Application of selenoprotein P as target spot in tumor diagnosis and treatment

Abstract

The invention belongs to the technical field of medical biological detection, and provides application of selenoprotein P as a target spot in tumor diagnosis and treatment. The invention specifically provides application of a reagent for detecting the expression level of selenoprotein P in preparation of a tumor diagnosis kit, provides a corresponding detection kit, and further provides application of the reagent as a target spot in preparation of tumor treatment drugs and a corresponding pharmaceutical composition. The invention discloses a key function of the Selenop in interaction of the RTMs and the tumor cells, and the key function is specifically shown as that the Selenop is transferred from the RTMs to the tumor cells, lipid oxidation is inhibited, and the tumor cells are protected from ferroptosis. Based on the discovery, the invention provides an application prospect of Selenop as a tumor treatment target, and provides a potential strategy and method for tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological detection and biological medicine, and relates to the application of selenoprotein P as a biological diagnostic marker and a target point, and provides the application of selenoprotein P as a target point in diagnosis and treatment of tumors. BACKGROUND

[0002] Macrophages play an important role in the tumor microenvironment (TME), and their functional heterogeneity is closely related to tumor progression. Resident tissue macrophages (RTMs) and monocyte-derived macrophages (MDMs) play complex and multifaceted roles in the tumor microenvironment, and both affect tumor occurrence, progression, immune escape and treatment response through different mechanisms. The functional differences of the two in tumors have not been fully elucidated. The research group recently found that RTMs are specifically enriched at the invasion front of pancreatic ductal adenocarcinoma, but the molecular mechanism of their regulation of tumor cell fate is still unclear.

[0003] Selenoprotein P (Selenop) is a selenium (Se)-rich secreted glycoprotein that is the main form of selenium transport and storage in the human body, and also has antioxidant and signal regulation functions. It plays a key role in various physiological and pathological processes, including tumor occurrence, metabolic regulation and immune response. Current research on Selenop mainly focuses on the following areas: (1) selenium metabolism regulation: as the main transport protein of selenium, it is responsible for transporting selenium synthesized by liver cells to organs with high selenium demand such as testes and brain, and synthesizing active proteins containing selenium selenocysteine through the Sec-tRNA[Ser]Sec-mediated UGA codon recoding mechanism; (2) antioxidant defense: maintaining cellular redox balance through thioredoxin reductase-like domain when GPX4 is deficient, and resisting endoplasmic reticulum stress in cooperation with the glutathione system; (3) immune regulation: regulating Th1 / Th2 balance: Selenop deficiency leads to reduced IFN-γ secretion and increased IL-4 levels, and mediates dendritic cell maturation through the LRP8 receptor; (4) disease association: neurodegenerative diseases: cerebrospinal fluid Selenop levels are negatively correlated with Alzheimer's disease Aβ plaque deposition; cardiovascular diseases: endothelial cell Selenop regulates vascular tone through the NO pathway; tumor dual role: shows tumor suppressor function in prostate cancer (through p53 activation), but promotes chemotherapy resistance in colorectal cancer.

[0004] The expression of Selenop in monocyte-derived macrophages (MDMs) is inhibited by the IL-4 / STAT6 pathway, and the high expression of Selenop in RTMs may be related to tissue-specific epigenetic modifications (such as H3K27ac modification), and existing research has not revealed the intercellular transfer mechanism of Selenop and its regulation of ferroptosis. SUMMARY

[0005] The present application is based on the above research, and provides the application of selenoprotein P as a target in the diagnosis and treatment of tumors. Specifically, it relates to the function of selenoprotein P (Selenop) in the interaction between resident tissue macrophages (RTMs) and tumor cells and its application in tumor treatment. The present application first found that Selenop is significantly negatively correlated with the degree of epithelial-mesenchymal transition (EMT) of pancreatic tumor cells, and tumor cells with high EMT degree have low expression of Selenop; and it is proved that RTMs inhibit lipid oxidation by high expression of Selenop and transfer to tumor cells with high EMT degree to protect tumor cells from ferroptosis, thereby promoting tumor EMT.

