LncRNA PVT1 accelerates malignant phenotypes of bladder cancer cells by modulating miR-194-5p/BCLAF1 axis as a ceRNA

Background: Numerous studies proved that long non-coding RNA (lncRNA) is involved in the progression of multifarious diseases, especially in some carcinomas. As a potential tumor biomarker, plasmacytoma variant translocation 1 gene (PVT1) is involved in the development and progression of multifarious cancers. Nevertheless, the intrinsic and concrete molecular mechanism of PVT1 in bladder cancer still remained unclear, which is also the dilemma faced in many non-coding RNA studies. Results: Our research revealed that PVT1 was significantly higher expression in bladder carcinoma specimens and cell lines. Further experiments indicated that knockdown or overexpression of PVT1 restrained or promoted the malignant phenotype and WNT/β-catenin signaling in bladder cancer cells. Meanwhile miR-194-5p was in contrast and miR-194-5p could partially reverse the function of PVT1 in malignant bladder tumor cells. As a microRNA sponge, PVT1 actively promotes the expression of b-cells lymphoma-2-associated transcription factor 1 (BCLAF1) to sponge miR-194-5p and subsequently increases malignant phenotypes of bladder cancer cells. Therefore, it performs a carcinogenic effect and miR-194-5p as the opposite function, and serves as an antioncogene in the bladder carcinomas pathogenesis. Conclusion: PVT1-miR-194-5p-BCLAF1 axis is involved in the malignant progression and development of bladder carcinomas. Experiments revealed that PVT1 has a significant regulatory effect on bladder cancer (BC) and can be used as a clinical diagnostic marker and a therapeutic molecular marker for patients suffering from BC. Methods: In urothelial bladder carcinoma specimens and cell lines, the relative expression levels of PVT1 and miR-194-5p were detected by quantitative reverse transcription PCR (RT-qPCR). Through experiments such as loss-function and over-expression, the biological effects of PVT1 and miR-194-5p on the proliferation, migration, apoptosis and tumorigenicity were explored in bladder cancer cells. Co-immunoprecipitation, proteomics experiments, dual luciferase reporter gene analysis, western blot and other methods were adopted to investigate the PVT1 potential mechanism in bladder carcinomas.


INTRODUCTION
As the premier malignant neoplasm in the urinary system, BC is a highly pathogenic malignant tumor that puts a heavy financial strain on patients and society [1][2][3][4]. BCs frequently recurs and even advanced malignant tumors often leads to the death of BC patients, so far it is difficult to be completely cured. Therefore, it is necessary to detect the potential mechanism of BC, so as to clarify its biological function in the occurrence and development of BC, and thus it can be transformed into a reasonable treatment strategy [5][6][7].
At present, the treatment for BC is still limited. Most BC pathological phenotypes are urothelial carcinomas, and the relatively ideal prognosis and high morbidity non-muscle-invasive BCs account for the majority, inversely, poor prognosis is the muscle-invasive BC [2,[8][9][10]. The non-invasive high-risk BC is used for transurethral cystectomy (TURBT), and the radical cystectomy is mainly applicable to muscular invasive high-risk bladder carcinomas, and the advanced tumors patients could mainly adopt targeted therapy or other conservative treatment measures [11][12][13]. However, the treatment effect of advanced tumor patients is still not ideal and the survival rate is lower. Therefore, there is an urgent need for alternative therapies and the development of more effective and safer therapies for bladder carcinoma is inevitable [5,[14][15][16]. The key molecular mechanisms of BC is thoroughly explored to conquer the bottleneck of clinical treatment in the future.
MicroRNAs (miRNAs) are 18-25 nucleotides' noncoding RNAs to regulate tumorigenesis and progression, which have been confirmed in various tumors [8,28,36]. MiRNAs are quite conserved in species evolution, which are found in plants, animals and fungi, and are only expressed in specific tissues and developmental stages. The tissue specificity and timing of microRNAs determine the functional specificity of tissues and cells, indicating that microRNAs act as various roles in term of the cell growth and development regulation [41,[50][51][52][53][54]. MiR-194-5p was reported in numerous tumors, including kidney carcinoma, colorectal cancer and glioma [55][56][57][58][59]. Nevertheless, the potential mechanisms of miR-194-5p in the malignant BC behaviors are thoroughly unknown. In our experiment, as a negative regulatory, miR-194-5p participated in the BC evolution.
A lot of literatures manifested lncRNAs function as miRNAs sponges [26,28,[35][36][37]56]. We reported the mutual correlation between PVT1 and miR-194-5p as miRNAs sponges in the bladder carcinomas. Our research could help us deeply broaden our horizons about the lncRNA-miRNA sponges' expression pattern and mutual roles of PVT1 and miR-194-5p in the BC.
In our study, we discovered that PVT1 is up-regulated in BC tissues and cells and miR-194-5p is converse. The relative expression levels of PVT1 and miR-194-5p are closely correlated with histological grade and TNM stage. In our study, we elucidated that PVT1 facilitated the progression of BC and miR-194-5p is converse.
Furthermore, it was found that PVT1 functions as a ceRNA-dependent manner to sponge miR-194-5p to tightly promote BCLAF1 expression. Meanwhile, our deepen study suggests that PVT1 acts as a significant tumor regulator and PVT1-miR-194-5p-BCLAF1 axis participates in BC progression, which emerges its possibly clinical application of BC as a meritoriously clinical diagnosis and treatment strategies. Maybe, our experiments can offer a novel direction for the exploration of BC therapeutic target and provide a more valid research method to detect tumor progression and treatment strategies for the ultimate aim of the precision cancer medicine.

