were presented in the form ofmean±standarddeviation. t-test was introduced for two-group comparison. A statistically significant difference was considered when p value was less than 0.05.


## 2.1. Bioinformatics Approaches

Expression data information of mature microRNAs (normal: 104, tumor: 1,103) and mRNAs (normal: 113, tumor: 1,109) of TCGA-BRCA was accessed from The Cancer Genome Atlas (TCGA) database. MicroRNA-139-5p expression was analyzed on basis of the downloaded data. R package “edgeR” was used to carry out differential expression analysis on mRNAs in TCGA-BRCA dataset with the normal group as the control and∣logFC∣>2, padj<0.01 as thresholds to acquire differentially expressed mRNAs (DEmRNAs). MicroRNA-139-5p target binding prediction was conducted by using targetScan, miRDB, and mirDIP databases, and the predicted mRNAs were intersected with upregulated DEmRNAs to screen the potential miR-139-5p targets. Correlation analysis was conducted to select miR-139-5p target mRNA presenting the highist negative correlation efficiency with miR-139-5p at expression level. Survival analysis of 11 target genes was processed by the GEPIA database.


## 2.2. Cell Cultivation

Human normal breast epithelial cell line MCF-10A (BNCC337734) and human BC cell lines MDA-MB-231 (BNCC337893), MCF7 (BNCC337656), HCC1937 (BNCC338509), and MX-1 (BNCC100280) were offered by BeNa Culture Collection (Beijing, China). MCF-10A, HCC1937, and MX-1 were grown in 90% RPMI-1640HyClone,USA+10% fetal bovine serum (FBS; Thermo Scientific HyClone, Beijing, China). MDA-MB-231 and MCF7 were cultivated in DMEM (Thermo, USA) with 10% FBS.


## 2.3. Transfection of Cells

MicroRNA-139-5p mimics, microRNA-139-5p inhibitor, oe-MEX3A, and corresponding negative controls were offered by GenePharma Company (Shanghai, P.R. China). Nontransfected control was presented to identify the transfection efficiency (FigureS1). 1.5 μL/mL Lipofectamine 2000 transfection reagent (ThermoFisher, USA) was utilized to get 50 nM gene segment or 1 μg plasmid transfected into BC cells. Experiments were carried out 24 h after cell culture, and transfection lasted for 48 h.


## 2.4. qRT-PCR

After total RNA was isolated by TRIzol reagent (ThemorFisher, USA), it was quantified using NanoDrop (ThermoFisher, USA). Reverse transcription of mRNAs and microRNA was performed by using the SuperScript III First-Strand Synthesis System kit (Invitrogen, USA) and NCode™ VILO™ microRNA cDNA Synthesis kit (Life Technologies). qRT-PCR analysis of mRNAs and microRNAs was conducted by using the SoFast™ EvaGreenH Supermix (Bio-Rad) and EXPRESS SYBR Green ER microRNA qRT-PCR kit (Life Technologies), respectively. U6 and GAPDH were used as internal control genes. The following were primer sequences: microRNA-139-5p forward: 5′-TCTACAGTGCACGTGTC-3′, reverse: 5′-GAATACCTCGGACCCTGC-3′; U6 forward: 5′-CTCGCTTCGGCAGCACATA-3′, reverse: 5′-AACGATTCACGAATTTGCGT-3′; MEX3A forward: 5′-CGGAGTGCACTCTGGCTTTGAG-3′, reverse: 5′-CAGAGGAGAAGAGCACGGAGGT-3′; GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′; reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′. MicroRNA or mRNA relative expression was analyzed by the 2-ΔΔCt method.


