Effects of ΔNp63 Gene Down-expression on Invasion of Bladder Carcinoma Cells in Vitro Peng Jing1#*, Jiaqiong Zou2# Purpose: This work aims to investigate the effects of ΔNp63 gene down-expression on invasion of bladder car- cinoma cells in vitro. Materials and Methods: Bladder carcinoma cell lines UM-UC-3 and 5637 were cultured. The expression plas- mids encoding ΔNp63 were constructed and transfected into UM-UC-3 and 5637 cells. The migration and adhe- sion of cells were detected. The expressions of ΔNp63 and invasion-related zonula occludens protein-1 (ZO-1) in cells were determined by real-time polymerase chain reaction (PCR) and western blot analysis. Confocal micros- copy was used to observe the location of ZO-1 in cells. Results: Results showed that the down-expression of ΔNp63 reduced the migration of UM-UC-3 and 5637 cells, decreased the heterogeneity adhesion, and increased homogeneous adhesion. After transfection with ΔNp63, the ZO-1 expression in cell membrane and cell cytoplasm was inhibited, also the ZO-1 mRNA and protein levels in cells were significantly decreased. Conclusion: This study indicates thatΔNp63 gene down-expression can reduce the invasion of bladder carcinoma cells in vitro. Keywords: ΔNp63; ZO-1; bladder cancer; invasion INTRODUCTION Bladder cancer is a multi-factor mixed and multiple genes involved disease. Accumulation of abnormal genotypes and the role of external environments even- tually leads to the occurrence of this disease. A previous study showed that p63 is present in all cell layers of pap- illary urothelial neoplasm(1), and other studies showed that ΔNp63 is expressed in some invasive carcinomas using immunoblotting and quantitative reverse tran- scriptase-polymerase chain reaction assays(2,3). ΔNp63 is an important member of p53 family, and the p63 gene located at chromosome 3q27-29 shows strong homolo- gy with the tumor suppressor gene p53(4). Although p63 owes high sequence and structural similarities with p53, their function and expression profiles are different. Wei et al(5) found that ΔNp63 is the predominant isoform during bladder development. Castillo-Martin et al (6) have characterized its role for bladder tumor progres- sion by a p63 positive basal/intermediate cells and "um- brella" cells. However, the role of ΔNp63 in bladder cancer cell lines is not clear. ΔNp63 isoform is selectively highly expressed in cell compartments of stratified and glandular epithelia(4,7). Our previous study(8) found that ΔNp63 is located in the nucleus. Silence of ΔNp63 suppressed the invasion and metastasis of UM-UC-3 cells and reduced claudin-1 expression. Claudin-1 is located in the cell membrane, especially in tight junctions. In this study, we focused 1Department of Pediatric Surgery, The Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P.R. China. 2Medical Laboratory of the First Affiliated Hospital of Chengdu Medical College,Chengdu. #Co-first authors *Correspondence: The Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan Tel: +86 0817 2262409. Fax: +86 0817 2262409. E-mail: 820128944@qq.com Received February 2020 & Accepted October 2020 on another tight junction associated protein-zonula oc- cludens protein-1 (ZO-1) and investigated the effects of ΔNp63 gene down-expression on ZO-1 expression and invasion of bladder carcinoma cells in vitro. MATERIALS AND METHODS Cell culture and transfection assay The human bladder carcinoma cell lines, UM-UC-3 and 5637, were purchased from the Institute of Cell Research of Chinese Academy of Sciences (Shanghai, China). The study was approved by the ethics commit- tee of North Sichuan Medical College, Nan Chong, China. UM-UC-3 Cells were cultured in MEM medium (Gibco Inc., CA, USA) supplemented with 10% fetal bovine serum (FBS; Sijixin Inc., Beijing, China) and 1% penicillin-streptomycin (Invitrogen, Shanghai, Chi- na); 5637 cells were cultured in RPMI-1640 medium (Gibco Inc., CA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were cultured at 37 oC with 5% CO2. The sh-ΔNp63 plasmid was kindly provided by Dr Yunfeng He (The First Affiliated