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Abstract

Recent studies underline the involvement of microRNAs in cancer development through induction of immune suppression milieu and evolution of drug resistance. The goal of this study was to evaluate the effects of miR-146a on regulatory T cells’ frequencies, T-lymphocyte proliferation, and cytokine expression as well as drug resistance in cancer cells. We found that miR-146a was overexpressed in colon cancer HT-29 cells. Peripheral blood mononuclear cells were obtained from healthy donors and were co-cultured with transfected HT-29 cells. Afterward, peripheral blood mononuclear cell proliferation, expression of anti-inflammatory cytokines, and regulatory T cells’ frequencies were assayed. Also, drug resistance in transfected HT-29 cells was analyzed following treatment with 5-fluorouracil and irinotecan. Overexpression of miR-146a increased transforming growth factor-β and interleukin-10 expressions and enhanced regulatory T cells’ frequencies in peripheral blood mononuclear cells. Also, the number of cells undergoing cell cycle arrest and apoptosis significantly decreased in transfected HT-29 cancer cells. In conclusion, upregulation of miR-146a plays an important role in enhancing immune suppression through increasing the regulatory T cells’ population. Also, our data indicated that colon cancer drug resistance is possibly associated with miR-146a overexpression.

Keywords

Colorectal cancer miR-146a immune regulation drug resistance

Introduction

Colorectal cancer is the third most common malignancy and the second leading cause of cancer-related deaths in the United States and other developed countries and equally affects men and women.^1,2 Through early detection and surgical resection, its 5-year survival rate is 90%, in which nearly 50% patients develop recurrent disease.³ Metastatic spread of chemotherapy-resistant cells to other organs, especially the liver, is the main cause of colon cancer deaths.⁴ Resistance to chemotherapy and immune suppressive milieu around the tumor cells remain major obstacles in effective anticancer treatments.^5,6

Numerous evidences indicate that cancer cells cause suppressive effects on the host immune system, particularly cell-mediated immune response resulting in relapse and progression of tumors.^7,8 Regulatory T cells (Tregs) are a heterogeneous subpopulation of T lymphocytes, which play a crucial role in tolerance maintaining.⁹ Tregs are CD4+ lymphocytes characterized by constitutive expression of CD25 and forkhead family transcription factor (Foxp3) that is the key regulatory gene for the development and function of CD4+ CD25+ Treg.¹⁰ Tregs inhibit the local immune response, decreasing T-lymphocyte proliferation and pro-inflammatory cytokine secretion which promote tumor progression.^11,12 Also, some studies showed that Tregs are resistant to conventional chemotherapy which may help tumor cells from immune evasion.¹³

One of the main choice treatments for colorectal cancer is chemotherapy using multiple anticancer drugs, such as irinotecan, oxaliplatin, and 5-fluorouracil (5FU).^14,15 Chemotherapy resistance has been attributed to many reasons including dysfunctional membrane transport, resistance to apoptosis, persistence of stem cell–like tumor cells, autophagy, and epigenetic changes.^16,17

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the level of transcription and translation and represent a pervasive feature of all cells.¹⁸ Recent investigations have shown that expression of distinct miRNAs differs in the colorectal cancer microenvironment, especially in cancer stem cells.^19,20 Cancer stem cells have recently been considered as a crucial player in cancer development and drug resistance. MiRNA dysregulation plays an important role in development of drug resistance through endogenous expression of noncoding RNA as functional regulators of gene expression.²¹ Also, miRNAs are considered main regulators of Treg homeostasis, population, and function as well as T-lymphocyte proliferation and cytokine production in normal and cancer tissues.^22,23 One of the main miRNAs overexpressed in Treg and cancer milieu is miR-146a.^11,24 MiR-146a is differentially expressed in diverse subtypes of T lymphocytes, such as Th1 and Th2 cells, naive T cells, and Tregs.¹¹ We profiled miRNAs in HT-29 colon cancer cell line–derived cancer stem cells compared to cancer cells, and data showed that miR-146a is significantly overexpressed in cancer stem cells (unpublished data). Also, Hwang et al.²⁵ reported that miR-146a stabilizes β-catenin and maintains Wnt signaling resulting in tumor growth.

