Role of miR-100-5p and CDC25A in breast carcinoma cells

Objective To inquiry about mechanism of miR-100-5p/CDC25A axis in breast carcinoma (BC), thus offering a new direction for BC targeted treatment. Methods qRT-PCR was employed to explore miR-100-5p and CDC25A mRNA levels. Western blot was employed for detecting protein expression of CDC25A. Targeting relationship of miR-100-5p and CDC25A was verified by dual-luciferase assay. In vitro experiments were used for assessment of cell functions. Results In BC tissue and cells, miR-100-5p was significantly lowly expressed (P < 0.05) while CDC25A was highly expressed. Besides, miR-100-5p downregulated CDC25A level. miR-100-5p had a marked influence on the prognosis of patients. The forced miR-100-5p expression hindered BC cell proliferation, migration and invasion, and facilitated cell apoptosis. Upregulated miR-100-5p weakened promotion of CDC25A on BC cell growth. Conclusion Together, these findings unveiled that CDC25A may be a key target of miR-100-5p that mediated progression of BC cells. Hence, miR-100-5p overexpression or CDC25A suppression may contribute to BC diagnosis.


INTRODUCTION
Breast carcinoma (BC) accounts for 7-10% of all solid malignancies (Feng et al., 2015). At present, the incidence and mortality rate of BC are increasing annually owing to the difficulty in the early diagnosis of BC caused by the complexity of endocrine system (Al-Hajj et al., 2003). Great improvements have been made in prognosis of BC patients in recent years. However, five-year survival rate of patients is still low, and postoperative malignant behaviors of BC cells are main causes of the disease (Anastasiadi et al., 2017). Therefore, it is of importance to make in-depth inquiry about mechanism of BC metastasis and actively search the target gene for the targeted therapy of BC.
CDC25 family members can regulate cell cycle (Brenner et al., 2014). CDC25 modulates cell cycle progression by hindering cyclin-dependent kinases (CDKs) phosphorylation, so as to activate the CDK complexes (Blomberg & Hoffmann, 1999). The high CDC25A expression has been found in different cancer types, and overexpression of CDC25A presents in approximately 50% of BC cases and implicates poor prognosis (Cangi et al., 2000). A previous study denoted that CDC25A regulates stem cell proliferation via targeting Sirtuin6 (SIRT6) in colorectal cancer (Liu et al., 2018). miR-365 strengthens radiosensitivity of non-small cell lung cancer (NSCLC) cells via targeting CDC25A (Li et al., 2019). miR-98-5p hinders osteosarcoma progression via targeting CDC25A (Liu & Cui, 2019). However, the regulatory mechanism of CDC25A in BC has not been explored.
By bioinformatics methods, we identified low miR-100-5p expression in BC. Modulatory role of miR-100-5p was investigated in BC via experiments, disclosing that miR-100-5p targeted CDC25A to restrain BC cell progression. Our investigation contributes to mechanistic understanding of BC progression, thereby providing targeted therapy for BC with novel theoretical basis.

Bioinformatics methods
Mature miRNA (104 normal samples, 1,103 tumor samples) and mRNA (113 samples, 1,109 samples) expression data of TCGA-BRCA dataset were obtained from TCGA (https://portal.gdc.cancer.gov/) database. Expression analysis and survival analysis were carried out on the target miRNA according to the obtained data. Differential mRNAs (DEmRNAs) were got through differential analysis by using ''edgeR'' package (|logFC|>2.0 and padj<0.01). Three databases miRDB, starBase, and mirDIP were used to predict target mRNAs of miR-100-5p. Candidate mRNAs were got by overlapping up-regulated DEmRNAs and predicted mRNAs of miR-100-5p, among which a mRNA having the highest correlation coefficient was picked as the objective of study.

qRT-PCR
Total RNA was extracted from cells by TRIzol kit (Invitrogen, Carlsbad, CA, USA) according to instructions. Concentration was assayed using NanoDrop 2000 system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA was generated by reversely transcribing miRNA with miScript IIRT kit (Qiagen, USA) and mRNA with PrimeScript RT Master Mix (Takara, Dalian, P.R. China) according to protocols. qRT-PCR was completed on Applied Biosystems R 7500 Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, MA) with miScript SYBR Green PCR Kit (Qiagen, Germany) and SYBR R Premix Ex Taq TM II (Takara Bio Inc., Shiga, Japan). PCR conditions were as follows: 95 • C 10 s, 60 • C 20 s, 72 • C 20 s, 45 cycles. 2 − Ct method was utilized for data analyzing. GAPDH and U6 were applied as endogenous controls. Primer sequences were exhibited in Table 1.

Clonogenic assay
Cells in varying transfection groups were treated with 0.25% trypsin, inoculated into 6-well plates (4 × 10 2 cells/well) and incubated in DMEM with 10% FBS for 2 weeks. Colonies were subjected to fixation with 95% methanol, 10 min of staining with 0.1% crystal violet, and rinsing with PBS, followed by cell colony counting.

Wound healing assay
After transfection, 1 × 10 5 BC cells (MCF-7) were placed into 6-well plates, and a pipette tip (200 µL) was employed to make a scratch through center of each well when cells grew to 80% confluence. Dispersed cells were discarded. Fresh mediums were employed to continuously culture the remaining cells. The migrated cells were observed and pictured at 0 and 24 h. Image J software was used for analysis. Migration width = wound healing width at 24 h -wound healing width at 0 h.

