Application of ANKRD18B inhibitors in the preparation of pancreatic cancer drugs

Abstract

This invention belongs to the field of biomedical technology and discloses the application of an ANKRD18B inhibitor in the preparation of pancreatic cancer drugs. The ANKRD18B gene is specifically highly expressed in the tumor tissues of metastatic pancreatic cancer patients, and pancreatic cancer patients with high ANKRD18B gene expression in their tissues have shorter overall survival and poorer prognosis. This invention discovers that siRNA can efficiently silence the expression of ANKRD18B in pancreatic cancer cells, effectively inhibiting the migration of pancreatic cancer cells and inducing apoptosis, thus demonstrating the preventive and therapeutic effect of ANKRD18B inhibitors on pancreatic cancer metastasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of ANKRD18B inhibitor in the preparation of pancreatic cancer drugs. BACKGROUND

[0002] Pancreatic cancer is one of the most malignant tumors in the digestive tract. According to the latest data released by the National Cancer Center of China, the incidence of pancreatic cancer in China has reached the 10th place, and the mortality rate has risen to the 6th place. In some large cities, the incidence and mortality of pancreatic cancer even reach the 7th and 5th places. It is reported that the average survival time of pancreatic cancer patients is less than 6 months, and the 5-year survival rate is less than 5%. Metastasis is an important biological characteristic of malignant tumors, and is also the main reason for the poor prognosis of pancreatic cancer patients. Therefore, to clarify the potential mechanism of pancreatic cancer metastasis and explore effective treatment methods is a problem to be solved in the treatment of pancreatic cancer.

[0003] With the development of tumor molecular biology research, molecular targeted therapy has become a new method for treating malignant tumors in addition to surgery, radiotherapy and chemotherapy. Unlike traditional chemotherapy, targeted therapy can prevent the growth, metastasis or invasion of cancer cells by interfering with specific target molecules required for cancer development, and has the advantages of strong specificity, obvious effect and small adverse reactions. In recent years, a large number of studies have proved that the invasion and metastasis of pancreatic cancer cells are regulated by multiple genes. However, there is no particularly effective target gene related to pancreatic cancer metastasis applied in clinical treatment. Therefore, finding effective biological target molecules for preventing and treating pancreatic cancer metastasis is a key problem to be solved, which is crucial for developing new effective prevention strategies.

[0004] ANKRD18B (ankyrin repeat domain 18B) is one of the members of the ankyrin repeat protein family, and little research has been done on the role of this gene in tumor occurrence and development. Some studies have found that high expression of ANKRD18B gene has the effect of inhibiting the growth of lung cancer cells, and has the biological characteristics of tumor suppressor genes. So far, the possible role of ANKRD18B gene in the prevention and treatment of pancreatic cancer has not been reported. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the technical problem to be solved by the present application is to provide the application of ANKRD18B inhibitor in the preparation of pancreatic cancer drugs. Studies have shown that the expression of ANKRD18B gene in tumor tissues of pancreatic cancer patients who have metastasized is significantly higher than that in tumor tissues of patients who have not metastasized. Moreover, high expression of ANKRD18B gene is related to poor prognosis of pancreatic cancer patients. Inhibition of the expression of ANKRD18B gene in pancreatic cancer cells can effectively inhibit cell migration and induce apoptosis.

[0006] The application provides application of the ANKRD18B inhibitor in preparation of a pancreatic cancer drug.

[0007] Based on the inhibitory effect of the ANKRD18B inhibitor on the migration of human pancreatic cancer cells and the apoptosis of human pancreatic cancer cells induced by the ANKRD18B inhibitor, the application also provides application of the ANKRD18B inhibitor in preparation of a drug for preventing and treating human pancreatic cancer.

[0008] In the application, the ANKRD18B inhibitor refers to various gene silencing technologies targeting ANKRD18B, including adenovirus-mediated shRNA interference technology or lentivirus-mediated CrisprCAS9 technology, and further, the ANKRD18B inhibitor is ANKRD18B siRNA, ANKRD18B shRNA, coding DNA of shRNA or ANKRD18B sgRNA. The ANKRD18B siRNA is a small interfering RNA targeting ANKRD18B, which can cause degradation of mRNA and silencing of the ANKRD18B gene. The ANKRD18B shRNA is a short hairpin RNA targeting ANKRD18B, which can be cloned into an expression vector and express short interfering RNA, so as to realize silencing of the ANKRD18B gene. The ANKRD18B sgRNA is a guide RNA targeting the ANKRD18B gene, and the sgRNA is an important part of the Crispr gene knockout system, which can be combined with cas9 protein to guide the cas9 enzyme to target the genomic DNA to cut the ANKRD18B gene.

