microRNA-30d mediated breast cancer invasion, migration, and EMT by targeting KLF11 and activating STAT3 pathway

Mingli Han | Yimeng Wang | Guangcheng Guo| Lin Li | Dongwei Dou | Xin Ge|Pengwei Lv|Fang Wang| Yuanting Gu
1 Department of Breast Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
2 The Key Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
3 Department of Geriatric Endocrinology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Breast cancer, the most prevalent malignancy, is the main cause of cancer-related deaths in women with tumors and causes over 500 000 deaths annually among women with breast cancer worldwide.1,2 According to the literature, when breast cancers are invasive, the cancer will invade the normal pericarcinomatous tissue, and metastasize to other parts of the body, such as the lymph nodes.3 After treatment for breast cancer, including hormone therapy, radiation therapy, chemotherapy, and surgery, approximately 30-75% ofpatients may experience a recurrence; moreover, most patients with a recurrence have distant metastases.4,5 It has been well documented that tumor metastasis is commonly resistant to most therapeutic drugs and possesses a surgically inoperable nature, thus resulting in breast cancer deaths.6 Although a number of strategies have been examined to reduce the metastasis of breast cancer cells, determination of the mechanism of breast cancer cell metastasis remains a significant challenge.
Evidence has indicated that microRNAs (miRNAs) contribute to breast cancer cell metastasis.7 miRNAs,post-transcriptional gene regulators, are small (∼22 nucleo- tides) endogenous non-coding RNAs.8,9 The first miRNAexpression profiling involved breast cancer tissue in 2005.10 Accumulating data have suggested that several miRNAs are widely deregulated in human breast cancer and play pivotal roles in cancer therapeutic resistance, etiology, progression and cancer metastasis.11–13
miR-30d and miR-124a were reported to serve as novelprognostic biomarkers and therapeutic targets of breast cancer in patients with type 2 diabetes mellitus.14,15 It is shown by other studies that the miR-30 microRNA family (miR-30a,-30b, -30c-1, -30c-2,-30d, -30e) is extensively expressed in multiple cancer cells and plays diverse roles in regulating tumor growth and metastasis.16 miR-30a and -30d were reported as tumor suppressors in lung cancer, and miR-30a silences suppressed ovarian cancer cell proliferation andmigration; moreover, miR-30d promoted melanoma cell invasion and immunosuppression.17–20 The specific function and molecular mechanism of miR-30d in breast cancer cell growth and metastasis has not been addressed. Evidence indicated that miR-30 suppressed krüppel-like factor 11(KLF11) expression in hepatic stellate cells,21 and Faryna et al22 showed that KLF11 was hypermethylated in breast cancer; this hypermethylation may be associated with low expression and cancer metastases. Thus, we speculated that the mechanisms by which miR-30d contributes to oncogene- sis include the low expression of KLF-11.
In the present study, we define for the first time that miR- 30d has been implicated in breast cancer cell growth, migration, invasion and EMT and how miR-30d affects the progression of breast cancer. We showed that miR-30d silencing inhibits growth and metastasis in breast cancer cells. Subsequent experiments suggested that KLF-11 is a direct target of miR-30d, and miR-30d activates STAT3 expression though inhibiting KLF-11 expression. These novel results dissect the role and mechanism of miR-30d in breast cancer cell growth and metastasis.

2.1 | Cell culture and transfection
The human breast cancer cell lines BT474, MDA-MB-231, HCC197, and MDA-MB-468 and the non-tumor mammary gland MCF10A cell line were all obtained from ATCC (Manassas, VA). All cancer cells were cultured in DMEM medium with 10% FBS and supplemented with 100 µg/mL streptomycin and 100 units/mL penicillin at 37°C in a 5% CO2 atmosphere. MCF10A cells were cultured in DMEM/F12 medium with 5% horse serum, 20 ng/mL EGF, 0.01 mg/mL insulin, 100 ng/mL cholera toxin, and 0.5 µg/mL hydrocorti- sone. miR-30d mimic oligonucleotides (Ambion, Foster City, CA), miR-30d inhibitor oligonucleotides (Exiqon, Vedbaek,Denmark) and corresponding negative control (NC) and anti- negative control (anti-NC) were transfected to BT474 and MDA-MB-231 cells by using RNAimax transfection reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. BT474 cells were co-transfected with miR-30d mimic and pcDNA3.1-KLF-11 for 48 h by using Lipofect- amine 3000 (Invitrogen).

