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Long-chain non-coding RNA IncRNA-BcrAR and application thereof in cell canceration resistance

A long-chain non-coding, pmscv-lncRNA-bcrar technology, applied in non-coding RNA and its application fields, to achieve the effect of promoting cell apoptosis and inhibiting tumor growth

Active Publication Date: 2014-05-14
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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  • Long-chain non-coding RNA IncRNA-BcrAR and application thereof in cell canceration resistance
  • Long-chain non-coding RNA IncRNA-BcrAR and application thereof in cell canceration resistance
  • Long-chain non-coding RNA IncRNA-BcrAR and application thereof in cell canceration resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0027] Using lncRNA chip to detect the expression of lncRNA in K562 cells, the expression of lncRNA-BcrAR was significantly up-regulated after artificially interfering with the Bcr-Abl gene in K562 cells, suggesting that lncRNA-BcrAR may be involved in the process of 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 Chromosome 11; 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 expression of shRNA by lentiviral vector pSIH-H1 was used to interfere with the Bcr-Abl gene in K562 cells. Synthesize the single-stranded DNA shown in the following sequen...

Embodiment 2

[0046] Example 2, the application of lncRNA-BcrAR in anti-chronic myelogenous leukemia

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

[0048] Total RNA was extracted from K562 cells, cDNA was synthesized by reverse transcription, primers for Northern blot probe amplification were designed, and cDNA was used as a template for PCR amplification 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 product was recovered, subjected to isotope labeling, and the length of lncRNA-BcrAR in K562 cells was detected by Northern blot method (Invitrogen). Such as figure 2 As shown, lncRNA-BcrAR exists in the form of transcripts with a length of about 3670 nt in K562 cells, and the infection of pSIH-Bcr-Abl shRNA packaging virus can significantly up-regulate the expression of lncRNA-BcrAR in K562 cells.

[0052] 2. E...

Embodiment 3

[0068] Example 3, the application of lncRNA-BcrAR in anti-A-MuLV-induced mouse leukemia

[0069] 1. Establishment of BC44 cell line (A-MuLV transformed mouse leukemia cells) overexpressing lncRNA-BcrAR

[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 plasmid 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, with a final concentration of 100 units / mL penicillin, 100 units / mL streptomycin and 10% fetal bovine serum (Gibco) added to the complete medium, placed at 37°C, saturated humidity, 5 %CO 2 concentration in the cell culture incubator. After 48-96 h, the cell supernatant was collected, and the supernatant contained pseudoviral particles encoding lncRNA-BcrAR.

[0072] 2. Cell infection

[0073] Collect, filter the virus liq...

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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 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-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 mouse lymphocytes, thereby triggering the generation of acute lymphoma in mice. Bcr-Abl is a fusion gene formed by the junction of Bcr and the C-terminus of c-Abl due to chromosomal translocation in human cells, and its expression product is a fusion oncoprotein. Bcr-Abl is the most critical oncogene that induces human chronic myelogenous leukemia (CML), and can also cause malignant transformation of human lymphocytes. Despite years of research, the molecular mechanism of Abl-induced cell ca...

Claims

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Application Information

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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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