## 2.5. Western Blotting

RIPA buffer solution (Solarbio, Beijing, China) was used to isolate total proteins in each group. The concentration of extracted samples was determined by the BCA assay kit (Abcam, Cambridge, UK). Separated by 10% SDS-PAGE, samples were transferred onto PVDF membrane (Invitrogen, USA), followed by incubation with primary antibodies. Then, proteins were treated with a secondary antibody (goat anti-rabbit IgG (1 : 2000, ab205718, Abcam, UK)) for 1 h. Protein levels were detected by the BeyoECL Chemiluminescence Kit (Beyotime Institute of Biotechnology). In this study, primary antibiotics were rabbit anti-MEX3A (1 : 500, ab79046, Abcam, UK) and rabbit anti-GAPDH (1 : 2500, ab9485, Abcam, UK).


## 2.6. CCK-8 Assay

Transfected cells were inoculated into a 96-well plate (2×103 cells/well). By using the CCK-8 kit, cell proliferation was assessed (Dojindo, Kumamoto, Japan) after cell cultivation. Optical density at 450 nm presented cell proliferative ability.


## 2.7. Cell Migration and Invasion Assays

Cells were placed in a 6-well plate (1×105 cells/well). When cells reached 100% confluence, a 10 μL pipette was used to create a scratch area in the central region of the monolayer cells. Then, cells were placed into a medium containing 1% FBS. At 0 h and 30 h, images of cell migration were taken and analyzed.After cell transfection, MDA-MB-231 and MCF7 cells were suspended in a serum-free medium. Next, suspended cells were seeded to the upper side of the chamber (Corning Incorporated, Corning, NY, USA). The bottom side was injected the medium with 10% FBS. After cell incubation, invasive cells were treated with 4% formaldehyde solution and 0.25% crystal violet. Afterwards, cells were washed twice with PBS and air-dried. Number of invading cells was observed by using an inverted microscope (Olympus Corporation, Tokyo, Japan).


## 2.8. Cell Apoptosis Detection

Transfected cells were prepared and double stained through using the PI-Annexin-V apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Apoptosis status was analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).


## 2.9. Dual-Luciferase Reporter Gene Detection

Wild-type (wt) and mutant-type (mut) sequences of MEX3A-3′UTR were subcloned into PGL3-M (Promega, Madison, WI, USA). MicroRNA-139-5p mimics or NC mimics were cotransfected into MDA-MB-231 cells with PGL3-3′UTR-wt or PGL3-MEX3A-3′UTR-mut. Luciferase activity was quantified using a dual-luciferase reporter gene system (Promega, USA).


## 2.10. Data Analysis

SPSS 22.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. Data were presented in the form ofmean±standarddeviation. t-test was introduced for two-group comparison. A statistically significant difference was considered when p value was less than 0.05.


## 3. Results

### 3.1. Significant Downregulation of MicroRNA-139-5p in BC

It is suggested by numerous studies that microRNA-139-5p is crucial in tumor progression [15, 16]. Hence, we attempted to illustrate functions of microRNA-139-5p in BC. TCGA-BRCA data indicated that the expression level of microRNA-139-5p in BC tissue was notably reduced (Figure 1(a)). Subsequent qRT-PCR result exhibited the same result (Figure 1(b)). MDA-MB-231 and MCF7 cells were used for cell functional assays later.Figure 1
Significant microRNA-139-5p downregulation in BC: (a) boxplot of microRNA-139-5p expression in normal (green) and tumor tissues (red); (b) qRT-PCR was used to detect the expression of microRNA-139-5p in human normal breast epithelial cell line MCF-10A and BC cell lines MDA-MB-231, MCF7, HCC1937, and MX-1.∗p<0.05; ∗∗∗∗p<0.0001.
(a)(b)


