LncRNA RP11-551L14.4 suppresses breast cancer development by inhibiting the expression of miR-4472

Tissue specimens

In this study, 36 pairs of breast cancer tissues (ICD-10 code: 2C60) and corresponding adjacent non-tumor tissues were collected from surgically treated breast cancer cases (median age, 56 years old; age range, 45–73 years old) at the Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University between January 2020 and August 2021. All tissues are stored in liquid nitrogen. Patients with other diseases were excluded and only patients with a single breast cancer were retained. The collection of tissue specimens was approved by the Ethics Committee of Chongqing Medical University (Approval No. 20200613). All patients have signed informed consent.

Cell culture

The human breast cancer cell lines (MDA-MB-453, MCF7, BT474, T47D, ZR-75-1) and the normal human breast epithelial cell line (MCF-10A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). ZR-75-1, T47D and BT474 were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Waltham, MA, USA); MDA-MB-453, MCF7 and MCF-10A were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco; Thermo Fisher Scientific) containing 10% FBS. All of them were maintained in an incubator at 37 °C with 5% CO₂.

Transfection assay

The lentivirus containing RP11-551L14.4 overexpression sequences (RP11-551L14.4) and the negative control (NC) were purchased from GenePharma (Shanghai, China). Cells were seeded into 6-well plates and infected with lentivirus. After 48 h, cells were harvested and subjected to Reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blotting and cell function assays.

Bioinformatics analysis. The expression profile for RP11-551L14.4 was obtained from Lnc2Cancer 3.0 database (http://bio-bigdata.hrbmu.edu.cn/lnc2cancer/) (Gao et al., 2021). The survival analysis for RP11-551L14.4 was obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/index.html) (Tang et al., 2017). The LncBase Predicted v.2 database (http://diana.imis.athena-innovation.gr) (Paraskevopoulou & Hatzigeorgiou, 2016) was used to predict the target miRNA of RP11-551L14.4.

qRT-PCR

Total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed into complementary Deoxy-ribose Nucleic Acid (cDNA) using PrimeScript RT Master Mix (TaKaRa, Otsu, Shiga, Japan). qRT-PCR was conducted using SYBR Premix Ex TaqTM (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer’s protocol. The primers are listed below: RP11-551L14.4: F: 5′-CTTGGCCTTCTCCATAACCA-3′, R: 5′-GACTGCTATGGAGGCTGAGG-3′; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): F: 5′-AAGGTGAAGGTCGGAGTCAAC-3′, R: 5′-GGGGTCATTGATGGCAACAATA-3′; RGMA: F: 5′- AACCAGCAGATCGACTTCCAG-3′, R: 5′- ACGGCTGTCTCGTATGGGA -3′; U6: F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′; miR-4472: F: 5′-AAAACAACACCCCCCACC-3′, R: 5′-CTCAACTGGTGTCGTGGA -3′; GAPDH was used for normalization for RP11-551L14.4 and RGMA mRNA. U6 was used for normalization for miR-4472.

Cell proliferation assay

After infection, T47D and BT474 cells were seeded into 96-well plates at 3,000 cells per well and maintained over 120 h duration, 20 µL cell counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) was added to each well and incubated for 2 h. Optical density (OD) value was measured at a wavelength of 450 nm by the spectrophotometer.

Colony formation assay

The infected T47D and BT474 cells were collected and seeded at 1,000 cells into 6-well plates and maintained for 14 days. The cells were washed three times with phosphate-buffered saline (PBS; Thermo Fisher Scientific), then fixed in methanol for 30 min and stained with with crystal violet staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Colonies were photographed and counted.

Cell cycle assay

The infected T47D and BT474 cells were harvested, washed with (PBS; Thermo Fisher Scientific) and then stained with propidium iodide (PI) solution for 30 min. Flow cytometry analyses were performed by using a flow cytometer (FACScan^®; BD Biosciences) and the Modfit LT software (Verity Software House, US).

Luciferase reporter assay

The binding sites of RP11-551L14.4 and miR-4472 were predicted with the LncBase Predicted v.2 database. The RP11-551L14.4-wild-type (RP11-551L14.4-WT) luciferase reporter gene plasmid and the RP11-551L14.4-mutant (RP11-551L14.4-MUT) reporter gene plasmid were constructed by GenePharma (Shanghai, China). T47D cells were co-transfected with miR-4472 mimic or miR-NC, and with RP11-551L14.4-WT or RP11-551L14.4-MUT reporter gene plasmid using Lipofectamine 3000 reagent (Invitrogen). After incubated for 48 h, the intensity of the fluorescence was measured using dual-luciferase activity assay according to the manufacturer protocol (Promega, Madison, WI, USA). The following sequences were used: miR-4472 mimic, 5′-GGUGGGGGGUGUUGUUUU-3′; miR-NC mimic, 5 ′-UUGUCCGAACGUGUCACGU-3′.

Western blotting

Total proteins were isolated with RIPA buffer (Thermo Fisher Scientific). After separated by 10% SDS-PAGE, proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Then membranes were blocked with 5% BSA for 2 h at room temperature and incubated with primary antibodies at 4 °C for 12 h. The antibodies are as follows: RGMA (cat. no. ab169761), CDK4 (cat. no. ab108357), CDK6 (cat. no. ab124821), Cyclin D1 (cat. no. ab16663), Cyclin D2 (cat. no. ab207604), GAPDH (cat. no. ab9485) (all 1:2,000 dilution; Abcam, Cambridge, UK). After washing with TBST-Tween-20), the membranes were incubated with secondary antibodies for 2 h. The antibodies are as follows: HRP-conjugated goat anti-rabbit IgG (cat. no. ab150077) (both 1:10,000 dilution; Abcam, Inc.). Protein bands were visualized using ECL (GE Healthcare, Chicago, IL, USA). GAPDH was used as a loading control.

