MicroRNA-200c Overexpression in Cancer-Associated Fibroblasts Reduces the Invasive Properties of Breast Cancer Cells: An Approach to Molecular Therapy

Background The most common malignancy is breast cancer, among women in the world. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Cancer associated broblasts (CAFs) play a critical role to support tumor cells in all aspect of cancer development such as cell proliferation, migration and angiogenesis. MiRNAs are one of the regulatory molecules that regulate the genes contributing to cell growth, differentiation, migration and apoptosis. Based on other studies, miR-200c, as a tumor suppressor, has low expression levels in cancer associated broblasts. In this investigation, effect of miR-200c overexpression was evaluated on proliferation, migration and angiogenesis of TNBC cells. Methods The broblasts were isolated from normal and cancer breast tissue. MiR-200c expression was assessed using RT PCR in cancer associated broblasts (CAFs) and normal brobalasts (NFs) and then, were transfected using miR-200c. Finally, its effect on proliferation, migration and angiogenesis of TNBC cells were evaluated. Our results conrm that in presence of miR-200c transfected broblasts, the proliferation, migration and angiogenesis of cancer cells signicantly decreased. This effect may be due to the reduction of growth factors provided by CAFs after miRNAs dysregulation.


Background
Breast cancer (BC) is the most common cancer and one of the main reasons of death among women (1 in 3 cases). Triple-negative breast cancer (TNBC) is the most aggressive subtype of BC, with earlier recurrence and poor prognosis. This subtype has a larger size tumor with faster growth and metastasis to farther organs. It is identi ed by lack of estrogen receptor (ER), progesterone receptor (PR), and hormone epidermal growth factor receptor 2 (HER2) (1,2). Both environmental and genetic factors can interact to increase the risk of BC (3).
In recent years, the crucial role of tumor microenvironment (TME), also known as tumor stroma, has increasingly been elucidated (4). The modern concept is that cancer cells do manifest the disease only through collaborative interactions with their supporting stroma. TME plays a main role in many tumorigenic processes such as proliferative signaling, angiogenesis, invasion and metastasis that consequently lead to patient's death (5). TME consists of four elements: tumor vessels, immune cells, extracellular matrix (ECM) and broblasts, also known as tumor-associated broblasts (TAF) or cancer associated broblast ( CAF) (4,6).
Fibroblasts are the most abundant cells in stroma contributed to many biologic processes such as secretion of ECM components, including growth factor and signal factors, wound healing and provide support for tissue cells in in ammation. Through these processes, quiescent broblasts undergo alternations, become active and convert to myo broblast (7,8). Similar to myo broblast, CAFs support the tumor by providing suitable conditions under which the tumor can survive, grow, evade from the immune system and become metastatic (9). CAFs are the main source of factors such as TGFβ, EGF, α-SMA and VEGF, which participate in tumor progress, angiogenesis, migration and metastasis (10,11).
The mechanism of broblast activation in TME is not completely understood. However, recent studies have con rmed that mutations and dysregulation of regulatory genes in stroma cells lead to the secretion of factors in uencing tumor progression (6,12). One of the regulatory genes are miRNAs that play an important role in the transformation of normal broblast to CAF (12). miRNAs are small noncoding RNAs that regulate the genes contributing to cell growth, differentiation, migration and apoptosis (13,14). Based on recent reports, 11 groups of miRNAs are deregulated in CAF compared to normal broblast (NF) such as the miRNA-200 family, which cause secretion of supporting factors for tumor progression and development (15). Studies have revealed that miR-200c, as a tumor suppressor, has low expression levels in cancer associated broblasts that may prompt the invasion, migration and angiogenesis ability of cancer cells (16)(17)(18).
The present research was launched to assess the rate of proliferation, angiogenesis and migration of TNBC cell line after indirect co-culture with CAF and NF, overexpressing miR-200c, as compared to control cells.

2-1 Isolation of broblasts from normal and cancer breast tissue
Tumor and normal tissues specimens were obtained from stage II TNBC patients between the ages of 30 and 50 years undergoing lumpectomy and mastectomy at Ordibehesht surgery clinic, Isfahan, Iran.
Written informed consent was obtained from each participants before surgery. The proposal was approved by the ethics committee of Isfahan University of Medical Sciences. The tumor samples were isolated from tumor zone (within tumor boundary) and normal samples isolated from normal zone (at least 10 mm distal from tumor boundary) and were taken to lab in Dulbecco's Modi ed Eagle's medium (DMEM)/F12 + 10% FBS medium, sliced and digested with enzyme mixture contained 160µg/ml of Collagenase IV and 25µg/ml Hyaluronidase (19). Digested samples were incubated at 37º with 5% CO 2 overnight and then cultured in DMEM/F12 + 10% FBS for about a week.