### 3.2. MicroRNA-139-5p Upregulation Suppresses Migration and Proliferation and Accelerates Apoptosis of BC Cells

Results of qRT-PCR displayed that transfected microRNA-139-5p mimics obviously elevated the expression level of microRNA-139-5p which was reduced notably by the microRNA-139-5p inhibitor (Figures2(a) and 2(f)), which suggested that the transfection efficiency in each treatment group was great enough for subsequent experiments. As displayed in Figure 2(b) and Figure 2(g), microRNA-139-5p overexpression blocked BC cell proliferative ability; on the contrary, knockdown of microRNA-139-5p enhanced it. Then, we found through wound healing assay that microRNA-139-5p suppressed cell migration (Figures 2(c) and 2(h)). Similarly, Transwell results displayed that microRNA-139-5p had an inhibiting effect on the invasion of BC cells (Figures 2(d) and 2(i)). Finally, in order to determine whether cell apoptosis could be affected by microRNA-139-5p, a flow cytometry assay was used for apoptosis evaluation. The results indicated that the apoptosis rate of BC cells increased after transfection of microRNA-139-5p mimics and reduced after microRNA-139-5p inhibitor treatment (Figures 2(e) and 2(j)). It could draw a conclusion that microRNA-139-5p overexpression repressed the progression of BC cells and promoted apoptosis.Figure 2
MicroRNA-139-5p affects BC-related cell behaviors: (a, f) microRNA-139-5p transfection efficiency in BC examined by qRT-PCR; (b, g) proliferation status was assessed using the CCK-8 assay; (c, h) migration level was tested by wound healing assay (40x); (d, i) invasive ability was tested by Transwell assay (100x); (e, j) apoptotic level was evaluated by flow cytometry.∗p<0.05.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.3. MEX3A Is a Target of MicroRNA-139-5p in BC

EdgeR differential analysis showed that 1,405 genes were upregulated and 757 were downregulated (Figure3(a)). Then, targets of microRNA-139-5p were analyzed based on several public databases. Predicted targets were intersected with 1,405 upregulated DEmRNAs to obtain 11 target genes with targeted binding sites of microRNA-139-5p (Figure 3(b)). Survival analysis showed that the effect of target gene on survival time was not significant (Figure S2). The correlation analysis exhibited that the MEX3A presented the highest Pearson correlation coefficient with microRNA-139-5p and they were significantly negatively correlated (Figure 3(c)). Furthermore, MEX3A was noted to be significantly overexpressed in BC tissue (Figure 3(d)). At a cellular level, results of MEX3A were also prominently upregulated in BC cells (Figures 3(e) and 3(f)).Figure 3
MEX3A is targeted by microRNA-139-5p in BC: (a) volcano map of DEmRNAs, red and green dots indicate up- and downregulated DEmRNAs, respectively; (b) Venn diagram of intersection of predicted targets of microRNA-139-5p and upregulated DEmRNAs; (c) Pearson correlation analysis of microRNA-139-5p and its predicted targets; (d) boxplot of MEX3A expression in the normal group (green) and the tumor group (red); (e) qRT-PCR was used to test mRNA expression of MEX3A in MCF-10A, MDA-MB-231, MCF7, HCC1937, and MX-1 cells; (f) Western blotting was used to measure MEX3A protein expression in each cell line; (g) binding sites of microRNA-139-5p and MEX3A; (h) dual-luciferase detection was used to test whether microRNA-139-5p binds to MEX3A mRNA; (i, j) MEX3A mRNA and protein expression levels.∗p<0.05; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Binding sites of microRNA-139-5p on MEX3A mRNA were predicted by the TargetScan database (Figure3(g)). Subsequently, dual-luciferase assay detection observed that luciferase activity of the wild-type group could be significantly reduced under overexpression of microRNA-139-5p, while no effects on luciferase activity could be observed in the mutant group; therefore, we confirmed that MEX3A could be one target of microRNA-139-5p (Figure 3(h)). Next, we detected the regulation of microRNA-139-5p on MEX3A expression and noted that the upregulation of microRNA-139-5p restrained the expression level of MEX3A at mRNA and protein manners (Figures 3(i) and 3(j)).


