# Downregulation of Long Noncoding RNA LUCAT1 Suppresses the Migration and Invasion of Bladder Cancer by Targeting miR-181c-5p

Authors: Yifan Chen; Wentao Zhang; Liliang Shen; Aimaitiaji Kadier; Jianhua Huang; Ruiliang Wang; Pengfei Wu; Xudong Yao
Journal: BioMed Research International
 (2020)

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## Abstract

Purpose. The long noncoding RNA LUCAT1 (lung cancer-associated transcript 1) has been reported to be highly expressed in bladder cancer samples, but its role and molecular mechanisms need to be elucidated. Methods. Bioinformatics methods show that miR-181c-5p is a target of LUCAT1. Here, we aimed to reveal whether LUCAT1 participates in the development of bladder cancer via targeting miR-181c-5p. The expression levels of LUCAT1 and miR-181c-5p were detected by RT-PCR technology in bladder cells and tissues. The effects of the LUCAT1/miR-181c-5p axis on cell proliferation, migration, invasion, and apoptosis were tested by CCK-8, wound healing, Transwell chambers, and flow cytometry assays. The expressions of apoptosis/migration-related proteins were detected by western blotting assays. Results. The results demonstrated that LUCAT1 was overexpressed in bladder cancer tissue and cells, while miR-181c-5p showed a low expression pattern as compared to normal bladder cells and tissues. Cell proliferation, migration, and invasion capacities were significantly impaired, and cell apoptosis was enhanced when LUCAT1 was silenced in UM-UC-3 and T24 cell lines, but this effect was abolished by miR-181c-5p downregulation. In addition, miR-181c-5p downregulation impaired LUCAT1 downregulation which mediated the decreased expressions of Bcl2 and N-cadherin and the increased expressions of Bax and E-cadherin. Moreover, we found that KRAS was a direct target of miR-181c-5p and was under the positive regulation of LUCAT1. Conclusion. Collectively, this study reveals that knockdown of LUCAT1 inhibits the migration and invasion of bladder cancer cells in a miR-181c-5p-dependent manner, which may be related to KRAS downregulation.


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## Body

## 1. Introduction

Bladder cancer is a common malignant tumor which occurs in the urinary system [1] and ranks the fourth most common malignant cancer among males [2]. Muscle-invasive bladder cancer (MIBC) accounts for 25-40% of bladder cancer cases and can spread to the pelvic lymph nodes and even the visceral organs [3]. Metastasis is a complex multistep process which contains cancer cell dissemination, transport, and colonization expansion [4, 5]. However, the molecular mechanisms underlying bladder cancer cell metastasis and invasion still remain incompletely clear.Long noncoding RNAs (lncRNAs) are transcripts which contain >200 nucleotides in length with no protein-coding capacity [6]. lncRNAs can modulate gene expression at chromatin organizational, transcriptional, or posttranscriptional levels [7]. Lots of studies have found that many lncRNAs are aberrantly expressed in cancers including bladder cancer, where they functions as tumor suppressors or oncogenes [6, 8, 9]. For example, the lncRNA zinc finger E-box-binding homeobox 1-antisense 1 (ZEB1-AS1) was reported to be overexpressed in bladder cancer, and ZEB1-AS1 repression significantly inhibited cell migration and invasion [10]. Hu et al. [11] demonstrated that lncRNA prostrate androgen-regulated transcript-1 (PART1) showed a high expression pattern in bladder cancer tissues, and overexpression caused significant enhancements in cell proliferation and invasion ability and an obvious repression in cell apoptosis. Cao et al. [12] reported that the expression of LUCAT1 (lung cancer-associated transcript 1) was significantly increased in bladder cancer samples, especially in the high-grade bladder cancer samples, and it may promote bladder cancer progression. However, the detailed roles and mechanisms underlying LUCAT1 in the migration and invasion still remain unknown.Increasing evidences have demonstrated that lncRNAs can act as competitive endogenous RNAs (ceRNA) of microRNAs (miRNAs) via 3′ untranslated region (UTR) or 5′ UTR and thereafter regulate gene expression and are strongly implicated in the modulation of biological processes, such as cell growth, survival, apoptosis, migration, and invasion [13, 14]. Using starBase, we identified that miR-181c-5p was a predicted target of LUCAT1, and the miR-181c-5p low expression level predicted a poor prognosis in bladder cancer, indicating that miR-181c-5p might play a role in LUCAT1-mediated bladder cancer progression.In the current study, we aimed to explore the functions of LUCAT1 in the migration and invasion of bladder cancer, as well as reveal miR-181c-5p roles in this process.


## 2. Materials and Methods

### 2.1. Bioinformatics Methods

Bioinformatics software starBase (http://starbase.sysu.edu.cn) was used to analyze the relationship between the expression levels of LUCAT1 and miR-181c-5p and the overall survival rates in patients with bladder cancer, as well as to evaluate the association between the expression levels of LUCAT1 and miR-181c-5p in bladder cancer cases. The binding sites between miR-181c-5p were predicted by using starBase, while the binding sites between miR-181c-5p and KRAS were predicted by using the TargetScan (http://www.targetscan.org/vert_71/) and miRDB (http://www.mirdb.org/cgi-bin/search.cgi).


### 2.2. Tissue Samples

Ninety bladder cancer tissues and the paired peritumoral nontumor tissues were derived from patients with bladder cancer who received bladdertomy in Shanghai Tenth People’s Hospital between February 2015 and February 2018. Informed consent has been obtained from every patient. This study has been performed in accordance with the Helsinki Declaration and was approved by the ethical committee of Shanghai Tenth People’s Hospital.