2-2 Cell lines
BT-20. In 1958, BT-20 cell line derived from a 74-year-old Caucasian female with TNBC breast cancer, caused by invasive ductal carcinoma in mammary glands.
MDA-MB-231. This is an epithelial, human breast cancer cell line that is highly aggressive and invasive. MDA_MB-231 cell line was established from a pleural effusion of a 51-year-old Caucasian female with TNBC breast cancer.
HUVEC. The human umbilical vein endothelial cells, HUVEC, are endothelial like cells that are usually used for angiogenesis assays.
All the cell lines were purchased from Pasteur Institute of Iran, cultured in DMEM + 10% FBS medium and incubated at 37º with 5% CO 2 for further assays.

2-3 Immunocytochemistry (ICC)
Alpha actin smooth muscle (α-SMA) is known as a speci c marker widely expressed in broblast (20). To identify CAFs and NFs, the α-SMA (cat no. ab9654. Abcam) biomarker antibody were used and analyzed by ICC test based on the company instructions.

2-5 Real-time RT-PCR analysis
Trizol reagent (Invitrogen, USA) use to RNA extraction, cDNA was synthesized and expression levels of mature has-mR-200c-3p was evaluated in NF, CAF, miR-200c transfected NF and miR-200c transfected CAF cells with ExiLENT SYBR ® Green master mix kit (Exiqon, Denmark) using StepOne Plus™ real-time PCR (Applied Biosystems, USA). Fold change was calculated using Livak method and ready to use primers were purchased from Exiqon and the SNORDs was used as a house keeping gene to normalize the data.

2-6 Co-culture of isolated broblastand TNBC cell lines (BT-20 and MDA-MB-231)
Study plan. There were ve sets of triplicates in this study. The rst set was TNBC breast cancer cells (BT-20 and MDA-MC-231) that were not co-cultured with broblast which served as the negative control. The second set was co-cultured with un-transfected NF. The third set was co-cultured with scramble transfected NF. The fourth set was co-cultured with miR-200c transfected NF. The fth set was cocultured with un-transfected CAF. The other set was co-cultured with scramble transfected CAF and the last set was co-cultured with transfected CAF. For co-culture, 3 ×10 3 CAF and NF cells were plated in the upper well of transwell and 2×10 3 BT-20, MDA-MB-231 or HUVEC cells were seeded in lower well.
MTT assay. To investigate the proliferation of BT-20 and MDA-MB-231 cells after co-culturing with broblast, based on sets described above, MTT test was carried out. Two days after co-culturing, the upper wells were removed, then 50 µl MTT solution (5 mg/ml) (Sigma-Aldrich Co., USA) was added to each culture well. After 4-hour incubation, 200µl DMSO was added and incubated for more 30 min, and the absorbance was read at 570 nm by ELISA reader.
Wound healing assay. The directional cell migration of BT-20 and MDA-MB-231 cells was evaluated using wound healing assay after co-culturing with broblast, according to the groupings described above. BT-20 and MDA-MB-231 cells were plated. After 24 h, the monolayer was scratched by a sterile pipette tip and the wells were obliterated of debris. Later, the co-culture was performed based on study plan section and allowed the cells to close the wound for 24, 48 and 72 h. Wound closure was measured with microscopic photography in 10 randomized eld. The results were reported and analyzed by the imageJ software (version 1.52i).
HUVEC capillary tube formation assay. To run the assay, 100µl of Geltrex (Cat No. A1413202 Gibco) was added to the lower plate of transwell to form a 3D vessel-like tubes, then 10 5 HUVEC cells were seeded top of the Geltrex. Co-culture was performed based on study plan section and allowed the HUVEC cells to form tubes after about 24 and 48 h. Image analysis was carried out using the Angioquant software (version v1.33). The size, length and number of branches were measured and reported.

2-7 Statistical analysis
The data were analyzed using one-way ANOVA and Mann-Whitney U test. All results were shown as mean ± SD and P < 0.05 was considered as statistically signi cant.

