miR-29a suppresses MCF-7 cell growth by downregulating tumor necrosis factor receptor 1

Abstract

Tumor necrosis factor receptor 1 is the main receptor mediating many tumor necrosis factor-alpha–induced cellular events. Some studies have shown that tumor necrosis factor receptor 1 promotes tumorigenesis by activating nuclear factor-kappa B signaling pathway, while other studies have confirmed that tumor necrosis factor receptor 1 plays an inhibitory role in tumors growth by inducing apoptosis in breast cancer. Therefore, the function of tumor necrosis factor receptor 1 in breast cancer requires clarification. In this study, we first found that tumor necrosis factor receptor 1 was significantly increased in human breast cancer tissues and cell lines, and knockdown of tumor necrosis factor receptor 1 by small interfering RNA inhibited cell proliferation by arresting the cell cycle and inducing apoptosis. In addition, miR-29a was predicted as a regulator of tumor necrosis factor receptor 1 by TargetScan and was shown to be inversely correlated with tumor necrosis factor receptor 1 expression in human breast cancer tissues and cell lines. Luciferase reporter assay further confirmed that miR-29a negatively regulated tumor necrosis factor receptor 1 expression by binding to the 3′ untranslated region. In our functional study, miR-29a overexpression remarkably suppressed cell proliferation and colony formation, arrested the cell cycle, and induced apoptosis in MCF-7 cell. Furthermore, in combination with tumor necrosis factor receptor 1 transfection, miR-29a significantly reversed the oncogenic role caused by tumor necrosis factor receptor 1 in MCF-7 cell. In addition, we demonstrated that miR-29a suppressed MCF-7 cell growth by inactivating the nuclear factor-kappa B signaling pathway and by decreasing cyclinD1 and Bcl-2/Bax protein levels. Taken together, our results suggest that miR-29a is an important regulator of tumor necrosis factor receptor 1 expression in breast cancer and functions as a tumor suppressor by targeting tumor necrosis factor receptor 1 to influence the growth of MCF-7 cell.

Keywords

Tumor necrosis factor receptor 1 miR-29a MCF-7 cell

Introduction

Despite some achievements in diagnostic and therapeutic measures, breast cancer is still the leading cause of cancer death among women worldwide.¹ According to the global cancer project (GOLBOCAN2012), an estimated 1.67 million breast cancer cases were diagnosed and 0.52 million cancer-related deaths were reported in 2012.² To a certain extent, high incidence of this disease is mainly due to the poor understanding of the mechanism of the cancer. Therefore, it is essential to elucidate the molecular mechanism of tumorigenesis to develop appropriate treatment modalities for breast cancer patients.

Recently, numerous evidence have shown that chronic inflammation in the tumor microenvironment contributes to growth as well as progression of cancers.^3–5 Tumor necrosis factor-alpha (TNF-α) is an important inflammatory cytokine involved in various cancers development.^6,7 It has been demonstrated that TNF-α participates in activating multiple cell signaling pathways that link inflammation, survival, and evolution toward breast cancer.^8,9 TNF-α exerts its biological function by binding to two distinct TNF receptors, TNFR1 and TNFR2. Of them, TNFR1 is the main receptor that mediates many TNF-α-induced cellular events and has a key role in tumorigenesis.^10–12 Studies have shown that TNFR1 enhances proliferation of C4HD murine mammary tumor cells and T47D human cell line by activating nuclear factor-kappa B (NF-κB) signaling pathway, and blockage of TNFR1 with specific antibodies is enough to impair biological effect of TNFR1 on breast cancer cells growth.¹³ However, results from other studies have demonstrated that activation of TNFR1 leads to the recruitment of tumor necrosis factor receptor type 1–associated death domain protein (TRADD) and Fas-associated death domain (FADD), which induces the activation of caspase-8, mitogen-activated protein kinase (MAPK) and ultimately cell death in breast cancer.^14–16 Therefore, it is necessary to further elucidate function of TNFR1 in tumorigenesis of breast cancer.

