Exploring the five different genes associated with PKCα in bladder cancer based on gene expression microarray

Abstract Much progress has been made in understanding the mechanism of bladder cancer (BC) progression. Protein kinase C‐α (PKCα) is overexpressed in many kinds of cancers. Additionally, PKCα is considered an oncogene that regulates proliferation, invasion, migration, apoptosis and cell cycle in multiple cancers. However, the mechanism underlying how these cellular processes are regulated by PKCα remains unknown. In the present study, we used PKCα siRNA to knock down PKCα gene expression and found that down‐regulation of PKCα could significantly inhibit cell proliferation, migration and invasion and induce apoptosis and G1/S cell cycle arrest in vitro. Overexpression of PKCα promotes tumour growth in vivo. We applied cDNA microarray technology to detect the differential gene expression in J82 cells with PKCα knockdown and found that five key genes (BIRC2, BIRC3, CDK4, TRAF1 and BMP4) were involved in proliferation and apoptosis according to GO analysis and pathway analyses. Correlation analysis revealed a moderate positive correlation between PKCα expression and the expression of five downstream genes. BIRC2 and BIRC3 inhibit apoptosis, whereas CDK4, TRAF1 and BMP4 promote proliferation. Essentially, all five of these target genes participated in proliferation, and apoptosis was regulated by PKCα via the NF‐kB signalling pathway.

tumour (TURBT). 2,3 Therefore, it is critical to identify highly sensitive diagnostic and prognostic markers for the treatment of this disease.
Protein kinase C-α (PKCα), also named PRKCA, is a member of the protein kinase C family. Accumulating evidence has demonstrated that PKCα is overexpressed in many kinds of cancers, such as breast, lung, renal, and colorectal cancers. [4][5][6] In addition, PKCα is considered to be an oncogene and regulates the proliferation, invasion, migration and apoptosis of tumour cells via activation of the mTOR-signalling pathway. 7 In addition to its tumorigenic activity, PKCα is involved in diverse functions in cell development and has been implicated in many pathological processes, such as inflammation, oxidative stress, myelodysplastic syndromes and diabetic nephropathy. 8 Our previous study demonstrated that PKCα played a significant role in tumorigenesis of BC. In the present study, we aimed to identify the pathogenic mechanisms underlying the antineoplastic effects induced by PKCα knockdown. First, we employed cDNA microarray technology to detect the expression of target genes on a genome-wide level using BC cells with PKCα knockdown. Using GO and KEGG enrichment analyses, we found that the expression of all five target genes was positively correlated with PKCα expression and demonstrated that all five target genes were regulated by PKCα via the NF-kB signalling pathway. All these findings contribute to our knowledge regarding BC progression and to the development of a novel therapy for BC.

| Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted using an miRcute miRNA Isolation Kit (TIANGEN, Beijing) in accordance with the manufacturer's instructions. For RT-PCR, 1500 ng of extracted RNA was directly reversetranscribed using Prime Script RT Master Mix (Takara, Dalian) and random primers. To quantify the amounts of mRNA, real-time PCR analyses were quantified by SYBR ® Premix Ex Taq™ Kit (Takara).
GAPDH expression was considered to be internal control. All analyses were performed using a Thermal Cycler Dice™ Real-Time TP800 System (Takara, Kyoto, Japan). The ΔΔCT method was employed to calculate the relative expression of different genes. The primers used are listed in Table 2.

| RTCA (real-time cell assay)
Transfected cells were washed with phosphate-buffered saline (PBS) and dissociated with trypsin. Then, the cells were planted in cell culture E-plates of 3500 cells per well and cultured in a humidified incubator. Cell growth curves were automatically recorded in real time with an xCELLigence System (Roche Applied Sciences), and the cell index was monitored for 3 days.

| Transwell assay
Transwell assays were used to assess the invasion and migration of BC cells by using or without using Matrigel, respectively. In all, 2 × 10 4 BC cells (T24 and UMUC3) were suspended in 200 µL serumfree RPMI 1640 medium and incubated in incubator. After 24 hour, the non-invading or non-migratory cells were removed with a cotton tip, and the remaining cells on the bottom were stained with 0.1% crystal violet.
Rabbit anti-Ki67 antibodies were purchased from Cell Signaling Technology Company. IHC analysis was used in accordance with the procedure. Images were obtained at 200× or 400× magnification.

| Statistical analysis
All data are expressed as the mean ± SD for three independent experiments: *P < 0.05, **P < 0.01 and ***P < 0.001. Data analyses were carried out using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA) and SPSS software ver. 20.0 (SPSS, Inc, Chicago, IL, USA). Probability (P) values < 0.05, as calculated using Student's t test as appropriate, were considered statistically significant. The expression of clinical samples data detected by RT-PCR, presented as Mann-Whitney U test was used for significant analysis.

| PKCα expression is up-regulated and exerts an oncogenic role in bladder cancer in vitro
As accumulating evidence has proven that PKCα is overexpressed in multiple cancers, we first re-confirmed whether PKCα expression was up-regulated in BC. We collected 20 pairs of BC tissues and normal adjacent tissues and analysed them by qRT-PCR, and analysed 20 pairs of BC tissues and normal adjacent tissues by Western blotting. The results showed that PKCα was overexpressed in BC TA B L E 2 Real-time PCR primer sequences

| PKCα plays an oncogenic role in bladder cancer in vivo
As demonstrated by the oncogenic role of PKCα in BC, we next de-

| Gene expression microarray analyses of PKCα target genes in bladder cancer cells
Having confirmed that PKCα participated in cellular function in BC cells, we next explored the mechanisms underlying how PKCα regulates cell function. We used cDNA microarray technology to detect target gene expression levels on a genome-wide scale using BC cells with PKCα knockdown. As a result, 500 genes were identified as being differentially expressed (Q < 0.05, P < 0.05, differential gene = 500) in PKCα knockdown cells ( Figure 3A-B). GO analysis and pathway analysis were performed with the target genes of PKCα, and biological processes and molecular functions for GO analysis,

| Analysis of the correlation between the identified key genes and PKCα in bladder cancer tissues
To explore the role of five key genes in BC, we first assessed the expression of these genes in bladder cancer cells, and the Western blotting results showed that all five genes were overexpressed in J82, T24 and UMUC3 cells compared with that in SV-HUC-1 cells ( Figure 4A).

