MiR-191-5p inhibits KLF6 to promote epithelial-mesenchymal transition in breast cancer

Abstract

BACKGROUND:

MicroRNAs (miRNAs) exert certain functions in the development of several cancers and can be a potential hallmark for cancer diagnosis and prognosis. MiR-191-5p has been proven to have high expression in breast cancer (BC), while its biological role and potential regulatory mechanisms in BC remain an open issue.

OBJECTIVE:

Bioinformatics was utilized to assay miR-191-5p level in BC tissues and predict its downstream target gene as well as the enriched signaling pathways of the target gene.

METHODS:

qRT-PCR was carried out to assay miR-191-5p and KLF6 levels in BC cells as well as miR-191-5p level in blood-derived exosomes from BC patients. Western blot was to examine the expression of proteins linked with cell adhesion, epithelial-mesenchymal transition (EMT), and exosome markers. A dual luciferase reporter assay was utilized to verify the interaction between miR-191-5p and KLF6. Abilities of cell phenotypes of BC cells were detected by CCK8, Transwell, and cell adhesion assay, separately.

RESULTS:

Upregulated miR-191-5p expression and downregulated KLF6 expression were observed in BC cells. There was a targeting relationship between miR-191-5p and KLF6. MiR-191-5p negatively regulated KLF6 to promote EMT and malignant progression of BC cells. Additionally, we described a dramatically high level of miR-191-5p in the blood exosomes of BC patients.

CONCLUSION:

MiR-191-5p advances the EMT of BC by targeting KLF6, indicating that miR-191-5p and KLF6 may be new biomarkers for BC.

Keywords

microRNAs KLF6 protein breast neoplasms cell adhesion epithelial-mesenchymal transition

1. Introduction

Breast cancer (BC), the most common cancer in women worldwide [1, 2], posts daunting challenges to women’s health globally [3]. Current treatments for BC generally include cell decompression surgery, radiotherapy, targeted endocrine/molecular therapy, immune checkpoint inhibitors therapy, and neoadjuvant chemotherapy [4, 5, 6]. In recent years, though advances in early diagnosis and treatment strategies have markedly improved BC patients’ survival and prognosis [7, 8], some BC patients still develop metastases during diagnosis and treatment, along with a grim 5-year survival rate of 26% [9, 10]. Metastasis is a major feature of malignancy and a leading cause of BC-related deaths [11, 12], while cell migration and motility are enhanced in epithelial cells during epithelial-mesenchymal transition (EMT), which is indispensable for cancer cell metastasis [13]. At present, how EMT functions in BC progression has been ascertained. SKA2 inducing BC invasion and metastasis through EMT [14]. HH/GLI1 axis regulates migration and invasion of BC cells by altering levels of EMT markers [15]. Inhibition of EMT in BC cells can reduce BC invasion and drug resistance [16]. It is thus clear that EMT functions a crucial role in BC metastasis. Therefore, it is a burning issue to fully dig into the molecular mechanism, mine effective molecular targets, find suitable biomarkers of BC metastasis, and provide a theoretical foundation for BC clinical treatment.

