WO2023226159A1 - Use of pim1 sirna in preparation of drug for treating arsenic-induced malignant cell transformation diseases - Google Patents

Abstract

The present invention relates to the use of PIM1 siRNA in the preparation of a drug for treating arsenic-induced malignant cell transformation diseases. It has been found that in the chemical carcinogenic process (in the process of the arsenic-induced HaCaT malignant cell transformation), the protein of cancer gene PIM1 is continuously and highly expressed since the cells are continuously infected for 14 generations. The PIM1 siRNA can inhibit the arsenic-induced malignant cell transformation by means of inhibiting the generation of hydrogen peroxide and superoxides, thereby prolonging the cell multiplication time and reducing the cell migration rate and the formation number of clonal colonies of soft agar.

Description

Application of a PIM1 siRNA in the preparation of drugs for the treatment of diseases caused by arsenic-induced malignant transformation of cells Technical field

The invention belongs to the field of biomedicine technology, and particularly refers to the application of a PIM1 siRNA in the preparation of drugs for the treatment of diseases caused by arsenic-induced malignant transformation of cells.

Background technique

Inorganic arsenic and its compounds are one of the 10 chemicals of major public health concern listed by the World Health Organization (WHO). The International Agency for Research on Cancer (IARC) has determined arsenic to be a Group 1 carcinogen (human carcinogen). The United States The Environmental Protection Agency (USEPA) classifies it as a Class A carcinogen (human carcinogen with high carcinogenic hazard). However, research on the molecular mechanism of arsenic carcinogenesis has lagged behind for a long time. The increasing identification and understanding of the molecular pathways altered in cancer disease paves the way for exploring the mechanisms of carcinogenesis and for further treatment of cancer. Therefore, the changes in molecular pathways and mechanisms during arsenic carcinogenesis require more in-depth study.

The oncogene PIM1 is located on chromosome 17 and is widely involved in a variety of biological activities. Some proteins and pathways with oncogenic properties have been identified as targets of the protein kinase activity encoded by the oncogene PIM1, such as the regulation of the cell cycle and the control of apoptosis. PIM1 can promote cell cycle progression and mitosis at various stages, such as phosphorylating Cdc25A, Cdc25C, etc. In addition to controlling cell growth pathways, PIM1 prevents apoptosis and thus may serve as an oncogenic survival factor. PIM1 affects the activity of different transcription factors, such as c-Myb, c-Myc, etc. Furthermore, PIM1 plays a role in hypoxia-induced chemoresistance. Alterations in PIM1 signaling have been widely found in various human tumor diseases. In hematologic malignancies, PIM1 expression is associated with poor prognosis in various leukemias, connective tissue lymphomas, and diffuse large B-cell lymphoma; in solid tumors, overexpression of PIM1 has been detected in bladder and prostate cancer specimens. expression, which is also associated with poor prognosis and response to treatment, and similar findings have been found in esophageal and gastric cancers. In cell culture models and patient gastric epithelial cells, PIM1 expression is up-regulated after infection with Helicobacter pylori; in head and neck cancer, PIM1 is highly expressed.

Contents of the invention

In order to solve the above technical problems, the present invention provides an application of PIM1 siRNA in the preparation of drugs for treating diseases caused by arsenic-induced malignant transformation of cells.

The first object of the present invention is to provide an application of PIM1 siRNA in the preparation of drugs for the treatment of diseases caused by arsenic-induced malignant transformation of cells. The PIM1 siRNA includes three different siRNAs, the sequences of the three siRNAs are as follows:

sc-36225A:

Justice chain: 5′-CCCAUAGAUACUCUUCUtt-3′

Antisense strand: 5′-AGAAGAGAGUAUCUAUGGGtt-3′

sc-36225B:

Sense strand: 5′-GUUGGCAUGGUAGUAUACatt-3′

Antisense strand: 5′-UGUAUACUACCAUGCCAACtt-3′

sc-36225C:

Sense strand: 5′-UUGGCAUGGUAGUAUACAAtt-3′

Antisense strand: 5′-UUGUAUACUACCAUGCCAatt-3′

In one embodiment of the invention, said cells are selected from HaCaT.