2.2 | Cell viability assay
BT474 and MDA-MB-231 cells were seeded into 96-well plates and transfected with miR-30d mimic and inhibitors. Cell viability was performed using a Cell Counting Kit (CCK8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

2.3 | Cell apoptosis
BT474 and MDA-MB-231 cells were seeded into a 96-well plate and transfected as above. Cell apoptosis was determined by an ELISA-based cell death detection kit (Roche, Palo Alto, CA) according to a previously described method.23 During apoptosis, histone-associated DNA fragments were generated and the quantitated DNA fragments were used to confirm cell apoptosis.

2.4 | Quantitative real-time PCR (qRT-PCR)
Total RNA from BT474 and MDA-MB-231 cells was extracted using Trizol reagent (Invitrogen). For gene expres- sion, cDNA was generated and qRT-PCR was performed using the SYBR green Supermix (Bio-Rad, Richmond, CA) on the ABI 7900 HT Real-Time PCR detection system. GAPDH was used for normalization. For miR-30d expression, the TaqMan miRNA Assay kit (ThermoFisher Scientific; Waltham, MA) was used. RNA U6 was used as a housekeeping gene. The following primers were used in this study: KLF1124 forward, 5′-ACGGTCTTGGCGGCCTAG-3′, and reverse, 5′-ACTTT- CATCAAAACCAGCCTCC-3′; and STAT325 forward, 5′-CTGGCC- TTTGGTGTTGAAAT-3′, and reverse, 5′-AA GGCACCCACAGAAACAAC-3′.

2.5 | Western blotting
Breast cancer cells were homogenized in ice-cold lysis buffer as described previously.26 Extracted proteins were separated by 10% SDS-PAGE. Then, proteins were transferred to nitrocellulose membranes and blocked with 5% milk. The following antibodies were used in this study: Rabbit polyclonal to KLF-11 (1:500), Bcl-2 (1:500) and Bax (1:500), Mouse monoclonal to E-cadherin (1:1000), Vimentin (1:1000) and Rabbit polyclonal to N-cadherin (1:1000). HRP- conjugated Goat Anti-Rabbit IgG at a dilution of 1/2000 andHRP-conjugated Rabbit Anti-Mouse IgG at a dilution of 1/2000 were used as the secondary antibodies.

2.6 | Caspase-3/7 activities assay
Caspase-Glo 3/7 kit (Promega, Madison, WI) was used to determine the activities of caspase-3/7 according to the manufacturer’s instructions. Caspase-Glo 3/7 reagent was added to breast cancer cells and the luminescence intensities were measured.

2.7 | Luciferase reporter assay
The 3′-UTR fragment of KLF-11 gene (wt-KLF-11 3′-UTR) and the corresponding mutated sequence (mut-KLF-11 3′- UTR) were synthesized and cloned into the Dual-Luciferase
reporter vector (Promega). The miR-30d mimic and wild-type or mutated reporter vectors were co-transfected into HEK 293T cells and the luciferase activities were detected by using Dual-Luciferase Assay System (Promega).

2.8 | Transwell assay
Cell migration and invasion was measured using the transwell assay, as described previously.26 BT474 and MDA-MB-231 cells (1 × 105) were seeded in the upper chamber of Matrigel-coated transwell for migration and the uncoated transwell for invasion. The medium in the lower chamber containing 10% serum acted as a chemoattractant, with 0.1% crystal violet used to stain the migrating or invading cells; the average number was counted using a microscope.

