The circular rna circfarsa sponges microrna 330 5p in tumor cells with bladder cancer phenotype

(2022) 22:373 Fang et al BMC Cancer https://doi.org/10.1186/s12885-022-09467-7 Open Access RESEARCH The circular RNA circFARSA sponges microRNA‑330‑5p in tumor cells with bladder cancer phenotype Chen Fang†, Xin Huang†, Jun Dai, Wei He, Le Xu and Fukang Sun*  Abstract  Background:  Circular RNAs (circRNAs) modulate gene expression in various malignancies However, their roles in the occurrence of bladder cancer (BC) and their underlying mechanisms of action are currently unclear Methods:  We measured levels of the circRNA phenylalanyl-tRNA synthetase subunit alpha (circFARSA) and target microRNAs (miRNAs/miRs) in BC tissues and cell lines using quantitative polymerase chain reactions The functions of circFARSA in tumor formation were examined in mice with BC xenografts in vivo and in BC cells via determination of their proliferation, activity, apoptosis, metastasis, and invasion in vitro using cell counting kit-8 assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry, western blotting, Transwell assays, and cell wound healing assays Interactions between miR-330 and circFARSA were predicted and confirmed by bioinformatic processing and dual-luciferase reporter gene assays, respectively Expression profiles of miR-330 targets in BC cells were assessed via western blotting Results:  circFARSA expression was markedly upregulated in BC tissues and cell lines compared with that in normal bladder samples Silencing circFARSA expression decreased BC cell proliferation, invasion, and migration but induced their apoptosis in vitro Downregulating circFARSA expression slowed tumor growth in vivo and directly sponged miR-330 and inhibited its function in BC cells in vitro Inhibiting miR-330 expression abolished the regulatory effects of circFARSA silencing on the tumor phenotypes of BC cells Conclusions:  circFARSA expression is upregulated and exerts oncogenic functions in BC by sponging miR-330 Keywords:  Bladder cancer, Tumor phenotype, circFARSA, miR-330, Proliferation, Invasion Background Bladder cancer (BC) is a prevalent malignant tumor worldwide, affecting ~430,000 individuals in 2012 [1] Among all diagnosed BC tumors, ~ 75% not invade muscle, 70% are likely to relapse, and 25% progress to muscle invasion [2] These features result in low 5-year survival rates for patients Thus, the fundamental *Correspondence: sfkang66@163.com † Chen Fang and Xin Huang contributed equally to this work and should be considered as equal firstcoauthors Department of Urology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No 197, Ruijing 2nd Road, Shanghai 200025, China processes underlying the occurrence, development, and metastasis of BC should be elucidated Non-coding RNAs (ncRNAs) are functional RNAs that not encode proteins and are abundant in living organisms They play crucial roles in various biological processes in BC [3] For instance, microRNAs (miRNAs/ miRs) regulate the metastasis, invasion, and chemical sensitivity of BC [4, 5] Long ncRNAs also significantly affect tumorigenesis, growth, apoptosis, and metastasis in BC [6–8], and roles of circular RNAs (circRNAs) in cancer have recently been highlighted © The Author(s) 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/ The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Fang et al BMC Cancer (2022) 22:373 circRNAs lack 5’-3’ polarity and polyadenylated tails, and form  covalently closed continuous  loops [9] They are highly conserved in various species and their expression is frequently tissue- and developmental stage-specific [10] Moreover, circRNAs are significantly enriched in numerous biological and pathological processes, such as proliferation, migration, invasion, metabolism, cell cycle progression, and carcinomatous changes [11, 12] circRNAs function as potential biomarkers of hepatocarcinoma [13], lung carcinoma [14], breast carcinoma [15], colorectal cancer [16] and stomach cancer, [17] However, their specific role in BC remans obscure Levels of the circRNA phenylalanyl-tRNA synthetase subunit alpha (circFARSA), a