Results
3-1 The broblasts isolated from normal and cancer breast tissues exhibit α-SMA marker. A combination of cells exists in isolated stromal cells from primary breast tissues that the main component of which are broblasts. The stromal broblasts isolated from normal and cancer breast tissues display features of NFs and CAFs. After 1 week, the cells gradually got out of the tissue sections (Fig 1Ag). Uniform broblasts initiate to grow after serial passage of primary cells (Fig 1B). The stromal broblasts separated from cancer and normal tissues expressed the activated myo broblast marker a-SMA based on ICC test results (Fig 1C). The results of previous studies indicated that at low passages cultured in vitro, the isolated broblasts reserve features of CAFs and NFs (21).
Six hours after transfection of broblast with miR-200c and scramble, the rate of transfection was evaluated using uorescence microscopy (Nikon Inverted Microscope-Japan) (Fig 2A,B) and ow cytometry (FACS Analysis-Becton, Dickinson and Company). The percentage of the cells containing scramble conjugated with FITC was about 78% (Fig 2C,D).
The level of miR-200c in CAF was lower than that of NF, but this difference was not signi cant (Fig. 3A).
After 24 h of transfection, the level of miR-200c was elevated to 4.6-fold in the miR-200c transfected NF cells compared to NF (Fig. 3B) and 3.5-fold in miR-200c transfected CAF cells compared to CAF (Fig. 3C).
Therefore, this results robustly con rmed e ciency of transfection.

3-4 MiR-200c-untransfected broblasts induced TNBC cell proliferation more than transfected broblasts.
To investigate the effect of mir-200c level changes in CAF on cancer cells, the proliferation of BT-20 and MDA-MB-231 cells was assessed after co-culturing with NF, miR-200c-transfected NF, CAF, miR-200ctransfected CAF and without any co-culture.
Cell proliferation of MDA-MB-231 cells signi cantly increase compared to control cells after co-culturing with NF and CAF (P= 0.01 and P=0.001, respectively). This results con rm the supportive role of broblast in cancer cell proliferation. This rate is more signi cant for those cancer cells co-cultured with CAF than NF. On the other hand, proliferation of BT-20 and MDA-MB-231 cells signi cantly decreased after co-culturing with miR-200c-transfected NF (P=0.000) and miR-200c-transfected CAF (P=0.000) compared to un-transfected ones (Fig 4). This results con rm that miR-200c has tumor suppressor role for cancer cells. The scramble transfected CAF and NF did not have signi cant effect on TNBC cell proliferation compared to un-transfected CAF and NF (P>0.05).
A wound healing assay was carried out under indirect co-culture conditions to con rm the effects of CAF, NF, miR-200c transfected CAF and NF stimulation on the BT-20 and MDA-MB-231 cell migration. The extent of the scratched zone narrowed when MDA-MB-231 and BT-20 cells were co-cultured with NFs, and this site was almost closed when MDA-MB-231 and BT-20 cells were co-cultured with CAFs within 72 hours (P=0.000) (Fig 5)(The microscopic images related to 72h after scratch are not shown). On the other hand, the size of the zone was broader when the cells were co-cultured with miR-200c transfected NF than untransfected NF (P=0.005) and this change was more signi cant in the cells co-cultured with miR-200c-transfected CAF than untransfected T-CAF (P=0.000) (Fig 5). The scramble transfected CAF and NF did not have signi cant effect on TNBC cell migration compared to un-transfected CAF and NF (P>0.05).
3-6 Angiogenesis in HUVEC cells is signi cantly decreased after co-culturing with miR-200c transfected NF and miR-200c transfected CAF compared to the un-transfected ones.
The anti-angiogenic effect of miR-200c overexpressing NF and CAF was evaluated after co-culturing with HUVEC tumor cells. We performed the in vitro HUVEC capillary tube formation assay. Figure 6 shows that HUVECs co-cultured with NF and CAF markedly produced capillary-like tubes compared with HUVECs cocultured with control cells (P = 0.05). In contrast, the angiogenesis notably suppressed in HUVEC cells after co-culturing with miR-200c transfected NF and CAF (P=0.000). Hence, poor vascularization involving small size, length and few branches of tubes was detected. This results con rm the suppressing role of miR-200c on angiogenesis stimulating behavior. The scramble transfected CAF and NF did not have signi cant effect on HUVEC cell angiogenesis compared to un-transfected CAF and NF (P>0.05).