Up-regulated expression of PVT1 and downregulated expression of miR-194-5p in BCs
The relative expression level of PVT1 and miR-194-5p was measured through RT-qPCR in BC samples. Compared to para-carcinoma tissues, the relative expression level of PVT1 was significantly increased about 2.28 times (43 of 70) of BC samples (P<0.001) ( Figure 1A, 1B), and the relative expression level of miR-194-5p was significantly decreased about 42.92% (46 of 70) of BC samples (P=0.023) ( Figure  1C, 1D). Compared to SV-HUC-1, the relative expression level of PVT1 was up-regulated in both BC cells, T24 about 3.76 times (P < 0.001) and UM-UC-3 about 2.84 times (P=0.004) ( Figure 1E), and the relative expression level of miR-194-5p was decreased in both BC cells, T24 about 44.16% (P=0.003) and UM-UC-3 about 63.77% (P = 0.001) ( Figure 1F). PVT1 expression levels were statistically positively correlated with miR-194-5p expression levels in BC samples and cells ( Figure 1G, 1H). As following in Table 1, the up-regulated expression of PVT1 was closely associated with histological grade (P = 0.014), and TNM stage (P< 0.001) in bladder carcinomas, the low expression of miR-194-5p was significantly related to histological grade (P=0.006) and TNM stage (P<0.001). But sex, age, tumor size, and lymph node metastasis were no obvious correlation with the relative expression levels of PVT1 and miR-194-5p. Our results manifested that PVT1 could act as the oncogene and miR-194-5p could act as the antioncogene in BCs.
EdU was elucidated cell proliferation as well. Compared with control group, EdU positive T24 and UM-UC-3 cells in si-PVT1 group were reduced and pcDNA3.1-PVT1 group was reversed after transfection in SV-HUC-1 cell line.
Our study manifested that the knockdown of PVT1 restrained bladder cell lines proliferation and overexpression PVT1 facilitated bladder cell lines proliferation.
Cell migration was detected after the transfection of siRNA and plasmids by scratch assay. Scratch assay that revealed that the ratio of the relative migration was decreased about 41.29% in T24 (P = 0.001) ( Figure 3A) and decreased about 42.18% in UM-UC-3 (P = 0.004) ( Figure 3B) in si-PVT1 group. The ratio of the relative migration in the pcDNA3.1-PVT1 group was upregulated about 2.032 times in SV-HUC-1 (P<0.001) ( Figure 3C).
Our results concluded that the knockdown of PVT1 restrained bladder cell lines migration. Overexpression PVT1 expression facilitated bladder cell lines migration.
Cell apoptosis was measured with flow cytometry assay. Compared with control groups, the ratios of apoptosis were up-regulated about 7.91 times in T24 (P<0.001) and increased about 4.99 times in UM-UC-3 (P=0.011) ( Figure 3D, 3E) after transfection si-PVT1.
Compared with control groups, the ratios of apoptosis were dramatically reduced about 0.332 times in SV-HUC-1 (P=0.003) ( Figure 3F) after the transfection of pcDNA3.1-PVT1.
In brief, the knockdown of PVT1 facilitated bladder cell lines apoptosis and overexpression PVT1 restrained bladder cell lines apoptosis. after transfection miR-194-5p inhibitor. Ultimately, over-expression of miR-194-5p accelerated BC cells apoptosis and down-regulation of mi-194-5p restrained BC cells apoptosis.
We used bioinformatics databases to predict BCLAF1 with miR-194-5p possible mutual binding sites, which was shown as Figure 6D. The predicted binding sites and binding effects were manifested through luciferase reporter assay.
We identified that protein is associated with BCLAF1 through immunoprecipitation (IP/co-IP) assays. CDC20 was detected to be a BCLAF1-binding protein in the bladder cell lines ( Figure 6E). At the same time, IP pyrolysis products are used for proteomics detection (Figures 6Ka-f), which was furthermore proved that BCLAF1 bound multiple functional proteins in BC cells, and KEGG, GO and other analysis were performed (Also as shown as the GO.anno,KEGG.anno and Subcellular.localization_anno), and we further revealed that BCLAF1 could interact with CDC20.
Our study proved that the relative expression level of PVT1 was closely related to BCLAF1 expression and down-regulation of PVT1 could reduce BCLAF1 expression in BC cells. Our further experiments confirmed that restrained PVT1 referred to the WNT signaling in BC cells ( Figure 6F). Western blotting was used to detect the WNT signaling that is associated downstream genes expression. Knockdown of PVT1 decreased CD44, MET, cyclinD1, CDC20, BCLAF1 and β-catenin expression in BC cells. Therefore, the down-regulation of PVT1 inhibited the progression of BC cells through inhibition of the Wnt/β-catenin signaling pathway because β-catenin was the vital protein in this signaling pathway.
We must confirm whether PVT1 increased BCLAF1 expression via miR-194-5p-dependent manner in BC cells. Our studies proved that over-expression of miR-194-5p reduced BCLAF1 expression and miR-194-5p inhibition up-regulated expression of BCLAF1 in BC cells ( Figure 6G). Our experiments indicated that PVT1 could closely promote BCLAF1 expression via sponging miR-194-5p in BC cells.
The knockdown of PVT1 caused the reduction of MMP-2 and MMP-9 ( Figure 6H). Thus, we concluded that the knockdown of PVT1 suppressed the migration of BC cell lines, which is in accordance with that after transfection miR-194-5p inhibition and mimics. Overexpression miR-194-5p decreased MPP2 and MPP9 expression and the knockdown of miR-194-5p increased MPP2 and MPP9 expression in BC cells ( Figure 6I). Therefore, we concluded that PVT1 and miR-194-5p referred to the migration of BC cell lines.

