This study was approved by the Ethics Committee of Youjiang Medical University for Nationalities, Baise, China. A total of 72 primary breast cancer tissues and adjacent non-tumor tissues were collected from Affiliated Hospital of Youjiang Medical University for Nationalities between March, 2013 to April, 2015. The clinical information of patients involved in this study was summarized in Table I. All informed consents were obtained. No patients received radiation therapy or chemotherapy before surgical resection. Tissues were immediately snap-frozen in liquid nitrogen after surgical resection, and stored in liquid nitrogen before use.

Human breast cancer cell line MCF-7 was purchased from Cell bank of Chinese Academy of Sciences, Shanghai, China. Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) in a 37°C humidified atmosphere of 5% CO₂.

Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific), and converted into cDNA using PrimeScript 1st Strand cDNA Synthesis kit (Takara Bio Inc., Tokyo, Japan). For miR expression detection, miRNA qPCR Detection kit (GeneCopoeia, Rockville, MD, USA) was used to conduct Real-Time PCR on ABI 7300 plus thermocycler (Thermo Fisher Scientific). U6 gene was used as an internal control. For mRNA expression detection, SYBR-Green I Real-Time PCR kit (Biomics, Nantong, China) was used to conduct Real-Time PCR on ABI 7300 plus thermocycler. The reaction condition was 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The relative expression was analyzed by the 2^−ΔΔCq method.

Bioinformatics analysis was performed to predict the potential target genes of miR-22 using Targetscan 3.1 online software (http://www.targetscan.org), according to the manufacture's instruction.

Luciferase reporter gene assay was conducted to confirm the targeting relationship between miR-22 and SIRT1. In briefly, the mutant type (MT) of SIRT1 3′UTR lacking complimentarity with miR-22 binding sequence was constructed using QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), according to the manufacture's instruction. The wild type (WT) or MT of SIRT1 3′UTR was cloned into the downstream of the firefly luciferase coding region of pMIR-GLOTM Luciferase vector (Promega Corp., Madison, WI, USA). MCF-7 cells were co-transfected with the WT- or MT-SIRT1-3′UTR luciferase reporter plasmid, and miR-NC or miR-22 mimic, respectively. The luciferase activity was detected after transfection for 48 h using the Dual Luciferase Reporter Assay System (Promega Corp), according to the manufacturer's instruction.

Lipofectamine® 2000 (Thermo Fisher Scientific) was used to conduct cell transfection, according to the manufacture's instruction. In briefly, MCF-7 cells were transfected with scramble miR mimic (miR-NC), miR-22 mimic, NC inhibitor, miR-22 inhibitor, non-specific siRNA (NC siRNA), SIRT1 siRNA, or co-transfected with miR-22 mimic and pcDNA3.1-SIRT1 ORF plasmid, respectively.

Cells were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), and protein was separated with 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime Institute of Biotechnology), which was then transferred onto polyvinylidene difluoride membrane (Thermo Fisher Scientific). The membrane was then incubated with PBS containing 5% non-fat milk (Yili, Beijing, China) overnight at 4°C. After washed with PBST for 3 times, the membrane was incubated with rabbit polyclonal anti-SIRT1 antibody (1:200; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-GAPDH antibody (1:200; Abcam) at room temperature for 3 h. After washed with PBST for 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (1:10,000; Abcam) at room temperature for 1 h. The immunoreactive band was detected using the enhanced chemiluminescence system (Thermo Fisher Scientific), according to the manufacture's instruction. The protein expression was measured using Image Pro Plus software (Media Cybernetics, Rockville, MD, USA).

MCF-7 cell suspension (5×10⁴ cells/well) was plated in a 96-well plate, and cultured for 12, 24, 48 or 72 h. After that, 10 µl of MTT (5 mg/ml) was added. Cells were incubated in a 37°C humidified atmosphere of 5% CO₂ for 4 h. Then, the supernatant was removed, and 100 µl of DMSO (Sigma, St. Louis, MO, USA) was added. The absorbance at 570 nm was examined using a microplate reader (Model 680; Bio-Rad, Berkeley, CA, USA).

Wound healing assay was conducted to examine the cell migration. MCF-7 cells in DMEM with 10% FBS were cultured to 100% confluence, and Mitomycin C (30 µg/ml; Yearthbio, Changsha, China) was added to inhibit cell proliferation. After that, the wound was created with a plastic scriber. Then, cells were washed with DPBS (Thermo Fisher Scientific) and incubated in DMEM in a 37°C humidified atmosphere of 5% CO₂ for 24 h. After that, DMEM was replaced with DMEM with 10% FBS, and then cultured in a 37°C humidified atmosphere of 5% CO₂ for 48 h. Cells were observed and photographed under a microscope (Nikon, Tokyo, Japan).

Transwell assay was conducted to examine cell invasion using the 24-well transwell chamber with a layer of matrigel (Chemicon, Temecula, CA, USA). MCF-7 cell suspension (containing 5×10⁵ cells) was added in the upper chamber, and DMEM containing 10% FBS was added into the lower chamber. After incubation in a 37°C humidified atmosphere of 5% CO₂ for 24 h, cells on the interior of the inserts were removed using a cotton-tipped swab. Invading cells on the lower surface of the membrane were stained with gentian violet (Sigma), rinsed by water, dried in air, and counted under a microscope (Nikon).