novel plasma circular RNA derived from exons 5–7 of the FARSA gene, are notably increased in different types of cancer [18] circFARSA binds to miR-330-5p (miR-330) and miR-336, sponges miRNA in colorectal cancer [19], boosts oncogene fatty acid synthase, and promotes A549 cell migration and invasion [18] Levels of circFARSA are increased in patients with non-small cell lung cancer (NSCLC); however, its involvement in bladder tumorigenesis remains obscure We used microarrays and quantitative polymerase chain reaction (qPCR) to compare the ectopic levels of circFARSA between normal bladder and BC tissues and cell lines We also examined the effects of circFARSA silencing on BC cell proliferation and invasion Our results confirmed that crosstalk across the standard circFARSA-miR-330 axis participates in the phenotypes of BC tumor cells Material and methods BC tissue specimens We obtained 30 BC and normal paracancerous tissue samples from patients with bladder urothelial carcinoma who were treated by bladder resection at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between 2016 and 2019 All samples were categorized according to the 2004 World Health Organization classification scheme for bladder neoplasms Table 1 shows the clinicopathological features of the patients Ethics The Ethics Committees of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine approved the study and all protocols involving experimental animals, which also complied with ARRIVE guidelines (http://​www.​ nc3rs.​org.​uk/​page.​asp?​id=​1357) All patients provided written, informed consent to participate in the study Page of 14 Table 1  Clinicopathological factors of bladder cancer patients Parameters Group CircFARSA expression P value High (n=15) Low (n=15) Gender Age (years) Tumor stage Tumor size Grade Lymph node metastasis * Male 12 Female < 55 ≥ 55 pTa-T1 pT2-T4 12 < 3.0 cm ≥ 3.0 cm 13 0.121 0.209 0.025* 0.018* Low 12 High 13 Absent 13 Present 10 0.011* 0.017* p < 0.05 Cell lines and cell culture T24, UM-UC-3, 5637, and J82 cell lines were derived from patients with urinary BC (American Type Culture Collection, Manassas, VA, USA) T24 cells were cultured in McCoy 5A (modified) medium, 5637 cells were cultured in Roswell Park Memorial Institute 1640 medium, and J82 and UM-UC-3 cells were cultured in Eagle Minimum Essential Medium (HyClone, Logan, UT, USA) All culture media were supplemented with 10% fructose 1,6-bisphosphate (HyClone) and 50 U/mL penicillin + 50 μg/mL streptomycin The cells were incubated at 37 °C under a 100% humidified, 5% ­CO2 atmosphere and routinely tested for mycoplasma infection Confluent cells were harvested with (0.25%; mM) trypsin-ethylenediaminetetraacetic acid (EDTA; Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA) Transfection of T24 and J82 cells Small hairpin (sh)-circFARSA, sh-negative control (NC), miR-330 mimic/inhibitor, and NC mimic/inhibitor were purchased from GenePharma (Shanghai, China) The circFARSA sequence was inserted into the Invitrogen pcDNA-3.1 vector (Thermo Fisher Scientific Inc.) to generate circ-FARSA-OE, which was then transfected into T24 and J82 cells using Invitrogen™ Lipofectamine® 3000 (Thermo Fisher Scientific Inc.) as described by the manufacturer Real‑time quantitative polymerase chain reaction (RT‑qPCR) Total RNA was isolated from the BC cell lines and tissue samples (100 mg) using Invitrogen™ TRIzol® (Thermo Fang et al BMC Cancer (2022) 22:373 Fisher Scientific Inc.) and reverse transcribed into complementary DNA using Invitrogen™ M-MLV First-Strand Kits and Oligo (dT) 20 primer Next, circFARSA, miR-330, and mRNAs were amplified using SYBR® Select Master Mix (all from Thermo Fisher Scientific Inc.), as described by the manufacturer The cycling conditions comprised denaturation for 10 at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 40 s at 60 °C The expression of targets was quantified using the ­2-ΔΔCT method with U6 or glyceraldehyde 3-phosphate dehydrogenase mRNA as the internal reference All samples were analyzed in triplicate Cell viability assays Cell viability was assayed