PKP1 siRNA, antibody, CRISPR-Cas composition and application thereof

Abstract

The disclosure firstly discovers that plakophilin 1 (PKP1) can be used as a marker for diagnosis / prognosis of squamous carcinoma of organ (such as pancreas) adenocarcinoma, and relates to application of PKP1 (such as DNA, mRNA or protein thereof) as the marker for diagnosis / prognosis of squamous carcinoma of organ (such as pancreas) adenocarcinoma. The present disclosure also relates to a composition for treating squamous carcinoma of adenocarcinoma of an organ (e.g., pancreas) using as an active ingredient a PKP1 gene expression inhibitor (e.g., siRNA, an antibody, a gene editing composition targeting the PKP1 gene (e.g., a CRISPR-Cas composition, etc.), and a method for diagnosing squamous carcinoma of adenocarcinoma by measuring PKP1.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a reagent for inhibiting the expression of a PKP1 gene, an antibody or a CRISPR-Cas composition, and a composition containing the reagent. BACKGROUND

[0002] As one of the malignant tumors with the highest mortality rate worldwide, pancreatic cancer has long been faced with the triple dilemma of "difficult early diagnosis, limited treatment options, and poor prognosis" in clinical diagnosis and treatment. According to clinical data statistics, most pancreatic cancer patients are in the local advanced or distant metastasis stage when diagnosed, with a 5-year survival rate of less than 10%, far exceeding other digestive system malignancies, and becoming a major disease that seriously threatens human life and health.

[0003] Currently, surgical resection is still the core of the clinical treatment of pancreatic cancer, but only about 15%-20% of patients have the conditions for radical resection when diagnosed, and the postoperative recurrence rate is as high as more than 70%, which significantly limits the surgical treatment. For patients who cannot be operated on, radiotherapy, targeted therapy, and immunotherapy, etc. comprehensive treatment methods become the main choice, but the existing schemes generally have problems such as "limited efficacy, strong side effects, and easy drug resistance": for example, although the gemcitabine-based chemotherapy regimen is the first-line standard treatment for advanced pancreatic cancer, the objective response rate is usually less than 20%, and it is easy to cause myelosuppression, gastrointestinal reactions and other serious adverse reactions; the approved targeted drugs (such as erlotinib) are only effective for patients with specific gene mutation subtypes, and the applicable population is narrow; and the immune checkpoint inhibitors in the treatment of pancreatic cancer have a "deserted immune environment" due to the tumor microenvironment, and the efficacy is far inferior to that of melanoma, lung cancer and other malignancies, which limits the clinical application.

[0004] There are more bottlenecks in the treatment of squamous cell carcinoma. In terms of targeted therapy, compared with adenocarcinoma, squamous cell carcinoma has fewer driver gene mutations, such as the rare occurrence of common mutations such as EGFR and ALK in lung squamous cell carcinoma, which makes it difficult to develop targeted drugs. Moreover, squamous cell carcinoma has high molecular heterogeneity, with large differences in cells between different patients and different regions of the same patient's tumor, making it difficult for a single targeted drug to cover and easily produce drug resistance. Although immunotherapy brings hope to some patients with squamous cell carcinoma, the overall response rate is not high. There are many immune suppressive factors in the tumor microenvironment, such as tumor-associated macrophages and regulatory T cells, which can weaken the immune killing, and the low tumor mutation load makes it difficult for the immune system to recognize and attack tumor cells. The effect of traditional chemotherapy and radiotherapy is also limited. Some squamous cell carcinomas do not respond well to chemotherapy, such as lung squamous cell carcinoma, which is less sensitive to chemotherapy than adenocarcinoma, and the side effects of chemotherapy seriously affect the patient's tolerance and quality of life. In radiotherapy, some squamous cell carcinomas are resistant, such as esophageal squamous cell carcinoma patients who are more likely to develop radiotherapy resistance due to specific gene mutations. In addition, the surgical resection of squamous cell carcinoma in some parts is difficult, such as head and neck squamous cell carcinoma, which is deep in location, with dense blood and lymphatic vessels and easy metastasis, lung portal squamous cell carcinoma is closely related to important structures, all of which pose challenges to surgery. Finally, the current treatment plan for squamous cell carcinoma is lacking in individualization, there are differences between Eastern and Western patients, and there are differences in radiotherapy sensitivity, prognosis, and other aspects between individual patients, making it difficult to achieve precise treatment.

[0005] With the development of molecular biology technology, small interfering RNA (siRNA) technology has shown great potential in the treatment of pancreatic cancer due to its strong sequence specificity and high efficiency in targeting gene expression. siRNA can specifically bind to target mRNA related to the development of pancreatic cancer, trigger the RNA interference (RNAi) pathway, and efficiently silence key pathogenic genes such as Kras mutant genes, Bcl-2 anti-apoptotic genes, and VEGF angiogenesis genes, thereby inhibiting pancreatic cancer cell proliferation, inducing cancer cell apoptosis, or blocking the formation of tumor microenvironment, providing a new direction for pancreatic cancer treatment. However, existing siRNA molecules for pancreatic cancer treatment still have obvious shortcomings: on the one hand, some siRNA molecules have low silencing efficiency for target genes, making it difficult to effectively inhibit the activation of pathogenic pathways, resulting in poor treatment effect; on the other hand, some siRNA molecules have off-target effects, which may non-specifically silence normal cell genes, causing potential side effects; in addition, even siRNA molecules with high silencing efficiency also have the characteristics of poor stability and easy degradation by nucleases, resulting in short half-life in the body and difficulty in sustained function. These problems limit the effectiveness of existing siRNA molecules in preclinical studies and preliminary applications of pancreatic cancer, and new siRNA molecules with higher target gene silencing efficiency, lower off-target effects, and stronger in vivo stability are urgently needed to break through the core bottleneck of current pancreatic cancer siRNA therapy and promote the clinical translation of this technology. SUMMARY

[0006] To develop an siRNA having a significant inhibitory effect on the PKP1 gene, the inventors designed the sense strand and the antisense strand of the double-stranded siRNA sequence, and surprisingly found that, in the case where a mismatch exists at a specific position of the sense strand and the antisense strand, a more superior inhibitory effect is exhibited. Based on this, the present application was made.

[0007] In one aspect, the present disclosure provides an siRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and the antisense strand comprises at least 15 consecutive nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 4, 6, 8.

[0008] In one aspect, the present disclosure provides a specific PKP1 antibody, which can bind to a specific antibody of the active binding domain of the PKP1 protein, and effectively inhibit the expression of PKP1 in a safe dose. In some embodiments, the antibody is purified by affinity chromatography method, and the PKP1 (including Head Domain) protein is used to immunize mice and the B cells of the immunized mice are extracted, and the B cells are fused with mouse myeloma cells under the induction of polyethylene glycol (PEG) or electrofusion technology to form hybridoma cells, and a monoclonal cell strain that only secretes an antibody against the target antigen is screened from the hybridoma cells, and the monoclonal cell strain is purified after being cultured and expanded.

[0009] In one aspect, the present disclosure provides a CRISPR-Cas composition for targeted editing of the PKP1 gene. In some embodiments, the CRISPR-Cas composition comprises a gRNA targeting the PKP1 gene, the gRNA comprising a polynucleotide sequence complementary to a targeting sequence of the PKP1 gene. In some embodiments, the gRNA targeting sequence is selected from a regulatory sequence or a coding sequence of the PKP1 gene.

[0010] In yet another aspect, the present disclosure provides a pharmaceutical composition for preventing or treating squamous metaplasia of adenocarcinoma (e.g., pancreatic cancer), which contains the siRNA, antibody, and gRNA agent of the present disclosure and a pharmaceutically acceptable carrier.

