Upregulation of miR-3658 in bladder cancer and tumor progression
Despite increasing advances in surgical techniques and adjuvant chemotherapies, bladder cancer remains the ninth leading cause of male malignancy-associated deaths worldwide. Several microRNAs (miRNAs) have been identified to be closely associated with the progression and prognosis of, and response to treatments in various human cancers. However, few studies have investigated the role of miR-3658 in bladder cancer. In this study, we examined the expression of miR-3658 in 96 pairs of bladder cancer tissues and adjacent non-tumor tissues via quantitative reverse-transcription polymerase chain reaction. Results showed that expression of miR-3658 was up-regulated in the bladder cancer tissues as compared with that in the corresponding control tissues (4.15 ± 2.78 vs 2.17 ± 1.14; P < 0.0001). Furthermore, higher miR-3658 expression was significantly associated with lymph node invasion, distant metastasis, histological grade, TNM stage, and tumor recurrence in bladder cancer (all P < 0.0001). miR-3658 expression was not associated with other clinicopathological variables such as age, gender, tumor size, and number (all P > 0.05). Our study revealed that miR-3658 overexpression is involved in tumor progression of bladder cancer, indicating that the miRNA possesses prognostic values.
Bladder cancer is a major unsolved public health problem, and is the ninth leading cause of male malignancy-related deaths. It is estimated that worldwide there are 429,800 new cases of and 165,100 deaths per year due to bladder cancer (Torre et al., 2015). Despite increasing advances in surgical techniques and adjuvant chemotherapies, prognosis of bladder cancer is extremely poor owing to high recurrence rate and metastatic potential (Herr, 1999; Sylvester et al., 2006; Cambier et al., 2016). Studies have shown that recurrence rate of bladder cancer is approximately 15-50%, and may even reach nearly 70% (Hisataki et al., 2000; Mehta et al., 2015). Therefore, it is vital to define novel prognostic biomarkers and new molecular drug targets in this disease.
MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs that are approximately 22 nucleotides in length. Studies have shown that miRNAs are closely associated with progression and prognosis of, and response to treatments in human cancer via transcriptional and post-transcriptional gene regulation (Calin and Croce, 2006; Meister, 2007). A single miRNA may target hundreds of mRNAs, and is therefore involved in multiple physiological and pathological processes, including cell proliferation, differentiation, apoptosis, and tumorigenesis (Calin et al., 2004). For example, miR-19 was identified to play crucial roles in rheumatoid arthritis and bladder cancer by targeting Toll-like receptor 2 and phosphatase and tensin homolog, respectively (Philippe et al., 2012; Feng et al., 2014). Accumulating evidence revealed that several miRNAs play important roles in the tumorigenesis and progression of various cancers by acting as tumor suppressors or oncogenes (Hede, 2005; Yu et al., 2010; Catto et al., 2011; Fan et al., 2013; Han et al., 2013; Drayton et al., 2014; Feng et al., 2014; Wang et al., 2013, 2015). By microarray analysis, we recently found several miRNAs to be associated with bladder cancer. Of these miRNAs, miR-3658 was significantly up-regulated with a 166-fold increase in expression as compared to that in the controls (Liu et al., 2016). Similarly, miR-3658 was reported to be over-expressed in multiple myeloma (Hao et al., 2015). However, few studies have examined miR-3658 in detail. The aberrant expression of miR-3658 and its correlations with clinical and pathological characteristics of bladder cancer are still poorly understood.
Therefore, the aim of the present study was to further explore the relationships between miR-3658 expression and bladder cancer.
MATERIAL AND METHODS
Patients and samples
The study consisted of 96 patients with urothelial carcinoma; disease was confirmed by surgical pathology. Between 2014 and 2015, patients who had undergone radical cystectomy at the Urology Department of the Second Hospital affiliated to Kunming Medical University were recruited into the study. Written informed consent was obtained from all subjects prior to enrollment, and the study protocol was approved by the local medical Ethics Committee. No patient had received preoperative chemotherapy or radiotherapy. For quantitative reverse-transcription polymerase chain reaction (qRT-PCR) we collected fresh bladder cancer tissues following radical cystectomy, and the corresponding adjacent non-tumor tissues from the macroscopic tumor margin were used as controls. All samples were placed immediately in liquid nitrogen and stored at -80°C until RNA extraction.
Clinicopathological parameters of the patients
All cases were classified according to the tumor-node-metastasis (TNM) system set by the Union International Cancer Control (UICC) in 2002. The urothelial carcinoma pathology-classification system (2004) set by the World Health Organization was used to classify tumor grade. The clinicopathological parameters for the 96 patients included age, gender, tumor size, tumor number, tumor occurrence (primary or recurrent), lymph node invasion, distant metastasis, histological grade, and TNM stage.