[0006] Specifically, the present application first proves that Selenop is significantly highly expressed in pancreatic cancer RTM through single-cell sequencing analysis, and reveals that the level of Selenop in pancreatic cancer cells is significantly negatively correlated with EMT and ferroptosis pathway at the preclinical and clinical levels; it is then verified that the selenium content in the pancreatic cancer region of mice and humans is significantly lower than that in the pericancer pancreatic tissue, indicating that the pancreatic cancer tissue is in a long-term low selenium level, and the selenium against cell lipid peroxidation may need to be obtained from the outside. Further animal experiments found that pancreatic cancer mice with Selenop gene defects in myeloid cells have longer survival time and better prognosis than wild-type mice, suggesting that Selenop in myeloid cells plays a role in promoting tumor EMT, and it is confirmed that Selenop protein can be transferred from macrophages to tumor cells to regulate tumor cell ferroptosis and EMT. Therefore, Selenop is a biomarker for the diagnosis of pancreatic cancer and a therapeutic target.

[0007] Specifically, the present application provides the following technical solutions:

[0008] In a first aspect of the present application, the application of selenoprotein P as a diagnostic marker is provided. Specifically, the application of a reagent for detecting the expression level of selenoprotein P in the preparation of a tumor diagnosis kit is provided.

[0009] Preferably, the tumor is a tumor in which the expression level of selenoprotein P is regulated by resident tissue macrophages (RTMs). The present application studies pancreatic cancer in the specific embodiment part, and other tumors that may be related to it, such as brain tumors (brain glioma), liver cancer, prostate cancer, colorectal cancer, lung cancer, breast cancer, bladder cancer, etc., are also within the scope of protection.

[0010] Preferably, the reagent for detecting selenoprotein P is a reagent for detecting the expression amount of selenoprotein P in a biological sample at the gene or protein expression level; the kit contains the reagent for detecting the expression amount of selenoprotein P in the biological sample.

[0011] Further optimization involves selecting reagents for detecting selenoprotein P in biological samples from one or more of the following detection techniques or methods: in situ hybridization, Northern blot, PCR, or immunohistochemistry.

[0012] Further preferred, the reagent for detecting the expression level of selenoprotein P in biological samples includes PCR primers with detection specificity for the selenoprotein P gene. The PCR primers with detection specificity for the selenoprotein P gene are shown in SEQ ID NO. 1-4 below.

[0013] In a second aspect, the present invention provides a product comprising the aforementioned substance for detecting selenoprotein P, the product having any one or more of the following uses: diagnosis or auxiliary diagnosis of tumors (such as pancreatic cancer); prognostic assessment or auxiliary prognostic assessment of tumors.

[0014] The product can be selected as a kit for detecting the expression level of selenoprotein P in biological samples at the gene or protein level. Preferably, it is a kit for detection at the gene level, consisting of a reverse transcription system, a primer system, and an amplification system. The primer system includes PCR primers as shown in SEQ ID NO. 1–4.

[0015] Selenin P-F1 primer: 5'-AGCTCTGCTTGTTACAAAGCC-3' (SEQ ID NO.1);

[0016] Selenin P-R1 primer: 5'-CAGGTCTTCCAATCTGGATGC-3' (SEQ ID NO.2);

[0017] Selenin P-F2 primer: 5'-CTTGTGTCTTCTGCAGGCAT-3' (SEQ ID NO.3);

[0018] Selenin P-R2 primer: 5'-GAGAGTCCAGACATCTATGCC-3' (SEQ ID NO.4).

[0019] Furthermore, the biological sample is selected from either tumor tissue obtained by puncture or circulating tumor cells collected from the patient's blood. Early diagnosis and prognostic assessment are achieved by collecting and detecting the level of selenoprotein P in tumor tissue or cells.