MicroRNAs (miRNAs) can bind to 3’UTR of mRNA and participate in stress response, cell cycle regulation, differentiation, migration, and apoptosis [17, 18, 19]. The function of miRNAs in BC progression has been studied before. MiR-454-3p stimulates the Wnt/ $\beta$ -catenin signaling through suppressing RPRD1A, thereby accelerating BC metastasis [7]. MiR-21 accelerates BC metastasis and tumor growth by stimulating the EMT process [20]. MiR-142-3p enhances paclitaxel sensitivity in drug-resistant BC by affecting the AKT-mTOR pathway via targeting GNB2 [21]. MiR-140-5p represses BC cells to migrate and invade via inhibition of the AKT/STAT3/NF- $\kappa$ B pathway [22]. MiR-191-5p is a key miRNA involved in cancer development. Zhou et al. [23] ascertained that miR-191-5p targets SATB1 to hinder the Wnt pathway, thereby suppressing malignant behaviors of lung adenocarcinoma cells. Chen et al. [24] reported that miR-191-5p exerts a suppressive effect on renal cell carcinoma (RCC), and down-regulation of miR-191-5p can raise the malignant behavior of RCC cells. Due to tumor heterogeneity, miR-191-5p is also a cancer promoter in certain types of cancer, miR-191-5p enhances the progression of osteosarcoma by targeting EGR1 to activate PI3K/AKT signaling pathway [25]. MiR-191-5p, sponged by lncRNA elNF1-AS1, inhibits colon cancer growth and metastasis by binding to TRIM5 [26]. Mounting research displays the potential roles of miRNAs in cancer diagnosis, treatment, and prognosis prediction [27, 28]. Strikingly, miR-191-5p was also ascertained to be a type of blood circulation-derived exosome [29]. In summary, miR-191-5p featured in tumor progression. However, studies on miR-191-5p in BC cell biology and metastasis remain an open issue. Therefore, this paper dived into how miR-191-5p functions in BC metastasis and its regulatory mechanism.

Here, we found up-regulation of miR-191-5p in BC. Through molecular experiments and cell experiments, we demonstrated that miR-191-5p had a downstream regulatory gene (KLF6), and miR-191-5p enhanced cell adhesion and EMT in BC cells by regulating KLF6. Viewed in total, this paper revealed the molecular mechanism by which miR-191-5p/KLF6 affected BC metastasis, providing fresh knowledge on the mechanism of BC development and a theoretical ground for BC treatment, indicating miR-191-5p and KLF6 to be the feasible therapeutic targets for BC.

2. Materials and methods

2.1 Sample collection

Blood samples were gathered from 10 patients (aged 45–65 years) diagnosed with BC at Zigong Fourth People’s Hospital from July to September 2022. All of them did not receive any preoperative curing and no other underlying diseases were detected. Blood samples were also gathered from 10 healthy subjects between the ages of 45 and 65. All samples were immediately centrifuged to eliminate cellular debris. The resulting blood samples were preserved at $-$ 80 ${{}^{\circ}}$ C. This study was agreed by the ethics committee of Zigong Fourth People’s Hospital, and all subjects provided informed consent.

2.2 Bioinformatics analysis

From TCGA (https://portal.gdc.cancer.gov/), BC mRNA expression data (Normal: 113, Tumor: 1109) and miRNA expression data (Normal: 104, Tumor: 1103) were acquired. EdgeR differential analysis ( $|$ logFC $|$ $>$ 1, padj $<$ 0.05) was utilized to obtain mRNAs and miRNAs that were differentially expressed, and target miRNAs were determined by the literature review. Starbase (https://starbase.sysu.edu.cn/starbase2/browseClipSeq.php) was utilized to predict target genes of target miRNA, which were intersected with the differentially down-regulated mRNAs, and then the correlation between miRNA and target gene was calculated. The miRWalk database was utilized for target binding site prediction of target miRNA to mRNA, and target genes were subject to pathway enrichment analysis on GSEA software.

2.3 Cell culture

Breast epithelial cells MCF-10A (CRL-10317 ${}^{\text{TM}}$ ) and BC cells MCF-7 (CRL-3435 ${}^{\text{TM}}$ ), SKBR3 (HTB-30 ${}^{\text{TM}}$ ), and MDA-MB-231 (CRM-HTB-26 ${}^{\text{TM}}$ ) were bought from ATCC. These cell lines were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) at 37 ${{}^{\circ}}$ C with 5% CO ${}_{2}$ . The cultural conditions were carried out with reference to the literature [30, 31].