### 3.4. MicroRNA-139-5p Modulates Progression of BC Cells via MEX3A

To investigate whether MEX3A played a vital role in microRNA-139-5p suppressing BC, we studied MDA-MB-231 and MCF7 cell lines after transfection. qRT-PCR result exhibited that the upregulation of microRNA-139-5p prominently reduced the mRNA level of MEX3A, while the mRNA level of MEX3A was restored after upregulating MEX3A (Figure4(a)). Western blotting also showed the same trend in the protein expression of MEX3A as the results of qRT-PCR (Figure 4(b)). Then, the CCK-8 assay was carried out on cells in each transfection group, and it was noted that the upregulation of microRNA-139-5p inhibited cell proliferation, which was reversed partially by the upregulation of MEX3A (Figure 4(c)). Cell migration and invasion assays were employed to find that upregulating microRNA-139-5p could restrain BC cell growth. However, when MDA-MB-231 and MCF7 cells cotransfected with microRNA-139-5p mimics and oe-MEX3A, the migratory and invasive abilities were significantly elevated in comparison with cells only treated with microRNA-139-5p mimics (Figures 4(d) and 4(e)). Finally, microRNA-139-5p overexpression elevated the apoptosis level of BC cells. However, the apoptosis level of BC cells with overexpressed microRNA-139-5p and MEX3A was significantly reduced (Figure 4(f)). These statistics indicated that MEX3A exerted a significant role in the inhibiting effect of microRNA-139-5p on BC progression.Figure 4
MicroRNA-139-5p/MEX3A regulates the progression of BC cells: (a, b) MEX3A mRNA and protein expression in BC cells in treatment groups; (c) cell proliferation status tested by CCK-8; (d) migration level of BC cells in treatment groups (40x) determined by wound healing assay; (e) invasion of BC cells in treatment groups analyzed by Transwell assay (100x); (f) apoptosis of BC cells in treatment groups evaluated by flow cytometry.∗p<0.05.
(a)(b)(c)(d)(e)(f)


## 3.1. Significant Downregulation of MicroRNA-139-5p in BC

It is suggested by numerous studies that microRNA-139-5p is crucial in tumor progression [15, 16]. Hence, we attempted to illustrate functions of microRNA-139-5p in BC. TCGA-BRCA data indicated that the expression level of microRNA-139-5p in BC tissue was notably reduced (Figure 1(a)). Subsequent qRT-PCR result exhibited the same result (Figure 1(b)). MDA-MB-231 and MCF7 cells were used for cell functional assays later.Figure 1
Significant microRNA-139-5p downregulation in BC: (a) boxplot of microRNA-139-5p expression in normal (green) and tumor tissues (red); (b) qRT-PCR was used to detect the expression of microRNA-139-5p in human normal breast epithelial cell line MCF-10A and BC cell lines MDA-MB-231, MCF7, HCC1937, and MX-1.∗p<0.05; ∗∗∗∗p<0.0001.
(a)(b)


## 3.2. MicroRNA-139-5p Upregulation Suppresses Migration and Proliferation and Accelerates Apoptosis of BC Cells

Results of qRT-PCR displayed that transfected microRNA-139-5p mimics obviously elevated the expression level of microRNA-139-5p which was reduced notably by the microRNA-139-5p inhibitor (Figures2(a) and 2(f)), which suggested that the transfection efficiency in each treatment group was great enough for subsequent experiments. As displayed in Figure 2(b) and Figure 2(g), microRNA-139-5p overexpression blocked BC cell proliferative ability; on the contrary, knockdown of microRNA-139-5p enhanced it. Then, we found through wound healing assay that microRNA-139-5p suppressed cell migration (Figures 2(c) and 2(h)). Similarly, Transwell results displayed that microRNA-139-5p had an inhibiting effect on the invasion of BC cells (Figures 2(d) and 2(i)). Finally, in order to determine whether cell apoptosis could be affected by microRNA-139-5p, a flow cytometry assay was used for apoptosis evaluation. The results indicated that the apoptosis rate of BC cells increased after transfection of microRNA-139-5p mimics and reduced after microRNA-139-5p inhibitor treatment (Figures 2(e) and 2(j)). It could draw a conclusion that microRNA-139-5p overexpression repressed the progression of BC cells and promoted apoptosis.Figure 2
MicroRNA-139-5p affects BC-related cell behaviors: (a, f) microRNA-139-5p transfection efficiency in BC examined by qRT-PCR; (b, g) proliferation status was assessed using the CCK-8 assay; (c, h) migration level was tested by wound healing assay (40x); (d, i) invasive ability was tested by Transwell assay (100x); (e, j) apoptotic level was evaluated by flow cytometry.∗p<0.05.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.3. MEX3A Is a Target of MicroRNA-139-5p in BC