In one embodiment of the invention, the disease is cutaneous basal cell carcinoma or cutaneous squamous cell carcinoma.

The second object of the present invention is to provide a recombinant vector, including a sequence capable of transcribing the PIM1 siRNA, and the sequence is embedded in the vector.

The third object of the present invention is to provide a pharmaceutical composition, including the drug or the recombinant vector.

In one embodiment of the present invention, a pharmaceutically or pharmacologically acceptable carrier is also included.

In one embodiment of the present invention, the carrier is selected from one of disintegrants, diluents, lubricants, adhesives, wetting agents, flavoring agents, suspending agents, surfactants and preservatives, or Various.

In one embodiment of the present invention, the pharmaceutical composition is in the form of tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts or injections.

The fourth object of the present invention is to provide a kit, including the drug or the recombinant vector or the pharmaceutical composition.

The fifth object of the present invention is to provide the application of the kit in preparing drugs for treating diseases caused by malignant transformation of cells caused by arsenic.

The above technical solution of the present invention has the following advantages compared with the existing technology:

The present invention's research has found that during the chemical carcinogenesis process (the process of malignant transformation of HaCaT cells caused by arsenic), the protein of oncogene PIM1 continues to be highly expressed starting from the 14th generation of continuous exposure of cells. PIM1 siRNA can inhibit the production of hydrogen peroxide and superoxide. , thereby prolonging cell doubling time, reducing cell migration rate and soft agar colony formation number, and inhibiting malignant transformation of cells caused by arsenic.

By transfecting T-HaCaT with PIM1 siRNA, the present invention found that the soft agar colony formation ability and cell migration ability were reduced, the cell doubling time was increased, and the malignant transformation indicators of cells were reversed, indicating that the continuous activation of PIM1 can promote the occurrence and development of cancer.

At the same time, the present invention found that after transfecting T-HaCaT cells with PIM1 siRNA, the expression level of NRF2 was reduced. Further detection of hydrogen peroxide and superoxide levels found that the levels of hydrogen peroxide and superoxide increased significantly. Activation of PIM1 can promote the expression of NRF2 and promote the occurrence and development of cancer. Further research on the role of PIM1 found that PIM1 may regulate the expression of the redox-sensitive nuclear transcription factor NRF2. NRF2 maintains the level of reactive oxygen species in cells by enhancing the antioxidant capacity of cells.

Description of the drawings

In order to make the content of the present invention easier to understand clearly, the present invention will be further described in detail below based on specific embodiments of the present invention and in conjunction with the accompanying drawings, wherein

Figure 1 shows the expression changes of PIM1 during the malignant transformation of HaCaT cells caused by NaAsO ₂ in Example 1 of the present invention; wherein (A) PIM1 protein Western results chart (B) PIM1 protein quantitative analysis results;

Figure 2 is the effect of PIM1 siRNA on PIM1 protein of T-HaCaT cells induced by NaAsO ₂ in Example 1 of the present invention, in which (A) PIM1 protein Western results diagram; (B) Quantitative analysis results of PIM1 protein;

Figure 3 is the effect of PIM1 siRNA on NRF2 protein in T-HaCaT cells induced by NaAsO ₂ in Example 1 of the present invention, wherein, (A) NRF2 protein Western results diagram; (B) NRF2 protein quantitative analysis results;

Figure 4 is the effect of PIM1 siRNA on reactive oxygen species in T-HaCaT cells caused by NaAsO ₂ in Example 2 of the present invention, in which (A) the experimental results of hydrogen peroxide levels; (B) the experimental results of superoxide levels;

Figure 5 is the effect of PIM1 siRNA on the doubling time of HaCaT cells malignantly transformed by NaAsO ₂ in Example 4 of the present invention;

Figure 6 is the effect of PIM1 siRNA on the migration ability of HaCaT cells malignantly transformed by NaAsO ₂ in Example 5 of the present invention; wherein, (A) cell scratch experiment results; (B) relative cell mobility;

Figure 7 is the effect of PIM1 siRNA on the anchorage-independent growth of HaCaT cells malignantly transformed by NaAsO ₂ in Example 6 of the present invention; wherein, (A) Cell soft agar cloning experimental results; (B) Cell soft agar cloning quantitative analysis result.