RNA extraction and qRT-PCR
Total RNA was extracted from frozen tissues, using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer protocol. The 260/280 OD was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Houston, TX, USA) to assess the concentration and purity of RNA extracted from the samples. Small nucleolar RNA (snRNA) U6 was used as an internal standard for normalization. To determine miR-3658 and snRNA U6 expression, qRT-PCR was performed using the SYBR PrimeScript miRNA RT-PCR kit (Takara Biotechnology Co. Ltd., Dalian, China); 2 μL RNA was added to the RT reaction system, and 2 μL cDNA served as the template for PCR amplification. Reactions were carried out in triplicates on an Applied Biosystems 7500 Real-Time PCR systems (Applied Biosystems, Carlsbad, CA, USA). All real-time PCR primers were synthesized by Sangon (Sangon Biotech, Shanghai, China). The primer sequences were as follows: miR-3658 forward: 5'-GTGGGGGTTTAAGAAAACACCAT-3' miR-3658 and reverse: 5'-GTGCAGGGTCCGAGGT-3'; U6 forward: 5'-GCTTCGGCAGCACATATACTAAAAT-3' and U6 reverse: 5'-GTGCAGGGTCCGAGGT-3'. The qRT-PCR cycling parameters were as follows: pre-denaturation at 95°C for 30 s; 40 cycles of denaturation at 95°C for 5 s, annealing and extension at 60°C for 20 s. Amplification specificity was confirmed by analyzing the melting curve. The cycle threshold (Ct) value was calculated, and the 2-ΔΔCt method [ΔCt = Ct (miR3658) - Ct (U6-snRNA)] was followed to quantify the relative expression of miR-3658 (Livak and Schmittgen, 2001).
Data are reported as means ± SD. Comparisons of miR-3658 expression in bladder cancer and adjacent non-tumor tissues were performed by the Student t-test. Correlations between miR-3658 expression and the clinicopathological parameters were assessed by the two-sample Student t-test. Statistical analyses were performed using the SPSS 19.0 software (version 19.0, IBM, Armonk, NY, USA) for Windows, and P < 0.05 was considered statistically significant.
MiR-3658 expression in bladder cancer tissues
The OD 260/280 ratios of all RNA samples ranged between 1.8 and 2.2, which confirmed that the purity and quality of RNA was adequate for subsequent experiments. We examined miR-3658 expression in 96 pairs of bladder cancer and adjacent non-tumor tissues by qRT-PCR. Results showed that expression levels of miR-3658 were significantly increased in the bladder cancer tissues as compared with that in the corresponding adjacent non-tumor tissues (4.15 ± 2.78 vs 2.17 ± 1.14), with a median 1.91-fold increase (t = 6.46, P < 0.0001) (Figure 1).
Comparison of miR-3658 expression in bladder cancer tissues and corresponding adjacent non-tumor tissues. Data are reported as means ± SD and analyzed by the Student t-test. *P ˂ 0.0001.
Correlation between miR-3658 expression and clinicopathological parameters
The clinicopathological parameters of 96 patients with bladder cancer are summarized in Table 1. To further investigate the correlation between miR-3658 expression and the clinicopathological parameters, the relative expression levels of miR-3658 in the bladder cancer tissues and the corresponding control tissues were analyzed. The analysis showed that high expression of miR-3658 in bladder cancer was significantly associated with aggressive tumor phenotypes such as tumor recurrence (t = 7.172, P < 0.0001), lymph node invasion (t = 11.739, P < 0.0001), distant metastasis (t = 6.497, P < 0.0001), histological grade (t = 22.021, P < 0.0001), and TNM stage (t = 5.722, P < 0.0001). However, miR-3658 expression level in bladder cancer tissue was not associated with other clinicopathological variables such as age, gender, and tumor size and number (all P > 0.05). These results are summarized in Table 2. In brief, our data indicate that miR-3658 was significantly up-regulated in the bladder cancer tissues, and its overexpression was closely associated with the clinicopathological features of tumor progression in the patients with bladder cancer.
Clinical pathological parameters of 96 patients with bladder cancer.
|Clinical features||N (%)|
|Mean age (years, mean ± SD)||45.61 ± 10.55|
|Radical cystectomy||96 (100)|
|≥3 cm||53 (55.2)|
|<3 cm||43 (44.8)|
|Tumor histological grade|
|Lymph node invasion|
Correlation between miR-3658 expression and different clinical pathological parameters of bladder cancer.
|Parameters||N||Relative miR-3658 level||t value||P value|
|≥50||36||3.22 ± 1.28|
|<50||60||3.76 ± 1.82||1.701||0.092|
|Male||74||2.61 ± 1.21|
|Female||22||2.53 ± 0.92||0.286||0.775|
|Single||42||3.27 ± 1.92|
|Multiple||54||3.55 ± 0.79||0.888||0.378|
|≥3 cm||53||2.82 ± 0.27|
|<3 cm||43||2.62 ± 1.56||0.831||0.411|
|Tumor histological grade|
|G1 + G2||49||1.19 ± 0.32|
|G3||47||4.10 ± 0.85||22.021||<0.0001|
|T1 + T2||61||2.14 ± 2.03|
|T3 + T4||35||3.98 ± 1.12||5.722||<0.0001|
|Lymph node invasion|
|Positive||64||2.14 ± 0.77|
|Negative||32||3.98 ± 0.62||11.739||<0.0001|
|Positive||86||2.03 ± 0.84|
|Negative||10||3.83 ± 0.72||6.497||<0.0001|
|Primary||63||1.48 ± 0.56|
|Recurrent||33||3.93 ± 1.92||7.172||<0.0001|
Data are reported as means ± SD.
