LncRNA PLAC2 upregulates miR-663 to downregulate TGF-β1 and suppress bladder cancer cell migration and invasion

The present study enrolled 56 BC patients from 166 BC patients admitted to the first affiliated hospital of Wannan Medical College between July 2010 and December 2013. Inclusion criteria were:1) newly diagnosed cases; 2) patients were willing to participate in the 5-year follow-up and completed follow-up. Exclusion criteria were: 1) recurrent BC; 2) other clinical disorders were observed; 3) any therapies were initiated within 3 months before admission; 4) previous or family history of malignancies; 4) patients lost or died of other causes during follow-up. The 56 patients were staged according to the AJCC criteria, and 12, 15, 15 and 14 cases were classified into stage I-IV, respectively. Based on the criteria of urothelial carcinoma grading established by American College of Surgeons, the 56 patients included 26 cases with high grade and 30 cases with low grade. This study was approved by the Ethics committee of aforementioned hospital. All patients were informed of the details of experiments and possible publication of this study, and they all signed the informed consent.

All patients were diagnosed by histopathological biopsy. During biopsy, BC and adjacent (within 3 cm around tumors) non-cancer tissues (0.08–0.12 g) were collected from 3 different positions of the tumor. All tissues were confirmed by histopathological examinations.

Human BC cell line HT-1197 (ATCC, USA) was used. Eagle’s Minimum Essential Medium was mixed with 10% fetal bovine serum (FBS) and was used as the culture medium. Cells were cultivated at 37 °C with 5% CO₂. PLAC2 and TGF-β1 expression vectors were constructed using pcDNA3.1 vector (Sangon, Shanghai, China). Negative control miRNA and miR-663 mimic were purchased from Sigma-Aldrich (USA). Inhibitor negative control and miR-663 inhibitor were also purchased from Sigma-Aldrich (USA). HT-1197 cells were harvested at the confluence of 70–90%. Next, 10 nM of LAC2 and TGF-β1 expression vector, 10 nM of empty pcDNA3.1 vector (negative control, NC), 30 nM of negative control (NC) miRNA and miR-663 mimic, or 30 nM of inhibitor negative control (NC) and miR-663 inhibitor were transfected into 10⁵ cells using lipofectamine 2000 transfection reagent (Sigma-Aldrich, USA). Cells without transfections were used as the control (C). the following experiments were performed using cells collected at 24 h post-transfection.

HT-1197 cells as well as tissues (ground in liquid nitrogen before use) were mixed with VWR Life Science RiboZol™ RNA Extraction Reagent (VWR, USA) to extract total RNAs. Following DNase I digestion to remove genomic DNA, AMV Reverse Transcriptase (Canvax Biotech, USA) was used to synthesize cDNA through reverse transcription. The qPCR mixtures were prepared using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems). The expression of PLAC2 and TGF-β1 were determined with GAPDH as the endogenous control. HT-1197 cells as well as tissues were also used to extract miRNAs using mirVana miRNA Isolation Kit (Thermo Fisher Scientific). Following miRNA reverse transcriptions using MystiCq® microRNA cDNA Synthesis Mix (Sigma-Aldrich, USA), all qPCR reaction mixtures were prepared using miScript SYBR Green PCR Kit (QIAGEN, Germany). The expression of miR-663 was analyzed with U6 as endogenous control. Three biological replicates were included in each experiment and data were processed using 2^-ΔΔCT method. Primer sequences were: TGF-β1: (forward) 5′-TACCATGCCAACTTCTGTCTGGGA-3′ and (reverse) 5′-ATGTTGGACAACTGCTCCACCTTG-3′; PLAC2 (forward) 5′-AATGTCTTGGCCTTGAATGA-3′ and (reverse) 5′-CAAACTCAGGGATACATGGA-3′; GAPDH (forward) 5′-GCACCGTCAAGGCTGAGAAC-3′ and (reverse) 5′-TGGTGAAGACGCCAGTGGA-3′; miR-663: (forward) 5′-AGGCGGGGCGCCGCGGGACCGC-3′. U6 primers and miR-663 reverse primers were included in the kit.

HT-1197 cells (3 × 10⁴) were harvested at 24 h after transfections and mixed with 1 ml serum-free Eagle’s Minimum Essential Medium to prepare single cell suspensions. Cells were transferred to the upper Transwell chamber (0.1 ml per well) and the lower chamber was filled with Eagle’s Minimum Essential Medium (20% FBS). The membrane was coated with Matrigel (356,234, Millipore, USA) before invasion assay to mimic invasion condition. In migrtaion assay, uncoated membranes were used. Cells were cultivated at 37 °C with 5% CO₂ for 8 h, and membranes were cleaned and stained with 1% crystal violet (Sigma-Aldrich, USA) at 22 °C for 15 min. Stained cells were observed under an optical microscope.

