MiR-138-5p improves the chemosensitivity of MDA-MB-231 breast cancer cell line to paclitaxel

Chemotherapy is a predominant strategy for breast cancer (BC) treatment and paclitaxel (PTX) has been known as a conventional chemotherapeutic drug. However, insensitivity of BC cells to PTX limits the anti-tumor effects of this agent. MicroRNAs are closely related to BC which are suggested as therapeutic factors in the combination therapy of BC. We examined the possible efficacy of miR-138-5p restoration in combination with PTX to impove BC treatment. The human breast cancer cell line MDA-MB-231 was transfected with miR-138-5p mimics and treated with PTX, in a combined or separate manner. The MTT assay was accomplished to determine inhibitory doses of PTX. Annexin V/PI assay and DAPI staining were applied to evaluate apoptosis. Flow cytometry was applied to determine cells arrested in different phases of the cell-cycle. Expression levels of molecular factors involved in cell migration, proliferation, apoptosis, and cell cycle were determined via western blotting and qRT-PCR. MiR-138-5p combined with PTX suppressed cell migration via modulating MMP2, E-cadherin, and vimentin and sustained colony formation and proliferation by downregulation of the PI3K/AKT pathway. qRT-PCR showed that miR-138-5p increases BC chemosensitivity to PTX by regulating the apoptosis factors, including Bcl-2, Bax, Caspase 3, and Caspase 9. Moreover, miR-138-5p restoration and paclitaxel therapy combined arrest the cells in the sub-G1 and G1 phases of cell cycle by regulating p21, CCND1, and CDK4. Restored miR-138-5p intensified the chemosensitivity of MDA-MB-231 cell line to PTX, and the combination of miR-138-5p with PTX might represent a novel approach in BC treatment.


Introduction
Breast cancer (BC) is the most common cancer in women and remains a global problem of public health [1].Chemotherapy is predominantly used for breast cancer.There are several classes of chemo-therapeutics, including alkylating agents, antimetabolites, immunological elements, hormonal components, and mitotic deprivation [2].Paclitaxel (PTX), approved by the FDA (Food and Drug Administration) in 1994, is an anticancer drug that is performed in BC treatment [3].PTX, as an anti-mitotic drug, is frequently used in the treatment of metastatic breast cancer as first-line therapy that causes mitotic arrest by restaining dynamics of mitotic microtubules [4], which leads to cell death during mitotic arrest or exit mitosis without cell division forming tetraploid cells [5][6][7].PTX causes creating a 4 C DNA peak in flowcytometry, which is called 'G 2 /M' arrest and also initiates different signaling pathways leading to apoptosis [8].However, high concentrations of PTX could result in chemoresistance in BC patients [9].The resistance of BC cells to chemotherapeutics like PTX may be result from disequilibrium in various signaling pathways, mutations in specific genes, and epigenetic dysregulations, including downregulation of microRNAs [10].Previous studies have introduced various types of microRNAs which are deregulated in BC cells [11].MicroRNAs could be either oncogenic miRNA (oncomiR) or tumor suppressor miRNA (tsmiR).Tumor suppressor miRNAs are downregulated in various cancers like BC.Therefore, microRNA replacement therapy could effectively increase the efficacy of chemotherapeatics used in BC treatment [12].MiR-138-5p is a known strong tsmiR whose anti-cancerous effects on various cancers, including BC, have been proved by several researches [13][14][15].A study on pancreatic cancer showed that miR-138-5p restoration, by targeting vimentin, could increase the chemosensitivity of cancerous cells to 5FU (5fluorouracil) [16].Moreover, recent studies demonstrated that miR-138-5p improves the sensitivity of cervical cancer cells to Cisplatin and Glioblastoma (GBM) cells to temozolomide (TMZ) by targeting H2AX and Survivin, respectively [17,18].As well, the most recent study revealed that MiR-138-5p could inhibit prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B [19].We showed in our previous research that miR-138-5p has several anti-tumorigenic effects on BC cells by targeting PD-L1 (programmed cell death ligand 1) and suppressing cell proliferation and migration and the induction of apoptosis [15].Recent investigations have proved the synergistic effects of miR-424-5p and miR-383-5p on elevating the chemosensitivity of BC cells to PTX [20,21].However, there is no report about the impression of miR-138-5p restoration on the chemosensitivity of BC cells to PTX.In this study, we hypothesized that miR-138-5p might increase the toxic effects of PTX in the MDA-MB-231 breast cancer cell line.Our data represented that miR-138-5p could enhance the cytotoxic effects of lower doses of PTX on BC cell migration, apoptosis, cell proliferation, colony formation, and cell cycle.The mentioned findings suggest that miR-138-5p restoration and paclitaxel therapy combined might be a novel therapeutic approach that improves the efficacy of chemotherapy in BC treatment.