Presently, numerous microRNAs, 18–25 nucleotides in length, have been identified that regulate most human gene expression at the post-transcriptional level and play very vital role in tumorigenesis.^17–19 By binding to the 3′ untranslated region (3′ UTR) of target mRNAs, microRNAs result in the silencing of target genes as a negative regulator.^20,21 Increasing evidence have demonstrated that microRNAs act as either oncogenes or tumor suppressors in human cancers.^22,23 More specifically, miR-29a, a member of miR-29 family, mostly acts as a tumor suppressor by regulating apoptosis or inhibiting NF-κB signaling pathway at the transcriptional level in human cancers, including gastric cancer, cervical cancer, and prostate cancer.^24–26 In breast cancer, miR-29a was shown to play an inhibitory role in tumorigenesis through downregulating B-Myb.²⁷ However, whether regulation of the growth of breast cancer by miR-29a was mediated by targeting TNFR1 has not been reported so far.

The aim of this study was to determine the role of TNFR1 in tumorigenesis of breast cancer, the relationship between miR-29a and TNFR1 expression in breast cancer, as well as the molecular mechanism of miR-29a regulating the growth of breast cancer through TNFR1, to provide a promising therapeutic target for breast cancer patients.

Materials and methods

Breast cancer samples and cell lines

A total of 20 pairs of primary breast cancer samples and surrounding normal mammary tissues were obtained from breast cancer patients who underwent surgery at the Breast Surgery of the Tumor Hospital in Mudanjiang. Written informed consent was obtained from each potential subject using a human-subject protocol approved by the institutional review board. All specimens were immediately snap-frozen in liquid nitrogen after surgical removal. Histological and pathological diagnosis was confirmed by a pathologist. Human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and normal mammary tissues were used as control. All cell lines were cultured in DMEM (Invitrogen, USA) containing 10% fetal bovine serum (FBS, Invitrogen) and were grown in a humidified 5% CO₂ incubator at 37°C.

Real-time polymerase chain reaction analysis of miR-29a and TNFR1 mRNA expression

The total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s protocol. The SYBR^® Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara, Japan) was used for real-time polymerase chain reaction (PCR) applications. The primers used in PCR reaction were as follows: TNFR1: 5′-GCCAGGAGAAACAGAAC AC-3′ (forward) and 5′-CCGTTGGTAGCGATACATTA-3′ (reverse). miR-29a: 5′-GCGGGACTGA TTTCTTTTGG-3′ (forward) and 5′-GTGCAGGGTCCGAGGTATTC-3′ (reverse). As an internal control, the primers of U6 were as follows: 5′-GTGCTCGCTTCGGCAGCACAT-3′ (forward) and 5′-TACCTT GCGAAGTGCTTAAAC-3′ (reverse). The 2^−ΔΔCT method was used to calculate the relative expression of different genes.

Oligonucleotides transfection

miR-29a mimic or scramble miR-29a mimic (miR-negative control (NC)) and small interfering RNA (siRNA)-TNFR1 or scramble siRNA-TNFR1 (miR-negative control (NC)) were designed and synthesized by Wanleibio (ShenYang, China). According to the manufacturer’s protocol, these oligonucleotides were transiently transfected into MCF-7 cell using Lipofectamine™ 2000 (Invitrogen) at a final concentration of 100 nM. After 72-h transfection, cells were harvested for further analysis. The sequences of siRNA-TNFR1 were 5′-GCCAGGAGAAACAGAAC AC-3′ (forward) and 5′-CCGTTGGTAGCGATACATTA-3′ (reverse).

Vector construction

The wild-type 3′ UTR region of human TNFR1 mRNA was amplified by PCR and cloned into pmirGLO vector (Promega, USA) downstream of the luciferase reporter gene and designated as pmirGLO-TNFR1-WT. The primers for pmirGLO-TNFR1-WT were as follows: 5′-CGCTAGCGCAG CTCTAAGGACCGTC-3′ (forward) and 5′-GTCGACGGCTCAGGGACGAACCAG-3′ (reverse). For mutated-type 3′ UTR construct, 5′-GUG-3′ region of wild-type 3′ UTR was substituted with a 5′-CAC-3′ fragment. The mutated sequences were inserted into the luciferase reporter and named as pmirGLO-TNFR1-MT. The primers for pmirGLO-TNFR1-MT were as follows: 5′-CACCTAACCCCTCGATGTACAT-3′ (forward) and 5′-GTGCAAGTAGGCGGCTGCTA-3′ (reverse). In addition, the coding sequence of TNFR1 was amplified from the complementary DNA (cDNA) and cloned into pcDNA3.1 (−) (Invitrogen).