using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays In brief, cells were incubated with 20 μL of MTT (0.5 mg/ mL) in 96-well plates, and the supernatant was discarded Dimethyl sulfoxide (150 μL) was added to the wells, which were then rotated for 10 to dissolve formazan Absorbance was measured at 540 nm using an Infinite M200 fluorescence microplate reader (Tecan Group AG., Männedorf, Switzerland) Cell proliferation assays Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) assays (Tongren, Beijing, China) Cells were seeded at a density of × ­103/well in 96-well plates and cultured for 0, 24, and 48 h One hour before the culture endpoint, CCK-8 reagent was added to the wells Cell proliferation rates were determined as optical density at 450 nm in each well determined using the Infinite M200 microplate reader Transwell migration assays Trypsinized cells were harvested and rinsed with D-Hanks balanced salt solution Culture inserts with 8-µm pores or Matrigel inserts were placed in 24-well plates; thereafter, F-12 medium (400 μL) supplemented with 10% fetal bovine serum containing hepatocyte growth factor (20 ng/mL) was added to the lower chamber Cells (2 × ­105/well) were seeded into the upper chamber and incubated for 20 h Cells that migrated through the pores were stained with crystal violet, and assessed via microscopy Wound healing assay Confluent cells in 6-well plates were wounded by scraping with a 10 μL pipette tip The ratios (%) of cells that migrated into the wound were assessed via microscopy and were calculated as the width of the wound at 48 h divided by that at h Page of 14 Western blotting Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (pH 8.0) and cOmplete™ Protease Inhibitor Cocktail (Roche Holdings AG, Basel, Switzerland) Intracellular protein concentrations were then determined using Bicinchoninic Acid Kits Next, the proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (MilliporeSigma Co., Ltd., Burlington, MA, USA) The blots were cut prior to hybridization with respective antibodies The membranes were incubated with primary antibodies overnight at ºC then non-specific protein binding was blocked with Tris-buffered saline-Tween 20 (TBST) The membranes were incubated with secondary antibodies at room temperature for h, thoroughly washed with TBST Thereafter, bands on the membranes were visualized using Super Signal West Femto Maximum Sensitivity Substrate Kits from (Thermo Fisher Scientific Inc.) Dual‑luciferase reporter assays (DLRAs) Wild-type (WT) and mutant (MUT) circFARSA plasmids were generated by amplifying the WT/MUT sequences of circFARSA and inserting them into the luciferase reporter plasmid pGL3 T24 cells were cotransfected with WT/MUT circFARSA and miR-330/ NC mimics Relative luminescence emission was quantified using Dual-Luciferase Assay Kits (Promega Corp., Madison, WI, USA) 48 h later Xenograft assays in vivo Four-week-old BALB/c nude mice (n = 8), (Vital River Laboratory Animal Technology Co., Beijing, China) were raised under specific-pathogen-free conditions Xenograft models were established by subcutaneously injecting the axillae of the mice with ~ × ­107 transfected and control T24 cells Tumor growth was monitored using a digital caliper, and the size was calculated as 0.5 × L × ­W2 The mice were euthanized weeks after the injection, and tumors were surgically excised, photographed and weighed Statistical analysis All data were analyzed via chi-square tests, Student t-tests, or one-way analysis of variance using SPSS 20.0 (IBM Corp., Armonk, NY, USA) All results are shown as means ± standard error Values with P < 0.05 were regarded as statistically significant Results Levels of circFARSA in BC tissues and cell lines We used a microarray to analyze differentially expressed circRNAs in BC and normal bladder tissues (n = Fang et al BMC Cancer (2022) 22:373 each) to determine how circRNA affects BC development Among several dysregulated circRNAs, circFARSA expression was upregulated in BC compared with that in normal healthy controls (Fig.  1A) Consistent with these findings, the qPCR data also revealed a marked increase in circFARSA