Application of stomatin protein and its coding gene in diagnosis and treatment of lung cancer

A gene and protein technology is applied in the application field of stomatin protein and its encoding gene in the diagnosis and treatment of lung cancer, which can solve the problems of unclear function and function of stomatin.

Inactive Publication Date: 2019-04-19
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role and function of stomatin in tumorigenesis and progression are still unclear

Method used

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  • Application of stomatin protein and its coding gene in diagnosis and treatment of lung cancer
  • Application of stomatin protein and its coding gene in diagnosis and treatment of lung cancer
  • Application of stomatin protein and its coding gene in diagnosis and treatment of lung cancer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1, cell preparation

[0091] 1. Cell preparation for transient knockdown of stomatin expression

[0092] 1. Design siRNA for the stomatin gene, the sequence is as follows:

[0093] Sense strand (5'-3'): GGAAUGUUCUGGGCACCAA dTdT;

[0094] Antisense strand (3'-5'): dTdT CCUUACAAGACCCGUGGUU.

[0095] 2. Artificially synthesize the siRNA in step 1.

[0096] 3. Take the siRNA synthesized in step 2, and use the small RNA transfection kit (Ribobio, Cat. No.: C10511-1) to transfect A549 cells (refer to the kit manual for the method), and set up the cells in the experimental group transfected with siRNA and those without transfection. The cells of the control group transfected with siRNA were harvested after 48 hours for detection or follow-up experiments.

[0097] 4. Take the siRNA synthesized in step 2, and use the small RNA transfection kit to transfect H520 cells (refer to the kit manual for the method), set up the experimental group cells transfected with siR...

Embodiment 2

[0118] Embodiment 2, the effect of stomatin on tumor cell proliferation

[0119] 1. MTT experiment

[0120] The proliferation of A549-sinc, A549-sistom, H520-sinc and H520-sistom was detected by MTT method.

[0121] Seed cells in a 96-well plate, 1000-5000 cells per well (set up 5 experimental wells for each cell and 1 well for blank control wells without inoculating cells), start detection on the first day after the cells adhere to the wall, and detect for 5 days in total , the detection method is as follows:

[0122] (1) Take a 96-well plate, discard the supernatant, and add 100 μl of MTT solution to each well;

[0123] (2) Place in the incubator, and develop color for 4 hours;

[0124] (3) Discard the supernatant, add 150 μl DMSO to each well, and shake for 10 minutes to dissolve the crystals;

[0125] (4) 490nm wavelength, detect the OD value of each well (the OD value of the experimental well - the OD value of the blank control well).

[0126] The result is as figure ...

Embodiment 3

[0136] Embodiment 3, the impact of stomatin on tumor cell metastasis and spread

[0137] one,

[0138] A 24-well plate scratch experiment was performed on A549-sistom cells and A549-sinc cells.

[0139] Take a 24-well plate full of cells, first draw three horizontal lines with a marker on the bottom of the well plate; draw three vertical lines with a pipette tip vertically and not tilted; wash three times with PBS, add serum-free medium, and culture at 37°C, respectively. Take more than 5 identical fields of view at 0h and 24h.

[0140] After 24 hours, the scratches healed as Figure 6 As shown in A (the field of view magnification is 100 times), calculate the migration distance of moving cells in 8 randomly selected fields of view, and the results are as follows Figure 6 Shown in B.

[0141] two,

[0142] Cell migration experiments were performed on A549-shstom cells and A549-shnc cells.

[0143] (1) Before preparing the cell suspension, starve the cells for 12-24 hour...

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Abstract

The invention discloses an application of stomatin protein and its coding gene in diagnosis and treatment of lung cancer. The invention provides the application of stomatin gene expression in preparation of a product; and the use of the product is to inhibit tumor cell metastasis. The invention also protects the application of a substance for inhibiting expression of the stomatin gene in the preparation of the product; and the purpose of the product is to inhibit the tumor cell proliferation. The experiment proves that the overexpression of the stomatin gene can promote the growth and proliferation of lung cancer cells, can significantly inhibit the vertical and horizontal migration and diffusion ability, highly suggests that the stomatin gene plays an important role in the development oflung cancer, can not only play the role of oncogenes to promote tumor proliferation and growth, but also has the function of inhibiting the tumor development and inhibiting the tumor metastasis. The invention provides a new target for the diagnosis and treatment of lung cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a stomatin protein and its coding gene in the diagnosis and treatment of lung cancer. Background technique [0002] Lung cancer is the most common cause of cancer-related death worldwide. Although the technology of early diagnosis and comprehensive treatment of lung cancer has been greatly developed in recent years, it has little effect on the overall survival rate of lung cancer patients. Therefore, the identification of new key genes and their functional mechanisms is crucial to the exploration and development of new methods for the diagnosis and treatment of lung cancer. [0003] Stomatin is an important lipid raft protein, which can interact with various ion channel proteins and transporters on the surface of cell membranes, and is an important component of cell membranes. However, the role and function of stomatin in tumorigenesis and development are still u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/713A61P35/04A61P35/00G01N33/68
CPCA61K31/713A61K48/005G01N33/6812
Inventor 马洁袁伟安华英隋晨光
Owner BEIJING HOSPITAL
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