MicroRNA-22 Suppresses Breast Cancer Cell Growth and Increases Paclitaxel Sensitivity by Targeting NRAS.

In recent study, microRNAs have various important functions in diverse biological processes and progression of cancer. In human breast cancer, microRNA-22 has been reported to be downregulated. However, molecular mechanism of microRNA-22 in breast cancer progression and chemosensitivity has not been well studied. In our study, these results demonstrated that microRNA-22 expression levels were significantly reduced in 40 pairs of human breast cancer tissues when compared to normal tissues. Enforced expression of microRNA-22 inhibited activity of cell proliferation and cell migration in breast cancer cells. Furthermore, microRNA-22 targeted NRAS proto-oncogene, GTPase (NRAS) in breast cancer cells. The expression levels of NRAS in human clinical specimens were higher in breast cancer tissues when compared to normal tissues. Moreover, microRNA-22 sensitized breast cancer cells to paclitaxel by regulation of NRAS. Our results then demonstrated that microRNA-22 functioned as a tumor suppressor microRNA and indicated potential application for the diagnosis and treatment of cancer in the future.

Introduction

Breast cancer in women is the most common type of cancer worldwide, which represents the second leading cause of cancer-related mortality.[1,2] Breast cancer has broad molecular and pathological heterogeneity subtypes, which reflected poor prognosis in some subtypes.[3,4] Chemotherapy and targeted therapies in breast cancer clinical treatment aim to reduce activity of cell survival, cell growth, and metastasis, while even more induce apoptosis in cells. Efficacy of clinical treatment is limited by development of resistance and subsequent tumor progression.[5-7] Mounting studies have suggested that noncoding microRNAs (miRNAs) play important roles during the development of breast cancer.

MicroRNAs are small, noncoding RNAs which are 19 ∼ 22 nucleotides in length. MicroRNAs play significant role at posttranscriptional levels in the regulation of gene expression.[8,9] By binding to the 3′-untranslated region (3′-UTR) of targeted messenger RNA (mRNA), miRNA emerges mRNA degradation or translational repression, then suppresses protein synthesis. MicroRNA-22 (miR-22) in several cancer types can affect tumor cell growth, cell migration, cell invasion, and apoptosis,[10-14] which functioned as a tumor suppressor gene. Several genes have been identified as miR-22 target genes, including, SP1, SIRT1, MMP14, YWHAZ, and Snail.[10-14] However, the function of miR-22 and the underlying mechanism in regulating breast cancer are still to be investigated.

RAS genes are the most frequently activated oncogenes in cancers. RAS family (including KRAS, NRAS, and HRAS) represent a family of small G proteins which are frequently activated in various human tumors. NRAS have been reported to involve in cellular signal transduction. NRAS altered in various cancers and has potential important roles in the regulation of cancer cell survival, cell proliferation, cell migration, cell invasion, and angiogenesis.[15,16] NRAS promotes tumor progression through activation of downstream signal pathways, including phosphatidylinositol4,5-bisphosphate 3- kinase/AKT serine/threonine kinase (PI3K/AKT), mitogen-activated protein kinase (MAPK/ERK), and nuclear factor kappa B (NF-κB) pathways.[17-20] Generally, silencing of NRAS functioned as an efficient therapeutic strategy in tumors, and novel therapeutic strategies are urgently needed and far from optimal.

Previous studies have reported that miR-22 plays roles in breast cancer via affecting different downstream pathways. Our study aimed to further identify the molecular mechanisms of miR-22. Our results in this study showed that miR-22 was significantly downregulated in human breast cancer samples when compared to normal samples. MicroRNA-22 inhibited activity of cell proliferation and cell migration through suppressing NRAS target. Moreover, miR-22 induced breast cancer cells more sensitive to paclitaxel through NRAS. Generally, our study revealed novel molecular mechanism of miR-22 in breast cancer, which possessed a novel strategy of miR-22-based therapeutics.

Methods and Materials

Clinical Tissues

Human breast cancer and matched normal tissue samples were collected from patients, with the informed consent from Jining First People’s Hospital, who are undergoing a surgical procedure. Parts of samples were snap-frozen in liquid nitrogen, while parts were fixed for histological examination. All samples in this study were histologically classified by clinical pathologist. These experiments have been approved by the ethics committees of Jining First People’s Hospital.

Cell Culture and Cell Transfection

MCF7, MDM231 (human breast cancer cell line), and HEK293T were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium. Cells were incubated at 37°C in an atmosphere of 5% CO2. MicroRNA-22 mimics and miR-NC were synthesized by GenePharma (Shanghai, China). According to the manufacturer’s instructions, indicated cells were transfected with miR-22 or miR-NC using Lipofectamine 3000 reagent (Invitrogen, USA).