2.4 Extraction of exosomes

Referring to a previous report [32], differential centrifugation was utilized to isolate exosomes. Briefly, to collect the supernatant, blood samples were taken, and centrifuged at 300 $\times$ g for 10 min, then at 2000 $\times$ g for 10 min. After that, samples were centrifuged again at 10,000 $\times$ g (4 ${{}^{\circ}}$ C) for 30 min to eliminate large particles. The supernatant was collected by centrifugation at 4 ${{}^{\circ}}$ C and 100,000 $\times$ g (Beckman Ti70) for 70 min to gather exosomes. After discarding the supernatant, protein contamination was removed by resuspension with 12 mL of PBS. Finally, pure exosomes were obtained by centrifugation at 100,000 $\times$ g for 70 min.

2.5 Cell transfection

MiR-191-5p mimic (miR-mimic) and negative control (NC) mimic-NC; miRNA inhibitor (ASO-miR) and NC ASO-NC, pcDNA3.1-KLF6 recombinant plasmid (oe-KLF6) and the blank pcDNA3.1 plasmid (oe-NC) as its NC were all acquired from RiboBio (China). MiR-mimic, ASO-miR, pcDNA3.1-KLF6, pcDNA3.1, and the corresponding NCs were transfected into BC cells utilizing the Lipofectamine 2000 kit (Thermo Fisher, USA).

2.6 qRT-PCR

Total RNA extraction was processed by utilizing TRIzol reagent (Invitrogen, USA). 500 $\mu$ L of chloroform was added to 1 mL of TRIzol and mixed with the cell lysate. The upper aqueous phase was mixed with an equal amount of pre-chilled isopropanol, allowed to stand for 30 s, and then centrifuged at 4 ${{}^{\circ}}$ C. After the supernatant was aspirated and removed, RNA was rinsed with ethanol (75%), air-dried and diluted with 30 $\mu$ L diethyl pyrocarbonate-treated distilled water (Beyotime, China). The obtained RNA was reversely transcribed to obtain cDNA by utilizing AMV reverse transcriptase (TaKaRa, Japan). HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme, China) was utilized to assay the expression of miR-191-5p and KLF6 by qRT-PCR on an ABI 7500 real-time PCR detection system (Applied Biosystems, USA) using cDNA as template. U6 and $\beta$ -actin were internal references for miR-191-5p and KLF6, separately. A total of three replicates of each sample were used to analyze the gene expression by 2 ${}^{-\Delta\Delta\text{CT}}$ method. Table 1 displays primer sequences.

Table 1
The qRT-PCR primers sequences

Primer	Forward	Reverse
miR-191-5p	5’-ACTGGATACGACCAGCTGCT-3’	5’-TGCGCCAACGGAATCCCAAAAGC-3’
KLF6	5’-AGTTAACCAGGCACTTCCG-3’	5’-CTTTTAGCCTACAGGATCCACC-3’
U6	5’-GCTTCGGCAGCACATATACTAAAAT-3’	5’-CTCAACTGGTGTCGTGGA-3’
$\beta$ -actin	5’-AGAAGGCTGGGGCTCATTTG-3’	5’-AGGGGCCATCCACAGTCTTC-3’

2.7 Western blot (WB)

After protein extraction using RIPA lysis buffer, protein concentration was tested by BCA. Proteins were added into an SDS-PAGE gel for electrophoretic separation. Proteins were transferred onto PVDF membranes and then sealed with skimmed milk (5%) for 2 h at room temperature. Subsequently, primary antibodies: (anti-KLF6, anti- $\beta$ -actin, anti-VCAM-1, anti-ICAM-1, anti-N-cadherin, anti-E-cadherin, anti-vimentin, anti-CD9, anti-CD81, and anti-FLOT1) were cultivated with PVDF membranes overnight at 4 ${{}^{\circ}}$ C. Membranes were then added with goat anti-rabbit IgG HRP secondary antibody (Abcam, UK) for 1 h of culture at room temperature. Detection was performed by chemiluminescence and imaged on the AlphaView analysis system (ProteinSimple, USA). Quantitative analysis of protein bands was performed by ImageJ software. The antibodies were all bought from Abcam, UK.

