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Application of lncRNA TRALR (long-chain non-coding RNA TRALR)

A pair-and-reagent technology, applied in the application field of long-chain non-coding RNATRALR, can solve the problem that the specific mechanism has not been elucidated, and achieve the effect of important promotion and application prospect, profound clinical significance, and increasing sensitivity.

Inactive Publication Date: 2018-09-07
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latest research shows that in addition to MGMT, the abnormality of various signaling pathways such as epidermal growth factor receptor (EGFR) in tumor cells is involved in the occurrence of TMZ resistance, but the specific mechanism has not yet been elucidated.

Method used

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  • Application of lncRNA TRALR (long-chain non-coding RNA TRALR)
  • Application of lncRNA TRALR (long-chain non-coding RNA TRALR)
  • Application of lncRNA TRALR (long-chain non-coding RNA TRALR)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 is to construct temozolomide-resistant T98G and U343 glioma cell lines

[0045] (1) IC50 was detected by MTS method: the cells were divided into 1×10 3 Seed in a 96-well plate at a density of / well, add a gradient concentration of temozolomide solution, and incubate at 37°C, 5% CO 2 Incubate for 72 h in an incubator. Add 20 μl MTS to each well. After incubating in the incubator for 2 h, the absorbance of each well was measured at 450 nm in a microplate reader. IC50 was calculated by SPSS software.

[0046] (2) Cell cycle detection by flow cytometry: the cells were divided into 1×10 6 The density was seeded in a 6-well plate, and temozolomide was added to incubate for 24 h. Digest the cells with trypsin, centrifuge at 1,000rpm for 4min after the digestion is terminated, and discard the supernatant. Wash the cells with ice-cold PBS, centrifuge at 1,000rpm for 4min, and discard the supernatant. Resuspend the cells in 100 μl PBS, slowly add 1ml 70-80% ethan...

Embodiment 2

[0047] Example 2 RT-PCR screening and verification of differentially expressed lncRNAs in glioma TMZ-resistant cells

[0048] (1) Extraction of total cellular RNA (TRIzol method)

[0049] The cells were seeded in 6-well plates and incubated in a 37°C, 5% CO2 incubator. After the cells were completely attached to the wall, 1000 μl TRIzol was added to each well. After standing still until completely dissolved, transfer to a 1.5ml EP tube. Add 0.2ml of chloroform, shake vigorously for 15 seconds, and place at room temperature for 3 minutes. Centrifuge at 4°C and 12000rpm for 15min, and transfer the supernatant to a new EP tube. Add 0.5ml of isopropanol and let stand at room temperature for 10min. After centrifugation at 4°C and 12,000 rpm for 10 minutes, a gelatinous precipitate appeared at the bottom of the EP tube. Discard the supernatant, wash the RNA pellet with 1ml 75℅ ethanol, and centrifuge at 4°C, 7500rpm for 5min, discard the supernatant again. After air-drying for...

Embodiment 3

[0072] Example 3 Smart Silencer Knockdown of lncRNA TRALR Causes G2 / M Phase Arrest and Inhibits Proliferation

[0073] 1) Inoculate cells: inoculate 1×10 5 ~5×10 5 Put the cells into the culture wells of a 24-well plate containing an appropriate amount of complete medium, so that the cell density at the time of transfection can reach 30-50%.

[0074] 2) Transfection step

[0075] For each transfection sample, prepare as follows:

[0076] a. Dilute lncRNASmart Silencer: use 30μl 1X riboFECT TM Dilute 2.5μl 20μM RiboTM lncRNASmart Silencer stock solution with CP Buffer and mix gently. The Smart Silencer sequence is as follows:

[0077] No. Target sequence (5'-3')

[0078] 1CCACCACTACAAAGCTTAA

[0079] 2CCTGTACATACCCAGATAA

[0080] 3CCTCTAACTAACATGACTT

[0081] 4GTAGGCACCTGACTTCGTGG

[0082] 5CTTGTGCAGGTGACAGGGAA

[0083] 6ACAGATTCGTGAGTGCCAGG

[0084] b. Mixture preparation: add 3μl riboFECT TM CP Reagent, gently pipette to mix, and incubate at room temperature for ...

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Abstract

The invention discloses an application of lncRNA TRALR (long-chain non-coding RNA TRALR). According to the research, lncRNA sequencing shows that lncRNA TRALR has abnormal high expression in a temozolomide-resistant brain glioma cell line, by means of specific interference to expression of lncRNA TRALR, BMI1 can be obviously inhibited and p21 level can be remarkably up-regulated, cell cycle arrestand aging are promoted, and sensitivity of brain glioma to temozolomide is increased. Therefore, a reagent for detecting the expression of lncRNA TRALR can be used for preparing preparations for brain glioma diagnosis and prognosis or sensitivity detection of patients with brain glioma, powerful molecular biology basis is provided for diagnosis, prognosis and chemotherapy sensitivity prediction of the patients with brain glioma, and far-reaching clinical significance and great popularization and application prospect are achieved.

Description

technical field [0001] The invention belongs to the technical field of tumor molecular biology, and specifically relates to the application of a long-chain non-coding RNA TRALR. Background technique [0002] Glioma is the most common malignant tumor of the central nervous system, accounting for about 80% of primary malignant brain tumors. According to the European Neuro-Oncology Association (EANO) statistics, the annual incidence of glioma in the world is about 6 / 100,000 . The treatment of glioma is a worldwide problem, especially for malignant glioblastoma. Even if the optimal treatment plan is adopted, the prognosis of patients is still very unsatisfactory, and the average survival period is only 13.3-15 months. Temozolomide (TMZ) is a new drug with better efficacy in the treatment of glioma that has emerged in the past two decades. It has been approved by the US FDA and recommended by the National Comprehensive Cancer Network guidelines and has become the preferred chemo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 龚志成颜元良徐志杰陈曦霍雷李学军曾双双钱龙熊小明
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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