A long non-coding RNA lncRNA-BcrAR and its application in anti-carcinogenesis

A long-chain non-coding, pmscv-lncrna-bcrar technology, applied in the field of non-coding RNA and its application, to promote cell apoptosis and inhibit tumor growth

Active Publication Date: 2016-01-20
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report about the role of long non-coding RNA in the process of Abl-mediated cell carcinogenesis, and the application of long non-coding RNA in anti-Abl-induced leukemia

Method used

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  • A long non-coding RNA lncRNA-BcrAR and its application in anti-carcinogenesis
  • A long non-coding RNA lncRNA-BcrAR and its application in anti-carcinogenesis
  • A long non-coding RNA lncRNA-BcrAR and its application in anti-carcinogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Expression of lncRNA-BcrAR in human chronic myeloid leukemia cell line

[0026] 1. Expression of lncRNA-BcrAR in human chronic myeloid leukemia cell line K562

[0027] The expression of lncRNA in K562 cells was detected by lncRNA chip. After artificially interfering with the Bcr-Abl gene in K562 cells, the expression of lncRNA-BcrAR was significantly up-regulated, suggesting that lncRNA-BcrAR may be involved in the malignant transformation of cells induced by Bcr-Abl. The nucleotide sequence shown in SEQ ID NO: 3 encodes the lncRNA-BcrAR gene, and the gene position is Chromosome11; 5265784-5269453.

[0028] 2. Construction of K562 cell line interfering with Bcr-Abl gene

[0029] 1. Construction of plasmid pSIH-Bcr-Abl shRNA carrying Bcr-Abl specific shRNA

[0030] The lentiviral vector pSIH-H1 was used to express shRNA to interfere with the Bcr-Abl gene in K562 cells. Synthesize the single-stranded DNA shown in the following sequence 1 (SEQ ID NO: 1) and t...

Embodiment 2

[0046] Example 2. Application of lncRNA-BcrAR in anti-chronic myeloid leukemia

[0047] 1. Northern blot to determine the existence of lncRNA-BcrAR in K562 cells

[0048] Total RNA from K562 cells was extracted, reverse transcribed to synthesize cDNA, and primers for Northern blot probe amplification were designed. Using cDNA as a template, PCR amplification was performed to obtain PCR amplification products.

[0049] lncRNA-BcrAR-NB-F: 5'-TTAGCAGTAACTGCTGAATTCCTGG-3';

[0050] lncRNA-BcrAR-NB-R: 5'-AAGGGGAAAACTGGGTTTTATTAC-3'.

[0051] The PCR amplification products were recovered and labeled with isotopes, and the length of lncRNA-BcrAR in K562 cells was detected by Northern blot method (Invitrogen). like figure 2 As shown, lncRNA-BcrAR exists in the form of a transcript with a length of about 3670 nt in K562 cells, and infection of pSIH-Bcr-AblshRNA packaging virus can significantly upregulate the expression of lncRNA-BcrAR in K562 cells.

[0052] 2. Establishment of o...

Embodiment 3

[0068] Example 3. Application of lncRNA-BcrAR in anti-A-MuLV-induced mouse leukemia

[0069] 1. Establishment of lncRNA-BcrARBC44 cell line (A-MuLV transformed mouse leukemia cells)

[0070] 1. Virus packaging

[0071] According to the instructions of Lipofectamine (Invitrogen) transfection reagent, the plasmid pMSCV-lncRNA-BcrAR carrying lncRNA-BcrAR and the packaging plasmids pCL-Eco (IMGENEX) and VSVG (Invitrogen) were co-transfected into the packaging cell line 293T packaging virus. 293T cells were cultured in DMEM (Gibco) medium, complete medium supplemented with final concentrations of 100units / mL penicillin, 100units / mL streptomycin and 10% fetal bovine serum (Gibco), placed at 37°C, saturated humidity, 5% CO 2 concentration of cells in an incubator. The cell supernatant was collected after 48-96 h, and the supernatant contained pseudovirus particles encoding lncRNA-BcrAR.

[0072] 2. Cell infection

[0073] Collect, filter the virus solution, add it to a 15ml tube ...

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Abstract

The invention relates to a long-chain non-coding RNA IncRNA-BcrAR and application thereof in cell canceration resistance. IncRNA-BcrAR is in low-level expression in a human chronic myelogenous leukemia cell line K562 with positive Bcr-Abl, the K562 cell line with overexpressed IncRNA-BcrAR is constructed, the action of IncRNA-BcrAR on Bcr-Abl induced cell neoplastic transformation is observed, experiments prove that the IncRNA-BcrAR overexpression can obviously promote K562 cell apoptosis induced by Imatinib (therapeutic drug of Abl positive leukemia patients) and can remarkably inhibit tumor growth induced by K562 cells in a naked mouse body; and besides, the IncRNA-BcrAR overexpression can remarkably promote A-MuLV transformed mouse leukemia cell BC44 apoptosis induced by Imatinib. The IncRNA-BcrAR has important effect on Bcr-Abl and v-Abl mediated cell canceration resistance, and the long-chain non-coding RNA IncRNA-BcrAR provides new molecular marker and drug target for diagnosis and treatment of Abl induced leukemia.

Description

technical field [0001] The present invention relates to a non-coding RNA and its application, in particular to a long-chain non-coding RNA lncRNA-BcrAR and its application in anti-cell carcinogenesis. Background technique [0002] Abl is an important oncogene that can induce malignant transformation of various cells including lymphocytes, including v-Abl, Bcr-Abl and Tel-Abl. v-Abl is an oncogene of Abelson murine leukemia virus (A-MuLV for short). A-MuLV mainly induces malignant transformation of murine lymphocytes, thereby triggering the generation of acute lymphoma in mice. Bcr-Abl is a fusion gene formed by joining Bcr and c-Abl C-terminus due to chromosomal translocation of human cells, and its expression product is a fusion oncoprotein. Bcr-Abl is the most critical oncogene to induce human chronic myelogenous leukemia (CML), and it can also cause malignant transformation of human lymphocytes. Despite years of research, the molecular mechanism of Abl-induced carcinog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63A61K48/00A61P35/00
Inventor 陈吉龙郭桂杰池晓娟陈志龙
Owner FUJIAN AGRI & FORESTRY UNIV
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