Non-muscle invasive bladder cancer (NMIBC), muscle invasive bladder cancer (MIBC), and metastatic lesions are all forms of urinary bladder cancer. Transitional cell carcinoma of the bladder (TCC) is the most frequent kind of bladder cancer.It manifests itself in two separate types, each with a distinct clinical and biological characteristics. 

The majority of patients have tumors that have not spread to the muscles, with muscle invasive tumors constituting the remaining 30 percent of cases. However, recurrence is common, even in those with a superficial illness, and it is linked to the emergence of a muscle-invasive disease (MID) [1]. 

Multidomain Cas family scaffolding protein, human enhancer of filamentation 1 (HEF1) [2,3]. The HEF1 protein was shown to play a role in the control of multiple different cellular processes, including adhesion, polarity, motility, and invasion [4].It modulates the protein complexes that direct cancer cell differentiation and invasion, and it is a member of the family of Crk-associated substrates [5].

IL 1 is a significant cytokine that has been linked to cancer progression in a number of different contexts. This is due to the fact that it is a crucial pro-inflammatory cytokine with the ability to control the production of several molecules involved in inflammation. IL-6 is the most significant cytokine in human inflammation regulation because it activates the STAT3 signaling pathways [6]. Because of its role in tumor formation and metastasis, the IL-6 signaling pathway may be linked to a poor outcome in a wide range of cancers [7]. Interleukin 8 is a chemokine that belongs to the CXC chemokine family. It was first shown to be a powerful chemo attractant for neutrophils and lymphocytes, and this was the basis of its discovery. IL-8 has also been shown to promote angiogenesis, tumor development, invasion, and possible metastasis in cancer, according to subsequent research. Recently, a number of studies that examined the relationship between elevated blood IL-8 levels and various cancers were published and were also awarded [8].

T-helper cells release the inflammatory cytokine interleukin 17 in response to the signalling protein interleukin 23. The IL-17R cell surface receptor, of which there are at least three versions (IL17RA, IL17RB, and IL17RC), is where IL-17 exerts its effects. By stimulating the production of chemokines in a number of organs and tissues, IL-17 operates as a potent mediator in delayed-type responses. In response to pathogen invasion, IL-17 signals mobilise monocytes and neutrophils to the site of inflammation, a process called phagocytosis. Animal studies suggest that IL-17 works in conjunction with TNF and IL-1 to promote inflammation.  TGF- β1 has a critical function in immune system regulation, and it exhibits a range of actions depending on the cell type or stage of development. TGF- β 1 is secreted by the majority of immune cells. As a result, the current study concentrated on the underlying processes of IL-1, IL-6, IL-8, IL-17 and TGF, as well as their potential utility in addressing the need for aggressive bladder cancer treatment. The current investigation attempted to analyse the expression of IL1, IL6, IL8, IL 17, and TGF in individuals with bladder cancer, as well as the expression levels of the HEF1 gene in samples of urothelial carcinoma. 

Materials and Methods

Patient characteristics: This retrospective analysis comprised fifty instances of paraffin-embedded urothelial carcinoma tissue (10 felales and 40males), ranging in age from 23 to 90 years.As a control group, fifty specimens of nontumorous bladder lesions were evaluated. Based on a consensus achieved at a 1998 meeting of the International Society of Urological Pathology (ISUP) and endorsed by the WHO in 2004, the tumours were classed histologically [9] into high-grade and low-grade papillary urothelial carcinoma. Tumors were staged according to the American Joint Committee of 2002 TNM classification was updated in 2009 (7th   version) [9].

