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Application of reagents for promoting the expression of circular RNA circRNF13 in the preparation of drugs for the treatment of squamous cell carcinoma of the tongue

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.

Active Publication Date: 2019-10-18
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 promoting the expression of circular RNA circRNF13 in the preparation of drugs for the treatment of squamous cell carcinoma of the tongue
  • Application of reagents for promoting the expression of circular RNA circRNF13 in the preparation of drugs for the treatment of squamous cell carcinoma of the tongue
  • Application of reagents for promoting the expression of circular RNA circRNF13 in the preparation of drugs for the treatment of squamous cell carcinoma of the tongue

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses application of a reagent capable of promoting circular RNA (Ribonucleic Acid) circRNF13 expression to preparation of a medicine for treating tongue squamous cell carcinoma. A research proves that circRNF13 is overexpressed in tongue squamous cell carcinoma cells and the proliferation of the tongue squamous cell carcinoma cells can be inhibited. Therefore, a circRNF13 overexpression preparation is used for treating the tongue squamous cell carcinoma and has profound clinical significance and an important popularization and application prospect.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to the application of a reagent for promoting the expression of circular RNA circRNF13 in the preparation of drugs for treating 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 non-coding RNA (long non-codin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66A61K31/7105A61P35/00
CPCA61K31/7105A61P35/00C12N15/85Y02A50/30
Inventor 曾朝阳熊芳张姗姗龚朝建郭灿刘凌云熊炜王裕民莫勇真张文玲李小玲李桂源
Owner CENT SOUTH UNIV
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