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Long-chain non-coding RNA (Ribonucleic Acid) correlated to occurrence and development of human hepatocellular carcinoma, amplification detection method and application

A long-chain non-coding, human hepatocellular carcinoma technology, applied in biochemical equipment and methods, microbial assay/inspection, DNA/RNA fragments, etc., can solve problems such as biological functions and molecular mechanisms that remain to be elucidated

Active Publication Date: 2017-08-08
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological functions and molecular mechanisms of lncRNAs in human HCC remain to be elucidated

Method used

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  • Long-chain non-coding RNA (Ribonucleic Acid) correlated to occurrence and development of human hepatocellular carcinoma, amplification detection method and application
  • Long-chain non-coding RNA (Ribonucleic Acid) correlated to occurrence and development of human hepatocellular carcinoma, amplification detection method and application
  • Long-chain non-coding RNA (Ribonucleic Acid) correlated to occurrence and development of human hepatocellular carcinoma, amplification detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preliminary screening of new genes related to hepatocellular carcinoma by bioinformatics

[0034] The cDNA xProfiler of the Cancer Genome Analysis Project CGAP platform was used together with the BLAST and DDD (Digital Differential Display) tools of the National Center for Biotechnology Information NCBI platform.

[0035] (1) Use the cDNA xProfiler tool of the CGAP platform:

[0036] Pool A selects all cDNA libraries related to liver cancer;

[0037] Pool B selects all cancer and non-cancer cDNA libraries except liver cancer.

[0038] (2) Select 1142 unknown EST sequences in the differential comparison results of the cDNA library, compare them in the Genbank database, exclude known repetitive genes and genome contamination sequences, and obtain 5 EST sequences that can be used for the next step of screening, Excluding the fragments with no specific bands amplified by PCR in the 3 EST sequences, leaving HCCL2 and HCCL5 for comparison, it was found that HCCL5...

Embodiment 2

[0039] Embodiment 2: PCR amplification full-length gene

[0040] 1) Preparation and extraction of human hepatocellular carcinoma HepG2 cell RNA;

[0041] 2) Using RNA as a template to synthesize a 3'RACE cDNA template, use the following primers to perform PCR to obtain a 3'RACE product;

[0042] The 3'RACE primer 3L3 nucleotide sequence of HCCL5 is as SEQ ID NO.2 in the sequence listing;

[0043] 3'RACE nested PCR primer of HCCL5: 3L3N nucleotide sequence as SEQ ID NO.3 in the sequence listing

[0044] 3) Synthesize 5'RACE cDNA template using RNA as a template, and use the following primers to perform PCR to obtain 5'RACE products;

[0045] The 5' RACE primer 5L3 nucleotide sequence of HCCL5 is as SEQ ID NO.4 in the sequence listing;

[0046] The 5' RACE nested PCR primer 5L3N nucleotide sequence of HCCL5 is as SEQ ID NO.5 in the sequence listing;

[0047] 4) By splicing 5'RACE and 3'RACE sequences, the full-length sequence was obtained.

[0048] According to the conventi...

Embodiment 3

[0049] Example 3: Obtaining the full length of lncRNA HCCL5 gene and construction of eukaryotic expression vector

[0050] Design PCR primers to amplify the full length of lncRNA HCCL5, add Hind III and Xho I restriction sites to the primers respectively, and connect the obtained fragments to the pcDNA3.1(+) eukaryotic expression vector, see nucleosides for the nucleotide sequence of the amplification primers Acid sequence list SEQID NO.2, NO.5. The amplified fragments were inserted into the pcDNA3.1(+) eukaryotic expression vector after conventional digestion, ligation and other operations. The obtained recombinant plasmid was confirmed by DNA sequencing, and the result showed that the recombinant plasmid was correct and could be used for follow-up research.

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Abstract

The invention discloses long-chain non-coding RNA (Ribonucleic Acid) correlated to occurrence and development of human hepatocellular carcinoma, an amplification detection method and application. A new long-chain non-coding RNA gene sequence is found and cloned for the first time and an experiment proves that specific expression of a gene in the hepatocellular carcinoma is correlated to malignant degree and metastatic ability of the hepatocellular carcinoma; the gene has usages of auxiliary diagnosis of the hepatocellular carcinoma, gene therapy of the hepatocellular carcinoma and prognostic prediction of the hepatocellular carcinoma. The invention further provides an RNAi (Ribonucleic Acid interfere) target sequence of the gene and a short hairpin segment for RNAi disturbance; the RNAi target sequence and the short hairpin segment have a capability of inhibiting the expression of the gene and have the usages of the auxiliary diagnosis and gene therapy of the hepatocellular carcinoma.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and specifically relates to a long-chain non-coding RNA related to the occurrence and development of human hepatocellular carcinoma, an amplification detection method and an application thereof. Background technique [0002] Hepatocellular carcinoma (HCC) is a common malignant tumor, accounting for more than 90% of the total primary liver cancer (Liver Cancer). It is estimated that no less than one million new patients are diagnosed every year in the world, accounting for the sixth place in the incidence of malignant tumors and the second place in the death rate. About 600,000 people die of liver cancer every year. According to several death retrospective surveys in my country, liver cancer in the 1970s, 1990s and early 21st century in China accounted for the top three in all malignant tumor deaths, and it was one of the main diseases that threatened health and life. In my country, HCC mos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/113A61K48/00A61K31/7088A61P35/00
CPCC12N15/113C12N2310/14C12N2310/531C12N2320/30C12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 彭丽李官成蒋斌元袁小青
Owner CENT SOUTH UNIV
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