PVT1 accelerated malignant phenotypes of BC cells via BCLAF1-dependent manner.
We should confirm whether PVT1 accelerated malignant phenotypes via BCLAF1-dependent manner in BC cells. Further experiments proved that knockdown of BCLAF1 dramatically reversed the promotion of BCLAF1 expression induced by over-expression of PVT1 in BC cells ( Figure 6J). Moreover, the knockdown of BCLAF1 reversed BC cells proliferation promotion ( Figure 7A-7D) induced by over-expression of PVT1. And BCLAF1 knockdown could reverse BC cells migration ( Figure 7E, 7F) induced by over-expression of PVT1. Meanwhile, knockdown of BCLAF1 could notably reverse BC cells apoptosis suppression ( Figure  7G, 7H) induced by over-expression of PVT1. Our studies proved that PVT1 could accelerate malignant phenotypes of BC cells via BCLAF1-dependent manner.

Knockdown of PVT1 suppressed BC cells tumorigenicity
The generation of xenograft was used to confirm whether PVT1 promoted the tumorigenicity of BC cells. Our experiments proved that the knockdown of PVT1 could restrain the tumorigenicity of BCs in vivo ( Figure 9A-9F). Solid tumors that were obtained from mice were displayed in Figure 9A. It was confirmed that the relative expression of PVT1 was reduced in LV-shPVT1 groups compared with LV-shNC group of BC cells in vivo ( Figure 9B). Tumor weight was lessened in LV-shPVT1 groups than LV-shNC groups in vivo ( Figure 9C). Tumor growth was slower in LV-shPVT1 groups than LV-shNC groups in vivo ( Figure 9D). Our experiments revealed that the knockdown of PVT1 could reduce CD44, MET, cyclinD1, CDC20, BCLAF1 and β-catenin expression of BC cells in vivo ( Figure  9E). IHC experiments proved that the knockdown of PVT1 restrained Ki-67 and BCLAF1 expression ( Figure  9F) of BC cells in vivo. Our experiments proved that PVT1 facilitated the tumorigenicity of BC via upregulating BCLAF1.
As simulated diagram shown as Figure 10, PVT1 acts as a microRNA sponge that actively promotes the expression of BCLAF1 to sponge miR-194-5p and
LncRNAs also refer to tumor proliferation, apoptosis, invasion and migration [40,42,46,56,59], which broaden our knowledge about the biological behavior of diseases especially in bladder carcinomas. Meanwhile, miRNAs are is a kind of classic and potential biomarkers and key regulators in some hot spots included tumor, and play similar roles, including  H). The apoptotic cells were measured after co-transfection with si-C+NC, si-BCLAF1+miR-194-5p inhibitor or mimics by flow cytometry analysis (I, J). (*P < 0.05, **P < 0.01). AGING oncogenes, tumor suppressor genes in tumors, regulating the proliferation, migration, apoptosis and other important biological behaviors of tumor cells [17,18,20,23]. Various literatures concluded that lncRNAs were closely related with miRNAs, and our study further confirmed the mutual relationship with them [26, 28, 35-37, 57, 59].
This report elucidated the mutual function between PVT1 and miR-194-5p in bladder carcinomas. Our study revealed that PVT1 sponging miR-194-5p could be the biomarker and therapeutic target for the diagnosis and treatment in the bladder carcinomas.
