MicroRNA-129-1-3p Represses the Progression of Triple-Negative Breast Cancer by Targeting the GRIN2D Gene

The aberrant expression of miRNA is strongly linked to numerous stages of triple-negative breast cancer (TNBC) progression, and it plays an indispensable role in the process from tumor onset and progress to invasion and metastasis. In this study, we first transfected miR-129-1-3p mimics and inhibitor into MDA-MB-231 TNBC cells, respectively. Then, we assessed the pathological role of miR-129-1-3p in MDA-MB-231 cells. The results showed that miR-129-1-3p were successfully inserted into MDA-MB-231 cells. Besides, miR-129-1-3p could distinctively repress the growth, migration along with infiltration of MDA-MB-231 cells, which might be related to the inhibition of GRIN2D expression. Our results indicate that miR-129-1-3p was illustrated to serve as a tumor repressor via targeting GRIN2D in TNBC cells and highlight that the restoration of miR-129-1-3p might be a new treatment target for TNBC.


Introduction
TNBC (triple-negative breast cancer) is an aggressive subclass of breast cancer (BC), representing around 10-20% of all invasive BCs [1,2]. As TNBC does not respond to endocrine treatment and always shows low effectiveness to targeted therapy, chemotherapy is still the most effective method of preventing cancer cell growth along with metastasis [3]. Nevertheless, because of the adverse side effects of chemotherapy, research into new safe and effective drugs or methods for TNBC is continuing.
The importance of milk calcium in newborns' growth and development requires that the mammary glands are intrinsically connected with Ca 2+ influx. The expression of some calcium channels and calcium pumps that transport Ca 2+ into milk will change in some BCs [4]. The calcium channels and calcium pumps may be specific to different molecular subtypes of BC [5]. Research evidence illustrates that Ca 2+ influx has a vital role in cellular growth, migration, angiogenesis, and multidrug resistance for BC [6][7][8][9][10][11].
Investigations have documented that miRNAs may play diverse roles in different kinds of cancers, with some acting as tumor repressor genes and others as oncogenes, depending on the pathway or gene they modulate [12][13][14]. miR-129-1-3p is one of the mature miR-129 family members, which plays an indispensable role in tumor onset and progress [15]. It is also known that miR-129-1-3p can interfere with tumor occurrence and prognosis through acting on diverse molecular targets in serous ovarian cancer, gastric cancer, as well as non-small cell lung cancer [16][17][18].
miR-129-1-3p role in TNBC biology remains unclear. Hence, we purposed to study the mediating role of miRNA-129-1-3p in repressing the growth, migration, as well as infiltration of TNBC cells to supply evidence for the development of new medication for the disease.

Materials and Methods
The details of the reagents and materials used in this study are in Appendix I.  The cells were inoculated with a serum-free medium enriched with 0.5-5 μM Fluo-3 AM at 20-37°C for 15-60 minutes, the fluorescent probe was loaded, and the cells were rinsed once or twice with PBS. After washing, the solution was incubated for another 20-30 minutes to ensure that Fluo-3 AM was wholly transformed into Fluo in the cells. DAPI solution (C1005, Beyotime, China) was introduced, and then subsequently incubated at RT (room temperature) for five minutes. We rinsed the cells in PBS twice, and then observed with a fluorescence microscope, and pictures were taken. The expression rate of Ca 2+ positive cells was calculated as Ca 2+ positive cells in the field divided by all cells in the field ×100%.

Western
Blot. The cell proteins were extracted with cell lysate, and the protein concentration detected. SDS-PAGE separation gel and SDS-PAGE laminated gel were prepared according to the required molecular weights of the proteins. The gel was extracted by electrophoresis and membrane transfer and sealed, and then the primary antibody was introduced and inoculated overnight at 4°C or at 25°C for 2 hours, and the secondary antibody was incubated at 25°C for 2 hours. The PVDF film was taken out, dropwise ECL chemiluminescence super-sensitive color reagent (A liquid: B liquid =1 : 1, P0018FM, Beyotime, China) was added to it, and the film was placed in a cassette to expose and develop the photograph. The ImageJ software was adopted to analyze the gray value of each group of bands and do the statistics. 2.6. Data Analysis. The data were processed in the SPSS Statistics for Windows, V.17.0. Chicago: SPSS Inc. All results for continuous variables are given as mean ± standard deviation (SD). The t-test was implemented for comparison between the two groups, with P < :05 signifying statistical significance.