2.9 | Statistical analysis
Results in this paper were performed three times, in triplicate, and are displayed as the mean ± SD and were enforced by the SPSS version 13.0 (SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) and Student’s t-test were used to statistical analysis. A P < 0.05 was considered statistically significant. 3 | RESULTS 3.1 | miR-30d is increased in breast cancer cell lines Evidence indicated that the expression of miR-30d was increased in multiple types of cancers,27 including breast cancer with type 2 diabetes mellitus.14,15 Firstly, we found that miR-30d expression was markedly increased in breast cancer cell lines BT474, MDA-MB-231, HCC197, and MDA-MB-468 compared with the control normal cellsMCF-10A (Figure 1). The increase was most obvious in MDA-MB-231 cells and was poorly increased in BT474 cells. Thus, we chose these two cell lines for further investigation. 3.2 | miR-30d increased breast cancer cell survival and inhibits its apoptosis Next, BT474 and MDA-MB-231 cells were transfected with miR-30d mimics and inhibitors. As shown in Figure 2A, miR- 30d mimics promote BT474 and MDA-MB-231 cell survival and the miR-30d inhibitor inhibits cell survival. Further studies suggested that the miR-30d mimic inhibits BT474 and MDA-MB-231 cell apoptosis, increased the level of anti- apoptosis protein Bcl-2, and decreased expression of the pro- apoptosis protein Bax, while the miR-30d inhibitor reversed these effects (Figures 2B and 2C). Caspase-3/7 activity was measured by Caspase-Glo 3/7 kit; the results indicated that miR-30d mimic significant suppressed caspase-3/7 activity, whereas miR-30d inhibitor increased caspase-3/7 activity (Figure 2D). 3.3 | miR-30d increased breast cancer cell migration, invasion, and EMT Given the known role of the miR-30 family in cell migration and invasion in triple-negative breast cancer cells, we speculated that miR-30d may have similar effects in breast cancer cells. To test this, we used transwell assay to measure the effects of miR-30d mimic and inhibitor on BT474 and MDA-MB-231 cell migration and invasion. Results indicate that miR-30d mimic significantly increased BT474 and MDA-MB-231 cell migration and invasion, whereas the miR-30d inhibitor suppressed this effect (Figures 3A and 3B). Emerging studies have revealed that the epithelial-mesen- chymal transition (EMT) process has emerged as a possible mechanism in cell metastasis.28 Epithelial (E-cadherin) and mesenchymal (Vimentin and N-cadherin) markers has been detected by Western blot, and the results showed that miR-30d mimic resulted in the loss of E-cadherin and the increased of Vimentin and N-cadherin, compared with the NC group. However, miR-30d inhibitor reverse these effects by increased E-cadherin and decreased Vimentin and N-cadherin expression (Figure 3C). 3.4 | KLF-11 and STAT3 was regulated by miR-30d A recent study found that miR-30 suppressed krüppel-like factor 11 (KLF-11) expression in hepatic stellate cells21 and inhibits miR-30 decreased STAT3 expression in glioma stem cells.29 We first determined the expression of KLF11 andSTAT3 in breast cancer cells transfected with miR-30d mimic or miR-30d inhibitor. RT-PCR results showed that the miR- 30d mimic remarkably decreased KLF-11 gene expression and increased STAT3 level, whereas miR-30d inhibitor up- regulated the KLF-11 gene level and down-regulated the STAT3 level (Figure 4A). The effects of miR-30d mimic and inhibitor on KLF-11 and STAT3 protein expression has a similarly tendency to the gene expression (Figure 4B). Moreover, KLF11 is a selected putative mRNA targets of miR-30d in the TargetScan (v6.2) (http://www.targetscan. org/). Thus, we believed that KLF11 may be a direct target ofmiR-30d. To test this, we co-transfected the miR-30d mimic and KLF-11 3′-UTR (wide-type or mutant type) into HEK293 cells. Compared with the NC group, we observed a significantdecline of relative luciferase activity in the miR-30d mimic group. More importantly, when mutated the 3′-UTR region of KLF-11, there has no obvious change of relative luciferase activity between the NC group and miR-30d mimic group (Figure 4C). 