[0011] In yet another aspect, the present disclosure provides a method of diagnosing, preventing, or treating squamous metaplasia of adenocarcinoma (e.g., pancreatic cancer), the method comprising administering an effective amount of the siRNA, antibody, and gRNA agent of the present disclosure and / or the pharmaceutical composition of the present disclosure to a subject suffering from squamous metaplasia of adenocarcinoma (e.g., pancreatic cancer).

[0012] In yet another aspect, the present disclosure provides a method of inhibiting expression of PKP1 in a cell, the method comprising contacting an effective dose of the siRNA, antibody and gRNA reagent of the present disclosure, and / or the pharmaceutical composition of the present disclosure with the cell.

[0013] In yet another aspect, the present disclosure provides a cell comprising the siRNA, antibody and gRNA reagent of the present disclosure, and / or the pharmaceutical composition of the present disclosure in the cell.

[0014] In yet another aspect, the present disclosure provides a vector comprising the siRNA, antibody and gRNA reagent of the present disclosure.

[0015] In yet another aspect, the present disclosure provides use of PKP1 as a squamous metaplasia marker or tumor target molecule of adenocarcinoma.

[0016] Incorporated by reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference herein.

[0017] Beneficial effects (1) The present disclosure first discovers that PKP1 can be used as a marker for diagnosis / prognosis of squamous metaplasia of pancreatic adenocarcinoma. Since the high correlation between the expression of PKP1 and squamous metaplasia of pancreatic (or other organs) adenocarcinoma is confirmed, the diagnosis of pancreatic adenocarcinoma by measuring the expression level of PKP1 gene has excellent effect. Clinically, the degree of squamous metaplasia of tumor tissue of patients can be accurately evaluated by detecting the expression of PKP1 in tumor tissue, and squamous metaplasia is closely related to disease progression, recurrence risk and survival prognosis. Moreover, the treatment plan of adenocarcinoma with squamous metaplasia is different from that of simple adenocarcinoma. The detection of PKP1 expression level provides a scientific basis for individualized treatment plan adjustment and follow-up strategy formulation, and further improves the clinical diagnosis and treatment and prognosis management system of this kind of malignant tumor.

[0018] (2) Precise targeting of pathogenesis, strong inhibition of tumor progression.

[0019] The present disclosure confirms the core regulatory role of PKP1 gene in the process of squamous metaplasia of pancreatic adenocarcinoma and other organ adenocarcinoma, and by inhibiting the expression of PKP1 gene, the proliferation pathway of malignant tumor cells can be directly blocked at the molecular level, and the migration ability and distant metastasis potential of tumor cells can be significantly inhibited. This mechanism has high specificity, only acts on tumor cells expressing PKP1, avoids non-specific damage to normal cells, greatly reduces the toxic and side effects of traditional treatment methods (such as radiotherapy and chemotherapy), and improves the safety and tolerance of treatment. (3) Fill in the detection technology gap, improve the clinical diagnosis dilemma. The present disclosure provides a specific detection method for pancreatic squamous carcinoma and other organ adenocarcinoma squamous carcinoma, which effectively fills the clinical short board of "lack of detection method and insufficient diagnosis basis" for this type of malignant tumor. Compared with the existing traditional diagnosis method relying on pathological morphology observation, the detection method can more accurately and efficiently identify pancreatic squamous carcinoma and other organ adenocarcinoma lesions, help the clinic to achieve "early detection and early diagnosis", provide key support for the timely development of subsequent treatment plan, and significantly reduce the risk of missing the treatment window period due to delayed diagnosis. (4) Optimize treatment plan and improve prognosis evaluation system The combination of siRNA, antibody and gRNA reagents provided by the present disclosure and existing tumor treatment methods (such as surgery, radiotherapy and chemotherapy, targeted therapy, etc.) can form a "synergistic" treatment effect: by inhibiting the expression of PKP1, the sensitivity of tumor cells to existing treatment methods is enhanced, tumor drug resistance is effectively overcome, and the treatment response rate and cure rate of patients are significantly improved, especially for patients with advanced or refractory pancreatic adenocarcinoma squamous carcinoma and other organ adenocarcinoma squamous carcinoma, providing a new treatment option. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The expression of PKP1 mRNA in pancreatic cancer and normal pancreatic tissue is described. (A) is the analysis of the expression level of PKP1 mRNA in pancreatic cancer tissue (T) and paracancer and normal pancreatic tissue (N) by GEPIA database; (B) is the Kaplan-Meier survival analysis of pancreatic cancer in the high and low expression groups of PKP1 based on the median of PKP1 in the GEPIA database; (C) is the analysis of the expression level of PKP1 protein in pancreatic cancer tissue (T) and paracancer tissue (N) by UALCAN database; (D) is the comparison of the protein expression of PKP1 in different Grade classification of pancreatic cancer by UALCAN database. The data is expressed as mean ± SD, the difference between two groups is analyzed by Student's t test, *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 2The expression of squamous carcinoma markers (TP63, KRT5, KRT6A and KRT6B) and PKP1 in different cell subpopulations is described. Using the scRNA-seq dataset of pancreatic squamous carcinoma (GSM5032772), the expression of squamous carcinoma markers and PKP1 in each cell subpopulation is analyzed. (A) UMAP plot of cell clustering; (B) UMAP plot of TP63 gene expression level; (C) UMAP plot of KRT5 gene expression level; (D) UMAP plot of KRT6A gene expression level; (E) UMAP plot of SERPINB3 gene expression level; (F) UMAP plot of PKP1 gene expression level.

[0022] Figure 3 A specific PKP1 antibody is described, which is verified by Western blot. The antibody purifies the PKP1 (including Head Domain) protein by affinity chromatography, immunizes mice with it, and extracts the B cells of the immunized mouse spleen, fuses the B cells with mouse myeloma cells under the induction of polyethylene glycol (PEG) or electrofusion technology to form hybridoma cells, screens monoclonal cell strains that only secrete antibodies against the target antigen from the hybridoma cells, and then purifies the specific antibodies that can bind the active binding domain of the PKP1 protein after expanding the culture. The specific antibody can recognize the target protein in the normal / knockdown state, and the band size is consistent with the theoretical molecular weight of PKP1 (82 kDa).

[0023] Figure 4 The expression of PKP1 and p63 proteins is detected by Western blot. (A) is based on the Bulk RNA-seq data of mouse pancreatic adenoid squamous carcinoma (GSE18116), the correlation between the mRNA expression levels of PKP1 and the markers of mouse squamous carcinoma (Krt5, Krt6a, Krt6b and Tp63) is analyzed; (B) is based on the Bulk RNA-seq data of human pancreatic adenoid squamous carcinoma (GSE241266), the correlation between the mRNA expression levels of PKP1 and the markers of human squamous carcinoma (KRT5, KRT6A, SERPINB3 and TP63) is analyzed; (C) is a Western blot analysis of the protein expression of p63, one of the key transcription factors of squamous carcinoma, and PKP1 in 5 pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, Hs766T and PK-59). The correlation coefficient (r) and p value are calculated by Pearson correlation analysis, *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 5The knockdown effect of siPKP1-1, siPKP1-2 and siPKP1-3 on the protein expression of PKP1 is described. (A) is based on BxPC3 cells, using Western blot method, the knockdown efficiency of 3 PKP1 specific siRNAs is verified, siPKP1-3 is the known sequence of commercial PKP1 siRNA. (B) PKP1 knockdown and overexpression in vitro cell model is constructed using siPKP-1 (siPKP1) and overexpression plasmid of PKP1 (oePKP1), Western blot analysis of PKP1 protein expression after siRNA knockdown of PKP1 (siPKP1) in BxPC-3 and Hs766T; and overexpression of PKP1 (oePKP1) after overexpression plasmid in AsPC-1 and Hs766T.