### 2.3. Cell Lines and Culture Conditions

One human epithelial immortalized uroepithelium cell line, SV-HUC-1, and four human bladder cancer cell lines, UM-UC-3, 5637, T24, and SW780, were purchased from the American Type Collection Culture (ATCC, Manassas, VA, USA). Among them, UM-UC-3, T24, and SW780 cell lines were derived from transitional cell carcinoma, and 5637 cells were derived from grade II carcinoma. SV-HUC-1 cells were grown in F-12K Medium (ATCC), UM-UC-3 cells were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC), 5637 cells were grown in RPMI-1640 medium (ATCC), T24 cells were cultured in McCoy’s 5a medium (ATCC), and SW780 cells were maintained in Leibovitz’s L-15 medium (ATCC), supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA).


### 2.4. Cell Transfection

Small interfering RNAs (siRNAs) used to downregulate LUCAT1 expression (si-LUCAT1), as well as the inhibitor used to silence miR-181c-5p, and their negative control vectors (si-NC, inhibitor-NC) were synthesized by GenePharma (Shanghai, China). The above vectors were introduced into bladder cancer T24 and UM-UC-3 cells via cell transfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) referring to the manufacturer’s descriptions.


### 2.5. Quantitative Real-Time PCR (RT-PCR) Analysis

Total RNA extracted from cells and tissues using the RNAPure Tissue & Cell Kit (DNase I) in accordance with the manufacturer’s instructions (CWBIO, Beijing, China). Then, a total of 1μg RNA was submitted to the reverse transcription of miRNA and mRNA using stem-loop primers and random primers with TaKaRa system (Dalian, China) according to the manufacturer’s instructions. Subsequently, the RT-PCR was carried out using a TaqMan Universal Master Mix II kit on a Bio-Rad detection system (Bio-Rad, Hercules, CA). GAPDH and U6 expression levels are served as internal references to normalize mRNA and miRNA expressions, respectively. Primers were listed as follows.LUCAT1: forward- (F-) 5′-CCTCACAAGAAGCTCACCCA-3′, reverse- (R-) 5′-CAGCATGTAGCCCATGGTAGA-3′; KRAS: F-5′-TAGGCAAGAGTGCCTTGACG-3′, R-5′-CCCTCCCCAGTCCTCATGTA-3′; and GAPDH: F-5′-CCACTAGGCGCTCACTGTTCTC-3′, R-5′-ACTCCGACCTTCACCTTCCC-3′. If the expression was lower than the median value, miR-181c-5p was considered as the low expression and vice versa. Similarly, LUCAT1 was considered to express at a high level when its expression was higher than the median value and vice versa.


### 2.6. Western Blotting Assay

Total protein extraction from cells was carried out by using the RIPA lysis buffer (Sangon Biotech, Shanghai, China) containing protease inhibitor (Beyotime, Shanghai, China), according to the manufacturer’s description. After quantification with a BCA Protein Kit (Bio-Rad Laboratories, CA, USA), 30μg proteins of each sample was loaded and separated by 10% polyacrylamide gels and was transferred into the polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were then incubated with 5% nonfat milk and probed with the primary antibodies overnight at 4°C, including Bcl2 (1 : 1000 dilution; No. #15071, Cell Signaling Technology), Bax (1 : 1000 dilution; No. #2772, Cell Signaling Technology), E-cadherin (1 : 1000 dilution; No. #3195, Cell Signaling Technology), N-cadherin (1 : 1000 dilution; No. #4061, Cell Signaling Technology), KRAS (1 : 1000 dilution; No. ab180772, Abcam, Cambridge, MA, USA), and GAPDH (1 : 2000 dilution; No. #2118, Cell Signaling Technology). After the incubation with the corresponding second antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), the protein expression levels were determined on a western blotting imaging and quantitative system (Bio-Rad, Hertfordshire, UK). Protein quantification was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) after background subtraction, with GAPDH level as an internal reference.


### 2.7. Luciferase Gene Reporter Assay

The wild type and the mutant type of the 3′UTR of LUCAT1 and KRSA were cloned into the pGL3 vector (Promega, Madison, WI, USA), which were called as LUCAT1/KRAS-WT or LUCAT1/KRAS-MUT, respectively. For the luciferase gene reporter assay, T24 and UM-UC-3 cells were cotransfected with the WT/MUT vector and mimic-miR-181c-5p/mimic-NC. The luciferase activity was determined through a dual-luciferase assay system (Promega) based on the manufacturer’s descriptions.


### 2.8. Cell Counting Kit-8 (CCK-8) Assay

The CCK-8 assay was used to detect cell proliferation. T24 and UM-UC-3 cells were seeded in 96-well plates at a density of3×103 cells/well and cultured at 37°C overnight, then the cells were given different cell transfections, including si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. After 1, 2, 3, 4, or 5 days of cell transfection, the culture medium was replaced with 10 μl CCK-8 reagent (Beyotime, Beijing, China) and 90 μl fresh medium. Then, the cells were incubated at 37°C for another 4 hours. The absorbance at 450 nm was measured using a plate reader (model 680; Bio-Rad, Hertfordshire, UK).


### 2.9. Flow Cytometry Assay

Flow cytometry assay was carried out to detect cell apoptosis. In brief, cells were transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. Following 48 hours of incubation at 37°C, the cells were harvested and subjected to apoptosis detection by using the Annexin V(FITC)/propidium iodide (PI) apoptosis detection kit (Dojindo, Japan). The cell apoptosis rate was determined by using the flow cytometry (Beckman Coulter, CA, USA) and analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA).


