Up-stream open reading frame 45aa-uORF nucleotide sequence of PTEN gene, and applications of polypeptide encoded by 45aa-uORF nucleotide sequence

A 45aa-uorf, nucleotide sequence technology, applied in the fields of application, gene therapy, genetic engineering, etc., can solve problems such as blank research and achieve the effect of reducing expression

Active Publication Date: 2018-12-18
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in the research of smORF in other species (Zanet et al., 2016), the research of smORF in human cancer is still blank

Method used

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  • Up-stream open reading frame 45aa-uORF nucleotide sequence of PTEN gene, and applications of polypeptide encoded by 45aa-uORF nucleotide sequence
  • Up-stream open reading frame 45aa-uORF nucleotide sequence of PTEN gene, and applications of polypeptide encoded by 45aa-uORF nucleotide sequence
  • Up-stream open reading frame 45aa-uORF nucleotide sequence of PTEN gene, and applications of polypeptide encoded by 45aa-uORF nucleotide sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Endogenous expression and identification of micropeptides encoded by 45aa-uORF

[0049] Through ORF finder (http: / / www.bioinformatics.org / sms / orf_find.html), a 138nt open reading frame (Open Reading Frame, ORF) was found at the 5`UTR of the first intron region of PTEN mRNA (NM_000314) , can encode a short peptide of 45 amino acids, which we named: 45aa-uORF, and the short peptide encoded by it is named (PTTN-45aa). The molecular weight of the protein is predicted to be about 5KD by the protein molecular weight prediction software http: / / www.bio-soft.net / sms / prot_mw.html. According to the amino acid sequence of 45aa-uORF, a polyclonal antibody that specifically recognizes the micropeptide and can be detected by western blot is designed. The specific method is as follows: by chemically synthesizing polypeptides, a segment of the MRDGGGRGPEPLSAC amino acid sequence of 45aa-uORF was synthesized as an immunogen, injected and immunized into New Zealand white rabbit...

Embodiment 2

[0072] Example 2 Determination of Cellular Biological Function of 45aa-uORF Micropeptide

[0073] 1) Construction of a stable cell line overexpressing 45aa-uORF

[0074] Synthesize a vector that overexpresses 45aa-uORF-GFP (vector backbone pCDH-CMV-MCS-EF1-Puro), and transfect it with the lentiviral backbone vector PSPAX2, PMD2G into HEK-293T cells at a ratio of 4:3:1 to package the lentivirus . After 48 hours and 72 hours, the medium supernatant with lentivirus was collected and then infected with U251 cells (8ug / ml polybrene was added to increase the infection efficiency). After puromycin (1mg / mL) was selected for three days, puromycin was removed, and then the cells were expanded normally . Cell transfection steps: 500,000 cells of 293T cells were inoculated in 6-well culture plates, and transfection could be carried out after 24 hours of cell attachment; before transfection, 100 microliters of serum-free medium DMEM and plasmids were prepared as a mixture; Mix 100 micro...

Embodiment 3

[0085] Example 3 Verifies the ability of micropeptides to enter cells

[0086] The U251 cells were inoculated on glass slides at a density of 50%, and the cells were treated with different concentrations of PTEN-45aa micropeptides linked to FITC fluorescent groups (the micropeptides were dissolved in DMSO at a concentration of 20 μg / μL) for 4 h, and then irradiated. Formalin fixed and photographed with fluorescence microscope observation. It was found that for PTEN-45aa micropeptide, 20 μM concentration can enter U251 cells smoothly within 4 hours, and all cells in the field of view have fluorescence ( Figure 4 ).

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to an up-stream open reading frame (uORF) of a phosphatase and tensin homolog (PTEN) gene, and applications of a protein encoded by the uORF. According to the present invention, the 5'UTR of PTEN has a potential 138-base open reading frame (45aa-uORF), wherein the 138-base open reading frame can encode a short peptide having 45 amino acids (named PTEN-45aa), and the short peptide has important effects in the occurrence and development process of tumors; and the new diagnosis, the new treatment method and the new drug screening platform are provided for diseases associated with PTEN expression regulation, particularly gliomas. The invention further provides a polypeptide for treating diseases associated with PTEN expression regulation.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an upstream open reading frame (uORF, up-stream open reading frame) of a PTEN (phosphatase and tensin homolog) gene and an application of the encoded protein thereof. Background technique [0002] Malignant tumors of the central nervous system (CNS) have one of the worst prognosis types among all human tumors, resulting in the highest potential life loss among tumors (up to about 20 years) (Rouse et al., 2016). Among them, malignant glioma (Malignant Glioma, MG) is the most common, with an annual incidence of about 7.2 / 100,000 (Sturm et al., 2014). Gliomas are divided into WHO grades I-IV according to histological morphology, among which grade IV is also called glioblastoma multiforme (GBM) (Louis et al., 2016a), accounting for half of malignant gliomas Above, the prognosis is extremely poor. Even after the most timely and effective surgery, radiotherapy and chemotherapy, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16G01N33/574A61K31/713A61K48/00A61P35/00
CPCA61K31/713C12N9/16C12Q1/6886G01N33/574A61P35/00G01N33/57496C12Q2600/158G01N2333/916C12N9/22A61K38/465A61K38/1703C07K14/435A61K48/00C12Y301/03067
Inventor 张弩
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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