Expression vector of long-chain non-coding RNALINC00472, tumor suppression reagent and application thereof

A technology of long-chain non-coding and expression vectors, applied in the field of expression vectors of long-chain non-coding RNA LINC00472, which can solve the problems of unknown function of lncRNA and achieve the effects of prolonging the action time, inhibiting growth, and high transfection efficiency

Active Publication Date: 2017-06-23
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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  • Expression vector of long-chain non-coding RNALINC00472, tumor suppression reagent and application thereof
  • Expression vector of long-chain non-coding RNALINC00472, tumor suppression reagent and application thereof
  • Expression vector of long-chain non-coding RNALINC00472, tumor suppression reagent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1, the TP53 gene was introduced into nasopharyngeal carcinoma cells, and the expression of lncRNA LINC00472 was significantly upregulated

[0055] 1. Materials and methods:

[0056] 1.1 Reagents and kits

[0057] Common biochemical reagents such as agarose (agrose) and gel recovery kits were purchased from Shanghai Huashun Biological Engineering Co., Ltd. Mini Kit (Qiagen) extraction kit extracts RNA with improved quality, SuperScript TM The III (Invitrogen) kit reverse transcribed RNA into cDNA. The Luciferase Reporter Gene Detection Kit Dual-LuciferaseReporter Assay System was purchased from Promega. The nasopharyngeal carcinoma cells HNE2 used in the present invention are preserved by the Cancer Institute of Central South University. The RPMI1640 medium and fetal bovine serum used for cell culture, and the trypsin used for digesting cells are all products of Gibco, USA. The lncRNA chip is a product of Agilent, the chip size is 4*180K, and there are 4650...

Embodiment 2

[0087] Example 2, lncRNA LINC00472 inhibits nasopharyngeal carcinoma cell cycle arrest and induces apoptosis

[0088] 1. Materials and methods

[0089] 1.1 Reagents and kits

[0090] Restriction endonucleases Nhe I, EcoR V and T4 DNA ligase were purchased from TakaRa Company; TRIZOL TMReagent (Invitrogen); Plasmid Extraction Kit, Gel Recovery Kit (OMEGA); Reverse Transcription Kit (Promega); Proteinase K, DNase I, RNAsin, RNase A (GBICOL Company); Tetramethylazolazolium Blue ( MTT, Sigma); antibiotic G418 (Ameresc).

[0091] 1.2 Construction of pcDNA3.1-LINC00472 eukaryotic expression vector

[0092] We chose pcDNA3.1 blank vector (from Invitrogen Company) to construct the overexpression vector of LINC00472. We selected Nhe I and EcoR V restriction sites for restriction digestion of pcDNA3.1 vector and inserted LINC00472 sequence into this site.

[0093] The steps to construct the pcDNA3.1-LINC00472 eukaryotic vector are as follows:

[0094] 1) Using the cDNA of HNE2 cel...

Embodiment 3

[0143] Example 3, LINC00472 inhibits the growth of nasopharyngeal carcinoma cells in a nude mouse transplantation tumor model

[0144] 1. Materials and methods

[0145] Eight male BALB / C nude mice, 4 weeks old, weighing 19±2g, were purchased from Shanghai Slack Experimental Animal Co., Ltd. All nude mice passed the quality inspection and were bred under specific pathogen-free (SPF) conditions in the Experimental Animal Department of Central South University.

[0146] The construction of LINC00472 eukaryotic expression vector and the preparation, cell culture and transfection of polylysine-modified silicon nanoparticles are the same as in Example 2.

[0147] HNE2 cells transfected with LINC00472 or pcDNA3.1 blank vector, and HNE2 cells (Mock) without any treatment were taken 2×10 6 Inject into the subcutaneous of the armpits of nude mice respectively, observe and compare the growth of the tumors, measure the size of the transplanted tumors with a vernier caliper across the sk...

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Abstract

The invention discloses an expression vector of long-chain non-coding RNA LINC00472, a tumor suppression reagent and application thereof, that is to apply the tumor suppression reagent to preparation of preparations for suppressing tumor cell growth and multiplication. According to the IncRNA sequence, an eukaryotic expression vector used for overexpression of LINC00472 is designed and synthesized, the vector is loaded on silicon nano-particles modified by polylysine to prepare nanospheres, so that LINC00472 can be successfully expressed in a nasopharyngeal carcinoma cell line, and growth of nasopharyngeal carcinoma cells is suppressed. The silicon nano-particles modified by polylysine can protect the LINC00472 vector against nuclease degradation, action time can be prolonged, transfection efficiency is higher, and further development and application can be better achieved.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to an expression vector of long-chain non-coding RNA LINC00472, a tumor suppressor agent and applications thereof. Background technique [0002] Nasopharyngeal carcinoma is a common malignant tumor of the head and neck with a 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] Since the TP53 gene (encoding p53 protein) was cloned in 1979, it has been one of the most striking and important tumor suppressor genes in the field of tumor molecular biology research. A large amount of evidence shows that TP53 gene is widely involved in the occurrence and development of various malignant tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85A61K31/7105A61P35/00
CPCA61K31/7115C12N15/1135C12N15/85C12N2310/10C12N2800/107
Inventor 曾朝阳李桂源李小玲熊炜龚朝建张姗姗郭灿熊芳廖前进周钰娟周鸣王贺冉
Owner CENT SOUTH UNIV
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