### 2.10. Wound Healing Assay

Wound healing assay was used to assess cell migration ability. Cells were first plated in 6-well plates at a concentration of5×105 cells/ml and cultured at 37°C overnight, followed by transfection with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. Next, the wounds were made using 20 μl pipette tips when cell confluence was reached at 100%, and the gloating cells were removed via PBS washing. Images of cell movement to the scratch area were taken every 6–12 hours using a microscope.


### 2.11. Transwell Chamber Assay

Transwell chambers with 8μm polycarbonate filters (BD Bioscience, San Diego, CA, USA) were applied for cell invasion assessment. In the procedure, chambers were coated with Matrigel on the lower side. Then, approximately 2×105 T24 or UM-UC-3 cells were resuspended with FBS-free medium and were planted in the upper chamber with 600 μl medium supplemented with 20% FBS in the lower chamber. Following 48 hours of incubation at 37°C, cells in the top of the membranes were removed using cotton buds and cells at the bottom of the membrane were fixed and stained with 0.2% crystal violet (Solarbio, Beijing, China). The stained cells were counted under a light microscope (magnification: 200x) to assess cell invasiveness.


### 2.12. Statistical Analysis

Each experiment in the current study was performed in triplicate. Statistical significance comparison between two groups and multiple groups was carried out using Student’st test and one-way ANOVA, respectively. Data analysis was performed by using the SPSS (version 23.0, SPSS Inc. Chicago, IL, USA). The P value < 0.05 was considered as statistically significance.We followed the methods of Chen et al. [15].


## 2.1. Bioinformatics Methods

Bioinformatics software starBase (http://starbase.sysu.edu.cn) was used to analyze the relationship between the expression levels of LUCAT1 and miR-181c-5p and the overall survival rates in patients with bladder cancer, as well as to evaluate the association between the expression levels of LUCAT1 and miR-181c-5p in bladder cancer cases. The binding sites between miR-181c-5p were predicted by using starBase, while the binding sites between miR-181c-5p and KRAS were predicted by using the TargetScan (http://www.targetscan.org/vert_71/) and miRDB (http://www.mirdb.org/cgi-bin/search.cgi).


## 2.2. Tissue Samples

Ninety bladder cancer tissues and the paired peritumoral nontumor tissues were derived from patients with bladder cancer who received bladdertomy in Shanghai Tenth People’s Hospital between February 2015 and February 2018. Informed consent has been obtained from every patient. This study has been performed in accordance with the Helsinki Declaration and was approved by the ethical committee of Shanghai Tenth People’s Hospital.


## 2.3. Cell Lines and Culture Conditions

One human epithelial immortalized uroepithelium cell line, SV-HUC-1, and four human bladder cancer cell lines, UM-UC-3, 5637, T24, and SW780, were purchased from the American Type Collection Culture (ATCC, Manassas, VA, USA). Among them, UM-UC-3, T24, and SW780 cell lines were derived from transitional cell carcinoma, and 5637 cells were derived from grade II carcinoma. SV-HUC-1 cells were grown in F-12K Medium (ATCC), UM-UC-3 cells were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC), 5637 cells were grown in RPMI-1640 medium (ATCC), T24 cells were cultured in McCoy’s 5a medium (ATCC), and SW780 cells were maintained in Leibovitz’s L-15 medium (ATCC), supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA).


## 2.4. Cell Transfection

Small interfering RNAs (siRNAs) used to downregulate LUCAT1 expression (si-LUCAT1), as well as the inhibitor used to silence miR-181c-5p, and their negative control vectors (si-NC, inhibitor-NC) were synthesized by GenePharma (Shanghai, China). The above vectors were introduced into bladder cancer T24 and UM-UC-3 cells via cell transfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) referring to the manufacturer’s descriptions.


## 2.5. Quantitative Real-Time PCR (RT-PCR) Analysis

Total RNA extracted from cells and tissues using the RNAPure Tissue & Cell Kit (DNase I) in accordance with the manufacturer’s instructions (CWBIO, Beijing, China). Then, a total of 1μg RNA was submitted to the reverse transcription of miRNA and mRNA using stem-loop primers and random primers with TaKaRa system (Dalian, China) according to the manufacturer’s instructions. Subsequently, the RT-PCR was carried out using a TaqMan Universal Master Mix II kit on a Bio-Rad detection system (Bio-Rad, Hercules, CA). GAPDH and U6 expression levels are served as internal references to normalize mRNA and miRNA expressions, respectively. Primers were listed as follows.LUCAT1: forward- (F-) 5′-CCTCACAAGAAGCTCACCCA-3′, reverse- (R-) 5′-CAGCATGTAGCCCATGGTAGA-3′; KRAS: F-5′-TAGGCAAGAGTGCCTTGACG-3′, R-5′-CCCTCCCCAGTCCTCATGTA-3′; and GAPDH: F-5′-CCACTAGGCGCTCACTGTTCTC-3′, R-5′-ACTCCGACCTTCACCTTCCC-3′. If the expression was lower than the median value, miR-181c-5p was considered as the low expression and vice versa. Similarly, LUCAT1 was considered to express at a high level when its expression was higher than the median value and vice versa.