Luciferase reporter assay

MCF-7 cell seeded in 96-well plates in triplicate was cotransfected with pmirGLO-TNFR1-WT or pmirGLO-TNFR1-MT and miR-29a mimic or NC using Lipofectamine 2000 in accordance with the manufacturer’s procedure. Cells were collected at 72 h post-transfection, and luciferase activity was measured using a dual-luciferase reporter assay kit (Promega, USA) and recorded by a chemiluminescence meter (Promega).

Western blot assay

Protein was extracted from the cells at 72 h post-transfection using a protein extraction reagent (Takara, Japan), and the protein concentration was measured using the BCA protein assay kit (Takara, Japan). Extracts (50 µg) were transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblots with antibodies to TNFR1 (1:500 dilution), Bax (1:500 dilution), Bcl-2 (1:500 dilution), cyclinD1 (1:500 dilution), and NF-κBp65 (1:500 dilution). β-actin was used as an internal control.

Proliferation assay

The proliferation of MCF-7 cell was assessed by the Cell Counting Kit-8 (CCK-8) assay kit (Invitrogen). Approximately 1 × 10⁴ cells were seeded in each well of 96-well plates, and 10 µL of CCK-8 was added to 90 µL of culture medium. After incubation at 37°C for 2.5 h, the absorbance was detected at 450 nm, and the OD450 value was correlated with live cell numbers.

Colony formation assay

The assay was conducted as previously described.²⁴ Briefly, MCF-7 cell was digested with trypsin and suspended into a single cell status. A total of 5 × 10³cells from each group were cultured in the 60-mm-diameter culture dish with 10% FBS for 14 days. The colonies were fixed and stained with 0.5% crystal violet for 15 min, and then washed three times. The number of colonies in 10 random view fields was counted under a microscope, and the average representing the 95% confident region was achieved.

The cell cycle and apoptosis assay

MCF-7 cell was transfected with miR-29a mimic, siRNA-TNFR1, TNFR1 vector, miR-29a+ TNFR1, and NC, respectively, and untreated cells were used as blank control. Cells were harvested after 72 h and stained with propidium iodide (PI, Sigma, USA) for the cell cycle analysis. For apoptosis, flow cytometry was also performed for detection using the Annexin V/PI apoptosis detection kit (Takara, Japan) according to the manufacturer’s protocol.

Statistical analysis

SPSS software was used for statistical analysis. Continuous data are presented as the mean ± standard deviation (SD) and were compared between two groups using Student’s unpaired t test. The linear correlation coefficient (Pearson’s r) was calculated to determine the correlation between TNFR1 and miR-29a expression in paired tissues. A p value of <0.05 was considered to be statistically significant. All graphs were generated with GraphPad Prism Version 5.0.

Results

TNFR1 expression was significantly upregulated in human breast cancer tissues and cell lines

To determine the expression of TNFR1 in breast cancer, we first detected TNFR1 mRNA expression by real-time PCR in human breast cancer tissues. As shown in Figure 1(a), TNFR1 mRNA expression was significantly upregulated in human breast cancer tissues compared with adjacent normal tissues. In contrast to normal tissues pooled from 20 samples, high levels of TNFR1 mRNA expression were also detected in human breast cancer cell lines MCF-7, MDA-MB-231, and T47D. In particular, MCF-7 cell showed the highest levels of TNFR1 mRNA expression and was used for subsequent studies (Figure 1(b)).

Figure 1.

TNFR1 expression was significantly upregulated in human breast cancer tissues and cell lines. (a) Relative TNFR1 mRNA expression in human breast cancer tissues and their corresponding non-tumor tissues (normal) was detected using real-time PCR and normalized to U6 snRNA. (b) Relative TNFR1 mRNA expression in human breast cancer cell lines MCF-7, MDA-MB-231, and T47D was detected using real-time PCR, and corresponding non-tumor tissues pooled from 20 samples (normal) were used as control. Results are the mean ± SD in triplicate.

