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Human PKMYT1AR gene and application thereof

A gene and gene expression technology, applied in the field of genes and their new applications, can solve the problem of unclear potential mechanism of specific downstream target genes

Active Publication Date: 2022-03-25
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how PKMYT1 expression is upregulated in lung cancer and the underlying mechanisms of its specific downstream target genes remain unclear.

Method used

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  • Human PKMYT1AR gene and application thereof
  • Human PKMYT1AR gene and application thereof
  • Human PKMYT1AR gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: Network database application

[0077] 1. Screening of research object PKMYT1AR

[0078] By downloading 3 data sets in the GEO (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi) database, the data sets and their research objects are (1) GSE81089 lung cancer and paracancerous tissues (2) GSE144520: Transcriptome sequencing gene expression dataset of A549 cells and cisplatin-resistance A549 / DPP cells; (3) GSE157427: Transcriptome sequencing gene expression data of lung cancer cells and lung cancer stem cells Set, logFC>2, p<0.01 in the data set were screened separately, and the obtained significantly up-regulated lncRNAs were taken as their common intersection.

[0079] The result is as figure 1 As shown, a total of 3 lncRNAs were obtained, including LINC01224, NEAT1 and ENST00000595422 (PKMYT1AR). LINC01224 and NEAT, which have been reported in many literatures, were removed, and ENST00000595422 was selected as the follow-up research object.

[0080] 2. Ana...

Embodiment 2

[0086] Embodiment 2: fluorescent quantitative PCR (qRT-PCR)

[0087] After the cells A549 and SPC-A1 are cultured until the density reaches 80-90%, discard the medium, wash once with PBS, add 1mL Trizol on ice, fully lyse, blow evenly with a pipette gun, and transfer to RNA-free 1.5mL centrifuge tube for enzyme; place the collected Trizol lysed cell solution at 4°C and centrifuge at 12000g for 5min, transfer the supernatant to a new 1.5mL centrifuge tube with a pipette gun; add 200μL chloroform, mix thoroughly and let stand for 5min , and then placed in a centrifuge at 4°C at 12,000g for 15 minutes. After centrifugation, it was divided into three layers. Transfer the upper layer of RNA to a new 1.5mL centrifuge tube; Place in a centrifuge at 4°C, centrifuge at 12000g for 10min; discard the supernatant, add 1mL of 75% absolute ethanol prepared with DEPC water and mix upside down, then centrifuge at 7500g for 5min at 4°C; discard the supernatant, and dry the precipitate until T...

Embodiment 3

[0090] Embodiment 3: RNA nucleoplasm separation experiment

[0091] Prepare about 10 7For A549 and SPC-A1 cells, discard the medium, wash with PBS, trypsinize the cells, resuspend in PBS and place on ice, add 500 μL cell lysate to treat, let stand on ice for about 10 minutes, and centrifuge the sample for 5 minutes (500g, 4°C) and put it on ice to separate the cytoplasm and nucleus of the cells. The supernatant is the cytoplasm and the precipitate is the nucleus. Add 500 μL of ice-bathed cell lysate to the In the nuclear precipitation part, shake and mix vigorously to lyse the nucleus. After the nuclear material is dissolved, perform RNA extraction and PCR analysis. In the subsequent qPCR analysis, U1 is used as the nucleus control, and β-actin is used as the cytoplasmic control; the results are as follows: Figure 4 In B, it shows that PKMYT1AR exists in both the cytoplasm and nucleus in A549 cells, but is mainly localized in the cytoplasm.

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Abstract

The invention discloses a human PKMYT1AR gene. The nucleotide sequence of the human PKMYT1AR gene is as shown in SEQ ID NO: 1; the reagent for detecting the expression quantity of the human PKMYT1AR gene is applied to preparation of the non-small cell lung cancer clinical diagnosis reagent, the expression level of the human PKMYT1AR gene is in negative correlation with clinical prognosis of the non-small cell lung cancer, and experimental results show that the expression of the human PKMYT1AR gene in a non-small cell lung cancer cell line is higher than that of normal pulmonary epithelial cells; after the PKMYT1AR gene is knocked down, the proliferation of a non-small cell lung cancer cell line is obviously inhibited, and the cell cycle is retarded in a G0 / G1 phase; the killing effect of clinical platinum chemotherapeutic drugs for non-small cell lung cancer on tumor cells can be obviously improved; the invention reveals that the PKMYT1AR gene is a potential risk gene of the non-small cell lung cancer, and PKMYT1AR expression inhibition is combined with a radiotherapy or chemotherapy drug DDP for use, so that the clinical treatment effect of the non-small cell lung cancer is enhanced.

Description

technical field [0001] The present invention relates to a gene and its new application, especially the human PKMYT1AR gene and its application in tumor clinical diagnosis and drug screening, in particular to the application of human PKMYT1AR gene in non-small cell lung cancer clinical diagnosis and drug screening. Background technique [0002] Lung cancer is a fatal malignant tumor originating from bronchial mucosa or glands, which can be divided into small cell lung cancer (SCLC: small cell lung cancer) and non-small cell lung cancer (NSCLC: non-small cell lung cancer). SCLC and NSCLC account for approximately 20% and 80% of lung cancer cases, respectively, while NSCLC can be further subdivided into adenocarcinoma (LUAD), squamous cell carcinoma (LUSC) and large cell lung cancer. The early treatment of lung cancer is a comprehensive treatment based on surgery. Multidisciplinary treatment plays an important role in the treatment of advanced non-small cell lung cancer. Radiot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6886C12N15/11A61K31/713A61P35/00
CPCC12N15/1137C12Q1/6886C12N15/113A61P35/00C12N2310/11C12Y207/11001C12Q2600/158C12Q2600/178C12Q2600/136C12Q2600/118C12N2310/14C12N2310/113C12N2320/32C12N2310/531Y02A50/30
Inventor 杨翠萍陈勇彬熊秋霞
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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