CircPVT1 Promotes Bladder Cancer Progression by Acting as a ceRNA for miR-140-3p to Target TRPS1

Background: Bladder cancer (BC) is one of the most malignancy tumor in the urinary system. Therefore, further studies are needed to revealed the molecular mechanism of BC progression and development. Previous study demonstrated that the deregulation of circRNAs can regulate cell biological functions in tumorigenesis and development. However, the roles of circPVT1 in BC have not yet been revealed. Materials and methods: The expression level of circPVT1, miR-140-3p, and TRPS1 were measured by RT-PCR in BC tissues and cells; dual luciferase reporter and RIP assay showed that circRNA served as a sponge for miRNA, and miRNA could target mRNA. In vitro, effects of overexpression circPVT1 or sh-circPVT1 can regulate BC cells’ proliferation, migration, invasion were detected by CCK-8 assay, wound healing assay, and transwell assay. Results: Our research demonstrated that the expression of circPVT1 was upregulated in BC tissues and cell lines, and increased in metastatic tissues compared to that of non-metastatic tissues. CircPVT1 sponging miR-140-3p to target TRPS1 was revealed by dual luciferase reporter and RIP assay. In addition, the expression level of miR-140-3p was reduced in BC tumor tissues, and TRPS1 was signi�cantly increased in BC tumor tissues. Pearson correlation analysis showed that miR-140-3p with circPVT1 and TRPS1 as compare with miR-140-3p have a moderately negative correlation, and there was a moderately positive correlation between circPVT1 with TRPS1. Further, cytological studies found that circPVT1 enhance BC cells’ proliferation, migration, and invasion by targeting TRPS1 via miR-140-3p. Conclusion: CircPVT1 plays a tumor enhancement role in BC and that can effectively promote cell proliferation, migration, invasion and EMT by targeting the miR-140-3p/TRPS1 axis. CircPVT1 may be a novel potential treatment and diagnosis biomarker in BC.


Background
Bladder cancer(BC) is a common malignant tumor of the urinary system.Globally, there will be an estimated 549,000 new cases diagnosed and BC has caused approximately 200,000 deaths in 2018, the incidence of BC in men is more than about four times that in women. [1]Currently, surgery, radiotherapy and chemotherapy are still the main therapies for BC, Due to its high incidence, and recurrence and metastasis rates, the ve-year survival rate for patients with BC remains low. [2,3] owever, there are insu cient studies focused on early BC diagnosis and speci c biomarkers.
In our study, we demonstrate that circPVT1 is more highly expressed in BC tissues and cell lines than in the relevant controls.CircPVT1 directly targets miR-140-3p and regulate downstream the expression of TRPS1.Cytological studies found that circPVT1 regulates proliferation, migration, invasion, and (epithelial to mesenchymal transition) EMT in BC cells by targeting the miR-140-3p/TRPS1 axis.Taken together, these results suggest that circPVT1 may serves as a novel potential treatment and diagnosis biomarker in BC.

Bioinformatics analysis
We used online databases (circinteractome, starbase and circbank) to nd potential miRNA of circPVT1 and the starbase databases was used to predict the target genes of miR-140-3p.

Clinical Specimens
39 pairs of BC clinical specimens and adjacent normal tumors were collected from the First A liated Hospital of Jiamusi University.None of them had undergone chemotherapy, radiotherapy or any anticancer treatment before surgery and all of the clinical specimens were immediately ash-frozen in liquid nitrogen during surgery until RNA extraction.
2.5 CCK-8 assay BC cells proliferation were determined by CCK-8 assay (Dojindo, Japan) following the manufacturer's instructions, a density of 1×10 4 T24 and EJ cells were plated into 96-well plates.After 24h, 10 μL of CCK-8 solution was combined and incubated without light for 2h at 37℃.The absorbance was measured at a wavelength of 450 nm using the BioTek (Winooski,USA) microplate spectrophotometer.
2.6 Wound healing assay 1×10 4 cells/well T24 cells or EJ cells was seeded into 6-well plates.When the cells reached 90% con uency, the tip of a 200 μL pipette was used to scratch the cell monolayer and then fresh serous medium was used to wash the plates three times.After 24 h, the wound width was calculated using Image J software.

