tRNA-derived fragment (tRF) related to non-small cell lung cancer (NSCLC) and application of tRF

A kit and inhibitor technology, applied in tRF and its application fields, can solve the problem of limited molecular markers for the diagnosis of non-small cell lung cancer

Active Publication Date: 2019-11-19
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a tRF (AS-tDR-007333) related to NSCLC and its application, aiming to solve the technical problem of limited molecular markers for the diagnosis of non-small cell lung cancer

Method used

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  • tRNA-derived fragment (tRF) related to non-small cell lung cancer (NSCLC) and application of tRF
  • tRNA-derived fragment (tRF) related to non-small cell lung cancer (NSCLC) and application of tRF
  • tRNA-derived fragment (tRF) related to non-small cell lung cancer (NSCLC) and application of tRF

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Discovery of AS-tDR-007333

[0061] 1. Selection of clinical research samples

[0062] (1) NSCLC cases diagnosed by pathology;

[0063] (2) The case has not undergone surgery, radiotherapy and chemotherapy before blood collection;

[0064] (3) A healthy control group matched with the age of the cases.

[0065] Blood collection time: preoperative blood is collected before anesthesia, and postoperative blood is collected 3-5 days after operation.

[0066] Collection standard: Collect 10ml of peripheral blood uniformly using blood collection tubes containing EDTA anticoagulant, mix them upside down, and put them in a 4°C refrigerator for temporary storage as soon as possible. Centrifuge within 12 hours (3000rpm, 5 minutes), use a disposable sterile dropper or pipette gun in the ultra-clean workbench to collect the upper layer of plasma and the lower layer of blood cells respectively, and place them in a 2ml external screw-cap cryopreservation tube. Mark the p...

Embodiment 2

[0080] Example 2 Analysis of expression differences of AS-tDR-007333

[0081] 1. Extraction of cellular RNA

[0082] (1) After washing the cells twice with PBS, add 1ml TRIzol reagent and let stand at room temperature for 10 minutes;

[0083] (2) Add 200 μl chloroform, close the cap tightly, shake vigorously for 30 seconds, and place at room temperature for 10 minutes;

[0084] (3) Centrifuge at 12000g, 4°C for 20 minutes, absorb the upper aqueous phase, add a new 1.5ml centrifuge tube, add 600μl pre-cooled isopropanol to precipitate RNA, mix well, and place at room temperature for 10 minutes;

[0085] (4) Centrifuge at 12000g, 4°C for 10 minutes, discard the supernatant;

[0086] (5) Use 75% ethanol prepared in DEPC water to slowly and gently adhere to the wall and add to the centrifuge tube to clean the RNA;

[0087] (6) Centrifuge at 7500g at 4°C for 10 minutes, suck up the ethanol in the tube, dry at room temperature for 5-10 minutes, and add 10-30 μl of DEPC water;

...

Embodiment 3

[0118] Example 3 Effect of overexpression or underexpression of AS-tDR-007333 on cell proliferation

[0119] 1. The effect of AS-tDR-007333 on the proliferation of NSCLC cell PC9.

[0120] Transfect the overexpressed single chain of AS-tDR-007333 (SEQ ID No.1) and its negative control AS-tDR-007333-NC (SEQ ID No.4) into PC9 cell lines respectively, culture for 48 hours, and adjust the cell density 2.5x10 4 / ml, spread into a 96-well plate at a concentration of 5000 cells / well. Set aside 5 wells in each plate as blank control wells.

[0121] Put the cells in the incubator for 6-12 hours; start testing after the cells adhere to the wall, which is recorded as the D0 time point; take 24 hours as the recording point, and so on as D1, D2, D3; 3.5ml complete medium + After 350 μl of CCK-8 reagent was thoroughly mixed, it was added to the experimental wells and blank control wells at a volume of 110 μl per well. After adding the samples, incubate in an incubator for 1 hour; take o...

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Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a tRNA-derived fragment (tRF) related to non-small cell lung cancer (NSCLC) and application of the tRF.Through experimental analysis, the tRF (AS-tDR-007333, the nucleotide sequence is shown in SEQ ID No.1) is significant in cancer-promoting activity, and the expression of the tRF in NSLC tissue, in plasma of NSCLC patients and in NSLC cells is significantly higher than that of the tRF in para-carcinoma tissue, healthy human plasma and normal bronchial epithelial cells. The results show that the tRF has the ability to significantly promote the tumor activity of NSCLC cells; and the expression of the tRF is inhibited to significantly inhibit the proliferation of the NSCLC cells.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to tRF related to NSCLC and its application. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world and the greatest hazard to human health. About 1.6 million people die from lung cancer every year in the world; among them, non-small cell lung cancer (NSCLC) accounts for about 80% of the total number of lung cancers, and is the most common cause of death from cancer. In China, lung cancer is not only the most common malignant tumor, but also its morbidity and mortality have been on the rise in the past 20 years. According to the forecast of the World Health Organization, if effective prevention and control measures cannot be taken, there will be more than one million new lung cancer patients in China every year by 2025, which will seriously threaten the lives and health of Chinese people...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/6886A61K31/7105A61P35/00
CPCA61K31/7105A61P35/00C12N15/113C12Q1/6886C12Q2600/118C12Q2600/178
Inventor 翟日洪杨文瀚钱有辉和琪涵
Owner SHENZHEN UNIV
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