Use of MBOAT1 in the preparation of a reagent for treating cancer

Abstract

The present invention relates to the field of cancer treatment, specifically, to the use of MOBAT1 in the treatment of cancer. More specifically, it relates to the use of MOBAT1 in the treatment of pancreatic cancer. The present invention provides the use of MOBAT1 in the preparation of a reagent for treating cancer.

Description

Technical Field

[0001] The present invention relates to the field of cancer treatment, and specifically, to the use of MBOAT1 in the treatment of cancer. More specifically, it relates to the use of MBOAT1 in the treatment of pancreatic cancer. Background Art

[0002] Pancreatic cancer is a highly dangerous digestive system tumor with hidden early manifestations, rapid late progression, and extremely poor prognosis, and is also known as the "king of cancers". Pancreatic ductal adenocarcinoma is the most common pathological type, and the median survival time of patients is only about six months. Globally, the incidence and mortality of pancreatic cancer are increasing year by year. The American Cancer Society predicts that pancreatic cancer will become the second leading cause of cancer-related death in 2030. In China, the latest statistical data on pancreatic cancer shows that from 1990 to 2017, the number of new cases of pancreatic cancer in China increased by 230.08%, and the standardized mortality rate increased by 47.51%. After years of investment and research, although certain progress has been made in the diagnosis and treatment of pancreatic cancer, the prognosis of pancreatic cancer patients has not been substantially improved. So far, the overall five-year survival rate of pancreatic cancer is still less than 10%.

[0003] Therefore, screening for new therapeutic targets is an urgent task in the basic and translational research of pancreatic cancer, and has very important scientific significance and application value. There is a need in this field for such new therapeutic targets to provide new options for the treatment of pancreatic cancer. Summary of the Invention

[0004] In view of this, on the one hand, the present invention provides the use of MBOAT1 in the preparation of a reagent for treating cancer.

[0005] According to the present invention, the MBOAT1 gene is derived from humans, and its Genbank accession number is NM_001080480.

[0006] Furthermore, the reagent is an MBOAT1 gene inhibitor.

[0007] According to a preferred embodiment of the present invention, the MBOAT1 gene inhibitor refers to a molecule or preparation that is prepared or screened using the MBOAT1 gene as a target and has an inhibitory effect on the MBOAT1 gene. The inhibitory effect includes, but is not limited to: inhibiting the activity of the MBOAT1 gene, or inhibiting the transcription or expression of the MBOAT1 gene.

[0008] According to a preferred embodiment of the present invention, the MBOAT1 gene inhibitor is a nucleic acid molecule, nucleic acid construct, lentivirus, antibody, or small molecule compound.

[0009] More preferably, the nucleic acid molecule is double-stranded RNA or shRNA.

[0010] Further preferably, the MBOAT1 gene target sequence acted on by the nucleic acid molecule is shown in any one of SEQ ID NO: 1-5.

[0011] Further preferably, the nucleotide sequence of the shRNA is shown in any one of SEQ ID NO: 6-11.

[0012] According to the present invention, the anti-cancer drug has one or more of the following functions: inhibiting the proliferation rate of cancer cells, changing the cancer cell cycle distribution, promoting cancer cell apoptosis, and inhibiting cancer cell clone formation.

[0013] Preferably according to the present invention, the anti-cancer drug necessarily includes an MBOAT1 gene inhibitor, and the MBOAT1 gene inhibitor is used as the sole active ingredient or one of the active ingredients for the aforementioned functions.

[0014] According to the present invention, the cancer is pancreatic cancer.

[0015] Further, the cancer is pancreatic ductal adenocarcinoma.

[0016] In a second aspect, a nucleic acid molecule is provided. The nucleic acid molecule is an MBOAT1 gene inhibitor and can reduce the expression of the MBOAT1 gene in pancreatic cancer cells, including:

[0017] a) Double-stranded RNA, wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the MBOAT1 gene under stringent conditions; or

[0018] b) shRNA, wherein the shRNA contains a nucleotide sequence capable of hybridizing with the MBOAT1 gene under stringent conditions.

[0019] According to the present invention, the double-stranded RNA comprises a first strand and a second strand. The first strand and the second strand are complementary to form an RNA dimer together, and the sequence of the first strand is the same as the MBOAT1 gene target sequence; the shRNA includes a sense strand fragment and an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment. The sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is the same as the MBOAT1 gene target sequence.

