Application of photosensitive channel protein XXM 2.0 in preparation of medicine for preventing or treating esophageal squamous carcinoma

Abstract

The invention discloses application of photosensitive channel protein XXM 2.0 in preparation of a medicine for preventing or treating esophageal squamous carcinoma, and belongs to the technical field of esophageal squamous carcinoma treatment. The activity and migration rate of esophageal squamous carcinoma cells are inhibited through the photosensitive channel protein XXM 2.0 activated by blue light. The phototoxicity problem can be avoided by activating the photosensitive channel protein XXM 2.0 in tumor cells through proper blue light, in addition, the activity and migration rate of esophageal squamous carcinoma cells can be remarkably inhibited, and a new research direction is developed for prevention and treatment of esophageal squamous carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of esophageal squamous cell carcinoma treatment, and particularly relates to the use of photosensitive channel protein XXM 2.0 in the preparation of drugs for preventing or treating esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer is the eleventh most common cancer globally and the seventh leading cause of cancer death. According to case classification, esophageal cancer can be divided into esophageal squamous-cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). In China, more than 90% of cases are esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is characterized by high malignancy and poor prognosis. Currently, surgical treatment is the main treatment method for esophageal squamous cell carcinoma, but the 5-year survival rate has remained between 30% and 40% for nearly 30 years. Therefore, it is necessary to further explore the internal mechanism of the occurrence and development of esophageal squamous cell carcinoma to provide new technical support for the basic research of esophageal squamous cell carcinoma.

[0003] Calcium ions are important second messengers within cells and play important roles in various aspects of cell physiological functions. The maintenance of calcium ion homeostasis is crucial for the growth and survival of tumor cells. Some studies have shown that calcium channel genes are highly expressed in various tumor cells and can promote the proliferation and migration of tumor cells by regulating calcium ion influx. Calcium channel proteins (such as ORAI1 and STIM2) are highly expressed in melanoma and can regulate store-operated calcium channels (SOCE), which have a significant impact on the migration and invasion ability of tumor cells. However, excessive intracellular calcium ion concentration can lead to mitochondrial dysfunction and endoplasmic reticulum stress, thereby triggering apoptosis. The activation of certain calcium channels increases the intracellular calcium ion concentration, which in turn inhibits cell proliferation and induces apoptosis. Calcium ion overload also leads to an increase in the intracellular oxidative stress level, further promoting apoptosis. Calcium ions have a dual role in cell physiological processes, being able to both promote cell proliferation and induce apoptosis. This dual role mainly depends on factors such as calcium ion concentration, cell type, and cell culture environment. This characteristic of calcium ions has important research value in the fields of tumor treatment and physiological regulation.

[0004] Optogenetics technology is a novel biological tool that combines optics and genetics. By expressing photosensitive proteins in cells, tissues, and organisms, and activating the photosensitive proteins with light of a specific wavelength, different functions can be studied. The optogenetic tool XXM2.0 regulates the transmembrane flow of calcium ions in response to blue light and has been applied in the field of botany, but there is no report on its role in esophageal squamous cell carcinoma cell lines. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides the use of photosensitive channel protein XXM 2.0 in the preparation of a drug for preventing or treating esophageal squamous cell carcinoma. The photosensitive channel protein XXM 2.0 can be activated by blue light and inhibits the cell viability and migration rate of esophageal squamous cell carcinoma.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:

[0007] The object of the present invention is to provide the use of photosensitive channel protein XXM 2.0 in the preparation of a drug for preventing or treating esophageal squamous cell carcinoma.

[0008] The photosensitive channel protein XXM 2.0 used in the present invention is further optimized based on the ChR2-XXM (D156H) mutant. On the basis of XXM 1.1, XXM 2.0 introduced the H134Q mutation, enhancing the photocurrent and calcium ion conduction ability. In addition, the truncation of 11 amino acids at the N-terminus of the structure of XXM 2.0, and the addition of an endoplasmic reticulum export signal peptide and a plasma membrane targeting signal peptide further enhanced the membrane localization and photocurrent.

[0009] Furthermore, the photosensitive channel protein XXM 2.0 inhibits the cell viability and migration rate of esophageal squamous cell carcinoma under light illumination conditions.

[0010] Furthermore, the light illumination conditions are blue light with a wavelength of 450 - 500 nm and a light intensity of 2.0 - 3.0 mW / cm 2 .