[0025] Figure 6 The effect of knockdown of PKP1 (siPKP1) expression on the expression level of p63 protein in BxPC-3 and Hs766T cells is described. Western blot analysis of p63 protein expression after knockdown of PKP1 (siPKP1) expression in BxPC-3 and Hs766T.

[0026] Figure 7 The effect of knockdown of PKP1 (siPKP1) expression on the proliferation of BxPC-3 and Hs766T cells is described. CCK8 analysis of the change in cell proliferation ability after knockdown of PKP1 (siPKP1) in BxPC-3 and Hs766T and overexpression of PKP1 (oePKP1) in AsPC-1 and Hs766T.

[0027] Figure 8 The effect of knockdown of PKP1 expression on the growth rate of tumors in BxPC-3 cells is described. After knockdown of PKP1 (siPKP1) in BxPC-3, the cells were injected subcutaneously into nude mice, (A) growth curve of subcutaneous tumor-bearing; (B) tumor weight column chart; (C) tumor photo. Data are expressed as mean ± SD, differences between two groups were analyzed by Student's t test, *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 9The effect of overexpression of PKP1 on tumor growth rate in Panc02 cells is described. After overexpression of PKP1 (oePKP1) in Panc02, the cells were injected into the subcutaneous and pancreas of C57BL / 6J mice, (A) Comparison of the photos, growth curve and column chart of tumor weight of C57BL / 6J mice subcutaneous tumor of Panc02 cells; (B) Comparison of the photos and column chart of tumor weight of C57BL / 6J mice orthotopic pancreatic tumor of Panc02 cells. Data are expressed as mean ± SD, the difference between the two groups was analyzed by Student's t test, *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 10 The decrease in the number of invasive cells after knockdown of PKP1 in BxPC-3 and Hs766T cells is described. Representative pictures and statistical column chart of cell invasion experiment after knockdown of PKP1 (siPKP1) in (A) BxPC-3 and (B) Hs766T cells; photos and statistical column chart of cell invasion experiment after overexpression of PKP1 (oePKP1) in (C) AsPC-1 and (D) Hs766T cells. Data are expressed as mean ± SD, the difference between the two groups was analyzed by Student's t test, *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 11 The cell scratch closure rate when PKP1 is knocked down in BxPC-3 and Hs766T cells is described. Representative pictures and statistical column chart of cell scratch experiment after knockdown of PKP1 (siPKP1) in (A) BxPC-3 and Hs766T cells; and representative pictures and statistical column chart of cell scratch experiment after overexpression of PKP1 (oePKP1) in (B) AsPC-1 and Hs766T cells. Data are expressed as mean ± SD, the difference between the two groups was analyzed by Student's t test, *p<0.05, **p<0.01, ***p<0.001.

[0031] Figure 12 The effect of overexpression of PKP1 (oePKP1) on the metastatic foci and tumor area of Panc02 cells on the liver is described. Among them, (A) Photos of liver metastasis of C57BL / 6J mice of Panc02 cells, (B) Liver weight ratio and (C) Representative pictures of H&E staining of liver tissue. Data are expressed as mean ± SD, the difference between the two groups was analyzed by Student's t test, *p<0.05, **p<0.01, ***p<0.001.

[0032] Figure 13The expression of PKP1 mRNA in other non-squamous carcinomas (rectal adenocarcinoma, gastric adenocarcinoma, thyroid carcinoma, prostate carcinoma, lung adenocarcinoma, hepatocellular carcinoma, colon adenocarcinoma and cholangiocarcinoma) is described. As can be seen from the figure, the expression of PKP1 mRNA in other non-squamous carcinomas does not change significantly (p>0.05).

[0033] Figure 14 The expression of PKP1 mRNA in other organ adenosquamous carcinomas (thymoma, bladder urothelial carcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma and endometrial adenocarcinoma and esophageal carcinoma) is described, and as can be seen from the figure, the expression of PKP1 mRNA in other organ adenosquamous carcinomas (thymoma, bladder urothelial carcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma and endometrial adenocarcinoma and esophageal carcinoma) is significantly higher (p<0.05) Figure 14 A-E), is increased in skin melanoma, and the survival rate of patients in the high expression group of PKP1 is significantly lower than that of patients in the low expression group of PKP1 (p<0.05) Figure 14 F).

[0034] Figure 15 The process of molecular feature analysis of PKP1 gene in pancreatic cancer is shown: input PKP1 gene in the "Proteomics" module of UALCAN platform, the system automatically completes the box plot analysis of protein expression difference between tumor and normal tissue in TCGA-PAAD cohort, and by activating the "Subgroup Analysis" function, the "Grade" parameter is selected to generate expression comparison under different pathological grades, and finally the expression characteristics of PKP1 at mRNA and protein levels and its potential correlation with clinical prognosis are determined. DETAILED DESCRIPTION

[0035] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present disclosure, and are not intended to limit the present disclosure in any aspect.

[0036] DEFINITIONS In the foregoing and hereinafter, capital letters C, G, U, A represent the base composition of nucleotides, unless otherwise specified.

[0037] In the context herein, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand each pair with a base on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or in RNA, uracil (U)); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner.

[0038] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "essentially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0039] In the above and below, a nucleotide sequence has "nucleotide difference" with another nucleotide sequence, meaning that the base type of the nucleotide at the same position is changed in the former compared to the latter, e.g., in the latter a nucleotide base is A, while in the former the corresponding nucleotide base at the same position is U, C, G or T, then it is recognized that there is nucleotide difference between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference is also recognized to occur at a position when an abasic nucleotide or its equivalent is substituted for the nucleotide at the original position.

[0040] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0041] As used herein, the term "subject," as used herein, refers to any animal, such as a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, a human, a non-human primate (e.g., a rhesus or other type of macaque monkey), a mouse, a pig, a horse, a donkey, a cow, a sheep, a rat, or any species of poultry.

[0042] As used herein, “treatment” refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding the fact that the subject can still be afflicted with the underlying disorder.

[0043] As used herein, “prevention” refers to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain “prophylactic benefit,” the siRNA, antibody and gRNA conjugate or pharmaceutical composition can be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though the diagnosis of the disease can not have been made.

[0044] The term “antibody” generally refers to an immunoglobulin that is reactive to a specified protein or peptide or fragment thereof. The antibody can be from any class of antibody, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (K) or lambda (l). The antibodies of the present application can be derived from any species. The term “antibody” can include intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule that exhibits the desired biological activity.

[0045] The following are described in detail in turn.

[0046] Embodiments The following describes in detail the specific embodiments of the present disclosure. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present disclosure, and are not intended to limit the present disclosure.

[0047] In the present disclosure, PKP1 is located at the region of 1q32.1 of human chromosome 1, the site information of which is as follows: Homo sapiens Chromosome 1, Assembly GRCh 38.p14, NC_00001.11: the sequence of its transcript (NM_001005337.3) (mRNA) is shown in SEQ ID NO. 1; the sequence of its amino acid is shown in SEQ ID NO. 2. SUMMARY The present disclosure provides a composition for treating pancreatic squamous carcinoma with a plakophilin 1 (PKP1) gene expression inhibitor as an active ingredient, and a method for diagnosing pancreatic adenocarcinoma by measuring PKP1.

[0048] siRNA In one aspect, the present disclosure provides an siRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and the antisense strand comprises at least 15 consecutive nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 4, 6, 8.

[0049] In some embodiments, the siRNA comprises at least one nucleotide mismatch (e.g., 1 or more) between the sense strand and the antisense strand.

[0050] In some embodiments, the sense strand of the siRNA agent is selected from any one of SEQ ID NOs: 3, 5, 7; and the antisense strand is selected from any one of SEQ ID NOs: 4, 6, 8.

