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RNA interference target RNA of long non-coding RNA related to human melanoma cells and application thereof

A melanoma cell and RNA interference technology, which is applied in the field of RNA interference target RNA and its application of long non-coding RNA related to human melanoma cells, and can solve problems such as tumor diagnosis and gene therapy impact

Inactive Publication Date: 2013-04-17
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These research results indicate that lncRNA plays an important role in cell proliferation, differentiation and carcinogenesis, and the discovery of tumor-associated lncRNA may have a significant impact on tumor diagnosis and gene therapy

Method used

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  • RNA interference target RNA of long non-coding RNA related to human melanoma cells and application thereof
  • RNA interference target RNA of long non-coding RNA related to human melanoma cells and application thereof
  • RNA interference target RNA of long non-coding RNA related to human melanoma cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Cloning and analysis of lncRNA-HN3

[0023] 1. Reagents

[0024] 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 BD Clontech Company of the United States; PCR primers were synthesized by Invitrogen Company.

[0025] 2. Bacterial strains, cell lines and vectors

[0026] 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.

[0027] 3. Experimental method

[002...

Embodiment 2

[0105] Example 2: Quantitative PCR detection of the expression of lncRNA-HN3 in human malignant melanoma tissues and adjacent tissues

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

[0107] The conventional reagents, bacterial strains, cell strains and vectors are all 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 (Invitrogen) (the method is the same as 3.1.1 in Example 1). Take 1 μg of the total RNA of the melanoma tissue and the total RNA of the corresponding paracancerous tissue, 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 corresp...

Embodiment 3

[0116] Example 3: Overexpression of lncRNA-HN3 promotes malignant growth characteristics of melanoma cells

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

[0118] The conventional 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 antibiotic 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 From Sigma Corporation of America.

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

[0121] BALB / c nude mice, SPF (Specific Pathogen-free, specific pathogen-free) grade, were 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-HN...

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Abstract

The invention discloses RNA interference (RNAi) target RNA of long non-coding RNA related to human melanoma cells and an application thereof and particularly provides RNAi target RNA of the long non-coding RNA related to the human melanoma cells, short-hairpin RNA (shRNA) of the interference target, shRNA coded small interference RNA (siRNA), sense strand DNA and antisense strand DNA of the coding shRNA, recombinant plasmids of the coding shRNA and the application thereof in the preparation of medicament for treating melanoma diseases.

Description

technical field [0001] The invention belongs to the professional field of biomedicine, and relates to a novel lncRNA belonging to a human lncRNA, its RNA interference (RNA interference, RNAi) target sequence, a short-hairpin RNA (short-hairpin RNA, shRNA) sequence, and a small RNA encoded by shRNA. Molecular interference RNA (small interference RNA, siRNA), sense and antisense strand DNA encoding shRNA, eukaryotic recombinant plasmid encoding shRNA, and their use in the preparation of reagents for diagnosing melanoma diseases and the preparation of drugs for treating melanoma diseases. 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 genomic DNA that does not encode proteins was originally considered to be nonfunctional and garbage in organisms, often called "Junk DNA". However, current research shows that most of these junk DNA can be transcribed...

Claims

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Application Information

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