Immunohistochemistry (IHC)

All 6 mm thick paraffin-embedded sections from both patients and controls were deparaffinized by heating the slides vertically in an oven for 1 hour, then washing them for 20 minutes in xylene and an ethanol series (100 percent, 95 percent, 75 percent, and 50 percent, all for 5 minutes each). For 20 minutes, sections were incubated with 3 percent H2O2 to inhibit endogenous peroxidase activity (BioGenex, CA, USA). Antigen retrieval was performed for 2 minutes at 97°C in a microwave using Citra Plus (BioGenex, CA, USA). Before applying primary, slices were treated with protein blocks from EMD Millipore (IL, USA). Untreated tumor portions served as both positive and negative controls. Chemicon, Temecula, CA, USA, provided the human lung positive control. The experiments used non-treated cancer tissues as negative controls. It was necessary to dye all samples more than once, and the results were extremely repeatable.

Immunohistochemical Scoring

Two histopathologists independently assessed the immunostained sections without knowing who the patient was or how serious the sickness was. Disagreements were debated under a microscope with many heads until a solution was found for the matter at hand. If there was a brown staining in the nucleus, it was thought to be positive for bladder cancer. Rahbar  et al. [10] examined 1000 cells in ten high-power fields for 24 hours to measure protein expression. As expected, four classifications were established based on the percentage of positive cells. They are as below: "grade 1 <25%; grade 2 ≥25%–50%; grade 3 ≥51%–75%; grade 4 ≥76%".

Molecular test

Extracted total RNA was processed with DNase I enzyme utilising samples (DNase I enzyme kit) and carried out in accordance with the protocol outlined by the Promega company, USA. This was done to eliminate any remaining traces of genomic DNA that could have been present. Then we used the M- MLV Reverse Transcriptase kit (Bioneer. Korea) containing random hexamer primer to synthesize cDNA from total RNA. In the presence of a random hexamer primer, mRNA serves as a template for the synthesis of single-stranded DNA by the enzyme reverse transcriptase (annealing to total RNA). After that, the single-stranded DNA molecule may be utilized as a guide to make a complementary pair of strands. Real-time polymerase chain reaction was then performed on the cDNA using HEF1-specific primers. A 5 μL cDNA, 2.5 μmol/L forward primer and 2.5 μmol/L reverse primer 2.5 Taq Man probe 2.5 DEPC water were added to Accu Power ® Plus Dual StarTM qPCR Pre Mix (Bioneer. Korea) in a total volume of 50 µL. The following RT reaction procedure was used: denatured at 95°C for 5 minutes, then 95°C for 20 minutes, then 60°C for 30 seconds. HEF1's forward and reverse primers included the following sequences: 5′ ATCTTGGCCATCAACAAGCC-3′ and 5′ TGCGTTGGTGTTGATGGTTG-3′. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers included the following sequences: 5′ ACGACCACTTTGTCAAGCTC-3′ (forward) and 5′ TTCCTCTTGTGCTCTTGCTG-3′ (reverse); (Bioneer company, Korea). The average 2−∆Ct, where ∆Ct (cycle threshold) = Ct - CtGAPDH, was used to determine the quantity of FOXM1 messenger RNA (mRNA) in comparison to GAPDH.

Statistical Analysis

Two software tools were used to summarize, display, and analyses data. These included the T-test, one-way ANOVA, and Chi-square test using an alpha value of<0.05 in Microsoft Office Excel 2007, and SPSS 18 for statistical analysis in the social sciences.

Determine of Cytokines

The pathologist classified the 30 paraffin-embedded bladder tumor sections into four grades, as shown in table 1 and figure 1, which correlate to different degrees of expression in bladder cells basing on the grade of cancer.

From the 30 samples tested, 16 (57%) were positive for IL 1, whereas just 4 (14.28%) were positive for any of the other three grades. Interleukin-1 beta (IL-1 β) has been shown to have a significant role in the initiation and progression of tumours in a wide range of malignancies, and these results are in line with those reported by Chavey  et al. [11]. In addition, Jin  et al. [12] analyzed how elevated interleukin-1beta (IL-1 β) levels correlate with breast cancer's aggressiveness and grade.

Takafumi  et al. [13] observed that the IL1 β gene was connected with higher adherence of the ovarian cell line to human mesothelial cells when they analyzed gene expression with a putative mechanistic impact. A strong association between IL-1 β expression and survival in the long run was discovered using immunohistochemistry on ovarian carcinoma samples.

