MiR-324-5P Functions as a Novel Serum Biomarker and Modulates Breast Cancer Malignant Activity


 Introduction: The best management practices for patients with breast cancer（BC） involve early detection and deterioration prevention. Therefore, it is important to study the occurrence of BC and its development mechanism and explore more sensitive diagnostic and prognostic indicators.Objective: Our study explored the significance and the serum miR-324-5p application in the diagnosis and prognosis of BC and evaluated possible molecular mechanisms.Methods: MiR-324-5p expressions in the serum and tissue of patients with BC were observed by real-time fluorescence quantitative polymerase chain reaction (PCR). Next, the miR-324-5p diagnostic ability in BC was evaluated by the receiver operating characteristic curve. Kaplan-Meier analyzed the relationship between expression of miR-324-5P and prognosis. The Cox regression model evaluated the serum miR-324-5P prognostic value in predicting disease-free survival (DFS) and overall survival (OS). In functional studies, cell counting Kit-8 assay and Cloning experiment were employed to assess BC cells’ proliferation level. Transwell assays and wound healing assays were performed to determine the effect of miR-324-5p on BC cell invasion and migration. In addition, the flow cytometry test was conducted to determine the effect of miR-324-5p on the apoptotic rate of BC cells. Finally, bioinformatics tools were employed to detect the potential target genes of miR-324-5p in BC.Results: miR-324-5p expression is up-regulated in in BC cells, which is related to tumor stage（p=0.0291)and age( p=0.0278). In addition, a BC diagnosis based on serum miR-324-5p levels had an 66% sensitivity, 78% specificity, and the area under the curve was approximately 0.7581. DFS (p=0.027) and OS (p<0.017) in the low miR-324-5P expression group were higher than those in the high expression group. The tumor node metastasis staging、Ki-67 and miR-324-5P were independent prognostic factors for the survival of patients with BC. In addition, miR-324-5p had a positive effect on BC cell invasion and proliferation, thereby preventing apoptosis. Finally, bioinformatics synthesis showed that the miR-324-5p target genes in BC are cysteine-rich hydrophobic domain 1 (CHIC1) and kruppel-like factor 7 (KLF7).Conclusion: MiR-324-5p may perform its oncogenic functions by interacting with the CHIC1 and KLF7 genes, and its existence in serum could serve as a diagnostic and prognostic biomarker of BC.


Introduction
Breast cancer (BC), is one of the most common malignancies in women and a leading cause of death in the 21st century. It is expected to become a primary obstacle to extended life expectancy [1]. However, there is a lack of accurate and early diagnostic and screening methods because of limited progress in BC molecular mechanisms determination. Thus, many patients with BC missed the opportunity for radical resection [2]. Therefore, studies should focus on BC molecular mechanisms to determine new biomarkers capable of identifying and predicting genes as potential treatment targets.
Micro-ribonucleic acid (miRNA) is a non-coding RNA composed of 21-25 nucleotides, widely distributed in the mammalian genome [3]. Thus far, the cancer cells regulation by miRNAs has been identi ed in many processes, such as cell proliferation, apoptosis [4], angiogenesis [5], radiosensitivity [6], and migration [7]. The miRNAs abnormal expression in malignant tumors would impact the carcinogenic expression or tumor suppressor genes during tumorigenesis and metastasis [8]. Research has revealed that miR-937-5p overexpression can regulate the cell proliferation and cell cycle via the Wnt signaling pathways [9]. Noyans et al. found that the miR-770-5p down-regulation could explain the epithelial-mesenchymal transition in BC [10]. Overall, these studies revealed the miRNAs important role in BC. Hence, abnormal miRNA expression may be a good biological marker for BC diagnosis and prognosis; besides, the involved miRNAs may also serve as potential treatment targets.
MiR-324-5p has been shown to either inhibit or promote human cancer. For example, it is inhibitory for gallbladder [11] and colorectal cancer [12] but can show carcinogenic effects in thyroid papillary [13], pancreatic [14], and gastric cancer [15]. However, whether the expression levels of miR-324-5p can serve as a predictor for BC development remains to be revealed. Therefore, this study evaluated the miR-324-5p relative expression levels in the serum and tissues of patients with BC and studied the correlation between the miR-3324-5p expression levels and patients with BC clinicopathological features. In addition, the miR-324-5P feasibility as a marker for BC diagnosis and prognosis was evaluated. Finally, we studied miR-324-5p for its functional effects on BC and analyzed its potential target genes. People's Hospital.. Among them, the patient was newly diagnosed as a female patient with BC, aged 27 to 73 years old. Blood was collected from all patients before they received any form of treatment. Collect whole blood into anticoagulant blood collection tubes. Following that, the tubes were gently inverted 4 to 5 times to mix the anticoagulant with the blood. Quickly transfer to a separate cryopreservation tube or centrifuge tube, store in -80°C refrigerator.
The tissue samples included 41 pairs of BC and adjacent tissues obtained from female patients, all of which were newly diagnosed BC in Shaoxing People's Hospital,aged 35 to 72 years. The tissue specimens were quick-frozen in liquid nitrogen within 30 minutes of in vitro and then transferred to the -80°C refrigerator for storage.All experimenters signed a written informed consent form.

