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Human cervical carcinoma metastasis relevant new long chain non-coding RNA sequence, separation method and uses thereof

A human cervical cancer, a new long-chain technology, applied in the field of tumor molecular biology, can solve the problems of diagnosis and gene therapy, lncRNA non-conservation, etc.

Inactive Publication Date: 2015-10-14
CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of studies have shown that lncRNA is extremely unconserved in biological evolution, and its expression specificity in tissue and developmental stages is significantly higher than that of protein-coding genes. The possible side effects of gene intervention therapy targeting tumor-associated lncRNA will be less than those of protein-coding genes. Encoding gene targets, the discovery of tumor-associated lncRNA may have a significant impact on tumor diagnosis and gene therapy

Method used

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  • Human cervical carcinoma metastasis relevant new long chain non-coding RNA sequence, separation method and uses thereof
  • Human cervical carcinoma metastasis relevant new long chain non-coding RNA sequence, separation method and uses thereof
  • Human cervical carcinoma metastasis relevant new long chain non-coding RNA sequence, separation method and uses thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preliminary screening of new genes related to cervical cancer by bioinformatics

[0040] cDNA xProfiler using the Cancer Genome Profiling Project CGAP platform with the US National The BLAST and DDD (Digital Differential Display) tools of the NCBI platform of the Biotechnology Information Center jointly screened.

[0041] 1) Use the cDNA xProfiler tool of the CGAP platform:

[0042] Pool A selects all cDNA libraries related to cervical cancer;

[0043] Pool B selects all cancer and non-cancer cDNA libraries except cervical cancer.

[0044] 2) Select 100 unknown EST sequences from the differential comparison results of the cDNA library and compare them in the Genbank database to exclude known repetitive genes and genome contamination sequences. The fragments with non-specific bands were amplified by PCR in the 9 EST sequences, and the C3 fragment was left for RACE to amplify its full-length sequence ( figure 1 ).

Embodiment 2

[0045] Embodiment 2: PCR amplification full-length gene

[0046] 1) Prepare and extract human cervical cancer cell RNA;

[0047] 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;

[0048] 3' RACE primer 3C3 nucleotide sequence of C3 such as sequence in the table SEQ ID No.2;

[0049] 3' RACE nested PCR primers for C3: 3C3N nucleotide sequence such as sequence in the table SEQ ID No.3

[0050] 3) Synthesize 5'RACE cDNA template using RNA as a template, and use the following primers to perform PCR to obtain 5'RACE products;

[0051] The 5' RACE primer 5C3 nucleotide sequence of C3 such as sequence in the table SEQ ID No.4;

[0052] C3 5' RACE nested PCR primer 5C3N nucleotide sequence such as sequence in the table SEQ ID No.5;

[0053] 4) By splicing 5'RACE and 3'RACE sequences, the full-length sequence was obtained.

[0054] According to the conventional RACE method, prepare RACE-re...

Embodiment 3

[0055] Example 3: Obtaining the full length of lncRNA-C3 gene and construction of eukaryotic expression vector

[0056] Design PCR primers to amplify the full length of lncRNA-C3, add BamH I and Hind III restriction sites to the primers, and connect the resulting fragments to the pcDNA3.1(+) eukaryotic expression vector, see the nucleotide sequence of the amplification primers Nucleotide sequence Form S EQ ID 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 digested with double enzymes ( image 3 A), DNA sequencing to determine ( image 3 B), the result shows that the recombinant plasmid is correct and can be used for follow-up research.

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Abstract

The present invention relates to a human cervical carcinoma metastasis relevant new long chain non-coding RNA sequence, a target sequence for RNA interference, a short hairpin sequence for encoding interference target, a RNAi target sequence containing the long chain non-coding RNA sequence, a shRNA short hairpin nucleic acid sequence recombinant vector and a ecombinant. The present invention further relates to a separation method and uses of the new long chain non-coding RNA sequence. According to the present invention, the long chain non-coding RNA sequence is obtained from the cervical carcinoma related cDNA library through RACE amplification through a bioinformatics method, and the function and the uses of the RNA sequence are verified through sequencing, hybridization, cell proliferation, cell invasion and other tests; the sequence-specific shRNA for RNAi, the siRNA sequence and the recombinant vector are designed, and the functions of the siRNA and the shRNA are verified; and the long chain non-coding RNA sequence, the RNAi interference target and the shRNA short hairpin sequence provide important significance in early diagnosis and treatment of cervical 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 gene sequence, a method for separating the sequence, an RNA interference target sequence of the gene, a short hairpin fragment for RNA interference and uses thereof. Background technique [0002] Cervical cancer is the third most common malignancy in women worldwide after breast cancer and colorectal cancer, and one of the most common female reproductive system malignancies in developing countries after breast cancer. In recent years, due to factors such as environmental pollution, increased social work pressure, and poor hygiene habits in life, the incidence of cervical cancer has increased significantly, and the trend of younger patients is becoming more and more obvious, and 85% of new cases are developing. China, so it has good social value for cervical cancer research. [0003] Tumor metastasis is an important biological feature ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12Q1/68A61K48/00A61K31/7088A61P35/00
Inventor 李官成蒋斌元房淑娟孙瑞利秦长飞李跃辉潘曦
Owner CENT SOUTH UNIV
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