PVT1 expression was significantly up-regulated in BC samples and cell lines. Most importantly, PVT1 expression was closely related to histological grade and TNM stage in the bladder carcinomas. Our further experiments proved that the down-regulation of PVT1 expression could restrain BC cells proliferation or migration and up-regulate apoptosis and so on. The overexpression of PVT1 accelerated cell proliferation, migration and anti-apoptosis in SV-HUC-1. Our further experiments of the nude mice animals illustrated that the knockdown of PVT1 inhibited tumorigenicity and lessened tumor volume and weight in vivo.
LncRNAs could act as sponges to be saturated with miRNAs. However, these different expressions of lncRNAs and miRNAs in various cancers could lead to diverse binding effects. Bioinformatics predicted potential sites to study the mechanism of PVT1 in bladder carcinomas. MiR-194-5p was the underlying targets to be predicted and has been verified. MiR-194-5p was the up-regulation induced by knockdown of PVT1. Moreover, miR-194-5p was down-regulated in BC samples and cell lines. Histological grade and TNM stage were closely related to miR-194-5p expression. MiR-194-5p mimics restrained bladder carcinoma cell progression, however miR-194-5p inhibitor was converse. MiR-194-5p acted as the tumor suppressor gene through regulating BCLAF1 expression. In addition, the knockdown of PVT1 induced miR-194-5p over-expression and dramatically restrained the malignant behaviors of BC cells. Down-regulated miR-194-5p partially rescued the suppression induced by knockdown of PVT1. Our studies displayed that PVT1 could sponge miR-194-5p to participate in the progression of the bladder carcinomas.
As we all known, miRNAs are through the indirect role of protein regulation of life activities and the target genes are regulated by binding to the 3'UTR of specific mRNAs, inducing degradation or transcriptional suppression of the target genes. We predicted potential target genes of miR-194-5p with biological software and explored its mechanism in the bladder carcinomas.
Luciferase reporter assay proved that BCLAF1 was the common target gene of miR-194-5p and BCLAF1 played the important role in regulating the evolution of tumors. Various literatures have reported BCLAF1 roles in some carcinomas' tumorigenesis, such as hepatocellular carcinoma [60] and acute myeloid leukemias tumorigenesis [61].  In all, our studies manifested that PVT1 downregulation could up-regulate miR-194-5p expression and subsequently restrain BCLAF1 expression as the ceRNAdependent manner. Furthermore, up-regulated miR-194-5p reversed BCLAF1 expression and BCLAF1 reduction reversed the malignant BC cells phenotypes promotion induced by overexpression of PVT1. The experiments opened up a new thought for novel molecular targets of human carcinomas, particularly in the bladder carcinoma, which broadened our comprehension on lncRNA-miRNA-proteins methods in illness evolution, and provided us an orientation to ulterior explore the disease's occurrence mechanism, and provide theoretical basis for the new treatment as well. That could be transformed into more fresh and valid remedial method for BC, which could be brought more brilliant prospects for mankind to radically cure BC thus altering the current diagnostic and therapeutic bottleneck for numerous cancer patients, especially advanced BCs in the future.