[0020] Preferably according to the present invention, the double-stranded RNA is small interfering RNA (siRNA).

[0021] Preferably according to the present invention, the nucleotide sequence of the shRNA is shown in any one of SEQ ID NO: 6-11.

[0022] According to the present invention, the shRNA can be processed by enzymatic cleavage to become siRNA, and then play a role in specifically silencing the expression of the MBOAT1 gene in pancreatic cancer cells.

[0023] In a third aspect of the present invention, there is provided a nucleic acid construct, which is an inhibitor of the MBOAT1 gene and contains a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule and can express the shRNA.

[0024] Preferably according to the present invention, the nucleic acid construct is obtained by cloning the gene fragment encoding the shRNA in the aforementioned nucleic acid molecule into a vector.

[0025] Preferably according to the present invention, the vector is a lentiviral vector.

[0026] Preferably according to the present invention, the nucleic acid construct further contains a promoter sequence and / or a nucleotide sequence encoding a detectable marker in tumor cells; more preferably, the detectable marker is green fluorescent protein (GFP).

[0027] After the MBOAT1 gene interfering nucleic acid construct is packaged into infectious virus particles through virus packaging, it infects tumor cells, and then transcribes the shRNA of the present invention. Through steps such as enzymatic cleavage processing, siRNA is finally obtained for specifically silencing the expression of the MBOAT1 gene.

[0028] In a fourth aspect of the present invention, there is provided a lentivirus, which is an inhibitor of the MBOAT1 gene and is obtained by virus packaging of the aforementioned nucleic acid construct with the assistance of a lentiviral packaging plasmid and a cell line.

[0029] In a fifth aspect of the present invention, there is provided a drug for treating pancreatic cancer, including one or more of the aforementioned MBOAT1 gene inhibitors.

[0030] Preferably according to the present invention, the MBOAT1 gene inhibitor is the only active ingredient or one of the active ingredients of the drug for treating pancreatic cancer.

[0031] The form of the drug for treating pancreatic cancer is not particularly limited and can be in the form of a solid, liquid, gel, semi-liquid or aerosol substance. Among them, the dosage form of the drug is any clinically or pharmaceutically acceptable dosage form. For example, but not limited to, the dosage form of the drug is powder, injection, capsule, oral liquid, tablet, dripping pill, spray.

[0032] Furthermore, the drug further includes an instruction manual.

[0033] Furthermore, the instruction manual contains usage information of the drug for treating cancer, such as the effective amount required for treating cancer, the number of administrations, the interval time, etc.

[0034] In the sixth aspect of the present invention, there is provided a combined therapeutic drug for pancreatic cancer, comprising one or more of the above-mentioned MBOAT1 gene inhibitors and at least one other therapeutic drug for pancreatic cancer.

[0035] Further, the drug is paclitaxel, gemcitabine, various targeted antibodies, etc.

[0036] Even further, the drug is gemcitabine. Brief Description of the Drawings

[0037] Figure 1 A. Expression of MBOAT1 mRNA in human pancreatic cancer tissues and adjacent tissues. B. H&E staining and MBOAT1 immunohistochemical staining results of normal mouse pancreatic tissues, low-grade pancreatic duct intraepithelial neoplasia, high-grade pancreatic duct intraepithelial neoplasia, and mouse adenocarcinoma tissues; scale bar: 100 μm. C. Expression of MBOAT1 protein in human pancreatic cancer tissues and paired adjacent tissues. D. Scoring of MBOAT1 expression in a human pancreatic cancer tissue microarray. E. Relationship between the expression of MBOAT1 and the overall survival prognosis of patients in the pancreatic cancer patient cohort of Renji Hospital. F. Relationship between the expression of MBOAT1 and the overall survival prognosis of patients in the TCGA pancreatic cancer patient cohort;

[0038] Figure 2 A. Protein expression levels of MBOAT1 in adenocarcinoma cell lines AsPC-1, Capan-1, Patu8988, PANC-1, and control cell lines HPNE and HPDE. B. Interference efficiency of MBOAT1 expression in AsPC-1 and Capan-1 cell lines. C. Changes in cell viability of pancreatic duct adenocarcinoma AsPC-1 and Capan-1 cells in the MBOAT1 interference group and the control group. D-E. Changes in colony formation and statistical results of AsPC-1 and Capan-1 cells in the MBOAT1 interference group and the control group. F. Changes in EdU fluorescence dye infiltration of AsPC-1 and Capan-1 cells in the MBOAT1 interference group and the control group. Scale bar is 50 μm;

