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Application of alkbh1 gene and its expression product in the preparation of kits for diagnosing tumors and drugs for treating tumors

A technology of gene expression and reagents, applied in the fields of biotechnology and medicine, can solve the problem of ALKBH1 mechanism of action without literature reports, etc., to achieve weakened cell migration rate and cell invasion ability, increase cell migration rate and cell invasion ability, and increase the level of modification Effect

Active Publication Date: 2021-06-04
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological function and possible mechanism of ALKBH1 in tumors have not been reported in the literature.

Method used

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  • Application of alkbh1 gene and its expression product in the preparation of kits for diagnosing tumors and drugs for treating tumors
  • Application of alkbh1 gene and its expression product in the preparation of kits for diagnosing tumors and drugs for treating tumors
  • Application of alkbh1 gene and its expression product in the preparation of kits for diagnosing tumors and drugs for treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: RT-PCR reaction detection of ALKBH1 gene expression in gastric cancer and liver cancer tissues.

[0036] The specific experimental plan is as follows:

[0037] 1. RNA extraction

[0038] 1) Tissue processing: Take about 10 mg of tissue and add 1 ml Trizol, homogenize with a homogenizer; centrifuge for 15 minutes at 12000 g, and take the supernatant.

[0039] 2) Add 200ul chloroform to the supernatant, mix vigorously up and down for half a minute, and let stand for 3 minutes.

[0040] 3) Centrifuge at 12,000g for 15 minutes at 4°C. At this time, it can be seen that the lysate is divided into three layers: the upper layer is RNA in the aqueous phase; the middle layer is DNA, lipids, etc.; the lower layer is cell residues, proteins, polysaccharides, etc.

[0041]4) Take the supernatant into a new EP tube; add an equal volume of isopropanol, mix well, let stand for 10 minutes, and then centrifuge at 12000g for 10 minutes at 4°C.

[0042] 5) Carefully remove th...

Embodiment 2

[0053] Example 2: Construction of ALKBH1 overexpression / silencing cells

[0054] (1) Digest the cells into a single-cell suspension, count, and use 5×10 5 The concentration of cells / well was inoculated into 6-well plates at 37°C, 5% CO 2 Incubate overnight in the incubator.

[0055] (2) Dissolve 2 μg of the overexpressed ALKBH1 plasmid in 250 μl opti-MEM; dissolve siALKBH1 in RNase-free deionized water to a final concentration of 20 μM, take 20 μl and dissolve it in 250 μl opti-MEM, mix well and let stand.

[0056] Among them, the relevant primer sequences for constructing overexpressed ALKBH1 plasmids are as follows:

[0057] Sense: 5'-CGGGGTACCGCGAGATGGGGAAGATGGCAGC-3' (SEQ ID NO: 1)

[0058] Anti-sense: 5'-CGGAATTCTTACTTGTCATCGTCGTCCTTGTAGTCGCTGTCAGGGTTTATCCTGGC-3' (SEQ ID NO: 2).

[0059] Wherein, the related siRNA sequence is as follows:

[0060] siALKBH1#1-sense:5'-GCAAGCCUAUGGACUCAAATT-3' (SEQ ID NO:5)

[0061] siALKBH1#1-anti-sense: 5'-UUUGAGUCCAUAGGCUUGCTT-3' (S...

Embodiment 3

[0072] Example 3: Verification that ALKBH1 is associated with cellular DNA 6mA levels

[0073] (1) DNA of tumor cells overexpressing / silencing ALKBH1 gene was extracted, denatured at 95° C. for 10 minutes, and cooled on ice for 5 minutes.

[0074] (2) Drop the DNA on the nitrocellulose membrane and dry it at room temperature for 5 minutes.

[0075] (3) Dry the membrane at 80°C for 2 hours, and then seal it with blocking solution at room temperature for 1.5 hours.

[0076] (4) Incubate the membrane with the specific 6ma antibody overnight at 4°C.

[0077] (5) Incubate the membrane with HRP-conjugated secondary antibody at room temperature for 1.5h, and then expose it in the dark room.

[0078] see results image 3 , 7 , the DNA 6mA level of tumor cell lines with up-regulated ALKBH1 expression decreased, and the DNA 6mA level of tumor cell lines with down-regulated ALKBH1 expression increased. These results suggest that ALKBH1 can regulate the DNA 6mA level of tumor cells. ...

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Abstract

The invention provides the application of human ALKBH1 gene in the preparation of products for diagnosing and / or treating tumors. The present invention finds that the expression level of the ALKBH1 gene is significantly increased by detecting the expression of the ALKBH1 gene in tumor tissues of gastric cancer and liver cancer patients. Compared with the control group, the DNA 6mA modification level, proliferation ability, cell migration rate and cell invasion ability of the liver cancer and ovarian cancer cell lines constructed by constructing ALKBH1 gene overexpression / silencing were significantly changed. The ALKBH1 gene and its expression products are used as markers for diagnosing tumors, making tumor diagnosis more accurate and rapid, and as target genes for preparing tumor drugs, providing new therapeutic targets and therapeutic approaches for treating tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, and specifically relates to the application of ALKBH1 gene and its expression product in preparing a kit for diagnosing tumors and medicines for treating tumors. Background technique [0002] Tumor is a major disease that threatens human health, and its occurrence and development is a multi-step process. Although clinical treatment programs have been continuously improved, the five-year survival rate of patients is still very low. Early diagnosis and treatment are the key to improving the survival rate of cancer patients. The occurrence and development of tumors involves the activation of oncogenes and the inactivation of tumor suppressor genes. By detecting the changed oncogenes and tumor suppressor genes, the occurrence of tumors can be detected early at the molecular level, thereby creating opportunities for timely diagnosis and treatment. . [0003] With the deepening of tumor rese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886G01N33/574A61K48/00A61K31/713A61P35/00
CPCA61K31/713C12Q1/6886C12Q2600/158C12Q2600/178G01N33/57438G01N33/57446G01N33/57449
Inventor 晏光荣肖传乐朱嵩焦军严兆坤何铭辉韩悦罗雪峰
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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