In the figure, ^* P<0.05, compared with cells in the passage control group, the difference is statistically significant. ^# P<0.05, compared with Con siRNA, the difference is statistically significant.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.

1. The HaCaT cell line used in the present invention was purchased from China Type Culture Collection Center and Wuhan University Collection Center.

2. After HaCaT cells were continuously treated with 0.1 μM NaAsO ₂ for 35 generations, the cells underwent malignant transformation, which was manifested as a significant shortening of cell doubling time, a significant increase in cell migration rate, and a significant increase in soft agar colony formation ability. At this time, cells were defined as cells malignantly transformed by 0.1 μM NaAsO ₂ (T-HaCaT).

3. Reagents used in the present invention: PIM1 siRNA (sc-36225, Santa Cruz, USA), Control siRNA (sc-37007, Santa Cruz, USA), siRNA Transfection Reagent (sc-29528, Santa Cruz, USA), siRNA Transfection Medium (sc- 36868, Santa Cruz, United States). Other reagents are conventional reagents.

4. The PIM1 siRNA of the present invention includes three different siRNAs, and the sequences of the three siRNAs are as follows:

sc-36225A:

Justice chain: 5′-CCCAUAGAUACUCUUCUtt-3′

Antisense strand: 5′-AGAAGAGAGUAUCUAUGGGtt-3′

sc-36225B:

Sense strand: 5′-GUUGGCAUGGUAGUAUACatt-3′

Antisense strand: 5′-UGUAUACUACCAUGCCAACtt-3′

sc-36225C:

Sense strand: 5′-UUGGCAUGGUAGUAUACAAtt-3′

Antisense strand: 5′-UUGUAUACUACCAUGCCAatt-3′.

5. Kit used in the present invention:

Example 1 Cell culture and arsenic contamination

The culture medium used for culturing cells was DMEM complete culture medium, and the culture conditions were 5% CO ₂ and 37°C in a constant temperature incubator. During cell passaging, pour off the medium liquid in the cell culture dish, wash the cells twice with PBS, then add 1.5 mL of trypsin digestion solution containing EDTA, digest on a 37°C heating plate for 6 minutes, and observe under a microscope. When the cell morphology changes and becomes floating away from the culture dish, add 2 mL of culture medium to terminate digestion. Use a pipette to transfer the cell suspension into a sterile centrifuge tube and centrifuge (1000g for 3 minutes at room temperature). Discard the supernatant and leave the cell sediment at the bottom of the sterile centrifuge tube. Use a pipette to add 1mL of culture medium and slowly blow it away. Cells were resuspended and subcultured at a ratio of 1:2. Use DMEM medium with a final concentration of 0.1 μM _NaAsO2 to culture to 35 generations (about 18 weeks), and establish a passage control group of cells that are not stained with arsenic.

Example 2 PIM1 siRNA treatment of malignantly transformed HaCaT cells

Malignantly transformed HaCaT cells and normal passage control cells were seeded in a 6-well plate, and subsequent processing was performed after the cells adhered. First prepare the PIM1 siRNA transfection reagent, and prepare the solution according to the amount of each culture dish. Each culture dish A solution: 12 μL PIM1 siRNA or Con siRNA, add 988 μL siRNA transfection medium; each culture dish B solution: 12 μL siRNA transfection reagent , add 988μL siRNA transfection medium. Mix liquid A and liquid B thoroughly to obtain a mixture containing transfection reagent and PIM1 siRNA or Con siRNA, and incubate in the dark for 20 minutes. Then wash the cells once with PBS and then with 2 mL of siRNA transfection medium. Aspirate the liquid. Add 2 mL of siRNA transfection reagent mixture to each culture dish and place it in a constant temperature incubator with 5% CO ₂ and 37°C. Incubate cells for 6 h. After 6 hours, absorb the liquid in the culture dish, add an appropriate amount of normal cell culture medium, and detect relevant indicators after 24-72 hours. The experimental results are shown in Figure 1-3.