TNFR1 downregulation inhibited cell proliferation and promoted apoptosis in MCF-7 cell

To determine the potential role of TNFR1 in breast cancer, siRNA-TNFR1 oligonucleotides were synthesized and transfected into MCF-7 cell. As shown in Figure 2(a), siRNA-TNFR1 could efficiently downregulate TNFR1 protein expression compared with NC or untreated cells (Control). In a Cell Counting Kit-8 (CCK-8) cell proliferation assay, TNFR1 knockdown significantly inhibited cell proliferation compared to controls (Figure 2(b)). The following cell cycle assay also supported the results, because downregulation of TNFR1 arrested G0/G1 phase transition and the percentage of cells in the G0/G1 phase was increased from 69.3% to 79.8% (Figure 2(c)). Furthermore, we observed that knockdown of TNFR1 expression via siRNA in MCF-7 cell led to a significant increase in apoptosis rate compared to controls. Collectively, the results demonstrated that suppression of TNFR1 activity might attenuate MCF-7 cell growth by arresting the cell cycle and promoting apoptosis.

Figure 2.

TNFR1 downregulation inhibited cell proliferation and promoted apoptosis in MCF-7 cell. (a) siRNA-TNFR1 oligonucleotides were transfected into MCF-7 cell and efficiently downregulated TNFR1 protein expression by western blot compared with NC or Control. (b) CCK-8 assay showed the effect of siRNA-TNFR1 transfection on MCF-7 cell proliferation. (c) Flow cytometry detected MCF-7 cell in GO/G1, Sand G2 phase. (d) Annexin V-FITC apoptosis detection analyzed the effect of siRNA-TNFR1 transfection on MCF-7 cell apoptosis by flow cytometry. Control represents untreated cells. Results are the mean ± SD in triplicate.

Predicted miR-29a was inversely correlated with TNFR1 expression in human breast cancer tissues and cell lines

To elucidate the molecular mechanisms by which TNFR1 promoted the growth of breast cancer cells, we used TargetScan (http://www.targetscan.org/) to predict miRNAs that targeted TNFR1. Among the candidate target genes, we mainly focused on miR-29a, which has been shown to inhibit tumorigenesis through arresting the cell cycle and inducing apoptosis. To observe expression of miR-29a, we performed real-time PCR on human breast cancer tissues and MCF-7, MDA-MB-231, and T47D cells. We found that both breast cancer tissues and cell lines expressed lower miR-29a levels compared with adjacent normal tissues. In particular, MCF-7 cell with the highest TNFR1 mRNA levels expressed the lowest miR-29a levels (Figure 3(a) and (b)). Moreover, there was an inverse correlation between miR-29a and TNFR1 mRNA expression in these samples (Figure 3(c)). These results implied that TNFR1 might be a direct target of miR-29a in breast cancer.

Figure 3.

miR-29a was lowly expressed and inversely correlated with TNFR1 in human breast cancer tissues and cell lines. (a) Relative expression of miR-29a in human breast cancer tissues and their corresponding non-tumor tissues (normal) was detected by real-time PCR and normalized to U6 snRNA. (b) Relative expression of miR-29a in human breast cancer cell lines MCF-7, MDA-MB-231, and T47D was detected by real-time PCR, and corresponding non-tumor tissues pooled from 20 samples (normal) were used as control. (c) The linear regression between miR-29a and TNFR1 mRNA expression in 20 human breast cancer tissues was analyzed. Results are the mean ± SD in triplicate.

miR-29a targeted TNFR1 by binding to the 3′ UTR

To validate that TNFR1 was indeed directly targeted by miR-29a, a luciferase reporter containing the wild-type (pmirGLO-TNFR1-WT) or mutant (pmirGLO-TNFR1-MT) 3′ UTR of TNFR1 was constructed (Figure 4(a)). After cotransfection with a miR-29a mimic, the luciferase activity was significantly decreased in the cells transfected with pmirGLO-TNFR1-WT, while the luciferase activity was not remarkably altered in the cells transfected with pmirGLO-TNFR1-MT, suggesting that miR-29a targeted TNFR1 by binding to the 3′ UTR (Figure 4(b)). To further confirm whether the altered miR-29a could affect endogenous TNFR1 protein expression in breast cancer, a miR-29a mimic and NC were transfected into MCF-7 cell, respectively. Results from western blot assay indicated that miR-29a overexpression significantly inhibited TNFR1 protein expression compared with controls (Figure 4(c)). In brief, miR-29a directly targeted TNFR1 mRNA and regulated TNFR1 protein expression post-transcriptionally.