Cell line culture
Human Breast Cancer cell line MDA-MB-231 was purchased from Pasteur Institute's Cell Bank of Iran (Iran) and cultured in RPMI-1640 culture medium (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillinstreptomycin (Sigma-Aldrich, Merck, Germany) and cultivated in the incubator with the temperature of 37 °C and 5% CO2.

Cell viability assay
To identify the inhibitory doses of PTX on the viability of MDA-MB-231 cells after transfection with miR-138-5p and treatment with PTX in a combined or separate manner, an MTT assay was applied.Briefly, transfected cells were seeded into the 96-well plates at a total of 1 × 10 4 cells per well and, after 24 h incubation, treated with different doses of PTX (0-12.5 µg / ml).The cells were incubated at 37 °C for 24 h followed by washing with PBS and incubating with 50 µl of 2 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h at 37 °C.In the next stage, the medium of each well was replaced with 150 µl of DMSO (dimethyl sulfoxide) and incubated for further 30 min at 37 °C.Finally, the optical absorbance of each well was determined at the 570 nm wavelength by the Eliza reader (Tecanmicroplate, Switzerland).All experiments were conducted three times in a triplicate manner.

RNA extraction and qRT-PCR
Cellular RNAs were extracted from breast cancer cells by TRIzol reagent (GeneAll, Korea), and their purity and concentration were determined by NanoDrop spectrophotometer (TermoFisher).Then cDNAs were synthesized from mRNAs using BioFACT Kit (BR631-096, Korea).Finally, qRT-PCR was carried out using a BioFACT 2×SYBR green kit in an ABI Biosystem instrument (USA) to evaluate the transcript levels of Caspase 3, Caspase 8, Caspase 9, BAX, Bcl-2, P21, CCND1, and CDK4.The fold changes of gene expression were analyzed using the 2 −ΔΔCt formula and β-actin was used as an internal control.Primer sequences were listed in Table 1.

Cell cycle
BC cells were transfected with the miR-138-5p or NC-miR mimics, and seeded at the initial numbers of 3 × 10 5 cells/ well in the 6-well plates.After 24 h incubation, the cells were treated with PTX in a combined or separate manner and kept in an incubator for 24 h at 37 °C.Thereafter, cells were detached, washed and fixed with PBS, and chilled at 70% ethanol for 24 h.Next, the cells were treated with RNase A (Bioneer, Korea) and incubated with PI at 25 °C in a dark place for 30 min.The cellular populations in different cell cycle phases were determined using MACSQuant flowcytometry, and FlowJo software was applied to analyze the data.

Statistical analysis
GraphPad Prism version 6.0 (GraphPad, San Diego, CA, USA) was applied for analyzing all data.To determine the

Wound healing assay
MDA-MB-231 cells were separately transfected with the NC-miR and miR-138-5p mimics and cultured at the initial number of 2 × 10 5 cells in each well of 24-well plates.The cells, after 24 h incubation at 37 °C, were treated with PTX and incubated for a further 24 h.The wound area was scratched using a sterile 100 µl pipette tip on a cellular monolayer, and removing of dead cells was done by the medium exchange.In the following, to evaluate the migratory potential of cells, the inverted microscope (Optika, XDS-3, Italy) was applied to measure the width of the wound at 0, 24, and 48 h after scratching.

Apoptosis assay using annexinV/PI
The rate of apoptosis induced by miR-138-5p, PTX, or their combination was determined using Annexin V/PI kit (BD Biosciences).Briefly, a number of 3 × 10 5 transfected cells were seeded into each well of the 6-well plate and incubated for 24 h at 37 °C, followed by treatment with PTX in a separate or combined manner.Then, the cells were harvested after further 24 h incubation, and other steps, including washing with PBS and staining with Annexin V/PI, were done.The apoptotic cells were distinguished by the MAC-SQuant 10 flow-cytometry, and obtained data were analyzed by FlowJo software 7.6 (TreeStar Inc, USA).

