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Long-chain non-coding RNA for regulating and controlling proto-oncogene MYB and application of long-chain non-coding RNA

A long-chain non-coding, proto-oncogene technology, applied to long-chain non-coding RNA. It can solve the problems of unclear function of lncRNA and unresearched effect on leukemia.

Pending Publication Date: 2022-03-11
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, although the research on lncRNA has progressed rapidly, the functions of most lncRNAs are still unclear. In the present invention, a transcribed lncRNA was found in the 96k region upstream of MYB. The effect of this lncRNA on the MYB gene and The effect on leukemia has not been studied

Method used

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  • Long-chain non-coding RNA for regulating and controlling proto-oncogene MYB and application of long-chain non-coding RNA
  • Long-chain non-coding RNA for regulating and controlling proto-oncogene MYB and application of long-chain non-coding RNA
  • Long-chain non-coding RNA for regulating and controlling proto-oncogene MYB and application of long-chain non-coding RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 RACE technology clones the full-length sequence of long non-coding RNA

[0047] (1) Total RNA extraction

[0048] Total RNA was extracted from K562 cells using the Trizol method, and RNase-free H was added 2 O, in a water bath at 55°C for 10 min to completely dissolve the RNA, and store in a freezer at -80°C.

[0049] (2) RACE first-strand cDNA synthesis

[0050] Using the Takara company provided RACE 5' / 3'Kit kit was used for synthesis.

[0051] The synthesized 3'-RACE-Ready cDNA and 5'-RACE-Ready cDNA products were diluted with one volume of distilled water and stored at -20°C.

[0052] (3) RACE-PCR reaction

[0053] In the first round of RACE-PCR, the universal primer UPM and the 5'-end or 3'-end gene-specific primers (gene-specific primer, GSP) were used for PCR amplification.

[0054] The PCR primer sequence of the long-chain non-coding RNA (hereinafter referred to as lncRNAMYB-96KS):

[0055] 5'RACE-MYB-96KS-GSP: 5'-gtgcctttaaggcaccctgcatacatgat-...

Embodiment 2

[0095] Example 2 Detection of the expression level of lncRNA MYB-96KS in leukemia cell lines

[0096] (1) Cell culture

[0097] Human erythroleukemia K562 cells, human acute myeloid leukemia U937 cells, human promyelocytic acute leukemia HL60 cells, Hela cells and 293T cells were purchased from ATCC (American Type Culture Collection). K562, U937, and HL60 cells were cultured with RPMI 1640basic (Gibco, #C11875500BT) medium, supplemented with 10% final concentration of fetal bovine serum (Gibco, #10099-141C) and 1% final concentration of streptomycin and penicillin (HyClone, #SV30010). Hela and 293T cells were cultured with DMEM (Gibco, #C11965500BT) medium plus 10% fetal bovine serum and 1% streptomycin and penicillin. The inoculated cells were placed at a temperature of 37°C, CO 2 Cultured in a cell culture incubator with a volume fraction of 5%.

[0098] (2) Extraction of total RNA

[0099] Cells were cultured in a 10cm cell culture dish. K562, U937, and HL60 cells were...

Embodiment 3

[0122] Example 3 lncRNA MYB-96KS overexpression and knockdown vector construction

[0123] The full-length sequence of lncRNA MYB-96KS obtained from Example 1 was used to construct overexpression and knockdown vectors. Dilute the T vector plasmid in Example 1 to 1 ng / μL, and use it as a template for subcloning PCR.

[0124] 1. Construction of pLVX-MYB-96KS overexpression vector

[0125] (1) Use the pLVX-IRES-Neo plasmid to construct the pLVX-MYB-96KS overexpression vector. First, two restriction endonuclease sites were selected according to the sequences of the pLVX-IRES-Neo plasmid and MYB-96KS, EcoRI and XbaI respectively. Then design subcloning primers with enzyme cleavage sites, the sequence is as follows:

[0126] MYB-96KS-EcoRI-F:

[0127] 5′-ggaattcgaagcatcagatgaagagggaagg-3′

[0128] MYB-96KS-XbaI-R:

[0129] 5′-gactagtttcttttttctttttttttctttttttttttgccatggtctagtc-3′

[0130] (2) Add restriction site PCR

[0131] reaction system:

[0132]

[0133] Reaction ...

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Abstract

The invention relates to molecular biology, in particular to long-chain non-coding RNA (Ribonucleic Acid) for regulating and controlling a protooncogene MYB and cDNA (Complementary Deoxyribonucleic Acid) thereof. The long-chain non-coding RNA or the cDNA thereof can regulate and control the expression of the MYB gene, and can be used as a tumor molecular marker or a target spot of a drug; the proliferation of leukemia cells can be regulated and controlled, and the gene can be used as a marker for leukemia detection and a target spot for treatment. The invention also discloses a recombinant vector and application thereof.

Description

technical field [0001] The invention relates to the fields of molecular biology and medicine, specifically a long-chain non-coding RNA that regulates the proto-oncogene MYB. Background technique [0002] MYB is a proto-oncogene that widely exists in eukaryotes and is highly conserved in evolution. Its main biological effect is to regulate cell proliferation and differentiation. Many studies have shown that MYB is highly expressed in leukemia and can act as a transcription factor to regulate the proliferation, differentiation, and invasion of cancer cells, and is the driving factor of leukemia. Therefore, MYB plays an important role in the detection and treatment of leukemia. [0003] At present, chemotherapy and bone marrow transplantation are the main treatments for leukemia. Chemotherapy has many side effects and is prone to clinical recurrence. However, bone marrow transplantation is expensive and has few sources of bone marrow, resulting in a low overall survival rate o...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/867C12Q1/6886
CPCC12N15/1135C12N15/86C12Q1/6886C12N2310/11C12N2740/15043C12N2800/107C12Q2600/158C12Q2600/178C12Q2600/136
Inventor 韩兵社张俊芳王玉成李梦佳张泽辉
Owner SHANGHAI OCEAN UNIV
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