CN114948984A

CN114948984A - Application of PIM1siRNA in preparation of medicine for treating arsenic-induced cell malignant transformation disease

Info

Publication number: CN114948984A
Application number: CN202210576566.3A
Authority: CN
Inventors: 吴昊; 安艳; 张晓云; 杨乾磊
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2022-08-30
Also published as: WO2023226159A1

Abstract

The invention provides an application of PIM1siRNA in preparation of a medicament for treating arsenic-induced cell malignant transformation diseases. The research of the invention finds that in the chemical carcinogenic process (in the arsenic-induced HaCaT cell malignant transformation process), the protein of the oncogene PIM1 is continuously and highly expressed from 14 generations of cells infected with virus continuously, and PIM1siRNA can prolong the cell doubling time by inhibiting the generation of hydrogen peroxide and superoxide, reduce the cell migration rate and the number of formed soft agar clone colonies and inhibit the arsenic-induced cell malignant transformation.

Description

Application of PIM1siRNA in preparation of medicine for treating arsenic-induced cell malignant transformation disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of PIM1siRNA in preparation of a medicine for treating arsenic-induced cell malignant transformation diseases.

Background

Inorganic arsenic and its compounds are among the 10 chemicals listed by the World Health Organization (WHO) that are attracting major public health concerns, and arsenic is identified by the international agency for research on cancer (IARC) as a category 1 carcinogen (human carcinogen) and classified by the United States Environmental Protection Agency (USEPA) as a category a carcinogen (human carcinogen with high carcinogenic hazard). However, the molecular mechanisms of arsenic carcinogenesis have been long lagged behind. The increasing identification and understanding of altered molecular pathways in cancer diseases paves the way to explore oncogenic mechanisms and to further treat cancer. Therefore, the molecular pathway changes and mechanisms in arsenic carcinogenesis require more intensive research.

The oncogene PIM1 is located on chromosome 17, and is widely involved in various biological activities. Several proteins and pathways with oncogenic properties have been identified as targets for protein kinase activity encoded by the oncogene PIM1, such as regulation of the cell cycle and control of apoptosis. PIM1 can promote cell cycle progression as well as mitosis at various stages, such as phosphorylated Cdc25A, Cdc25C, and the like. In addition to controlling cell growth pathways, PIM1 prevents apoptosis and thus acts as an oncogenic survival factor. PIM1 affects the activity of different transcription factors, such as c-Myb, c-Myc, etc. In addition, PIM1 plays a role in hypoxia-induced chemoresistance. Alterations in PIM1 signaling are widely observed in various neoplastic diseases in humans. In hematological malignancies, PIM1 expression is associated with poor prognosis in various leukemias, connective tissue lymphomas, and diffuse large B-cell lymphomas; in solid tumors, overexpression of PIM1 has been detected in bladder and prostate cancer specimens, where poor prognosis and response to treatment are also associated, and esophageal cancer is similarly found with gastric cancer. PIM1 expression was upregulated following infection with helicobacter pylori in cell culture models as well as in patient gastric epithelial cells; in head and neck cancer, PIM1 is highly expressed.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of PIM1siRNA in preparing a medicament for treating arsenic-induced cell malignant transformation diseases.

The first purpose of the invention is to provide an application of PIM1siRNA in preparing a medicine for treating arsenic-induced cell malignant transformation diseases. The PIM1siRNA comprises three different sirnas, wherein the sequences of the three sirnas are as follows:

sc-36225A:

sense chain 5'-CCCAUAGAUACUCUCUUCUtt-3'

Antisense strand 5'-AGAAGAGAGUAUCUAUGGGtt-3'

sc-36225B:

Sense chain 5'-GUUGGCAUGGUAGUAUACAtt-3'

Antisense strand 5'-UGUAUACUACCAUGCCAACtt-3'

sc-36225C:

Sense chain 5'-UUGGCAUGGUAGUAUACAAtt-3'

Antisense strand 5'-UUGUAUACUACCAUGCCAAtt-3'

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

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

The second purpose of the invention is to provide a recombinant vector, which comprises a sequence capable of transcribing the PIM1siRNA, wherein the sequence is embedded into the vector.

