A migratory body marker

Abstract

The present invention provides a migrasome marker for liver cancer cell lines, belonging to the field of biomedicine. The present invention first discovered that CD151 can serve as a new migrasome marker in liver cancer cell lines and influences migrasome generation. The study of CD151 as a migrasome marker in liver cancer cell lines is of great significance for the regulation of migrasome formation and for studying processes involved in active cell migration, such as embryonic development, immune response, and tumor metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a migrasome marker. Background Art

[0002] Migrasomes are single-layer membrane vesicles with a diameter of 0.5-3 μm that form at the ends or intersections of contractile filaments produced at the tail of a cell during directional cell migration. The process by which cells produce migrasomes is called migratory exocytosis. After a cell migrates away, the migrasome remains in place until it ruptures or is engulfed by other cells, thereby transferring substances between cells. Current research on migrasomes has revealed that they play an important role in signaling during active cell migration processes, such as embryonic development, immune responses, and tumor metastasis.

[0003] Migrasome formation depends on cell migration. Studies have shown that when migration increases, the number of migrasomes increases, and vice versa. In cultured cells, cell migration depends on adhesion to the extracellular matrix. Migrasomes also adhere to the extracellular matrix, so adhesion molecules may play a role in migrasome formation. Mass spectrometry analysis has shown that integrin α5β1 is enriched on migrasomes. Live cell imaging studies have shown that integrins are localized to the site of migrasome formation and appear on contractile filaments before migrasomes. Furthermore, three-dimensional imaging revealed that integrins are located at the base of migrasomes. These spatiotemporal distributions indicate that integrins are essential for migrasome formation. Mammalian integrins have 18 α and 8 β subunits, and different integrins bind to different extracellular matrix proteins. Migrasome formation is determined by the binding of integrins to their specific extracellular matrix. For L929 cells, which are rich in integrin α5β1, fibronectin is the optimal extracellular matrix for promoting migrasome formation. This mechanism of migrasome formation, which relies on the regulation of the integrin-extracellular matrix pairing network, provides an important theoretical basis for the specificity of migrasomes as signal transduction mediators in vivo. At the same time, the integrins on migrasomes also provide specificity for their entry into specific receptor cells.

[0004] To accurately study the formation of migratoria, Huang et al. designed an in vitro migratoria reconstitution system that generates giant unilamellar vesicles (GUVs) using purified TSPAN4-GFP, cholesterol, and other lipids. To simulate cell adhesion and migration, biotinylated unilamellar vesicles were first attached to the bottom of a streptavidin-coated flow chamber. A directional liquid flow was then applied within the chamber to provide mechanical force, dislodging the unilamellar vesicles from their adhesion points. This method successfully reconstructed migratoria formation, with both TSPAN4 and cholesterol accumulating on the reconstituted migratoria. In the absence of TSPAN4 or cholesterol in the system, migratoria could not be reconstituted, with TSPAN4 or cholesterol being randomly distributed on the artificially generated filaments. Huang et al. also employed an alternative system in which a glass needle attached to the unilamellar vesicles applied a tensile force, rather than a directional liquid flow. Using this method, they successfully elongated tubular structures resembling contractile filaments from TSPAN4-GFP-infused unilamellar vesicles. During the tube pulling process, microdomains of TEM aggregate to form macrodomains of TEMA, which spontaneously expand on the membrane tube to form a migratoria-like structure. Unilamellar vesicles lacking TSPAN4-GFP or cholesterol are unable to reconstitute migratoria-like structures. These two in vitro reconstitution systems demonstrate that TSPAN4 and cholesterol are necessary and sufficient for migratoria formation. Furthermore, the study demonstrates that the initial formation of TEMAs on the contractile filaments increases the bending stiffness of the membrane to a greater degree than that found elsewhere on the contractile filaments, leading to spontaneous expansion at these locations to form migratoria. This research provides the theoretical foundation for the mechanism of migratoria formation and pioneers the study of migratoria function centered on TSPAN4 and TEMAs, providing a foundation for subsequent studies of the regulation of migratoria formation.

[0005] Currently, TSPAN4 is primarily used as a migrasome marker in NRK cells. However, in pan-cancer cells, TSPAN4 expression is low, making it inadequate for migrasome research. Therefore, developing new migrasome markers is crucial for understanding the regulation of migrasome development. Summary of the Invention

[0006] In view of the above-mentioned deficiencies, the present invention provides a new migrasome marker.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides the use of CD151 as a migratoria marker.

[0009] In another aspect, the present invention provides a migratoria marker, wherein the migratoria marker is CD151.

[0010] In another aspect, the present invention provides a use of a reagent for detecting CD151 in preparing a migratoria detection kit.

[0011] Specifically, the reagents for detecting CD151 include but are not limited to specific antibodies, probes, primers and / or fluorescent reagents for detecting CD151.

[0012] In another aspect, the present invention provides a migrator detection kit, comprising a reagent for detecting CD151.

[0013] Specifically, the reagents for detecting CD151 include but are not limited to specific antibodies, probes, primers and / or fluorescent reagents for detecting CD151.

