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Applications of long non-coding RNA LINC00673

A long-chain non-coding, reagent technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial assay/inspection, etc., can solve problems such as unknown lncRNA function

Active Publication Date: 2018-10-26
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more than 40,000 human lncRNAs have been cloned, but the functions of most lncRNAs are unknown

Method used

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  • Applications of long non-coding RNA LINC00673
  • Applications of long non-coding RNA LINC00673
  • Applications of long non-coding RNA LINC00673

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, real-time fluorescence quantitative method detection confirmed the up-regulation of LINC00673 in oral tumors

[0036] 1. Materials and methods:

[0037] Total RNA was extracted from 15 normal oral mucosa and 15 oral tumor tissues, and 2 μg of RNA was reverse-transcribed into cDNA, followed by real-time fluorescent quantitative PCR.

[0038] Forward primer: 5'-TTCTCCTGTAACGTGTGGCC-3',

[0039] Reverse primer: 5'-CTGGTGGGAATGTGGATCAGT-3'.

[0040] Internal reference gene GAPDH-specific PCR primers for control:

[0041] Forward primer 5'-ACCACAGTCCATGCCATCAC-3'

[0042] Reverse primer 5'-TCCACCACCCCTGTTGCTGTA-3'.

[0043] Real-time fluorescence quantitative PCR reaction system

[0044]

[0045] Real-time fluorescent quantitative PCR reaction steps

[0046]

[0047] After the reaction, the amplification curve and melting curve of real-time fluorescent quantitative PCR were confirmed, and the expression intensity of each gene was standardized according...

Embodiment 2

[0050] Example 2, in situ hybridization detection found that the expression of LINC00673 in oral tumors is related to the prognosis of patients

[0051] 1. Material method

[0052] 1.1 Oligonucleotide probes for in situ hybridization of oral tumors and normal control tissues

[0053] Based on the LINC00673 gene sequence analysis, three oligonucleotide probes with the best hybridization parameters and the best specificity were designed. In addition, the housekeeping gene GAPDH was used as a positive control.

[0054] Oligonucleotide probes for in situ hybridization detection of LINC00673 expression:

[0055] LINC00673 Probe 1:

[0056] 5'-GAAAAACCTCTTGCACCACCTTAGTCTCCAAAGA-3';

[0057] LINC00673 Probe 2: 5'-CTTTCCTGTTCTTTTCTCTACCCTTCCTGACTAG-3';

[0058] LINC00673 Probe 3: 5'-CATGAAGTAATAATAAAGGTTCCGCTTATCAACC-3'.

[0059] Positive control probe (to detect the housekeeping gene GAPDH):

[0060] GAPDH probe 1: 5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3';

[0061] GAPDH probe 2: ...

Embodiment 3

[0124] Example 3, siRNA interferes with the expression of LINC00673

[0125] 1. Material method

[0126] 1.1 Reagents and kits

[0127] TRIZOL TM Reagent (Invitrogen);

[0128] Reverse transcription kit (#A3500, Promega);

[0129] Antibiotic G418 (Ameresc).

[0130] 1.2 Design of shRNA

[0131] Firstly, input LINC00673 sequence into Invitrogen's Block-It RNAi designer software to find the best siRNA target of this lncRNA, and select the best 2 corresponding target sequences as follows:

[0132] siRNA-1::5'-CCAGUUGUCCUUGACUGCAUGGUUU-3',

[0133] siRNA-2: 5'-AGGGAACCACAGGAUUCCAUGUGAU-3'

[0134] Negative control Scramble sequence: 5'-GACACGCGACUUGUACCAC-3'.

[0135] 1.3 Cell culture and transfection

[0136] The oral cancer cell line Tca8113 was purchased from the Cell Center of Central South University. The RPMI 1640 medium and fetal bovine serum used for cell culture, and the trypsin used for digesting cells were all products of Gibco, USA.

[0137] Oral cancer cell...

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Abstract

The invention discloses application of long non-coding RNA (LncRNA) LINC00673 to preparation of a prognosis preparation for oral tumor patients and particularly to preparation of a kit for predicting prognosis of oral tumor patients through a real-time fluorescent quantitative determination method. Studies verify that LncRNA LINC00673 is in up-regulated expression in oral tumor tissues, the oral tumor patients with highly expressed LncRNA LINC00673 are poorer in prognosis than those with lowly expressed LncRNA LINC00673, and accordingly using expression of the LncRNA LINC00673 for predicting the prognosis of the oral tumor patients can provide strong molecular biology bases for prognosis of the oral tumor patients and has far-reaching clinical significance and promising popularization and application prospect.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to an application method of long-chain non-coding RNA LINC00673. Background technique [0002] Oral and maxillofacial malignancies are common head and neck malignancies with high incidence, prone to cervical lymph node metastasis and poor prognosis. Studies have shown that the occurrence and development of this tumor is a complex process involving multiple genes, multiple steps, and multiple stages, in which the inactivation of tumor suppressor genes and the activation of oncogenes play a key role. [0003] Long non-coding RNAs (long non-coding RNAs, lncRNAs) are a class of RNAs molecules with a length of more than 200 nt, lack of a specific and complete open reading frame, and little or no protein coding function. Recent studies have shown that lncRNAs participate in the regulation of important life activities such as growth, development, aging and death of organ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/6886
CPCC12N15/113C12N2310/10C12Q1/686C12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178C12Q2561/113C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 龚朝建郭灿曾朝阳熊炜李桂源喻建军刘彦李小玲张姗姗李夏雨向波李征
Owner CENT SOUTH UNIV
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