Bladder cancer is one of the most common urogenital tumors, and shows poor prognosis owing to cancer recurrence. Prevention of tumor recurrence and inhibition of disease progression are challenges that need to be overcome in the treatment of bladder cancer. Currently, clinicopathological parameters such as tumor stage, tumor grade, tumor number, and prior histories of tumor recurrence are used to evaluate prognosis of bladder cancer. However, their sensitivity and reliability have recently been questioned (Sanguedolce et al., 2015). Therefore, novel molecular markers are urgently needed to improve the prediction accuracy of disease progression and prognosis in bladder cancer, and can be used to explore more efficient, individualized therapeutic strategies.
miRNAs are single-strand, non-protein-coding RNA molecules. Studies have shown an association between miRNA deregulation in various tumors and clinicopathological characteristics, including clinical outcomes. It was further suggested that miRNAs can act as oncogenes or anti-oncogenes, and are involved in cellular proliferation, differentiation, apoptosis, tumor invasion, and metastasis (Hede, 2005; Yu et al., 2010; Catto et al., 2011; Fan et al., 2013; Han et al., 2013; Drayton et al., 2014; Feng et al., 2014; Wang et al., 2013, 2015Yamada et al., 2011; Han et al., 2011, 2013Yoshino et al., 2013; Drayton et al., 2014; Feng et al., 2014; Inoguchi et al., 2014; Wang et al., 2010, 2014Zhang et al., 2013, 2014ab). These studies suggest that miRNAs may be useful as prognostic biomarkers and therapeutic targets in bladder cancer. Therefore, it is an important first step to explore miRNAs expression and its association with clinicopathological characteristics of bladder cancer.
Human miR-3658 is located on chromosome 1, and its reference mature sequence is 5'-UUUAAGAAAACACCAUGGAGAU-3' (GenBank accession No. MIMAT0018078), according to the miRBase database. Meiri et al. (2010) first identified miR-3658 via high throughput sequencing in RNA extracted from 23 breast, bladder, colon, and lung solid-tumor specimens. Subsequently, Hao et al. (2015) found that miR-3658 is dysregulated in multiple myeloma (MM) serum samples, via miRCURY LNA microRNA arrays and qRT-PCR; they showed that the RQ value (2-ΔΔCt) of miR-3658 was significantly increased in the MM group as compared with that in the healthy donor group (1.74 vs 0.12, P = 0.025). These findings indicated that miR-3658 plays an important role in the development and progression of various tumors.
However, the role of miR-3658 in bladder cancer remains unknown. To explore miR-3658 expression and its clinical significance in bladder cancers, we measured miR-3658 expression in 96 pairs of bladder cancer tissues and their adjacent non-tumor tissues. We found that miR-3658 was significantly up-regulated in the bladder cancer tissues. In addition, we also found that higher miR-3658 expression was closely associated with aggressive clinicopathological characteristics of bladder cancer, such as high tumor grade, poor TNM stage, lymph node, and distant metastasis, as well as tumor recurrence. These data suggested that miR-3658 may act as an oncogenic miRNA, and may be used as potential prognostic biomarker for bladder cancers.
In this study, we indicated the potential connection between miR-3658 and aggressive clinicopathological characteristics of bladder cancer for the first time. However, the precise mechanisms of miR-3658 in gene regulation, such as its target genes and gene-gene interactions, remain unclear. Recently, Cipollini et al. (2014) computationally predicted that the alleles of rs1819698, in strong linkage disequilibrium with rs1417608, disrupt the binding site for miR-3658 (DeltaS 0.571). Single nucleotide polymorphism of rs1417608 located in the 3'-untranslated regions of the hormone regulation gene 3-beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) was associated with a nearly 2-fold increase in bladder cancer risk (Andrew et al., 2012). HSD3B2 encodes NAD+-dependent microsomal enzymes that stimulate biosynthesis and inactivation of many types of steroid hormones. Simard et al. (2005) reported that HSD3B2 is predominantly expressed in the adrenal glands and gonads, and its deficiency could affect the synthesis of adrenal glands and gonads. Morbidity due to bladder cancer is approximately three times more prevalent in males than in females (Kaufman et al., 2009; Torre et al., 2015), but the reasons remain unknown. Miyamoto et al. (2012) reported that both androgens and androgen receptors are involved in the occurrence and development of bladder cancer. However, our data showed that miR-3658 expression in bladder cancer tissues was not associated with gender (P = 0.775), suggesting that functions of miR-3658 may target downstream genes in the gene-regulatory network.
We utilized the web tool in the TargetScan7.0 database (
In conclusion, we report that up-regulation of miR-3658 is closely associated with aggressive clinicopathological characteristics such as lymph node invasion, distant metastasis, tumor recurrence, poor histological grade, and high TNM stage in bladder cancer. This indicates that miR-3658 possesses potential prognostic values. However, further investigations should be performed to better understand the significance of this miRNA and its role in bladder cancer.