TERT Promoter Mutation And SMYD3 Expression In Urothelial Carcinoma

BackgroundUrothelial carcinoma is one of the most common malignant tumor of urinary system in recent years, and its incidence is increasing in worldwide, But at present its pathogenesis was not yet clear. The regulation of telomere length and telomerase activity in human aging and plays an important role in diseases such as tumor. Telomeres exists in eukaryotic chromosomes, which is composed of tandem repetitive DNA (8-20 KB) sequence (TTAGGG) and related proteins. Telomeres could provide the transcription of DNA buffer and plays an important role in protect the chromosomes from fusion and degradation, chromosomal location, copying, protection, regulation of cell growth and life. Telomerase is a ribonucleoprotein composed of proteins and RNA, which is a RNA dependent DNA polymerase, telomere fragment can be added to the end of the telomere. As telomerase to maintain the necessary structure and function components-telomere binding protein plays an important role, and one of the most important is telomerase protein complex, which is made up of TRF1 (TTAGGG repeats factor 1), TRF2, TIN2, RAP2, TPP1 and POT1, can control the 3’end and t-ring form. Telomerase function is mainly composed of telomerase reverse transcriptase (TERT) and telomerase RNA component through the synthesis of telomere repeat sequences in the end of the chromosome. TERT gene on chromosome 5p15.33, which can carry as much as 2 times normal somatic cells TERT gene copy, the abnormal will appear when gene reduce or increase replication. TERT copied increase will lead to the occurrence of tumor. More than 30% of human malignant tumors carry more than three times TERT replication. TERT promoter is rich in GC, lack of TATA box and CAAT box, it contains at least five SP1 binding sites, two E-boxes and one transcription start site, which can be combined with multifunctional transcription factor TFII-I, studies have shown that TERT transcription mechanism is controlled by multiple factors, such as c-Myc, SP1, Survivin, STAT3, EWS-ETS, HIF-1 alpha, Mad/Max, P53, WT1, Rb, TGF-beta/Smad, API. TERT promoter point mutations plays an important role in the process of the activation of telomerase. Recent studies have shed light on the importance of epigenetic events in the carcinogenesis and progression of the urinary system, including facilitation of PI3K-AKT signaling by histone-modifying enzymes. SET and MYND domain-containing protein 3(SMYD3) is a histone methyltransferase (HMT), which recognizes and occupies the binding motif/s in the promoter region of its downstream target genes, and di-/tri-methylates H3-K4, thereby leading to transcriptional activation. Expression of SMYD3 is undetectable or very weak in many types of normal human tissues, whereas over-expression of SMYD3 has been shown to be associated with development and progression of colorectal, hepatocellular and breast cancer. And SMYD3 is critical for these carcinomas. In this study we explore TERT promoter mutation and SMYD3 expression in urothelial carcinoma and the correlation between TERT promoter/SMYD3 and clinicopatholog-ical parameters.ObjectiveIn this study, we will detect TERT promoter mutation and the expression of histone methylation enzyme SMYD3 expression in the patients with urothelial carcinoma, and the relationship between TERT promoter/SMYD3 and clinicopathological parameters including prognosis of the urothelial carcinoma patients. Through the experiments in vitro, we aim to do the preliminary study on the biological function and mechanism of SMYD3 in bladder cancer.MethodsUsing Sanger Squencing to detect 98 cases of renal pelvis carcinoma,122 cases of ureteral carcinoma,182 cases of bladder urothelial carcinoma tissue specimens, normal adjacent tissue and 20 cases of ureteral carcinoma patient’s preoperative urine, 16 cases of renal pelvis carcinoma preoperative urine,102 cases of bladder cancer patient’s preoperative urine and 46 cases postoperative urine. Analysis the urothelial cancer TERT promoter mutation and the relationship between TERT promoter mutant of carcinoma and clinical pathological; Detect FGFR3-7/0/15 mutation in bladder cancer and analyze its correlation with TERT promoter mutation.We detected the expression of SMYD3 mRNA using real-time quantitative PCR (Realtime-PCR) in 25 bladder cancer specimens. And the SMYD3 protein expression was determined using western blot and immunohistochemistry. Associations between SMYD3 immunostaining level and clinicopathological parameters were analyzed with ^2-test. In vitro, two typical cell line, T24 and 5637 bladder cancer cell line, were employed to exam the involvement of SMYD3 in BC using small interfering RNA (siRNA) and short hairpin RNA (shRNA). After in acquring reliable interference effects, we detected the SMYD3 expression in BC cells using Western Blot. We determined the relationship between SMYD3 and BC cell proliferation using cloning formation experiment(Colony formation assay) and Xenograft model of BC in vivo. And we using transwell cell migration (Cell migration assay), invasion (Matrigel invasion assay) experiment detected the relationship of migration and invasion between SMYD3 and BC cell; Then, we detected the cell apoptosis and cycle change in BC cell after SMYD3 was interfered by siRNA or shRNA using flow cytometry assay and Western Blot. At last, we detected