levels in the BC compared with that in normal healthy bladder cells (Fig.  1B), thus confirming our preliminary results (data not shown) Therefore, circFARSA was further investigated The qPCR results showed elevated circFARSA expression in UM-UC-3, T24, J82, and 5637 compared with that in NC cells (Fig. 1C) These findings show that circFARSA expression is upregulated in BC tissues and suggest that it is involved in BC development Effects of circFARSA silencing on cell viability and proliferation We transfected T24 and J82 cells with shRNA-circFARSA to reduce circFARSA levels and with shRNA-NC (control) to evaluate the role of circFARSA in BC cell viability and proliferation circFARSA expression evidently decreased in cells transfected with shRNA-circFARSA for 48 h (Fig. 2A and B) The MTT assay results showed reduced cell viability after transfection with shRNAcircFARSA compared with NC cells (Fig.  2C and D) Furthermore, the results of CCK-8 assays showed that Page of 14 circFARSA silencing resulted in decreased T24 and J82 cell proliferation rates at 24 and 48 h after transfection with shRNA-circFARSA compared with that in NC cells (Fig. 2E and F) These data suggest that circFARSA expression is essential for maintaining BC cell viability Effects of circFARSA silencing on BC cell apoptosis We assessed whether circFARSA silencing represses BC cell viability by inducing the apoptotic pathway We analyzed cells stained with annexin V and propidium iodide using flow cytometry (FCM) and detected apoptotic factors using western blotting The FCM data showed that circFARSA silencing in T24 and J82 cells resulted in upregulated annexin V-positive cells compared with NC cells (Fig. 3A and B) The western blots revealed elevated levels of the pro-apoptotic  proteins Bax and Bad and reduced levels of anti-apoptotic proteins Bcl-2 and BclxL in BC cells after circFARSA silencing compared with that in NC cells (Fig. 3C and D) Effect of circFARSA silencing on BC cell invasion and migration We assessed the effects of circFARSA silencing on BC cell metastasis The migration and invasive capacities Fig. 1  Increased circFARSA expression in BC tissues and cell lines A Heatmap of normalized circRNA expression in BC tissue samples (n = 3) from patients and in normal bladder tissues (n = 3) Green and red correspond to low and high circRNA expression, respectively B qPCR findings of circFARSA levels in BC and normal bladder specimens (n = 30 each), BC 5637, J82, T24, and UM-UC-3 cell lines, and normal control cells Results as shown as the mean ± standard deviation (SD) *P < 0.05 and †P < 0.01 vs indicated groups Fang et al BMC Cancer (2022) 22:373 Page of 14 Fig. 2  Viability and proliferation of T24 and J82 cells were reduced by circFARSA silencing T24 and J82 cells were transfected with shRNA-circFARSA and shRNA-NC for 48 h A, B Expression of circFARSA in T24 and J82 cells determined via qPCR C, D) T24 and J82 cell viability (E, F) T24 and J82 cell proliferation rates at 0, 24, and 48 h after transfection Results are shown as means ± SDs *P < 0.05 and †P < 0.01 vs indicated groups of T24 and J82 cells were determined by migration and wound healing assays We found that circFARSA level depletion decreased the invasive capacities (Fig.  4A and B) and attenuated the migratory rates (Fig. 4C and D) of both cell types, suggesting that circFARSA expression is critical for maintaining BC cell metastasis circFARSA sponges miR‑330 miR-330 is a target of circFARSA in lung and colorectal cancers [18, 19] Our bioinformatic prediction revealed putative miR-330 binding sites in circFARSA complementarity to the seed region (Fig. 5A) We then explored the direct effects of circFARSA expression on miR-330 using DLRAs Transfection with the miR-330 mimic bound to WT circFARSA and decreased the luciferase activity of T24 cells to approximately 60% (Fig.  5B) We examined miR-330 levels in BC tissue specimens and cell lines using qPCR The results indicated significantly decreased miR-330 levels in BC specimens and cell lines compared with NC tissues and cells (Fig. 5C and D) T24 and J82 cells transfected with