Regulatory roles of lncRNA PANDAR in breast cancer cell proliferation

Human breast invasive ductal carcinoma Michigan cancer foundation 7 (MCF-7) cells were obtained from the American Type Culture Collection (catalog No. HTB-22), HEK-293T cells (catalog No. GNHu17) were obtained from the China Center for Type Culture Collection of the National Collection of Authenticated Cell Cultures Cell Bank affiliated with the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and these were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 mg/mL). Cells were incubated in culture medium under a humidified atmosphere of 5% CO₂ in air at 37 °C. Cell identity had been confirmed by short tandem repeat DNA profiling, and PCR had been used to confirm the absence of any mycoplasma contamination.

The genes of lncRNAs, including TCONS00068220 (XR_928848.2), which is the same gene as LOC105375819, for HOX transcript antisense RNA (HOTAIR, NR_003716.3), ciR-has_circ_0001073, BX647792, and PANDAR (NR_109836.1), were amplified from human genomic DNA isolated from MCF-7 cells. lncRNA were then cloned into the pLVX-Puro vector to construct a series of lncRNA plasmids. Circular RNA ciR-has_circ_0001073 was cloned into pLCDH-ciR vector. Primer sequences are listed as follows: HOTAIR forward (F): CAAGCTTCGAATTACGAATTCGACTCGCCTGTGCTCTGGAGCTTG; HOTAIR reverse (R): TTATCTAGAGTCGCGGGATCGGAAAATGCATCCAGATATTAATAT; BX647792 F: CAAGCTTCGAATTACGAATTCGGCCAGAAGGGGAGGAAATTGGAA; BX647792 R: TTATCTAGAGTCGCGGGATCGCCAGTTTTTCCAACTTCCCCTTTC; LOC105375819 F: CAAGCTTCGAATTACGAATTCCCGACTTTCACTTATCAGACCTTG; LOC105375819 R: TTATCTAGAGTCGCGGGATCGGGTGAGCAGTTTTTATTAACCTGT; PANDAR F: CAAGCTTCGAATTACGAATTCTTTCAGGAATGCCGCAGATGTACA; PANDAR R TTATCTAGAGTCGCGGGATCGCAGTGGCTCACGCCTGTAATCTCA; ciR-hsa_circ_0001073 F: ACCTCCATAGAAGATTCTAGAGAGTTCTAAAATTAAACTATGTGG; ciR-hsa_ circ_0001073 R: CTCAGCGCCACAGCAGAATTCGTTAATTGAACAGATTTTATTTTA.

The human and mouse E-cad gene promoters (Nos. 42081 and 61798) reporter plasmids were purchased from Addgene. lncRNA expression vectors, reporter plasmid containing mouse E-cad promoter, and internal control plasmid pRL-TK were cotransfected into HEK-293T cells. The pRL-TK vector served as a reference for normalizing transfection efficiency. At 48 h after transfection, dual-luciferase assays were performed by using a dual-luciferase reporter assay kit (Promega), according to the manufacturer's instructions. The ratio of firefly-to-Renilla luciferase activity was calculated and results were normalized to the corresponding negative control. Data are represented as the mean ± standard deviation (SD) of triplicate assays. Significant differences from controls are indicated by *P < 0.05, **P < 0.01.

LentiX-293 cells were seeded into a 10 cm dish at 2.5 × 10⁶ cells/dish. On the next day, a mixture of 5 μg pLVX-PANDAR or pLVX-Puro, 3.75 μg psPAX2 (catalog No. 12260; Addgene), and 1.25 μg pMD2.G (catalog No. 12259; Addgene) were diluted into 1 mL opti-MEM medium (Invitrogen); then, 30 μL PEI solution (1 mg/mL, catalog No. 23966; Polyscience) was added and mixed thoroughly. The mixture was incubated at room temperature for 25 min to prepare the transfection complexes. Then, the mixture was added dropwise into the abovementioned LentiX-293 cells. At 48 h after transfection, the supernatants were collected and centrifuged at 40,000 × g to collect lentivirus pellets. We used 1 mL DMEM medium to resuspend the lentivirus. MCF-7 cells (1 × 10⁶ cells in a 60 mm dish) were infected with each lentivirus at a multiplicity of infection (MOI) of 1. At 48 h after infection, stable cell lines were selected by adding 1 μg/mL puromycin (Sigma).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The cDNA was synthesized through reverse-transcription reaction with Rever-Tra-Ace-α-Transcriptase (Toyobo) and subsequently amplified by PCR using SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Bio). The primer sequences used in quantitative real-time polymerase chain reaction (qRT-PCR) are listed as follows: GAPDH QRT F: CCCATGTTCGTCATGGGTGT; GAPDH QRT R: TGGTCATGAGTCCTTCCACGATA; PANDAR F: TCAGGAATGCCGCAGATGTA; PANDAR R: GACCGTGTCTGGAGGATGCC.