2.8 Cell counting kit-8 (CCK-8) assay

Cells were placed into 96-well plates for 0, 24, 48, 72, and 96 h of incubation. After that, each well of the plates with 10 $\mu$ L of CCK-8 solution addition was cultivated for 2 h. A microplate reader (BioTex, USA) was utilized to detect absorbance at 450 nm. Three biological replicates were done for each set of experiments.

2.9 Transwell migration/invasion assay

For migration detection, cells in serum-free medium were plated into inserts of the Transwell chamber. After incubation for 24 h, the cells left in the upper layer were removed with cotton swabs. The remained cells were fixed with methanol and stained with 0.1% crystal violet. Migrating cells were observed and counted under an optical microscope.

For invasion detection, the Transwell membrane was pre-coated with matrix gel at 4 ${{}^{\circ}}$ C for 12 h. Cells in serum-free medium were plated in inserts of a Matrigel-coated Transwell chamber. Then, a culture medium containing 10% FBS was supplemented to the lower chamber. After 24 h, non-invaded cells were removed, and invaded cells underwent 15 min of fixation with 70% ethanol and 20 min of staining with 0.1% crystal violet. Finally, images were taken by an optical microscope.

2.10 Dual-luciferase reporter gene assay

PmirGLO-KLF6-3’-UTR-WT and pmirGLO-KLF6-3’-UTR-MUT luciferase reporter plasmids (GenePharma, China) were constructed. Then, wild-type or mutant KLF6-WT/KLF6-MUT plasmids and miR-191-5p mimic/mimic-NC were co-transfected into MDA-MB-231 cells. After 48 h, the activities of firefly luciferase and Renilla luciferase in each group were assayed by dual luciferase analysis system (Promega, USA). The experiment was done in triplicate.

2.11 Cell adhesion assays

For detecting the adhesion ability, BC cells (2 $\times$ 10 ${}^{4}$ cells/well) were plated into 96-well plates and cultivated at 37 ${}^{\circ}$ C for 4 h. After that, the plate was rinsed twice with PBS, sequentially fixed and stained with cold methanol and 0.5% crystal violet, and finally rinsed with PBS for 5 times. The number of adherent cells was photographed and counted in 5 random fields and normalized to the number of specific controls [33].

2.12 Electron microscopy

The isolated exosomes were detected by transmission electron microscopy (TEM). A drop of purified exosomes (about 10 $\mu$ L) was immobilized with 1% glutaraldehyde for 10 min and then mounted on a carbon-coated copper grid. After staining with 1% uranyl acetate and drying, the copper grid was placed on HT7700 TEM (Hitachi, Japan). Images of exosomes were taken and their sizes were calculated [32].

2.13 Statistical analysis

One-way analysis of variance or $t$ -test was utilized for analysis. Mean $\pm$ standard deviation was utilized to express data. SPSS Statistics 21 software was introduced for statistical analyses. Statistical significance was identified as $P<$ 0.05.

3. Results

3.1 MiR-191-5p is notably up-regulated in BC tissues and cells

The expression data of BC miRNA were gained from TCGA database, and differentially expressed mRNAs were obtained by EdgeR differential analysis. The target miRNA was identified as miR-191-5p by consulting the literature and differential data. Bioinformatics analysis showed that miR-191-5p was up-regulated in BC tissues versus normal tissues (Fig. 1A). Afterward, miR-191-5p expression in BC and normal breast epithelial cell lines was assessed by qRT-PCR, which showed higher expression in BC cell lines (Fig. 1B). These findings added up to the statement that miR-191-5p was notably increased in BC tissues and cells, and may modulate BC development.

Figure 1.

Up-regulated expression of miR-191-5p in BC tissues and cells. (A) Violin plot of miR-191-5p expression in BC tissues (pink) and normal tissues (green); (B) qRT-PCR was utilized to assay the expression of miR-191-5p in BC cell lines (MCF-7, SKBR3, and MDA-MB-231) and normal breast epithelial cell line (MCF-10A); ${}^{*}P<$ 0.05.

Figure 2.

