Role of a schistosoma haematobium specific microRNA as a predictive and prognostic tool for bilharzial bladder cancer in Egypt

Urinary bladder cancer is a common malignancy in Egypt, thus reliable methodologies are required for screening and early detection. In this study, we analyzed the gene expression of a Schistosoma hematobium specific microRNA “Sha-miR-71a” and mitogen-associated protein kinase-3 (MAPK-3) in the urine samples of 50 bladder cancer patients and 50 patients with benign bilharzial cystitis. Fifty control subjects were also tested. Indirect hemagglutination test (IHA) diagnosed 70% of studied cancer cases as bilharzial associated bladder cancer (BBC), while histopathological examination detected only 18%. Urinary Sha-miR-71a & MAPK-3 revealed enhanced expression in BBC (p-value = 0.001) compared to non-bilharzial bladder cancer (NBBC) cases. Patients with chronic bilharzial cystitis exhibited a significant increase in gene expression compared to those with acute infection (p-value = 0.001). Sha-miR-71a and MAPK-3 showed good sensitivity and specificity in the diagnosis of BBC when analyzed by the receiver operating characteristic (ROC) curve. They were also prognostic regarding malignancy grade. Both biomarkers showed a positive correlation. Our results revealed that IHA is a reliable test in the diagnosis of bilharziasis associated with bladder cancer, and that Sha-miR-71a and MAPK-3 provide non-invasive specific biomarkers to diagnose BBC, as well as a potential role in testing bilharzial patients for risk to develop cancer.

MicroRNAs (miRNAs) are key regulators in post-transcriptional suppression. They are small non-coding RNAs, approximately 22 nucleotides in length 11 . Their aberrant expression has been recently used as novel biomarkers detected in the urine of BBC and NBBC patients 12 . Moreover, the role of some small RNA in carcinogenesis and refining prognosis in cell lines have been elucidated 13 . It has been reported that some miRNAs can predict treatment merit and prognosis 10 .
As known as the Schistosoma specific miRNAs has an important role in the underlying pathogenesis of schistosomiasis as well as the parasite development, this drew the attention to the possibility of their applications in diagnostics 14 .
Recent studies have provided some valuable information about the small RNA complement of Schistosoma hematobium 15,16 . Sha-miR-71a is a Schistosoma hematobium specific micro RNA with the highest level of transcription in adult worm with no sex bias 16 . Such novel discoveries will facilitate further studies on the parasite genome and host-parasite cross-talk. Early detection for bilharziasis and subsequently decreasing cancer risk is the most efficient strategy for fighting bladder cancer in endemic areas.
Our study is based on the fact that bladder cancer is prevalent in Egypt, with bilharziasis being a neglected disease, though schistosomiasis is an important cause of oncogenesis. Bladder cancer is a preventable cancer if we can reach a non-invasive protocol for early detection, better follow up, and a reliable prognostic marker that can improve management plan and hence an improved outcome. Inspite of the very limited data about the small non coding RNAs sequences of this neglected parasite, we aim that this study and other subsequent studies would enlighten this field of research.

Patients and methods
Study design. A multi-center, prospective, cross-sectional study was conducted between February 2019 and January 2020 at the National Institute of Urology and Nephrology, Ain Shams University Hospitals, and Badr Teaching Hospital, Egypt. Patient selection. One hundred patients with urinary symptoms (hematuria, urinary retention, dysuria… etc.), who were planned to undergo cystoscopy for suspicion of bladder cancer (BC) or benign bilharzial cystitis (Bil), were selected.
Demographic data and medical history were recorded for each patient. Informed consent was obtained from all participants.
Two minors were included in this study (13&15 year old), and an informed consent was obtained from their parents.
Study groups. Patients were classified as: • 50 cases with bladder cancer were chosen based on cystoscopy and histopathologic examination.
• 50 patients with bilharzial cystitis were also included based on positive ova in urine and/or positive IHA tests in collected urine and blood samples respectively.
Exclusion criteria. In bladder cancer group Patients who have received chemotherapy, or with previously diagnosed other malignancy within the past 5 years. Control group of 50 healthy volunteers were asked to provide urine and blood samples.