[0051] In some embodiments, the sense strand and the antisense strand of the siRNA are as set forth in SEQ ID NOs: 3, 4, respectively, or as set forth in SEQ ID NOs: 5, 6, respectively, or as set forth in SEQ ID NOs: 7, 8, respectively.

[0052] In some embodiments, the sense strand or the antisense strand of the siRNA comprises at least one modification.

[0053] Pharmaceutical composition In one aspect, the present disclosure provides a pharmaceutical composition capable of being used for preventing or treating pancreatic cancer, which contains the siRNA, antibody, and gRNA agent of the present disclosure and a pharmaceutically acceptable carrier.

[0054] In some embodiments, the weight ratio of the siRNA, antibody, and gRNA agent to the pharmaceutically acceptable carrier is 1: (1-500); optionally, the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1: (1-50).

[0055] In the present disclosure, the pharmaceutically acceptable carrier can be one or more of the carriers conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and derivatives thereof. In some embodiments, the pharmaceutically acceptable carrier contains an organic amine, a helper lipid, and a pegylated lipid.

[0056] In some embodiments, the pharmaceutical composition can be a liquid formulation, such as an injection solution.

[0057] In some embodiments, the pharmaceutical composition can also be a lyophilized powder injection, which is mixed with a liquid excipient to prepare a liquid formulation when administered.

[0058] In some embodiments, the liquid formulation can be, but is not limited to, used for subcutaneous, intramuscular, or intravenous injection administration, and can also be, but is not limited to, administered to the lung by spraying or administered to other organ tissues (e.g., the liver) by pulmonary spray. In some embodiments, the pharmaceutical composition is used for intravenous injection administration.

[0059] Treatment In one aspect, the present disclosure provides a method of diagnosing, preventing, or treating pancreatic cancer, the method comprising administering an effective amount of the siRNA, antibody, and gRNA reagent of the present disclosure or the pharmaceutical composition of the present disclosure to a subject having squamous metaplasia of adenocarcinoma (e.g., pancreatic cancer).

[0060] In yet another aspect, the present disclosure also provides a product of the siRNA, antibody and gRNA reagent of the present disclosure for use in the preparation of a medicament for evaluating the efficacy of squamous metaplasia treatment of adenocarcinoma (e.g., pancreatic cancer) and monitoring the efficacy of the treatment.

[0061] Cell In one aspect, the present disclosure provides a cell comprising the siRNA reagent of the present disclosure, and / or the pharmaceutical composition of the present disclosure described in claim 7 or 8.

[0062] In yet another aspect, the present disclosure provides a method of inhibiting the expression of PKP1 in a cell, the method comprising contacting an effective dose of the siRNA reagent of the present disclosure, and / or the pharmaceutical composition of the present disclosure with the adenocarcinoma (e.g., pancreatic cancer) squamous metaplasia.

[0063] Vector In one aspect, the present disclosure provides a vector comprising the siRNA, antibody and gRNA reagent of the present disclosure.

[0064] Other types of PKP1 gene expression inhibitors The PKP1 gene expression inhibitors described herein include all agents capable of inhibiting the expression of the PKP1 gene, including antibodies, gene editing compositions targeting the PKP1 gene (e.g., CRISPR-Cas compositions).

[0065] In one aspect, the present disclosure provides a specific PKP1 antibody that can bind to the active binding domain of the PKP1 protein and effectively inhibit the expression of PKP1 at a safe dose.

[0066] In one aspect, the present disclosure provides a CRISPR-Cas composition targeting the editing of the PKP1 gene.

[0067] In some embodiments, the CRISPR-Cas composition comprises a gRNA targeting the PKP1 gene, the gRNA comprising a polynucleotide sequence complementary to a targeting sequence of the PKP1 gene.

[0068] In some embodiments, the gRNA targeting sequence is selected from a regulatory sequence or a coding sequence of the PKP1 gene.

[0069] Kit In yet another aspect, the present disclosure provides a kit comprising an effective amount of the siRNA, antibody and gRNA and / or pharmaceutical composition of the present disclosure.

[0070] In some embodiments, the kits described herein can provide the siRNA, antibody, and gRNA in one container. In some embodiments, the kits described herein can comprise one container providing a pharmaceutically acceptable excipient. In some embodiments, the kits can further comprise other ingredients, such as stabilizers or preservatives, etc. In some embodiments, the kits described herein can comprise at least one other therapeutic agent in a container different from the container providing the siRNA, antibody, and gRNA described herein. In some embodiments, the kits can comprise instructions for mixing the siRNA, antibody, and gRNA with a pharmaceutically acceptable carrier and / or excipient or other ingredients, if any.

[0071] In the kits of the present disclosure, the siRNA, antibody, and gRNA and pharmaceutically acceptable carrier and / or excipient and the siRNA, antibody, and gRNA, pharmaceutical composition and / or siRNA, antibody, and gRNA conjugate, and / or pharmaceutically acceptable excipient can be provided in any form, such as liquid form, dry form, or lyophilized form.

[0072] In some embodiments, the siRNA, antibody, and gRNA and pharmaceutically acceptable carrier and / or excipient and the pharmaceutical composition and / or siRNA, antibody, and gRNA conjugate and optional pharmaceutically acceptable excipient are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kits of the present disclosure. In yet another aspect, the present disclosure also provides the use of PKP1 as a molecular marker in a kit for the diagnosis or prognosis of pancreatic or other organ cancer.

[0073] In yet another aspect, the present disclosure also provides the use of PKP1 as a marker for guiding the treatment of pancreatic or other organ cancer patients.

[0074] In some embodiments, the diagnosis of pancreatic cancer includes the diagnosis of cancer, the diagnosis of cancer stage, the diagnosis of cancer grade, the diagnosis of cancer molecule, the diagnosis of cancer distant metastasis, and / or the diagnosis of cancer lymph node metastasis.

[0075] The application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the application, not to limit the scope of the application. The experimental methods in the following examples are conventional methods, and no special instructions are given. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0076] AsPC-1 cells, CFPAC-1 cells and Hs766T cells were purchased from Guangzhou Sike Biotechnology Co., Ltd.; BxPC-3 cells and PK-59 cells were purchased from Guangzhou Zhibang Biotechnology Co., Ltd. All cell lines were added with complete culture medium containing 10% fetal bovine serum and cultured in a humidified incubator at 37°C and 5% CO2.

[0077] Anti-PKP1 (PA5-29098) antibody was purchased from Invitrogen Corporation; Anti-p63 (13109) antibody was purchased from CST Corporation.

[0078] PKP1 plasmid (PKP1 overexpression plasmid, oePKP1) and blank plasmid (oeCON) as a control were purchased from Jin Srisi Technology Co., Ltd. The PKP1 overexpression plasmid cloning vector backbone was pcDNA3.1(+)-C-DYK, CMV was selected as a strong promoter, EcoRI and XbaI were selected for cleavage, and human PKP1 gene sequence was added after the promoter sequence, with a length of 2181 nt. Ampicillin-containing medium can be used as a screening condition. After the PKP1 overexpression plasmid E. coli grew to an appropriate concentration, plasmid extraction was performed. The plasmid extraction reagent (NucleoBond Xtra Midi Plus EF, 740422.50) used was purchased from Guangzhou Branch of Genbio International Trading (Shanghai) Co., Ltd. (MACHEREY-NAGEL).

[0079] Matrigel (#356234) required for ectopic xenograft tumor experiment was purchased from Becton, Dickinson and Company, USA. CCK8 (#CK04) was purchased from Dojindo Corporation.

[0080] Female nude mice for ectopic xenograft tumor model and C57BL / 6 mice for ectopic and orthotopic allograft tumor model were purchased from Shanghai South Model Organisms Technology Co., Ltd. Mice were used at 4 weeks old. All animals were raised in the South China University of Technology animal facility, which was approved by the National Laboratory Animal Care Evaluation and Accreditation Agency according to current regulations and standards.