## 2.6. Western Blotting Assay

Total protein extraction from cells was carried out by using the RIPA lysis buffer (Sangon Biotech, Shanghai, China) containing protease inhibitor (Beyotime, Shanghai, China), according to the manufacturer’s description. After quantification with a BCA Protein Kit (Bio-Rad Laboratories, CA, USA), 30μg proteins of each sample was loaded and separated by 10% polyacrylamide gels and was transferred into the polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were then incubated with 5% nonfat milk and probed with the primary antibodies overnight at 4°C, including Bcl2 (1 : 1000 dilution; No. #15071, Cell Signaling Technology), Bax (1 : 1000 dilution; No. #2772, Cell Signaling Technology), E-cadherin (1 : 1000 dilution; No. #3195, Cell Signaling Technology), N-cadherin (1 : 1000 dilution; No. #4061, Cell Signaling Technology), KRAS (1 : 1000 dilution; No. ab180772, Abcam, Cambridge, MA, USA), and GAPDH (1 : 2000 dilution; No. #2118, Cell Signaling Technology). After the incubation with the corresponding second antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), the protein expression levels were determined on a western blotting imaging and quantitative system (Bio-Rad, Hertfordshire, UK). Protein quantification was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) after background subtraction, with GAPDH level as an internal reference.


## 2.7. Luciferase Gene Reporter Assay

The wild type and the mutant type of the 3′UTR of LUCAT1 and KRSA were cloned into the pGL3 vector (Promega, Madison, WI, USA), which were called as LUCAT1/KRAS-WT or LUCAT1/KRAS-MUT, respectively. For the luciferase gene reporter assay, T24 and UM-UC-3 cells were cotransfected with the WT/MUT vector and mimic-miR-181c-5p/mimic-NC. The luciferase activity was determined through a dual-luciferase assay system (Promega) based on the manufacturer’s descriptions.


## 2.8. Cell Counting Kit-8 (CCK-8) Assay

The CCK-8 assay was used to detect cell proliferation. T24 and UM-UC-3 cells were seeded in 96-well plates at a density of3×103 cells/well and cultured at 37°C overnight, then the cells were given different cell transfections, including si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. After 1, 2, 3, 4, or 5 days of cell transfection, the culture medium was replaced with 10 μl CCK-8 reagent (Beyotime, Beijing, China) and 90 μl fresh medium. Then, the cells were incubated at 37°C for another 4 hours. The absorbance at 450 nm was measured using a plate reader (model 680; Bio-Rad, Hertfordshire, UK).


## 2.9. Flow Cytometry Assay

Flow cytometry assay was carried out to detect cell apoptosis. In brief, cells were transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. Following 48 hours of incubation at 37°C, the cells were harvested and subjected to apoptosis detection by using the Annexin V(FITC)/propidium iodide (PI) apoptosis detection kit (Dojindo, Japan). The cell apoptosis rate was determined by using the flow cytometry (Beckman Coulter, CA, USA) and analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA).


## 2.10. Wound Healing Assay

Wound healing assay was used to assess cell migration ability. Cells were first plated in 6-well plates at a concentration of5×105 cells/ml and cultured at 37°C overnight, followed by transfection with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p. Next, the wounds were made using 20 μl pipette tips when cell confluence was reached at 100%, and the gloating cells were removed via PBS washing. Images of cell movement to the scratch area were taken every 6–12 hours using a microscope.


## 2.11. Transwell Chamber Assay

Transwell chambers with 8μm polycarbonate filters (BD Bioscience, San Diego, CA, USA) were applied for cell invasion assessment. In the procedure, chambers were coated with Matrigel on the lower side. Then, approximately 2×105 T24 or UM-UC-3 cells were resuspended with FBS-free medium and were planted in the upper chamber with 600 μl medium supplemented with 20% FBS in the lower chamber. Following 48 hours of incubation at 37°C, cells in the top of the membranes were removed using cotton buds and cells at the bottom of the membrane were fixed and stained with 0.2% crystal violet (Solarbio, Beijing, China). The stained cells were counted under a light microscope (magnification: 200x) to assess cell invasiveness.


## 2.12. Statistical Analysis

Each experiment in the current study was performed in triplicate. Statistical significance comparison between two groups and multiple groups was carried out using Student’st test and one-way ANOVA, respectively. Data analysis was performed by using the SPSS (version 23.0, SPSS Inc. Chicago, IL, USA). The P value < 0.05 was considered as statistically significance.We followed the methods of Chen et al. [15].