Figure 4.

miR-29a targeted TNFR1 by binding to the 3′ UTR. (a) Chematic representation of miR-29a target binding site in the TNFR1 mRNA 3′ UTR was identified by the microinspector prediction program. Wild-type (WT) and mutation (MT) of 3′ UTR in seed sequences were indicated. (b) Dual luciferase reporter assay was performed. The expression of the reporter containing TNFR1 3′ UTR was suppressed by miR-29a, but not in the mutated construction. (c) Western blot assay was used to examine the effect of miR-29a on the endogenous TNFR1 expression in MCF-7 cell. Control represents untreated cells. Results are the mean ± SD in triplicate.

miR-29a played a suppressive role in the regulation of MCF-7 cell growth

To determine the potential role of miR-29a on MCF-7 cell proliferation, a CCK-8 assay was performed. As shown in Figure 5(a), transfection of miR-29a mimic significantly decreased proliferation of MCF-7 cell compared with NC or untreated cells. Similarly, the number of colony formation in cells transfected with miR-29a mimic was lower than that in cells transfected with NC or untreated cells (Figure 5(b)). Moreover, we found that MCF-7 cell transfected with miR-29a mimic has a higher percentage of cells in the G0/G1 phase relative to controls (80.1% vs 70.2%) (Figure 5(c)). Next, we performed Annexin V/PI double-staining and flow cytometry analysis to investigate the role of miR-29a on apoptosis. Results showed that miR-29a mimic transfection resulted in an increase in apoptosis rate compared with NC or untreated cells, suggesting that miR-29a promoted apoptosis in MCF-7 cell (Figure 5(d)). Collectively, these data demonstrated that miR-29a might play a suppressive role in MCF-7 cell growth.

Figure 5.

miR-29a played a suppressive role in the regulation of MCF-7 cell growth. CCK-8 assay (a) and colony formation assay (b) were used to measure the effect of miR-29a on MCF-7 cell proliferation. (c) Flow cytometry analysis was used to analyze MCF-7 cell in GO/G1, S, G2 phase. (d) Annexin V-FITC apoptosis detection was used to examine the effect of miR-29a on MCF-7 cell apoptosis. Control represents untreated cells. Results are the mean ± SD in triplicate.

miR-29a exerted its suppressive function by targeting TNFR1 in MCF-7 cell

To examine whether miR-29a exerted its suppressive function through its target gene TNFR1, we ectopically expressed TNFR1 (using a TNFR1 expression vector) together with a miR-29a mimic in MCF-7 cell. As shown in Figure 6(a), ectopically expressed TNFR1 significantly enhanced MCF-7 cell proliferation. When MCF-7 cell was transfected with both TNFR1 vector and miR-29a mimic, their proliferation was partially rescued compared with those transfected with TNFR1 vector alone. The cell cycle assay further confirmed that transfection of miR-29a mimic attenuated TNFR1 induction of cell progression in MCF-7 cell (Figure 6(b)). In addition, we also found that apoptosis inhibition caused by TNFR1 was reversed by cotransfection with miR-29a (Figure 6(c)). Taken together, these results suggested that miR-29a exerted its suppressive function by targeting TNFR1 in MCF-7 cell.

Figure 6.