The third purpose of the invention is to provide a pharmaceutical composition comprising the drug or the recombinant vector.

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

In one embodiment of the invention, the carrier is selected from one or more of disintegrants, diluents, lubricants, binders, wetting agents, flavouring agents, suspending agents, surfactants and preservatives.

In one embodiment of the present invention, the pharmaceutical composition is in the form of tablet, capsule, soft capsule, granule, pill, oral liquid, emulsion, dry suspension, dry extract or injection.

The fourth purpose of the invention is to provide a kit comprising the medicament or the recombinant vector or the pharmaceutical composition.

The fifth purpose of the invention is to provide the application of the kit in preparing the medicine for treating arsenic-caused cell malignant transformation diseases.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the research of the invention finds that in the chemical carcinogenic process (in the arsenic-induced HaCaT cell malignant transformation process), the protein of the oncogene PIM1 is continuously and highly expressed from 14 generations of cells infected with virus continuously, and PIM1siRNA can prolong the cell doubling time, reduce the cell migration rate and the number of formed soft agar clone colonies and inhibit the arsenic-induced cell malignant transformation.

After the T-HaCaT is transfected with PIM1siRNA, the invention discovers that the soft agar clone forming ability and the cell migration ability are reduced, the cell doubling time is increased, and the cell malignant transformation index is reversed, which indicates that the continuous activation of PIM1 can promote the generation and development of cancer.

Meanwhile, after the T-HaCaT cell is transfected with PIM1siRNA, the expression level of NRF2 is reduced, and the levels of hydrogen peroxide and superoxide are obviously increased by further detecting the levels of hydrogen peroxide and superoxide. Activation of PIM1 can promote expression of NRF2 and can promote the development of cancer. With further studies on the effect of PIM1, it was found that PIM1 might regulate the expression of the redox-sensitive nuclear transcription factor NRF 2. NRF2 maintains reactive oxygen species levels in cells by enhancing the antioxidant capacity of the cells.

Drawings

In order that the present disclosure may be more readily understood, a more particular description of the disclosure will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings

FIG. 1 is NaAsO in example 1 of the present invention ₂ The expression of PIM1 is changed during the malignant transformation of HaCaT cells; wherein (A) Western result of PIM1 protein is shown in (B) quantitative analysis result of PIM1 protein;

FIG. 2 is PIM1siRNA to NaAsO in example 1 of the present invention ₂ The influence of the PIM1 protein of the T-HaCaT cell, wherein (A) a Western result chart of the PIM1 protein; (B) quantitative analysis results of PIM1 protein;

FIG. 3 is PIM1siRNA to NaAsO in example 1 of the present invention ₂ The influence of the NRF2 protein of the T-HaCaT cell, wherein (A) a Western result chart of the NRF2 protein; (B) the result of quantitative analysis of NRF2 protein;

FIG. 4 is PIM1siRNA to NaAsO in example 2 of the present invention ₂ The influence of the active oxygen of the T-HaCaT cells, wherein (A) a hydrogen peroxide horizontal experiment result chart; (B) experimental results of superoxide levels;

FIG. 5 is PIM1siRNA to NaAsO in example 4 of the present invention ₂ The effect of doubling time of the resulting malignantly transformed HaCaT cells;

FIG. 6 is PIM1siRNA to NaAsO in example 5 of the present invention ₂ The effect of the migratory capacity of the resulting malignantly transformed HaCaT cells; wherein, (A) cell scratch test result chart; (B) relative mobility of cells;

FIG. 7 is PIM1siRNA to NaAsO in example 6 of the present invention ₂ The resulting malignantly transformed HaCaT cells anchor the influence of independent growth; wherein, (A) cell soft agar clone experiment result chart; (B) and (5) quantitatively analyzing the cell soft agar clone.