## 3. Results

### 3.1. Analysis of the Expression Patterns and Clinical Values of LUCAT1 and miR-181c-5p in Bladder Cancer

To explore the function of the LUCAT1/miR-181c-5p axis in the progression of bladder cancer, we first applied bioinformatics methods to predict the relationship between LUCAT1/miR-181c-5p expression levels and patients’ overall survival. The results showed that both the low expression level of miR-181c-5p (Figure1(a)) and the high expression level of LUCAT1 (Figure 1(b)) predicted shorter overall survival in patients with bladder carcinoma. In addition, the miR-181c-5p expression was negatively associated with LUCAT1 expression in a total of 408 bladder cancer samples (Figure 1(c)), suggesting that there might be a link between miR-181c-5p and LUCAT1 in bladder cancer. We then performed the RT-PCR assay to detect the expression profiles of LUCAT1 and miR-181c-5p in bladder cancer cell lines and tissues. Compared with that of the SV-HUC-1 cells, the expression of LUCAT1 was significantly increased, and miR-181c-5p expression was reduced in the UM-UC-3, 5637, T24, and SW780 cell lines, with UM-UC-3 and T24 showing the highest (Figure 2(a)) or lowest (Figure 2(b)) and chosen for the following experiments. Consistently, LUCAT1 expression was obviously increased in bladder cancer tissues, while miR-181c-5p expression was reduced compared to the adjacent nontumor tissues (Figures 2(c) and 2(d)). And miR-181c-5p expression showed a negative association with LUCAT1 expression in 90 tested bladder cancer samples (Figure 2(e)). Moreover, both the high expression of LUCAT1 and the low expression of miR-181c-5p showed a close relationship with the poor prognosis in bladder cancer cases (Figures 2(f) and 2(g)).Figure 1
Bioinformatics method analysis of the clinical value of LUCAT1 and miR-181c-5p expression levels in predicting the prognosis of bladder cancer. (a, b) starBase was used to evaluate the relationship between the expression patterns of LUCAT1 and miR-181c-5p and the overall survival in bladder cancer patients. (c) starBase was used to evaluate the association between the expression levels of miR-181c-5p and LUCAT1 in bladder cancer cases.
(a)(b)(c)Figure 2
Evaluation of the expression and clinical value of miR-181c-5p and LUCAT1 in bladder cancer. (a, b) RT-PCR analysis of the expression of LUCAT1 and miR-181c-5p in SV-HUC-1, T24, UM-UC-3, 5637, and SW780 cell lines. (c, d) The expression patterns of LUCAT1 and miR-181c-5p in 90 paired bladder cancer tissues and normal tissues were detected by RT-PCR assay. (e) The association between LUCAT1 and miR-181c-5p expression patterns in 90 matched bladder cancer tissues and nontumor tissues was determined by the Pearson correlation test. (f, g) The relationship between LUCAT1 and miR-181c-5p expression levels and patients’ overall survival was detected by the Kaplan-Meier (∗P<0.05).
(a)(b)(c)(d)(e)(f)(g)


### 3.2. miR-181c-5p Is a Direct Target of LUCAT1 in Bladder Cancer Cells

Then, we evaluate the relationship between miR-181c-5p and LUCAT1 via the luciferase gene reporter assay. The putative binding sites between LUCAT1 and miR-181c-5p were shown in Figure3(a). The transfection of mimic-miR-181c-5p significantly elevated miR-181c-5p expression in both T24 and UM-UC-3 cell lines (Figures 3(b) and 3(d)). Compared with the control group, the luciferase activity of LUCAT1-WT was significantly decreased when miR-181c-5p was overexpressed in T24 and UM-UC-3 cells (Figures 3(c) and 3(e)). These results confirm that LUCAT1 can sponge miR-181c-5p in bladder cancer cells.Figure 3
miR-181c-5p was a target of LUCAT1 in bladder cancer cells. (a) The wild type (WT) or mutated type (MUT) of the binding sites in LUCAT1 with miR-181c-5p. (b) miR-181c-5p expression was detected by RT-PCR after T24 cells were transfected with mimic-NC or mimic-miR-181c-5p. (c) Luciferase gene reporter experiment was applied to evaluate the relationship between miR-181c-5p and LUCAT1 in T24 cells. (d) miR-181c-5p expression was detected by RT-PCR after T24 cells were transfected with mimic-NC or mimic-miR-181c-5p. (e) Luciferase gene reporter experiment was applied to evaluate the relationship between miR-181c-5p and LUCAT1 in T24 cells (n=3, ∗P<0.05).
(a)(b)(c)(d)(e)


### 3.3. Knockdown of LUCAT1 Represses Cell Growth, Migration, and Invasion via Targeting miR-181c-5p in Bladder Cancer

We then determined the roles of the LUCAT1/miR-181c-5p axis in the progression of bladder cancerin vitro. siRNA transfection induced a significant reduction in the expression levels of LUCAT1 in both T24 and UM-UC-3 cell lines (Figures 4(a) and 4(b)). Knockdown of LUCAT1 apparently decreased cell proliferation (Figures 4(c) and 4(d)), migration (Figures 4(g) and 4(h)), and invasion (Figures 4(i) and 4(j)) and induced cell apoptosis (Figures 4(e) and 4(f)). However, miR-181c-5p downregulation induced obvious enhancements in cell proliferation (Figures 4(c) and 4(d)), migration (Figures 4(g) and 4(h)), and invasion (Figures 4(i) and 4(j)) and induced a reduction in cell apoptosis (Figures 4(e) and 4(f)), as well as neutralized the above effects mediated by LUCAT1 (Figures 4(c)–4(j)). In addition, downregulation of miR-181c-5p abolished si-LUCAT1-mediated downregulation of Bcl2 and N-cadherin and upregulation of Bax and E-cadherin in both T24 and UM-UC-3 cell lines (Figures 5(a) and 5(b)). These results demonstrate that downregulation of LUCAT1 inhibited the progression of bladder cancer via targeting miR-181c-5p.Figure 4
LUCAT1 downregulation repressed cell proliferation, migration, and invasion and induced cell apoptosis in a miR-181c-5p-dependent manner. (a, b) The expression of LUCAT1 was detected by RT-PCR assay after T24 and UM-UC-3 cells were transfected with si-LUCAT1 or si-NC (n=3, ∗P<0.05). T24 and UM-UC-3 cells were transiently transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p and thereby submitted to the following assays. (c, d) CCK-8 assay was used to detect cell proliferation ability. (e, f) Flow cytometry assay was carried out to assess cell apoptosis populations. (g, h) Wound healing assay was used to detect cell migration capacity. (i, j) Transwell chambers coated with Matrigel were applied to detect cell invasion (n=3; ∗P<0.05, compared with si-NC+inhibitor-NC group; #P<0.05, compared with si-LUCAT1+inhibitor-NC group).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 5
LUCAT1 downregulation decreased the expression of Bcl2 and N-cadherin and increased the Bax and E-cadherin expression via targeting miR-181c-5p in bladder cancer cells. (a, b) After T24 and UM-UC-3 cells were transiently transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p, western blotting technology was applied to test the protein expressions of Bcl2, Bax, N-cadherin, and E-cadherin (n=3, ∗P<0.05, compared with si-NC+inhibitor-NC group; #P<0.05, compared with si-LUCAT1+inhibitor-NC group).
(a)(b)