The aim of this study was to evaluate the effects of miR-146a overexpression on the Treg population and on peripheral blood mononuclear cell (PBMC) proliferation and expression of transforming growth factor (TGF)-β and interleukin (IL)-10 as a possibly way tumor cells escape surveillance. Also, we investigated the effect of overexpression of miR-146a on sensitivity of cancer cells to common chemotherapy drugs.

Materials and methods

Cell lines and culture conditions

The human colonic adenocarcinoma cell lines HT-29 and HEK-293T were purchased from Iranian Biological Resource Center (IBRC, Tehran, Iran). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified incubator at 37°C and 5% CO₂. Cells were suspended using trypsin (0.25% trypsin/ethylenediaminetetraacetic acid (EDTA; Gibco , USA) for 3–5 min).

Production of lentivirus vectors

Lentivirus-based vectors for miR-146a (pLenti-III-miR-GFP), negative control (pLenti-III-miR-GFP-Blank), envelop plasmid pMD2G, codes for the broad range VSV-G envelope, and packaging plasmid PAX2 were obtained from Applied Biological Materials (Richmond, BC, Canada). Viral particles were generated by calcium phosphate–mediated transfection of HEK-293T cells (Tronolab Protocol, 2007). Briefly, 5 × 10⁶ 293T cells were plated in a 10-cm² plate in 10 mL of the medium and transfected with 21 µg vector plasmid including miR-146a or blank, 10.5 µg pMD2G plasmid, and 21 µg psPAX2 plasmid. The medium was removed around 14–16 h after transfection and replaced with 10 mL of fresh pre-heated virus collecting medium. Lentiviral particles were collected at 24, 48, and 72 h after transfection from supernatant of HEK-293T cells, concentrated using polyethylene glycol (PEG; molecular weight (MW): 8000), and stored at −80°C.

Vector titration

Titers of virus were determined by infection of HEK-293T cells using serial dilutions. Briefly, 6 × 10⁴ 293T cells per 2 mL media per well were plated in a 12-well plate. Serial dilutions of the virus were made in 1.5 mL Eppendorf tubes and then were added to the pre-plated HEK-293T cells. After 72 h, green fluorescent protein (GFP)-expressing cells were quantified by flow cytometry for each dilution to determine transducing units (TU) per milliliter. The following formula was used for calculating titer (TU) per milliliter: TU/mL = (seeded cells (count at day 1) × %GFP-positive cells) × 1000/µL of vector.

Lentiviral vector transduction

The human colonic adenocarcinoma cell line HT-29 was cultured at 5 × 10⁴ cells/cm² in a 24-well culture plate for 24 h and then transduced with viruses containing miR-146a and blank vector at a multiplicity of infection (MOI) of 5. Virus supernatants were removed after 24 h, and 1 mL of fresh medium was added to the cells. GFP expression was visualized using a fluorescence microscope 3 days post-transduction.

Co-culture of PBMCs and transfected HT-29 with miR-146a

For co-culture, HT-29 transfected with miR-146a, blank, and HT-29 cancer cells was plated in a 24-well plate, and PBMCs were added to different groups to analyze percentage of Tregs, PBMC proliferation, and cytokine expression. The ratio of PBMCs to transfected cells in co-cultures was 5:1. PBMCs were collected and washed by centrifugation. The cell pellet was re-suspended in 1% FBS medium, and the appropriate number of mononuclear cells was added to each well on top of the adhered transfected HT-29 cells. Also, PBMCs were cultured with miR-146a-transfected HT-29 and blank for 48 h for evaluating TGF-β and IL-10 expressions. To assess Tregs’ frequencies, co-culture duration of PBMC and transfected HT-29 was 7 days.

Proliferation assay

Peripheral whole blood samples from different healthy donors were collected in EDTA tubes, and PBMCs were isolated using Ficoll–Hypaque gradients (Sigma, St Louis, MO, USA). The obtained PBMCs were cultured in six-well plates using RPMI 1640 culture medium in the presence of 10% FBS at 37°C and in a 5% CO₂ humidified atmosphere. To evaluate cell proliferation, PBMCs were labeled before co-culture with transfected HT-29 cells with miR-146a and stained with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 µM; Invitrogen, Eugene, OR, USA) according to the manufacturer’s instructions and maintained in culture for 24, 48, 72, and 96 h. Then, the cells were harvested and washed twice with 2 mL of phosphate-buffered saline (PBS) containing 1% bovine serum albumin.