Establishment of Stable Cell Lines

Following the manufacturer’s manual, lentivirus carrying miR-22 or miR-NC was packaged in HEK293T cells with packaging kit (Thermo Fisher Scientific, USA). To obtain stable cell lines, indicated cells were infected by lentivirus carrying miR-22 or miR-NC, and cells were treated with polybrene (Sigma-Aldrich, USA) and selected by puromycin (Sigma-Aldrich, USA) for at least 2 weeks.

Quantitative Reverse Transcription Polymerase Chain Reaction

RNAs in this study were extracted from indicated samples using TRIzol reagent (Takara, Dalian, China). According to the manufacturer’s instructions, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for miR-22 were tested relative to expression of U6. Moreover, qRT-PCR of 200 to 500 ng total RNA was performed using SYBR Green Kit (Takara, Dalian, China) on a 7500 HT system. Fold changes in this study were calculated by relative quantification (2−▵▵ ). Experiments of qRT-PCR were carried out in triplicate.

Cell Proliferation Assay

Cells were treated as indicated and seeded in 96-well plates with a density of 2000 per well. At time point of 24, 48, 72 and 96 hours, CCK-8 (Dojindo, Japan) reagents were added into each well, which was followed by 1- to 2-hour incubation. Absorbance at optical density (OD) 450 nm was then determined to evaluate the indicated proliferation activity of cells.

Migration Assay

According to the manufacturer’s instructions, the indicated effect of miR-22 on cell migration was tested with migration chambers (BD Biosciences, USA); 5 × 104 transfected cells were applied in the upper well of chamber containing serum-free DMEM, while DMEM was added to the lower chamber. After 18 to 24 hours, any noninvading cells were removed and indicated cells were fixed and stained. Images were captured and experiments were conducted in triplicate.

Western Blotting

Cells were harvested and lyzed in Radio-Immunoprecipitation Assay (RIPA) buffer supplemented with protease inhibitors phenylmethanesulfonyl fluoride (PMSF) on ice for 30 minutes. After centrifugation for 15 minutes, protein concentrations were determined by the Bicinchoninic acid (BCA) kit (Beyotime, China) and then measured by Western blotting. The membrane was tested with NRAS antibody (Cell Signaling Technology, Danvers, Massachusetts) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Atlanta, Georgia).

Luciferase Reporter Assay

In this study, 3′-UTR of NRAS containing the miR-22 binding site was amplified. To generate MT miR-22 binding site, 2 nucleotides of the miR-22 binding site were substituted. The complementary sequence in the 3′-UTR of NRAS (GGCAGCU) was replaced by GCGACGU. The PCR products were inserted into pMIR-reporter plasmid and then validated by DNA sequencing. Indicated plasmids with miR-22 or miR-NC were transfected into MCF7 cells in 24-well plates. After 24 to 48 hours, luciferase assays were performed using the Dual Luciferase Reporter Assay System (Promega, Promega, Wisconsin).

In Vitro Chemosensitivity Assay

Cells were usually seeded at a density of 5000 cells per well in a 96-well plate. Freshly prepared paclitaxel was added with the final concentration ranging from 1 nM to 32 nM (Sigma-Aldrich, USA). Forty-eight hours later, cell viability with different group were measured by CCK-8.

Apoptosis Assay

For Annexin V staining, phycoerythrin-Annexin V, propidium iodide, and binding buffer were added to the samples. Fifteen minutes later, samples were analyzed by flow cytometry (FACS Canto II; BD Biosciences), and these data were analyzed by FlowJo software 7.6.

Caspase-3 Activity Assay

According to the manufacturer’s protocol, activity of caspase-3 was determined using Beyotime caspase-3 activity kit. This assay was performed on 96-well plates with cell lysate, reaction buffer, and caspase-3 substrate, and maintained at 37°C for 2 hours, then measured at OD 405 nm.

Statistical Analysis

All experiments in this study were performed in triplicate and data were analyzed by t test with GraphPad Prism 5 software (La Jolla, California); P < .05 was considered as statistically significant. The correlation between miR-22 and NRAS in breast cancer tissues was analyzed with Pearson rank test.

Results

MicroRNA-22 Is Downregulated in Breast Cancer Samples

In our study, we tested miR-22 expression levels in 40 pairs of breast cancer samples and normal samples, which Figure 1A investigated that the miR-22 expression levels in breast cancer samples were significantly lower when compared to normal samples. Moreover, miR-22 expression levels in World Health Organization stage III-IV breast cancer samples were significantly lower than those in stage I as well as stage II; the result indicated that miR-22 expression may have some correlation with breast cancer progression (Figure 1B). Generally, miR-22 with lower expression levels in patients with breast cancer could predict poor prognosis for breast cancer, to be one potential new biomarker in cancer.