[0039] Figure 3A. Tumor formation of MBOAT1 interference group and control group of pancreatic ductal adenocarcinoma AsPC-1 cells in the subcutaneous tissue of nude mice; the scale bar is 1 cm. B-C. Statistical results of the tumor growth curve (B) and weight (C) of AsPC-1 cells in the subcutaneous tissue of nude mice in the MBOAT1 interference group and the control group; D-E. Immunohistochemical staining of MBOAT1 (D) and PCNA (E) in the subcutaneous tumors of the MBOAT1 interference group and the control group; the scale bar is 100 μm. F. In vivo small animal imaging results and overall survival curves of the pancreas in C57BL6 / J mice in the control group, MBOAT1 interference group, gemcitabine chemotherapy group, and MBOAT1 interference combined chemotherapy group. These results further clarify that MBOAT1 plays a very important role in the occurrence and development of pancreatic ductal adenocarcinoma, suggesting that interfering with the expression of MBOAT1 can inhibit the growth of pancreatic cancer. Detailed implementation manners

[0040] The present invention will be specifically described below in combination with specific implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0041] Example 1: High expression of MBOAT1 in the present invention is positively correlated with poor prognosis of pancreatic cancer

[0042] The applicant first used bioinformatics analysis to search for genes with significantly up-regulated expression in pancreatic ductal adenocarcinoma and found the acyltransferase MBOAT1. Verification was carried out using multiple pancreatic cancer GEO databases (GSE15471, GSE16515, and GSE28735) and Renji pancreatic cancer sequencing data, and it was found that the mRNA level of MBOAT1 was significantly up-regulated in pancreatic ductal adenocarcinoma tissues compared with adjacent tissues ( Figure 1 A). Subsequently, immunohistochemical staining was performed on pancreatic tissues from pancreatic cancer spontaneous KPC model mice, and it was found that the protein expression level of MBOAT1 was significantly up-regulated in low-grade and high-grade pancreatic duct intraepithelial neoplasia and pancreatic ductal adenocarcinoma tissues of mice compared with normal mouse pancreatic tissues ( Figure 1 B). Further immunohistochemical staining also found that the protein expression of MBOAT1 was significantly higher in human pancreatic cancer tissues than in paired adjacent tissues ( Figure 1 C). Using a tissue microarray of 111 pancreatic cancer tissues established by Renji Hospital, immunohistochemical staining was performed, and according to the different expression intensities of MBOAT1, it was divided into a low-expression group (including samples scored as - and +) and a high-expression group (including samples scored as ++ and +++) ( Figure 1D). In the overall survival prognosis analysis of clinical patients in the low-expression group and high-expression group, the applicant found that pancreatic cancer patients with high expression of MBOAT1 had a shorter overall survival time (Figure 1E). Further analysis of the pancreatic cancer patient cohort in the TCGA database based on MBOAT1 expression also found that high expression of MBOAT1 predicted poor prognosis of patients ( Figure 1 F).

[0043] Example 2. Interfering with MBOAT1 expression inhibits the proliferation of pancreatic cancer cells

[0044] To further study the biological function of MBOAT1 in pancreatic ductal adenocarcinoma tumor cells, the applicant first detected the protein expression level of MBOAT1 in pancreatic cancer cell lines AsPC-1, Capan-1, Patu8988, PANC-1 and control cell lines HPNE and HPDE by WB. The results showed that the protein expression level of MBOAT1 in pancreatic cancer cell lines was significantly higher than that in pancreatic cancer control cell lines ( Figure 2 A). Subsequently, the applicant selected AsPC-1 and Capan-1 cells with the highest relative expression of MBOAT1 for subsequent functional experiments. Lentiviruses stably expressing interfering MBOAT1 shRNA (SEQ ID NO.6 and 7) were used to infect AsPC-1 and Capan-1 cells to construct a cell line with silenced MBOAT1 expression. WB was further used to analyze the expression of MBOAT1 in MBOAT1 interference group cells and control group cells to verify the interference efficiency of shRNA ( Figure 2 B). Then, through the CCK-8 cell viability experiment, it was found that the viability of AsPC-1 and Capan-1 cells with interfering MBOAT1 expression was significantly lower than that of the control group ( Figure 2 C). Similarly, the results of the colony formation experiment also showed that interfering with the expression of MBOAT1 could inhibit the colony formation of pancreatic cancer cells ( Figure 2 D, E). The applicant further used the EdU fluorescence kit to detect the change in the rate of EdU dye uptake by AsPC-1 and Capan-1 cells in the MBOAT1 interference group and the control group, and found that interfering with MBOAT1 could significantly inhibit the rate of EdU dye uptake by pancreatic ductal adenocarcinoma cells ( Figure 2 F). The results of the above in vitro cell experiments suggest that MBOAT1 is closely related to the proliferation, colony formation and DNA replication of pancreatic ductal adenocarcinoma cells.