Flow cytometry

Tregs were analyzed with the Treg detection kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s protocol, to assess Treg differentiation in the presence of transfected HT-29 with miR-146a, blank, and HT-29 cancer cells 7 days after co-culture (described above). Briefly, after co-culture, harvested PBMCs were stained with anti-FOX-P3-PE, anti-CD25-PE-cy7, and anti-CD4-APC (BD Biosciences). Flow cytometry was performed using the FACS Canto II (BD Biosciences), and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

Quantitative real-time reverse transcriptase polymerase chain reaction

To assess miR-146a expression in transfected and control groups, total RNA was extracted using miRCURY™ RNA Isolation Kit (Exiqon, Vedbaek, Denmark), and complementary DNA (cDNA) was synthesized by stem-loop primers by Universal cDNA Synthesis Kit II (Exiqon), according to the manufacturer’s instructions, and stored at −20°C until use. Expression of miRNAs was measured using the SYBR Green Master Mix (Exiqon), according to the manufacturer’s protocol. MiRNA expression was quantified using the 2^−ΔΔCT method. The primers used for stem-loop reverse transcriptase polymerase chain reaction (RT-PCR) for miR-146 and SNORD47 (internal control), as well as primers for polymerase chain reaction (PCR) are listed in Supplementary Table S1. To evaluate the cytokine expression changes after co-culture, PBMCs were harvested from different groups and total RNA was extracted using High Pure RNA Isolation Kit (Roche, Switzerland) according to the manufacturer’s instructions. cDNA was synthesized by Sensiscript^® Reverse Transcription Kit (Qiagen, Hilden, Germany). Quantitative RT-PCR was performed using the LightCycler FastStart DNA Master PLUS SYBR GreenI (Qiagen), according to the manufacturer’s protocol, on Roche LightCycler version 3.5. The primer sequences and PCR conditions are summarized in Supplementary Table S1. The relative amount of each messenger RNA (mRNA) was normalized to HPRT.The fold change from experiments relative to the control was calculated using 2^−ΔΔCT method.

Drug resistance

5FU and irinotecan, as routine chemotherapy drugs, were used to evaluate the resistance of transfected HT-29 cells with miR-146a compared to HT-29 colon cancer cells to chemotherapy reagents. HT-29 and transfected HT-29 cells were cultured in 24-well plates. After reaching 80% confluency, 5FU and irinotecan were provided in a stock sterile saline solution of 25, 50, 100, and 200 ng/mL and were added in different groups. After 48 h, apoptosis and cell cycling were analyzed.

Apoptosis and cell cycle assays

Apoptotic cell death was confirmed in selected experiments with Annexin V-PE–7AAD double staining. HT-29 and transfected HT-29 cells were treated with various concentrations of 5FU or irinotecan. After 48 h, apoptosis was detected using an Annexin V-PE Apoptosis Detection Kit (ebioscience, USA). Cells were stained with Annexin V-PE and 7AAD and analyzed by flow cytometry (FACS Can II; BD Biosciences). The results were expressed as the ratio of apoptotic to total cells. Cell cycle phase distribution was analyzed by flow cytometry using propidium iodide (PI) staining. Briefly, after cell fixation in 70% ethanol, cells were re-suspended in PBS with 0.25% Triton X. After three washing steps, cells were incubated with RNaseA (100 µg/mL) at 37°C and PI was added at 10 µg/mL directly before flow cytometric analysis.

Statistical analysis

Data were expressed as percentages, mean values, and standard deviations. Groups were compared using the one-way analysis of variance (ANOVA) with post hoc Tukey’s analysis. p value of less than 0.05 denoted a statistically significant difference. GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analyses.