Apoptosis test by DAPI staining
In proof of induced apoptosis, chromatin fragmentation was determined using DAPI staining in treated cells.In summary, transfected cells were calculated and cultivated at the initial concentration of 15 × 10 3 cells per well in 96-well plates.The cells were treated with PTX after overnight incubation and cultured at 37 °C for a further 24 h.After that, the cells were washed three times with PBS, and fixing with 4% paraformaldehyde was performed for one h.Then, penetrating of cell membranes was accomplished using Triton-X-100 (0.1%) for 10 min, followed by washing with PBS and staining with 60 µl of 0.1% DAPI for 15 min in a dark place.Eventually, chromatin fragmentation, as a sign of apoptosis, was determined by the cytation 5 cell imaging system (Biotek, USA).

Colony formation assay
To explore the inhibitory effect of miR-138-5p and PTX on colony-forming ability, transfected BC cells were seeded into the 6-well plates at a density of 5 × 10³ cells per well and treated with PTX in a combined or separate manner after 24 h incubation.After incubation for 12 days, the cells MDA-MB-231 cells in our previous study [15].Here, cells were incubated with various concentrations of PTX (0 to 12.5 µg/ml) for 24 h, and the cell viability was determined using an MTT assay to indicate the inhibitory doses of PTX.According to our results, 5 µg/ml and 10 µg/ml of PTX decreased the viability of BC cells to 75% and 50%, respectively.Therefore, 5 µg/ml and 10 µg/ml of PTX were selected as IC 25 and IC 50 , respectively (Fig. 1:A).

MiR-138-5p amplified the toxicity of PTX in lower doses
Data from MTT assay displayed decreased survival rates of BC cells by 25%, 50%, 40%, 51% and 70% in the PTX (IC25), PTX (IC50), miR-138-5p, miR-138-5p + PTX (IC25) and miR-138-5p + PTX (IC50) groups compared to the N-control group respectively (Fig. 1:B).Besides, the miR-138-5p + PTX (IC25) group showed a significantly less cell survival rate rather than the PTX (IC25) group or the miR-138-5p group alone, which means PTX (IC25) combined with miR-138-5p approximately has toxicity as the same of PTX (IC50).Treatment with 10 µg/ml of PTX (IC 50 ), separately or in combination with miR-138-5p, was highly toxic and led to more than 50% cell death.So, two statistical significance between two groups, Student's paired t-test was used, and the statistical significance among several groups was calculated and analyzed by the Kruskalwallis test of one-way ANOVA or multiple comparisons test of Two-way ANOVA.Mean ± S.E.M was used to express all data.All experiments were performed in a triplicate and repeated three times separately.

IC 25 and IC 50 determination of PTX in MDA-MB-231 cells
As reported in the previous study, the MDA-MB-231 cell line was selected as the main cell line to restore miR-138-5p between four different breast cancer cell lines (MDA-MB-468, MDA-MB-231, SKBR-3, and MCF-7) based on the least level of miR-138-5p transcript and the most level of PD-L1 expression [15].Our experiments were continued using the MDA-MB-231 cells as the main cell line to explore the effects of miR-138-5p restoration and paclitaxel therapy combined.A 40 pmol concentration of miR-138-5p mimics was introduced as an optimal transfection dose in

miR-138-5p enhanced the suppressive effects of PTX on colony formation and cell proliferation in MDA-MB-231 cells
To investigate the effects of miR-138-5p and PTX in combination or a separate manner on cloning and cell expansion, the colony formation and western blotting assays were employed.In our previous study, the inhibitory effects of miR-138-5p on colony formation and cell proliferation was observed.Here, analyzed data suggested that PTX could reduce the colony forming and cell proliferating rates via downregulation of the PI3K/AKT pathway.Moreover, colony formation and the PI3K/AKT pathway were suppressed more significantly in the miR-138-5p + PTX (IC25) group versus other groups (p < 0.0001) (Figs. 4 and 5).

miR-138-5p and PTX induced apoptosis in MDA-MB-231 cells synergistically
Data obtained from flow-cytometry and DAPI staining illustrated induced apoptosis in all MDA-MB-231 treated groups compared to the N-control one.Also, significant increased early apoptosis (25.5% apoptotic cells) and increased total apoptosis (36.6% apoptotic cells) were observed in the miR-138-5p + PTX (IC25) group versus other groups (p < 0.0001) (Fig. 6).Furthermore, a remarkable increase in chromatin fragmentation was detected in the miR-138-5p + PTX groups treated with the mentioned dose were removed from the next tests.

