Application of reagent capable of promoting circular RNA (Ribonucleic Acid) circRNF13 expression to preparation of medicine for treating tongue squamous cell carcinoma
A tongue squamous cell carcinoma, ring-shaped technology, applied in the field of tumor molecular biology, can solve problems such as biological function and mechanism of action that have not been reported.
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Embodiment 1
[0046] Example 1, PCR sequencing determined the full-length sequence of circRNF13 in tongue squamous cell carcinoma cells
[0047] 1. Materials and methods
[0048] 1.1 Cell lines
[0049] Tongue squamous cell carcinoma cells Tca8113 and Cal27 were purchased from the Cell Center of Central South University and cultured under conventional conditions.
[0050] 1.2 Reagents and kits
[0051] TRIZOL TM Reagent (Invitrogen); Gel Recovery Kit (OMEGA); Reverse Transcription Kit (Promega); Proteinase K, DNase I, RNAsin, RNase A (GBICOL Company); Enzyme (Takara).
[0052] 1.3 Real-time quantitative PCR detection of circRNF13 expression in tongue squamous cell carcinoma cells
[0053] Total RNA was extracted, and 1 μg of RNA was reverse-transcribed into cDNA, followed by real-time fluorescent quantitative PCR. The forward primer of circRNF13 is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2, and the reverse primer is 5-GTGTAGACTTGTGTGGCTGA-3. As shown in SEQ NO:3.
[0054] The...
Embodiment 2
[0083] Example 2, circRNF13 inhibits cell cycle arrest and induces apoptosis in tongue squamous cell carcinoma cells
[0084] 1. Materials and methods
[0085] 1.1 Reagents and kits
[0086] Restriction enzymes Cla I and Sac II and T4DNA ligase were purchased from TakaRa Company; TRIZOL TM Reagent (Invitrogen); Plasmid Extraction Kit, Gel Recovery Kit (OMEGA); Reverse Transcription Kit (Promega); Proteinase K, DNase I, RNAsin, RNase A (GBICOL Company); Tetramethylazolazolium Blue ( MTT, Sigma); antibiotic G418 (Ameresc); cell cycle detection kit (Invitrogen), apoptosis detection kit (Invitrogene).
[0087] 1.2 Induction and culture of tongue squamous cell carcinoma drug-resistant cells
[0088] In the cell culture medium, a low dose of cisplatin was added, and the concentration of cisplatin was gradually increased. After a long period of induction culture, a drug-resistant cell line resistant to cisplatin was finally obtained.
[0089] 1.3 Construction of circRNF13 eukaryo...
Embodiment 3
[0171] Example 3, real-time fluorescent quantitative PCR method detection confirmed the down-regulation of circRNF13 in tongue squamous cell carcinoma
[0172] 1. Materials and methods:
[0173] 28 adjacent tongue squamous cell carcinoma tissues and 28 tongue squamous cell carcinoma tissues were collected, total RNA was extracted, 1 μg RNA was reverse transcribed into cDNA, and real-time fluorescent quantitative PCR was performed. The forward primer of circRNF13 is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2, and the reverse primer is 5-GTGTAGACTTGTGTGGCTGA-3. As shown in SEQ NO:3.
[0174] The GAPDH forward primer used for the control is 5'-ACCACAGTCCATGCCATCAC-3' as shown in SEQ NO:4, and the reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' as shown in SEQ NO:5.
[0175] Real-time fluorescence quantitative PCR reaction system
[0176]
[0177] Real-time fluorescent quantitative PCR reaction steps
[0178]
[0179]5 Plate read
[0180] 6 82°C 30sec
[0181] 7 Plate re...
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