[0081] Example 1. Design of siRNA sequence and exploration of PKP1 expression in human pancreatic cancer PKP1 is located in the region of 1q32.1 of human chromosome 1, and the site information is as follows: Homo sapiens Chromosome 1, Assembly GRCh 38.p14, NC00001.11, the transcript (NM_001005337.3) (mRNA) sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

[0082] The specific targeting siRNA and blank control siCON designed in the application are synthesized by GenePharma, wherein the siRNA sequence of PKP1 is shown in the following table:

[0083] Further, the applicant determines the mRNA expression level comparison of PKP1 in pancreatic cancer tissue (T) and paracancer and normal pancreatic tissue (N) through the "Expression DIY" module in the GEPIA database (http: / / gepia.cancer-pku.cn / ); the correlation between the expression level of PKP1 gene and the overall survival of PAAD patients is studied by using the "Survival Plots" module. The protein expression level comparison of PKP1 in pancreatic cancer tissue (T) and paracancer and normal pancreatic tissue (N) and the comparison of protein expression in different Grade classification of pancreatic cancer are explored by using the "Proteomics" module in the UALCAN database (https: / / ualcan.path.uab.edu / ). The specific determination process is as follows (the specific reference process is shown in Figure 14 1. Access module: open the UALCAN website → select "Proteomics" in the top menu → input the gene name PKP1.

[0084] 2. Select the cancer type: select "Pancreatic cancer" (PAAD) in the result page.

[0085] 3. Analysis content (1) Basic comparison: automatically generate the box plot of the expression level of PKP1 protein in tumor (T) vs. normal / paracancer (N) tissue.

[0086] (2) Subgroup analysis: click "Subgroup Analysis" → select the "Grade" parameter to generate the expression comparison under different pathological grades.

[0087] 4. Key reference details Characteristics of case samples in the database: ​(1) Tumor samples: Source: Primary pancreatic ductal adenocarcinoma (PDAC) tissues surgically resected.

[0088] Clinical association: Each sample is associated with clinicopathological data (e.g., grade, stage, survival information).

[0089] Control samples: Cancer-adjacent tissue: Tissue >2 cm from the tumor margin (pathologically confirmed without cancer cell infiltration); Healthy tissue: Normal pancreas from organ donors or non-pancreatic cancer surgery patients.

[0090] (2) Determination of Grade Standard: All grades are based on the AJCC (American Joint Committee on Cancer) pathological grading system, where G1 represents well-differentiated (cancer cells close to normal morphology); G2 represents moderately differentiated; G3 represents poorly differentiated (highly atypical cells); and G4 represents undifferentiated (rarely seen, often classified as G3).

[0091] Grade data source: Grade information is reviewed and confirmed by pathologists at CPTAC partner hospitals and integrated into the metadata.

[0092] (3) Detection method of PKP1 protein / mRNA expression Technical platform: High-throughput mass spectrometry (LC-MS / MS), ELISA, immunohistochemistry, and PCR / qPCR. Quantitative principle: Peptide labeling: Use TMT (Tandem Mass Tag) isotope to label proteolytic peptides from different samples; Liquid chromatography separation: Mixed peptides are separated by liquid chromatography according to their physical and chemical properties; Mass spectrometry detection: Primary mass spectrometry measures peptide mass, secondary mass spectrometry measures fragmented peptides, and analyzes fragment ion sequences.

[0093] (4) Database matching: Compare fragment spectra with human protein databases to identify proteins (such as PKP1).

[0094] Expression calculation: Relative quantification: Based on the intensity of TMT reporter ions, calculate the relative abundance of PKP1 peptides in tumor vs. normal samples; Standardization: Data is batch corrected and globally normalized.

[0095] 5. Example of result interpretation (1) T vs N expression comparison: If the box plot shows that the median PKP1 expression in the tumor group is significantly higher than that in the normal group (p<0.01), it suggests that PKP1 may play a pro-cancer role in pancreatic cancer.

[0096] (2) Grade comparison: If the expression of PKP1 in group G3 is greater than that in group G1 (p<0.05), it indicates that high expression of PKP1 is associated with low differentiation (high malignancy) and may serve as a marker of poor prognosis.

[0097] By following the steps above, the protein expression profile and clinical relevance of PKP1 in pancreatic cancer can be quickly obtained without conducting experiments, providing crucial clues for subsequent research.

[0098] The result is as follows Figure 1 As shown in the figure, PKP1 mRNA is significantly highly expressed in pancreatic cancer (p<0.05). Figure 1 A), correspondingly, its protein level was also significantly elevated in pancreatic cancer (p<0.001) ( Figure 1 C). Furthermore, compared to the normal group, the protein expression level of PKP1 in Grade 1 showed no significant change, while Grade 2 (p<0.001) and Grade 3 (p<0.01) showed significant upregulation. Only one case of Grade 4 was observed, and it was not considered. Figure 1 D). Meanwhile, the survival rate of patients in the PKP1 high expression group was significantly lower than that of patients in the PKP1 low expression group (p<0.05). Figure 1 B).

[0099] Furthermore, the applicant also confirmed the expression of PKP1 in human pancreatic squamous cell carcinoma. Specifically, scRNA-seq data (GSM5032772) of human pancreatic squamous cell carcinoma were obtained from the GEO database and preprocessed using the "Seurat" software package in R to explore the expression of squamous cell carcinoma markers (TP63, KRT5, KRT6A, and KRT6B) and PKP1 in different cell subpopulations. Experimental results are shown below. Figure 2 : UMAP mapping of squamous cell carcinoma markers (TP63, KRT5, KRT6A and SERPIB) Figure 2 (BE) The study found that the cancer cell subset in the data contained almost only squamous cell carcinomas and no adenocarcinomas, while PKP1 was highly expressed in the cancer cell subset. Further, the applicant determined the relationship between PKP1 and squamous carcinoma markers. Specifically, the applicant obtained Bulk RNA-seq data of mouse pancreatic adenosquamous carcinoma (GSE18116) and Bulk RNA-seq data of human pancreatic adenosquamous carcinoma (GSE241266) from GEO database, and analyzed the correlation between PKP1 and squamous carcinoma markers mRNA levels by Pearson correlation. The total protein of various pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, Hs766T and PK-59) was collected, and the expression of PKP1 and p63 protein was detected by Western blot (determination results can be seen in Figure 4 ).

[0100] PKP1 is highly positively correlated with the mRNA expression level of the marker of pancreatic squamous carcinoma. In the GSE181166 data set, the Pearson correlation coefficient of PKP1 and TP63 mRNA expression level reached 0.99 (p<0.001), and the Pearson correlation coefficients of PKP1 and KRT5, KRT6A and KRT6B mRNA expression level reached 0.99 (p<0.001), 0.98 (p<0.001) and 0.88 (p<0.01) respectively Figure 4 A); similarly, in the GSE241226 data set, although the Pearson correlation coefficients of PKP1 and KRT6A and SERPINB3 mRNA expression level were low and not significant, the Pearson correlation coefficients of PKP1 and TP63 and KRT5 mRNA expression level were still very high, and the correlation coefficients were 0.98 (p<0.001) and 0.97 (p<0.001) respectively Figure 4 B). In addition, p63 is only expressed in BxPC-3 and Hs766T cells, so BxPC-3 and Hs766T cells may both have the potential of squamous metaplasia, or even be derived from pancreatic squamous carcinoma. Similarly, PKP1 is only expressed in these two cells Figure 4 C), and the expression trend is similar to that of p63, and the expression in BxPC-3 cells is higher than that in Hs766T cells.