CONCLUSIONS
The experiments proved that PVT1 could sponge miR-194-5p to closely promote BCLAF1 expression and subsequently accelerate the malignant BC cells' phenotypes, and therefore could act as the carcinogene in the BCs. Our experiments could provide some useful directions to further exploration on its pathogenesis of the progression and development of the BC. In conclusion, the experiments revealed that PVT1-miR-194-5p-BCLAF1 axis could play the significant roles in the progression and development of BCs. PVT1 and miR-194-5p are novel and important tumor biomarkers, and could be some underlying diagnostic biomarkers and remedial targets for malignant BC in the future.

Patient specimens
Our study included BC patients who received tumorectomy. We quick-freeze the BC specimens and paired normal peritumoral specimens in liquid nitrogen quickly after resection. Every one patient has signed the written informed consent. The first affiliated hospital of Soochow University research ethics committee approved the experiment.

Cell lines and cell culture
T24, UM-UC-3 and SV-HUC-1 were obtained from the Institute of Cell Biology, Chinese Academy of Sciences in Shanghai. T24 and UM-UC-3 cells were cultivated in DMEM (Gibco, USA). SV-HUC-1 cells were cultivated in F12 (Life technologies, USA). 1% antibiotics (100 U/ml penicillin and 100 μg /ml streptomycin sulfates) and 10% FBS were blended in DMEM and F12. The atmosphere of the incubator is at 37° C and 5% CO2.

RT-qPCR
Based on the product descriptions, the whole RNA was extracted from the specimens or the transfected cells using TRIzol reagent (Invitrogen, USA). The cDNA was synthesized from total RNA applying the Prime Script RT Reagent Kit with gDNA Eraser (Takara, Japan). The relative expression levels of PVT1 were measured by RT-qPCR with SYBR Premix Ex Taq II (Takara) on the CFX96 sequence detection system (Bio-Rad). The primer sequences were displayed in Supplementary Table 1. The endogenous controls were Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA. The relative quantification method (2 -ΔΔCt ) was adopted to calculate the expressions that were normalized to endogenous controls.