The oncogenic role induced by TNFR1 was partially rescued by contransfection with miR-29a. (a) CCK-8 assay examined the effect of miR-29a in combination with TNFR1 transfection on MCF-7 cell proliferation. (b) Flow cytometry detected the effect of miR-29a in combination with TNFR1 transfection on MCF-7 cell cycle distribution. (c) Annexin V-FITC apoptosis detection assessed the effect of miR-29a in combination with TNFR1 transfection on MCF-7 cell apoptosis. Control represents untreated cell. Results are the mean ± SD in triplicate.

miR-29a inactivated NF-κB signaling pathway by TNFR1 in MCF-7 cell

Previous studies have demonstrated that activation of the NF-κB signaling pathway mediated by TNFR1 contributes to both proliferation promotion and apoptosis inhibition in breast cancer cells.¹³ In addition, NF-κBp65 is a key component in the NF-κB signaling pathway, and cyclinD1 and Bcl-2/Bax are major regulators involved in cell proliferation and apoptosis in the NF-κB signaling pathway.^28,29 Therefore, to test whether miR-29a/TNFR1 regulates MCF-7 cell growth through NF-κB signaling pathway, we examined the protein expression of NF-κBp65, cylcin D1, and Bcl-2/Bax in MCF-7 cell. As demonstrated in Figure 7(a), NF-κBp65 protein expression was significantly enhanced in MCF-7 cell transfected with TNFR1 vector, while NF-κBp65 protein expression was significantly reduced in MCF-7 cell transfected with miR-29a mimic. Moreover, cotransfection with miR-29a mimic and TNFR1 vector could abolish upregulation of NF-κBp65 protein expression mediated by TNFR1 vector alone. Consistent with these findings, miR-29a mimic transfection significantly resulted in the decrease of cyclinD1 and Bcl-2/Bax protein expression, and attenuated TNFR1 induced the increase of cyclinD1 and Bcl-2/Bax protein expression by addition of TNFR1 vector (Figure 7(b) and (c)). Altogether, these results indicated that miR-29a inactivated NF-κB signaling pathway through regulation of TNFR1 expression in the growth of MCF-7 cell.

Figure 7.

miR-29a functioned as a tumor suppressor by inactivating NF-κB signaling pathway and decreasing cyclinD1 and Bci-2/Bax protein levels. (a) NF-κBp65 protein expression by western blot was detected in MCF-7 cell transfected with TNFR1, miR-29a, or miR-29a+ TNFR1. (b) CyclinD1 protein expression by western blot was detected in MCF-7 cell transfected with TNFR1, miR-29a, or miR-29a+ TNFR1. (c) Bci-2/Bax protein by western blot was detected in MCF-7 cell transfected with TNFR1, miR-29a, or miR-29a+ TNFR1. Control represents untreated cell. Results are the mean ± SD in triplicate.

Discussion

At present, function of TNFR1 in tumorigenesis remains controversial.^30,31 Some studies confirm that TNFR1 can promote proliferation by activating the NF-κB signaling pathway, whereas other studies show that TNFR1 can induce apoptosis through a series of caspase-dependent signaling in certain cell lines.^32,33 Therefore, the role of TNFR1 expression in tumorigenesis of breast cancer is necessary to be studied further. In this study, by real-time PCR assay, we found that expression level of TNFR1 mRNA was relatively high in human breast cancer tissues and cell lines (MCF-7, MDA-MB-231 and T47D) (Figure 1(a) and (b)). Especially, MCF-7 cell expressed the highest level of TNFR1 mRNA among cell lines. So we mainly focused on studying the role of TNFR1 in MCF-7 cell. We performed series of assays in MCF-7 cell and observed that knockdown of TNFR1 by siRNA in MCF-7 cell significantly inhibited cell proliferation, arrested the cell cycle transition, and induced apoptosis (Figure 2(b)–(d)), suggesting that TNFR1 acted as a tumor promoter in the growth of MCF-7 cell. A recent report has also confirmed that TNFR1 is overexpressed in ovarian cancer, and knockdown of TNFR1 dramatically attenuates malignant phenotypes, including proliferation and colony growth further extending the oncogenic role of TNFR1.³⁴