EdgeR differential analysis showed that 1,405 genes were upregulated and 757 were downregulated (Figure3(a)). Then, targets of microRNA-139-5p were analyzed based on several public databases. Predicted targets were intersected with 1,405 upregulated DEmRNAs to obtain 11 target genes with targeted binding sites of microRNA-139-5p (Figure 3(b)). Survival analysis showed that the effect of target gene on survival time was not significant (Figure S2). The correlation analysis exhibited that the MEX3A presented the highest Pearson correlation coefficient with microRNA-139-5p and they were significantly negatively correlated (Figure 3(c)). Furthermore, MEX3A was noted to be significantly overexpressed in BC tissue (Figure 3(d)). At a cellular level, results of MEX3A were also prominently upregulated in BC cells (Figures 3(e) and 3(f)).Figure 3
MEX3A is targeted by microRNA-139-5p in BC: (a) volcano map of DEmRNAs, red and green dots indicate up- and downregulated DEmRNAs, respectively; (b) Venn diagram of intersection of predicted targets of microRNA-139-5p and upregulated DEmRNAs; (c) Pearson correlation analysis of microRNA-139-5p and its predicted targets; (d) boxplot of MEX3A expression in the normal group (green) and the tumor group (red); (e) qRT-PCR was used to test mRNA expression of MEX3A in MCF-10A, MDA-MB-231, MCF7, HCC1937, and MX-1 cells; (f) Western blotting was used to measure MEX3A protein expression in each cell line; (g) binding sites of microRNA-139-5p and MEX3A; (h) dual-luciferase detection was used to test whether microRNA-139-5p binds to MEX3A mRNA; (i, j) MEX3A mRNA and protein expression levels.∗p<0.05; ∗∗∗∗p<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Binding sites of microRNA-139-5p on MEX3A mRNA were predicted by the TargetScan database (Figure3(g)). Subsequently, dual-luciferase assay detection observed that luciferase activity of the wild-type group could be significantly reduced under overexpression of microRNA-139-5p, while no effects on luciferase activity could be observed in the mutant group; therefore, we confirmed that MEX3A could be one target of microRNA-139-5p (Figure 3(h)). Next, we detected the regulation of microRNA-139-5p on MEX3A expression and noted that the upregulation of microRNA-139-5p restrained the expression level of MEX3A at mRNA and protein manners (Figures 3(i) and 3(j)).