Results

Establishment of miR-146a-overexpressing HT-29 cells

The expression of recombinant lentiviral vector containing miR-146a and blank was examined in HEK-293T cell lines by fluorescence microscopy 48 h after transfection of cells with PsPAX2, PMD2-G, pLenti-III-mir-GFP-blank, and pLenti-III-mir-GFP-has-miR-146a (Figure 1S). GFP protein was used as reporter gene in miR-146a and blank vectors. The results revealed that miR-146a was expressed in HEK-293T cells. The virus particles derived from the transfected HEK-293T, which contained miR-146a and blank vectors, were used for HT-29 cell transduction. The consistent overexpression of mature miR-146a in transduced HT-29 cells was confirmed by fluorescence microscopy and RT-PCR. The results revealed that the expression of miR-146a in the miR-146a transduced HT-29 cells’ group was significantly increased compared with control groups (p < 0.01; Figure 1(a) and (b)).

Figure 1.

(a) Consistent expression of miR-146a in HT-29 cells 48 h after transfection. A: Un-transfected HT-29 cells. B, C, and D: transfected HT-29 cells with miR-146a in dose-dependent manner of virus particles. (b) Real-time PCR documentation of miR-146a expression in HT-29 cells 48 h after transfection. *p < 0.05; **p < 0.01; ***p < 0.001.

PBMC proliferation after co-culture with transfected HT-29 cells

We evaluated the proliferation of PBMCs 24, 48, 72, and 96 h after co-culture of CFSE-labeled PBMC with transfected HT-29 to determine the effects of miR-146a overexpression on PBMC proliferation. Proliferation capacity of PBMCs was not remarkably changed in transfected HT-29 cells with miR-146a groups compared to that in blank and control groups (Figure 2).

Figure 2.

Proliferation assay of PBMC 72 h after co-culture with transfected HT-29 cells with miR-146a using CFSE method. (a) PBMC population based on SSC and FSC characters. (b) Un-proliferated PBMC. (c) Proliferation of PBMC after exposure to PHA. (d) Proliferation of PBMC in presence of untransfected HT-29 cells. (e) Proliferation of PBMC in presence of transfected HT-29 cells with miR-146a. (f) Proliferation of PBMC in presence of transfected HT-29 cells with blank vector. Bottom figure: percentage of divided cells in different groups.

Increased Tregs’ frequencies following co-culture of transfected HT-29 cells with PBMC

The frequencies of Tregs in PBMCs following co-culture with transfected HT-29 cells were determined by flow cytometry. After 7 days of co-culture, the results revealed that overexpression of miR-146a in HT-29 cells significantly increased the Tregs’ frequencies in PBMCs compared to the blank and control groups (p < 0.01; Figure 3). These data showed that miR-146a plays an important role in immune suppression in the tumor microenvironment.

Figure 3.

Regulatory T cells’ frequencies in PBMC following co-culture with different groups of cancer cells. (a) PBMC and their isotype controls. (b) Tregs’ frequencies in PBMC after co-culture with HT-29 cells without transfection. (c) Tregs’ frequencies in PBMC population following co-culture with transfected HT-29 cancer cells with miR-146a. (d) Tregs’ frequencies in PBMC population following co-culture with transfected HT-29 cancer cells with blank vector. (e) Tregs’ frequencies in PBMC population without any treatment as control group. (f) The results revealed Tregs’ frequencies in PBMC co-cultured with transfected HT-29 cancer cells with miR-146a significantly increased compared to the all control groups (p < 0.001). Also, Tregs’ frequencies in PBMC co-cultured with HT-29 and transfected HT-29 cells with blank vector remarkably increased compared to PBMC without treatment (p < 0.05 and p < 0.01, respectively).

Expression level of anti-inflammatory cytokines in PBMC following co-culture with transfected HT-29 cells with miR-146a

A cytokine milieu that suppresses immune response is crucial in the maintenance and progression of cancer cells. To determine TGF-β and IL-10 expression in PBMC following co-culture with transfected HT-29 cells, expression of mentioned cytokines was analyzed by RT-PCR. As shown in Figure 4(a) and (b), the expression levels of TGF-β and IL-10 in PBMC cultured with transfected HT-29 cells significantly increased compared with that in blank and control groups (p < 0.01 and p < 0.05, respectively).

Figure 4.

(a) Expression of TGF-β in PBMC following co-culture in different groups. (b) Expression of IL-10 in PBMC following co-culture in different groups. *p < 0.05; **p < 0.01; ***p < 0.001.

