Relapse and Survival in Bladder Cancer Patients Undergoing microRNA-129 and microRNA-145 Assays

Objective: The lack of indicators to measure tumor’s invasive biological behavior is an important issue. The aim of this study was to examine the effect of miRNAs 129 and 145 on tumor progression as well as patient survival. Method: Seventy five breast cancer (BC) patients and 75 controls were included in this research. Two miRNA expressions were estimated using real-time PCR. Biomarkers for BC detection was tested using ROC curves and AUC. Result: miR-129 and miR-145 expressions were significant. miR-129 and miR-145 classifiers (AUC = 0.943 and 0.748, respectively) help diagnose BC. Unlike miR-145, miR-129 did not affect the Kaplan–Meier survival curve analysis for progression-free survival at the end of the trial. The development of transitional cell carcinoma disease was found to have a strong correlation with miR-145 in both univariate and multivariate Cox regression analyses. Additionally, infiltrating + invasive urothelial carcinoma was also found to be correlated with miR-145. Conversely, elevated miR-129 expression in BC patients did not lead to an increase in cancer-specific recurrence or mortality, as observed in both univariate and multivariate Cox regression studies. Conclusion: The miRNA signature can help detect survival-associated miRNAs and develop BC miRNA therapeutics.


Materials and Methods
In this study, 150 participants were included: 75 with BC and 75 healthy volunteers confirmed using cystoscopy.This research was conducted at the Clinical Urology Department, Faculty of Medicine, Menoufia University, from November 2021 to December 2022.U6 controlled miR-145 and miR129.The 2_DDCt technique (Livak and Schmittgen, 2001) evaluated miR-145-5p and miR129-5p levels on an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA, USA).

Statistical Analysis
Computer data were analyzed using IBM SPSS 20.0.A percentage and number represented quantitative data.Only BC deaths were examined.At the last follow-up, censored patients did not attain the endpoint.The survival curves were generated using the Kaplan-Meier method.Survival distribution tests were conducted on subgroups using the log-rank method.Multivariate Cox proportional hazards regression analysis examined miRNA levels' predictive power after controlling for other factors.Significant differences between populations were observed at confidence levels above 95% (p < 0.05) in all two-sided tests.The survival study used Fisher's Exact or Monte Carlo correction, Student's t-test, Mann-Whitney test, and Chi-square test.Data significance was at the level of 5%.

Results
The participants included 75 BC patients (mean: 68.93 ± 8.31 years), with 51 males and 24 females.Of 75 healthy volunteers, 47 males and 28 females were free of chronic diseases and had a mean age of 67.19 ± 6.14 years.Moreover, 75 BC cases were grouped by age (45-73 years) and smoking (25 negative and 50 positive).BC patients and controls shared gender, age, and smoking rates.
Significant variations in urea, creatinine, and Hb levels were found between patients and controls (p ≤ 0.001).Table 2 shows that the case group had significantly lower miR-129 and miR-145 expressions than the control group (p = 0.001).

Discussion
According to the latest data from the Egyptian National Cancer Institute registry, the prevalence of BC is 10.1% [20].BC, which is the sixth most common cancer in the    [21].Complexity and limited therapeutic efficacy make BC, the most common urinary system cancer, worse [22].Thus, BC mechanisms and new treatments must be understood.Ta, T1, and T2-4 are muscle-invasive cancers, and malignancies exist.Long-term follow-up shows that benign, noninvasive papillary tumors seldom become muscle-invasive, up to 60% [23].Many high-throughput studies examined genetic alterations and gene expression in BC progression [3].miRNAs regulate mammalian gene expression and physiology [24,25].Numerous studies demonstrated that improperly produced miRNAs disrupt well-controlled cellular RNA networks, promoting cancer cell growth, progression, and metastasis.Cancer cells' aberrant miRNAs and RNA network modifications explain growth and metastasis.BC cell growth requires dysregulated miRNAs [14].In the above scenario, we examined miR-129 and miR-145 expression levels as diagnostic, prognostic, and therapeutic biomarkers.
According to Cohen and Brown [38], 40% hematuria was found.Rafique and Javed [39] found 78.6% hematuria, while Ragab et al. [40] reported 72.5%.Gupta et al. [41] found painless hematuria in 40% of BC patients.The majority of BC patients who undergo cystoscopy and provide sufficient urine samples have microhematuria.The tumors were histologically classified using the WHO (1999)/ISUP urothelial neoplasm grading system [42].First-line testing includes cystoscopy and urine cytology.Cystoscopy is the most effective method for staging and diagnosing BC.Cancers can be classified into high-grade and low-grade categories [43].Based on aggressiveness, low-grade cancer cells grow slowly, seem normal, and operate like healthy cells, whereas high-grade cells expand quickly, look disordered, and are more likely to go into the bladder muscle layer.
CT helps in determining the localization of tumors [47].CT displays BC diagnostics, tumor stage, and therapeutic selection vascular structure.Liu et al. [48] reported tumor CT size ranging from ≤3 cm for 60% to >3 cm for 40%.CT accuracy depends on bladder tumor lesion size.Many studies revealed that microRNAs (miRNAs/ miRs) promote oncological and nononcological illnesses through biological signaling networks.miRNAs are now of interest to BC researchers [49].Small, noncoding RNAs impede translation, lowering target gene expression.In this study, we investigated the effects of miR-129 on gene expression [50].The study found that BC patients had less miR-129 and miR-145 expression than those in controls.
Screening and therapeutic follow-up should be replaced with routine miR-129 and miR-145 deployment.Urine or plasma tests can detect noninvasive symptoms.Furthermore, we need more participants to confirm our findings.The miRNA signature may improve survivalassociated miRNA research and BLC miRNA target-based therapies.
In conclusion, human BC malignancies depended on miR-145 and miR-129 for carcinogenesis, progression, histological pattern, grade, CT tumor size, recurrence, survival, and mortality.It may also be a future BC biomarker used for screening, prognosis, and identifying treatment targets.

Figure 2 .
Figure 2. Kaplan-Meier Survival Curve for Progression-Free Survival with MiRNA 129 and 145 a and b.No significant difference in progression-free survival between patients with low and high micro-RNA 129 expression was found at the end of the study (P > 0.246, Mean= 11.250, 77.5% & 11.429, 88.6%), while there was a significant difference in progression-free survival between patients with low and high micro-RNA 145 expression at the end of the study (P<0.001,mean 10.718, 66.7% & 12.0, 100%).

Table 3 .
Cox Regression Univariate and Multivariate for Case Group Relapse Parameters ).

Table 4 .
Univariate and Multivariate Cox Regression Analysis for Case Group Mortality Parameters