Hospital, Chongqing Medical University, Chongqing, China) . The structure consisting of two 19 bp stem-tar- geting ΔNp63 mRNA, a 9 bp loop and a short poly(A) 6 sequence. The sequences of two oligonucleotides were as follows: forward,5'-GATCCGTGCCCAGACT- CAATTTAGTTTCAAGACGACTAAATTGAGTCT- UROLOGICAL ONCOLOGY Urology Journal/Vol 18 No. 4/ July-August 2021/ pp. 404-410. [DOI: 10.22037/uj.v16i7.5991] GGGCATTTTGTCTTCA AGACGACTAAATT- GAGTCTGGGCATTTTTTGTCGACA-3' and reverse, 5'-AGCTTGTCGACAAAAAATGCCCAGACT- CAATTTAGTCGTCTTGAAACTAAATTGAGTCT- GGGCACG-3'. The sequences of the vector plasmid were as follows: forward, 5'-GATCCGACTTCATAA- GGCGCATGCTTCAAGACGGCATGCGCCT- TATGAAGTCTTTTTTGTCGACA-3' and reverse, 5'-AGCTTGTCGACAAAAAAGACTTCATAA- GGCGCATGCCGTCTTGAAGCATGCGCCT- TATAAGTCG-3'. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the man- ufacturer's instructions. Cell wound healing assay Cells were plated in six well plates for the wound heal- ing assay. A wound was created on the monolayer cells when the cells reached 90% confluence by scraping a gap using a micropipette tip. The 5637 cells plate was then washed with serum-free RPMI-1640 medium to clean the dissociated cells, and UM-UN-3 cells were washed by serum-free MEM medium. 5637 cells were then incubated with serum-free RPMI-1640 medium at 37 oC in 5% CO 2 , and UM-UN-3 cells were then incu- bated with serum-free MEM medium at 37 oC in 5% CO2. Cells that migrated into the unit length area were counted five times for each group at 0, 12, 24 and 48 h following scraping. Cell homogeneous adhesion assay Cell homogeneous adhesion assay could indicate the ad- hesion ability of 5637 and UM-UC-3 cells, which could indirectly reflect the invasion ability of tumor cells. Cells were plated in 48 well plates for the homogeneous adhesion assay. The culture medium was sucked out, followed by twice washing with phosphate-buffered sa- line (PBS) to remove the suspended cells, then the cells reached 90% confluence. 5637 cells were re-suspend- ed with RPMI640 medium and UM-UN-3 cells with DMEM medium. The cell re-suspension concentration in each group was 1×105/mL. 200 ul cells were added to a 48-well plate incubated at 37 oC in 5% CO 2 for 8 h. The non-adherent cells were sucked out, followed by washing with PBS twice. All non-adherent cells were counted. The number of homogeneity adherent cells was equal to seeded 200 ul cells minus non-adherent cells. Each group was repeated for four times. Cell heterogeneity adhesion assay Cell heterogeneity adhesion assay could verify the ad- hesion ability between tumor cells and matrix, which indirectly reflected the invasion of cells. Cells (1×105 / mL) were added into a 96-well plate covered with colla- gen IV and incubated at 37 oC in 5% CO 2 for 120 min. The plate was washed with PBS to clean the dissociated cells. Approximately 20 μl of 5 mg/mL MTT (Sigma Aldrich Inc., MO, USA) was added to the culture medi- um. Following incubation for 10 min at room tempera- ture, the culture medium was removed, and then 200 μl dimethylsulfoxide was added to each well. Absorbance (A value) was measured at 570 nm. Each sample was assayed four times. Real-time polymerase chain reaction (PCR) Total RNA was isolated using an RNeasy mini kit (Qia- gen Inc., Hilden, Germany) and treated with DNase I (Qiagen Inc., Hilden, Germany). Real-time PCR was conducted using an iCycler Bio-Rad Laboratories, Inc., PA, USA) with an iQ SYBR-Green Supermix (Bio- Rad), according to the manufacturer's instructions. The ΔNp63 primer and β-actin as described previously (8). The ZO-1 primer was as follows: ZO-1, 5’-TC- CAGTCCCTTACCTTTCGC-3’ (sense) and 5’-CCC TGGGTGACTAACGGC-3’ (antisense). The PCR con- ΔNp63 gene and invasion in bladder cancer-Jing et al. Urological Oncology 405 Figure 1. Down-expression of ΔNp63 reduced the migration of cells. 