[0101] Example 2. PKP1 knockdown and overexpression experiment of pancreatic cancer cell lines This example carried out PKP1 knockdown and overexpression experiment of pancreatic cancer cell lines, including the following steps: 1. Plating BxPC-3, AsPC-1 and Hs766T cells in exponential growth phase were collected, and the cells were resuspended with complete culture medium; the density of the cell suspension was adjusted to 5 x 105 cells / mL, 1 mL of the adjusted cell suspension was added to each well of a 6-well plate, and 1 mL of fresh complete culture medium was supplemented; and the plate was cultured in an incubator overnight.

[0102] 2. Transfection The siRNA (siRNA-NC (siCON) and siRNA-PKP1 (siPKP1-1, siPKP1-2 and siPKP1-3)) and plasmid (pcDNA3.1 (oeCON) and pcPKP1 (oePKP1)) were taken out from a -20 °C refrigerator, thawed at room temperature and placed in a clean bench; 4 sterile 1.5 mL EP tubes were taken, 6 μL of PEI solution was added to each tube, and then 5 μL of siRNA-NC, siRNA-PKP1, pcDNA3.1 and pcPKP1 was added respectively; 200 μL of PBS was added to each tube, mixed and stood for 15 min for standby; a 6-well plate was taken out, the cell waste liquid was discarded, and 1 mL of PBS was added to each well; the cells were washed once and the PBS was discarded; 1 mL of basal medium was added to each well, and the mixed transfection reagent was added to the corresponding wells, mixed and cultured in a cell incubator for 6 h; the 6-well plate was taken out, the waste liquid was discarded, 2 mL of complete culture medium was added to each well, and the plate was cultured in a cell incubator for 48 h; the 6-well plate was taken out, the waste liquid was discarded; 1 mL of PBS was added to each well, the cells were washed once and the PBS was discarded, and the washing was repeated twice.

[0103] 3. Collection of total cell protein.

[0104] The experimental results are shown in Figure 5 and Figure 6 In BxPC-3 cells, siPKP1-1, siPKP1-2 and siPKP1-3 reduced the protein expression of PKP1 with the same knockdown efficiency (A), and siPKP1-1 was selected as a representative siPKP1 for subsequent experiments. In BxPC-3 and Hs766T cells, the protein expression of PKP1 was significantly inhibited by siPKP1 (B); similarly, in AsPC-1 and Hs766T cells, the protein expression of PKP1 was significantly overexpressed by oePKP1 (B). In addition, in BxPC-3 and Hs766T cells, the expression level of p63 was significantly inhibited by PKP1 (C). Figure 5 A), and siPKP1-1 was selected as a representative siPKP1 for subsequent experiments. In BxPC-3 and Hs766T cells, the protein expression of PKP1 was significantly inhibited by siPKP1 (B); similarly, in AsPC-1 and Hs766T cells, the protein expression of PKP1 was significantly overexpressed by oePKP1 (B). In addition, in BxPC-3 and Hs766T cells, the expression level of p63 was significantly inhibited by PKP1 (C). Figure 5 B). Figure 6 .

[0105] Example 3. Effect of PKP1 overexpression on the proliferation of pancreatic cancer cell lines This embodiment determined the effect of PKP1 overexpression on the proliferation of pancreatic cancer cell lines by measuring BxPC-3 and Hs766T cells. The specific steps are as follows: 1. Plating: Collect cells in the exponential growth phase, resuspend the cells in complete culture medium, and adjust the cell density to 1×10⁻⁶. 5 cells / mL; seed 100 μL of the adjusted cell suspension into each well of a 96-well plate; incubate overnight in an incubator.

[0106] 2. Transfection: Transfect cells according to the method in Example 4, wherein only 7 μL of the prepared transfection reagent is needed per well.

[0107] 3. CCK-8 assay: Discard the waste liquid in the 96-well plate, add 110 μL of the prepared CCK-8 working reagent to each well, incubate in an incubator for 2 h, and then measure the OD value at 450 nm wavelength using a microplate reader; count from the time of adding the transfection reagent, detect at 7 time points (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h).

[0108] 4. Data processing: Calculate the mean and standard deviation of OD for each group and plot the growth curve.

[0109] Experimental results are as follows Figure 7 As shown, compared with the siCON group, the proliferation rate of BxPC-3 and Hs766T cells was significantly reduced after PKP1 knockdown; for the PKP1 overexpressing cell lines, the proliferation rate of AsPC-1 and Hs766T cells was significantly increased compared with the oeCON group.

[0110] Example 4. Mouse pancreatic cancer xenograft experiment The effect of PKP1 expression on mouse pancreatic cancer xenografts was determined by knocking down PKP1 in BxPC-3 cells. The specific steps are as follows: 1. Cell preparation: Seed cells in 10 cm dishes; Transfection was performed as described in Example 4, using 6 times the amount of transfection reagent as in a 6-well plate; trypsin digestion and cell pellet collection was performed; cells were resuspended in 1 mL PBS, centrifuged again at 1000 rpm for 5 min, and washed. The cell density was then adjusted to 1×10⁷ cells / mL; one-third volume of the cell suspension was added to the matrix gel, mixed well, and aliquoted into sterile 1.5 mL EP tubes, 100 μL per tube, and stored on ice for later use.

[0111] 2. Animal grouping and cell inoculation: Nude mice or C57BL / 6 mice were divided into experimental and control groups; mice were anesthetized by intraperitoneal injection of afodin (15 μL / g); the skin on the right side of the back was grasped with forceps, and 100 μL of cell suspension (1×10⁻⁶) was injected into each mouse. 6For subcutaneous tumor, the tumor volume was measured every 3 days (the length of the longest diameter and the shortest diameter of the tumor were measured, and the tumor volume = the longest diameter x the shortest diameter2x 0.52). For orthotopic tumor, the pancreas was exposed by laparotomy, and the cells were injected into the tail of the pancreas.

[0112] 3. Tumor monitoring: The tumor volume was measured every 3 days (the length of the longest diameter and the shortest diameter of the tumor were measured, and the tumor volume = the longest diameter x the shortest diameter2x 0.52).

[0113] 4. Endpoint processing: When the maximum tumor volume reached 1000 mm3, the mice were decapitated, and the tumor tissue was stripped and weighed; the tumor tissue was fixed with 4% paraformaldehyde for subsequent experiments.

[0114] The experimental results are shown in Figure 8 and Figure 9 After knocking down PKP1 in BxPC-3 cells, the tumor growth rate was significantly reduced compared with the siCON group. By the 42nd day, the tumor volume of the siPKP1 group was only 34.52% of the siCON group (p < 0.05), and the weight was only 49.83% (p < 0.05) (Fig. 1A). Figure 8 After overexpressing PKP1 in Panc02 cells, the tumor growth rate was significantly increased compared with the oeCON group. By the 18th day, the tumor volume of the oePKP1 group was 1.98 times that of the oeCON group (p < 0.001), and the weight was 2.17 times (p < 0.001) (Fig. 1B). Figure 9 The results of pancreatic orthotopic tumor were consistent with those of Fig. 1A. The tumor volume of the oePKP1 group was significantly larger than that of the oeCON group, and the weight was 1.44 times that of the oeCON group (p < 0.05) (Fig. 1C). Figure 9

[0115] Example 5. Invasion and migration experiment of pancreatic cancer cell line By knocking down the expression of PKP1 in BxPC-3 and Hs766T cells, the effects on the number of invasive cells and the cell scratch closure rate were determined, and the specific steps are as follows: 1. Invasion experiment: (1) Dilute the Matrigel and the basal medium at a ratio of 1:8 for standby; add 60 μL of Matrigel solution to each well of the upper chamber of Transwewll; place Transwewll in the cell culture incubator for 3 h; discard the excess liquid in the upper chamber of Transwewll, and add 100 μL of basal medium to each well, and then place it back in the incubator for 30 min; discard the excess liquid in the upper chamber of Transwewll for standby.