Samples collection.
i. Urine samples all subjects were asked to provide urine samples before instrumentation or bladder tumor excision. Freshly voided urine samples (100 ml) were collected in sterile containers. ii. Blood samples 3 ml were withdrawn from patients using sterile labeled vacutainers. iii. Tissue samples forty-eight transurethral cystoscopic resection of bladder tumor (TURBT) samples and two radical cystectomy samples were included.
Urine examination. Urine samples were microscopically examined for detection of bilharzial ova.
Anti-bilharzial antibodies. Blood  Statistical analysis. Data analysis was performed using Statistical Package for the Social Sciences software (SPSS, Version 19, and Chicago, IL, USA). Quantitative data were summarized using mean, median, standard deviation (SD), minimum and maximum, while frequency/count and relative frequency/percentage were used for categorical data. Comparisons were performed using chi-square, t-tests or ANOVA tests to examine the relation between qualitative variables. p-value < 0.05 was considered significant.

Results
Patients'data. Sixty-seven men and 33 women were included in this study. The mean age for the cancer group is 61 ± 10 years, while for the bilharzial cystitis group it is 32.5 ± 13. Out of the 50 bladder cancer patients, 80% were males. The clinicopathologic and demographic characteristics are shown in Table 1.

Microscopic urine examination and serodiagnosis of bilharziasis by IHA. Urine and serological
examinations of the samples confirmed the absence of bilharzial infestation in the healthy control group. On the other hand, 70% of patients (35 out of 50), diagnosed with bladder cancer (by cystoscopy and histopathological examination) were positive for anti-Schistosoma antibodies (BBC) by the IHA test, with 51.4% having high antibody titer (> 1/640). Bilharzial ova were detected microscopically in 44% of the bladder cancer group, 86% of which showed positive indirect hemagglutinin test (38% of bladder cancer cases) ( Supplementary Fig. S1).
Regarding the bilharzial cystitis group, bilharzial ova was detected in 58% of cases and IHA was found positive in 42% (Table 1).
Histopathological examination. Cystitis group. In inflammatory mass lesions, Schistosoma ova were histologically detected in only nine cases. While features of proliferative cystitis with Brunn's nests, cystitis cystica, and cystitis glandularis, with, or without, dysplasia were noted in 50 cases (Fig. 1).
Bladder cancer group. Forty-eight samples were provided by transurethral resection of bladder tumor (TURBT) while two were radical cystectomy specimens. Six cases were pure squamous cell carcinoma (SCC), four lowgrade superficial, non-invasive papillary transitional cell carcinoma and 40 cases high-grade transitional cell www.nature.com/scientificreports/ carcinoma (TCC), High grade TCC attributed to 80% of the cancer cases, six of which were associated with squamous features (Fig. 1).
Regarding the staging, seven cases were Ta, only one case was T1, 34 cases were T2, and 8 cases were T3b. As to the nodal status, the two radical cystectomy cases were representing 80% of the cancer cases, six of which show associated squamous features (Fig. 1).  www.nature.com/scientificreports/ Regarding the staging, seven cases were Ta, only one case was T1, 34 cases were T2, and 8 cases were T3b. As regards nodal status, the two radical cystectomy cases were N0. Bilharziasis was histologically confirmed in only 9 cases (18%).
Urinary Sha-miR-71a gene expression levels in the studied groups. Comparing the expression levels of miR-71a (indicated by RQ values) between studied groups, the BC group showed higher RQ level (median: 15.8), compared to benign bilharzial cystitis group (median: 0.73) with significance value p = 0.003. Comparing gene expression levels of miR-71a in BBC and NBBC, a highly significant difference was noted, with enhanced expression in the BBC group. Within the bladder cancer group, transitional cell carcinoma cases showed enhanced gene expression of Sha-miR-71a compared to those with squamous cell carcinoma, and cases with high-grade malignancy varied significantly in gene expression of Sha-miR-71a compared to those with low-grade malignancy (Fig. 2).
Urinary Sha-miR-71a was more expressed in patients with chronic bilharzial infection, and in those with high IHA titer compared to those with recent infection and low antibody titer respectively, showing a highly significant difference (p = 0.001) ( Table 2).
Urinary MAPK3 gene expression levels in the studied groups. MAPK-3 gene was significantly expressed in the bladder cancer group, compared to bilharzial cystitis group. Testing the differential gene expression of MAPK-3 in BBC and NBBC, a highly significant difference was noticed with enhanced expression in the www.nature.com/scientificreports/ BBC group. Within the bladder cancer group, transitional cell carcinoma cases had enhanced gene expression of MAPK-3 compared to those with squamous cell carcinoma, and cases with high-grade malignancy varied significantly in gene expression of MAPK-3 compared to those with low-grade malignancy (Fig. 2). Urinary MAPK-3 was more expressed in patients with chronic bilharzial infection, and in those with high IHA titer compared to those with recent infection and low antibody titer respectively, showing a highly significant difference (p value = 0.001) ( Table 2).
Pearson's correlation analysis showed a strong correlation between Sha-miR-71a and its gene target MAPK-3 in both the bilharzial bladder cancer group and the bilharzial cystitis group, (Fig. 4).

