lncRNA LLNLR-299G3.1 related to esophageal squamous cell carcinoma (ESCC) and application thereof

A kit and inhibitor technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as limited clinical application of lncRNA molecules

Active Publication Date: 2020-01-03
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide an ESCC-related lncRNA LLNLR-299G3.1 and its applicatio

Method used

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  • lncRNA LLNLR-299G3.1 related to esophageal squamous cell carcinoma (ESCC) and application thereof
  • lncRNA LLNLR-299G3.1 related to esophageal squamous cell carcinoma (ESCC) and application thereof
  • lncRNA LLNLR-299G3.1 related to esophageal squamous cell carcinoma (ESCC) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Discovery of LLNLR-299G3.1

[0048] 1. Selection of research objects

[0049] Pathologically confirmed ESCC cases, age-matched patients with esophagitis confirmed by digestive endoscopy and healthy controls were collected from the Department of Thoracic Surgery of Shenzhen People's Hospital. All research subjects signed the informed consent. The research protocol was approved by the Medical Ethics Committee (approval number: 2016001).

[0050] 2. Comparison of plasma exosomal lncRNA expression profiles of research subjects (ESCC patients, esophagitis patients, healthy controls)

[0051] Peripheral blood was drawn from the research subjects, allowed to stand at 4°C for 1 hour, centrifuged at 1000g for 10 minutes to separate the plasma, and the separated plasma was stored at -80°C until testing.

[0052] Plasma exosomes were isolated using an exosome isolation kit (Ribo-Exosome-Isolation-Kit C10110). The main steps included: centrifugation at 2,000×g for 20 m...

Embodiment 2

[0064] Example 2 Study on the relationship between the expression of LLNLR-299G3.1 and the proliferation and migration of ESCC cells

[0065] 1. Construction of LLNLR-299G3.1 overexpression stable transfection strain

[0066] According to the sequence (SEQ ID No.1) of LLNLR-299G3.1 in GeneBank, primers (SEQ ID No.2 and SEQ ID No.3) were designed to amplify human full-length LLNLR-299G3.1, and the sequence was directly synthesized into The lentiviral shuttle plasmid vector is the pHBLV-CMV-MCS-3flag-EF1-ZsGreen-T2A-PURO vector (map as Figure 5 shown) above. Among them, LLNLR-299G3.1 is inserted between EcoRI and BamHI.

[0067] First amplify the target gene by PCR, then digest the above-mentioned lentiviral shuttle plasmid vector with EcoRI and BamHI endonucleases, and configure the ligation system according to the ratio of the target gene to vector 3:1-8:1 (vector vector 1 μl; target gene 1 μl ; T4ligase 0.5μl; 10×ligase Buffer 1μl; ddH 2 O To 10μl), 14~16℃, 16hrs to comp...

Embodiment 3

[0082] Example 3 Antisense oligonucleotide (ASO) of LLNLR-299G3.1 inhibits the growth of ESCC transplanted tumor in nude mice

[0083] In this example, liposome nanoparticles were used to deliver antisense oligonucleotides against LLNLR-299G3.1 (one of ASO, ASO1, ASO2 or ASO3) to inhibit the growth of ESCC transplanted tumors in nude mice. Animals were divided into groups: ESCC cells TE1 in the logarithmic growth phase were taken, and 1×10 cells were subcutaneously injected on the back of nude mice 7 cells / mouse to construct an ESCC animal model. When the tumor volume is about 30mm 3 Afterwards, the animals were randomly divided into 4 groups, 5 in each group, and were injected with ASO empty-liposome nanoparticle-linked polypeptide (i.e. Figure 10 Middle label 2: plCSA-NNPs), ASO-liposome nanoparticles (ie Figure 10 Middle label 3: ANPs); ASO-liposome nanoparticle-linked polypeptide (ie Figure 10 Middle label 4: plCSA-ANPs), and normal saline (blank control, ie Figur...

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Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to lncRNA LLNLR-299G3.1 related to esophageal squamous cell carcinoma (ESCC) and an application thereof. According to the invention, the lncRNA (LLNLR-299 G3.1) with obvious cancer promoting activity is proved by experimental analysis, so that a new target point is provided for a new scheme of individualized accurate treatment of ESCC. Expression of the lncRNA in plasma exudates of ESCC patients and in ESCC cells is significantly higher than expression in plasma exudates of healthy people and in normal esophageal epithelial cells respectively. The research results show that the lncRNA has obvious capability of promoting tumor activity of ESCC cells, and through inhibition of expression of the lncRNA in cells and a nude mouse transplanted tumor model, proliferation and migration capability of ESCC cells can be obviously inhibited.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to an lncRNA LLNLR-299G3.1 related to ESCC and its application. Background technique [0002] Esophageal cancer is a malignant tumor that occurs in the esophageal epithelium, and its mortality rate ranks fourth and sixth among all tumors in China and the world, respectively. my country is the country with the highest morbidity and mortality rate of esophageal cancer in the world. Among the more than 500,000 newly discovered esophageal cancer patients in the world every year, more than 50% of the cases occur in China, and 90% of them are esophageal squamous cell carcinoma (esophageal squamous cell carcinoma). squamous cell carcinoma, ESCC). At present, the overall 5-year survival rate of esophageal cancer is only about 10%, but the 5-year survival rate of stage I esophageal cancer after surgical resection can reach 90%, while the five-year survival rate of sta...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113
CPCC12Q1/6886C12Q2600/178C12Q2600/118
Inventor 翟日洪杨林范秀军田莉宋译张宝珍
Owner SHENZHEN UNIV
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