MiR-138-5p improved the PTX-induced inhibition of BC cell migration
Using wound healing assay, the migratory potential of MDA-MB-231 cells after transfection with miR-138-5p and, or treatment with PTX (IC25) was explored for 0, 24 and 48 h after scratching via inverted microscope.As mentioned in our previous study, the migration to the wound area was sustained in miR-138-5p-overexpressing cells by downregulation of MMP9, MMP2, vimentin, and upregulation of E-cadherin [15].Here, results indicated that cell migration was repressed in all treatment groups compared to N-control one.As well, the migration rate of MDA-MB-231 cells was decreased more significantly in cells transfected with miR-138-5p and treated with PTX (miR-138-5p + PTX (IC25) group), compared to a separate manner (Fig. 2).Furthermore, the western blotting assay represented a more significantly decreased level of MMP2 protein and increased level of E-cadherin protein in the miR-138-5p + PTX (IC25) group against the miR-138-5p group (p < 0.01, p < 0.0001) or the PTX (IC25) group alone (p < 0.0001, p < 0.001) (Fig. 3).respectively.Interestingly, in the miR-138-5p + PTX (IC25) group, the percentage of arrested cells in the G 2 /M phase significantly decreased into 53.2% while 19.9% and 14.6% of them were arrested in the sub-G 1 and G 1 phases, respectively (Fig. 8: A, B).In order to investigate the molecular mechanism underlying cell-cycle arrest, the transcript levels of several factors involved in cell-cycle, including Cyclindependent kinase 4 (CDK4), cyclin D1 (CCND1), and p21 (cyclin-dependent kinase inhibitor 1), were determined using qRT-PCR.According to the analyzed data from qRT-PCR, there was no significant difference between the PTX (IC25) group and the N-control group in transcript levels of mentioned factors.However, considerable downregulation in CDK4 and CCND1 and remarkable upregulation of p21 were observed in both the miR-138-5p and miR-138-5p + PTX (IC25) groups (Fig. 8: C).

miR-138-5p in combination with PTX led to arrest the cell cycle in the sub-G 1 and G 1 phases in MDA-MB-231 cells
As it was mentioned in introduction section, PTX can arrest the cell cycle in the G 2 /M phase.Here, using cell cycle assay, we detected 79.9% of PTX (IC25) cells arrested in the G 2 /M phase.Also, 22.9% and 37.7% of cells related to the miR-138-5p group were arrested in the sub-G 1 and G 1 phases, MMP2: matrix metalloproteinase-2, E-cadherin: epithelial cadherin resistance to paclitaxel-induced apoptosis [27].MicroR-NAs are involved in various cellular processes and their deregulation might influence on cellular behaviors leading to multiple disorders and cancers including BC [28].Multiple studies have introduced various microRNAs involved in improving the chemosensitivity of cancer cells.For example, miR-142-3p improves PTX sensitivity in resistant breast cancer by inhibiting autophagy through the GNB2-AKT-mTOR Pathway [29].On the opposite side, several tumor-promoting microRNAs are upregulated in cancers which mediate PTX resistance; upregulation of miRNA-4262 can mediate PTX resistance via PTEN downregulation and activation of the PI3K/Akt signaling pathway [30].Also, microRNA-155-5p contributes to paclitaxel resistance via TP53INP1 in human breast cancer [31].miR-138-5p as a tumor suppressor miRNA has different roles in biological processes indicated in our previous study; It was demonstrated that miR-138-5p is downregulated in BC cells and its overexpression has multiple effects on BC cells including inhibition of cell proliferation and cell migration and induction of opoptosis via targeting PD-L1 [15].Several studies have evidenced that miR-138-5p could improve the chemosensitivity of cancer cells to different chemotherapy agents [16][17][18].Recently, Liang, M., et al. have reported that miR-138-5p restoration could sensitize PTX-resistant epithelial