[0009] The application also provides that the drug further comprises a cationic liposome. In the application, the cationic liposome Lipofectamine3000 is used as a transfection reagent.

[0010] In some embodiments, the ANKRD18B inhibitor is siRNA targeting ANKRD18B, which is composed of a complementary sense strand and an antisense strand, wherein the sense strand is a nucleotide sequence as shown in SEQ ID NO. 1, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO. 2; or the sense strand is a nucleotide sequence as shown in SEQ ID NO. 3, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO. 4; or the sense strand is a nucleotide sequence as shown in SEQ ID NO. 5, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO. 6.

[0011] Further, the adopted ANKRD18B inhibitor is an siRNA against ANKRD18B, which is composed of a complementary sense strand and an antisense strand.

[0012] Further, the sense strand of the siRNA of ANKRD18B is a nucleotide sequence as shown in SEQ ID NO. 1, and the antisense strand of the siRNA of ANKRD18B is a nucleotide sequence as shown in SEQ ID NO. 2.

[0013] Further, the sense strand of the siRNA of ANKRD18B is a nucleotide sequence as shown in SEQ ID NO. 3, and the antisense strand of the siRNA of ANKRD18B is a nucleotide sequence as shown in SEQ ID NO. 4.

[0014] Further, the sense strand of the siRNA of ANKRD18B is a nucleotide sequence as shown in SEQ ID NO. 5, and the antisense strand of the siRNA of ANKRD18B is a nucleotide sequence as shown in SEQ ID NO. 6.

[0015] Further, the pancreatic cancer cell is a human pancreatic cancer SW1990 cell.

[0016] The drug of the present application further comprises other therapeutic agents selected from other drugs for treating pancreatic cancer. The dosage form of the drug is an injection solution or a powder injection for injection.

[0017] Advantages and beneficial effects of the present application:

[0018] The present application first discovers that high expression of ANKRD18B gene is related to metastasis of pancreatic cancer. When the expression of ANKRD18B gene is inhibited, the migration of pancreatic cancer cells can be effectively inhibited and apoptosis can be promoted, indicating that ANKRD18B can be used as an important molecular target for preventing and treating metastasis of pancreatic cancer, and can provide a powerful means for targeted treatment of pancreatic cancer. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 FIG. 1 shows the expression difference of ANKRD18B gene in tumor tissues of patients with primary pancreatic cancer and tumor tissues of patients with metastatic pancreatic cancer in ICGC database;

[0020] Figure 2 FIG. 4 shows the effect of expression of ANKRD18B gene on survival rate of patients with pancreatic cancer analyzed by GEPIA in TCGA database;

[0021] Figure 3 FIG. 6 shows the effect of knockdown of expression of ANKRD18B gene on migration of pancreatic cancer cells;

[0022] Figure 4 This study demonstrates the effect of knocking down the expression of the ANKRD18B gene on apoptosis in pancreatic cancer cells. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the reagent manufacturer.

[0024] Example 1: Differences in ANKRD18B gene expression between tumor tissues from patients with in situ pancreatic cancer and those with metastatic pancreatic cancer in the ICGC database.

[0025] The ICGC (International Cancer Genome Consortium) database collected tumor data from 50 different cancer types (or subtypes), including gene aberrations, somatic mutations, epigenetic modifications, and clinical data. This study obtained RNA-seq data (level 3) and corresponding clinical information from tumor tissues of 222 Canadian pancreatic cancer patients from the ICGC database (https: / / dcc.icgc.org / releases / current / Projects). Statistical analysis of the RNA-seq data was performed using R software v4.0.3. The expression of the ANKRD18B gene in tumor tissues from patients with orthotopic pancreatic cancer and those with metastatic pancreatic cancer is shown below. Figure 1 As shown, the horizontal axis represents different groups of samples, and the vertical axis represents the expression distribution of the gene. The significance of the two groups of samples was determined by the Wilcox test. The results showed that the expression of ANKRD18B in the tumor tissue of patients with metastatic pancreatic cancer was significantly higher than that in the tumor tissue of patients with orthotopic pancreatic cancer (p<0.0001).

