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Cyclic RNA circ-ZKSCAN1 use

A circular and versatile technology, applied in the field of circular RNA, can solve the problems of lack of research on liver cancer, achieve the effect of increased cell migration rate and cell invasion ability, and accurate diagnosis of liver cancer

Active Publication Date: 2016-04-27
GUANGZHOU FOREVERGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that ZKSCAN1 can promote the invasion and metastasis of gastric cancer, but it has not been studied in liver cancer

Method used

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  • Cyclic RNA circ-ZKSCAN1 use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: RT-PCR reaction detection of circ-ZKSCAN1 gene expression in liver cancer tissue.

[0035] The specific experimental plan is as follows:

[0036] 1. RNA extraction

[0037] 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.

[0038] 2) Add 200ul chloroform to the supernatant, mix vigorously up and down for half a minute, and let stand for 3 minutes.

[0039] 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.

[0040]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.

[0041] 5) Carefully remove the supernatant, ...

Embodiment 2

[0052] Example 2: Detection of the expression of circ-ZKSCAN1 in liver cancer by QPCR

[0053] 1. RNA extraction: with embodiment 1;

[0054] 2. cDNA reverse transcription: same as Example 1;

[0055] 3. QPCR amplification experiment

[0056] 1) Experimental system:

[0057]

[0058] The circ-ZKSCAN1 divergent primer of Example 1 was used to amplify the circular RNA circ-ZKSCAN1, and the hsaGAPDHconvergent primer of Example 1 was used to amplify the internal reference gene.

[0059] 2) Reaction conditions:

[0060] Step 1: 95°C for 2 minutes

[0061] Step 2 (40 cycles): 95°C for 3 seconds, 60°C for 30 seconds

[0062] The third step 60-95 ℃ melting curve

[0063] 3) Amplify the target gene on the machine

[0064] 4) qPCR relative quantitative results

[0065] The formula for calculating the relative expression of the target gene is: 2-ΔΔCt=2-[(ΔCt)Test-(ΔCt)Control]. Ct object is the Ct value of the target gene, and Ct housekeeper is the Ct value of the housekeepin...

Embodiment 3

[0068] Example 3: Construction of shcirc-ZKSCAN1 lentivirus and its stable cell line

[0069] 1. Construction of shcirc-ZKSCAN1 lentiviral vector: The shRNA sequence was synthesized in Shanghai Jierui Company, the sequence was annealed into a double-stranded DNA fragment, and inserted into the LV008 (pLL-U6-shRNA-CMV-GFP-Puro) vector through the multiple cloning site , recombinant plasmids were identified by sequencing. The nucleotide sense strand and antisense strand sequences of shcirc-ZKSCAN1 are shown in SEQ ID NO:10 and 11. The nucleotide sense strand and antisense strand sequences of the Control negative control are shown in SEQ ID NO: 12 and 13.

[0070] 2. Lentiviral packaging

[0071] (1) 24 hours before transfection, 293T cells in the logarithmic growth phase were digested with trypsin, passaged to a 10 cm cell culture dish, and cultured in a 37° C., 5% CO2 incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection. Cell sta...

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Abstract

The present invention provides cyclic RNA circ-ZKSCAN1 use, the objective existence of circ-ZKSCAN1 gene is first confirmed; expression status of the circ-ZKSCAN1 gene in a patient with hepatocarcinoma is detected to find that the expression level of the circ-ZKSCAN1 gene is significantly reduced; hepatoma cells transfected with circ-ZKSCAN1 gene shRNA sequence are compared with control hepatoma cells transfected with an empty vector to find that the proliferation rate, cell migration ratio and cell invasion ability and the like are significantly improved. The circ-ZKSCAN1 gene and a circ-ZKSCAN1 gene expression product are used as a hepatoma diagnostic marker, so that hepatoma diagnosis is more accurate and rapid, and the circ-ZKSCAN1 gene as a target gene for preparing hepatoma treatment drugs provides a new therapeutic target and therapeutic approach.

Description

technical field [0001] The present invention relates to a circular RNA, in particular to the use of a circular RNA circ-ZKSCAN1. 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. One of the main reasons is the difficul...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P35/00C12Q1/68
CPCC12N15/113C12N2310/14C12Q1/6886C12Q2600/106
Inventor 罗景燕刘波姚志成李伟琴赖炳权
Owner GUANGZHOU FOREVERGEN BIOTECH CO LTD
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