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Application of EphrinA1 protein in preparation of drug for inhibiting tumor cell invasion and metastasis

A tumor cell and protein technology, applied in antineoplastic drugs, drug combinations, pharmaceutical formulations, etc., can solve the problems of lack of early diagnosis, poor prognosis, and death of gastric cancer patients, and achieve the ability to inhibit metastasis, tumor metastasis, and invasion Effect

Inactive Publication Date: 2019-03-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the insidious onset of gastric cancer and the lack of effective means for early diagnosis, most patients are in the middle and late stages when they are discovered, so the prognosis is extremely poor, especially for patients with distant metastasis, whose 5-year survival rate is only about 5%, which is an advanced stage. One of the leading causes of death in gastric cancer patients

Method used

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  • Application of EphrinA1 protein in preparation of drug for inhibiting tumor cell invasion and metastasis
  • Application of EphrinA1 protein in preparation of drug for inhibiting tumor cell invasion and metastasis
  • Application of EphrinA1 protein in preparation of drug for inhibiting tumor cell invasion and metastasis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. EphrinA1 is significantly highly expressed in human gastric cancer tissues and gastric cancer cell lines

[0043] 1. Expression of EphrinA1 in various human tissues and human gastric cancer cell lines

[0044] 1.1 The tissue is ground into a powder with a mortar at a very low temperature, and the total RNA of the powdered tissue is separated and extracted using TRIZOL reagent (purchased from Invitrogen, USA), and performed in accordance with the standard operation of the reagent manual.

[0045] 1.2 Take a PCR tube A and add 800ng total RNA, make up DEPC water to a total volume of 10μL, and mix. Take another PCR tube B and add 2μL of 10xRT buffer, 2μL of 10x random primer, 0.8μL of dNTP, 1μL of RNA reverse transcriptase, 4.2μL of DEPC water, and mix. Add the mixture of tube B to tube A, mix well, and perform reverse transcription reaction using a PCR machine (purchased from American ABI). The procedure is as follows:

[0046] 25℃

10min

37℃

120min

85℃

5min

[...

Embodiment 2

[0057] Example 2, RNA interference technology knocking down the expression of EphrinA1 can significantly inhibit the invasion of gastric cancer cells

[0058] 1. Cell transfection with siRNA

[0059] Through the siRNA design software, design and synthesize two siRNAs specifically targeting EphrinA1 and silence its expression (purchased from Shanghai Jima, China). Take the cells in the logarithmic growth phase and plate them. When the cell density reaches about 50%, use the transfection system in the following table to dilute Lipo RNAi MAX (purchased from Invitrogen, US) and siRNA with OPTI medium (purchased from Gibco, US). Add the diluted siRNA to the Lipo RNAi MAX tube, mix well and let it stand for 5 minutes, then add it to the cell culture medium, shake well, and change the medium after 24.

[0060] Component

24-well

6-well

SiRNA-lipid complex per well

50ul

250ul

Final siRNA used per well

5pmol

25pmol

Final Lipo RNAi MAX used per well

1.5ul

7.5ul

[0061] 2. Cell prol...

Embodiment 3

[0077] Example 3. Stable knockdown of EphrinA1 can significantly inhibit the invasion ability of gastric cancer cells

[0078] 1. Lentiviral vector construction

[0079] 1.1 Design and synthesis shEphrinA1

[0080] According to the shRNA design principles, with EphrinA1RNA as the target sequence, two target site sequences for EphrinA1 were designed, and the corresponding forward and reverse sequences (purchased from Shanghai Shenggong, China) were synthesized, respectively:

[0081]

[0082] 1.2 Annealing:

[0083] Take 100pM single-stranded DNA, add 10x annealing buffer, and add 50uL ddH2O. Put it in the water that has just been boiled and let it slowly cool to room temperature.

[0084] The shRNA lentiviral expression vector of EphrinA1 was constructed through the steps of restriction digestion, linking, identification, and sequencing.

[0085] 2. Lentivirus packaging

[0086] 2.1 Spread HEK293T cells into T25 flasks, and when the cell density reaches 80-90%, perform transfection.

[008...

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Abstract

The invention discloses application of EphrinA1 in tumor development, metastasis, treatment and prognosis of patients. The invention discloses that EphrinA1 is involved in the process of tumor growth,invasion and metastasis. The high expression of EphrinA1 in a gastric cancer tissue leads to poor prognosis of gastric cancer patients, and targeted knockdown or knockout of EphrinA1 expression can significantly inhibit the invasion ability of gastric cancer cells and the metastasis ability of the gastric cancer cells in mice. The invention utilizes a function that an antibody targeting EphrinA1blocks EphrinA1, and can effectively inhibit the invasion ability of the gastric cancer cells. The invention finds that EphrinA1 has an important application value in tumor therapy and can be used asa target for antitumor drugs.

Description

Technical field [0001] The invention relates to the application of EphrinA1 protein in the preparation of drugs for inhibiting tumor cell invasion and metastasis Background technique [0002] The latest global cancer statistics in 2018 estimated the incidence and mortality of 36 cancers in 185 countries around the world. The report shows that in 2018, the number of new cancer cases worldwide will reach 18.1 million, and the number of deaths will reach 9.6 million. It is expected to be the leading cause of death in the 21st century. The five cancers with the highest incidence rates in both sexes are lung cancer (11.6% of the total number of cancers), female breast cancer (11.6%), colorectal cancer (10.2%), prostate cancer (7.1%) and gastric cancer (5.7 %). The five cancers with the highest mortality rate are lung cancer (18.4% of total cancer deaths), colorectal cancer (9.2%), gastric cancer (8.2%), liver cancer (8.2%) and breast cancer (6.6%) . This result shows that the globa...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P35/00
CPCA61K45/00A61P35/00
Inventor 周天华卓巍李爽刘易曼
Owner ZHEJIANG UNIV
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