| PKCα affects cell proliferation and apoptosis in an NF-kB-dependent manner in bladder cancer cells
According to GO analysis and KEGG pathway analysis, we found five key genes that were associated with cell proliferation and apoptosis, which participates in the NF-kB signalling pathway and cancer-related pathways. To further determine whether the oncogenic role of PKCα in BC depends on the NF-kB signalling pathway, we transfected cells with PKCα siRNA (PKCα si), TNF-α (20 ng/mL) or both (P + T). Interestingly, we observed that all five genes were regulated by the PKCα/NF-kB axis according to Western blotting ( Figure 5A). Additionally, we performed the RTCA and flow cytometry and observed that the activation of the NF-kB signalling pathway significantly promoted proliferation and reduced apoptosis of PKCαdepleted T24 and UMUC3 cells ( Figure 5B-C).

| The five identified genes affect cell function via the PKCα/NF-kB axis
Given the association between the PKCα/NF-kB axis and both pro- combined with TNF-α activation significantly induced apoptosis compared with TNF-α group in T24 and UMUC3 cells ( Figure 6A-B).

| D ISCUSS I ON
Though much progress has been achieved in understanding the mechanism of tumorigenesis, many targeted therapies are emerging was the first kinase considered to be an important regulator of the cell cycle, which could induce G1/S cell cycle transition or G2/M cell cycle transition. 12 However, emerging evidence has demonstrated that CDK4 is involved in cellular processes other than cell cycle.
CDK4 is also essential for regulating proliferation, migration, invasion and apoptosis. 13 Dysregulation of CDK4 could lead to multiple pathological processes, diseases or even cancer. Many cancers, including breast cancer, acute myeloid leukaemia, non-small-cell lung cancer and ovarian cancer, have been found to be associated with the dysregulation of CDK4. Accumulating studies revealed that aberrant CDK4 could participate in many cellular pathways associated with malignancies, including the PI3K/Akt signalling pathway in lung cancer, the Wnt/β-catenin signalling pathway in BC and the JAK-STAT signalling pathway in gastric cancer. [14][15][16] Among these, the mechanism by which CDK4 contributes to breast cancer progression has been thoroughly studied, and CDK4 inhibition has been applied to a wide range of human cancers. [17][18][19] Therefore, developing inhibitors targeting CDK4 and understanding their anticancer effects have garnered increasing interest in recent years. 20,21 In this study, we found a moderate positive correlation between the expression of PKCα and that of CDK4. Furthermore, PKCα knockdown significantly inhibited BC cell proliferation through the NF-kB/CDK4 axis.
TRAF1 (tumour necrosis factor receptor-associated factor 1) is a member of the TNF receptor (TNFR) family, which plays significant roles in cell signal transduction, and is considered to be a key adaptor molecule. Some studies indicate that TRAF2 could recruit BIRC2 and BIRC3 to form a protein complex that can facilitate the recruitment of TRAF1. 22 All these processes contribute to TNF-induced activation of the TRAF1 signalling pathway. New research has also shown that TRAF1 affects the cell cycle in cancer in a CDK4-dependent manner. An increasing number of studies revealed that TRAF1 could participate in many cellular pathways associated with pathophysiological processes; these pathways include the P-38 MAPK, NF-kB and Akt signalling pathways. 23,24 Because of its extensive functionality in pathophysiological processes, TRAF1 has been reported to be involved in various diseases, including ulcerative colitis, rheumatoid arthritis, hepatic steatosis and, especially, various cancers. 25,26 In the present study, we found a moderate positive correlation between the expression of PKCα and that of TRAF1. Furthermore, PKCα knockdown significantly inhibited BC cell proliferation through the NF-kB/TRAF1 axis.
BMP4 (bone morphogenetic protein-4) belongs to the BMP family, which was originally considered to comprise proteins that induce new bone formation. The BMP family was eventually reported to be a member of the transforming growth factor b (TGF-b) superfamily, whose members play key roles in signal transduction. 27 In addition to its significant role in skeletogenesis, BMP4 was revealed to be an important regulator in many diseases, such as Alzheimer's disease, pathological cardiac hypertrophy/heart failure, diabetic nephropathy and multiple cancers. BMP4 has emerged in many cancers, including breast cancer, prostate cancer, colorectal cancer and non-small-cell lung cancer. 28 In some cancers, BMP4 was also reported to be associated with poor prognosis. 29,30 In the present study, we found a F I G U R E 6 The five key genes affect cell functions regulated by the PKCα/NF-kB axis. (A and B) T24 and UMUC3 cells were transfected with BIRC2 or BIRC3 siRNA, TNF-α (20 ng/mL) or both. Apoptosis was estimated in T24 and UMUC3 cells by using flow cytometry. (C to H) T24 and UMUC3 cells were transfected with CDK4, TRAF1 or BMP4 siRNA, TNF-α (20 ng/mL) or both. Proliferation in treated T24 and UMUC3 cells was detected by EdU or RTCAs