Expression vector and anti-tumor reagent of long-chain non-coded RNALOC401317, and applications of expression vector and anti-tumor reagent

A long-chain non-coding, expression vector technology, applied in the field of long-chain non-coding RNA LOC401317 expression vector, can solve the problem of unknown lncRNA function, and achieve the effect of prolonging the action time, high transfection efficiency, and inhibiting growth.

Active Publication Date: 2015-03-04
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 and anti-tumor reagent of long-chain non-coded RNALOC401317, and applications of expression vector and anti-tumor reagent
  • Expression vector and anti-tumor reagent of long-chain non-coded RNALOC401317, and applications of expression vector and anti-tumor reagent
  • Expression vector and anti-tumor reagent of long-chain non-coded RNALOC401317, and applications of expression vector and anti-tumor reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1, the TP53 gene was introduced into nasopharyngeal carcinoma cells, and the expression of lncRNA LOC401317 was significantly upregulated

[0059] 1. Materials and methods:

[0060] 1.1 Reagents and kits

[0061] 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 Assay Kit (Dual-Luciferase Reporter 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 46506 lnc...

Embodiment 2

[0092] Example 2, lncRNA LOC401317 inhibits nasopharyngeal carcinoma cell cycle arrest and induces apoptosis

[0093] 1. Materials and methods

[0094] 1.1 Reagents and kits

[0095] Restriction enzymes Nhe I and EcoR I and T4DNA ligase were purchased from TakaRa Company; TRIZOL TM Reagent (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).

[0096] 1.2 Construction of pcDNA3.1-LOC401317 eukaryotic expression vector

[0097] We chose pcDNA3.1 blank vector (from Invitrogen Company) to construct the overexpression vector of LOC401317. We chose Nhe I and EcoR I restriction sites for digestion of pcDNA3.1 vector and inserted LOC401317 sequence into this site.

[0098] The steps to construct pcDNA3.1-LOC401317 eukaryotic vector are as follows:

[0099] 1) Using the cDNA of HNE2 cells transfected with T...

Embodiment 3

[0150] Example 3, LOC401317 inhibits the growth of nasopharyngeal carcinoma cells in a nude mouse transplantation tumor model

[0151] 1. Materials and methods

[0152] Thirty 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.

[0153] The construction of LOC401317 eukaryotic expression vector and the preparation, cell culture and transfection of polylysine-modified silicon nanoparticles are the same as in Example 2.

[0154] Take 2×10 HNE2 cells transfected with LOC401317 or pcDNA3.1 blank vector, and HNE2 cells (Mock) without any treatment 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 skin fr...

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Abstract

The invention discloses an expression vector and an anti-tumor reagent of long-chain non-coded RNALOC401317, and applications of the expression vector and the anti-tumor reagent, namely the applications in preparation of a preparation for inhibiting growth and proliferation of tumor cells. According to a sequence of the lncRNA, a eukaryotic expression vector for overexpression of LOC401317 is designed and synthesized, and loaded on polylysine modified silicon nano-particles to prepare nano-spheres, so that the LOC401317 is successfully expressed in a nasopharynx cancer cell line and the growth of the nasopharynx cancer cells is inhibited. The polylysine modified silicon nano-particles mentioned in the invention can protect the LOC401317 vector from degradation of nuclease and prolong the action time, and thus a higher transfection efficiency is realized and further development and application are facilitated.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and specifically relates to an expression vector of long-chain non-coding RNA LOC401317, 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 tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/63C12N15/66A61P35/00
Inventor 熊炜李桂源曾朝阳龚朝建李小玲廖前进陈攀曾勇张姗姗向娟娟周艳宏李征
Owner CENT SOUTH UNIV
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