Cell culture
Human BC cell lines T-47D, MCF-7, Hs578T and non-tumorigenic epithelial cell line MCF-10A are provided by the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Among them, MCF-10A cells use DMEM/F12 medium (Gibco), and additionally add 5% horse serum (HyClone), insulin, epidermal growth factor, and hydrocortisone. Other cell lines use DMEM medium with 10% fetal bovine serum (Hyclone). All cells were cultured at 37°C and 5% CO 2 .

Transfection
3 × 10 5 MCF-7 cells were inoculated in one well of the six-well plate. These were then transfected at a nal concentration of 50 nM miR-324-5p mimics or with the appropriate miRNA mimic control using lipofectamine 6000 transfection reagent (Beyotime Biotechnology). The Hs578T cells were seeded at 2×10 5 cells per well into 6-well plates and were transfected with miR-324-5p inhibitors at a nal concentration of 100 nM or with the appropriate miRNA inhibitor control using Lipofectamine 6000 transfection. All subsequent functional assays were performed 48-72h post-transfection. Guangzhou RiboBio provides transfection reagents.

RNA isolation and quantitative real-time PCR (RT-qPCR)
We chose to use Trizol (Haoxin) to collect total RNA from cultured cells and tissues. Serum miRNAs was harvested from each 200μl serum sample by using a Serum/plasma miRNA extraction and separation kit (TianGen). The total RNA then underwent reverse transcription using Prime ScriptTM RT reagent Kit with gDNA Eraser. For microRNAs detection, stem-loop RT-PCR was carried out by using the cDNA product as templates by SYBR® Premix Ex Taq™ (TaKaRa) in the Light Cycler 480 System (Roche Diagnostics). For internal reference, primers of U6 and U6. miR-324-5p were obtained from RiboBio.

Wound healing assay
When the cell density after transfection reaches 90-95%, Scratch with 200 µl grab head and wash the residual cells on the scratch with sterile PBS. Then, add fresh medium with 1% FBS into the 6-well plates, and microscopic images were taken over the plates at 0-24 h. The wound healing rates were calculated and compared to the width at 0 h.

Transwell assay
For migration assays, MCF-7 cells and Hs578T cells (5×10 4 cells/well) were cultured in the upper compartment of the transwell chamber (BD Biosciences) in serum-free medium. The lower chamber contains 600µl medium (containing 10% fetal bovine serum). The transwell chambers were coated with 50 µl Matrigel, and cell invasion was evaluated at a dilution of 1:5 in serum-free DMEM. After transfection place the transfected cells in the upper part of the chamber. Stain the cells for 48 hours. Finally, ve random elds under the uorescence microscope (Olympus Corporation) were selected for each group and had their cell numbers counted.

Cell apoptosis assay
Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen Biotech) was used to detect cell apoptosis. After 24 hours of transfection, 1×10 5 cells were made into 100μl solution with culture medium and transferred to a centrifuge tube.Flow cytometry detection was performed for Annexin V-FITC and PI using a LSRFortessa-Flow Cytometer (Becton Dickinson).

Bioinformatics analysis
The expression of miR-324-5p in BC is searched for online database The Cancer Genome Atlas. The miR-324-5p target gene is combined with Targetscan, miRDB and miRtarbase databases. Inclusion criteria requires at least three databases. The expression of miR-324-5p and the correlation between such expression and BC were analyzed via StarBase and GEPIA.