Data were expressed as mean ± standard deviation. The association between miR-22 expression and clinical characteristics in breast cancer were analyzed using Chi-square test. The difference between two groups was analyzed using Student t-test. SPSS18.0 software (SPSS, Inc., Chicago, IL, USA) was used to conduct statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

In the present study, we firstly examined the miR-22 expression in breast cancer. As shown in Fig. 1A, the miR-22 levels were significantly reduced in breast cancer tissues compared with adjacent non-tumor tissues. Moreover, the miR-22 levels were much lower in stage III–IV breast cancer, when compared with stage I–II breast cancer (Fig. 1B), suggesting that downregulation of miR-22 may contribute to the malignant progression of breast cancer. To further confirm these findings, we divided them into high miR-22 group and low miR-22 group, according to the mean value to miR-22 expression as the cutoff. As indicated in Table I, low expression of miR-22 was significantly associated with the poor differentiation, metastasis, and advanced clinical stage, but not with age or tumor size (Table I). These findings suggest that downregulation of miR-22 may contribute to the malignant progression of breast cancer.

As miRs function through regulating their target genes, we further performed bioinformatics analysis to predict the potential target gene of miR-22 using Targetscan software. As indicated in Fig. 2A, SIRT1 was a putative target gene of miR-22. To confirm this targeting relationship, the WT-SIRT1-3′UTR and MT-SIRT1-3′UTR luciferase reporter plasmids were constructed, respectively (Fig. 2B and C). Luciferase reporter gene assay data indicated that the luciferase activity was significantly decreased in MCF-7 cells co-transfected with miR-22 mimics and WT-SIRT1-3′UTR vector, which was eliminated by transfection with the MT-SIRT1-3′UTR vector (Fig. 2D), indicating that miR-22 can directly bind to the 3′UTR of SIRT1 mRNA. Therefore, SIRT1 is a target gene of miR-22 in MCF-7 cells.

As miRs generally show suppressive effects on the protein expression of their target genes, we then studied the effects of miR-22 on SIRT1 expression in MCF-7 cells. MCF-7 cells were transfected with miR-22 mimic to upregulate its expression, and transfection with miR-NC was used as the control group. After transfection, the miR-22 expression was significantly increased in the miR-22 group compared with the miR-NC group (Fig. 3A). After that, we conducted western blot to determine the protein expression of SIRT1. As indicated in Fig. 3B, overexpression of miR-22 reduced the protein levels of SIRT1. To further confirm these findings, MCF-7 cells were transfected with miR-22 inhibitor to decrease its expression, and transfection with NC inhibitor was used as the control group. As shown in Fig. 3C, the miR-22 levels were significantly reduced in the miR-22 inhibitor group compared with the NC inhibitor group. Moreover, knockdown of miR-22 increased the protein expression of SIRT1 (Fig. 3D). Accordingly, we demonstrate that the protein expression of SIRT1 is negatively regulated by miR-22. After that, we further examined the expression of SIRT1 in breast cancer tissues. qPCR data showed that the SIRT1 levels were significantly higher in breast cancer tissues compared with adjacent non-tumor tissues (Fig. 3E). Moreover, the mRNA levels of SIRT1 were higher in stage III–IV breast cancer, when compared with stage I–II breast cancer. Based on these above data, we suggest that the increased expression of SIRT1 in breast cancer may be due to the downregulation of miR-22.

We then studied the regulatory roles of miR-22 in the regulation of breast cancer growth and metastasis in vitro. MTT assay, wound healing assay and transwell assay were conducted to examine the cell proliferation, migration and invasion, respectively. As indicated in Fig. 4A-C, ectopic expression of miR-22 led to a significant decrease in the proliferation, migration and invasion of MCF-7 cells, suggesting that miR-22 may have suppressive effects on breast cancer growth and metastasis.

As we found that SIRT1 was upregulated in breast cancer and negatively regulated by miR-22 in MCF-7 cells, we speculated that SIRT1 might be involved in the miR-22-mediated proliferation, migration and invasion of MCF-7 cells. To verify this speculation, miR-22-overexpressing MCF-7 cells were transfected with pcDNA3.1-SIRT1 expression plasmid. After transfection, the protein levels of SIRT1 were remarkably increased in the miR-22+SIRT1 group compared with the miR-22 group (Fig. 5A). Further investigation showed that the proliferation, migration and invasion of MCF-7 cells were also upregulated in the miR-22+SIRT1 group, when compared with those in the miR-22 group, indicating that restoration of SIRT1 attenuated the suppressive effects of miR-22 on the malignant phenotypes of MCF-7 cells (Fig. 5B-D). Taken these data together, we suggest that the tumor suppressive role of miR-22 in MCF-7 cells is, partly at least, through directly inhibiting the protein expression of SIRT1.