Discussion
Tumor microenvironment, act as a supporting compartment in cancer process by providing a dynamic and physiologic niche for tumor. CAF, as a main functional cell in TME, produces and secretes growth factors, angiogenic factors which are a key player in EMT process and chemokines and cytokines that help migration and invasion of tumor leading to metastasis (22). Moreover, CAFs facilitate tumor cell movement and migration due to remodeling the ECM structure via changes in E-cadherin production, cellcell and cell-ECM junctions (22,23).
Previous studies have shown that CAFs can provide a perfect niche for the tumor to develop and grow (24). Similar lines of evidence were found in our study and the supportive role of CAFs was con rmed, they can trigger the early steps of tumor development and cell proliferation. Our observation showed that cancer cells grow better and faster in the presence of CAFs compared to NF. This may be due to of quiescent nature of normal broblasts.
The co-culture of NF/CAF with BT-20 and MDA-MB-231 cells revealed that migration rate of cancer cells was increased compared to when cancer cells were grown alone. However, the increase was more pronounced in the cancer cells co-cultured with CAFs than NFs. After about 72 h, the wound had not completely healed in in the cancer cells co-cultured with NF, while it completely healed when co-cultured with CAF.
Angiogenesis in tumor is a complex process that depends on several angiogenic factors such as VEGF (25). The present study demonstrates that angiogenesis is improved in the vicinity of the CAF, where the secretion of VEGF is high, as also con rmed in the past (26).
Generally, the manipulation of gene regulation in cancer microenvironment is an attractive therapeutic strategy for reducing proliferation, migration and angiogenesis. Fibroblast components are produced under control of genes, which are, in turn, regulated by miRNAs. Observations have identi ed about 11 dysregulated miRNAs in CAFs, whose target genes mostly participate in proliferation, angiogenesis, cell differentiation, migration, secretion and cell adhesion (15). In this study, we focused on miR-200c, one of the main member of miR-200 family, which is down-regulated in CAF.
MiR-200 family target many genes such as MSN, FN1, MARCKS, QKI, FGD1, LOX, KDR, PAG1,ZEB and SIP1 which have an important role in TGFβ-induced EMT process, cell adherences, migration and invasion (27)(28)(29). In addition, these family members are known to be tumor suppressor and the lack of these molecules could lead to aggressive phenotype in many cancers such as gastric (30) glioma (31) lung (32) and breast (33). Some evidence demonstrate that miR-200 family are downregulated in breast cancer CAFs compared to NF (34).
There is much evidence regarding the regulatory effects of miR-200c on the nature of cancer (35). miR-200c regulates insulin receptor substrate 1 (IRS1) post transcriptionally and its over expression may be retard to the growth of prostate cancer cells (36). Likewise, overexpression of miR-200c in melanoma cells remarkably reduce the proliferation (37). Some studies indicated miR-200c overexpression in breast cancer cells, like MDA-MB-231, can reduce the EMT and migration (38,39). Our observation revealed that miR-200c transfected CAFs and NFs co-cultured with TNBC basal and luminal breast cancer notably reduced growth and proliferation of tumor. Accordingly, the features of cancer cells which were cocultured with CAFs showed a signi cant difference compared to the group co-cultured with miR-200c transfected CAFs. However, this gap was less signi cant between the group co-cultured with NF and that co-cultured with miR-200c transfected NF.
Mir-200 family cooperate in tumor invasion and metastasis through inhibition of ZEB1 and ZEB2 gene expression (40). MiR-200c plays also an effective part in cancer metastasis via interference in TGFβ pathway (41). Here, we observed that transfection of CAFs with miR-200c can dramatically reduce breast cancer cell migration and invasion ability. Furthermore, overexpression of miR-200c in NF may also lead to deceased malignant cells invasion, nevertheless its effect is negligible. According to the wound assay, the scratch in the CAF co-cultured group was healed in a very short time while, the miR-200c-transfected CAF co-cultured group needed a much longer time for healing. The same pattern was observed in the NFs, but with a milder slope.
Increasing studies has revealed the therapeutic role of miR-200c on tumor angiogenesis. Accordingly, miR-200c can inhibit the tumor vacuolization (42). Our observation showed that transfection CAFs with miR-200c signi cantly decreased the angiogenesis in HUVEC cells. As it was found in angiogenesis test, CAF co-cultured group formed more tubes which were bigger in size and more branched than the control group. In addition, with a little difference, the NF co-cultured group showed the same results. Other ndings testify the supportive role of some CAF biomarkers such as miR-200c (43).

Conclusion
These results con rm that transfection of CAFs with miR-200c may limit variable aspects of breast cancer development such as growth, migration and angiogenesis. In this way, miR-200c can be regarded as a potential therapeutic tool for breast cancer in the future.

Consent for publication: Not applicable
Availability of data and materials section: Not applicable Funding: This study was funded by Isfahan University of Medical Sciences deputy of research (grant number 396159). The funding body was used to buy consumables materials.
Author contribution statement: The authors, NS and LS contributed in practical work, acquisition of data, or analysis and interpretation of data.
The author, MRH contributed in sample preparing. The author, SHJ contributed in conception and design, or acquisition of data, or analysis and interpretation of data