[0026] Example 2: The effect of ANKRD18B gene expression on the survival rate of pancreatic cancer patients

[0027] The overall survival (OS) between the high and low expression groups of the ANKRD18B gene in the PAAD (pancreatic cancer) dataset included in TCGA was analyzed using the GEPIA website (http: / / gepia.cancer-pku.cn). The parameters were set as follows: group cutoff value, median; confidence interval, 95%; log-rank p-value, <0.05. Figure 2The figure shows the effect of ANKRD18B gene expression on overall survival (OS) in pancreatic cancer patients. HR represents the risk coefficient of the high-expression group relative to the low-expression group. If HR>1, it indicates that the gene is a risk factor (higher expression, worse prognosis); if HR<1, it indicates that the gene is a protective factor (higher expression, better prognosis). The results show that high expression of the ANKRD18B gene is a high-risk factor for poor prognosis in pancreatic cancer patients.

[0028] Example 3: Effect of knockdown of ANKRD18B gene expression on pancreatic cancer cell migration

[0029] (1) Cell Culture

[0030] Human pancreatic cancer SW1990 cell line was purchased from ATCC. The cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2.

[0031] (2) siRNA transfection

[0032] 2×10 5 SW1990 cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 24 h, and then transfected according to the Lipofectamine 3000 instructions.

[0033] The following are three sequences used to knock down ANKRD18B gene expression:

[0034] siRNA1#

[0035] The justice chain is 5'-ACGUUUCAGGAGAAUAAUGGC-3' (SEQ ID NO.1)

[0036] The antisense chain is 5'-CAUUAUUCUCCUGAAACGUGG-3' (SEQ ID NO.2)

[0037] siRNA2#

[0038] The justice chain is 5'-ACAACAAAAAGCAUAAUCCUC-3' (SEQ ID NO.3)

[0039] The antisense chain is 5'-GGAUUAUGCUUUUUGUUGUGA-3' (SEQ ID NO.4)

[0040] siRNA3#

[0041] The justice chain is 5'-UAAAAUUUCCAAACAUCUCAU-3' (SEQ ID NO.5)

[0042] The antisense chain is 5'-GAGAUGUUUGGAAAUUUUAUG-3' (SEQ ID NO.6)

[0043] (3) Detection of ANKRD18B protein expression

[0044] SW1990 cells were seeded in 6-well plates and transfected with siRNA for 48 hours. Total protein was extracted, and the expression of ANKRD18B in different experimental groups was detected by Western blot. Electrophoresis conditions were: S1 80V, 45 min; S2 110V, approximately 1.5 h. After electrophoresis, a suitable size PVDF membrane was cut and soaked in a small amount of methanol. A sponge, filter paper, gel, and PVDF membrane were placed on the black side of the transfer clamp, avoiding air bubbles. Another layer of filter paper and sponge was then added. The clamp was assembled and placed in the transfer tank, pre-cooled transfer buffer was added, and the power was turned on. The transfer program was set to 0.35A for 1.5 h. After transfer, the membrane was placed in a container, and an appropriate amount of 5% skim milk was added. The membrane was incubated on a shaker for 1 h, and then washed three times with TBST. The washed membrane was incubated overnight at 4°C with diluted ANKRD18B antibody (ThermoFisher, 1:600) on a shaker, followed by three washes with TBST. Then, it was incubated with secondary antibody at room temperature for 1.5 h, followed by three washes with TBST. Finally, the band grayscale values ​​were analyzed using ECL development. Experimental results are shown below. Figure 3 All three siRNAs significantly downregulated the expression of ANKRD18B protein, with siRNA3# being the most significant.

[0045] (4) Scratch test

[0046] The effect of the ANKRD18B gene on the migration of SW1990 pancreatic cancer cells was investigated using a cell scratch assay. The expression of the ANKRD18B gene in SW1990 cells was knocked down by transfection with ANKRD18B siRNA1#, ANKRD18B siRNA2#, and ANKRD18B siRNA3#, respectively, to study the effect of the ANKRD18B gene on the migration of pancreatic cancer cells.

