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Long non-coding RNA sequence of human melanoma cell specific expression and application thereof

A melanoma cell, non-coding technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, anti-tumor drugs, etc., can solve the problem that lncRNA is in its infancy

Inactive Publication Date: 2010-01-06
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the research in the field of ncRNA is small molecule ncRNA, while the research on lncRNA is still in its infancy.

Method used

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  • Long non-coding RNA sequence of human melanoma cell specific expression and application thereof
  • Long non-coding RNA sequence of human melanoma cell specific expression and application thereof
  • Long non-coding RNA sequence of human melanoma cell specific expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Cloning and analysis of lncRNA-UN

[0024] 1. Reagents

[0025]Trizol reagent was purchased from Invitrogen Company of the United States; MMLV reverse transcriptase, DNA polymerase, pfu enzyme, Nde I enzyme, Xho I enzyme and RNase inhibitor were all purchased from Lithuanian Fermentas Company; T7 RNA polymerase was purchased from NEB Company of the United States; random primers were purchased from From Promega Company of the United States; TALON metal ion resin was purchased from BDClontech Company of the United States; PCR primers were synthesized by Invitrogen Company.

[0026] 2. Bacterial strains, cell lines and vectors

[0027] Human malignant melanoma cells yusac were purchased from American Type Culture Collection (ATCC); E.coli BL2 strain was purchased from Tianjin Tianli Technology Co., Ltd.; PCR-blunt-TOPO vector was purchased from Invitrogen, USA; pET-28a(+) Vectors were purchased from Novagen, Germany.

[0028] 3. Experimental method

[0029] ...

Embodiment 2

[0105] Example 2: Quantitative PCR detection of the expression of lncRNA-UN in human malignant melanoma tissues and adjacent tissues

[0106] 1. Reagents, strains, cell lines and vectors

[0107] Routine reagents, bacterial strains, cell strains and carriers are the same as in Example 1.

[0108] QuantiTect SYBR Green PCR kit was purchased from Qiagen, Germany.

[0109] 2. Experimental method

[0110] 2.1.1 Synthesized total cDNA

[0111] Using human melanoma tissue and corresponding paracancerous tissue as materials, total RNA was extracted from melanoma tissue and corresponding paracancerous tissue with Trizol reagent (the method is the same as 3.1.1 in Implementation 1). Take 1 μg of the total RNA of the melanoma tissue and the total RNA of the corresponding paracancerous tissue respectively, and carry out reverse transcription reaction with random primers (the method is the same as 3.1.2 in Example 1) to obtain the total cDNA of the melanoma tissue and the total cDNA of...

Embodiment 3

[0116] Example 3: Effect of overexpression of lncRNA-UN on the growth of melanoma cells

[0117] 1. Reagents, strains, cell lines and vectors

[0118] Routine reagents, bacterial strains, cell strains and carriers are the same as in Example 1.

[0119] The pcDNA-3.1 eukaryotic expression vector, lipofectamine 2000 transfection reagent, and G418 antibiotics were all purchased from Invitrogen Company of the United States; DMEM medium and fetal bovine serum were purchased from Gibico Company of the United States; nitro blue tetrazolium (NBT) was purchased from American Sigma Corporation.

[0120] Perfection 4990 flatbed scanner was purchased from Epson Corporation of Japan.

[0121] BALB / c nude mice, SPF (Specific Pathogen-free, no specific pathogens) grade, purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0122] 2. Experimental method

[0123] 2.1 Construction of monoclonal cell lines stably and highly expressing lncRNA-UN

[0124] 2.1.1 Construction of eukaryo...

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Abstract

The invention discloses a long non-coding RNA (lnc RNA-UN) of human melanoma cell specific expression, an RNA interfering target spot thereof, a short hairpin RNA (shRNA) of the RNA interfering target spot, a small interfering RNA (siRNA), a sense strand DNA and an antisense strand DNA of the coding short hairpin RNA and a recombinant plasmid of a transcription short hairpin RNA. Proved by experiments, the lnc RNA-UN is highly expressed in a melanoma tissue and not expressed in a peficancerous tissue and can be applied to preparing a reagent for diagnosing melanoma diseases. Proved by the experiments, the recombinant plasmid of the transcription short hairpin RNA expresses the shRNA and the siRNA in a melanoma cell and can obviously degrade the cell endogenous lncRNA-UN and restrain the malignant growth of the melanoma cell, thus the shRNA and the siRNA of the RNA interfering target spot of the lncRNA-UN and the recombinant plasmid of the transcription short hairpin RNA can be applied to medicaments for treating the melanoma diseases.

Description

technical field [0001] The present invention belongs to the field of biomedicine, and in particular relates to a long non-coding RNA (long non-coding RNA, referred to as lncRNA, hereinafter "lncRNA" means long non-coding RNA) specifically expressed in human melanoma cells and its application. Background technique [0002] There are about 20,000 to 30,000 genes that encode proteins in the human body, accounting for only 2% of the human genome, and the remaining 98% of the genomic DNA that does not encode proteins was originally considered to be nonfunctional and garbage in organisms, commonly known as "Junk DNA". However, current research shows that most of these junk DNA can be transcribed to produce non-coding RNA (non-coding RNA, ncRNA for short). According to the size of mature transcripts, ncRNA can be divided into small molecule ncRNA (such as siRNA, miRNA, piRNA, etc.), medium-length ncRNA (70-200nt) and lncRNA (long≥200nt). At present, most of the research in the fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/79C12Q1/68A61K48/00A61P35/00C12N15/113
Inventor 宋旭李灵俸婷婷连莹莹张广丰
Owner SICHUAN UNIV
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