PDE1A, a tumor treatment target and diagnostic biomarker, and a kit and application thereof

Abstract

The present invention discloses a PDE1A target and diagnostic biomarker, as well as a kit and application thereof, belonging to the technical field of tumor diagnosis and treatment. By detecting PDE1A and utilizing its differential expression in NSCLC tissue and adjacent adjacent tissue, the present invention specifically identifies NSCLC. Furthermore, by targeting PDE1A, NSCLC metastasis can be significantly inhibited, making it a target for anti-metastatic drug therapy. Therefore, PDE1A, as a molecular marker for tumor therapy and markers, is applied in tumor treatment and diagnosis, providing new ideas and directions for therapeutic-related drugs and diagnostic products.

Description

Technical Field

[0001] The present application relates to the technical field of tumor diagnosis, and in particular to a PDE1A, a kit, and an application thereof, which are tumor treatment targets and diagnostic biomarkers. Background Art

[0002] In recent years, despite continuous advancements in surgical, radiotherapy, and chemotherapy for lung cancer, and breakthroughs in treatment options including molecularly targeted therapies and immunotherapy, treatment rates for most patients with advanced lung cancer have remained relatively stable, with the five-year survival rate remaining at only 16%, a near-perfect improvement. Lung cancer has become one of the leading malignant tumors threatening the lives and health of the nation. Metastasis is believed to be the primary cause of the high mortality rate from lung cancer. A study showed that nearly 80% of lung cancer patients present with metastatic disease at stage II, III, or IV at diagnosis. For patients with metastatic disease, traditional surgery, radiotherapy, chemotherapy, and emerging biological therapies offer minimal benefit. For patients diagnosed early, while treatment can effectively alleviate disease progression, the risk of recurrence and metastasis remains high. Reducing the incidence of metastasis is crucial for reducing mortality in NSCLC patients. Therefore, in-depth understanding of the molecular mechanisms of NSCLC metastasis and the development of reliable prevention and treatment strategies are among the most pressing challenges in lung cancer research.

[0003] PDE1A (phosphodiesterase 1A) is a subtype of the PDE family member PDE1, along with PDE1B and PDE1C. PDE1A is primarily composed of Ca2+ and calmodulin (CaM). Its primary function is through calcium-calmodulin binding to the CaM domain of PDE1, relieving N-terminal autoinhibition at the catalytic site and promoting enzyme activity. As the earliest discovered isoenzyme, PDE1 can hydrolyze both cGMP and cAMP. PDE1A is commonly found in the vascular smooth muscle system and plays a role in regulating tone, while PDE1B is primarily found in the nervous system and is involved in memory and immune regulation. PDE1C is found in both systems and participates in the biological functions of the vascular smooth muscle system.

[0004] PDE1A hydrolyzes cAMP, reducing its intracellular expression, thereby regulating various physiological processes in the body. Reducing PDE1A expression can attenuate leukemia cell proliferation, affect the cell cycle, and induce apoptosis. Tehranolide, a natural sesquiterpene lactone with a peroxide group, has been found by Noori et al. to inhibit CaM activity, further inhibiting PDE1A activation. This leads to intracellular cAMP accumulation and PKA activation, ultimately inhibiting cancer cell proliferation.

[0005] Therefore, exploring the specific role and molecular mechanism of PDE1A in the occurrence and development of non-small cell lung cancer, developing new early diagnostic markers and feasible therapeutic targets, and providing potential clinical diagnostic and therapeutic background for the diagnosis and treatment of patients with non-small cell lung cancer. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a PDE1A, a kit and an application thereof for use as a tumor treatment target and diagnostic biomarker.

[0007] According to a first aspect of an embodiment of the present invention, there is provided a PDE1A used as a tumor treatment target and diagnostic biomarker, characterized in that the base sequence of the PDE1A is shown in SEQ ID NO.1.

[0008] According to a second aspect of an embodiment of the present invention, a kit for detecting the expression level of a tumor marker in a sample is provided, comprising a molecular probe pair specifically complementary to the PDE1A of claim 1 and / or a primer pair for amplifying the PDE1A of claim 1.

[0009] Furthermore, the primer pair is selected from at least one of the following primer pairs 1 to 9;

[0010] Among them, the base sequence of primer pair 1 is shown in SEQ ID NOs. 2 and 3; the base sequence of primer pair 2 is shown in SEQ ID NOs. 4 and 5; the base sequence of primer pair 3 is shown in SEQ ID NOs. 6 and 7; the base sequence of primer pair 4 is shown in SEQ ID NOs. 8 and 9; the base sequence of primer pair 5 is shown in SEQ ID NOs. 10 and 11; the base sequence of primer pair 6 is shown in SEQ ID NOs. 12 and 13; the base sequence of primer pair 7 is shown in SEQ ID NOs. 14 and 15; the base sequence of primer pair 8 is shown in SEQ ID NOs. 16 and 17; and the base sequence of primer pair 9 is shown in SEQ ID NOs. 18 and 19.