miR-129-1-3p
Repressed the Growth of MDA-MB-231 Cells. As illustrated in Figure 2(a), in contrast to the controls, the clone formation ability of cells in the NC groups was not dramatically different (P > :05). The mimics of miR-129-1-3p dramatically diminished the clone formation rate of cells (P < :01) in contrast with the mimics NC. Besides, miR-129-1-3p inhibitor dramatically increased the rate of cell clone formation (P < :01) relative to the inhibitor NC. As shown in Figure 2(b), the survival rate of MDA-MB-231 cell transfects of miR-129-1-3p mimics was dramatically reduced (P < :01) in contrast with that of the mimics NC cell transfects. The MDA-MB-231 cell transfects of miR-129-1-3p suppressor exhibited dramatically elevated the survival rate of cells than inhibitor NC cell transfects (P < :01).
3.3. miR-129-1-3p Dampened the Migration along with Infiltration of MDA-MB-231 Cells. As illustrated in Figure 3, in contrast with the controls, there was no remarkable difference in growth and migration ability between the mimics NC group and the inhibitor NC group (P > :05). Relative to mimics NC cell transfects, the repair area of the scratch area was remarkably decreased in miR-129-1-3p mimic cell transfects (P < :01). The miR-129-1-3p suppressor remarkably increased the repair area of the scratch area and fused the cells in contrast with inhibitor NC (P < :01).
As depicted in Figure 4, relative to the mimic NC cell transfects, the number of invading cells in miR-129-1-3p mimics was remarkably decreased (P < :01). In contrast with inhibitor NC, the miR-129-1-3p inhibitor group remarkably increased the number of cells descending through the matrix gel (P < :01).

miR-129-1-3p
Repressed the Content of Calcium in MDA-MB-231 Cells. As illustrated in Figure 6, compared to controls, there was no remarkable difference in the Ca 2+ positive cell expression rate in the NC transfects (P > :05). In contrast with the mimics NC cell transfects, the rate of expression of Ca 2+ positive cells in the miR-129-1-3p mimic transfects was dramatically reduced (P < :01). The rate of expression of Ca 2+ positive cells in the transfects of miR-129-1-3p inhibitor was remarkably increased (P < :01) in contrast to the inhibitor NC transfects. Furthermore, the protein contents of GRIN2D in the miR-129-1-3p mimic transfects were remarkably decreased (P < :01) than in the controls, while in the miR-129-1-3p suppressor transfects they were remarkably elevated (P < :01) ( Figure 5).