Transwell invasion assay
First, 9.6 mg/ml Matrigel matrix (356234, BD Company, USA) diluted with serum-free medium at 1:8. Fifty µL of dilution was added to the Transwell upper chambers for 30 min. After transfected for 48 h, BC cells were suspended in 200 µl serum-free DMEM (1 × 10 5 cells/mL) and were filled into the upper chamber, while medium with 15% FBS was filled into lower chamber. After being cultured for 48 h at 37 • C, cells that did not pass membranes were removed using a cotton swab, while cells in lower chamber were subjected to 0.1% crystal violet for staining. A microscope was implicated to random 4 fields, and invaded cells were counted and photographed.

Western blot
Cells were lysed by radioimmunoprecipitation assay buffer (RIPA; Sigma-Aldrich), and protein concentration was assessed by Pierce BCA (Thermo, USA) protein assay kit. Total proteins were isolated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich). After blocking, membranes were cultivated overnight with primary antibodies, including rabbit anti-CDC25A and rabbit anti-GAPDH. Subsequently, PBS + 0.1% Tween-20 (PBST) was taken to rinse membranes 3×10 min. Afterward, secondary antibody goat anti-rabbit IgG H&L (HRP) (ab205718) was incubated with membranes at room temperature for 1 h, and then membranes were rinsed with PBST 3×10 min. All protein bands were visualized by chemiluminescence reaction (Bio-Rad, Hercules, CA, USA), followed by analysis by Image Lab (Bio-Rad). All antibodies were from Abcam (China).

Flow cytometry
BC cells in logarithmic growth phase were transfected. Forty-eight h later, cells were collected. Cell suspension (100 µL) was incubated 15 min in dark with Annexin-V-FITC (5 µL, KeyGen Biotech, Nanjing, China), and then propidium iodide (2.5 µL, PI) was utilized for cell staining. FACS Calibur (BD Biosciences, San Jose, CA, USA) was adopted to measure cell apoptosis. Flowjo software (Tree Star Corp, San Carlos, CA, USA) was recommended for data analysis.

Statistical analysis
All data were processed by GraphPad Prism 6.0 (La Jolla, CA). Each experiment was repeated in triplicate, including 3 technical replicates and 3 biological replicates. The results were presented by mean ± standard deviation (SD). To compare differences among three or more groups, one-way analysis of variance, followed by Bonferroni test was used. Student's t -test was used for significance test between the two groups. P < 0.05 was accepted as significant.

DISCUSSION
With 1.3 million cases increasing annually, BC is a symbolic cancer in female's daily life (Qin & Liu, 2019). A recent study has illustrated that miRNAs might be potential therapeutic targets for BC (Luan et al., 2017). miRNAs are regarded as effective modulators of cell proliferation, metastasis, translation and tumorigenesis (Zhou et al., 2017). There is a correlation between miRNAs and mRNAs that makes specific cure of certain cancers possible at molecular level (Zhou et al., 2013). Present studies disclosed that miR-100-5p is aberrantly expressed in several cancers, modulating cancer cell malignant behaviors. For instance, miR-100-5p is lowly expressed in prostate cancer cells, and it down-regulates mTOR to hamper progression of prostate cancer cells (Ye, Li & Wang, 2020). In chordoma tissue, miR-100-5p is down-regulated, while enforced miR-100-5p expression can hinder chordoma growth, and it can partially hinder chordoma cell malignant behaviors via EMT suppression (Zhang et al., 2020). Wang, Tao & Bian (2021) unveiled that miR-100-5p may be a new biomarker for patients with skin melanoma, and it may be related to survival time of patients. Moreover, Annalisa et al. (Petrelli, Bellomo et al. 2020) elucidated that miR-100 can be used as a novel biomarker for patients suffering luminal BC. Nonetheless, research on role and modulatory mechanism of miR-100-5p in BC is in high unmet need. In this study, we first carried out bioinformatics analysis, revealing low miR-100-5p expression in BC tissue. qRT-PCR detection also found that miR-100-5p was decreased in BC cells. Additionally, we noted that overexpression of miR-100-5p inhibited progression of MCF-7 cells as well, indicating that miR-100-5p had a cancer-suppressive effect on BC cells, congruous with earlier investigations.
To further elucidate modulatory mechanism of miR-100-5p in progression of BC, CDC25A was predicted to be a target of miR-100-5p, which was unveiled to be highly expressed in BC through bioinformatics analysis. CDC25A is a key regulatory factor in progression of cell cycle and checkpoint response (Jin, 2011). miR-365 facilitates radio-sensitivity of NSCLC cells in vitro and in vivo by targeting CDC25A (Ding et al., 2019). miR-122-5p has an inverse modulatory impact on CDC25A expression, which is conducive to the improvement of the prognosis of cervical cancer patients (Ding et al., 2019). miR-449a targets CDC25A to noticeably reduce cell proliferation and invasion, while inducing cell apoptosis in endometrial carcinoma (Ye et al., 2014). Here, we uncovered that miR-100-5p downregulated CDC25A expression. Overexpressing CDC25A facilitated BC cell malignant progression. In addition, rescue experiment suggested that miR-100-5p hampered BC cell progression by targeting CDC25A.
In conclusion, we validated the inhibitory effect of miR-100-5p on malignant behaviors of BC cells. Besides, miR-100-5p suppresses development of BC through reducing CDC25A expression. Our study helps us to gain more awareness of role of miR-100-5p in BC, and brings additional insight into the exploration of novel targeted therapy for BC. Nevertheless, there are several limitations. For instance, it is necessary to further investigate the involvement of signaling pathways downstream of miR-100-5p/CDC25A. Besides, direct relationship of miR-100-5p and CDC25A, as well as their molecular mechanism in regulating biological behaviors are still elusive. These shortcomings need to be resolved in further studies.