Discussion
Bladder cancer in Egypt has its unique features, in part because of Schistosoma hematobuim's infestation. Its high prevalence requires the continuous search for more specific, accurate and non-invasive biomarkers for early detection and better prognostic outcomes.
There are several misbeliefs concerning its association with bilharziasis, and subsequently the lines of management 19 . Published researches in Egypt are either old studies suggesting the prevalence of SCC 20 , recent studies claiming control of schistosomiasis 21 , or recent studies suggesting that there is no difference between BBC and NBBC cancer regarding disease pathogenesis, prognosis, and management [22][23][24] .
Hereby, we have decided to search for the possible defects in the methodology of previous studies and to fill the literature gaps that have led to unreal proposed facts affecting our daily practice.
Firstly, urine and blood samples were collected from patients suspected of bilharzial bladder cystitis, attending the urology clinic in Badr Teaching Hospital, National Urology Institute, and Ain Shams Teaching Hospitals. Only those with positive ova in urine and/or positive IHA tests were included in this group, which reached 50 samples. After centrifugation of urine samples, the cell pellet was washed with PBS and kept at − 80 °C after adding RNAlater (Qiagen, USA) for further genetic study.
Microscopic examination of urine samples confirmed the presence of ova in 29 out of the 50 patients. The other 21 cases showed positive IHA tests, with no ova detected by microscopic urine examination. Accordingly, this group was sub-classified into acute and chronic cystitis. We postulate that these 2 tests should be done as a screening, complementing each other in endemic areas to cover the spectrum of acute and chronic infestations. It is noteworthy that 100% of these patients came from rural areas. Only nine cases were histologically confirmed as bilharzial in the cystoscopic biopsies reflecting the marginal role of histopathology in the grouping of cases as bilharzial and non-bilharzial.
In our study, cases of bilharzial cystitis included two 13 and 15-year old boys reflecting new bilharzial infestations. Noteworthy, our cases were referred to hospitals in Cairo, where the possibility of receiving patients of rural residency acquiring this infestation is much less than in other rural hospitals. This should draw attention to the importance of having a national screening program. There should be a sensitive method to detect the actual burden of bilharziasis in Egypt, especially with the persistent prevalence of bladder cancer 19 .
Secondly, we collected urine and blood samples from patients who were candidates for cystoscopic resection/ biopsy due to suspicion of bladder cancer (hematuria and bladder mass by U/S). Samples of this group were for patients attending urology clinics at the National urology institute and Ain Shams teaching hospitals. Cystoscopic specimens were histopathologically examined and 50 cases with proved bladder cancer were included in the study. The majority of cancer cases (80%) were males. This is aligned with other studies confirming the prevalence of such neoplasia among males 19,25 . As regards the age, cancer was reported about three decades later than that of the cystitis group (61 ± 10 years for the cancer group versus 32.5 ± 13 for the cystitis group). This is attributed Table 2. Comparative analysis between the expression level of miR-71a and MAPK-3 in patients with bilharzial cystitis. n number, χ 2 Chi-square test value, HS highly significant difference, S significant difference, NS non significant difference.