## 3.4. MicroRNA-139-5p Modulates Progression of BC Cells via MEX3A

To investigate whether MEX3A played a vital role in microRNA-139-5p suppressing BC, we studied MDA-MB-231 and MCF7 cell lines after transfection. qRT-PCR result exhibited that the upregulation of microRNA-139-5p prominently reduced the mRNA level of MEX3A, while the mRNA level of MEX3A was restored after upregulating MEX3A (Figure4(a)). Western blotting also showed the same trend in the protein expression of MEX3A as the results of qRT-PCR (Figure 4(b)). Then, the CCK-8 assay was carried out on cells in each transfection group, and it was noted that the upregulation of microRNA-139-5p inhibited cell proliferation, which was reversed partially by the upregulation of MEX3A (Figure 4(c)). Cell migration and invasion assays were employed to find that upregulating microRNA-139-5p could restrain BC cell growth. However, when MDA-MB-231 and MCF7 cells cotransfected with microRNA-139-5p mimics and oe-MEX3A, the migratory and invasive abilities were significantly elevated in comparison with cells only treated with microRNA-139-5p mimics (Figures 4(d) and 4(e)). Finally, microRNA-139-5p overexpression elevated the apoptosis level of BC cells. However, the apoptosis level of BC cells with overexpressed microRNA-139-5p and MEX3A was significantly reduced (Figure 4(f)). These statistics indicated that MEX3A exerted a significant role in the inhibiting effect of microRNA-139-5p on BC progression.Figure 4
MicroRNA-139-5p/MEX3A regulates the progression of BC cells: (a, b) MEX3A mRNA and protein expression in BC cells in treatment groups; (c) cell proliferation status tested by CCK-8; (d) migration level of BC cells in treatment groups (40x) determined by wound healing assay; (e) invasion of BC cells in treatment groups analyzed by Transwell assay (100x); (f) apoptosis of BC cells in treatment groups evaluated by flow cytometry.∗p<0.05.
(a)(b)(c)(d)(e)(f)


## 4. Discussion

Multiple studies indicate that dysregulation of microRNAs is clinically linked with tumor growth and chemotherapy resistance, leading to high incidence and mortality in patients [17–19]. Previous literature has discovered that microRNA-139-5p is prominently lowly expressed in many cancers [20, 21]. Similarly, we noted that microRNA-139-5p was remarkably downregulated in BC. Through functional experiments, upregulated microRNA-139-5p performed a suppressive function in proliferation and migration and facilitate apoptosis of BC cells, which was the same as the results of Zhang et al. [22]. MicroRNA-139-5p can regulate the development of other cancers. For instance, microRNA-139-5p targets NOTCH1 to inhibit colorectal cancer cell proliferation and metastasis and promote apoptosis [23]. Combined with the above findings, we believe that microRNA-139-5p exerts tumor-suppression functions in BC .MEX3 protein is crucial in self-renewal and differentiation and is also involved in the development of cancer cells. For instance, MEX3A expression in gastric cancer tissue is dramatically higher when it is compared with that in adjacent normal tissue, and knockout of hMEX-3A reduces the colony formation ability of gastric cancer cells as well as significantly affects the survival of cancer cells [24]. In colon cancer, MEX3A is involved in regulating colorectal epithelial homeostasis, injury response, and malignant transformation and is significantly upregulated [25]. In this study, we mainly explored its regulatory effect on BC cell functions. The results presented that MEX3A was overexpressed in BC and was significantly negatively correlated with microRNA-139-5p. We also noted that overexpression of microRNA-139-5p restrained the expression of MEX3A directly through binding to the 3′UTR of MEX3A. Moreover, functional analysis suggested that microRNA-139-5p and MEX3A overexpression attenuated the suppressive effect of overexpressing microRNA-139-5p alone on BC cell growth. On the above basis, we discovered that inhibition of microRNA-139-5p on BC cells was partially achieved by targeting MEX3A.Our study demonstrated remarkable downregulation of microRNA-139-5p in BC, and abnormal expression of microRNA-139-5p inhibited malignant behaviors of BC cells by directly targeting MEX3A. Combined with our current results and previous findings, we proposed microRNA-139-5p regulates BC development as a tumor suppressor. This study enables us to further understand the mechanism of microRNA-139-5p in BC, which can develop new approaches for BC treatment. Although the experimental results in this paper are sufficient to prove the regulatory relationship between microRNA-139-5p and MEX3A, there are still some weaknesses. For instance, due to limited conditions, no clinical studies were conducted to further explore the influence of microRNA on the prognosis of patients in clinical practice, and no clinical tissue was utilized to further validate the relationship between microRNA-139-5p and MEX3A.



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*Source: 6591541-2021-11-02.xml*