[0011] Furthermore, the blue light wavelength is 470 nm and the light intensity is 2.55 mW / cm 2 .

[0012] Under the above blue light modulation parameters, the activation efficiency of the XXM 2.0 photosensitive channel protein on esophageal squamous cell carcinoma cells is relatively high, which can further increase the transmembrane flow of calcium ions and will not cause physiological damage to the cells. When not within the above blue light modulation parameter range, it will not only lead to a low activation efficiency of the photosensitive channel protein but also cause phototoxicity.

[0013] Another object of the present invention is to provide a vector, which comprises a nucleic acid encoding the above-mentioned photosensitive channel protein XXM 2.0.

[0014] Another object of the present invention is to provide an expression system, which comprises the above-mentioned vector.

[0015] Furthermore, the expression system comprises the photosensitive channel protein XXM 2.0, the fluorescent protein eYFP, a signal peptide and a vector.

[0016] Furthermore, the vector is a lentiviral expression vector.

[0017] Furthermore, the lentiviral expression vector includes the promoter EF1a, the vector resistance Ampicillin, and the selection marker Puromycin.

[0018] Another object of the present invention is to provide the use of activating the activity of the photosensitive channel protein XXM 2.0 in the preparation of a drug for treating esophageal squamous cell carcinoma.

[0019] Another object of the present invention is to provide a photodynamic drug for treating esophageal squamous cell carcinoma, which uses the above-mentioned photosensitive channel protein XXM 2.0 or the vector as an active ingredient.

[0020] Furthermore, the drug also includes its pharmaceutically acceptable adjuvants.

[0021] Furthermore, the drug is in the form of powder, liquid preparation, capsule, tablet or pill.

[0022] Another object of the present invention is to provide an illumination system, which includes a light source, a light shutter and a radiator. The light source is located above the light shutter, and the radiator is located above the light source.

[0023] Furthermore, the light source includes a circuit board and several LED light strips; the circuit board is outside the light shutter, and six light strips are adhered to the radiator.

[0024] Furthermore, each light strip is a blue LED light strip.

[0025] Furthermore, measure the temperature of the culture environment during illumination to avoid the influence of phototoxicity on cells.

[0026] Furthermore, perform light stimulation on cells at the optimal temperature.

[0027] Advantages of the present invention:

[0028] By activating the photosensitive channel protein XXM 2.0 in tumor cells with appropriate blue light, the present invention can avoid the problem of phototoxicity. In addition, it can significantly inhibit the viability and migration rate of esophageal squamous cell carcinoma cells, and develop a new research direction for the prevention and treatment of esophageal squamous cell carcinoma. Description of the Drawings

[0029] Figure 1 It is the structural diagram of the lentiviral expression vector pLVX-XXM 2.0;

[0030] Figure 2 It is the expression of the photosensitive channel protein XXM 2.0 in 293T cells;

[0031] Figure 3 It is the membrane localization diagram of the photosensitive channel protein XXM 2.0;

[0032] Figure 4 Figure showing the expression results of the photosensitive channel protein XXM 2.0 in tumor cells detected by qRT-PCR;

[0033] Figure 5 Figure showing the change results of the calcium indicator R-GECO1 under blue light;

[0034] Figure 6 Schematic diagram of the lighting device developed in the present invention;

[0035] Figure 7 Figure showing the temperature change under lighting conditions;

[0036] Figure 8 Figure showing the detection results of the effect of photoactivated photosensitive channel protein XXM 2.0 on tumor cell viability;

[0037] Figure 9 Figure showing the detection results of the effect of photoactivated photosensitive channel protein XXM 2.0 on the migration rate of tumor cells. Detailed implementation manners

[0038] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0039] Example 1 Construction of tumor cells stably expressing XXM 2.0

[0040] 1. Construction of the lentiviral expression vector pLVX-XXM 2.0

[0041] The structure of the lentiviral expression vector pLVX-XXM 2.0 constructed in the present invention is as Figure 1 shown, which includes a cleavable N-terminal signal peptide Lucy-Rho (LR), a plasma membrane transport signal (T) from Kir2.1, an endoplasmic reticulum (ER) export signal (E) from Kir2.1, an enhanced yellow fluorescent protein (eYFP), pLVX (a lentiviral expression vector for mammalian cells), and a ChR2 mutant named XXM 2.0 (ChR2(HQ / DH)-(N)11aa).

