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Uses of circular RNA circ-PTPRA

A circular and versatile technology, applied in the fields of molecular biology and oncology, can solve the problems of pancreatic cancer without research, and achieve the effect of increasing cell growth rate, increasing apoptosis level, and reducing growth rate

Active Publication Date: 2021-05-04
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PTRPA encodes receptor tyrosine-protein phosphatase alpha (Receptor-typetyrosine-protein phosphatase alpha). It has been reported that PTPRA is upregulated in lung squamous cell carcinoma, which is associated with poor prognosis of lung cancer, but it has not been studied in pancreatic cancer

Method used

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  • Uses of circular RNA circ-PTPRA
  • Uses of circular RNA circ-PTPRA
  • Uses of circular RNA circ-PTPRA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: RT-PCR reaction detection of circ-PTPRA gene expression in pancreatic cancer tissue.

[0033] The specific experimental plan is as follows:

[0034] 1. RNA extraction

[0035] 1) Tissue processing: Take about 10 mg of tissue and add 1 mL of Trizol, homogenize with a homogenizer; centrifuge for 15 minutes at 12,000 g, and take the supernatant.

[0036] 2) Add 200 μL of chloroform to the supernatant, mix vigorously up and down for half a minute, and let stand for 3 minutes.

[0037] 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.

[0038] 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.

[0039] 5) Carefully remove the ...

Embodiment 2

[0055] Example 2: Detection of circ-PTPRA expression in pancreatic cancer by QPCR

[0056] 1, RNA extraction: with embodiment 1;

[0057] 2. cDNA reverse transcription: same as Example 1;

[0058] 3. QPCR amplification experiment

[0059] 1) Experimental system:

[0060]

[0061] 2) Reaction conditions:

[0062] Step 1: 95°C for 2 minutes

[0063] Step 2 (40 cycles): 95°C for 3 seconds, 60°C for 30 seconds

[0064] The third step 60 ~ 95 ℃ melting curve

[0065] 3) Amplify the target gene on the machine

[0066] 4) qPCR relative quantitative results

[0067] 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. △Ct=Ct Purpose-Ct Housekeeper, indicating the relative Ct value of the target gene of each sample relative to the housekeeping gene, △△Ct=(△Ct)Test-(△Ct)Control, indicating that the treatment group...

Embodiment 3

[0068] Example 3: Construction of circ-PTPRA interference fragment and overexpression plasmid

[0069] 1. Design of circ-PTPRA interference fragment: siRNA sequence was synthesized in Shanghai Gemma Company. The nucleotide sequence of the siRNA sense strand of circ-PTPRA is shown in SEQ ID NO:4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:5. The nucleotide sense strand sequence of the Control negative control is shown in SEQ ID NO:6, and the antisense strand sequence is shown in SEQ ID NO:7.

[0070] 2. Overexpression plasmid construction: Synthesize the complete linear sequence of circ-PTPRA, anneal the sequence into a double-stranded DNA fragment, and insert it into the LV-Circ vector through multiple cloning sites. The recombinant plasmid is identified by sequencing, and the Control negative control is the uninserted sequence The LV-Circ empty vector.

[0071] 3. Interference and overexpression efficiency verification:

[0072] 1) After the ...

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Abstract

The present invention discloses the use of circular RNA circ-PTPRA. The present invention first confirms the objective existence of circ-PTPRA; by detecting the expression of circ-PTPRA in pancreatic cancer patients, it is found that its expression level is significantly improved; The study of cell function found that the growth rate of pancreatic cancer cells overexpressing circ-PTPRA was increased and the level of apoptosis was down-regulated. circ‑PTPRA and its expression products are used as markers for the diagnosis of pancreatic cancer, making the diagnosis of pancreatic cancer more accurate and rapid, and as the target gene for the preparation of drugs for the treatment of pancreatic cancer, it provides a new therapeutic target and therapeutic approach for the treatment of pancreatic cancer.

Description

technical field [0001] The invention relates to the fields of molecular biology and oncology, in particular to the use of a circular RNA circ-PTPRA. Background technique [0002] Pancreatic cancer (Pancreatic cancer) is a malignant tumor with high malignancy and extremely poor prognosis. In the past 40 years, the 5-year survival rate of patients has been around 5%. Because the pancreas is a retroperitoneal organ and its location is hidden, it is difficult to detect early pancreatic cancer. Most patients are diagnosed at an advanced stage, and the mortality rate is extremely high. Therefore, it is called "the king of cancer" and is one of the top ten malignant diseases that cause population death. one of the tumors. In recent years, the incidence of pancreatic cancer has gradually increased. According to statistics, in 2015, there were 90,000 new cases of pancreatic cancer nationwide, and another 79,000 people died of pancreatic cancer. It has become one of the diseases that...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886A61K45/00A61K48/00A61K31/7088A61P35/00
CPCA61K31/7088A61K45/00C12Q1/6886C12Q2600/158C12Q2600/178
Inventor 张钰莹王敏殷晓煜罗景燕纪卫东
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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