Bilharzial cystitis
Acute infection (IHA negative) (n = 29) 0. www.nature.com/scientificreports/ to the long term complication of the chronic inflammatory process. This comes in agreement with previous data about the incidence of bladder cancer which increases with age, particularly between the age of 50 and 70 years 26 . Also, all our gathered cancer cases presented from urban areas, while patients presenting with the inflammatory lesions lived in rural areas. This could be attributed to the fact that bilharzial infestation requires an agricultural environment. Mass immigration from rural to urban areas can explain the presence of cancer in urban areas (Table 1).
Based on the results of the IHA test, cancer patients were classified as BBC and NBBC 17,19 . It is worthy to note that histopathologic confirmation of bilharziasis is reported in only 18% of cancer cases in comparison with 70% by IHA. This again reflects the poor sensitivity of histopathology as a diagnostic tool for bilharziasis 19 .
Using more than one method for the confirmation or exclusion of bilharziasis in the inflammatory and neoplastic cases brings a higher degree of accuracy in the categorization of the cases as bilharzial and non-bilharzial. This is different from what is published in the recent Egyptian articles. This variable was frequently neglected though it is an important parameter in the methodology 22 .
Limited knowledge of the genome of S. haematobium has for long hindered the discovery of this parasite which is considered neglected, compared to other schistosomes. One of the earliest studies in 2011 27 was able to silence two genes in S. haematobium using RNA interference. Such experiment pointed that it is possible to manipulate the parasite genes for the purpose of study and search for novel anti-parasite treatments.
The identification of molecular biomarkers like miRNAs has proved to be of value in recent years since genetic and epigenetic dysregulations are regarded as the main contributors in the initiation and progression of cancer 28 . Biomarkers have been approved by the FDA to be used in the detection of bladder cancer. However, one parameter is not enough to provide clinicians with the necessary information 29 . www.nature.com/scientificreports/ Increased knowledge of Schistosoma hematobium small non-coding RNA sequences 16 may provide insights into the parasite-host interactions which might enable the development of novel strategies to lessen its deleterious pathogenesis and progression to malignancy.
In this study, gene expression of a Schistosoma hematobium derived micro RNA "Sha-miR-71a" and its gene target MAPK-3 mitogen-activated protein kinase-3 16 was measured in urine samples of patients diagnosed with bladder cancer (BBC and NBBC) as well as patients with bilharzial cystitis using real-time reverse transcription RT-PCR. To our knowledge, this study is the first to investigate a Schistosoma hematobium-specific small noncoding RNA and its role in the pathogenesis of chronic cystitis and bladder cancer.
Sha-miR-71a is the most highly transcribed small non-coding microRNA in both males and females adult worm 16 . Using bioinformatics tools, miRanda v.3.3a 29 and PITA 16 , the gene targets of this microRNA in the host were determined and mitogen-activated protein kinase -3 "MAPK-3" was selected. This gene target was chosen because dysregulated MAPK pathway is involved in bladder cancer 30 . Also, Miranda 3.3a score was > 300 and PITA score was < − 10 indicating valid binding sites 16 . To assess its expression level in patients enrolled in this study. MAPK is known to have aberrant gene expression in bladder cancer and to play a role in tumorigenesis 10 . This raises the question about the role of Sha-miR-71a in the initiation of cancer by targeting the mitogenactivated protein kinase, a protein that enhances cellular proliferation.
Using the 2 −∆∆Ct method, relative gene expression was calculated. Both genes (Sha-miR-71a and MAPK-3) were found to be significantly expressed in cancer cases more than benign cystitis cases (Fig. 2). A highly significant difference was noted in the expression level of both genes between BBC &NBBC with enhanced expression in the BBC group. This indicates its specificity in the diagnosis of bilharzial bladder cancer.
