DNA sequences drive their various functions through post-transcriptional processes, using mRNA or lncRNA (long non-coding RNA), and this accommodates the gene network by using various RNA types. However, the tools necessary to regulate RNA molecules are few. Likewise, RNA knockdown techniques that can be artificially controlled have not been extensively explored. Here, we investigated a novel light-inducible synthetic system based on CRISPR-Cas13a that can be used for RNA knockdown and binding in cancer cells. Based on the techniques of synthetic molecular biology, we constructed a light sensor, which efficiently induced Cas13a protein expression after blue light illumination. We also chose a lncRNA, Metastasis-associated Lung Adenocarcinoma Transcript 1 (MALAT1), as the functional target and detected it in bladder cancer 5637 and T24 cells in order to demonstrate the application of our synthetic system. Fluorescence reporter assays and real-time quantitative PCR (qRT-PCR) were used to detect the expression of the target gene. Phenotypic experiments were also used to test the effects of our synthetic system in bladder cancers. The results showed that our synthetic light-switchable system could inhibit the expression of MALAT1, and the fluorescence activity of enhanced green fluorescent protein. Our novel system provides a new technique to study RNA functions in gene networks and for precise tumor treatments.
Numerous attempts for detection of circulating tumor cells (CTC) have been made to develop reliable assays for early diagnosis of cancers. In this study, we validated the application of folate receptor α (FRα) as the tumor marker to detect CTC through tumor-specific ligand PCR (LT-PCR) and assessed its utility for diagnosis of bladder transitional cell carcinoma (TCC). Immunohistochemistry for FRα was performed on ten bladder TCC tissues. Enzyme-linked immunosorbent assay (ELISA) for FRα was performed on both urine and serum specimens from bladder TCC patients (n = 64 and n = 20, respectively) and healthy volunteers (n = 20 and n = 23, respectively). Western blot analysis and qRT-PCR were performed to confirm the expression of FRα in bladder TCC cells. CTC values in 3-mL peripheral blood were measured in 57 bladder TCC patients, 48 healthy volunteers, and 15 subjects with benign urologic pathologies by the folate receptor α ligand-targeted PCR. We found that FRα protein was overexpressed in both bladder TCC cells and tissues. The levels of FRα mRNA were also much higher in bladder cancer cell lines 5637 and SW780 than those of leukocyte. Values of FRα were higher in both serum and urine specimens of bladder TCC patients than those of control. CTC values were also higher in 3-mL peripheral blood of bladder TCC patients than those of control (median 26.5 Cu/3 mL vs 14.0 Cu/3 mL). Area under the receiver operating characteristic (ROC) curve for bladder TCC detection was 0.819, 95 % CI (0.738-0.883). At the cutoff value of 15.43 Cu/3 mL, the sensitivity and the specificity for detecting bladder cancer are 82.14 and 61.9 %, respectively. We concluded that quantitation of CTCs through FRα ligand-PCR could be a promising method for noninvasive diagnosis of bladder TCC.
Emerging evidences have indicated that long non-coding RNAs (lncRNAs) are potential biomarkers, playing important roles in the development of cancer. LncRNA Activated in RCC with Sunitinib Resistance (lncARSR) is a novel lncRNA that functions as a potential biomarker and is involved in the progression of cancers. However, the clinical significance and molecular mechanism of lncARSR in bladder cancer (Bca) remains unknow. In this study, we discovered that lncARSR was significantly up-regulated in bladder cancer. In addition, increased expression of lncARSR was positively correlated with higher histological grade and larger tumor size. Further experiments demonstrated that suppression of lncARSR attenuated the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) process of Bca cells. Mechanistically, lncARSR was mainly located in the cytoplasm and acted as a miRNA sponge to positively modulate the expression of Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) via sponging miR-129-5p and subsequently promoted the proliferation and metastasis of Bca cells, thus playing an oncogenic role in Bca pathogenesis. In conclusion, our study indicated that lncARSR plays a critical regulatory role in Bca cells and lncARSR may serve as a potential diagnostic biomarker and therapeutic target for bladder cancer.
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