As can be seen from Figure 1, compared with the cells in the passage control group (cells not treated with NaAsO ₂ ), the expression level of PIM1 in HaCaT (T-HaCaT) cells caused by malignant transformation caused by 0.1 μM NaAsO ₂ significantly increased with the increase of exposure time. (P<0.05). The WesternBlot results and its quantitative analysis chart show that as the number of exposure generations of HaCaT cells treated with 0.1 μM NaAsO ₂ increases, the protein expression level of PIM1 continues to increase. Specifically, the protein expression level of PIM1 in the 14th generation is significantly lower than that in the 0th generation control group. There were statistically significant differences in PIM1 protein expression levels at passages 21, 28, and 35 compared with those at passage 0 and the normal passage control group. As shown in Figure 2, after T-HaCaT cells were transfected with PIM1 siRNA: compared with cells transfected with control group (Con siRNA), the expression level of PIM1 was significantly reduced (P<0.05), indicating that the transfection was successful. At the same time, as shown in Figure 3 It shows that compared with the cells in the passage control group (cells not treated with NaAsO ₂ ), the expression level of NRF2 in HaCaT (T-HaCaT) cells caused by malignant transformation caused by 0.1 μM NaAsO ₂ was significantly increased (P<0.05). After transfection with PIM1 siRNA, the expression level of NRF2 was significantly reduced compared with cells transfected with control group (Con siRNA) (P<0.05).

Example 3 Detection of hydrogen peroxide and superoxide in cells

Steps to test hydrogen peroxide levels:

(1) Sample preparation: collect cell culture fluid

(2) Preparation of standard substance: Dilute the standard substance with cell culture medium according to the instructions. The standard solution is diluted to 1, 2, 5, 10, 20, 50, and 100 micromol/L.

(3) Use a 96-well plate for detection, add 50 μL of sample or standard into each well, and then add twice the volume of hydrogen peroxide detection reagent.

(4) After mixing, let stand at room temperature for 30 minutes, and then use a microplate reader to detect the absorbance value at a wavelength of 560 nm.

(5) Calculate the concentration of hydrogen peroxide in the sample based on the standard curve. The experimental results are shown in Figure 4.

Steps to test superoxide levels:

(1) Sample preparation: Seed cells into a 96-well plate at a cell density of 10,000 cells/well.

(2) Discard the culture medium in the well and wash once with PBS.

(3) Prepare working solution according to 212μL per well (including 200μL detection buffer, 10μL WST-1, 2μL Catalase).

(4) Add 200 μL of prepared detection working solution to each well and incubate at 37°C for 3 minutes.

(5) Add 2 μL SOD to the two reserved detection wells to verify the entire system. The arrangement is as follows:

(6) Detect the absorbance value at 450nm. The experimental results are shown in Figure 4.

As shown in Figure 4, the levels of hydrogen peroxide and hydrogen superoxide were detected. There was no significant difference (P>0.05) in the levels of hydrogen peroxide and superoxide in T-HaCaT cells compared with the passaged control cells. After T-HaCaT was transfected with PIM1 siRNA, the levels of hydrogen peroxide and superoxide were detected and found to be significantly increased, with statistical differences (P<0.05). It shows that PIM1 siRNA can reverse the changes in T-HaCaT reactive oxygen species levels caused by 0.1 μM NaAsO ₂ .

Example 4 Cell Doubling Time Detection

Specific experimental steps for cell doubling time detection: Collect cells from the normal treatment group and 0.1 μM NaAsO ₂ treatment group by trypsin digestion, and seed them in a 24-well plate at 10,000 cells per well. After that, every 24 hours, cells from 3 wells in each group were counted separately, and the time required for the number of cells to double was calculated according to the formula: (t×lg2)/(lgNh-lgNi). t is the culture time, Nh is the number of cells after culture for t time (h), and Ni is the initial number of cells inoculated. The experimental results are shown in Figure 5.