MiR-324-5p expression in BC tissue
We rst analyzed miR-324-5p expression in 1,103 BC patients included in The Cancer Genome Atlas (TCGA) database ( Fig. 1A) to con rm whether the miR324-5P expression in BC was disordered. By comparing the results obtained from 41 BC breast lesion samples and 41 adjacent normal samples, we con rmed that miR-324-5p levels increased in BC (Fig. 1B). The miR-324-5P expression was relatively higher in patients with positive lymph node metastasis and advanced breast cancer Fig. 1C-D . Overall, the data showed that miR-324-5p might promote BC progression.

Expression and diagnostic value of serum miR-324-5p in BC.
The miR-324-5p relative expression in BC serum (n = 104) and normal serum (n = 50) was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which revealed that BC serum contained higher miR-324-5p levels than the normal serum ( Fig. 2A). After evaluating the potential of serum miR-324-5p levels as a diagnostic biomarker for BC, further evaluation results showed that the receiver operating characteristic (AUC) value for the miR-324-5p based diagnosis of BC was 0.7581 (95% con dence interval (CI: 0.6828-0.8335), the sensitivity was 66%, and the speci city was 78%. (Figure 2B).

Correlation between clinicopathological features and miR-324-5p expression in patients with BC
We analyzed whether miR-324-5p in serum had signi cant differential expressions due to different clinicopathological features. Patients were categorized into a high expression (miR-324-5p expression in serum > 4.36) and a low expression (miR-324-5p expression in serum < 4.36) groups. The results showed that the high miR-324-5p expression was not related to age, tumor size and lymph node metastasis , human epithelial growth factor receptor 2, estrogen receptor, progesterone receptor or type, Ki-67 but is correlated with the speci c TNM stage p=0.0291)and age( p=0.0278)( Table 1).

MiR-324-5p promoted BC cell migration and invasion
The wound healing results showed that the miR-324-5p up-regulation could stimulate wound closure after 24 h of scratch injury in MCF-7 cells. However, in Hs578T cells the inhibitory expression of miR-324-5p had a contrasting effect (Fig. 5A-B). In addition, attenuating the miR-324-5p expression can inhibit the migration and invasion of HS578T cells, while a miR-324-5p mimic can enhance MCF-7 cells, as shown in Fig. 5C-D. These results indicate that miR-324-5p has a positive effect on BC cells migration and invasion.