MiR-191-5p promotes the malignant phenotypes and EMT process in BC. (A) Cell viability was analyzed by CCK-8 assay; (B) The effect of each treatment on cell adhesion was assayed by cell adhesion assay; (C) The expression of cell adhesion-related proteins in different treatment groups; (D) The migration and invasion of SKBR3 and MDA-MB-231 cells in different treatment groups; (E) The expression of EMT-related proteins in different treatment groups. ${}^{*}P<$ 0.05.

Figure 3.

MiR-191-5p targets KLF6. (A) Upset plot of downstream target genes of miR-191-5p predicted by Starbase and differentially down-regulated mRNAs; (B) Pearson correlation between miR-191-5p and KLF6; (C) Violin plot of KLF6 expression in BC tissues (pink) and normal tissues (green); (D) The expression of KLF6 in BC cell lines (MCF-7, SKBR3, and MDA-MB-231) and normal breast epithelial cell line (MCF-10A) was detected by qRT-PCR; (E) Bioinformatics analysis predicted that KLF6 mRNA-3’UTR was targeted by miR-191-5p. (F) Binding relationship between miR-191-5p and KLF6; (G) Expression of KLF6 mRNA in MDA-MB-231 cells after transfection with ASO-NC/ASO-miR.

Figure 4.

MiR-191-5p inhibits KLF6 expression and promotes EMT in BC. (A) qRT-PCR was used to detect the expression of KLF6 in each treatment group; (B) CCK-8 was used to analyze the cell viability of each treatment group; (C) Cell adhesion assay was used to detect the effect of each treatment group on cell adhesion; (D) WB was used to detect the expression levels of adhesion-related proteins in different treatment groups; (E) GSEA pathway enrichment results of KLF6; (F) Transwell migration assay was used to detect the migration and invasion of MDA-MB-231 cells after different treatments; (G) WB was used to detect the expression of EMT-related proteins in different treatment groups. ${}^{*}$ vs mimic-NC+oe-NC, ${}^{\#}$ vs mimic-NC+oe-KLF6; ${}^{*}$ , ${}^{\#}P<$ 0.05.

Figure 5.

Isolation of blood-derived exosomes from BC patients and expression of miR-191-5p. (A) Source of miR-191-5p checked by EVmiRNA website; (B) TEM images of exosomes extracted from the blood of healthy volunteers and BC patients; (C) Exosomal markers CD9, CD81, and FLOT1 in blood samples of healthy volunteers and BC patients were detected by WB; (D) The expression of miR-191-5p in exosomes of normal and BC patients was detected by qRT-PCR. ${}^{*}P<$ 0.05.

3.2 Influence of miR-191-5p on EMT in BC

Through literature review, miR-191-5p turned up to be involved in the cell adhesion pathway [34], and studies have reported that FOCAL ADHESION can enhance EMT progression [35, 36]. Hence, we speculated that miR-191-5p may affect EMT progression. Since miR-191-5p had higher expression in MDA-MB-231 and lower in SKBR3, we utilized MDA-MB-231 and SKBR3 cells for miR-191-5p knockdown and overexpression treatments, separately. The modulatory role of miR-191-5p in BC was studied by transfecting mimic-NC/miR-mimic into SKBR3 cells, and ASO-NC/ASO-miR into MDA-MB-231 cells. The control group mimic-NC, miR-mimic notably enhanced SKBR3 cell viability, while ASO-miR hampered the viability of MDA-MB-231 cells, as CCK-8 assay illustrated (Fig. 2A). The outcomes of cell adhesion assay illustrated that in contrast to the control group, increased miR-191-5p boosted the adhesion of SKBR3 cells markedly, while ASO-miR markedly reduced the adhesion ability of MDA-MB-231 cells (Fig. 2B). WB was utilized to assess expression of cell adhesion-related proteins ICAM-1 and VCAM-1, with the outcomes showing that their expression was increased in miR-191-5p overexpression group but decreased in ASO-miR-treated cells (Fig. 2C). Transwell assay results unveiled that invasion and migration of cells overexpressing miR-191-5p were prominently enhanced, while the two abilities of cells in the ASO-miR treatment group were observably reduced (Fig. 2D). Finally, we utilized WB to measure EMT-related proteins expression, showing that E-cadherin expression was hindered and N-cadherin and Vimentin expression was prominently increased in miR-191-5p overexpressing cells, while expression changes of EMT-related proteins in ASO-miR treatment group were contrary (Fig. 2E). In summary, miR-191-5p accelerated BC cell phenotypes and EMT.