[0020] In the specific embodiments section of this invention, pancreatic cancer samples are used as the research object. Therefore, in practice, by collecting and detecting the level of selenoprotein P in pancreatic cancer tumor tissue or cells, early diagnosis and prognostic assessment of pancreatic cancer can be achieved.

[0021] A third aspect of the invention provides the application of selenoprotein P as a therapeutic target. Specifically, it provides the application of inhibiting or silencing selenoprotein P in the preparation of pancreatic cancer drugs.

[0022] The substance that inhibits or silences selenoprotein P is selected from any of the following:

[0023] (1) RNA interference molecules or antisense oligonucleotides that inhibit the expression of selenoprotein P, or recombinant expression vectors or transgenic cell lines containing them;

[0024] (2) Small molecule inhibitors;

[0025] (3) Substances that are used for lentiviral vector infection or gene knockout.

[0026] Preferably, the nucleic acid sequence corresponding to the antisense oligonucleotide is selected from any one or more combinations of nucleic acid sequences in Table 1 below:

[0027] Table 1 Antisense Oligonucleotide Sequences

[0028]

[0029]

[0030] A fourth aspect of the present invention provides a pharmaceutical composition for treating tumors, preferably a pharmaceutical composition for treating pancreatic cancer, comprising an active component and a pharmaceutically acceptable carrier, said active component being a substance that inhibits or silences selenoprotein P, selected from any of the following:

[0031] (1) RNA interference molecules or antisense oligonucleotides that inhibit the expression of selenoprotein P, or recombinant expression vectors or transgenic cell lines containing them;

[0032] (2) Small molecule inhibitors;

[0033] (3) Substances that are used for lentiviral vector infection or gene knockout.

[0034] The selection of nucleic acid sequences for RNA interference molecules or antisense oligonucleotides is shown in Table 1 above.

[0035] A fifth aspect of the present invention provides a product comprising at least one of the second, third, and fourth aspects. The product has at least one function of (1)-(3):

[0036] (1) Diagnosis and / or treatment of tumors such as pancreatic cancer in which tissue-resident macrophage RTMs regulate the expression level of selenoprotein P;

[0037] (2) Promotes lipid oxidation and ferroptosis in tumor cells;

[0038] (3) Inhibition of tumor cell EMT;

[0039] (4) Blocking the protective effect of RTMs on tumor cells.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] This invention is the first to discover a significant negative correlation between Selenop and the degree of epithelial-mesenchymal transition (EMT) in pancreatic tumor cells, with tumor cells exhibiting high EMT levels expressing low levels of Selenop. Furthermore, it demonstrates that RTMs promote tumor EMT by highly expressing Selenop and translocating into tumor cells with high EMT levels, thereby inhibiting lipid oxidation and protecting tumor cells from ferroptosis. This provides a novel mechanism and solution for pancreatic cancer research, possessing significant clinical and social benefits. Attached Figure Description

[0042] Figure 1 The results of single-cell sequencing analysis showed that Selenop was significantly overexpressed in pancreatic cancer RTMs: (A) secreted genes that were expressed more in RTMs than in MDMs and tumor cells, and (2) the relative expression levels of Selenop in pancreatic cancer RTMs and MDMs subsets.

[0043] Figure 2 The results showed that the expression level of SELENOP in pancreatic cancer cells was significantly negatively correlated with the EMT and ferroptosis pathway scores of tumor cells: where A is the single-cell sequencing result of mouse pancreatic cancer samples; and B is the single-cell sequencing result of human pancreatic cancer samples.

[0044] Figure 3 This demonstrates the use of laser ablation inductively coupled plasma triple quadrupole mass spectrometry to analyze pancreatic tissue from KPC mice, a preclinical model of spontaneous pancreatic cancer. Figure 4 A) Compared with clinical pancreatic cancer samples ( Figure 4 B) Pathological sections were subjected to spatial selenium imaging and quantitative analysis.