shRNA-circFARSA overexpressed miR-330 compared with that in the sh-NC group (Fig.  5E and F) These findings suggested that circFARSA functions as a miR-330 sponge in BC cells Effects of inhibition of miR‑330 expression on circFARSA silencing–mediated proliferation and metastasis We investigated the mechanism through which miR-330 acts on circFARSA-mediated proliferation and migration by simultaneously transfecting T24 and J82 cells with a miR-330 or NC inhibitor and shRNA-circFARSA to reduce miR-330 levels The qPCR results revealed markedly reduced miR-330 expression in BC cells depleted Fang et al BMC Cancer (2022) 22:373 Page of 14 Fig. 3  circFARSA silencing induced T24 and J82 cell apoptosis T24 and J82 cells were transfected with shRNA-circFARSA and shRNA-NC for 48 h A, B Proportions of apoptotic T24 and J82 cells determined via FCM C, D Levels of Bax, Bad, Bcl-2, and Bcl-xL proteins in T24 and J82 cells determined via western blotting Results are shown as the mean ± SD *P < 0.05 and †P < 0.01 vs indicated groups of circFARSA compared with that seen with the NC inhibitor (Fig. 6A and B) circFARSA expression did not change after inhibition of miR-330 expression However, the results of the CCK-8 assays showed that inhibition of miR-330 expression considerably recovered the proliferation of T24 and J82 cells with depleted circFARSA levels relative to cells transfected with the NC inhibitor (Fig. 6C and D) Moreover, inhibition of miR-330 expression ameliorated the proportions of apoptotic cells induced by circFARSA depletion (Fig.  6E and F) Transwell assays showed that inhibiting miR-330 expression led to a notable increase in the numbers of infiltrative T24 and J82 cells (Fig.  6G and H) The results of the wound healing assays showed that inhibition of miR-330 expression also markedly recovered the numbers of migrating T24 and J82 cells with silenced circFARSA (Fig. 6I and J) We examined the effects of upregulating miR-330 expression on the proliferation, invasion, and migration of T24 and J82 cells transfected with miR-330 mimic for 48 h The results of qPCR showed evident elevation of miR330 expression in both cell lines (Fig. 7A and B) Transfecting T24 and J82 cells with the miR-330 mimic reduced cell proliferation in CCK-8 assays (Fig.  7C and D), promoted cell apoptosis in FCM (Fig. 7E and F), and impaired invasion in Transwell assays (Fig.  7G and H) and migration (Fig. 7I and J) as efficiently as circFARSA silencing Therefore, miR-330 seems to function in the circFARSA-regulated proliferation and metastasis of T24 and J82 cells Fang et al BMC Cancer (2022) 22:373 Page of 14 Fig. 4  circFARSA silencing induced T24 and J82 cell invasion and migration T24 and J82 cells were transfected with shRNA-circFARSA and shRNA-NC for 48 h A, B Invasive and (C, D) migratory capacities of T24 and J82 cells determined via Transwell and wound healing assays, respectively Results are shown as the mean ± SD *P < 0.05 and †P < 0.01 vs indicated groups Several oncogenes are mediated by the circFARSA‑miR‑330 axis miR-330 inhibits tumors by targeting mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), sirtuin (SIRT6), integrin alpha-5 (ITGA5), p21-activated kinase (PAK1), mucin (MUC1), and NIN1 binding protein homolog (NOB1) [20–25] Here, we determined whether the levels of these proteins are controlled by the circFARSAmiR-330 axis Western blotting revealed that all these proteins were downregulated in cells transfected with shRNA-circFARSA Furthermore, inhibiting miR-330 ... J82 cells transfected with shRNA -circFARSA overexpressed miR -330 compared with that in the sh-NC group (Fig.  5E and F) These findings suggested that circFARSA functions as a miR -330 sponge in. .. (PAK1), mucin (MUC1), and NIN1 binding protein homolog (NOB1) [20–25] Here, we determined whether the levels of these proteins are controlled by the circFARSAmiR -330 axis Western blotting revealed... transfection with shRNA -circFARSA compared with that in NC cells (Fig. 2E and F) These data suggest that circFARSA expression is essential for maintaining BC cell viability Effects of? ?circFARSA silencing