Discussion
Breast Cancer (BC) is the cause of 25% of all death cases in women worldwide.One of the conventional chemotherapeutic agents is paclitaxel (PTX) [22] which has been used as a first line treatment in BC chemotherapy since 1994 [23].Also, Paclitaxel is employed in adjuvant settings.Recently, Ke-Da Yu, et al., have suggested a paclitaxel-plus-carboplatin regimen as a practical alternative adjuvant chemotherapy choice for patients with operable triple negative breast cancer [24].However, resistance to chemotherapeutic drugs is considered as the main obstacle in the BC chemotherapy and the insensitivity of BC cells to PTX limits its anti-tumor effects [25].On the other hand, PTX has several side effects on patients receiving higher doses of PTX [26].Dulcie Lai, et al., have suggested the possible mechanism of paclitaxel resistance; TAZ (transcriptional co-activator with PDZbinding motif) or WWTR1 is a transcriptional coactivator which acts as a downstream regulatory target in the Hippo signaling pathway and forms a complex with TREAD which translocate to the nucleus and binds to the promoter of Cyr61 or CTGF genes and promotes the expression of mentioned genes which subsequently Cyr61 or CTGF protein is secreted out of the cells and activates integrin heterodimers on the cell membrane leading to cell survival and of MMP2, vimentin, and E-cadherin and the inhibition of cell migration.Zhang, J., et al. discovered that 3'-UTR of vimentin mRNA is a direct target of miR-138-5p [38].It may be concluded that miR-138-5p would intensify the anti-migratory effect of PTX on BC cells through suppressing vimentin and modulating MMP2 and E-cadherin.In the following, it was disclosed that miR-138-5p in combination with PTX can reduce the cell proliferation and colony formation in MDA-MB-231 cells more significantly rather than the PTX (IC25) treatment alone.In continue, the expression levels of PI3K, p-PI3K, AKT and p-AKT involved in the PI3K/AKT pathway were evaluated to investigate the molecular mechanisms underlying the anti-proliferative effects of miR-138-5p and PTX on BC cells.Our data from western blotting showed significantly less levels of PI3K, p-PI3K, and AKT proteins in the miR-138-5p + PTX ovarian cancer cells via targeting cyclin-dependent kinase 6 (CDK6) [32].LncRNA UCA1 contributes to oxaliplatin (OXA) resistance and activation of the AKT/mTOR signaling pathway, through inhibition of miR-138-5p, in hepatocellular carcinoma cells [33].
This study confirmed the in vitro therapeutic effects of miR-138-5p restoration on the chemosensitivity of BC cells to PTX in MDA-MB-231 breast cancer cell line.Multiple researches have revealed the suppressive effects of PTX on cell migration in cancerous smooth muscle cells and BC cells [34][35][36][37].In our previous study, the inhibition of cell migration affected by miR-138-5p restoration was observed in MDA-MB-231 cells [15].Herein, we first explored the impression of miR-138-5p restoration and PTX therapy combined on cell migration and their sinergistic effect was observed through modulating the expression levels  [39].Also, it has been proved that CCND1 overexpression correlates with tumor progression [41] and the CCND1-CDK4 complex promotes the transition from G 1 to S phase by sustaining the retinoblastoma protein (pRb) [42].p21, a cyclin-dependent kinase inhibitor, inhibits all cyclin/CDK complexes and functions as a regulator of cell cycle progression in the G 1 and S phases [43][44][45].In this study, the significant reduced transcript levels of CDK4 and CCND1 were observed in (IC25) group rather than the miR-138-5p group or the PTX (IC25) one.Also, decreased number of colonies in the miR-138-5p + PTX (IC25) group versus the PTX (IC25) group reflected the synergistic impression of miR-138-5p restoration on the anti-mitotic effect of PTX in MDA-MB-231 cells.G. Li et al. have reported that PTX inhibits cell proliferation and promote cell apoptosis by downregulating the PI3K/AKT signaling pathway in MCF-7 cell line [39].Moreover, in our previous study, we found that miR-138-5p could suppress cell proliferation via downregulation of PI3K/AKT pathway [15].Also, Cui et al. discovered that Long non-coding RNA TRPM2-AS activate epidermal growth factor receptor (EGFR) and PI3K/AKT signaling pathway via sponging miRNA-138-5p in non-small cell lung cancer [40].Therefore, we may conclude that miR-138-5p and PTX, in a combined manner, display their synergistic effect on inhibiting cell proliferation through suppressing PI3K/AKT pathway.To further investigate the effects of miR-138-5p restoration and paclitaxel therapy combined