[0045] Figure 4 The study showed that targeted intervention with Selenop significantly prolonged the survival of pancreatic cancer animal models and inhibited the development of pancreatic cancer EMT: (A) shorter survival of pancreatic cancer mice with Selenop gene deficiency in myeloid cells compared with wild-type mice, and (B) EMT levels in tumor cells.

[0046] Figure 5 A schematic diagram illustrating the functional mechanism by which Selenop, derived from RTMs, regulates ferroptosis in tumor cells.

[0047] Figure 6The study demonstrated the impact of orthotopic implantation of pancreatic cancer cells into the pancreas on pancreatic tumors. Specifically, (A) the construction of RTM-specific Selenop conditional knockout mice (Mrc1) was shown. CreERT2 Selenop fl / fl The model was established by in situ implantation of pancreatic cancer cells into the pancreas. Immunofluorescence imaging analysis was used to evaluate the effect on pancreatic tumors. (B) In the co-culture system of macrophages and pancreatic cancer cells, macrophages were labeled with selenium-74 isotope, and the selenium-74 content in tumor cells was quantitatively detected. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0049] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0050] The present invention mainly focuses on the following aspects:

[0051] (1) At the preclinical and clinical levels, it was revealed that the level of Selenop in pancreatic cancer cells was significantly negatively correlated with EMT and ferroptosis pathway;

[0052] (2) Design Selenop-specific qPCR primer sequences for pancreatic cancer diagnosis;

[0053] (3) The development of space selenium element imaging and pathological diagnostic image integration analysis technology revealed that the selenium content in pancreatic cancer is significantly lower than that in pancreatic tissue;

[0054] (4) Develop and construct an RTM-specific Selenop gene knockout mouse model, Mrc1CreERT2Selenopfl / fl, and demonstrate in a preclinical pancreatic cancer animal model that targeted intervention with Selenop can significantly prolong the survival of pancreatic cancer animal models and inhibit EMT in pancreatic cancer.

[0055] (5) Develop an in vitro co-culture system combined with selenium isotope tracing to demonstrate that RTM protects tumor cells undergoing EMT from ferroptosis by delivering Selenop protein to tumor cells.

[0056] The specific steps are as follows:

[0057] 1. Single-cell sequencing analysis demonstrated that Selenop was significantly highly expressed in pancreatic cancer RTM.

[0058] Transcriptome differences among RTMs, MDMs, and tumor cells were analyzed using a mononuclear cell lineage tracing model, transcriptome sequencing, and single-cell sequencing. Secretory genes with higher expression in RTMs than in MDMs and tumor cells were screened, revealing that Selenop was specifically highly expressed in RTMs. Figure 1 ).

[0059] 2. Both preclinical and clinical studies revealed a significant negative correlation between Selenop levels in pancreatic cancer cells and EMT and ferroptosis pathways. Single-cell sequencing of preclinical pancreatic cancer models (mice) and clinical pancreatic cancer samples showed that the expression level of Selenop mRNA in tumor epithelial cells was significantly negatively correlated with the levels of EMT and ferroptosis pathways in the tumor. Figure 2 The results suggest that Selenop, which exerts antioxidant effects, inhibits cellular lipid oxidation, resists tumor ferroptosis, and promotes EMT. Selenop is a biomarker reflecting the sensitivity of clinical pancreatic cancer to EMT and ferroptosis.

[0060] 3. Design of Selenop-specific qPCR primer sequences for pancreatic cancer diagnosis.

[0061] Based on the above results, we developed and validated specific qPCR primer sequences for the detection of Selenop levels, which can be used to detect Selenop mRNA levels in pancreatic cancer samples as a biomarker for pancreatic cancer diagnosis and prognostic assessment.

[0062] Selenin P-F1 primer: 5'-AGCTCTGCTTGTTACAAAGCC-3' (SEQ ID NO.1);

[0063] Selenin P-R1 primer: 5'-CAGGTCTTCCAATCTGGATGC-3' (SEQ ID NO.2);

[0064] Selenin P-F2 primer: 5'-CTTGTGTCTTCTGCAGGCAT-3' (SEQ ID NO.3);

[0065] Selenin P-R2 primer: 5'-GAGAGTCCAGACATCTATGCC-3' (SEQ ID NO.4).