[0116] ​(2) Cells were plated and transfected, then collected; the cell density was adjusted to 2.5 x 105 cells / mL with basal medium, 200 μL of the cell suspension was added to each well in the upper chamber of the Transwell; 500 μL of complete medium was added to each well in the lower chamber of the Transwell; and the cells were incubated in an incubator for 24 h.

[0117] (3) The upper chamber of the Transwell was removed, the medium was discarded, and the cells inside the upper chamber were gently wiped off with a cotton swab; a new 24-well plate was taken, 600 μL of 4% paraformaldehyde was added to each well, and then the upper chamber was put back into the well to fix for 30 min; the fixing solution was discarded, 0.1% crystal violet was added to the lower chamber, the upper chamber was put back in, and the cells were stained for 10 min, and then washed with PBS for 3 times, 5 min each time.

[0118] (4) The cells were photographed under a microscope and saved.

[0119] 2. Migration experiment: (1) Cells were plated and transfected.

[0120] (2) After 48 h of transfection, a yellow tip (200 μL) was used to make a scratch; the cells were rinsed with PBS twice to remove the floating cells; the PBS was discarded, and basal medium was added.

[0121] (3) The scratch healing at 0 h, 6 h, 12 h, 24 h, and 48 h was photographed and recorded, and the changes in the scratch width between different groups were compared.

[0122] The experimental results are shown in Figure 10 and Figure 11 , in the invasion experiment Figure 10 , after knocking down PKP1 in BxPC-3 and Hs766T cells, the number of invaded cells decreased by 88.45% (p<0.001) and 90.48% (p<0.001), respectively; while after overexpressing PKP1 in AsPC-1 and Hs766T cells, the number of invaded cells increased by 9.24 times (p<0.01) and 6.63 times (p<0.01), respectively. In the migration experiment Figure 11 , after knocking down PKP1 in BxPC-3 and Hs766T cells, the scratch closure rate of the cells decreased significantly, in which the scratch closure rate of BxPC-3 cells decreased by 38.28% (p<0.05), and that of Hs766T cells decreased by 89.65% (p<0.001); after overexpressing PKP1 in AsPC-1 and Hs766T cells, the scratch closure rate of the cells increased significantly, in which the scratch closure rate of AsPC-1 cells increased by about 1.83 times (p<0.01), and that of Hs766T cells increased by about 1.48 times (p<0.05).

[0123] Example 6. Pancreatic cancer cell line invasion and migration experiment This example determines the effect of PKP1 overexpression on pancreatic cancer cell line invasion and migration by post-transplantation determination of Panc02 cells, and the specific experimental steps are as follows: 1. Cell preparation: The same as in Example 4, but instead of adding Matrigel, an equal volume of PBS is used.

[0124] 2. Animal grouping and cell inoculation: C57BL / 6 mice were divided into experimental and control groups; the mice were anesthetized by intraperitoneal injection of atropine (15 μL / g); the mouse liver was exposed by laparotomy; the submesenteric vein was found by following the hepatic portal vein, and the tumor cells were injected into it.

[0125] 3. End point treatment: After 3 weeks, the mice were sacrificed by decapitation, the body weight of the mice was recorded, and then the laparotomy was performed, the liver tissue was peeled off and weighed, and photographed; the liver tissue was fixed with 4% paraformaldehyde for subsequent experiments.

[0126] The experimental results are shown in Figure 14 After 21 days of injection of Panc02 cells into the superior mesenteric vein, the mice were sacrificed and the livers were removed, and it was found that there were more metastatic foci of Panc02 cells on the liver of the mice in the PKP1 overexpression group, and the tumor area was also significantly larger than that in the control group. Subsequently, the liver weight ratio of the mice in the oeCON and oePKP1 groups was calculated by weighing the body weight and liver tumor of the mice, and the results showed that the liver weight ratio of the oePKP1 group was significantly greater than that of the oeCON group (p<0.01).

[0127] Example 7. Exploration of PKP1 expression in human other non-squamous carcinomas and other organ adenosquamous carcinomas The applicant also explored the expression of PKP1 in human other non-squamous carcinomas and other organ adenosquamous carcinomas through the "Expression DIY" module in the GEPIA database (http: / / gepia.cancer-pku.cn / ).

[0128] 1. The mRNA expression levels of PKP1 in other non-squamous carcinoma tissues (T) and adjacent and normal tissues (N) were compared (Fig. 1): Figure 13 ): The mRNA expression of PKP1 in other non-squamous carcinomas (rectal adenocarcinoma, gastric adenocarcinoma, thyroid carcinoma, prostate cancer, lung adenocarcinoma, hepatocellular carcinoma, colon adenocarcinoma, and cholangiocarcinoma) showed no significant change (p>0.05) Figure 13 ).

[0129] 2. The expression of PKP1 in human other organ adenosquamous carcinomas (e.g., its relationship with survival and prognosis) was explored The expression levels of PKP1 mRNA in adenosquamous carcinoma (T) tissues, adjacent normal pancreatic tissues, and normal tissues were compared using the "Expression DIY" module in the GEPIA database (http: / / gepia.cancer-pku.cn / ). The association between PKP1 gene expression levels and overall survival in patients with adenosquamous carcinoma of other organs was studied using the "Survival Plots" module.

[0130] PKP1 mRNA was significantly overexpressed in adenosquamous carcinomas of other organs (thymoma, urothelial carcinoma of the bladder, squamous cell carcinoma of the lung, squamous cell carcinoma of the cervix, endometrial adenocarcinoma, and esophageal cancer) (p<0.05). Figure 14 PKP1 (acrylamide esterase) is elevated in cutaneous melanoma, and the survival rate of patients in the high PKP1 expression group is significantly lower than that of patients in the low PKP1 expression group (p<0.05). Figure 14 F).

[0131] Example 7: Utilizing other PKP1 inhibitors (antibodies) to influence adenosquamous carcinoma (predictive). Preparation of PKP1 antibodies (using conventional methods in the art; human antibodies can be prepared by a variety of methods known in the art, including the phage display method using antibody libraries derived from human immunoglobulin sequences described above (see U.S. Patents 4,444,887 and 4,716,111, and PCT Publications WO 98 / 46645, WO 98 / 60433, WO98 / 24893, WO 98 / 16664, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, the contents of each of which are incorporated herein by reference in their entirety). Alternatively, techniques for preparing human monoclonal antibodies can be used, such as those described in Cole et al., "Monoclonal Antibodies and Cancer Therapy" (Alan). R. Riss, (1985) and Boerner et al. (J. Immunol., 147(1):86-95, (1991)). Treatment of subjects with relevant diseases (e.g., adenocarcinoma) by administration (e.g., subcutaneous injection).

[0132] Example 8: Influence of other PKP1 inhibitors (CRISPR-Cas compositions) on adenosquamous carcinoma (predictive) The gRNA target sequence was determined by scanning the PKP1 gene (and taking into account the PAM of CRISPR-Cas, such as the 5'-NGG PAM of Cas9).

[0133] According to the gRNA sequence structure of CRISPR-Cas, a gRNA is designed, for example, when CRISPR-Cas9 is used, the gRNA includes a guide sequence complementary to the target sequence of the VEGFR2 gene, and a tracr domain including a tracr sequence and a tracr mate sequence.

[0134] The CRISPR-Cas composition is prepared, for example, the Cas protein and the gRNA are prepared, and the transfection is carried out by means of electroporation; or the Cas protein mRNA and the gRNA are prepared, and are delivered to the cells of the subject by LNP delivery or the like; or are delivered by a viral vector, so as to treat the disease related to the subject (for example, adenocarcinoma).