Transwell assay
1×10 4 cells/well T24 or EJ cells were seeded on the upper chambers with 2% serum medium and medium containing 20% serum medium were added to the lower chambers.Then, the cells were incubated at 37℃ for 24h.The migration cells number were counted to calculate the average number of migrated cells per plate.

Dual-luciferase reporter assay
Cells were cotransfected with circPVT1-miR-140-3p and TRPS1-miR-140-3p into the luciferase gene(wildtype or mutant-type), the speci c operation was carried out according to the manufacturer's protocol.After 48h, luciferase activities were calculated for each well using the Dual Luciferase Reporter Assay System (Promega).

RNA-binding protein immunoprecipitation (RIP)
RIP assay (Millipore, Billerica, MA) was carried out according to the manufacturer's instructions.The RIP lysate was obtained and centrifuged at 14,000 rpm for 10 minutes.Add 100 µL of the supernatant to the RIP immunoprecipitation buffer containing the magnetic bead-antibody complex, then the RNA was puri ed and obtained from TRIzol.Finally, analysis of immunoprecipitated RNA by RT-PCR.

Tumor xenografts
5×10 6 cells/well T24 were stably transfected with sh-circPVT1 plasmids or circPVT1 overexpression plasmids or negative control vector were subcutaneously injected into the upper back of nude mice.
Tumor weight was detected every 7 days.The volume of tumors were calculated using the following formula: V = 0.5 × L × W 2 .one month later, all animals were scari ed, and the tumor volume were recorded.The laboratory animals were approved by the medical laboratory animal ethics committee of Jiamusi University.Instructive notions with respect to caring for laboratory animals (which is released by the Ministry of Science and Technology of the People's Republic of China in September 30th, 2006.) were followed for the welfare of the animals.

Western blot assay
Proteins were extracted from cells using RIPA with proteinase inhibitors (Sigma-Aldrich, USA) and the concentrations of proteins were measured using BCA Protein Assay kits (Thermo Scienti c, MA).After separation by 10% SDS/PAGE, proteins were blocked with 10% non-fat milk for 1 h and immunoblotted with the primary antiodies at 4 °C overnight.Proteins were incubated with secondary antibodies for 1 hr.After TBST washing 3 times and protein levels were measured using the chemiluminescence image system (Bio-Rad, USA).

Statistic analysis
All data were analyzed by SPSS 20.0, P < 0.05 indicated statistical signi cant ndings.Data is represented as means ± standard, and the differences of the two groups' data were analyzed by Student's t-test; Pearson correlation analysis was used to evaluate the correlation among circPVT1, miR-140-3p and TRPS1.

Results
3.1 circPVT1 is up-regulated in BC patients and cell lines RT-PCR results showed that the expression of circPVT1 was higher in BC tissues compared to that in adjacent normal tumors(P<0.001,Fig. 1A), and was signi cantly increased in metastatic tissues compared to that of non-metastatic tissues(P<0.001,Fig. 1B).Besides, the expression of circPVT1 was signi cantly increased in T24, UMUC3, EJ, 5637 and J82 compared with SV-HUC-1(Fig.1C).The linear mRNA PVT1 expression was signi cantly decreased, but circPVT1 expression did not have no signi cant change after the RNase R treatment(Fig.1D).Then convergent primers and divergent primers were used to amplify linear or circRNA PVT1 by cDNA and genomic DNA (gDNA).RT-PCR results showed that divergent primers could amplify by cDNA but could not amplify by gDNA(Fig.1E).