the relationship between SMYD3 and PI3K-AKT pathway after SMYD3 was interfered by siRNA or shRNA in BC cell using Western Blot.ResultsHere we analyzed 220 UTUC patients [98 with renal pelvic carcinoma (RPC) and 122 with ureter carcinoma (UC)] and developed a Competitive Allele-Specific TaqMan PCR (castPCR) for urinary assay. We identified C228T or C250T mutations in 42 of 98 (43%) RPC and 23 of 122 (19%) UC tumors. Distant metastases were significantly correlated with UTUC patients harboring TERT promoter mutations (P= 0.001). C228T were detected in 6/10 and 9/10 of urine samples from patients with mutation-carrying tumors using Sanger sequencing and castPCR, respectively. When urine samples from 70 BC patients were analyzed together, the castPCR and Sanger sequencing sensitivity of urinary C228T assay was 89% and 50%, respectively (P< 0.001). Then, we analized tumors from 182 Han Chinese patients with UBC and urine samples from 102 patients for mutations in the TERT promoter, FGFR3 and TERT mRNA expression in tumors and/or urine. TERT promoter and FGFR3 mutations were identified in 87 of 182 (47.8%) and 7 of 102 (6.7%) UBC cases, respectively. In 46 urine samples from patients with TERT promoter mutation-carrying tumors, the mutant promoter was detected in 24 (52%) prior to operation and disappeared in most examined urine samples (80%) 1 week after operation. TERT mRNA was detected in urine derived from 46 of 49 patients (94%) that was analyzed before operation independently of the presence of TERT promoter mutations. Collectively, FGFR3 mutations occur at a very low rate in Han Chinese UBC.The results of real-time quantitative PCR experiment show that the expression level of SMYD3 mRNA in bladder cancer(BC) tissue (0.152+0.031) is significantly higher than that in adjacent to carcinoma (0.00039+0.0015). SMYD3 protein expression is strong in nucleus from 8 out of 65 (12%) cases and in cytoplasm of BC tumors from 58 out of 65 (89%) cases and while only 5 out of 65 (8%) of matched normal tissues exhibited a positive cytoplasmic staining (P< 0.01), and none of them were positive for nuclear staining. BC tissues at pT2, pT3 and pT4 expressed a higher level of SMYD3 than pTl and pTa tumors(P< 0.01). Moreover, significantly higher levels of SMYD3 expression were observed in G3 tumors compared to those in G2 and G1 ones (P= 0.032), although there was no significant difference between G1 and G2 (P = 0.53).we carried out western blot analysis of 25 freshly frozen BC tumors and their matched normal counterparts from the above patients. An enhanced SMYD3 expression was observed in 24 of 25 tumors compared with their corresponding non-cancerous tissues. In vitro, The proliferation of T24 and 5637 cells transfected with SMYD3 siRNA or stably expressing SMYD3 shRNA were monitored by cell counting or CCK8-assay. We observed that SMYD3 depletion with siRNA led to a significant increase in cell grow inhibition rate. Moreover, T24 and 5637 cells stably expressing SMYD3 shRNA had a significantly lower number of colonies than the same cells transfected with control shRNA. The efficient silencing of SMYD3 expression by siRNA and shRNA was verified. Consistent with the in vitro data, significantly smaller tumors were observed in mice receiving T24 and 5637 cells expressing SMYD3 shRNA 8-week after xenotransplantation. FACS analysis showed that depletion of SMYD3 by shRNA induced apoptosis in T24 and 5637 cells. SMYD3 knockdown led to a remarkable accumulation of cells in the GO/Gl phase, or blockade of DNA replication in the G1-S check point. With western blots on BC cells stably expressing SMYD3 shRNA/control shRNA, we found that SMYD3 exerted its anti-apoptosis effect through up-regulating the expression of Bcl-2 and the down-regulating the expression of p53, Bax, Bad. We further investigated some cell cycle regulators in BC cells and observed that SMYD3 depletion led to a decreased cyclinDl, CDK4, cyclinEl, CDK2 protein expression and enhanced p21, p27 expression. Cell migration and invasion assays indicate that cells expressing SMYD3 siRNA exhibited reduced ability to pass through the pore. Using a preliminary gene expression microarray analysis, we found that PI3K-AKT pathway was repressed by SMYD3 depletion in T24 and 5637 cells. SMYD3 knockdown by shRNA led to a reduced phosphorylated mTOR, which activates the downstream target P70S6K and 4E-BP1, and the activation of P70S6K leads to the regulation of genes involved in intermediary metabolism, protein accretion and cell growth.ConclusionTERT promoter mutations occur in UTUCs with a high frequency in RPCs and predict distant metastasis. castPCR assays of the mutation are a useful tool for urine-based diagnostics of urological malignancies. Han Chinese patients with UBC have relatively low TERT promoter mutation frequency compared with patients in Western countries, and simultaneous detection of both mutant TERT promoter and TERT mRNA improves sensitivity and specificity of urine-based diagnosis. FGFR3 mutations occur at a very low rate in Han Chinese UBC and cannot serve as diagnostic markers for Chinese patients. SMYD3 achieves its oncogenic role in BC tumorigenesis through inducing AKT activity. By linking the over-expression of SMYD3 in BC and SMYD3 induced AKT downstream gene expression, our study provide a unique avenue for controlling BC by direct inhibition of SMYD3 expression or activity.