Overexpression of miR-146a ameliorates the cancer cells’ resistance

To evaluate the effects of chemotherapy drugs on cancer cell growth following transfection of cells with miR-146a, cell cycling and apoptosis analyses were performed following 5FU and irinotecan addition in different doses. As shown in Figures 5 and 6, the cell cycle distributions for different groups of transfected and non-transfected HT-29 cultures that were treated with 25, 50, 100, and 200 ng/mL of irinotecan or 5FU changed (p < 0.001). The data showed that cell cycles were remarkably arrested in G0/G1 in control groups compared to that of miR-146a-transfected HT-29 cells in both drugs’ treatment. Also, the elevated miR-146a expression in the HT-29 cells ameliorated the cell proliferation in a dose-dependent manner when compared with the miRNA control following treatment with 5FU or irinotecan (Figures 5 and 6). Thus, overexpression of miR-146a enhances the drug resistance in HT-29 cells.

Figure 5.

Cell cycle of HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a. (a) Cell cycle of HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a without any treatment. (b) HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a after treatment with 5-flououracil. Cellular arrest in HT-29 cells and transfected HT-29 cells with blank vector significantly increased compared to the HT-29 cells transfected with miR-146a in doses of 100 and 200 ng/mL 5FU (p < 0.05 and p < 0.01, respectively). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6.

Cell cycle of HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a. (a) Cell cycle of HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a without any treatment. (b) HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a after treatment with irinotecan. Cellular arrest in HT-29 cells and transfected HT-29 cells with blank vector significantly increased compared to the HT-29 cells transfected with miR-146a in doses of 100 and 200 ng/mL irinotecan (p < 0.01). *p < 0.05; **p < 0.01; ***p < 0.001.

Cell death in 5FU- and irinotecan-treated transfected and non-transfected HT-29 cells was analyzed using the Annexin V detection kit. Percentages of Annexin V–positive cells in drug-treated HT-29 cancer cells and blank group were significantly higher compared to that in the miR-146a-transfected HT-29 cells exposed to different doses of 5FU or irinotecan in a dose-dependent manner (p < 0.001; Figures 7 and 8). These results showed that drug resistance in transfected HT-29 cells remained in high doses of chemotherapeutic agents and indicated that miR-146a induces drug resistance in colon cancer cells.

Figure 7.

Apoptosis assay in HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a. Apoptotic cells in transfected HT-29 cells with miR-146a remarkably decreased compared to control groups following treatment with 5FU at 100 and 200 ng/mL (p < 0.01 and p < 0.05, respectively). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8.

Apoptosis assay in HT-29 cells, transfected HT-29 cells with blank vector, and transfected HT-29 cells with miR-146a. Apoptotic cells in transfected HT-29 cells with miR-146a remarkably decreased compared to control groups following treatment with irinotecan at 25, 50, 100, and 200 ng/mL. *p < 0.05; **p < 0.01; ***p < 0.001.

Discussion

In this study, we evaluated the effects of miR-146a on PBMC populations and their cytokine expression. First, we successfully overexpressed miR-146a in HT-29 cell line. We showed that PBMC proliferation was not significantly changed by miR-146a. Conversely, PBMC co-culture with miR-146a-transfected HT-29 cells increased the Treg population in PBMCs. On the other hand, our data showed that the expression levels of the immune suppressing cytokines TGF-β and IL-10 increased in PBMCs in the presence of high levels of miR-146a.

Previous investigations have shown that miR-146a plays an important role in T cells’ polarization, proliferation, and secretion of cytokines.^24,26 miR-146a decreases interferon (IFN)-γ-producing T cells and Th1 subtype T cells. Conversely, expression of miR-146a results in amelioration of Treg, Th2 subtype, and memory T cells.¹¹ Various studies have indicated that miR-146a is involved in cancer progression and maintenance.²⁷ Overexpression of miR-146a is reported in papillary thyroid carcinoma, the highly metastatic human breast cancer cell line MDA-MB-231, and cervical cancer tissues, leading to remarkable downregulation of IRAK1 and TRAF6, inhibiting nuclear factor-κB (NF-κB) activity.^28,29 However, some researches pointed out that miR-146a acted as a tumor suppressor through reduction in ROCK1 and IRAK-1 protein levels in the hormone-refractory prostate carcinoma (HRPC) and pancreatic cancer cells, respectively.²⁴