circPVT1 regulates BC cells proliferation, migration, and invasion
CircPVT1 over-expression plasmid could signi cantly increased the expression of circPVT1, and sh-circPVT1 could signi cantly decreased the expression of circPVT1 in EJ and T24 cells(Fig.2A).However, there was no signi cant change PVT1 mRNA expression.CCK8 assay suggested that sh-circPVT1 induced the inhibition of cell proliferation and circPVT1 overexpression could promoted cell proliferation in EJ and T24 cells (Fig. 2B).Wound healing assay and transwell assay revealed that circPVT1 knockdown could suppressed invasion and migration, and circPVT1 overexpression could reversed the function of EJ and T24 cells,(Fig.2C, D, E, F).BALB/c nude mice results showed that mice injected with T24 cells circPVT1 knockdown had smaller average volume than the control groups, whereas overexpressing circPVT1 signi cantly increased tumor xenografts(Fig.2G, H).
We used starbase V3.0 databases (http://starbase.sysu.edu.cn/) to predict the target genes of miR-140-3p.Dual luciferase reporter assay con rmed that miR-140-3p considerably decreased the luciferase activity in the TRPS1-WT group but not in the TRPS1-MUT group when compared with the control group(Fig.3F, G).Furthermore, the expression of TRPS1 transfected with circPVT1 over-expression plasmid was up-regulated, and The expression of TRPS1 was down-regulated cotransfected with miR-140-3p mimics in EJ and T24 cells, miR-140-3p mimics can reverse TRPS1 overexpression function by circPVT1 (Fig. 3H).RT-PCR revealed that the expression of miR-140-3p was reduced and TRPS1 was signi cantly increasedin BC tumor tissues compared with their controls (Fig. 3I, J).

circPVT1 inhibits bladder cells proliferation, migration, invasion and EMT by targeting TRPS1 via miR-140-3p
The study also investigated whether or not circPVT1 directly target TRPS1 via miR-140-3p to induce EMT in BC cells.The results showed miR-140-3p inhibition could reverse the function of BC cells' proliferation, migration, and invasive by sh-circPVT1 (Fig. 4A,B,C), western blot results showed that the expression of TRPS1 protein had the similar results in BC cells.Furthermore, E-cadherin expression was signi cantly decreased, N-cadherin and Vimentin were increased by sh-circPVT1 and sh-TRPS1, and miR-140-3p inhibition could signi cantly decreased E-cadherin and increased N-cadherin, Vimentin expression(Fig.4D), and showed that miR-140-3p inhibition could neutralize the expression of EMT biomarker by sh-circPVT1 in BC cells(Fig.4D, E, F).

Discussion
BC is a heterogeneous disease, making it more di cult to study its diagnosis, treatment and pathogenesis.The overall e cacy of existing BC treatment options is limited, [15] and the pathogenesis of BC remains unclear.Studies have reported that circular RNA is involved in the pathogenesis of BC. [16][17][18] In our research, we found that circPVT1 expression was higher in BC tissues and cell lines and was signi cantly increased in metastatic tissues compared with non-metastatic tissues.
It is widely accepted that circRNA can binding to miRNA and miRNA can target mRNA were widely accepted.In gastric cancer, circSPECC1 regulates the growth and invasion by targeting miR-526b and the downstream KDM4A/YAP1 pathway. [19]Non-small cell lung cancer progression is regulated by circ_0020123 by sponging miR-488-3p. [20]It has been shown that circRNA can affect the migration, invasion and EMT in various tumors, including BC. [21][22][23][24][25] Dual luciferase reporter and RIP assay results showed that circPVT1 could sponge miR-140-3p, and that miR-140-3p could target TRPS1.Further, Cytological studies showed that the lower circPVT1 expression can effectively inhibit cell proliferation, migration, invasion, and the EMT in BC cells by targeting TRPS1 via miR-140-3p.
Studies have reported that EMT is the driving force for cancer cells metastasis, the EMT is an important mechanism in the early stage of cancer metastasis, to study the molecular mechanism of EMT is the key to improve tumor diagnosis and treatment. [26,27] he decreased expression of E-cadherin and Vimentin, N-cadherin and other proteins have increased which have been considered to be the most signi cant feature of EMT.The changes of these EMT markers have enabled cancer cells to obtain features that promote migration and invasion. [28]Similarly,our results reveal that the over-expression of circPVT1 can regulate the E-cadherin/N-cadherin pathway to inhibit the EMT in BC cells.