In the drawings, there is shown in the drawings, ^* P<0.05, the difference was statistically significant compared to the passage control cells. ^# P<0.05, the difference was statistically significant compared to Con siRNA.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

1, the HaCaT cell strain used in the invention is purchased from China center for type culture Collection, the Collection center of Wuhan university.

2，0.1μM NaAsO ₂ After HaCaT cells are continuously treated to 35 generations, the cells are subjected to malignant transformation, which shows that the cell doubling time is obviously shortened, the cell migration rate is obviously increased, and the soft agar clone forming capacity is obviously increased. The cells were defined at this time as 0.1. mu.M NaAsO ₂ Malignantly transformed cells (T-HaCaT).

3, reagents used in the invention: PIM1siRNA (sc-36225, Santa Cruz, USA), Control siRNA (sc-37007, Santa Cruz, USA), sirrnatransffection Reagent (sc-29528, Santa Cruz, USA), sirrnatransffection Medium (sc-36868, Santa Cruz, USA). The other reagents are conventional reagents.

4, the PIM1siRNA comprises three different siRNAs, wherein the sequences of the three siRNAs are as follows:

sc-36225A:

sense chain 5'-CCCAUAGAUACUCUCUUCUtt-3'

Antisense strand 5'-AGAAGAGAGUAUCUAUGGGtt-3'

sc-36225B:

Sense chain 5'-GUUGGCAUGGUAGUAUACAtt-3'

Antisense strand 5'-UGUAUACUACCAUGCCAACtt-3'

sc-36225C:

Sense chain 5'-UUGGCAUGGUAGUAUACAAtt-3'

Antisense strand 5'-UUGUAUACUACCAUGCCAAtt-3'.

5, the kit used in the invention:

example 1 cell culture and arsenic staining

The culture medium for culturing cells is DMEM complete culture medium, and the culture condition is 5% CO ₂ And a constant temperature incubator at 37 ℃. In the cell passage, the medium liquid in the cell culture dish was poured out, the cells were washed 2 times with PBS, 1.5mL of trypsin digestion solution containing EDTA was added, the cells were digested on a hot plate at 37 ℃ for 6min, observed under the mirror, and when the morphology of the cells changed and floated off the dish, 2mL of the medium was added to stop the digestion. Transferring the cell suspension into a sterile centrifuge tube by using a pipette gun, centrifuging (1000g at room temperature for 3min), discarding the supernatant, leaving the cell precipitate at the bottom of the sterile centrifuge tube, adding 1mL of culture medium by using the pipette, slowly blowing off the cells to resuspend the cells, and subculturing according to the ratio of 1: 2. The final concentration of NaAsO was 0.1. mu.M ₂ The DMEM medium of (1) was cultured to 35 passages (about 18 weeks), while passaging control cells without arsenic staining were established.

Example 2PIM 1siRNA treatment of malignantly transformed HaCaT cells

And (3) inoculating the malignant transformed HaCaT cells and the cells of the normal passage control group into a 6-well plate, and performing subsequent treatment after the cells are attached to the wall. First, a PIM1siRNA transfection reagent was prepared, and a solution was prepared according to the amount per dish, wherein a solution per dish: 12 μ L PIM1siRNA or Con siRNA, add 988 μ L siRNA transfection medium; per culture dish, liquid B: 12 μ L siRNA transfection reagent, 988 μ L siRNA transfection medium was added. After mixing well the solution A and the solution B, a mixture containing the transfection reagent and PIM1siRNA or Con siRNA was obtained and incubated for 20min in the dark. Then, the cells were washed once with PBS and once with 2mL siRNA transfection medium, aspirated, 2mL siRNA transfection reagent mixture was added to each dish, and placed in 5% CO ₂ And incubating the cells in a constant temperature incubator at 37 ℃ for 6 h. After 6h, sucking the liquid in the culture dish, adding a proper amount of normal cell culture medium, and detecting relevant indexes after 24-72 h. The results of the experiment are shown in FIGS. 1 to 3.

