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Circular RNA (Ribonucleic Acid) and application thereof

A kind of circular, purposeful technology, applied to circular RNA, can solve the problem that the function of circular RNA is poorly understood, and achieve the effect of inhibiting the proliferation of liver cancer cells

Active Publication Date: 2020-11-06
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about the role of circular RNAs in liver cancer. Therefore, it is necessary to conduct in-depth research on circular RNAs in hepatocellular carcinoma, and to screen out circular RNAs that can be used as therapeutic targets to realize the diagnosis and treatment of hepatocellular carcinoma.

Method used

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  • Circular RNA (Ribonucleic Acid) and application thereof
  • Circular RNA (Ribonucleic Acid) and application thereof
  • Circular RNA (Ribonucleic Acid) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]In this example, the inventor designed a specific primer pair capable of amplifying circDLC1 (CircBase ID: hsa_circ_0135718), and used the primer pair to amplify the circular RNA of the circDLC1 gene, and confirmed the splicing of the circular RNA by the method of generation sequencing site (back-splice site), and then through RNase R digestion experiments, it was determined that circDLC1 was expressed as a circular RNA molecule with a closed circular structure.

[0040] The specific experimental plan is as follows:

[0041] 1. Extraction and concentration determination of total cellular RNA

[0042] (1) When the cells in the 6-well plate exceed 90%, discard the old culture medium, wash once with PBS, add 1mL Trizol to each well, and then transfer to a 1.5mL EP tube;

[0043] (2) Place the EP tube at room temperature for 10 minutes so that the nucleic acid protein complex can be fully separated;

[0044] (3) Add 0.2mL chloroform to the EP tube, shake it for about 10s, ...

Embodiment 2

[0070] In this example, the expression difference of circDLC1 in the liver cancer tissue and the corresponding para-cancerous tissue was detected by fluorescent quantitative PCR (QPCR), and it was found that the expression of circDLC1 in the liver cancer tissue was significantly lower than that in the para-cancerous tissue, indicating that circDLC1 can be used for the diagnosis of liver cancer .

[0071] The specific experimental plan is as follows:

[0072] 1. Extraction and concentration determination of total tissue RNA

[0073] Approved by the Ethical Review Committee of Sichuan University, written informed consent was provided to each patient according to the policy of the committee. After the patients were fully informed and signed the informed consent, 40 cases of liver cancer tissues and paired normal paracancerous tissues. After clinical collection, the samples were stored in liquid nitrogen tanks by pathological laboratory staff, and then transferred to a -80°C ref...

Embodiment 3

[0101] In this example, by constructing circDLC1 on a lentiviral vector dedicated to circular RNA, a stable cell line overexpressing the gene was established. Through the CCK8 proliferation experiment, it was found that after circDLC1 was overexpressed, the proliferation ability of liver cancer cells was significantly inhibited. Transwell migration and invasion experiments confirmed that the migration and invasion ability of liver cancer cells were significantly inhibited after overexpression of circDLC1; the above experiments showed that the circDLC1 gene and its expression products can be used as therapeutic targets for the treatment of hepatocellular carcinoma.

[0102] The specific experimental plan is as follows:

[0103] 1. Lentiviral Infection

[0104] (1) Take out the lentivirus frozen at -80°C and place it on ice until it melts;

[0105] (2) Take out the 24-well plate on which the cells to be infected are laid one day in advance, and discard the old culture medium; ...

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Abstract

The invention discloses a circular RNA (Ribonucleic Acid) and application thereof. The cDNA (complementary deoxyribonucleic acid) sequence of the circular RNA is disclosed by SEQ ID NO:1, and the structure of the circular RNA is an end-to-end circular structure formed in a way that a nucleotide sequence disclosed by SEQ ID NO:1 is sheared after transcription is carried out. Through QPCR (quantitative polymerase chain reaction), an expression difference situation of circDLC1 in hepatocellular carcinoma tissues and para-carcinoma tissues is detected so as to discover that the expression of the circDLC1 in the hepatocellular carcinoma tissues is obviously lowered than the expression in the para-carcinoma tissues, and the circDLC1 can be indicated to be used for hepatocellular carcinoma diagnosis. A migration and invasion experiment can prove that after the circDLC1 is subjected to overexpression, hepatocellular carcinoma cell migration and invasion ability is obviously inhibited so as toindicate that the circDLC1 can be used as a treatment target spot for diagnosing and treating the hepatocellular carcinoma cells; and a naked mouse subcutaneous tumor model, a naked mouse hepatocellular carcinoma in situ model and a naked mouse advanced stage transferring model are constructed to prove that circDLC1 overexpression has an inhibition function for proliferation, invasion and transferof hepatocellular carcinoma cells in the body of the naked mouse.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a circular RNA, a kit containing the circular RNA, a pharmaceutical composition, and the application of the circular RNA as a therapeutic target in the diagnosis and treatment of hepatocellular carcinoma. Background technique [0002] Liver cancer is one of the most common malignant tumors worldwide. Primary hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is the main form of liver cancer, accounting for about 90% of primary liver cancer. Postoperative recurrence and metastasis of liver cancer are the main reasons for poor prognosis, and its five-year survival rate is less than 20%. [0003] Circular RNAs (circRNAs) are a class of non-coding RNAs that do not have a 5' end cap and a 3' end poly(A) tail, and form a circular structure with covalent bonds. Since circular RNA has a closed circular structure, it is not easy to be digested by RNase R enzyme, and it can exi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/6886C12Q1/6851A61K45/00A61P35/00A61P35/04
CPCA61K45/00A61P35/00A61P35/04C12N15/113C12N2310/532C12Q1/6851C12Q1/6886C12Q2600/136C12Q2600/158C12Q2531/113C12Q2525/307C12Q2521/107
Inventor 曾勇袁克非刘海灵徐琳兰天李慧
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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