HT-1197 cells (3 × 10⁵) were harvested at 24 h after transfections and mixed with 1 ml RIPA solution (Beyotime, Jiangsu, China) to extract total proteins. Proteins were denatured and subjected to 12% SDS-PAGE gel electrophoresis. Membranes were first incubated with 5% non-fat milk at 22 °C for 2 h, followed by incubation with TGF-β1 and GAPDH rabbit polyclonal primary antibodies at 4 °C overnight. Then membranes were incubated with IgG-HRP goat anti rabbit (1:1000, MBS435036, MyBioSource) secondary antibody at 22 °C for 2 h. Signals were developed using ECL (Sigma-Aldrich, USA) and Image J v1.46 software was used to process data.

Means values presented in this study were calculated using the data from 3 biological replicates. Differences between two types of tissue were compared using paired t test. ANOVA Tukey’s test was used to compare differences among different cell and patient groups. Correlations were analyzed using linear regression. The 56 BC patients were divided into high (n = 26) and low (n = 30) PLAC2 level groups (Youden’s index). Survival curves were plotted using K-M method and compared by log-rank test. Chi-squared test was performed to analyze the correlations between the expression levels of PLAC2 and patients’ clinical data. P < 0.05 was statistically significant.

The expression of PLAC2 in two types of tissue of BC patients (n = 56) were detected by RT-qPCR and the expression data were compared by paired t test. The data revealed that the expression levels of PLAC2 were significantly lower in BC tissues in comparison to that in non-cancer tissues (Fig. 1a p < 0.05). The expression levels of PLAC2 were slightly lower in high grade group than that in low grade group, but the difference was not significant (Fig. 1b, p < 0.05). Chi-squared test showed that the expression levels of PLAC2 were not significantly correlated with patients’ age, gender, multifocal tumors, CIS, cancer stages and tumor grades (Table 1).

The 56 patients were staged according to the AJCC criteria, and 12, 15, 15 and 14 cases were classified into stage I-IV, respectively. The expression levels of PLAC2 in BC were compared among different clinical stages using one-way ANOVA and Tukey test. No significant differences were observed among different clinical stages (Fig. 2a). In addition, survival curves showed that patients with low expression levels of PLAC2 had significantly worse overall survival rate (Fig. 2b). It is worth noting that the 56 patients included 26 cases with high grade and 30 cases with low grade. The expression levels of PLAC2 were slightly lower in high grade group in comparison to that in the low-grade group, but the difference was not significant (p < 0.05, data not shown).

The expression of TGF-β1 and miR-663 in BC tissues were also detected by RT-qPCR experiments. Correlations were analyzed by linear regression. It was observed that the expression of PLAC2 was positively correlated with the expression of miR-663 (Fig. 3a) and inversely correlated with TGF-β1 (Fig. 3b) across BC tissues.

After transfections, the expression levels of PLAC2, miR-663 and TGF-β1 were significantly altered at 24 h after transfections (Fig. 4a, p < 0.05), indicating the successful transfections. In was observed that overexpression of PLAC2 resulted in upregulation of miR-663, while overexpression of miR-663 showed no significantly changed expression levels of PLAC2 (Fig. 4b, p < 0.05). In addition, overexpression of PLAC2 and miR-663 resulted in the downregulation of TGF-β1, while miR-663 inhibitor attenuated the effects of overexpression of PLAC2 (Fig. 4c, p < 0.05). Moreover, overexpression of TGF-β1 did not significantly affect the expression of PLAC2 and miR-663 (Fig. 4d). Therefore, a novel PLAC2/miR-663/TGF-β1 pathway was characterized. It is worth noting that the targeting of TGF-β1 by miR-663 has been well established [12]. Transwell migration and invasion assays were performed to investigate the involvement of the PLAC2/miR-663/TGF-β1 pathway in regulating BC cell behaviors. It showed that overexpression of PLAC2 and miR-663 resulted in reduced rates of BC cell migration (Fig. 5a) and invasion (Fig. 5b), while overexpression of TGF-β1 resulted in increased rates (p < 0.05). In addition, miR-663 inhibitor reduced the effects of overexpressing PLAC2 (p < 0.05). It is worth noting that our preliminary CCK-8 assay resulted showed that PLAC2 had no significant effects on cell proliferation, apoptosis and stemness (data not shown).