[0014] In another aspect, the present invention provides a method for detecting migratoria, wherein the detection method is to obtain the expression level and / or location of migratoria by detecting CD151.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This study, published in the journal Cell Biology, demonstrates that CD151 can be used as a novel migrasome marker in liver cancer cell lines and influences migrasome formation. The study of CD151 as a migrasome marker in liver cancer cell lines is of great significance for understanding the regulation of migrasome formation and for studying processes involved in active cell migration, such as embryonic development, immune responses, and tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the database analysis results.

[0018] Figure 2 This is the confocal image of CD151 and Migrasome.

[0019] Figure 3 These are the results of CD151 SiRNA and TSPAN4 SiRNA knockdown and the confocal results of HCCLM3, MHCC-97H, MHCC-97L, Hep 3B, Hep G2, and Huh7 cells. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0021] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0022] Example 1. CD151 can specifically label Migrasomes

[0023] Depend on Figure 1 It can be seen that through analysis of databases such as TCGA and GEO, it was found that the expression of TSPAN4 in pan-cancer cells was low, while the expression of CD151 was relatively high.

[0024] Therefore, the relationship between CD151 and migratoria was further investigated using the following experimental methods.

[0025] Experimental method 1:

[0026] 1) Approximately 2.5 × 10^5 NRK-GFP cells were seeded into a 35 mm diameter confocal microplate (pre-incubated with 10 μg / ml Fibronectin at 37°C for 0.5-1 h). After 15-20 h of culture, migrasome staining was performed.

[0027] 2) CD151 expression in cells was detected by immunofluorescence technique, and then CD151-labeled structures were captured by confocal microscopy.

[0028] 3) Stain with a migrasome dye and image the migrasome structure using a confocal microscope. Then, merge the images and observe whether they are the same structure as the migrasomes stained with CD151 immunofluorescence.

[0029] Experimental method 2: Si RNA transfection

[0030] Six cell lines, HCCLM3, MHCC-97H, MHCC-97L, Hep 3B, Hep G2, and Huh7, were seeded in 24-well plates, and Si RNA transfection was started when the cell confluence reached 50%.

[0031] The Si RNA transfection steps are as follows (24-well plate, Si RNA transfection concentration is 50nM):

[0032] 1) Si RNA (CD151 SiRNA and TSPAN4 SiRNA, Sanbaoyi) was constructed from Keygene Biotechnology.

[0033] Table 1. Si RNA sequences

[0034]

[0035] 2) Dilute the Si RNA to 20 μM with enzyme-free water.

[0036] 3) Take 1.25μl of Si RNA and add it to 50μl of Opti-Medium to dilute it, blow gently 3-5 times, and let it stand for 5 minutes. Take 3μl of Lipo2000 and add it to 50μl of Opti-Medium to dilute it, blow gently 3-5 times, and let it stand for 5 minutes. Mix the two together, blow gently 3-5 times, and let it stand for 20 minutes.

[0037] 4) Add 400 μl of serum-free and double-antibody-free culture medium to a 24-well plate, then add 100 μl of the above mixture and shake well.

[0038] 5) After incubation at 37°C, 5% CO2 for 4-6 hours, replace the culture medium with serum-containing medium but without double antibody.

[0039] 6) Conduct subsequent verification and related experiments after 24-72 hours of culture.

[0040] The best-performing Si RNA was selected by QPCR and Western Blot. After transfection of the six cell lines described above with the best-performing Si RNA, approximately 2.5 × 10^5 cells were seeded into a 35 mm diameter confocal microplate (pre-incubated with 10 μg / ml Fibronectin at 37°C for 0.5-1 h). Migrasome staining was performed after approximately 15-20 h of incubation, followed by confocal microscopy.

[0041] Test results such as Figure 3 As shown by Figure 3It can be seen that after knockdown by CD151 siRNA and TSPAN4 siRNA, qPCR verification was performed (NC group: group without any treatment; Mock group: group without target siRNA; Si RNA group: group with target siRNA). Si2 had the best knockdown effect on CD151, and protein expression was also verified to prove the effectiveness of small interfering RNA (due to the low expression of TSPAN4, after multiple experiments, it was impossible to accurately measure TSPAN4 expression in L292 cells, so the effectiveness of TSPAN4 siRNA was ultimately verified only by WB protein level). In L929 cells, it was found that migrasomes were reduced after CD151 knockdown, and CD151 had a better effect than TSPAN4. The same experiment was performed in 6 liver cancer cell lines, and migrasome expression was reduced. This shows that CD151 plays a role in migrasomes in liver cancer cells that is comparable to TSPAN4, and CD151 can be used as a marker protein for migrasomes in liver cancer cells.

[0042] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a group of siRNAs in the preparation of a drug for reducing the content of migratory bodies in liver cancer cells, characterized in that: The sense strand of the siRNA is CCCUCAAGAGUGACUACAUCATT, and the antisense strand is UGAUGUAGUCAUCUUGAGGGTT; the drug is a drug that indirectly reduces the content of migratory bodies in liver cancer cells by knocking down the expression of CD151 protein; and the liver cancer cells are selected from one of HCCLM3, MHCC-97H, MHCC-97L, Hep 3B, Hep G2 or Huh7.

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