Discussion
BC is the most prevalent tumor that threatens the health of women worldwide [1]. Although there are increasingly new strategies to prevent BC, its incidence and recurrence rates are increasing, illustrating the critical need for early detection and treatment [2]. MiRNAs are widely expressed in eukaryotes, and they participate in tissues and organs development [16], involving the regulation of multiple functions, such as cell proliferation, apoptosis, invasive migration, metabolism, neuronal patterns, immune regulation, and cellular stress responses [17]. Thus, miRNAs have been identi ed as one of the important factors in human cancer [18]. Some studies report that many miRNAs are unbalanced in BC and either promote or inhibit BC occurrence and metastasis [19][20][21]. Research has shown that the expression of various miRNAs can be detected in 12 kinds of body uids, including serum, urine, and saliva, suggesting that miRNA could be detected in a liquid biopsy [22]. Mitchell et al. [23] initially described serum miRNAs, and subsequent in-depth studies suggested that serum miRNA can be used as a biomarker for various malignant tumors, including BC. For example, the diagnostic speci city of miR-195, which is up-regulated in the patients with BC serum, is approximately 90% [24]. In addition, upregulated serum miR-103 possesses 84% sensitivity and 70% speci city, demonstrating its diagnostic potential [25]. D. LI found serum exosomal miR-148a as a novel prognostic biomarker for breast cancer [26]. These ndings make serum miRNA a promising potential BC biomarker.
Studies have shown that miR-324-5p affects tumor occurrence and development by regulating target genes in various human cancers. MiR-324-5p promotes the progression of thyroid papillary cancer via the PTPRD/CEBPD [13]. In addition, miR-324-5p reduced the tumor protein expression, phosphatase, and tensin homolog and may improve gastric cancer`s invasion and proliferation ability [14]. Furthermore, Wan et al. [15] discovered that miR-324-5p promoted pancreatic cancer progression due to the tumor suppressor gene expression and kruppel-like factor 3.
In this study, we discovered that the miR-324-5p expression in BC tissues increased signi cantly compared to the normal breast tissues for the rst time. Furthermore, the miR-324-5p levels in the tissues of patients with BC and at an advanced stage of TNM or positive lymph node metastasis were higher than those in early-stage patients or negative lymph node metastasis. In addition, we noted that the expression trend of serum miR-324-5p levels was consistent with that in the tissues of patients with BC, suggesting a possibility of miR-324-5p as a BC biomarker. The role of serum miR-324-5P in the diagnosis and prognosis evaluation of breast cancer was explored. The receiver operating characteristic (AUC) of BC diagnosis based on miR-324-5p was 0.7208 and the sensitivity was 83%. The expression of miR-324-5p was related to TNM stage, progesterone receptor, tumor size and lymph node metastasis. In addition, patients with low expression of miR-324-5P signi cantly increaseded DFS and OS in 5 years. COX regression showed that miR-324-5p was an independent prognostic factor of OS in multivariate analysis of breast cancer. MiR-324-5P is a sensitive indicator, and its prognostic value is greater than the diagnostic value. The number of serum samples in this study was limited. As a result, the outcomes of the study need to be validated with a larger sample size.
In addition, the mechanism study of miR-324-5P on BC shows that it can promote proliferation, BC migration and inhibit apoptosis. MiRNAs inhibit the expression of multiple target genes. In this study, 10 genes from the target gene database were selected as candidates to validate miR-324-5p potential target genes and test the miR-324-5p predictive ability in BC. Furthermore, miR-324-5p was inversely correlated with CDS1, CHIC1, CUEDC2, KLF7, SLITRK4, and SP1, CHIC1, CUEDC2, and KLF7 were overexpressed and associated with better prognosis. In addition, we observed that CHIC1 and KLF7 from the GEPIA database were signi cantly lower in BC tissues than normal breast tissue. Currently, CHIC1-related diseases include ovarian epithelial tumors and Allan-Herndon-Dudley syndrome. [27]. KLF7 is known as the ubiquitous kruppel-like factor and is a key factor in the development of many cancers and an independent predictor of poor prognosis in several cancers [28]. In vitro, functional experiments have shown that KLF7 can affect tumor proliferation and migration [29][30][31]. Therefore, miR-324-5p may target CHIC1 and KLF7 to facilitate the progression of BC, resulting in a decreased survival rate of BC patients.

Conclusion
In conclusion, the serum miR-324-5p expression may be used as a biomarker for the BC diagnosis and prognosis. MiR-324-5p may promote the occurrence and development of BC cells by inhibiting the CHC1 and KLF7.

Declarations
Ethics approval and consent to participate Written informed consent was signed by all breast cancer patients and volunteers. This study was approved by the Ethics Committee of Shaoxing People's Hospital and conducted according to the guidelines of Helsinki Declaration.

Consent for publication
Not applicable

Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests      Relationship between miR-324-5P and survival.
(A) The disease-free survival in the miR-324-5P low expression group was higher than that in the miR-324-5P high expression group. (B) The overall survival in the miR-324-5P low expression group was higher than that in the miR-324-5P high expression group.  The effect of miR-324-5p in promoting cancer migration and invasion A-B The effect of miR-324-5p on the wound healing ability of Hs578T and MCF-7 cells for 24 h, determined by a scari cation test. C-D The effect of miR-324-5p on the invasive and migratory abilities of Hs578T and MCF-7 cells for 24 h, observed by transwell assays.

Figure 6
Identi cation of miR-324-5p targeted genes in BC. (A) Venn diagram of the intersection of three target gene prediction databases. (B-K) Correlation data between target genes and miR-324-5p in the starBase database.

Figure 7
Further authentication of the targeting relationship between target genes and miR-324-5p. A-E The prognostic analysis of miR-324-5p in relation to CDS1, CHIC1, CUEDC2, KLF7, SP1, and SLITRK4 shown by the Kaplan-Meier plotter. F-H The expression of target genes (CUEDC2, KLF7, and CHIC1) in BC, obtained from the GEPIA database.