5637 and UM-UC-3 cells were cultured and transfected with vector plasmid or sh-ΔNp63 plasmid for 48 hr, respectively. Transfected cells were used for scratch assay. Cells migrating to the unit length area after 0, 12, 24, and 48 hr scraping were counted (magnification×20). ditions were as follows: 94 oC for 4 min, followed by 35 cycles at 94 oC for 20 sec, 60 oC for 30 sec and 72 oC for 30 sec, with data acquisition during each cycle. Melting curve analysis was conducted following PCR cycling to verify the purity and quality of the PCR prod- uct. Western blot analysis The protein was quantified with the Bio-Rad protein colorimetric assay. Protein was separated using 8% sodium dodecyl sulfate polyacrylamide gel electro- phoresis following addition of the sample buffer to the cellular extract and boiling the samples at 95 oC for 5 min. The protein was transferred onto a polyvinylidene difluoride membrane (Millipore Inc., MA, USA) and the membrane was then blocked for 1 h at room temper- ature with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST). Then, the blots were washed Figure 2. Down-expression of ΔNp63 increased homogeneous adhesion of 5637 and UM-UC-3 cells. 5637 and UM-UC-3 cells or Cells transfected with vector plasmid or sh-ΔNp63 plasmid were used for homogeneous adhesion assay. The adherent cells were calculated. The data are shown as the mean ± SD (n = 4). ***, p < .001; ns, not significant. Figure 3. Down-expression of ΔNp63 reduced the heterogeneity adhesion of 5637 and UM-UC-3 cells. 5637 and UM-UC-3 cells or Cells transfected with vector plasmid or sh-ΔNp63 plasmid were used for heterogeneity adhesion assay. MTT assays were used to determine the adherent cells. The data are shown as the mean ± SD (n = 4). ***, p < .001; ns, not significant. ΔNp63 gene and invasion in bladder cancer-Jing et al. Vol 18 No 4 July-August 2021 406 and incubated overnight at 4˚C in TBST containing 1% BSA with primary antibodies against ΔNp63 (1: 200), ZO-1 (1: 200) and GAPDH (1: 3,000). The membranes were washed three times with TBST, incubated with goat anti-rabbit horseradish peroxidase-conjugated sec- ondary antibodies (1: 2,500 dilution in TBST contain- ing 1% BSA) for 120 min at room temperature and then washed three times with TBST. Following the chemilu- minescence reaction, bands were detected by exposing the blots to X-ray films for the appropriate time. For Urological Oncology 407 Figure 4. ZO-1 expression was inhibited in 5637 and UM-UC-3 cells transfected with sh-ΔNp63. ZO-1 and ΔNp63 expression were analyzed by immunofluorescence assays. Representative pictures of three independent experiments with consistent outcome are shown. Figure 5. ZO-1 expression both in mRNA and protein levels was decreased in cells transfected with sh-ΔNp63. ZO-1 and ΔNp63 ex- pression were analyzed by PCR and western blot assays. For 5637 cells: 1, negative control; 2, vector plasmid; 3, sh-ΔNp63 plasmid; For UM-UC-3 cells: 4, negative control; 5, vector plasmid; 6, sh-ΔNp63 plasmid. For western blot analysis, representative blots of three inde- pendent experiments with consistent outcome are shown. The data are shown as the mean ± SD (n = 3). ***, p < .001; ns, not significant. ΔNp63 gene and invasion in bladder cancer-Jing et al. quantitative analysis, bands were detected and evalu- ated densitometrically with UVP Gelatin image pro- cessing system Labworks 4.6 software and normalized against GAPDH density. Confocal microscopy Cells were seeded on polylysine (10 μg/mL) coated glass chamber slides at a density of 2,000 cells/chamber and washed, fixed in ice-cold 4% paraformaldehyde for 15 min and permeabilized in 100 mM phosphate buffer containing 0.2% Triton X-100 (Sigma-Aldrich Corp., MO, USA) for 4 min. Cells were then incubated with 5% bovine serum albumin (BSA; Sigma-Aldrich Corp., MO, USA) and immunolabeled with anti-ΔNp63 (1: 500; Santa Cruz Biotechnology Inc., CA, USA) and an- ti-ZO-1 antibodies (1: 500; Santa Cruz Biotechnology Inc., CA, USA) at room temperature for 1 h. Normal goat IgG instead of anti-ΔNp63 antibody was used in specific experiments to serve as the negative control. Following incubation with the primary antibodies, the cells were washed and incubated for 1 h with fluores- cein isothiocyanate-conjugated anti-ΔNp63 antibodies (1: 500; Santa Cruz Biotechnology