3.3 KLF6 is the downstream target gene of miR-191-5p

We utilized Starbase to predict its downstream target genes to study the mechanism of miR-191-5p in BC. The predicted results were overlapped with 2066 differentially down-regulated mRNAs to obtain 31 potential target mRNAs (Fig. 3A). Pearson correlation analysis found a negative correlation of KLF6 with miR-191-5p (cor $=-$ 0.249) (Fig. 3B), so it was determined to be the downstream target gene of miR-191-5p. As bioinformatics analysis revealed, its expression was observably hindered in BC tissues (Fig. 3C). Based on subsequent qRT-PCR analysis, KLF6 expression was markedly hindered in BC cell lines, consistent with bioinformatics results (Fig. 3D). Since the cell phenotype was more prominent in MDA-MB-231 cells, we next selected this cell line for experiments. Bind sites of miR-191-5p on KLF6 3’UTR were revealed by bioinformatics analysis (Fig. 3E). Next, we sought to determine whether KLF6 3’UTR is directly targeted by miR-191-5p. We utilized dual luciferase reporter assay to verify the binding relationship between the two, with results showing that increased miR-191-5p hampered luciferase activity of the KLF6-wt group, but had no influence on that of the KLF6-mut group (Fig. 3F), which indicated that miR-191-5p bound to the 3’UTR of KLF6 mRNA directly. Finally, qRT-PCR was utilized to assay KLF6 level in MDA-MB-231 cells after ASO-NC/ASO-miR treatment, whose results presented that KLF6 expression was dramatically increased in the ASO-miR treatment group (Fig. 3G). Thus, miR-191-5p downregulated KLF6 expression in BC cells.

3.4 MiR-191-5p promotes BC EMT via KLF6

For further exploration of the impact of miR-191-5p-mediated KLF6 on EMT in BC, we set up cell grouping (mimic-NC+oe-NC, mimic-NC+oe-KLF6, and miR-mimic+oe-KLF6) for subsequent experiments. Firstly, qRT-PCR was conducted to assay KLF6 expression in each treatment group, with results showing that KLF6 expression in mimic-NC+oe-KLF6 group was notably higher, while KLF6 expression in miR-mimic+oe-KLF6 treatment group returned to the level of control group (Fig. 4A). Subsequently, CCK-8 analysis in each group showed that cell viability was prominently reduced after KLF6 was overexpressed, and forced expression of miR-191-5p offset the impact of KLF6 overexpression on cell viability (Fig. 4B). Subsequently, cell adhesion was investigated, with results showing that cell adhesion ability of the mimic-NC+oe-KLF6 treatment group was reduced, and forced expression of KLF6 could offset the increased cell adhesion ability caused by miR-191-5p forced expression (Fig. 4C). Based on WB, KLF6 expression was dramatically down-regulated after forced expression, and KLF6 overexpression could offset impact of miR-191-5p forced expression on cell adhesion-related protein expression (Fig. 4D). GSEA presented that KLF6 was remarkably enriched in FOCAL ADHESION signaling (Fig. 4E) and might activate the activation of FOCAL ADHESION pathway. Relevant studies have revealed that the FOCAL ADHESION signaling pathway can accelerate EMT progression [37]. Based on the above analysis, we speculated the function of miR-191-5p in promoting BC by activating related adhesion proteins in the FOCAL ADHESION signaling pathway through KLF6. We then examined how cells migrated and invaded in different treatment groups. Cell migration and invasion were prominently decreased with overexpression of KLF6, and KLF6 overexpression notably offset the impact of increased miR-191-5p on cell migration and invasion, which could be seen in Transwell (Fig. 4F). Finally, we adopted WB to assay the expression of EMT-related proteins, with data showing that EMT-related proteins were prominently down-regulated in the mimic-NC+oe-KLF6 group, and KLF6 overexpression alleviated the up-regulation of EMT-related proteins caused by miR-191-5p overexpression (Fig. 4G). Taken together, miR-191-5p facilitated malignant behaviors and EMT of BC cells by targeting KLF6.