Foxp3+ Treg are crucial guardians of immune homeostasis. One of the key molecular mechanisms that control the functions of Treg is miR-mediated regulation.⁹ Lu et al.¹¹ demonstrated an indispensable role of miR-146a in Treg-mediated modulation. Expression of miR-146a in Tregs targeted STAT1, one of the main regulators of Th1-cell differentiation, and shifts naive T cells toward Th2 subtypes.¹¹ In this study, the results revealed that miR-146a increases Tregs’ frequencies and immune modulating cytokines including TGF-β and IL-10. These data are in parallel to previous results that indicated anti-inflammatory effects of miR-146a in tumor milieu and suppression of tumor necrosis factor alpha (TNFα) and IL-6.^30,31 MiR-146a overexpression inhibited STAT1 in PBMCs, leading to upregulation of TGF-β and IL-10 expressions. IFN-γ signaling normally leads to the expression of SMAD7 through the STAT1 pathway; however, miR-146a expression downregulated IFN-γ. Reduction in IFN-γ helps t increase TGF-β expression.³²

MiRNAs act as key regulators of T-cell differentiation, proliferation, and the acquisition of effector phenotype. Activation of NF-κB signaling induces miR-146a expression, leading to inhibition of T-cell activation and population expansion through target genes TRAF6 and IRAK1.²⁴ Proliferation capacity of PBMCs was not significantly changed in this case. These data may be related to complex cells that were used for experiments because PBMCs contain multiple cells that miR-146a might differentially affect. 5FU and irinotecan are two chemotherapeutic agents commonly used in treatment of colon cancer as they induce cancer cells’ apoptosis by different mechanisms.³³ One of the main obstacles in cancer therapy is cancer drug resistance. Resistance to chemotherapeutic agents is associated with various factors including miRNA expression profiles in cancer cells.^34,35 MiRNAs are involved in various biological and functional processes, and abnormal expression of miRNAs is reported in many human cancers. Various studies demonstrated that different miRNAs’ expression profiles are related to drug resistance. For example, miR-222 and miR-29a change the resistance of breast cancer cells to Adriamycin and docetaxel through regulating the phosphatase and tensin homolog (PTEN) protein as a tumor suppressor gene.³⁴ Sorrentino et al. reported that miR-335 and miR-130a enhance the sensitivity of ovarian cancer to carboplatin/cisplatin with paclitaxel. They argued that downregulation of miR-130a is associated with enhanced expression of macrophage colony-stimulating factor (M-CSF), which increases in an aggressive form of ovarian cancer.³⁵ Reduction of miR-128 in breast cancer cells induces chemotherapeutic resistance via Bmi-1 and ABCC5.³⁶ We demonstrated that miR-146a enhances drug resistance in colon cancer cell line (HT-29) to 5FU and irinotecan as common chemotherapeutic agents. Similarly, Pogribny et al. showed that expression of miR-146a is increased in drug-resistant cell lines of breast cancer. MiR-146a regulates the cellular levels of various proteins such as BCRA1, HOXD10, tumor suppressor p27, and estrogen receptor α.³⁷ These proteins play an important role in pathogenesis of cancer and cancer drug resistance.

In conclusion, this study demonstrates that overexpression of miR-146a is involved in different processes in the cancer microenvironment including enhancement of Tregs’ frequencies, increasing the secretion of anti-inflammatory cytokines, such as TGF-β and IL-10, and acquiring resistance to 5FU or irinotecan in colon cancer HT-29 cells. Further studies include the function analyses of its target pathways in vivo on immune response when miR-146a is overexpressed and investigation of T cells’ subtype proliferation.

Footnotes

Acknowledgements

The authors would like to thank the Stem Cell Technology Research Center for its good cooperation.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by Tarbiat Modares University and Digestive Disease Research Institute of Tehran University, Tehran, Iran.

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Supplementary Material

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MicroRNA-146a induces immune suppression and drug-resistant colorectal cancer cells