As can be seen from FIG. 1, cells of the passaged control group (without NaAsO) ₂ Treated cells) compared, 0.1 μ M NaAsO ₂ The expression level of PIM1 in the malignantly transformed HaCaT (T-HaCaT) cells is obviously increased along with the increase of the infection time (P<0.05). WesternBlot results and quantitative analysis show that with 0.1. mu.M NaAsO ₂ The protein expression level of the PIM1 shows a continuous rising trend when the number of infection generations of HaCaT cells is increased, which is specifically shown in that the protein expression level of the PIM1 generation is statistically different from that of a 0 generation control group, and the protein expression levels of the PIM1 generations are statistically different from that of the 0 generation control group and a normal generation control group. As shown in FIG. 2, after transfection of PIM1siRNA into T-HaCaT cells: compared with transfected control group (Con siRNA) cells, the expression level of PIM1 is obviously reduced (P)<0.05) indicating successful transfection, and as shown in fig. 3, compared to passaged control cells (without NaAsO) ₂ Treated cells) compared to 0.1 μ M NaAsO ₂ The expression level of NRF2 in malignantly transformed HaCaT (T-HaCaT) cells is remarkably increased (P)<0.05), after the T-HaCaT cells are transfected with PIM1siRNA, the expression level of NRF2 is obviously reduced compared with that of transfected control group (Con siRNA) (P)<0.05)。

Example 3 detection of Hydrogen peroxide and superoxide in cells

Hydrogen peroxide level detection step:

(1) preparation of samples: collecting cell culture fluid

(2) Preparation of standard product: standards were diluted with cell culture medium as indicated. The standard solution was diluted to 1, 2, 5, 10, 20, 50, 100 micromoles/liter.

(3) Assays were performed in 96-well plates, and 50 μ L of sample or standard was added to each well, followed by twice the volume of hydrogen peroxide detection reagent.

(4) After mixing, standing for 30min at room temperature, and detecting the absorbance value at the wavelength of 560nm by using an enzyme-labeling instrument.

(5) And calculating the hydrogen peroxide concentration in the sample according to the standard curve. The results of the experiment are shown in FIG. 4.

Detecting the superoxide level:

(1) preparation of samples: cells were seeded at a cell density of 10,000/well in 96-well plates.

(2) The culture medium was discarded from the wells and washed 1 time with PBS.

(3) Working solution (containing 200. mu.L of assay buffer, 10. mu.L of LWST-1, 2. mu.L of Catalase) was prepared at 212. mu.L per well.

(4) Add 200. mu.L of prepared detection working solution into each well, and carry out detection at 37 ℃ for 3 min.

(5) 2 μ L of SOD was added to the reserved 2 wells to verify the whole system. The arrangement is as follows:

(6) the absorbance value was measured at 450 nm. The results of the experiment are shown in FIG. 4.

As shown in FIG. 4, when the levels of hydrogen peroxide and superoxide are detected, the levels of hydrogen peroxide and superoxide of T-HaCaT cells are not significantly different from those of the cells of the passage control group (P)>0.05). After the T-HaCaT is transfected into PIM1siRNA, the levels of hydrogen peroxide and superoxide are detected to be remarkably increased and have statistical difference (P)<0.05). Indicating that PIM1siRNA can reverse 0.1. mu.M NaAsO ₂ Resulting T-HaCaT reactive oxygen species levels change.

Example 4 cell doubling time assay

The specific experimental steps of cell doubling time detection are as follows: pancreatin digestion to collect the normal treatment group, 0.1. mu.M NaAsO ₂ The group of cells was treated and seeded at 10,000 per well in 24-well plates. And then every 24h, counting the cells in 3 holes collected by each group, and calculating the time required for doubling the cell number according to a formula: (t × lg 2)/(lgNh-lgNi). t is the culture time, Nh is the number of cells after the culture time t (h), and Ni is the number of cells initially seeded. The results of the experiment are shown in FIG. 5.

