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Ringlike RNA circ-CCNY and application thereof

A circular and versatile technology, applied in the field of molecular biology and oncology, to achieve the effect of accurate diagnosis and slowed proliferation of liver cancer

Active Publication Date: 2016-12-14
GUANGZHOU FOREVERGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There have not been any reports about the circ-CCNY function yet

Method used

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  • Ringlike RNA circ-CCNY and application thereof
  • Ringlike RNA circ-CCNY and application thereof
  • Ringlike RNA circ-CCNY and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: RT-PCR reaction detection of circ-CCNY gene expression in liver cancer tissue.

[0031] The specific experimental plan is as follows:

[0032] 1. RNA extraction

[0033] 1) Tissue processing: Take about 10 mg of tissue and add 1 ml Trizol, homogenize with a homogenizer; centrifuge for 15 minutes at 12000 g, and take the supernatant.

[0034] 2) Add 200ul chloroform to the supernatant, mix vigorously up and down for half a minute, and let stand for 3 minutes.

[0035] 3) Centrifuge at 12000g for 15 minutes at 4°C. At this time, it can be seen that the lysate is divided into three layers: the upper layer is RNA in the aqueous phase; the middle layer is DNA, lipids, etc.; the lower layer is cell residues, proteins, polysaccharides, etc.

[0036] 4) Take the supernatant into a new EP tube; add an equal volume of isopropanol, mix well, let stand for 10 minutes, and then centrifuge at 12000g for 10 minutes at 4°C.

[0037] 5) Carefully remove the supernatant, be...

Embodiment 2

[0048] Example 2: Detecting the expression of circ-CCNY in liver cancer by QPCR

[0049] 1. RNA extraction: with embodiment 1;

[0050] 2. cDNA reverse transcription: same as Example 1;

[0051] 3. QPCR amplification experiment

[0052] 1) Experimental system:

[0053]

[0054] The circ-CCNY divergent primer of Example 1 was used to amplify the circular RNA circ-CCNY, and the hsaGAPDH convergent primer of Example 1 was used to amplify the internal reference gene.

[0055] 2) Reaction conditions:

[0056] Step 1: 95°C for 2 minutes

[0057] Step 2 (40 cycles): 95°C for 3 seconds, 60°C for 30 seconds

[0058] The third step 60-95 ℃ melting curve

[0059] 3) Amplify the target gene on the machine

[0060] 4) qPCR relative quantitative results

[0061] The formula for calculating the relative expression of the target gene is: 2-△△Ct=2-[(△Ct)Test-(△Ct)Control]. Ct object is the Ct value of the target gene, and Ct housekeeper is the Ct value of the housekeeping gene. △C...

Embodiment 3

[0064] Example 3: Overexpression of circ-CCNY lentivirus and its stable cell line construction

[0065] 1. Construction of overexpressed circ-CCNY lentiviral vector: Synthesize the full linear sequence of circ-CCNY, anneal the sequence into a double-stranded DNA fragment, insert it into the LV-Circ vector through multiple cloning sites, and identify the recombinant plasmid by sequencing, and the Control is negative The control is the LV-Circ empty vector without inserting the sequence.

[0066] 2. Lentiviral Packaging

[0067] (1) 24 hours before transfection, digest 293T cells in the logarithmic growth phase with trypsin, transfer to 10cm cell culture dish, 37°C, 5% CO 2 Cultured in an incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection. Cell state is critical for virus packaging, so good cell state and low passage times need to be guaranteed.

[0068] (2) Replace the cell culture medium with serum-free medium before transfect...

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Abstract

The invention provides ringlike RNA circ-CCNY and application thereof. The nucleotide sequence of the circ-CCNY gene is as shown in SEQ ID NO: 1; the expression level of the ringlike RNA circ-CCNY is obviously reduced by detecting the expression condition of the circ-CCNY gene in a liver cancer patient; and the multiplication rate of the ringlike RNA circ-CCNY is obviously reduced by comparing liver cancer cells for expressing the RNA circ-CCNY gene with control liver cancer cells. The ringlike RNA circ-CCNY gene and an expression product thereof serve as markers of diagnosing liver cancer, so that diagnosis of the liver cancer is more accurate and quicker, and the ringlike RNA circ-CCNY gene as a target gene for preparing drugs capable of curing the liver cancer provides a new therapeutic target and therapy pathway for curing the liver cancer.

Description

technical field [0001] The invention belongs to the fields of molecular biology and oncology, and in particular relates to a circular RNA circ-CCNY and its application. Background technique [0002] Primary hepatocellular carcinoma (Hepatocellular carcinoma, HCC, hereinafter referred to as liver cancer) is one of the most common malignant tumors worldwide. About 740,000 patients are newly diagnosed with hepatocellular carcinoma each year worldwide, and about 690,000 patients die of liver cancer at the same time. Among them, my country accounts for 55% of the annual new cases in the world, becoming one of the regions with the highest incidence of liver cancer in the world. [0003] Circular RNA (ciucular RNA, circRNA) is a new member of the RNA family that is different from traditional linear RNA. It is a non-coding RNA molecule that does not have a 5' end cap and a 3' end poly(A) tail, and forms a circular structure with a covalent bond. . The latest research shows that c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/7105A61P35/00C12Q1/68
CPCA61K31/7105A61K48/00A61P35/00C12N15/113C12Q1/68C12N2310/10C12N2320/30C12Q1/6886C12Q2600/158C12Q2600/178C12N2310/532
Inventor 罗景燕刘波姚志成唐毅李伟琴赖炳权
Owner GUANGZHOU FOREVERGEN BIOTECH CO LTD
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