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

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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

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

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Example Embodiment

[0030] Example 1: RT-PCR reaction to detect the expression of circ-NFATC3 gene in liver cancer tissue.

[0031] The specific experimental plan is as follows:

[0032] 1. RNA extraction

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

[0034] 2) Add 200ul of chloroform to the supernatant, mix up and down vigorously for half a minute, and let it 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 water-phase RNA; 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 centrifuge at 12000g for 10 minutes at 4°C.

[0037] 5) Carefully remove the supernatant, taking care not to lose t...

Example Embodiment

[0048] Example 2: QPCR detection of circ-NFATC3 expression in liver cancer

[0049] 1. RNA extraction: same as Example 1;

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

[0051] 3. QPCR amplification experiment

[0052] 1) Experimental system:

[0053]

[0054] The circ-NFATC3divergent primer of Example 1 was used to amplify the circular RNA circ-NFATC3, and the hsaGAPDH convergent primer of Example 1 was used to amplify the internal reference gene.

[0055] 2) Reaction conditions:

[0056] The first step: 95℃2min

[0057] The second step (40 cycles): 95°C for 3 seconds, 60°C for 30 seconds

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

[0059] 3) On the computer for target gene amplification

[0060] 4) qPCR relative quantitative results

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

Example Embodiment

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

[0065] 1. Overexpression circ-NFATC3 lentiviral vector construction: Synthesize the circ-NFATC3 linear full sequence in Shanghai Jierui Company, the sequence is annealed into a double-stranded DNA fragment, inserted into the LVminiCirc vector through multiple cloning sites, and the recombinant plasmid is identified by sequencing , Control negative control is an empty LVminiCirc vector with no sequence inserted.

[0066] 2. Lentivirus packaging

[0067] (1) 24h before transfection, digest 293T cells in logarithmic growth phase with trypsin and pass them to a 10cm cell culture dish at 37℃, 5% CO 2 Cultivate in an incubator. It can be used for transfection when the cell density reaches 70% to 80% in 24 hours. Cell state is very important for virus packaging, so it is necessary to ensure good cell state and fewer passages.

[0068] (2) Change the cell culture medium to serum-free medium be...

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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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