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Ring-shaped RNA circ-NFATC3 and application thereof

A circular and versatile technology, applied in the fields of molecular biology and oncology, can solve the problems of difficult early diagnosis, difficult early diagnosis of liver cancer, and low surgical cure rate, and achieve accurate diagnosis of liver cancer, reduced cell migration rate and cell invasion ability. Effect

Active Publication Date: 2016-12-07
GUANGZHOU FOREVERGEN BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, comprehensive treatment centered on surgical treatment is still the only hope for long-term survival of patients with primary liver cancer. However, the current treatment of liver cancer is difficult for early diagnosis, low surgical radical rate, high recurrence rate, and poor prognosis.
One of the main reasons is the difficulty of early diagnosis. As the only serological marker widely accepted for the diagnosis or detection of liver cancer, the sensitivity of AFP in the diagnosis of liver cancer can only reach about 50%. Many factors eventually lead to more than 70% of patients in the Liver cancer has entered the advanced stage at the time of first visit, thus losing the opportunity for radical surgical treatment including surgical resection or liver transplantation

Method used

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  • Ring-shaped RNA circ-NFATC3 and application thereof
  • Ring-shaped RNA circ-NFATC3 and application thereof
  • Ring-shaped RNA circ-NFATC3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: RT-PCR reaction detection of circ-NFATC3 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 12,000g 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,...

Embodiment 2

[0048] Example 2: Detecting the expression of circ-NFATC3 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-NFATC3 divergent primer in Example 1 was used to amplify circular RNA circ-NFATC3, and the hsaGAPDH convergent primer in 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. ...

Embodiment 3

[0064] Example 3: Overexpression of circ-NFATC3 lentivirus and its stable cell line construction

[0065] 1. Construction of overexpressed circ-NFATC3 lentiviral vector: The complete linear sequence of circ-NFATC3 was synthesized in Shanghai Jierui Company, and the sequence was annealed into a double-stranded DNA fragment, which was inserted into the LVminiCirc vector through multiple cloning sites, and the recombinant plasmid was identified by sequencing , Control Negative control is LVminiCirc empty vector without insert 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 cultur...

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Abstract

The invention provides ring-shaped RNA circ-NFATC3 and application thereof. The nucleotide sequence of a ring-shaped RNA circ-NFATC3 gene is as shown as SEQ ID NO:1. Besides, the invention provides a medicine composition, a liver cancer diagnosis kit and application of the circ-NFATC3 gene serving as a target gene to preparing medicine for treating liver cancer. By means of the circ-NFATC3 gene and expression products of the circ-NFATC3 gene as liver cancer diagnosis markers, liver cancer diagnosis is more accurate and quicker, and a new therapeutic target and a new treatment means are provided for liver cancer treatment by means of the circ-NFATC3 gene as the target gene for preparing medicine for treating 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-NFATC3 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] So far, comprehensive treatment centered on surgical treatment is still the only hope for long-term survival of patients with primary liver cancer. However, the current treatment of liver cancer is difficult for early diagnosis, low surgical radical rate, high recurrence rate, and poor prognosis...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61K31/7105A61P35/00C12Q1/68
Inventor 吴文哲何重华罗景燕殷豪杨畔赖炳权
Owner GUANGZHOU FOREVERGEN BIOTECH CO LTD
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