[0042] 2. Construction of a tumor cell line stably expressing XXM 2.0

[0043] Seed 293T cells in a 10-cm dish. After the cells adhered to the dish, replace the medium with serum-free and antibiotic-free medium and starve the cells for 1 h. Add 1 mL of the prepared transfection reagent to the cell culture dish. Solution A: 500 μL of DMEM without antibiotics + 10 μg of the target gene + 7.5 μg of PS.PAX2 + 2.5 μg of PMD2.G. Gently mix. Solution B: 30 μL of PEI + 500 μL of DMEM. Gently mix. Mix Solution A and Solution B gently, let stand for 20 min, and then add dropwise to the cell culture dish. Gently shake the culture dish during the dropping process to mix. Replace with fresh complete DMEM medium after 8 h. After observing the expression of fluorescence under an inverted fluorescence microscope, collect the medium containing virus particles at 24 and 48 h. Concentrate the virus solution using PEG8000 and then infect KYSE30 cells. Screen for stable transfected cells using 1 μg / mL puromycin. The expression of XXM 2.0 in HEK293T cells is as Figure 2 shown.

[0044] 3. Seed the KYSE30 cells expressing XXM 2.0 in a 35-mm glass-bottom confocal culture dish. After the cells adhered to the dish, observe the localization of XXM 2.0 in the cells using a laser confocal microscope. The results are shown in Figure 3 .

[0045] Figure 3 The detection results of

[0046] show that XXM 2.0 can be localized and expressed on the cell membrane when observed under a 488-nm laser, indicating that KYSE30 cells stably expressing XXM 2.0 have been successfully constructed.

[0046] Example 2 Detection of the Expression of XXM 2.0 in Tumor Cells

[0047] Seed the cells in the Ctrl group, eYFP group, and XXM 2.0 group (experimental group) in 6-well plates. Lyse the cells in each group after 12 h, extract RNA and perform reverse transcription, and detect the expression of XXM 2.0 in the cells using qPCR. The results are shown in Figure 4 .

[0048] As Figure 4 shown, the results show that XXM 2.0 is highly expressed in the KYSE30 tumor cells in the experimental group.

[0049] Example 3 Changes in the Calcium Indicator R-GECO1 under Blue Light

[0050] Co-transfect R-GECO1 and XXM 2.0 into 293T cells. 24 hours after cell seeding, perform transient transfection of the cells. Replace the medium 1 hour before transfection and add 1.5 mL of DMEM medium without serum and antibiotics to each well. Calculate the ratio in advance according to the plasmid concentration. Transfection DNA: liposomal nucleic acid transfection reagent = 1:2. For each well of cells, dilute 1 μg of DNA and 2 μL of Hieff liposomal nucleic acid transfection reagent with 250 μL of DMEM medium without serum and antibiotics respectively, and mix well. Mix the diluted DNA with the liposomal nucleic acid transfection reagent, gently mix well, and let it stand at room temperature for 20 minutes to form the DNA-liposome complex. Add 500 μL of the DNA-liposome complex to the cells and gently shake the culture plate to mix well. After 6 hours, discard the old medium and add the complete medium, and continue culturing. Transfer the co-transfected cells to a 35 mm glass dish for culture. According to the fluorescence characteristics of R-GECO1, select appropriate excitation light (560 - 590 nm) and emission light (620 - 660 nm) channels. Select the "time series" function in the microscope software and set the total shooting time. Find a suitable field of view under the microscope, start continuous shooting, first perform dark treatment for several seconds, and then stimulate the cells with blue light (2.7 mW / cm 2 ), to induce changes in calcium ion signals. Use the image analysis software ImageJ to quantitatively analyze the fluorescence intensity, and the results are as Figure 5 shown.

[0051] Example 4 Effects of blue light activation of XXM 2.0 on tumor cells

[0052] 1. Effects on cell viability

[0053] Use KYSE30 cells stably expressing XXM 2.0 with passage numbers of 3 - 6. When the cells grow to the best stage, digest the cells, resuspend them, dilute and count, and add the cell suspension to a 96-well plate, with a volume of 1 mL per well, approximately 10,000 cells per well. Experimental treatment: The plated cells are placed in a cell culture incubator with culture conditions of 5% CO 2 , 37 °C. After culturing for 12 hours, detect the cell viability after culturing for 1 hour under dark conditions and blue light irradiation conditions respectively.