Cell proliferation assays
CCK-8 (Beyotime, Shanghai) was applied to cell proliferation based on the product descriptions. AGING Cells were incubated in a 96-well plate for 24 hours and then respectively transfected with siRNAs or plasmids in the CCK-8 assays. 0, 24, 48 and 72 h after transfection, the absorbance in each well was measured at by a microplate reader (Bio-Rad, USA).

EdU incorporation assay
EdU (Ethynyl-2-deoxyuridine) Apollo DNA in vitro kit (RIBOBIO, Guangzhou) was also used for cell proliferation that was detected by EdU incorporation assay based on the product descriptions. In a word, cells transfected siRNA or plasmid and were incubated with 100 μl of 50 μM EdU per well for 2 h at 37° C, respectively. Cells were visualized under a fluorescence digital camera system.

Cell migration assay
The cells were cultivated into the 6-well plates in the incubator. About 90% confluence was obtained before the transfection siRNA or plasma transfected cells.
Use the sterilization 200ul pet tips to generate clean lines in 6-well plates. Use digital camera system to take photos in each well quickly. A day later, the picture was taken again. The travel distance is set at 0 and 24 hours.

Flow cytometry assay
SiRNAs or plasmid vectors were respectively transfected in bladder T24, UM-UC-3 and SV-HUC-1 cells. For 48 hours after transfection, cells were collected and resuspended in fixation fluid 5 µl of Annexin V-FIFC and 10 µl of propidium iodide were added to 195 µl of cell suspension. Flow cytometry (Beckman, USA) was applied to detect cell apoptosis.

Western blot analysis
Protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking in the 5% non-fat milk and the membranes were incubated overnight for at least 16h in 4° C with the primary antibody. At room temperature for 1-2h, the membranes were then incubated with a secondary antibody and enhanced chemiluminescence ECL kit (Beyotime Biotechnology, China) was visualized. βactin was the internal standard, and the antibodies were demonstrated in Supplementary Table 2.

Dual-luciferase assays
Based on the product descriptions, dual-Luciferase Reporter Assay System (Promega, USA) was used for the Dual-luciferase reporter assays. The binding and mutant sequences were respectively cloned into pmirGLO Dual-luciferase vectors (Fubio Biological Technology Co, Suzhou, China). PVT1, BCLAF1 WT or Mut constructed and co-transfected along with miR-194-5p mimics or NC, and then transfected with Lipofectamine 3000 and incubated for 48 h. Microplate reader was applied to measure the luciferase activities.

Mouse model experiments
Our experiment was approved by Institutional Ethics Review Board of Soochow University. 5-wk old male BALB/c nude mice were divided into two groups and each group included 5 mice. LV-PVT1 and LV-NC were purchased from Genechem (Shanghai, China). 2×10 6 UM-UC-3 cells were injected into the mice dorsal flank regions. Every 5 days, tumor growth was measured. The formula, a*b 2 /2 (a represents the long diameter and b refers to the short diameter), was used to calculate tumor volume. Mice were executed after 30 days and the subcutaneous weight of each tumor was measured.

Statistical analysis
Assays were performed in triplicate at least and data were shown as mean ± standard deviation (SD) of those biological replicates or samples. SPSS 20.0 software (IBM, Chicago, IL, USA) etc. were used to analyze assays' statistical analyses. Paired samples t-test was used to analyze the relative expression of PVT1 and miR-194-5p. ANOVA was used to analyze CCK-8 assay data. The independent samples t-test was used to analyze other data. P< 0.05 was regarded as the statistically significant one.

AUTHOR CONTRIBUTIONS
Mingwei Chen and Jianquan Hou designed the study; Mingwei Chen and Rongyuan Zhang, Chunyang Chen and KeKe Ding performed experiments; Mingwei Chen, Le Lu, Xuedong Wei and Yuhua Huang involved to collect data; Mingwei Chen wrote the manuscript; Mingwei Chen,Guangbo, Zhang and Jianquan Hou reviewed the paper.

CONFLICTS OF INTEREST
There is no conflicts of interest.