Conclusion
Overall, miR-138-5p restoration in combination with PTX therapy could improve the inhibition of cell migration, apoptosis induction, and the cell cycle arrest.Also, this combination decreases the potency of proliferation and colony-formation in BC cells synergistically.In conclusion, based on our information obtained from the current project, the combined therapy might be a new therapeutic approach to decrease the efficacious consuming PTX dose in chemotherapy and enhance the efficacy of BC treatment.However, further experiments are essential to detect precisely the unknown cellular pathways underlying these synergistic effects or signaling pathways involved in the reversion of paclitaxel resistance by miR-138-5p restoration.the miR-138-5p + PTX (IC25) group rather than the PTX (IC25) cells.Moreover, a remarkable up-regulation of p21 was detected in the miR-138-5p + PTX (IC25) group versus the PTX (IC25) group or miR-138-5p-transfected group.On the other hand, Liu et al. introduced CCND1 mRNA as a direct target of miR-138-5p in nasopharyngeal carcinoma cells [46].As well, miR-138-5p blocks the G 1 /S transition by suppressing the cell-cycle regulators in prostate cancer cells [47].Therefore, targeting of CCND1 mRNA by miR-138-5p restoration, downregulation of CDK4, and upregulation of p21 may explain significant high percentage of the sub-G1 and G1 arrested cells in the miR-138-5p + PTX (IC25) group compared to other groups.Overall, the combination of miR-138-5p with PTX may improve the efficacy of PTX as a chemotherapeutic drug in BC treatment.

Fig. 2
Fig. 2 MiR-138-5p improved the PTX-induced inhibition of BC cell migration.Cell migration was repressed in all groups compared to the N-control group and also the potency of migration was suppressed

Fig. 3
Fig. 3 miR-138-5p in combination with PTX had Synergistic effects on modulating the expression level of biomarkers involved in cell migration.A, B, C. Western blot significantly detected deceased protein levels of pro-MMP2 and active-MMP2 and increased level of E-cad-

Fig. 4
Fig. 4 miR-138-5p enhanced the suppressive effect of PTX on the colony formation.The Number of colonies in the miR-138-5p + PTX (IC25) group were significantly less than the PTX (IC25) group or the

Fig. 5
Fig. 5 miR-138-5p enhanced the suppressive effect of PTX on the cell proliferation.A, B, C. data obtained from western blotting represented remarkable decreased protein levels of AKT, PI3K and p-PI3K in the miR-138-5p + PTX (IC25) group versus the PTX (IC25) group or the miR-138-5p alone.(Multiple comparison Two-way ANOVA, ns

Fig. 7
Fig. 7 miR-138-5p and PTX amplified the chromatin fragmentation and modulated the expression level of apoptotic biomarkers.(A) DAPI staining revealed remarkable increase in chromatin fragmentation in the miR-138-5p + PTX (IC25) group compared to other groups.(B) The transcript levels of apoptotic biomarkers, including BAX/Bcl-2 ratio, Caspase3, and Caspase 9 were significantly overexpressed in

Fig. 8
Fig. 8 miR-138-5p in combination with PTX led to arrest the cell cycle in the sub-G 1 and G 1 phases.A, B. Cell-cycle assay detected 79.9% of PTX (IC25) cells arrested in the G 2 /M phase.In addition, 22.9% and 37.7% of miR-138-5p cells were arrested in the sub-G 1 and G 1 phases respectively.However, the percentage of arrested cells in the G 2 /M phase significantly decreased into 53.2% in the miR-138-5p + PTX (IC25) group and also 19.9% and 14.6% of mentioned cells were arrested in the sub-G 1 and G 1 phases respectively.C. The transcript levels of CCND1, CDK4, and p21 were evaluated by qRT-PCR which

Table 1
Primer Sequences