It is well known that miRNAs can act as tumor suppression by targeting specific oncogenes, and deregulation of tumor suppressive miRNAs may affect human tumorigenesis.^35,36 miR-29a has been showed to be downregulated in different types of cancers and has been predominately attributed to tumor-suppressing properties. In gastric cancer, miR-29a was significantly underexpressed, and it suppressed the tumor microvessel density by targeting vascular endothelial growth factor A (VEGF-A).³⁷ Also, miR-29a was reported to inhibit prostate cell proliferation and induce apoptosis via KDM5B protein regulation.³⁸ However, whether miR-29a affected the biological activity of breast cancer cell by targeting TNFR1 has not been demonstrated so far. Our study showed that miR-29a was significantly downregulated in human breast cancer tissues and cell lines, and there was an inverse relationship between miR-29a and TNFR1 expression (Figure 3(a)–(c)). Furthermore, luciferase reporter and western blot assays demonstrated that miR-29a suppressed the endogenous expression level of TNFR1 in MCF-7 cell by binding to the 3′ UTR of TNFR1 (Figure 4(b) and (c)), suggesting that miR-29a was a direct regulator of TNFR1 expression in MCF-7 cell. To determine the role of miR-29a on MCF-7 cell growth, miR-29a mimic was transfected into MCF-7 cell for functional assays. Results showed that the ectopic expression of miR-29a significantly inhibited cell proliferation and colony formation by inducing the cell cycle arrest and apoptosis (Figure 5(a)–(d)). Moreover, by cotransfection with TNFR1, miR-29a attenuated the oncogenic role induced by TNFR1 in MCF-7 cell (Figure 6(a)–(c)). Our findings demonstrated for the first time that, miR-29a indeed played a suppressive role in MCF-7 cell growth by targeting TNFR1.

NF-κB, a major transcription factor, is closely related to the growth and progression of cancers.^39,40 Studies have shown that the aberrant expression of TNFR1 leads to abnormal cellular growth and proliferation via the downstream NF-κB signaling pathway in numerous types of cancers.^41,42 Through TNFR1, stimulation of NF-κB signaling pathway controls the initiation and progression of human cervical cancer.⁴³ In experimental model of mouse breast cancer, TNFR1 has also been demonstrated to fully activate NF-κB transcriptional activation.¹³ Based on these, we investigated the activity of NF-κB signaling pathway in each group by examining NF-κBp65 protein levels in MCF-7 cell. In line with the previously mentioned studies, we found that the forced upregulation of TNFR1 significantly promoted the activity of NF-κB signaling pathway; however, this change was attenuated by cotransfection with miR-29a, possibly through downregulation of TNFR1 expression (Figure 7(a)). Furthermore, activation of NF-κB signaling pathway has anti-apoptosis and pro-proliferation effects on tumorigenesis.^44,45 This included regulation of genes such as cyclinD1 and Bcl-2/Bax among others.^46,47 Therefore, we further detected cyclinD1 and Bcl-2/Bax protein levels in MCF-7 cell. We found that TNFR1 vector transfection significantly upregulated cyclinD1 and Bcl-2/Bax protein levels, while cotransfection with miR-29a reversed this effect, suggesting that miR-29a decreased cyclinD1 and Bcl-2/Bax protein levels by downregulating TNFR1 (Figure 7(b) and (c)). These results revealed that NF-κB signaling pathway, cyclinD1, and Bcl-2/Bax played very vital role in ectopic expression of miR-29a to suppress MCF-7 cell growth through TNFR1. Inactivation of NF-κB signaling pathway and decrease of cyclinD1 and Bcl-2/Bax partially resulted in proliferation inhibition and apoptosis promotion of MCF-7 cell.

In summary, our study demonstrates that miR-29a is an important regulator of TNFR1 expression in breast cancer and functions as a tumor suppressor in MCF-7 cell growth by downregulating TNFR1. Therefore, miR-29a/TNFR1 may provide a promising therapeutic strategy for the treatment of breast cancer.

Footnotes

Author contribution

P.S. conceived the project, drafted the manuscript, and interpreted the data; Y.Z. and W.L. designed the experiments and carried out the majority of the experiments; L.L. and Y.L. were responsible for material support and image processing; L.C. and C.X. helped to collect clinical samples; F.Y. was responsible for data collection and statistical analysis. All authors read and approved the final manuscript.

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

This work was supported by grants from the Natural Science Foundation of Heilongjiang Province (H201377), the Key Project of the Heilongjiang Health Bureau (2007-015), and the National Natural Science Foundation of China (81371362).

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