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The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 10⁵ copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

MicroRNA (miRNA) can function as tumor suppressors or oncogenes, and also as potential specific cancer biomarkers; however, there are few published studies on miRNA in synovial sarcomas, and their function remains unclear. We transfected the OncomiR miRNA Precursor Virus Library into synovial sarcoma Fuji cells followed by a colony formation assay to identify miRNAs to confer an aggressive tumorigenicity, and identified miR-17-5p from the large colonies. MiR-17 was found to be induced by a chimeric oncoprotein SS18-SSX specific for synovial sarcoma, and all examined cases of human synovial sarcoma expressed miR-17, even at high levels in several cases. Overexpression of miR-17 in synovial sarcoma cells, Fuji and HS-SYII, increased colony forming ability in addition to cell growth, but not cell motility and invasion. Tumor volume formed in mice in vivo was significantly increased by miR-17 overexpression with a marked increase of MIB-1 index. According to PicTar and Miranda algorithms, which predicted CDKN1A (p21) as a putative target of miR-17, a luciferase assay was performed and revealed that miR-17 directly targets the 3-UTR of p21 mRNA. Indeed, p21 protein level was remarkably decreased by miR-17 overexpression in a p53-independent manner. It is noteworthy that miR-17 succeeded in suppressing doxorubicin-evoked higher expression of p21 and conferred the drug resistance. Meanwhile, introduction of anti-miR-17 in Fuji and HS-SYII cells significantly decreased cell growth, consistent with rescued expression of p21. Taken together, miR-17 promotes the tumor growth of synovial sarcomas by post-transcriptional suppression of p21, which may be amenable to innovative therapeutic targeting in synovial sarcoma.

MicroRNA (miRNA) can function as tumor suppressors or oncogenes, and also as potential specific cancer biomarkers; however, there are few published studies on miRNA in synovial sarcomas, and their function remains unclear. We transfected the OncomiR miRNA Precursor Virus Library into synovial sarcoma Fuji cells followed by a colony formation assay to identify miRNAs to confer an aggressive tumorigenicity, and identified miR-17-5p from the large colonies. MiR-17 was found to be induced by a chimeric oncoprotein SS18-SSX specific for synovial sarcoma, and all examined cases of human synovial sarcoma expressed miR-17, even at high levels in several cases. Overexpression of miR-17 in synovial sarcoma cells, Fuji and HS-SYII, increased colony forming ability in addition to cell growth, but not cell motility and invasion. Tumor volume formed in mice in vivo was significantly increased by miR-17 overexpression with a marked increase of MIB-1 index. According to PicTar and Miranda algorithms, which predicted CDKN1A (p21) as a putative target of miR-17, a luciferase assay was performed and revealed that miR-17 directly targets the 3-UTR of p21 mRNA. Indeed, p21 protein level was remarkably decreased by miR-17 overexpression in a p53-independent manner. It is noteworthy that miR-17 succeeded in suppressing doxorubicin-evoked higher expression of p21 and conferred the drug resistance. Meanwhile, introduction of anti-miR-17 in Fuji and HS-SYII cells significantly decreased cell growth, consistent with rescued expression of p21. Taken together, miR-17 promotes the tumor growth of synovial sarcomas by post-transcriptional suppression of p21, which may be amenable to innovative therapeutic targeting in synovial sarcoma.