In our study, grade I had a greater level of IL1 β expression (57.1 percent) than the upper grades (grades III&IV), which both had a 14.28 percent level of expression14.28 percent each.The findings of the present study contradict those of Chavey  et al. [11], who claimed that high-grade tumours had higher cytokine levels than low-grade tumours and that most cytokines had no relationship to patient age, histological type, tumour size, or lymph node status.

According to the data in table (3-7) for IL 8, upper grades (III-IV) only show positive expression in a rate of 27.2% whereas lower grades (I-II) only show it in a rate of 54.5%. The majority of the research found a relationship between this interleukin and various types of cancer. Snoussi  et al. [14] showed that breast cancer tissues had more IL-8 than normal tissue, which may be related to angiogenesis. They also observed that IL-8 released by tumour cells promotes endothelial cell growth and survival.

Instead of finding that non-invasive tumors produce more IL-8 and are not connected to tumor recurrence, Sabrina  et al. [8] observed the opposite. They also discovered a connection between poor prognosis and elevated levels of IL-8 expression. IL-8 may be critical for tumor progression, but not tumor promotion or start, as this result suggests. High levels of IL 17 expression were identified in 12 out of the 16 samples (or 54 percent of the total). When it came to expression 4, patients in grades 1 and 3 had the largest percentages (36.3% and 9.09 percent, respectively).

IL 17 is a multifunctional cytokine with the capacity to promote tumor development and spread. In bladder cancer, an increase in IL-17 generates a positive feedback loop that promotes malignant growth. Mohsen  et al. (2016) found a link between high IL 17 expression and early-stage cancer. In the case of TGF, the results of IHC staining show that roughly 12(50 percent) of cells in grade 1 are positive, while grade 4 has the lowest expression 2(8.33 percent).

Some IHC investigations have shown a different link between TGF- β 1 and certain diseases, while others have found distinct TGF- β 1 protein patterns. TGF has been shown to increase tumorigenicity in a variety of tumor forms. In vivo, prostate cancer cells, for example, exhibit a high amount of TGF. In addition, as previously reported, overexpression of TGF in a rat prostate cancer cell line boosted tumorigenicity. Apart from that, Kim  et al. [2] revealed the likely process of cancer beginning in their work. They also established a strong correlation between the loss of TGF β signaling receptor expression and the number of grading levels in prostate cancer.

HEF1 Genes Expression in Urothelial Carcinoma and Non-Tumorous Bladder Tissues

HEF1 gene expressions (fold change) were found to be 3.072+0.868 s.d. in nontumorous bladder tissue and 11.219+9.48 s.d. in malignant tissue, soHEF1 gene expression was considerably (p<0.0001) higher in urothelial carcinoma than in non-tumorous bladder tissues (table 1).

The Relevance of Bladder Cancer Stage with HEF1 Gene Expression.

To clarify the relationship between urothelial carcinoma stage and HEF1 gene expression, a one-way ANOVA was performed on the data. Stage Ta and stage T1 (P=0.314) and stage Ta and non-tumorous tissue (P=0.19) do not significantly vary from one another, but there is a considerable difference between stage T1 and stage T2 (P=0.034) and between stage T2 and stage Ta (p=0.029) ( table 4).The mean fold change of  the up-regulated cases was 7.413+ 2.180 for stage Ta, 10.943+5.705 for stage T1  and  15.006+12.045 s.d. for stage T2.

In the current study, we observed up- regulation of HEF1 in urothelial carcinoma  with 11.219 folds compared to the normal epithelial tissues (3.072 folds) (table  1) indicating that the expression level of HEF1correlates with the tumorigenesis of urothelial carcinoma. The results show that HEF1 expression is upregulated in urothelial carcinoma patients, which is consistent with the findings of Qi Zhang.

Analysis of variance (ANOVA) revealed a significant difference (P=0.004) (table.3) between low and high grade, with higher expression levels (fold change) in high grade (15.74) than in low grade (10.44), suggesting a role for up-regulation of HEF1 in the carcinogenesis and prognosis of bladder cancer. The findings of our study were consistent with those of Qi Zhang.