Inc., CA, USA) and Cy3-conjugated anti-ZO-1 antibodies (1: 500; Santa Cruz Biotechnology Inc., CA, USA) for 1 h. Addition- al washes were performed and the cells were mounted using fluorescent mounting medium (Applygen Tech- nologies, Inc., Beijing, China). Cells were viewed with a Leica SP2 upright microscope and the images were captured in LCS Light (Leica Science Lab, Berlin, Ger- many). Statistical analysis All statistical analysis was carried out using SPSS17.0 software (SPSS Inc., Chicago, IL, USA). Data were ex- pressed as mean±SD. One-way ANOVA was used to determine the levels of difference between all groups. P <.05 was considered as statistically significant. RESULTS Down-expression of ΔNp63 reduced the migration of cells Cell wound healing assay showed that, at 12 h, the densities of UM-UN-3 cells in negative control, vector plasmid, and sh-ΔNp63 plasmid groups were 14.2±3.7, 13.9 ± 3.3 and 6.2 ± 2.3 cells/mm2, respectively. At 24 h, the densities of UM-UN-3 cells in negative con- trol, vector plasmid, and sh-ΔNp63 plasmid groups were 22.0 ± 1.2, 18.2 ± 2.1 and 12.6 ± 1.4 cells/mm2, respectively. At 48 h, the densities of UM-UN-3 cells in negative control, vector plasmid, and sh-ΔNp63 plasmid groups were 35.2±1.7, 33.5±1.3 and 27.2±2.3 cells/mm2, respectively. At 12h, the densities of 5637 cells in negative control, vector plasmid, and sh-ΔNp63 plasmid groups were11.8 ± 3.7, 11.2 ± 3.3 and 8.2 ± 3.3 cells/mm2, respectively. At 24 h, the densities of UM- UN-3 cells in negative control, vector plasmid, and sh- ΔNp63 plasmid groups were 19.0±3.2, 16.2±3.1 and 11.6±2.4 cells/mm2, respectively. At 48 h, the densities of UM-UN-3 cells in negative control, vector plasmid, and sh-ΔNp63 plasmid groups were 30.2 ± 1.2, 27.5 ± 2.3 and 26.2 ± 2.1 cells/mm2, respectively. At each time point, the densities of UM-UN-3 and 5637 cells in sh-ΔNp63 plasmid group were significantly lower than that in other groups (P < .05). This indicated that the down-expression of ΔNp63 could reduce the migration of bladder cancer cells (Figure 1). Down-expression of ΔNp63 reduced the heterogeneity adhesion, but increased homogeneous adhesion of cells Cell homogeneous adhesion assay showed that the numbers of adherent 5637 cells in the negative con- trol, vector plasmid, and sh-ΔNp63 plasmid groups were 1020.25 ± 20.25,1025.5 ± 17.48, and 2012.75 ± 9.54 cells/ml, respectively. The numbers of adherent UM-UC-3 cells in the vector plasmid, negative control and sh-ΔNp63 plasmid groups were 1521.95 ± 35.45, 1536.35 ± 20.65, and 2475.45 ± 15.35 cells/ml, respec- tively (Figure 2). The cell heterogeneity adhesion as- say showed that the A values of 5637 cells in negative control, vector plasmid, and sh-ΔNp63 plasmid groups were 0.459 ± 0.035, 0.412 ± 0.014 and 0.295 ± 0.017, respectively. The A values of UM-UN-3 cells in neg- ative control, vector plasmid, and sh-ΔNp63 plasmid groups were 0.412 ± 0.017, 0.398 ± 0.013 and 0.267 ± 0.021, respectively (Figure 3). This indicated that, after transfection with ΔNp63, the heterogeneity adhesion capacity of bladder cancer cells was decreased. Taken together, these results indicated that down-expression of ΔNp63 inhibited the invasion ability of 5637 and UM-UC-3 cells. Location and expression of ZO-1 in cells Laser confocal microscopy showed that ZO-1 protein was mainly located in the cell membrane and cell cyto- plasm. Our results showed that, after transfection with sh-ΔNp63, ZO-1 expression was inhibited (Figure 4). Real-time PCR and western blot analysis demonstrat- ed that, after transfection with sh-ΔNp63, both ZO-1 mRNA and protein expression in 5637 and UM-UN-3 cells were significantly decreased (Figure 5). DISCUSSION Approximately 90% of cancers occur in epithelial orig- inal cells (9), so understanding the events that allow epithelial cells to progress towards tumorigenic path- ways is required. Usually, ΔNp63 is over-expressed in epithelial cancers, showing correlation with poor prognosis(11). Some studies(7,9,11,12) have focused on the signaling pathways regulated by ΔNp63 and studied p63 levels in the mature