[0066] 4. Development of space-based selenium imaging and pathological diagnostic image integration analysis technology revealed that the selenium content in pancreatic cancer is significantly lower than that in pancreatic tissue.

[0067] Selenop, as a major selenium transporter, was analyzed at the elemental level using laser ablation inductively coupled plasma triple quadrupole mass spectrometry (LASIK) to detect its content in pancreatic tissue from KPC mice, a preclinical model of spontaneous pancreatic cancer. Figure 3 A) Compared with clinical pancreatic cancer samples ( Figure 3 B) Pathological sections were subjected to spatial selenium imaging and quantitative analysis, which was then combined with corresponding pathological diagnostic images. It was found that at the mouse and human levels, the selenium content in pancreatic cancer regions was significantly lower than that in adjacent pancreatic tissue, indicating that pancreatic cancer tissue is chronically under selenium levels, and selenium, which combats cellular lipid peroxidation, may need to be obtained from external sources.

[0068] 5. Development and construction of RTM-specific Selenop gene knockout mice, Mrc1 CreERT2 Selenop fl / fl In preclinical animal models of pancreatic cancer, targeted intervention with Selenop significantly prolonged the survival of pancreatic cancer animal models and inhibited EMT (extracorporeal membrane oxygenation) in the development of pancreatic cancer.

[0069] By constructing a myeloid-specific knockout Lyz2 from Selenop Cre Selenop fl / fl In a mouse model, we found that pancreatic cancer mice with Selenop gene deficiency in myeloid cells had longer survival and better prognosis compared to wild-type mice. Figure 4 A). Tumor cell analysis revealed that Selenop gene-deficient mice in myeloid cells had lower EMT levels in tumor cells compared to wild-type mice. Figure 4 B) indicates that Selenop in myeloid cells plays a pro-tumor EMT role.

[0070] 6. Develop an in vitro co-culture system for binding selenium isotope tracing to demonstrate that RTM protects tumor cells undergoing EMT against ferroptosis by delivering Selenop protein to tumor cells.

[0071] A schematic diagram illustrating the functional mechanism by which Selenop derived from RTMs regulates tumor cell ferroptosis is shown below. Figure 5 By constructing RTM-specific Selenop conditional gene knockout mice, Mrc1... CreERT2 Selenop fl / fl The model was used to assess the impact on pancreatic tumors by in situ implantation of pancreatic cancer cells. Immunofluorescence imaging analysis showed that more tumor cells in the RTM-specific Selenop-deficient group underwent ferroptosis. Figure 6A) demonstrated that RTM expression of Selenop may promote tumor EMT by inhibiting lipid oxidation in tumor cells and protecting them from ferroptosis. In a co-culture system of wild-type macrophages and pancreatic cancer cells, experiments using selenium-74 isotope-labeled macrophages confirmed the transfer of selenium-74 from macrophages to tumor cells. Figure 6 B). Comparing the selenium 74 content of tumor cells in the co-culture system of wild-type and Selenop-deficient macrophage groups, the selenium 74 content of tumor cells in the Selenop-deficient group was significantly lower than that in the wild-type group ( Figure 6 (C) This study confirmed that the Selenop protein can transfer from macrophages to tumor cells, thereby regulating ferroptosis and EMT in tumor cells. Therefore, Selenop is a biomarker for the diagnosis of pancreatic cancer and a therapeutic target.

[0072] The above results indicate that selenoprotein P is specifically highly expressed in pancreatic cancer, and the level of Selenop in pancreatic cancer cells is significantly negatively correlated with the degree of EMT and ferroptosis pathway. Downregulation of selenoprotein P can promote lipid oxidation and ferroptosis in tumor cells, inhibit the degree of EMT in tumor cells, and block the protective effect of RTMs on tumor cells. These results suggest that there is a close correlation between selenoprotein P and the occurrence and development of pancreatic cancer, and therefore it can serve as a biomarker for tumor diagnosis and the selection of tumor treatment strategies.