Those in stage T2 exhibited a substantial (P=0.029) increase in HEF1 gene expression fold as compared to those in stage Ta. Also, it was clear that the level of HEF1 gene expression was significantly higher (P= 0.034) in stage T2 tumours than in stage T1 tumours (Table 4).

Table 1: The proportion and grade of IL1, IL 6, IL 8, IL 17, and TGF in BCP and in the control group

Table 2: Results of the relative expression of the FOXM1 and HEF1 genes in non-tumorous and cancerous bladder tissues

p<0.05 Highly significant difference

Table 3: HEF1 gene expression levels (mean of fold change) in non-tumorous bladder tissue and various stages of bladder cancer

*. At the 0.05 level, the difference in the mean is important.

Figure 1: Patients with bladder cancer were subjected to immunohistochemical labeling for IL 1, IL 6, IL 8, IL 17 and TGFin. (a) Immunohistochemical stain of IL-1. (b) Immunohistochemical stain of IL-6. (C) Immunohistochemical stain of IL-8. (D) Immunohistochemical staining of IL-17glioma with late antigen antibody. (E) Immunohistochemical stain of TGF

Acknowledgment

The authors would like to thank Dr. Qasim Sharhan for his technical assistance and scientific remarks.

1. Aldousari, S., and W. Kassouf. "Update on the Management of Non-Muscle Invasive Bladder Cancer." Cancer Urology Association Journal, vol. 4, 2010, pp. 56–64.

2. Kim, S.H., Forn et al. "Optimal Conditions for the Retrieval of CD4 and CD8 Antigens in Formalin-Fixed, Paraffin-Embedded Tissues." Journal of Molecular Histology, vol. 35, 2004, pp. 403–844.

3. Mohsen, M., Forn et al. "Breast Cancer in Iran: An Epidemiological Review." Asian Pacific Journal of Cancer Prevention, vol. 13, 2016, pp. 383–391.

4. Zhang, Q., Forn et al. "High Expression of HEF1 Is Associated with Poor Prognosis in Urinary Bladder Carcinoma." OncoTargets and Therapy, vol. 7, 2014, pp. 1319–1326.

5. Rahbar, A., Forn et al. "Human Cytomegalovirus Infection Levels in Glioblastoma Multiforme Are of Prognostic Value for Survival." Neuro-Oncology, vol. 57, 2013, pp. 36–42.

6. Chaturmohta, A., Forn et al. "Do Expression Profiles of Cytokines VEGF, TNF-α, IL-1β, IL-6 and IL-8 Correlate with Gallbladder Cancer?" Journal of Cancer Science and Clinical Oncology, vol. 2, 2015, pp. 1–8.

7. Sangai, T., Forn et al. "Biomarkers of Response to Akt Inhibitor MK-2206 in Breast Cancer." Clinical Cancer Research, vol. 18, 2012, pp. 5816–5828.

8. Reis, S.T., Forn et al. "Increased Expression of MMP-9 and IL-8 Are Correlated with Poor Prognosis of Bladder Cancer." Asian Pacific Journal of Cancer Prevention, vol. 8, 2012, pp. 12–18.

9. Robbins, and Cotran. Pathologic Basis of Disease. 9th ed., 2010, p. 979.

10. Chavey, C., Forn et al. "Oestrogen Receptor Negative Breast Cancers Exhibit High Cytokine Content." Breast Cancer Research, vol. 33, 2007, pp. 9–15.

11. Jin, Y., Forn et al. "Molecular and Genetic Analysis of JIL-1, a Novel Nuclear Kinase." Molecular Biology of the Cell, vol. 8, 1997, pp. 15–45.

12. Snoussi, K., Forn et al. "Genetic Variation in IL-8 Associated with Increased Risk and Poor Prognosis of Breast Carcinoma." Cancer Letters, vol. 67, 2006, pp. 16–21.