### 3.4. Downregulation of LUCAT1 Decreases KRAS Expression via Targeting miR-181c-5p in Bladder Cancer

To further reveal the underlying mechanism of LUCAT1 in the progression of bladder cancer, we then applied TargetScan to predict the target of miR-181c-5p. The results showed that KRAS was a predicted target of miR-181c-5p, and the putative binding sites are shown in Figure6(a). The luciferase gene reporter assay showed that overexpression of miR-181c-5p significantly reduced the luciferase activity, whereas this effect was abrogated when the binding sites were mutated (Figures 6(b) and 6(c)). In addition, downregulation of LUCAT1 significantly reduced KRAS expression at both mRNA and protein levels, whereas this effect was obviously abolished when miR-181c-5p was downregulated (Figures 6(d) and 6(e)). These results demonstrate that the downstream gene KRAS might be involved in LUCAT1/miR-181c-5p-mediated bladder cancer progression.Figure 6
KRAS was a target of miR-181c-5p in bladder cancer. (a) The wild type (WT) or mutated type (MUT) of binding sites between KRAS and miR-181c-5p. (b, c) Luciferase gene reporter experiment was used to evaluate the relationship between miR-181c-5p and KRAS in T24 and UM-UC-3 cell lines (n=3, ∗P<0.05). (d, e) The mRNA and protein expression levels of KRAS in differently transfected T24 and UM-UC-3 cells (n=3, ∗P<0.05, compared with si-NC+inhibitor-NC group; #P<0.05, compared with si-LUCAT1+inhibitor-NC group).
(a)(b)(c)(d)(e)


## 3.1. Analysis of the Expression Patterns and Clinical Values of LUCAT1 and miR-181c-5p in Bladder Cancer

To explore the function of the LUCAT1/miR-181c-5p axis in the progression of bladder cancer, we first applied bioinformatics methods to predict the relationship between LUCAT1/miR-181c-5p expression levels and patients’ overall survival. The results showed that both the low expression level of miR-181c-5p (Figure1(a)) and the high expression level of LUCAT1 (Figure 1(b)) predicted shorter overall survival in patients with bladder carcinoma. In addition, the miR-181c-5p expression was negatively associated with LUCAT1 expression in a total of 408 bladder cancer samples (Figure 1(c)), suggesting that there might be a link between miR-181c-5p and LUCAT1 in bladder cancer. We then performed the RT-PCR assay to detect the expression profiles of LUCAT1 and miR-181c-5p in bladder cancer cell lines and tissues. Compared with that of the SV-HUC-1 cells, the expression of LUCAT1 was significantly increased, and miR-181c-5p expression was reduced in the UM-UC-3, 5637, T24, and SW780 cell lines, with UM-UC-3 and T24 showing the highest (Figure 2(a)) or lowest (Figure 2(b)) and chosen for the following experiments. Consistently, LUCAT1 expression was obviously increased in bladder cancer tissues, while miR-181c-5p expression was reduced compared to the adjacent nontumor tissues (Figures 2(c) and 2(d)). And miR-181c-5p expression showed a negative association with LUCAT1 expression in 90 tested bladder cancer samples (Figure 2(e)). Moreover, both the high expression of LUCAT1 and the low expression of miR-181c-5p showed a close relationship with the poor prognosis in bladder cancer cases (Figures 2(f) and 2(g)).Figure 1
Bioinformatics method analysis of the clinical value of LUCAT1 and miR-181c-5p expression levels in predicting the prognosis of bladder cancer. (a, b) starBase was used to evaluate the relationship between the expression patterns of LUCAT1 and miR-181c-5p and the overall survival in bladder cancer patients. (c) starBase was used to evaluate the association between the expression levels of miR-181c-5p and LUCAT1 in bladder cancer cases.
(a)(b)(c)Figure 2
Evaluation of the expression and clinical value of miR-181c-5p and LUCAT1 in bladder cancer. (a, b) RT-PCR analysis of the expression of LUCAT1 and miR-181c-5p in SV-HUC-1, T24, UM-UC-3, 5637, and SW780 cell lines. (c, d) The expression patterns of LUCAT1 and miR-181c-5p in 90 paired bladder cancer tissues and normal tissues were detected by RT-PCR assay. (e) The association between LUCAT1 and miR-181c-5p expression patterns in 90 matched bladder cancer tissues and nontumor tissues was determined by the Pearson correlation test. (f, g) The relationship between LUCAT1 and miR-181c-5p expression levels and patients’ overall survival was detected by the Kaplan-Meier (∗P<0.05).
(a)(b)(c)(d)(e)(f)(g)