Statistical analysis

SPSS software (version 20.0; IBM Corp.) was used to perform the statistical analysis. Data are shown as the mean ± standard deviation. All comparisons between paired tumor and adjacent non-tumor tissues were performed with paired Student’s t-tests. Unpaired Student’s t-tests were performed for comparisons between two groups of cells. Cox proportional hazards model multivariate analyses were used to evaluate the significance of lncRNA RP11-551L14.4 expression and clinicopathological features. The overall survival or disease free survival was assessed using Kaplan–Meier survival analysis followed by the log-rank test. One-way ANOVA followed by Bonferroni’s correction was adopted to compare several groups. The gene expression correlation was assessed using the Pearson’s correlation coefficient test. P value<0.05 was considered statistically significant.

RP11-551L14.4 is low expressed in breast cancer tissues and cell lines

We used the Lnc2Cancer 3.0 database to analyze the expression of RP11-551L14.4 in breast cancer and found that RP11-551L14.4 were low expressed in breast cancer tissues compared to normal breast tissues (Fig. 1A) and the expression of RP11-551L14.4 decreased with advanced tumor stage (Fig. 1B). The Kaplan–Meier survival curves indicated that patients with high RP11-551L14.4 expression displayed favorable overall survival and disease-free survival (Fig. 1C). To verify the expression of RP11-551L14.4 in breast cancer, we collected 36 pairs of breast cancer tissues and adjacent tissues. The results of qRT-PCR showed that the expression of RP11-551L14.4 decreased in breast cancer tissues when compared to the adjacent tissues (Fig. 1D). Consistently, RP11-551L14.4 was found low expressed in breast cancer cell lines (MDA-MB-453, MCF7, BT474, T47D, ZR-75-1). Among them, RP11-551L14.4 has the lowest expression in T47D and BT474; therefore, the two cell lines were selected for subsequent experiments (Fig. 1E). Collectively, the results suggested that RP11-551L14.4 might act as a tumor suppressor in breast cancer.

RP11-551L14.4 overexpression inhibits breast cancer cells proliferation, colony formation and attenuates cell cycle

To investigate the function of RP11-551L14.4 in breast cancer, we constructed RP11-551L14.4 overexpression model using lentiviral vector. qRT-PCR was performed to detect the level of RP11-551L14.4 after transfecting the RP11-551L14.4 lentiviral vector in T47D and BT474 (Fig. 2A). Cell proliferation and clonogenic assays were analyzed by CCK-8 and cell cloning assays in RP11-551L14.4 overexpressing cell lines, respectively. The proliferation and colony-forming abilities of T47D and BT474 cells were significantly inhibited (Figs. 2B and 2C). Flow cytometry assay showed that the G₀/G₁ phase fraction of the RP11-551L14.4 group was more than that of the Control group, but less cells in the G₂/M phase (Fig. 2D). The results revealed that cell proliferation and cell cycle in RP11-551L14.4 overexpression group was remarkably suppressed compared with that in the Control group.

miR-4472 is a direct target of RP11-551L14.4

The LncBase Predicted v.2 database was used to investigate the underlying mechanisms by which RP11-551L14.4 exerts its function. miR-4472 was predicted as a potential target of RP11-551L14.4 (Fig. 3A). Dual luciferase reporter assay revealed that miR-4472 significantly impaired the luciferase activity of the wild type RP11-551L14.4 vector without impairing that of the mutant vector (Fig. 3B). In addition, miR-4472 was significantly upregulated in breast cancer cell lines (Fig. 3C). qRT-PCR showed that miR-4472 expression was attenuated after transfecting the RP11-551L14.4 lentiviral vector (Fig. 3D). Correlation analysis between RP11-551L14.4 and miR-4472 expression in breast cancer tissues revealed an inverse relationship (Fig. 3E). The above results suggested that miR-4472 can be targeted by RP11-551L14.4 through the binding site.

miR-4472 reverses the effects caused by RP11-551L14.4 in breast cancer cells

Considering that RP11-551L14.4 could negatively regulate the expression of miR-4472, we supposed that RP11-551L14.4-mediated downregulation of miR-4472 directly decreased breast cancer cell proliferation. A rescue experiment was conducted, both RP11-551L14.4-overexpressed vector and miR-4472 mimics were co-transfected into breast cancer cells. qRT-PCR was performed to detect the level of miR-4472 after transfecting miR-4472 mimics with or without RP11-551L14.4-overexpressed vector in T47D and BT474 (Fig. 4A). The rescue experiments showed that restoration of miR-4472 could abrogate RP11-551L14.4-induced suppression of proliferation in both T47D and BT474 cells (Figs. 4B–4D). Therefore, our results support the concept that RP11-551L14.4 function as a tumor gene suppressor since its effect could be reversed by miR-4472 in breast cancer cells.

RP11-551L14.4 promotes repulsive guidance molecule A (RGMA) expression

qRT-PCR and Western blot showed that RGMA expression was enhanced after transfecting the RP11-551L14.4 lentiviral vector (Figs. 5A and 5C). Correlation analysis between RP11-551L14.4 and RGMA mRNA expression in breast cancer tissues revealed a positive relationship (Fig. 5B). Western blot analysis showed that the expression of cell cycle related proteins CDK4, CDK6, Cyclin D1 and Cyclin D2 was attenuated in the RP11-551L14.4 group compared with the Control group (Fig. 5C).