3.5 | miR-30d mediate breast cancer cell growth, metastasis and EMT is dependent on KLF-11 and STAT3 expression To further demonstrated the mechanism of miR-30d in breast cancer cell growth and metastasis, we constructed miR-30d and KLF-11 overexpression BT474 cells; the Western blot assay suggested that miR-30d and KLF-11 overexpression signifi- cantly increased the expression of KLF-11 compared with miR-30d mimic group (Figure 5A). We also observed that KLF-11 overexpression inhibits miR-30d mimic-induced pSTAT3 expression; the results indicated that miR-30d inducedpSTAT3 expression is dependent on KLF-11 level (Figure 5A). As shown in Figure 5B–F, compared with miR-30d mimic group, KLF-11 overexpression inhibits miR-30d mediated cell survival, migration and invasion, promote cell apoptosis and caspase-3/7 activity. Moreover, above results suggested thatmiR-30d mimic activity pSTAT3, and SH-4-54 was previously shown act as an effective inhibitor for STAT3.30 Similarly, we observed that SH-4-54 (10 µM) treatment also reverse theeffects of miR-30d on cell survival, apoptosis, caspase-3/7 activity, migration, and invasion (Figure 5b–f). 4 | DISCUSSION This study investigates the function and mechanism of miR- 30d in breast cancer cell growth and metastasis. The first finding was that miR-30d is increased in types of breast cancer cell lines, promotes breast cancer cell survival, migration, invasion and EMT, inhibits cell apoptosis, and mediates apoptosis-related gene expression. However, miR- 30d inhibition reverses all effects of miR-30d mimics. Further studies have suggested that the roles of miR-30d in breast cancer cell growth and metastasis are dependent on the low level of KLF-11 and the high level of pSTAT3. Results in the present study are important for understanding the functionality and biological effects of miR-30d, and provides a new molecular target for breast cancer diagnosis and treatment. According to the published estimates, miR-30 family which comprising six molecules (miR-30a-f) is ubiquitously expressed and play different roles in different humans cell types.31 The miR- 30 family has been implicated in cancer cell biology, such as epithelial mesenchymal transition, cell proliferation, apoptosis, and invasion.19,32 miR-30d is a member of the miR-30 family, and acts as a double-edged sword in cancer cells. Yan et al33 showed that miR-30d overexpression inhibited tumor progression and suppressed lung cancer cell proliferation, migration and invasion, induced apoptosis and mediated the cell cycle. However, Naohito et al34 found that miR-30d could increase prostate cancer cell proliferation and invasion and act as a novel prognostic maker of prostate cancer. Moreover, there is convincing evidence that miR- 30d serves as a novel prognostic biomarker in breast cancer in patients with type 2 diabetes mellitus.14,15 Consistent these results, we found that miR-30d was increased in breast cancer cell lines and miR-30d overexpression promotes cell growth and metastasis whilst inhibiting cell apoptosis. In the present study, we demonstrated that KLF-11 is a direct target of miR-30d. It is shown by other studies that miR-30d could inhibit KLF-11 expression in hepatic stellate cells21; ourstudy also confirmed that miR-30d could inhibit KLF-11 expression. The krüppel-like factors belong to the Cys2/His2 zinc-finger DNA-binding protein family and play critical roles in the growth and development of types of tissues and cells, including cancer. It has been well documented that KLF-11 is involved in the progression of a wide variety of cancers, such as ovarian cancer and pancreatic cancer.35,36 Moreover, Wang et al35 also suggested that KLF11 promoter methylation in ovarian cancer significantly reduced KLF11 gene expression. Interestingly, Faryna et al22 showed that KLF11 was hyper- methylated and the hypermethylation was accompanied by the low expression of KLF-1. Further studies has confirmed that miR-30d increased the expression of STAT3 phosphorylation, and has any noticeable differences in total protein expression. These results indicated that miR-30d could mediated the activity of STAT3, but the level of total STAT3 was only marginally affected. However, the reason and mechanism of miR-30d was only marginally affected the level of total STAT3 is still unclear. 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