The harvested cells were pelleted and resuspended in sodium dodecyl sulfate (SDS) lysis buffer (50 mM pH 6.8 Tris-HCl, 1% SDS, 10% glycerol, and 20 mM dithiothreitol) and incubated on ice. The lysates were centrifuged at 8,000 × g (5 min, 4 °C). Subsequently, proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking nonspecific binding sites with 5% nonfat milk for 1 h at ambient room temperature, they were incubated overnight at 4 °C with mouse monoclonal primary antibodies against E-cad (1:1,000; catalog No. sc-21791, antibody ID AB_626777), N-cad (1:1,000; catalog No. sc-59987; antibody ID AB_781744), and β-actin (1:1,000, catalog No. sc-47778; antibody ID AB_2714189) (Santa Cruz Biotechnology). Then, horseradish peroxidase (HRP)-linked goat secondary anti-mouse antibodies (1:2,000; catalog No. A0216; antibody ID AB_2860575; Beyotime, China) were added at ambient room temperature and incubated with the proteins transferred to the membrane for 1 h. Immunoreactive signal was developed and visualized with enhanced chemiluminescence (ECL) reagents (Millipore).

A cell counting kit-8 (CCK-8) was used to assess cell viability. Briefly, MCF-7 stable cell line overexpressing PANDAR or control cells were seeded into 96-well plates at 2 × 10³ cells/well. Then, 10 μL CCK-8 reagents (Solarbio, China) were added per well for the indicated time, followed by further incubation at 37 °C for 2–4 h. Optical density was measured at 450 nm on a microplate reader (Bio-Rad).

Data are shown as means ± SD calculated by WPS Office 2019. The empirical data obtained from all experiments were fitted to histograms using GraphPad Prism 7 software. The experiments were performed in triplicate and repeated (more than once). A Student t test (two-tailed) was performed to compare parametric data. Differences with P < 0.05 were considered significant.

E-cad, a biomarker of epithelial cells, is downregulated in the EMT process. Therefore, lncRNAs altering E-cad expression are involved in carcinogenesis. We constructed a series of noncoding RNA expression vectors and verified their overexpression effects in HEK-293T cells (Figure 1A–E). Then, we screened noncoding RNAs that potentially regulated the E-cad gene promoter by dual-luciferase reporter assay, including HOTAIR, LOC105375819, BX647792, and PANDAR. The human E-cad promoter reporter plasmid and a series of lncRNAs or circular RNAs were cotransfected into HEK-293T cells. We found that the overexpression of ciR-hsa_circ_0001073 (1.148 ± 0.014) and LOC105375819 (1.205 ± 0.019) significantly increased the luciferase activity, whereas overexpression of PANDAR (0.897 ± 0.041) suppresses the luciferase activity (Figure 1F). This finding suggests that ciR-hsa_circ_0001073, LOC105375819, and PANDAR may regulate E-cad expression in human cells.

Figure 1

To explore which regulation mechanism is evolutionarily conservative, the mouse E-cad promoter reporter vector and a series of lncRNAs or circular RNAs were cotransfected into HEK-293T cells, respectively. This was done to assess whether the abilities of ciR-hsa_circ_0001073, LOC105375819, and PANDAR able to regulate E-cad transcription were evolutionarily conserved. LOC105375819 (1.219 ± 0.086) remarkably increased luciferase activity, which was reduced by PANDAR (0.692 ± 0.071) and BX647792 (0.566 ± 0.032) (Figure 1G). These findings indicated that LOC105375819 and PANDAR regulated the promoter activity of the E-cad gene with evolutionary conservatism.

Because PANDAR modulated E-cad promoter activity, we hypothesized that it might contribute to modulating the EMT process. First, PANDAR was overexpressed in MCF-7 cells by using lentivirus, and then qRT-PCR was used to detect the efficiency of overexpression of PANDAR (Figure 2A). Further, western blotting was performed to determine the expression profile of EMT-related proteins, including E-cad, and N-cad protein. As depicted in Figure 2B, an increased PANDAR expression led to elevated N-cad levels. However, it reduced E-cad levels compared with the control group, suggesting that it may potentially control EMT in breast cancer.

Figure 2

MCF-7 cell expressing PANDAR or corresponding control was seeded into 96-well plates with the same density, followed by the photomicrographs and CCK8 assay to quantitate cell proliferation at indicated times (Figure 3A, B). The overexpression of PANDAR significantly increased MCF-7 cell proliferation at day 3 post-plating (1.000 ± 0.120 vs 1.412 ± 0.105). This significant increase in MCF-7 cell proliferation suggests that PANDAR is an oncogene regulating breast cancer proliferation.

Figure 3