[0135] The sequence information related to the present disclosure is as follows: SEQ ID NO: 1, PKP1 mRNA sequence SEQ ID NO: 2, Protein sequence expressed by the PKP1 mRNA sequence MNHSPLKTALAYECFQDQDNSTLALPSDQKMKTGTSGRQRVQEQVMMTVKRQKSKSSQSSTLSHSNRGSMYDGLADNYNYGTTSRSSYYSKFQAGNGSWGYPIYNGTLKREPDNRRFSSYSQMENWSRHYPRGSCNTTGAGSDICFMQKIKASRSEPDLYCDPRGTLRKGTLGSKGQKTTQNRYSFYSTCSGQKAIKKCPVRPPSCASKQDPVYIPPISCNKDLSFGHSRASSKICSEDIECSGLTIPKAVQYLSSQDEKYQAIGAYYIQHTCFQDESAKQQVYQLGGICKLVDLLRSPNQNVQQAAAGALRNLVFRSTTNKLETRRQNGIREAVSLLRRTGNAEIQKQLTGLLWNLSSTDELKEELIADALPVLADRVIIPFSGWCDGNSNMSREVVDPEVFFNATGCLRNLSSADAGRQTMRNYSGLIDSLMAYVQNCVAASRCDDKSVENCMCVLHNLSYRLDAEVPTRYRQLEYNARNAYTEKSSTGCFSNKSDKMMNNNYDCPLPEEETNPKGSGWLYHSDAIRTYLNLMGKSKKDATLEACAGALQNLTASKGLMSSGMSQLIGLKEKGLPQIARLLQSGNSDVVRSGASLLSNMSRHPLLHRVMGNQVFPEVTRLLTSHTGNTSNSEDILSSACYTVRNLMASQPQLAKQYFSSSMLNNIINLCRSSASPKAAEAARLLLSDMWSSKELQGVLRQQGFDRNMLGTLAGANSLRNFTSRFSEQ ID NO: 3, siPKP1-1 sense strand GCAACUCCGAAGACAUCUUTT SEQ ID NO: 4, siPKP1-1 antisense strand AAGAUGUCUUCGGAGUUGCTT SEQ ID NO: 5, siPKP1-2 sense strand GGAUGAAUCUGCCAAGCAATT SEQ ID NO: 6, siPKP1-2 antisense strand UUGCUUGGCAGAUUCAUCCTT SEQ ID NO: 7, siPKP1-3 sense strand UUGUUCAUCAUCUUGUGAACAATT SEQ ID NO: 8, siPKP1-3 antisense strand GCGACAAGAUGAUGAACAATT The above describes some embodiments of the present disclosure in detail, but the present disclosure is not limited to the specific details in the above-described embodiments. Within the technical concept range of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection range of the present disclosure.

[0136] In addition, it should be noted that the various specific technical features described in the above-described some embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, various possible combination manners are not described again in the present disclosure.

[0137] In addition, various different embodiments of the present disclosure can also be combined in any manner as long as it does not deviate from the idea of the present disclosure, and it should also be considered as disclosed by the present disclosure.

Claims

1. An siRNA reagent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides having a nucleotide sequence of at least 3 nucleotides similar to any one of SEQ ID NO: 3, 5, 7, and the antisense strand comprises at least 15 consecutive nucleotides having a nucleotide sequence of at least 3 nucleotides similar to any one of SEQ ID NO: 4, 6, 8.

2. The siRNA reagent of claim 1, wherein the sense strand and antisense strand of the siRNA contain at least one nucleotide mismatch (e.g., one or more).

3. The siRNA reagent as described in claim 2, wherein the sense strand of the siRNA reagent is selected from any one of SEQ ID NO: 3, 5, and 7; and the antisense strand is selected from any one of SEQ ID NO: 4, 6, and 8; Optionally, the sense strand and antisense strand of the siRNA are as shown in SEQ ID NO:3, 4, SEQ ID NO:5, 6, or SEQ ID NO:7, 8, respectively.

4. The siRNA reagent of claim 3, wherein the sense strand or antisense strand of the siRNA contains at least one modification.

5. A specific PKP1 antibody that can bind to the active binding domain of the PKP1 protein, thereby effectively inhibiting PKP1 expression within a safe dose range.

6. A CRISPR-Cas composition for targeting and editing the PKP1 gene. In some embodiments, the CRISPR-Cas composition comprises a gRNA targeting the PKP1 gene, the gRNA comprising a polynucleotide sequence complementary to a targeting sequence of the PKP1 gene, optionally, the gRNA targeting sequence being selected from the regulatory sequence of the PKP1 gene or its coding sequence.

7. A pharmaceutical composition capable of preventing or treating squamous cell carcinoma of adenocarcinoma (e.g., pancreatic cancer), comprising siRNA, antibody, or CRISPR-Cas composition as described in any one of claims 1-6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the weight ratio of the siRNA reagent to the pharmaceutically acceptable carrier is 1:(1-500); optionally, the weight ratio of the siRNA, antibody, and gRNA to the pharmaceutically acceptable carrier is 1:(1-50); optionally, the pharmaceutically acceptable carrier contains organic amines, auxiliary lipids, and polyethylene glycol-modified lipids.

9. A method for diagnosing, preventing, or treating squamous cell carcinoma of (pancreatic) adenocarcinoma, the method comprising administering an effective amount of any one of claims 1-3 siRNA, antibody and gRNA reagent, or the pharmaceutical composition of claim 7 or 8 to a subject suffering from pancreatic cancer.

10. A method for inhibiting PKP1 expression in cells, the method comprising contacting the adenocarcinoma squamous cell carcinoma cells with an effective dose of siRNA, antibody and gRNA reagent of any one of claims 1-3, and / or pharmaceutical composition of claim 7 or 8.

11. A cell comprising the siRNA, antibody, and gRNA reagent of any one of claims 1-3, and / or the pharmaceutical composition of claim 7 or 8.

12. A vector comprising the siRNA, antibody, and gRNA reagent as described in any one of claims 1-3.

13. Application of PKP1 as a marker for adenocarcinoma or squamous cell carcinoma or as a tumor target molecule.

14. The application as described in claim 11, using PKP1 gene expression as a predictive marker for tumor immune escape and poor prognosis; Optionally, PKP1 gene expression can be determined by measuring the expression of DNA, mRNA, or protein.

15. As described in claim 11, the expression of PKP1 in tumor tissues and body fluids (e.g., blood or pancreatic juice) is positively correlated with the expression of P63, a marker of squamous metaplasia, and our study found that the expression of PKP1 can positively regulate the expression of P63. The increase in PKP1 expression in squamous metaplasia tissues occurs earlier than that of P63. The increased expression of PKP1 in adenocarcinoma tissues can serve as a diagnostic and predictive marker for squamous metaplasia of adenocarcinoma.

16. The application of claim 11 or 12, wherein the application includes: (1) As a diagnostic marker for squamous cell carcinoma of adenocarcinoma, or for the preparation of a kit for the diagnosis of squamous cell carcinoma of adenocarcinoma; (2) As a prognostic marker for squamous cell carcinoma of adenocarcinoma, or for the preparation of a kit for prognostic assessment of squamous cell carcinoma of adenocarcinoma; (3) Kits for preparing efficacy evaluation indicators and efficacy monitoring of drugs for the treatment of adenocarcinoma squamous cell carcinoma; (4) Used to prepare drug components for treating squamous cell carcinoma of adenocarcinoma.

17. A PKP1 inhibitor comprising a PKP1 antibody or a CRISPR-Cas composition that targets and edits the PKP1 gene; Optionally, the CRISPR-Cas composition includes a gRNA targeting the PKP1 gene, the gRNA comprising a polynucleotide sequence complementary to the targeting sequence of the PKP1 gene; Optionally, the gRNA targeting sequence is selected from the regulatory sequence of the PKP1 gene or its coding sequence.

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