Figure 1.

Micro-22 is significantly downregulated in breast cancer tissues. A, Relative miR-22 expression levels were analyzed by qRT-PCR in 40 pairs of human breast cancer tissues and adjacent normal tissues. U6 RNA level was used as an internal control. B, All samples were histologically classified by clinical pathologist. Relative expression levels of miR-22 in different stages of cancer tissues. Data represent mean (SD) of 3 replicates. *Significant difference at P < .05. **Significant difference at P < .01. qRT-PCR indicates quantitative reverse transcription-polymerase chain reaction.

Forced Expression of miR-22 Inhibits Activity of Cell Proliferation and Cell Migration in Breast Cancer Cells

In this study, we infected breast cancer cell lines Michigan Cancer Foundation – 7 (MCF7) and MDA-MB-231 (MDM231) with miR-22 or miR-negative control (NC) lentiviral to established stable cell lines, then followed by selection of puromycin (Figure 2A and B). Cell viability assay was conducted in indicated cell lines and indicated that the miR-22 significantly reduced the activity of cell proliferation in MCF7 as well as MDM231 (Figure 2C and D). We next investigated the function of miR-22 on activity of cell migration, which showed that overexpression of miR-22 decreased the migration ability of cancer cells (Figure 2E and F). Our results in this study showed that forced expression of miR-22 in breast cancer cells inhibits activity of cell proliferation as well as cell migration.

Figure 2.

Forced expression of miR-22 inhibits activity of cell proliferation and cell migration in breast cancer cells. A and B, Relative expression levels of miR-22 in MCF7/miR-22, MCF7/miR-NC, MDM231/miR-22, and MDM231/miR-NC stable cell lines were confirmed by qRT-PCR. C and D, Overexpression of miR-22 arrested cell proliferation in MCF7 and MDM231 cells. E and F, MiR-22 overexpression reduced cell migration in MCF7 and MDM231 cells. Scale bar = 20 μm. Data represent mean (SD) of 3 replicates. *Significant difference at P < .05. **Significant difference at P < .01. NC indicates negative control; SD, standard deviation; qRT-PCR; quantitative reverse transcription-polymerase chain reaction.

NRAS Is a Novel Target of miR-22

We analyzed the underlying mechanism of miR-22 with TargetScan (www.targetscan.org) in this study. Figure 3A shows that the 3′-UTR regions of NRAS contained binding site for the miR-22 seed region. Human NRAS 3′-UTR, which contains either wild-type (WT) or mutant (MT) miR-22 binding sequence, then was cloned in the pMIR-reporter vector. MCF7 cells were then transfected with either miR-22 or miR-NC mimics, plus 2 reporter plasmids. The luciferase activity in Figure 3B containing the NRAS 3′′UTR WT was significantly reduced by miR-22, while NRAS 3′-UTR MT showed no difference. Western blotting analysis in Figure 3C was conducted to test the expression of NRAS protein. NRAS protein expression was downregulated in miR-22-overexpressed cells. We next measured NRAS mRNA expression level in human breast cancer samples and normal samples, which showed that NRAS was significantly higher in cancer samples when compared to normal samples (Figure 4A). As shown in Figure 4B, Pearson rank correlation analysis showed that NRAS and miR-22 in breast cancer samples were inversely correlated (Pearson correlation r = −0.6133). Generally, our results in this study suggested that NRAS is a novel target of miR-22.

Figure 3.

NRAS is a novel target of miR-22. A, Sequence of the miR-22 binding site within the human NRAS 3′-UTR and a schematic diagram of the reporter construct showing the entire NRAS 3′-UTR sequence and the mutant NRAS 3′-UTR sequence. The mutant nucleotides of the NRAS 3′-UTR are labeled in red. B, Luciferase assay on MCF7 cells, which were cotransfected with miR-NC or miR-22, and a luciferase reporter containing the full length of NRAS 3′-UTR (WT) or a mutant (MT) harboring 4 mutant nucleotides of the miR-22 binding site. Luciferase activities were measured 24 hours posttransfection. MiR-22 markedly suppressed luciferase activity in NRAS 3′-UTR (WT) reporter constructs. C, The immunoblotting showed that expression levels of NRAS were decreased in cells with miR-22 overexpression. Data represent mean (SD) of 3 replicates. **Significant difference at P < .01. MT, mutant-type; NC; negative control; SD, standard deviation; UTR, untranslated region; WT, wild-type.

Figure 4.

