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Application of reagents for detecting circular RNA circRNF13 in preparation of prognostic preparations for patients with tongue squamous cell carcinoma

A detection reagent and tongue squamous cell carcinoma technology, which is applied in the field of tumor molecular biology and can solve the problems of biological function and mechanism of action that have not been reported.

Active Publication Date: 2020-05-22
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

We found that the expression of circRNF13 changed most significantly, suggesting that circRNF13 may be related to cisplatin resistance in tongue squamous cell carcinoma, but so far, the biological function and mechanism of circRNF13 have not been reported

Method used

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  • Application of reagents for detecting circular RNA circRNF13 in preparation of prognostic preparations for patients with tongue squamous cell carcinoma
  • Application of reagents for detecting circular RNA circRNF13 in preparation of prognostic preparations for patients with tongue squamous cell carcinoma
  • Application of reagents for detecting circular RNA circRNF13 in preparation of prognostic preparations for patients with tongue squamous cell carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1, PCR sequencing determined the full-length sequence of circRNF13 in tongue squamous cell carcinoma cells

[0039] 1. Materials and methods

[0040] 1.1 Cell lines

[0041] Tongue squamous cell carcinoma cells Tca8113 and Cal27 were purchased from the Cell Center of Central South University and cultured under conventional conditions.

[0042] 1.2 Reagents and kits

[0043] TRIZOL TM Reagent (Invitrogen); Gel Recovery Kit (OMEGA); Reverse Transcription Kit (Promega); Proteinase K, DNase I, RNAsin, RNase A (GBICOL Company); Enzyme (Takara).

[0044] 1.3 Real-time quantitative PCR detection of circRNF13 expression in tongue squamous cell carcinoma cells

[0045] 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.

[0046] The...

Embodiment 2

[0070] Example 2, circRNF13 inhibits cell cycle arrest and induces apoptosis in tongue squamous cell carcinoma cells

[0071] 1. Materials and methods

[0072] 1.1 Reagents and kits

[0073] 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).

[0074] 1.2 Induction and culture of tongue squamous cell carcinoma drug-resistant cells

[0075] 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.

[0076] 1.3 Construction of circRNF13 eukaryo...

Embodiment 3

[0153] Example 3, real-time fluorescent quantitative PCR method detection confirmed the down-regulation of circRNF13 in tongue squamous cell carcinoma

[0154] 1. Materials and methods:

[0155] 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.

[0156] 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.

[0157] Real-time fluorescence quantitative PCR reaction system

[0158]

[0159] Real-time fluorescent quantitative PCR reaction steps

[0160]

[0161] After the reaction, the amplification curve and melting...

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Abstract

The invention discloses application of a reagent for detecting cyclic RNA circRNF13 in preparing a prognosis preparation for tongue squamous cell carcinoma patients, in particular to a preparation forprognosis of the tongue squamous cell carcinoma patients by using a real-time fluorescence quantitative analysis method. The survival analysis of the expression of the circRNF13 in tongue squamous cell carcinoma tissues and the survival time and status of the patients shows that the average survival time of patients who do not express the circRNF13 in the tongue squamous cell carcinoma tissues issignificantly shorter than that of patients with higher expression of the circRNF13. The circRNF13 is a molecular marker associated with the prognosis of the tongue squamous cell carcinoma; the circRNF13 is low in expression or is not expressed, which causes poor prognosis of the patients.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and specifically relates to the application of a reagent for detecting circular RNA circRNF13 in tongue squamous cell carcinoma tissue in preparation of a prognostic preparation for tongue squamous cell carcinoma. Background technique [0002] Human Genome Project and its follow-up DNA Element Encyclopedia Project (The Encyclopedia of DNAElements Project, ENCODE) research results show that protein-coding gene sequences only account for 1-3% of the human genome sequence, while most of the human genome can be transcribed The sequence is non-coding RNA (non-coding RNA, ncRNA). Although non-coding RNA does not encode protein, it has been attracting much attention in the field of biomedical research due to its extensive involvement in the regulation of gene expression in cells. The most studied are linear ncRNA molecules, which can be divided into microRNA (microRNA, miRNA, 19-23nt) and long no...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 曾朝阳郭灿熊芳刘凌云熊炜张姗姗龚朝建王裕民莫勇真廖前进张文玲李桂源
Owner CENT SOUTH UNIV
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