Conclusion
In conclusion, this study demonstrated that circPVT1 was more highly expression in BC tissues and cell lines, and circPVT1 act as a sponge for miR-140-3p to regulate TRPS1.Moreover, cytological studies found that circPVT1 regulates cell proliferation, migration, invasion, and EMT by targeting TRPS1 via miR-140-3p in BC cells.Our results suggest that circPVT1 plays a tumor enhancement role in BC and that can effectively promote cell proliferation, migration, invasion and EMT by targeting the miR-140-3p/TRPS1 axis.circPVT1 may be a novel potential treatment and diagnosis biomarker in BC.

Declarations Ethics approval and consent to participate
This study was approved by the Ethics Committee of Jiamusi University.All BC patients provided written informed consent and the study abided by the right to privacy of human rights subjects.consent for publication Not applicable.Figure 2 circPVT1 regulates BC cells proliferation, migration, invasion A, RT-PCR assay showed circPVT1 overexpression plasmid was signi cantly increased the expression of circPVT1, and sh-circPVT1 could signi cantly decreased the expression of circPVT1 in EJ and T24 cells.B, CCK8 assay showed that circPVT1 overexpression induced to promoted cell proliferation and sh-circPVT1 could inhibition of cell proliferation in EJ and T24 cells C,D,E,F, Wound healing, transwell assays showed that over-expression of circPVT1 could signi cantly increased invasion, migration and circPVT1 knockdown signi cantly suppressed migration, invasion in EJ and T24 cells.G,H, BALB/c nude mice injected with T24 cells sh-circPVT1 had smaller volume and overexpressing circPVT1 could promoted tumor Xenografts.Data represent mean ± SD. *P < 0.05 compare with negative control.

Figures
CircPVT1 acts as a sponge for miR-140-3p, and TRPS1 is a direct target of miR-140-3p in BC cell A, bioinformatic analysis to search for miR-140-3p interact with circPVT1-MUT or circPVT1-WT.B, 16 miRNA mimics were co-transfected with the circPVT1 vector into T24 cells, the line means reduced at least half of the luciferase reporter activities.C, RNA pull-down assay for the luciferase activity of circPVT1-MUT or circPVT1-WT in T24cells co-transfected with 6 miRNA mimics.D, RIP assay for the amount of circPVT1 and miR-140-3p in T24 cells transfected with circPVT1 overexpression or sh-circPVT1 or negative control.E, qRT-PCR analysis of expression levels of miR-140-3p in EJ and T24 cells transfected with circPVT1 overexpression or sh-circPVT1 or negative control.F, bioinformatic analysis to search for miR-140-3p interact with TRPS1-MUT or TRPS1-WT.G, Luciferase reporter assay for the luciferase activity of TRPS1-MUT or TRPS1-WT in T24 cells co-transfected with miR-140-3p.H, RT-PCR analysis of the expression levels of TRPS1 transfected with sh-circPVT1 plasmid was down-regulated and circPVT1 over-expression was up-regulated compare with their control in EJ and T24 cells , the expression of TRPS1 transfected with miR-140-3p mimics was down-regulated in EJ and T24 cells compare with their control.I, QRT-PCR analysis of the expression levels of miR-140-3p in BC tissues compared with normal tissues.J, The expression levels of TRPS1 in BC tumor tissues compared with adjacent normal tissues.K, Pearson correlation was used for correlation analysis between circPVT1, miR-140-3p and TRPS1 in BC patients.