MicroRNA-22 induces breast cancer cells more sensitive to paclitaxel by inhibiting NRAS. A, The expression of NRAS in adjacent normal tissues and human breast cancer specimens was determined by qRT-PCR analysis, and fold changes were obtained from the ratio of NRAS to GAPDH levels. B, Spearman rank correlation analysis showed that the expression levels of NRAS and miR-22 in breast cancer tissues were inversely correlated. Data represent mean (SD) of 3 replicates. **Significant difference at P < .01. SD indicates standard deviation; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

MicroRNA-22 Induces Breast Cancer Cells More Sensitive to Paclitaxel by Inhibiting NRAS

Resistance to paclitaxel treatment, the major cause for the failure of drug-based treatment in cancers, it is urgent in clinical therapy to discover new strategies and increase paclitaxel effectiveness. Our results in Figure 5A showed that miR-22 increased chemosensitivity to paclitaxel in MCF7 cells. Cell growth rate with the treatment of 4 nM paclitaxel was tested by Cell Counting Kit-8 (CCK-8), and NRAS reversed the miR-22-induced breast cancer sensitivity to paclitaxel (Figure 5B and C). FACS analysis in Figure 5D was performed to investigate cell apoptosis rates. MicroRNA-22 and paclitaxel treatment in cells significantly induced activity of cell apoptosis, and NRAS partially abolished the effect of miR-22. We next investigated the activities of caspase-3, which were upregulated upon treatment of miR-22 and paclitaxel, whereas NRAS reduced activation of caspase-3 (Figure 5E). Our results in this study showed that miR-22 renders breast cancer cells sensitive to paclitaxel, combination of miR-22 and paclitaxel in breast cancer cells promoted apoptotic effect by targeting NRAS.

Figure 5.

MicroRNA-22 induces breast cancer cells more sensitive to paclitaxel by inhibiting NRAS. A, MCF7 cells stably expressing miR-NC or miR-22 were pretreated with paclitaxel for indicated concentrations, then subjected to CCK-8 assay. B and C, MCF7 cells stably expressing miR-NC, miR-22, or miR-22 forced expression of NRAS were pretreated with 4 nM of paclitaxel for indicated time points, then subjected to CCK-8 assay, apoptosis analyzed by flow cytometry (D) and caspase 3 assay (E). Data represent mean (SD) of 3 replicates. * or # P < .05. **P < .01. *Significant difference compared to control. #Significant difference compared to miR-22 forced expression of NRAS treatment. CCK-8 indicates Cell Counting Kit-8.

Discussion

MicroRNAs imply important roles in tumorigenesis by a number of mechanisms in recent studies, which indicated miRNAs have been shown to be correlated with clinical characteristics of various cancers and outcomes. The role of miRNAs in breast cancer and chemoresistance have also been studied. Such as, Zheng et al have studied that miR-125a-3p in estrogen receptor-positive breast cancer inhibits ERα and overrides tamoxifen resistance by targeting CDK3[21]; Niu et al have reported in human prostate cancer that insulin-like growth factor-I induces chemoresistance to docetaxel through inhibiting miR-143[22]; Pan et al have showed that microRNA-503 enhances drug sensitivity of MCF-7/ADR cells through suppressing avidin-biotin complex transport proteins and eIF4G,[23] while Xie et al showed that miR-519d in breast cancer stem cells damaged cisplatin resistance by downregulation of MCL-1.[24]

Previous studies implied that miR-22 is downregulated in several cancers including breast cancer. We found in this study that miR-22 was downregulated in breast cancer samples compared with normal samples, and miR-22 was inversely correlated with histopathologic grade. More interesting, we further found overexpression of miR-22 inhibited activity of cell proliferation and cell migration. Thus, we demonstrated that miR-22 regulated breast cancer growth, which provides new clinical strategies for breast cancer.

NRAS has long been identified to play an important role in cancer cell growth. In this study, NRAS oncogene was validated as target of miR-22. Firstly, miR-22 directly recognized NRAS 3′-UTR transcripts in luciferase reporter assay. Secondly, the NRAS expression in breast cancer cells was significantly decreased stably expressing miR-22. Thirdly, NRAS were upregulated in breast cancer tissues. These results in our study showed that miR-22 is a tumor suppressor through targeting NRAS.

Recently research have showed that miRNAs play essential roles in the progression of drug resistance.[25-29] In this study, these results showed that miR-22 promoted inhibition effects of paclitaxel. Flow Cytometer Assay showed that in breast cancer cells, miR-22 have higher apoptosis activity. Therefore, miR-22 restoration treatment may offer a novel strategy in chemoresistance.

In conclusion, this study demonstrated that miR-22 played an important role in breast cancer through regulation of NRAS. Although our study confirmed that miR-22 could inhibit progression of breast cancer by targeting NRAS, however, there might be many other targets of miR-22 which could affect tumorigenesis of breast cancer. Nonetheless, our study indicated miR-22 as a tumor suppressor through suppression of NRAS. Further studies are required to identify more molecular mechanisms of miR-22 and pathways in breast cancer.