3.5 MiR-191-5p is derived from blood exosomes of patients

MiR-191-5p was reported to be an exosome derived from blood circulation [29]. We learned that miR-191-5p is largely derived from blood by searching the EVmiRNA (http://bioinfo.life.hust.edu.cn/EVmiRNA#!/) (Fig. 5A). Then, blood exosomes of BC patients and healthy volunteers were isolated by differential centrifugation, and the morphology of exosomes was identified by TEM. They showed round or oval membranous vesicles (Fig. 5B). In addition, we applied WB to confirm the expression of exosome markers (CD9, CD81, FLOT1) in the blood of BC patients (Fig. 5C). Finally, we obtained total RNA from 10 ug exosomes and assayed miR-191-5p expression by qRT-PCR, with results showing that miR-191-5p had a higher level in blood exosomes of BC patients than in blood exosomes from healthy people (Fig. 5D). In summary, miR-191-5p had high expression in blood-derived exosomes of BC patients.

4. Discussion

EMT is concerned with tumor initiation, metastasis, and drug resistance [38, 39]. During EMT, epithelial cells acquire mesenchymal morphology by up-regulating mesenchymal marker expression and decreasing epithelial marker expression responsible for tight junction formation [40]. EMT featured in the progression of BC [41]. Cheng et al. [42] confirmed that WT1 suppresses the transcription of STIM1, which activates calcium channels to increase Ca ${}^{2+}$ entry, thereby promoting EMT and BC cell progression. Additionally, a close link between tumor cell adhesion and EMT was found [35, 36]. For example, lncRNA UNC5B-AS1 facilitates malignant progression and EMT of lung cancer cells via targeting miR-218-5p [43]. Additionally, both the occurrence of EMT and the change of cell adhesion ability feature in tumor metastasis. For example, miR-32 facilitates migration, invasion, and adhesion of esophageal squamous cell carcinoma (ESCC) cells and the EMT process through CXXC5, thereby promoting the metastasis of ESCC [44]. Dhawan [45] suggests that the ICAM-1-ICAM-1 homology interaction of BC cells with bone marrow mesenchymal stem cells has mediatory function on the metastatic expansion of cancer cells, shifting hematopoietic stem cells out of their niches. Here, we dived into the function of miR-191-5p/KLF6 in BC cell adhesion and EMT.

Having been reported to be one of the key miRNAs involved in cancer progression, miR-191-5p, whose high expression in ESCC, gastric cancer, and BC facilitating malignant progression of tumors has been reported by previous studies [46, 47, 48]. For example, miR-191-5p was down-regulated in liver cancer, which hampers EMT and cancer stemness [49]. Due to tumor heterogeneity, miR-191-5p is a tumor suppressor gene in renal and lung adenocarcinoma [23, 24], such as in BC, miR-191-5p inhibits BC cell apoptosis and adriamycin sensitivity by targeting SOX4 [50]. In lung cancer, forced expression of miR-191-5p suppresses proliferation and migration of A549 and H1650 cells [23]. Here, miR-191-5p was ascertained to have high expression in BC, whose malignant phenotypes were enhanced by the forced expression of miR-191-5p.