As shown in Figure 5, the doubling time of T-HaCaT cells was detected and found to be significantly lower than the doubling time of cells in the passage control group (P<0.05). After T-HaCaT cells were transfected with PIM1 siRNA, the cell doubling time was significantly increased compared with the cells in the transfected control group (P<0.05).

Example 5 Cell scratch experiment

Specific steps of cell scratch experiment:

1) Digest and collect cells and inoculate them into a six-well plate and place them in a cell culture incubator for culture.

2) After 24 hours of cell growth, use a 200 μL pipette tip to scratch perpendicularly to the plate well, making the scratch width as consistent as possible.

3) Aspirate off the old culture medium, rinse the cells three times with PBS, add serum-free culture medium, take pictures and continue to culture in the cell culture incubator.

4) Take pictures again after 48 hours. When taking pictures, select at least three scratch fields per hole, and use Image J software for quantitative analysis. The experimental results are shown in Figure 6.

As shown in Figure 6, the scratch experiment found that compared with the cells in the passage control group, the cell migration ability of T-HaCaT cells was significantly enhanced (P<0.05). After transfection with PIM1 siRNA, compared with the cells in the transfected control group, Cell migration ability was significantly reduced (P<0.05).

Example 6 Soft agar colony formation experiment

1) Preparation of agarose solution: Add an appropriate amount of agarose powder to double-distilled water to prepare an agarose solution with a concentration of 1.4%. After autoclaving, take a 42°C water bath for 2 hours and mix with the same volume of 2×DMEM complete culture medium to obtain a concentration of 0.7% agarose solution.

2) Add bottom glue: Use a pipette to absorb 2mL of 0.7% agarose solution and slowly add it to a 35mm petri dish. After solidification at room temperature, the bottom glue is obtained.

3) Add top glue: After counting the cells, adjust the cell concentration. Place 5,000 cells in DMEM complete medium and add an equal volume of 0.7% agarose solution to obtain a cell suspension with 0.35% agarose. , add 1.5mL to the bottom glue to become the top glue.

4) Culture: After the top gel is solidified, add 1.0 mL of DMEM complete culture medium, and continue to culture in a 5% CO ₂ , 37°C cell culture incubator for 4 weeks, changing the medium every 2 to 3 days.

5) Counting: Observe under a microscope and select colonies with cell diameters greater than 50 μm for counting. The experimental results are shown in Figure 7.

As shown in Figure 7, compared with cells in the passage control group, the anchorage-independent growth ability of T-HaCaT cells was significantly enhanced (P<0.05). After T-HaCaT cells were transfected with PIM1 siRNA, the anchorage-independent growth ability of the cells was significantly reduced compared with the transfected control group (P<0.05). The above experimental results show that PIM1 siRNA can reverse the malignant transformation phenotype of T-HaCaT cells caused by 0.1 μM NaAsO ₂ .

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications in different forms can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (10)

Application of a PIM1 siRNA in the preparation of drugs for the treatment of diseases caused by arsenic-induced malignant transformation of cells. The application according to claim 1, characterized in that the cells are selected from HaCaT. The application according to claim 1, wherein the disease is basal cell carcinoma of the skin or squamous cell carcinoma of the skin. A recombinant vector, characterized in that it includes a sequence capable of transcribing the PIM1 siRNA described in any one of claims 1-3, and the sequence is embedded in the vector. A pharmaceutical composition, characterized by comprising the drug described in any one of claims 1-3 or the recombinant vector described in claim 4. The pharmaceutical composition according to claim 5, further comprising a pharmaceutically or pharmacologically acceptable carrier. The pharmaceutical composition according to claim 6, characterized in that the carrier is selected from the group consisting of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants and preservatives. one or more of the agents. The pharmaceutical composition according to claim 5, characterized in that the dosage form of the pharmaceutical composition is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry infusions ointment or injection. A kit, characterized by comprising the medicine described in any one of claims 1-3, the recombinant vector described in claim 4, or the pharmaceutical composition described in any one of claims 5-8. Application of the kit described in claim 9 in the preparation of drugs for treating diseases caused by malignant transformation of cells caused by arsenic.

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