[0047] SW1990 cells transfected with siRNA for 48 hours were seeded into 6-well plates and cultured overnight, ensuring the cells covered the bottom of the plate as completely as possible. Using a 10 μL pipette tip aligned with a ruler (keeping the tip as vertical as possible, not tilted), a horizontal line was drawn perpendicularly to the back of the cell. The cells were washed three times with PBS to remove the drawn cells. The size of the scratch was recorded under a 100x inverted microscope and noted as the 0-hour scratch width. The cells were then cultured at 37°C and 5% CO2 for another 24 hours. The size of the scratch was recorded again under a 100x inverted microscope and noted as the 24-hour scratch width. Cell migration rate = (0-hour scratch width - 24-hour scratch width) / 2. Results are as follows. Figure 3As shown, the migration rate of cells in the three ANKRD18B gene knockdown groups was significantly lower than that of the control group cells after 24 h (P<0.001), indicating that silencing the expression of the ANKRD18B gene can effectively inhibit the migration of pancreatic cancer cells, suggesting that the ANKRD18B gene plays a key role in the migration of SW1990 cells.

[0048] Example 4: Effect of knockdown of ANKRD18B gene expression on apoptosis in pancreatic cancer cells

[0049] SW1990 cells were cultured in a humid environment at 37°C and 5% CO2 in high-glucose DMEM medium supplemented with 10% fetal bovine serum. The effect of ANKRD18B gene expression knockdown on pancreatic cancer cell apoptosis was investigated using siRNA3# knockdown as described in Example 3. SW1990 cells transfected with siRNA3# for 48 hours were seeded in 6-well plates and cultured for 48 hours. Apoptosis was then assessed by annexin V-FITC / PI staining. Cells were stained in 100 μL binding buffer containing 5 μL annexin V-FITC and 10 μL propidium iodide (20 ng / mL), then incubated at room temperature in the dark for 10-15 minutes, and apoptosis was detected by flow cytometry. Results are as follows: Figure 4 As shown, compared with the control group, the percentage of apoptosis in the ANKRD18B knockdown group was significantly increased (P<0.001). These results indicate that inhibiting the expression of the ANKRD18B gene in pancreatic cancer cells can promote apoptosis.

[0050] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the scope of the invention. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An application of an ANKRD18B inhibitor in preparing a drug for treating pancreatic cancer, wherein the drug for treating pancreatic cancer comprises one of a drug for inhibiting migration of pancreatic cancer cells, a drug for inducing apoptosis of pancreatic cancer cells and a drug for preventing and treating metastasis of pancreatic cancer. The ANKRD18B inhibitor is an ANKRD18B siRNA. A sense strand of the ANKRD18B siRNA is a nucleotide sequence as shown in SEQ ID NO. 1, and an antisense strand of the ANKRD18B siRNA is a nucleotide sequence as shown in SEQ ID NO.

2. 2.An application of an ANKRD18B inhibitor in preparing a drug for treating pancreatic cancer, wherein the drug for treating pancreatic cancer comprises one of a drug for inhibiting migration of pancreatic cancer cells, a drug for inducing apoptosis of pancreatic cancer cells and a drug for preventing and treating metastasis of pancreatic cancer. The ANKRD18B inhibitor is an ANKRD18B siRNA. A sense strand of the ANKRD18B siRNA is a nucleotide sequence as shown in SEQ ID NO. 3, and an antisense strand of the ANKRD18B siRNA is a nucleotide sequence as shown in SEQ ID NO.

4. 3.An application of an ANKRD18B inhibitor in preparing a drug for treating pancreatic cancer, wherein the drug for treating pancreatic cancer comprises one of a drug for inhibiting migration of pancreatic cancer cells, a drug for inducing apoptosis of pancreatic cancer cells and a drug for preventing and treating metastasis of pancreatic cancer. The ANKRD18B inhibitor is an ANKRD18B siRNA. A sense strand of the ANKRD18B siRNA is a nucleotide sequence as shown in SEQ ID NO. 5, and an antisense strand of the ANKRD18B siRNA is a nucleotide sequence as shown in SEQ ID NO.

6.

4. The use according to any one of claims 1 to 3, characterized in that, The pancreatic cancer cells are human pancreatic cancer SW1990 cells.

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