## 3.2. miR-181c-5p Is a Direct Target of LUCAT1 in Bladder Cancer Cells

Then, we evaluate the relationship between miR-181c-5p and LUCAT1 via the luciferase gene reporter assay. The putative binding sites between LUCAT1 and miR-181c-5p were shown in Figure3(a). The transfection of mimic-miR-181c-5p significantly elevated miR-181c-5p expression in both T24 and UM-UC-3 cell lines (Figures 3(b) and 3(d)). Compared with the control group, the luciferase activity of LUCAT1-WT was significantly decreased when miR-181c-5p was overexpressed in T24 and UM-UC-3 cells (Figures 3(c) and 3(e)). These results confirm that LUCAT1 can sponge miR-181c-5p in bladder cancer cells.Figure 3
miR-181c-5p was a target of LUCAT1 in bladder cancer cells. (a) The wild type (WT) or mutated type (MUT) of the binding sites in LUCAT1 with miR-181c-5p. (b) miR-181c-5p expression was detected by RT-PCR after T24 cells were transfected with mimic-NC or mimic-miR-181c-5p. (c) Luciferase gene reporter experiment was applied to evaluate the relationship between miR-181c-5p and LUCAT1 in T24 cells. (d) miR-181c-5p expression was detected by RT-PCR after T24 cells were transfected with mimic-NC or mimic-miR-181c-5p. (e) Luciferase gene reporter experiment was applied to evaluate the relationship between miR-181c-5p and LUCAT1 in T24 cells (n=3, ∗P<0.05).
(a)(b)(c)(d)(e)


## 3.3. Knockdown of LUCAT1 Represses Cell Growth, Migration, and Invasion via Targeting miR-181c-5p in Bladder Cancer

We then determined the roles of the LUCAT1/miR-181c-5p axis in the progression of bladder cancerin vitro. siRNA transfection induced a significant reduction in the expression levels of LUCAT1 in both T24 and UM-UC-3 cell lines (Figures 4(a) and 4(b)). Knockdown of LUCAT1 apparently decreased cell proliferation (Figures 4(c) and 4(d)), migration (Figures 4(g) and 4(h)), and invasion (Figures 4(i) and 4(j)) and induced cell apoptosis (Figures 4(e) and 4(f)). However, miR-181c-5p downregulation induced obvious enhancements in cell proliferation (Figures 4(c) and 4(d)), migration (Figures 4(g) and 4(h)), and invasion (Figures 4(i) and 4(j)) and induced a reduction in cell apoptosis (Figures 4(e) and 4(f)), as well as neutralized the above effects mediated by LUCAT1 (Figures 4(c)–4(j)). In addition, downregulation of miR-181c-5p abolished si-LUCAT1-mediated downregulation of Bcl2 and N-cadherin and upregulation of Bax and E-cadherin in both T24 and UM-UC-3 cell lines (Figures 5(a) and 5(b)). These results demonstrate that downregulation of LUCAT1 inhibited the progression of bladder cancer via targeting miR-181c-5p.Figure 4
LUCAT1 downregulation repressed cell proliferation, migration, and invasion and induced cell apoptosis in a miR-181c-5p-dependent manner. (a, b) The expression of LUCAT1 was detected by RT-PCR assay after T24 and UM-UC-3 cells were transfected with si-LUCAT1 or si-NC (n=3, ∗P<0.05). T24 and UM-UC-3 cells were transiently transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p and thereby submitted to the following assays. (c, d) CCK-8 assay was used to detect cell proliferation ability. (e, f) Flow cytometry assay was carried out to assess cell apoptosis populations. (g, h) Wound healing assay was used to detect cell migration capacity. (i, j) Transwell chambers coated with Matrigel were applied to detect cell invasion (n=3; ∗P<0.05, compared with si-NC+inhibitor-NC group; #P<0.05, compared with si-LUCAT1+inhibitor-NC group).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 5
LUCAT1 downregulation decreased the expression of Bcl2 and N-cadherin and increased the Bax and E-cadherin expression via targeting miR-181c-5p in bladder cancer cells. (a, b) After T24 and UM-UC-3 cells were transiently transfected with si-NC+inhibitor-NC, si-LUCAT1+inhibitor-NC, si-LUCAT1+inhibitor-miR-181c-5p, and si-NC+inhibitor-miR-181c-5p, western blotting technology was applied to test the protein expressions of Bcl2, Bax, N-cadherin, and E-cadherin (n=3, ∗P<0.05, compared with si-NC+inhibitor-NC group; #P<0.05, compared with si-LUCAT1+inhibitor-NC group).
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## 3.4. Downregulation of LUCAT1 Decreases KRAS Expression via Targeting miR-181c-5p in Bladder Cancer

To further reveal the underlying mechanism of LUCAT1 in the progression of bladder cancer, we then applied TargetScan to predict the target of miR-181c-5p. The results showed that KRAS was a predicted target of miR-181c-5p, and the putative binding sites are shown in Figure6(a). The luciferase gene reporter assay showed that overexpression of miR-181c-5p significantly reduced the luciferase activity, whereas this effect was abrogated when the binding sites were mutated (Figures 6(b) and 6(c)). In addition, downregulation of LUCAT1 significantly reduced KRAS expression at both mRNA and protein levels, whereas this effect was obviously abolished when miR-181c-5p was downregulated (Figures 6(d) and 6(e)). These results demonstrate that the downstream gene KRAS might be involved in LUCAT1/miR-181c-5p-mediated bladder cancer progression.Figure 6
KRAS was a target of miR-181c-5p in bladder cancer. (a) The wild type (WT) or mutated type (MUT) of binding sites between KRAS and miR-181c-5p. (b, c) Luciferase gene reporter experiment was used to evaluate the relationship between miR-181c-5p and KRAS in T24 and UM-UC-3 cell lines (n=3, ∗P<0.05). (d, e) The mRNA and protein expression levels of KRAS in differently transfected T24 and UM-UC-3 cells (n=3, ∗P<0.05, compared with si-NC+inhibitor-NC group; #P<0.05, compared with si-LUCAT1+inhibitor-NC group).
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## 4. Discussion

