Application of siRNA of long non-coding RNA LINC00673 and inhibition preparation

A long-chain non-coding and preparation technology, applied in the application field of siRNA, can solve the problems of unknown function of lncRNA

Active Publication Date: 2017-03-15
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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  • Application of siRNA of long non-coding RNA LINC00673 and inhibition preparation
  • Application of siRNA of long non-coding RNA LINC00673 and inhibition preparation
  • Application of siRNA of long non-coding RNA LINC00673 and inhibition preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1, real-time fluorescence quantitative method detection confirmed the down-regulation of LINC00673 expression in oral tumors

[0031] 1. Materials and methods:

[0032] 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.

[0033] Forward primer: 5'-TTCTCCTGTAACGTGTGGCC-3',

[0034] Reverse primer: 5'-CTGGTGGGAATGTGGATCAGT-3'.

[0035] Internal reference gene GAPDH specific PCR primers for control

[0036] Forward primer 5'-ACCACAGTCCATGCCATCAC-3'

[0037] Reverse primer 5'-TCCACCACCCCTGTTGCTGTA-3'.

[0038] Real-time fluorescence quantitative PCR reaction system

[0039]

[0040] Real-time fluorescent quantitative PCR reaction steps

[0041]

[0042] 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 standardiz...

Embodiment 2

[0045] Example 2, in situ hybridization detection found that the expression of LINC00673 in oral tumors is related to the prognosis of patients

[0046] 1. Material method

[0047] 1.1 Oligonucleotide probes for in situ hybridization of oral tumors and normal control tissues

[0048] Based on the LINC00673 gene sequence analysis, two 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.

[0049] Oligonucleotide probes for in situ hybridization detection of LINC00673 expression:

[0050] LINC00673 Probe 1:

[0051] 5'-GAAAAACCTCTTGCACCACCTTAGTCTCCAAAGA-3';

[0052] LINC00673 Probe 2: 5'-CTTTCCTGTTCTTTTCTCTACCCTTCCTGACTAG-3';

[0053] LINC00673 Probe 3: 5'-CATGAAGTAATAATAAAGGTTCCGCTTATCAACC-3'.

[0054] Positive control probe (to detect the housekeeping gene GAPDH):

[0055] GAPDH probe 1: 5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'

[0056] GAPDH probe 2: 5'-GT...

Embodiment 3

[0118] Example 3, siRNA interferes with the expression of LINC00673

[0119] 1. Material method

[0120] 1.1 Reagents and kits

[0121] TRIZOL TM Reagent (Invitrogen);

[0122] Reverse transcription kit (#A3500, Promega);

[0123] Antibiotic G418 (Ameresc).

[0124] 1.2 Design of shRNA

[0125] 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:

[0126] siRNA-1::5'-CCAGUUGUCCUUGACUGCAUGGUUU-3',

[0127] siRNA-2: 5'-AGGGAACCACAGGAUUCCAUGUGAU-3'

[0128] Negative control Scramble sequence: 5'-GACACGCGACUUGUACCAC-3'.

[0129] 1.3 Cell culture and transfection

[0130] 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.

[0131] Oral cancer cell...

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Abstract

The invention discloses application of siRNA of long non-coding RNA LINC00673 and an inhibition preparation. The sequence of the siRNA of a targeted LINC00673 is designed, synthesized, and transfected into oral cancer cell strains. It is proved that the expression of the targeted interference LINC00673 can obviously inhibit the invasion and transfer abilities of the oral cancer cells. The inhibition preparation provides a new and potential pathway for treating an oral cancer.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to an application method of siRNA 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...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/7105A61P35/00A61P35/04
CPCA61K31/7105
Inventor 曾朝阳郭灿熊炜李桂源喻建军刘彦李小玲龚朝建张姗姗张文玲周鸣周艳宏
Owner CENT SOUTH UNIV
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