BZW2 gene knockdown induces cell growth inhibition, G1 arrest and apoptosis in muscle‐invasive bladder cancers: A microarray pathway analysis

Abstract Bladder cancer is among the most common cancers all over the world. The function of basic leucine zipper and W2 domains 2 (BZW2) in tumour progression has been reported. However, the biological function of BZW2 in muscle‐invasive bladder cancers (MIBCs) remains to be determined. The aim of the present study was to reveal the expression and roles of BZW2 in human MIBCs and to explore the molecular mechanisms underlying these functions. Clinically, BZW2 expression was higher in MIBC tissues than the adjacent non‐tumour tissues. Knocking down BZW2 using shRNA inhibited cell proliferation and G1/S cell cycle progression in vitro, and induced apoptosis in both 5637 and T24 cells. Moreover, in vivo studies with mice xenograft models confirmed the anti‐proliferative effects of BZW2‐knockdown, providing a future therapeutic target. We also performed biochemical microarray analysis to identify the potential signalling pathways, disease states and functions which could be affected by suppressing BZW2 in MIBC cells. Collectively, our findings suggest BZW2 has an oncogenic role in MIBCs and serves as a promising target for molecular diagnosis and gene therapy.

extensively studied molecular aspects of bladder cancer. 9 For instances, increasing cyclin D1 positivity is regarded as a predictor of improved survival and of a lower progression rate in MIBCs. 10 Moreover, almost all MIBCs have defects in genes encoding proteins that control the G1 cell cycle checkpoint. 9 However, there is still no molecular biomarker to predict the progression of disease accurately. Therefore, it demands more efforts to explore the new molecular targets and underlying mechanism for bladder cancer, especially MIBCs.
Basic leucine zipper and W2 domains 2 (BZW2) is a member of the bZIP superfamily of transcription factors. 11 BZW2 is an evolutionary highly conserved protein and involved in cell-cell adhesion via cadherin binding. 12 BZW1, another member of the bZIP superfamily, has been recognized as a novel proliferation regulator in salivary mucoepidermoid carcinoma. 13 In contrast, there was little study reported on the potential role of BZW2 in cancers. Most recently, Cheng et al reported that BZW2 is up-regulated in osteosarcoma and its down-regulation inhibits cell growth by inactivating the Akt/mTOR signalling pathway, 11 suggesting BZW2 plays a potentially important role in osteosarcoma progression. In addition, a statistical analysis conducted on clinical patients (https:// www.proteinatlas.org/ENSG00000136261-BZW2/pathology) [14][15][16] showed that high expression of BZW2 is most common in urothelial cancer among a wide variety of different cancers ( Figure 1).
Nonetheless, it remains unclear about the exact role of BZW2 in context of MIBCs.
In the present study, we combined in vitro, in vivo, bioinformatics and clinical studies to explore the function and possible mechanism of BZW2 in MIBCs. We evaluated the expression level of BZW2 in clinical patients with advanced bladder cancer (of stage T2 and above), as well as in two different human MIBC cell lines (5637 and T24). We also assessed the effects of BZW2 knockdown on cell growth, cell cycle progression, cell death in vitro, as well as the tumour growth inhibition in vivo. The signalling pathways and disease states affected by BZW2 knockdown were further analysed, which could provide insights into the possible mechanism behind the BZW2 function in MIBCs.

| Cell line and cell culture
Human MIBC cell lines (T24 and 5637) and normal bladder epithelial cell line (SV-HUC-1) were purchased from the Cell Bank of Shanghai

| Animals
Male BALB/c Nude mice (6-8 weeks age) were obtained from F I G U R E 1 A statistical analysis conducted on clinical patients (https://www.proteinatlas.org/ENSG00000136261-BZW2/pathology). [14][15][16] For each cancer, colour-coded bars indicate the percentage of patients with high and medium protein expression level. The cancer types are colour-coded according to which type of normal organ the cancer originates from. Low or not detected protein expression results in a white bar.

| Plasmids construct and gene knockdown
The shRNA sequence corresponds to Homo sapiens basic leucine zipper and W2 domains 2 (BZW2), (GenBank accession number NM_014038). Duplex shRNA oligos were cloned into the AgeI and EcoRI sites in GV115. The sense shRNA strand of BZW2 was CCGGGCTGATGTTCTGAGCGAAGAACTCGAGTTCT TCGCTCAGAACATCAGCTTTTTG. The insert fidelity was confirmed by sequencing both strands with the following primers (forward: CCTATTTCCCATGATTCCTTCATA; reverse: GTAATA CGGTTATCCACGCG). BZW2 was knocked down with a GV115 based lentiviral vector. Lentiviruses were generated in HEK293T cells as described, 17 and then cells were infected with virus-containing supernatants. The alteration of expression of BZW2 was confirmed by real-time RT-PCR and Western blotting.