Within the bladder cancer group, transitional cell carcinoma cases had enhanced gene expression of Sha-miR-71a and MAPK-3 compared to those with squamous cell carcinoma. This reflects the changing pattern of bladder cancer in Egypt because the prevalent cancer in our cases is TCC (88%) where SCC is progressively declining. We do not believe that this is attributed to the control of bilharziasis for several reasons. Firstly; even with efficient control; the previous burden of bilharzia infestation a few decades before will lead to the emergence of new cancer cases nowadays. Secondly, new fresh cases are still being reported. Thirdly, 88% of patients who were classified as BBC were transitional cell carcinoma, and lastly, the expression of schistosoma specific microRNA (Sha-miR-71 a) was significantly increased in TCC cases compared to its expression in SCC cases. Also, noteworthy to mention that nodal metastasis was not reported in any of our cases. This could be attributed to the limiting host immune response 31 .
We also noticed that cases with high-grade malignancy varied significantly in gene expression of Sha-miR-71a and MAPK-3 compared to those with low-grade malignancy (Fig. 2) and expression levels were increased in high grade cancer (RQ value was significantly different from low grade cancer cases). This draws the attention to this micro RNA as having a prognostic value.
We should note here that the median RQ of both Sha-miR-71a and MAPK-3 significantly increased in cases with chronic bilharzial cystitis (cases with positive IHA and no ova in urine) compared to acute cases (Table 2), with MAPK-3 significantly rising in BBC (Fig. 2). This means that MAPK-3 and epigenetic regulator sha-miR-71a, both must be elevated to diagnose BBC. This finding can direct us to validate a protocol that ensures close follow up of cases with bilharzial cystitis for prevention and early cancer detection.
The ROC curve analysis revealed urinary sha-miR-71a and MAPK-3 to be diagnostic for BBC at a cut off 10.5 and 6.2 with 88%, 87% sensitivity and 100%, 100% specificity, respectively. They were also of prognostic www.nature.com/scientificreports/ value regarding malignancy grade at a cut off 10.5 and 6.2 with 87%, 88% sensitivity and 99%, 99% specificity, respectively. Both urinary biomarkers were found to be statistically correlated. They can be potential biomarkers providing a non-invasive, easy and specific tool that can be of diagnostic and prognostic value.
In previous studies 7,10,12 , genomic instability due to schistosomiasis and aberrant miRNAs produced by the host in response to schistosomiasis infection were studied. Thus such epigenetic regulators mediate disease pathogenesis and its carcinogenesis and can be used as potential biomarkers 32 . Another study was performed on Schistosoma japonicum miRNAs, their role in cancer initiation and how this can lead to new interventional strategies to halt ongoing pathogenesis 33 .
Dysregulated miRNAs and their possible targets involved pathogenesis of muscle-invasive bladder cancer have also been identified, which can be of value in modifying therapeutic strategies 10 . A miR-192-5p was described as a promising therapeutic target since its overexpression inhibited the growth of bladder cancer cells 32 .
In another study, urinary sFas was pointed to as a non-invasive diagnostic marker for BBC 34 . In a study in 2018 13 , a circular RNA (Cdr1) was used for therapeutic purposes, by targeting miR-135a, up-regulating p21 and thus decreasing proliferation of bladder cancer cells.
Also another study 35 suggested that aberrant DNA methylation of cancer genes, like RARb2 and APC can be of value as urinary molecular markers for early detection of bilharzial and non bilharzial bladder cancer.

Conclusion and recommendations
1. The oncogenic role of bilharziasis is underestimated in Egypt. 2. There is a changing pattern of bladder cancer in Egypt with increased levels of TCC and decreased levels of SCC. Schistosomiasis remains a major cause of tumorogenesis. 3. BBC is a preventable disease if reliable tools for screening are provided. 4. It is highly recommended to implement a national screening program for schistosomiasis with subsequent screening and continuous follow up of those with increased cancer risk. This approach could decrease the burden of bladder cancer in Egypt. 5. A pilot study on a small scale can provide the required needs as an assessment for a national program. 6. Urinary MAPK-3 &its epigenetic regulator, Sha-miR-71a can provide diagnostic non-invasive specific markers to differentiate BBC from NBBC. 7. A suggested plan for screening patients with cystitis is as follows: Perform IHA and microscopic urine examination, followed by urinary Sha-miR-71a and MAPK-3 genetic testing for positive cases. High expression levels should be considered at risk to develop cancer.