epidermis. ΔNp63 was the main isoform detected and expressed mainly in the ba- sal layers. Its expression was down regulated in well differentiated layers(13,14). In addition, ΔNp63 opposes the tumor suppressive effects of cellular senescence suggesting a role in oncogene initiation(15,16). Molecular mechanisms about the role of ΔNp63 in cell migration and invasion to date mainly comprised the identifica- tion of specific genes known to influence cell motility, including N-cadherin, E-cadherin, epithelial cell-cell adhesion molecule, and so on(17-19). Further research is still needed. Tight junctions proteins are important in effecting inva- sive phenotype of cancer cells, also important in influ- encing intracellular signaling pathways of these cells. Our previous study(8) found that the down-expression of ΔNp63 changed the cell adhesion, and there was correlation between ΔNp63 and claudin-1. Whether other tight junction associated proteins are involved in this process is still unknown. In this study, we fur- ther proved that ΔNp63 influence the invasion ability of bladder cancer cells partially through regulating the expression of ZO-1. Down expression of ΔNp63 leaded to decreased expression of ZO-1, which contributed to ΔNp63 gene and invasion in bladder cancer-Jing et al. Vol 18 No 4 July-August 2021 408 the impaired adhesive and invasive ability of bladder cancer cells transfected with sh-ΔNp63 plasmid. ZO-1 is membrane-associated guanylate kinase-family proteins presenting in tight junctions. In epithelial cells, ZO-1 is exclusively located at the zonula occludens which is composed of tight junctions. ZO-1 could pro- mote tumor cell invasion. Reduced expression of ZO-1 correlated with decreased proliferation and/or transfor- mation of epithelial cells(20-22). The depletion of ZO-1 in cultured epithelial cells resulted in a delay in barrier formation(23,24), and ZO-1 gene deletions were embryon- ic lethal in mice(25). ZO-1 has been reported to accumu- late transiently in the nucleus of proliferating cells(26), playing a role in cell differentiation rather than cell pro- liferation(27). How ΔNp63 influences cell-cell adhesion is still not well defined. We tried to demonstrate this by confocal microscopy and western blot analysis. Our results indicated that ZO-1 is located both in the cell membrane and cell cytoplasm. Confocal microscopy and western blot analysis showed that ZO-1 expression reduced in cells transfected with sh-ΔNp63. ΔNp63 silence in the human bladder carcinoma cell lines, UM- UC-3 and 5637, was confirmed by PCR and western blot assays. To date, we have demonstrated the down expression of ZO-1 and claudin-1 in ΔNp63-silenced cells. The regulatory role of ΔNp63 in cell adhesive ability was explored by wound healing and adhesion assays in vitro. It may also promote cell migration during tumor invasion and metastasis. In addition, ΔNp63 modulated extensive adhesive gene spectrum, including N-cadher- in, β4-integrin, and tight junction-associated protein (17,28). Although the role of p63 in tumor formation and progression has been well studied, as a member of p53 gene family, its role in tumors’ metastasis is complex and remains unclear. The role of ΔNp63 expression in urothelial carcinomas still remains to be elucidated (2,29). Because of rare mutations or allelic deletions of p63 gene in human bladder carcinomas(30), the loss of ΔNp63 mRNA may attribute to epigenetic alterations. Based on recent researches, ΔNp63 expression corre- lates with the severity of bladder cancer. In conclusion, ΔNp63 regulated the invasive ability of tumor cells partially through tight junction associated proteins, es- pecially ZO-1 in bladder cancer cells. This study lays the basis for further understanding on the role of p63 in tumors. CONCLUSIONS This study indicates thatΔNp63 gene down-expression can reduce the invasion of bladder carcinoma cells in vitro, laying the basis for further understanding of the role of p63 in tumors. 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ΔNp63 gene and invasion in bladder cancer-Jing et al.