Discussion
miRNA is involved in almost all aspects of cancer biology, including angiogenesis, drug resistance, apoptosis, proliferation, invasion, and metastasis. Studies have shown that miR-NAs may act as tumor suppressors or oncogenes in many cancers, depending on which pathway or gene they regulate. The pathogenesis of triple-negative breast cancer is complicated, with many influencing factors, and the prognosis is not good. More and more studies have shown that miRNAs are involved in regulating the occurrence and progression of breast cancer. Among them, miR-129-1-3p has been confirmed to play an important role in tumorigenesis and development. However, whether miR-129-1-3p is involved in the occurrence and development of breast cancer has not yet been reported. Herein, we studied the function of miR-129-1-3p in TNBC biology. In transfected MDA-MB-231 cells, CCK-8 and plate cloning experiments illustrated that miR-129-1-3p mimics could repress the growth of MDA-MB-231 cells and the ability to form clones, whereas miR-3 BioMed Research International 129-1-3p inhibitors have the opposite influence. The results illustrate that miR-129-1-3p may play a particular role in repressing TNBC occurrence and development. A similar function was found in research for ovarian cancer. Another report documented that miR-129-1-3p along with miR-129-2-3p levels were remarkably reduced in serous ovarian cancer, and miR-129-1-3p overexpression could considerably dampen the growth of SKOV3 cells, which suggested that restoration of miR-129-1-3p expression may be a new treatment strategy for ovarian cancer [16].
Tumor invasion or metastasis is one of the most critical signs of malignant tumors and the most important fatal factor for BC [20]. Wang et al. illustrated that miR-129-1-3p represses the migration of gastric cancer cells BGC-823 [17]. Herein, we established that miR-129-1-3p mimics repressed the potential of MDA-MB-231 cells to migrate and invade far away, while miR-129-1-3p suppressor enhanced MDA-MB-231 cells' ability to migrate, as well as invade far away. It indicates that miR-129-1-3p dampens the migration along with infiltration of MDA-MB-231 cells.
CD147 is a highly glycosylated transmembrane protein. Many studies have shown that CD147 is involved in multiple vital steps in cancer development and is excessively expressed in human tumors such as BC and ovarian cancer [5,21]. MMP-2 and MMP-9 have a strong ability to degrade the main components of the basement membrane. Various evidence indicate that MMP-2 along with MMP-9 are overexpressed in malignant tumors, including BC [22,23]. Besides, MMP-2 coupled with MMP-9 was reported to be involved in the adhesion, migration, as well as infiltration of MDA-MB-231 cells [24].
Furthermore, MMP-2 and MMP-9 can remarkably affect angiogenesis in malignant tumors by activating VEGF [25]. The VEGF is a remarkably specific vascular endothelial cell growth factor promoting elevated vascular permeability, vascular endothelial cell migration, growth, and angiogenesis. It participates in the occurrence and progression of many angiogenesis-dependent diseases, including cancer [26]. In addition, the increase in VEGF levels is also linked to tumor formation and drug resistance [27]. Herein, we detected the expression of MMP-9, CD147, MMP-2, as well as VEGF protein linked to tumor cell migration along with infiltration in MDA-MB-231 cells via Western blot experiment. The results showed that miR-129-1-3p mimics reduced the expression of CD147, MMP-2, MMP-9, and VEGF protein in MDA-MB-231 cells, whereas miR-129-1-3p inhibitor had the opposite influence. The above results further verified the results of cell scratches and transwell experiments, suggesting that miR-129-1-3p has a vital role in repressing the migration and infiltration of MDA-MB-231 cells.
Ca 2+ is also closely linked to BC cell growth [8], invasion and metastasis [9], angiogenesis [10], and multidrug resistance [11]. Kanwar et al. found in mouse BC metastasis model experiments that higher Ca 2+ levels increase the survival and metastasis ability of BC tumor cells [28]. Liu et al. documented that calcium channel inhibitors (SOCC) can inhibit the migration of MCF-7 cells, indicating that abnormal Ca 2+ levels in BC cells can affect their migration ability [29]. Herein, we established that mimics of miR-129-1-3p considerably repressed the intracellular Ca 2+ concentration of MDA-MB-231 cells and reduced the number of Ca 2+ positive cells. miR-129-1-3p inhibitor dramatically increased the intracellular Ca 2+ concentrations, as well as Ca 2+positive cell numbers expressed. This result indicates that the tumor suppressor effect of miR-129-1-3p is linked to the Ca 2+ cascade.
We have verified that the Ca 2+ -linked GRIN2D 3 ′ -UTR has a binding site with miR-129-1-3p, which means that miR-129-1-3p can target and negatively regulate the expression of GRIN2D [30]. GRIN2D constitutes a subunit of the glutamate receptor calcium channel. It is worth noting that the glutamate receptor is overexpressed in multiple cancers and plays a vital role in cancer cell growth [31]. Besides, GRIN2D can be used as a critical hub of dysregulated  BioMed Research International signaling pathways in cancer [32]. Ferguson et al. exhibited that GRIN2D is expressed explicitly in the blood vessels of colorectal cancer and can be used as a specific marker of colorectal cancer angiogenesis [33]. Herein, the expression of GRIN2D mRNA along with protein in MDA-MB-231 cells was detected via qRT-PCR and Western blot assays.
The data illustrated that miR-129-1-3p mimics could dramatically reduce the expression of GRIN2D mRNA and Ca 2+ linked GRIN2D protein in the MDA-MB-231 cells.
On the other hand, miR-129-1-3p inhibitor dramatically increased GRIN2D mRNA expression and Ca 2+ associated protein expression in MDA-MB-231 cells. This finding sup-ports the experimental data of Fluo-3 AM for the determination of Ca 2+ content in MDA-MB-231 cells. Combined with these results, we confirmed that miR-129-1-3p can inhibit the growth, migration, and infiltration of TNBC cells by dampening the expression of GRIN2D gene and protein, thereby reducing the content of Ca 2+ in TNBC cells.
The present study enriched the knowledge of the biological functions of miR-129-1-3p and laid a good foundation for the study of the pathogenesis of TNBC. miR-129-1-3p is expected to become an essential target for TNBC treatment with a broad application prospect. For the next research step, we plan to introduce GRIN2D overexpression

Data Availability
The data generated in this work can be accessed from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.