[0054] The blue light illumination equipment and illumination conditions are as follows:

[0055] Use a self-made blue light and green light pulse instrument in the laboratory to provide light stimulation for the cells. The light stimulation equipment is as Figure 6 shown. In the figure, A is a semi-sealed light box, B is an LED light strip with a specific wavelength, C provides a stable power supply, and D controls the light illumination time interval, which can provide stable light stimulation. Under blue light (470 nm, 2.5 mW / cm 2)Under the condition of , the cyclic light illumination mode of illuminating for 1 min and pausing for 2 min can maintain the temperature at 37 °C, and the results are as Figure 7 shown.

[0056] Cell viability detection process:

[0057] After cell culture, carefully aspirate the supernatant, add 100 μL of fresh complete medium and 10 μL of CCK8 solution, and prepare a zero adjustment well (only medium, serum and CCK8). After continuing to culture for 2 h, measure the absorbance value of each well at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the relative cell viability. The results are shown in Figure 8 .

[0058] As Figure 8 shown, the viability of KYSE30 cells with XXM 2.0 activated by blue light decreased significantly.

[0059] 2. Effect on cell migration

[0060] Use KYSE30 cells stably expressing XXM 2.0 with passage numbers of 3 - 6. When the cells grow to the optimal stage, digest the cells, resuspend them, dilute and count. Add the cell suspension into a 6-well plate, with a volume of 2 mL per well, approximately 500,000 cells per well; use a marker pen to draw equally spaced horizontal lines on the back of the cell plate in advance. Experimental treatment: The plated cells are placed in a cell culture incubator, and the culture conditions are 5% CO 2 , 37 °C. After culturing for 12 h, use a ruler as a guide and use a 10-μL pipette to create cell scratches (the flat surface of the pipette tip is drawn perpendicular to the plane of the culture plate across the cell layer. It is best to keep the force consistent and try to complete it in one go to ensure that the width of each scratch is the same), and the scratch direction is perpendicular to the marked line. Aspirate the medium, gently wash 3 times with PBS to fully wash away the floating cells. When washing with PBS, be gentle, add it slowly while adhering to the wall, and do not aspirate the adherent cells. Replace with serum-free medium and take a photo under the microscope. Irradiate and culture for 1 h under dark conditions and blue light irradiation conditions respectively (the blue light activation conditions are as described above). Wipe off the scratches on the back of the 6-well plate. Take a photo under a 4× objective lens to ensure that the scratches are centered and perpendicular, and pay attention to the consistent background. After taking the photos, the width of the scratch area can be measured using Image J to compare the cell migration speed. The results are shown in Figure 9 .

[0061] As Figure 9 shown, the migration ability of KYSE30 cells with XXM 2.0 activated by blue light decreased significantly.

[0062] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. Use of photosensitive channel protein XXM 2.0 in the preparation of drugs for preventing or treating esophageal squamous cell carcinoma.

2. The use according to claim 1, characterized in that The photosensitive channel protein XXM 2.0 inhibits the activity and migration rate of esophageal squamous cell carcinoma cells under light conditions.

3. The use according to claim 2, characterized in that: The illumination condition is blue light with a wavelength of 450-500nm and a light intensity of 2.0-3.0mW / cm 2 .

4. The use according to claim 3, characterized in that The blue light has a wavelength of 470 nm and a light intensity of 2.55 mW / cm 2 .

5. A carrier, characterized in that It comprises a nucleic acid encoding the light-sensitive channel protein XMM 2.0 described in claim 1.

6. Use of a preparation for activating the activity of photosensitive channel protein XXM 2.0 in the preparation of a drug for treating esophageal squamous cell carcinoma.

7. A photodynamic drug for treating esophageal squamous cell carcinoma, characterized in that: The active ingredient is the light-sensitive channel protein XXM 2.0 described in claim 1 or the carrier described in claim 5.

8. The photodynamic drug according to claim 7, characterized in that: The medicine also includes pharmaceutically acceptable adjuvants.

9. The photodynamic drug according to claim 7, characterized in that: The medicine is in the form of powder, liquid preparation, capsule, tablet or pill.

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