In recent years, studies have learned that KLF6 participates in regulating cell proliferation, differentiation, and angiogenesis [51]. It is also a critical regulator of tumor progression. Studies have ascertained that KLF6 is a tumor suppressor, and its inhibition can up-regulate the expression of TGF- $\beta$ and enhance EMT progression [52]. Moreover, the tumor suppressor function of KLF6 was validated in cancers like hepatocellular carcinoma, thyroid cancer, and ovarian cancer [53, 54, 55, 56]. Similar results were obtained in our study. KLF6 was significantly down-regulated and functioned as a cancer repressor gene in BC. Additionally, mechanism studies disclosed in ovarian cancer, lncRNA LINP1 accelerates invasion and proliferation of ovarian cancer cells through KLF6 [56]. Platelet releasing substance accelerates proliferation and hinders apoptosis of liver cancer cells by hindering KLF6 expression [53]. Here, we validated that KLF6 was a target downstream of miR-191-5p in BC, and miR-191-5p could suppress KLF6 expression to raise the cell phenotypes and EMT of BC cells. Besides, we also confirmed the expression of exosomal markers in the blood of BC patients, and miR-191-5p had high expression in blood-derived exosomes of BC patients. Studies have ascertained that miRNAs are abundant in body fluids (such as blood), which are routinely examined, and miRNA molecules can be circulated in free or exosome-encapsulated forms [57, 58]. Therefore, miR-191-5p in this study might be an ideal biomarker for the early identification of BC disease.

Our findings illustrated the increased miR-191-5p in BC tissues and blood exosomes of BC patients. Molecular and cell experiments illustrated the role of miR-191-5p in promoting metastasis in BC progression. Further studies ascertained that miR-191-5p ultimately elevated metastasis by down-regulating the expression of KLF6 in BC. Here, we delineated the regulatory relationship and mechanism of miR-191-5p and KLF6 in vitro only but did not further verify it at animal and clinical levels. Moreover, this study did not deeply mine the downstream target genes and potential signaling pathways of KLF6. Therefore, we intend to continue to explore the downstream signaling pathway of KLF6, and mine the effect of miR-191-5p/KLF6 axis and its downstream regulatory genes in BC. We revealed a new mechanism of the miR-191-5p/KLF6 axis in inducing EMT in BC, enabling us to obtain a clearer appreciation of the pathogenesis of BC and furnishing us with a theoretical underpinning for mining the pathogenesis of BC and the development of potential therapeutic targets.

Author contributions

(I) Conception and design: Ling Pan, Xuandi Yue; (II) Administrative support: Wenya Liu; (III) Provision of study materials: Ling Pan, Hao Zhao; (IV) Collection and assembly of data: Bin Chen, Wenya Liu; (V) Data analysis and interpretation: Hao Zhao, Xuandi Yue. All authors wrote the manuscript and approved the final version.

Funding

Not applicable.

Ethics approval

This study was reviewed and approved by the Ethics Committee of Zigong Fourth People’s Hospital (approval number 2022 (020)).

Data availability statement

The data in this study are available from the corresponding author on reasonable request.

Footnotes

Conflict of interest

The authors declare that they have no conflict of interest.

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MiR-191-5p inhibits KLF6 to promote epithelial-mesenchymal transition in breast cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Sample collection

2.2 Bioinformatics analysis

2.3 Cell culture

2.4 Extraction of exosomes

2.5 Cell transfection

2.6 qRT-PCR

Table 1 The qRT-PCR primers sequences

2.8 Cell counting kit-8 (CCK-8) assay

2.9 Transwell migration/invasion assay

2.10 Dual-luciferase reporter gene assay

2.11 Cell adhesion assays

2.12 Electron microscopy

2.13 Statistical analysis

3. Results

3.1 MiR-191-5p is notably up-regulated in BC tissues and cells

3.3 KLF6 is the downstream target gene of miR-191-5p

3.4 MiR-191-5p promotes BC EMT via KLF6

3.5 MiR-191-5p is derived from blood exosomes of patients

4. Discussion

Author contributions

Funding

Ethics approval

Data availability statement

Footnotes

Conflict of interest

References

Table 1
The qRT-PCR primers sequences