[0045] Example 3. Interfering with MBOAT1 expression inhibits the in vivo progression of pancreatic cancer in mice

[0046] Based on the results of previous in vitro cell function experiments, the applicant further studied the effect of MBOAT1 on the progression of pancreatic ductal adenocarcinoma in animals. The applicant first constructed subcutaneous tumors of pancreatic cancer and inoculated control cells of AsPC-1 and MBOAT1-interfered cells around the groin of nude mice by subcutaneous implantation. After 7 weeks, the subcutaneous tumors were taken for observation, and it was found that the tumor mass in the MBOAT1-interference group was significantly smaller than that in the control group ( Figure 3 A). By plotting the curve of tumor volume change, it was found that the tumor growth of mice in the MBOAT1-interference group was significantly slower than that of mice in the control group ( Figure 3 B). Further weighing of the subcutaneous tumors found that the tumor burden of mice in the interference group was significantly reduced ( Figure 3 C). Staining of tumor sections showed that the staining of the tumor cell proliferation marker PCNA (Proliferating Cell Nuclear Antigen) in the subcutaneous tumors of the interference group was also significantly weaker than that in the control group ( Figure 3 D-E). Subsequently, the applicant constructed an orthotopic model of pancreatic cancer. By the method of orthotopic implantation in the pancreas, the MBOAT1-interference group and control group cells of mouse pancreatic cancer cells KPC1199 were inoculated into the pancreas in situ of C57BL6 / J mice through insulin needles respectively. Then, the progression of in situ tumors was detected regularly by small animal imaging. The results showed that interfering with the expression of MBOAT1 could significantly inhibit the progression of in situ pancreatic cancer ( Figure 3 F). After that, the orthotopic model mice were treated with the chemotherapy drug gemcitabine (50 mg / kg, once every five days), and the progression of pancreatic cancer in mice was observed. It was found that interfering with MBOAT1 and gemcitabine chemotherapy could show a synergistic effect, further inhibiting the progression of in situ tumors and prolonging the overall survival time of mice ( Figure 3 F).

Claims

1. Use of an MBOAT1 gene inhibitor in the preparation of a drug for treating pancreatic cancer, wherein, The MBOAT1 gene inhibitor is the shRNA shown in SEQ ID NO.6 or 7.

2. A nucleic acid molecule, which is an MBOAT1 gene inhibitor and can reduce the expression of the MBOAT1 gene in pancreatic cancer cells. The MBOAT1 gene inhibitor is shRNA: The shRNA contains a nucleotide sequence capable of hybridizing with the MBOAT1 gene under stringent conditions, and the shRNA is shown in SEQ ID NO.6 or 7.

3. A nucleic acid construct, which is an MBOAT1 gene inhibitor and contains a gene fragment encoding the shRNA of the nucleic acid molecule described in claim 2 and can express the shRNA.

4. A lentivirus, which is an MBOAT1 gene inhibitor and is formed by virus packaging with the assistance of a lentivirus packaging plasmid and a cell line using the nucleic acid construct described in claim 3.

5. A drug for treating pancreatic cancer, comprising an MBOAT1 gene inhibitor, wherein, The MBOAT1 gene inhibitor is the shRNA shown in SEQ ID NO.6 and / or 7.

6. The drug according to claim 5, wherein, The drug also includes an instruction manual; wherein, the instruction manual includes usage information of the drug for treating cancer.

7. A combined therapeutic drug composition for pancreatic cancer, comprising the drug described in claim 5 or 6 and at least one other therapeutic drug for pancreatic cancer.

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