[0073] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. The application of a reagent for detecting the expression level of selenoprotein P in the preparation of tumor diagnostic kits, characterized in that, The tumor in question is one in which tissue-resident macrophages (RTMs) regulate the expression level of selenoprotein P.

2. The application according to claim 1, characterized in that, The reagent for detecting the expression level of selenoprotein P is a reagent for detecting the expression level of selenoprotein P in biological samples at the gene or protein level; the kit contains reagents for detecting the expression level of selenoprotein P in biological samples.

3. The application according to claim 2, characterized in that, The reagents used to detect selenoprotein P in biological samples are selected from one or more of the following detection techniques or methods: in situ hybridization, Northern blot, PCR, or immunohistochemistry. The PCR detection reagent includes PCR primers, the primer sequences of which are shown in SEQ ID NO.1~4.

4. The application according to claim 1, characterized in that, The tumor was selected from pancreatic cancer.

5. A tumor diagnostic kit, characterized in that, This kit contains reagents for detecting the content of selenoprotein P in biological samples, wherein the tumor is a tumor in which tissue-resident macrophage RTMs regulate the expression level of selenoprotein P.

6. The reagent kit according to claim 5, characterized in that, The kit consists of a reverse transcription system, a primer system, and an amplification system, wherein the primer system includes PCR primers as shown in SEQ ID NO.1 to 4.

7. The application of substances targeting selenoprotein P in the preparation of drugs for treating tumors, characterized in that, The tumor in question is one in which tissue-resident macrophages (RTMs) regulate the expression level of selenoprotein P.

8. Application of substances that inhibit or silence selenoprotein P in the preparation of drugs for treating pancreatic cancer.

9. The application according to claim 8, characterized in that, The substance that inhibits or silences selenoprotein P is selected from any of the following: (1) RNA interference molecules or antisense oligonucleotides that inhibit the expression of selenoprotein P, or recombinant expression vectors or transgenic cell lines containing them; (2) Small molecule inhibitors; (3) Substances that are used for lentiviral vector infection or gene knockout, The nucleic acid sequence corresponding to the antisense oligonucleotide is selected from any one or more combinations of the following nucleic acid sequences: SEQ ID NO.5~6, SEQ ID NO.7~8, SEQ ID NO.9~10, SEQ ID NO.11~12, SEQ ID NO.13~14, SEQ ID NO.15~16, SEQ ID NO.17~18, SEQ ID NO.19~20, SEQ ID NO.21~22, SEQ ID NO.23~24, SEQ ID NO.25~26, SEQ ID NO.27~28, SEQ ID NO.29~30, SEQ ID NO.31~32.

10. A pharmaceutical composition for treating pancreatic cancer, characterized in that, It includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a substance used to inhibit the expression level of selenoprotein P. The substance that inhibits the expression level of selenoprotein P is selected from any of the following: (1) RNA interference molecules or antisense oligonucleotides that inhibit the expression of selenoprotein P, or recombinant expression vectors or transgenic cell lines containing them; (2) Small molecule inhibitors; (3) Substances that are used for lentiviral vector infection or gene knockout, The nucleic acid sequence corresponding to the antisense oligonucleotide is selected from any one or more of the following nucleic acid sequences. Column combinations: SEQ ID NO.5 - 6, SEQ ID NO.7 - 8, SEQ ID NO.9 - 10, SEQ ID NO.11 - 12, SEQ ID NO.13 - 14, SEQ ID NO.15 - 16, SEQ ID NO.17 - 18, SEQ ID NO.19 - 20, SEQ ID NO.21 - 22, SEQ ID NO.23 - 24, SEQ ID NO.25 - 26, SEQ ID NO.27 - 28, SEQ ID NO.29 - 30, SEQ ID NO.31 - 32.

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