| Cell scratch test
The 5637 cells in logarithmic growth phase were treated with serum-free treatment for 24 hours, adjusted to a cell concentration of 5 × 10 5 mL −1 with RPMI-1640 medium containing 10% FBS, inoculated into 6-well plates and incubated in a 37°C, 5% CO 2 incubator. When the 5637 cells were grown to 90% confluence, the 6-well plate was removed for scratching. During the scratching process, a 10 μL tip was used to draw straight along the marking line, and the floating cells were washed away with PBS, and then the tumour cells were separately added to the medium of BZW2-knockdown (KD) group and normal control (NC) group. The incubation was continued in an incubator at 37°C in a 5% CO 2 atmosphere, after 24 hours, the degree of cell migration was observed under a fluorescence microscope. Three replicate wells were set per cell and each experiment was repeated three times.

| Cell cycle analysis
The effect of BZW2 knockdown on the cell cycle distribution was measured with PI staining according to previous publication. 19 After infection with shRNA, about 1 × 10 6 5637 or T24 cells were

| Western blot analysis
Western blotting was performed as described before. 20  . The X-ray film was developed by an enhanced chemo-luminescence system.

| RNA isolation and quantitative real-time PCR
Total RNA was extracted from cultured cells with TRIzol reagents according to the standard manual. RNA was reversely transcribed using RevertAid First Strand cDNA Synthesis Kit before quantification with spectrophotometry. Quantitative real-time PCR (qRT-PCR) was carried out on the Applied Biosystems Prism 7300 sequence detection system with Maxima SYBR Green/ROX qPCR Master Mix according to the manual. GAPDH was considered as an internal control. The specific primers used in real-time PCR were shown as below (Table 1).

| Statistical analysis
All experiments were performed in triplicate and the results are expressed as the mean ± SD. Data were analysed using SPSS software (Chicago, IL, USA). Differences with P-values < 0.05 were considered statistically significant.

| BZW2 is overexpressed in MIBC cell lines and MIBC tissues
Basic leucine zipper and W2 domains 2 (BZW2) has been recognized to exhibit oncogenic function in osteosarcoma. 11 With great inter-

| Knockdown of BZW2 inhibits cell growth and migration, and decreased the viable cell number in 5637 and T24 cells
To explore the functions of BZW2 in MIBCs, gene knockdown with shRNA was used against BZW2 in both human MIBC cell lines (5637 and T24). Prior to the evaluation, we first validated the effect of BZW2-knockdown by determining the expressions at both mRNA and protein levels with qRT-PCR and Western blotting, respectively. As shown in Figure 3A

| BZW2 knockdown induces G1-phase cell cycle arrest and apoptosis in 5637 and T24 cells
We

| In vivo study of anti-proliferative effects of BZW2 knockdown on MIBC xenograft models
Our results suggested that BZW2 could serve as a potential anti-  Figure 5D). These findings strongly support the essential role of BZW2 in the tumorigenesis of MIBCs, which provides a solid basis for the target-based therapies.

| Microarray pathway analysis of signalling and disease states in response to BZW2 knockdown
To explore the possible molecular mechanism underlying the anti- The volcano graph demonstrates the distribution of differentially expressed genes by fold change between the KD group and the NC group ( Figure 6A).
On the above basis, we used IPA tools (http://www.ingenuity. com) to identify differentially expressed canonical pathways by defining the enrichment P-value of the pathway. Signalling pathways associated with BZW2-knockdown cells were sequenced by their -Log(P-value) ( Figure 6B) 23 Notably, several pathways were found to be associated with cell cycle, including cyclins and cell cycle regulation, 24 oestrogen-mediated S-phase entry, 25

| The connection between the BZW2 and other genes of cell cycle regulators
For more in-depth exploration, we crossed the differential genes with several well-known genes involved in cell cycle regulation, and selected Cyclin A, Cyclin B, Cyclin D, Cyclin E and CDK1 genes to verify the link between BZW2 and other genes for cell cycle regulators. As expected, BZW2 knockdown significantly decreased the protein expression of above five genes compared with that in the control cells ( Figure 8A). And we validated the above results from mRNA levels using q-PCR analysis, which confirmed that the mRNA expression of the above five genes in BZW2-knockdown T24 and 5637 cells was significantly lower than that of the control cells (P < 0.001) ( Figure 8B-F). These results indicated that BZW2-knockdown may induce cell cycle arrest.

ACK N OWLED G EM ENTS
This study was supported by a grant from the National Natural Science Foundation of China (No.81573751).

CO N FLI C T O F I NTE R E S T
All the authors have no conflict of interest to declare.