As shown in FIG. 5, the doubling time of T-HaCaT cells was significantly reduced compared to that of the passage control group (P <0.05) when measured. After the T-HaCaT cells are transfected with PIM1siRNA, the cell doubling time of the cells is obviously increased compared with that of transfected control cells (P < 0.05).

Example 5 cell scratch test

The cell scratching test comprises the following specific steps:

1) digesting and collecting cells, inoculating the cells into a six-well plate, and culturing in a cell culture box.

2) After 24h the cells were full, 200 μ L of tip was used to scratch perpendicularly to the plate well to make the width of the scratch as uniform as possible.

3) And (4) sucking out the old culture medium, washing the cells for three times by PBS, adding the serum-free culture medium, photographing, and continuously placing the cells in a cell culture box for culture.

4) And (4) photographing again after 48h, selecting at least three scratch fields for each hole during photographing, and carrying out quantitative analysis by using Image J software, wherein the experimental result is shown in figure 6.

As shown in FIG. 6, the scratch test shows that the cell migration ability of T-HaCaT cells is obviously enhanced (P <0.05) compared with that of the cells of the passage control group, and the cell migration ability is obviously reduced (P <0.05) compared with that of the cells of the transfection control group after the PIM1siRNA is transfected.

Example 6 Soft agar colony formation experiment

1) Preparing an agarose solution: adding appropriate amount of agarose powder into double distilled water to obtain agarose solution with concentration of 1.4%, autoclaving, water bathing at 42 deg.C for 2 hr, and mixing with the same volume of 2 × DMEM complete culture medium to obtain agarose solution with concentration of 0.7%.

2) Adding a bottom layer adhesive: 2mL of 0.7% agarose solution is sucked by a pipette and slowly added into a 35mm culture dish, and after the agarose solution is solidified at room temperature, a bottom layer gel is obtained.

3) Adding a top layer adhesive: after counting the cells, adjusting the cell concentration, placing 5,000 cells in DMEM complete medium, adding an equal volume of 0.7% agarose solution to obtain a cell suspension with 0.35% agarose, and adding 1.5mL agarose to the bottom layer gel to obtain the top layer gel.

4) Culturing: adding 1.0mL DMEM complete medium after the top layer gel is solidified in 5% CO ₂ And continuously culturing in a 37 ℃ cell culture box for 4 weeks, and changing the culture solution every 2 to 3 days.

5) Counting: for microscopic observation, colonies with cell diameters greater than 50 μm were selected and counted. The results of the experiment are shown in FIG. 7.

As shown in FIG. 7, the anchorage-independent growth capacity of T-HaCaT cells was significantly enhanced (P) compared to that of passaged control cells<0.05). After transfection of PIM1siRNA to T-HaCaT cells, the independent growth capacity of the anchored cells is significantly reduced compared with that of the transfected control group (P)<0.05). The above experimental results show that PIM1siRNA can reverse 0.1. mu.M NaAsO ₂ Resulting in a malignant transformation phenotype of T-HaCaT cells.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. An application of PIM1siRNA in preparing the medicines for treating the As cell caused malignant transformation disease is disclosed.

2. Use according to claim 1, wherein the cells are selected from HaCaT.

3. Use according to claim 1, characterized in that the disease is basal cell carcinoma of the skin or squamous cell carcinoma of the skin.

4. A recombinant vector comprising a sequence capable of transcribing the PIM1siRNA of any one of claims 1-3, embedded in a vector.

5. A pharmaceutical composition comprising the agent of any one of claims 1 to 3 or the recombinant vector of claim 4.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically or pharmacologically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the carrier is selected from one or more of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants, and preservatives.

8. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is in the form of tablet, capsule, soft capsule, granule, pill, oral liquid, emulsion, dry suspension, dry extract or injection.

9. A kit comprising a medicament according to any one of claims 1 to 3 or a recombinant vector according to claim 4 or a pharmaceutical composition according to any one of claims 5 to 8.

10. Use of the kit of claim 9 for the manufacture of a medicament for the treatment of diseases involving malignant transformation of arsenic cells.