Although great efforts were made in the investigation of bladder cancer progression, it still remains as a serious burden in worldwide due to its high recurrence rate and metastasis [16]. In recent years, lncRNAs have attracted more and more attention for their potential to be served as novel biomarkers due to their critical regulation roles in carcinogenesis [8, 17]. Since the roles and mechanism underlying LUCAT1 in the occurrence and development of bladder cancer remain unknown, we conducted this study. The major finding of the current study was that LUCAT1 was overexpressed in bladder cancer cell lines and tissues, which was positively associated with the unfavorable prognosis. Further investigation revealed that downregulation of LUCAT1 significantly suppressed bladder cancer growth, migration, and invasion and induced cell apoptosis via interacting with miR-181c-5p.LUCAT1 is located on chromosome 5 and was first discovered in the airway epithelium of cigarette smokers [18]. Up to now, LUCAT1 has been shown to be implicated in the carcinogenesis of several kinds of cancers. LUCAT1 was first found to be overexpressed in non-small cancer lung cancer and closely associated with the poor prognosis; in addition, LUCAT1 downregulation significantly suppressed cell proliferation in vitro and in vivo via modulating p21 and p27 expressions [19]. Gao et al. [20] found that LUCAT1 was substantially overexpressed in glioma tissues and cells lines, and suppression of LUCAT1 obviously inhibited glioma cell proliferation and invasion capacities. Yoon et al. [21] reported that the expression level of LUCAT1 was increased in esophageal squamous cell carcinoma (ESCC), where it functioned as an oncogene to promote the progression of ESCC via upregulating the DNA methyltransferase 1 (DNMT1) expression. Liu et al. [22] found that LUCAT1 also showed a high expression pattern in prostate cancer tissues, and overexpression of LUCAT1 caused a significant enhancement in cell migration and invasion abilities via reducing KISS1 expression. The elevated expression of LUCAT1 was reported to be associated with tumor metastasis and the clinical stage, as well as low survival rate in ovarian cancer, and LUCAT1 repression significantly inhibited ovarian cancer cell proliferation, migration, and invasion, whereas it increased the cell apoptotic rate [23]. In addition, the increased expression of LUCAT1 induced by SP1 caused significant increases in cell proliferation, migration, and invasion via targeting miR-181a in cervical cancer [24]. All of the above findings illustrate that LUCAT1 presents a high expression profile and serves as an oncogene in cancers. Congruently, we observed that LUCAT1 was upregulated in bladder cancer tissues and cells, and the Kaplan-Meier analysis showed that patients with LUCAT1 high expression presented with a lower overall survival than patients with LUCAT1 low expression. In addition, we observed that downregulation of LUCAT1 with siRNA transfection significantly inhibited the in vitro proliferation, migration, and invasion and promoted the apoptosis rate of T24 and UM-UC-3 cells, indicating that LUCAT1 serves as an oncogene in bladder cancer.Using bioinformatics methods, we found that miR-181c-5p was a predicted target of LUCAT1, which was then verified by the luciferase gene reporter assay. In addition, our results demonstrated that miR-181c-5p was lowly expressed in bladder cancer tissues and cell lines, and depletion of miR-181c-5p significantly facilitated cell growth, migration, and invasion abilities, as well as reduced cell apoptosis in both UM-UC-3 and T24 cell lines, illustrating that miR-181c-5p functions as a tumor suppressor in bladder cancer. Moreover, miR-181c-5p downregulation abolished LUCAT1 downregulation roles in inhibiting bladder cancer migration and invasion, indicating that knockdown of LUCAT1 represses bladder cancer progression via targeting miR-181c-5p. Overexpression of miR-181c-5p has been demonstrated to weaken the enzalutamide resistance of castration-resistant prostate cancer cells, as well as repress cell invasion and tumorigenesis, suggesting that miR-181c-5p functions as a tumor suppressor in prostate cancer [25]. However, Sanchez-Diaz et al. [26] reported that inhibition of miR-181c-5p using antisense oligonucleotides caused the depletion of the cancer stem cell fraction and impairment of sphere formation in pediatric cancers, suggesting that miR-181c-5p might serve as an oncogene in pediatric cancers. The different functions of miR-181c-5p in cancers might be caused by the different tumor microenvironments, which should be elucidated in further studies.To further reveal the detailed mechanisms of LUCAT1 in bladder cancer metastasis, we also used bioinformatics methods and luciferase gene reporter assay to find that KRAS was a direct target of miR-181c-5p and under a positive regulation of LUCAT1 in bladder cancer. KRAS is identified to be an oncogene, which encodes a small GTPase that sits at the plasma membrane and transduces signals from activated growth factor receptors to multiple downstream signaling molecules. The activation of KRAS is strongly implicated in many steps of carcinogenesis, such as tumor cell survival, apoptosis, metastasis, metabolism, and drug resistance [27, 28]. However, we did not explore whether KRAS plays a role in LUCAT1/miR-181c-5p-invloved bladder cancer progression. The exact details of the LUCAT1/miR-181c-5p/KRAS axis in bladder cancer progression remain to be further determined.In conclusion, this study reveals a high expression pattern of LUCAT1 and a low expression pattern of miR-181c-5p in bladder cancer; knockdown of LUCAT1 inhibits the migration and invasion of bladder cancer cells in a miR-181c-5p-dependent manner. This study provides new marker and therapeutic target for bladder cancer diagnosis and treatment.



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*Source: 4817608-2020-11-17.xml*