[0011] Furthermore, the molecular probe pair is selected from at least one of the following molecular probe pairs 1 to 7;

[0012] Among them, the base sequence of molecular probe pair 1 is shown in SEQ ID NOs. 20 to 21; the base sequence of molecular probe pair 2 is shown in SEQ ID NOs. 22 to 23; the base sequence of molecular probe pair 3 is shown in SEQ ID NOs. 24 to 25; the base sequence of molecular probe pair 4 is shown in SEQ ID NOs. 26 to 27; the base sequence of molecular probe pair 5 is shown in SEQ ID NOs. 28 to 29; the base sequence of molecular probe pair 6 is shown in SEQ ID NOs. 30 to 31; and the base sequence of molecular probe pair 7 is shown in SEQ ID NOs. 32 to 33.

[0013] Furthermore, the test sample includes at least one of non-small cell lung cancer tissue and non-small cell lung cancer adjacent tissue.

[0014] According to a third aspect of the embodiments of the present invention, there is provided use of the PDE1A described in the first aspect in preparing a drug for preventing and / or treating tumors.

[0015] Furthermore, the tumor includes lung cancer, preferably, the tumor includes non-small cell lung cancer.

[0016] Furthermore, the tumor includes primary tumor and metastatic tumor.

[0017] According to a fourth aspect of an embodiment of the present invention, a reagent for detecting the expression level of a tumor marker in a sample is provided for use in preparing a kit for detecting non-small cell lung cancer, wherein the base sequence of the tumor marker PDE1A is shown in SEQ ID No. 1.

[0018] According to a fifth aspect of the embodiments of the present invention, a drug for preventing and / or treating tumors is provided, wherein the drug comprises the PDE1A described in the first aspect as an active ingredient.

[0019] The technical solutions provided by the embodiments of the present application may have the following beneficial effects:

[0020] The present invention can be used for diagnosis, prognosis evaluation or drug screening of non-small cell lung cancer by detecting the expression level of a marker, PDE1A, and utilizing the differential expression of this marker in non-small cell lung cancer tissue and adjacent adjacent tissues.

[0021] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are incorporated into and constitute a part of the specification, illustrate embodiments consistent with the present application, and together with the specification, serve to explain the principles of the present application. It should be understood that the following drawings illustrate only certain embodiments of the present invention and should not be considered as limiting the scope. It is clear that those skilled in the art can derive other relevant drawings based on these drawings without inventive effort.

[0023] Figure 1 Graphs showing the effect of PDE1A on overall survival of NSCLC patients provided in an embodiment of the present invention; A is a graph showing the relationship between PDE1A expression and risk prognosis of NSCLC patients; B is a graph showing the effect of PDE1A expression on survival of NSCLC patients;

[0024] Figure 2 The PDE1 provided in the embodiments of the present invention is involved in regulating the migration and motility of NSCLC cells and the EMT process. A is a Transwell assay showing changes in the migration and motility of NCI-H1299 cells after silencing PDE1A, PDE1B, and PDE1C; B is a statistical analysis of the experimental results in A.

[0025] Figure 3 A graph showing the protein changes of the metastasis suppressor protein E-cadherin detected by Western blot in A549 cells after silencing PDE1 provided in an embodiment of the present invention;

[0026] Figure 4 Figure 1 shows the migration and motility of parental cells T0 and highly metastatic NSCLC cells T3 provided in an embodiment of the present invention; A is a schematic diagram of the method for constructing highly metastatic cells T3 in a Transwell chamber; B is a representative graph of the migration ability experiment of NCI-H1299 and A549 cells T0 and T3;

[0027] Figure 5 The results of Western blot detection of PDE1A and metastasis-related protein expression in NCI-H1299 and A549 parental cells T0 and highly metastatic cells T3 provided in the embodiments of the present invention;

[0028] Figure 6 A is a graph showing the mRNA expression of metastasis-related proteins in parental cells T0 and highly metastatic cells T3 detected by qRT-PCR provided in an embodiment of the present invention; B is a graph showing the mRNA levels of E-cadherin, N-cadherin, and PDE1A in ANCI-H1299 cells T0 and T3; C is a graph showing the mRNA levels of E-cadherin, transcription factor Slug, and PDE1A in A549 cells T0 and T3;

[0029] Figure 7 The expression results of PDE1A in HELF and NSCLC cells provided in the examples of the present invention are shown in FIG.

[0030] Figure 8 Figure 1 is a graph showing the weakened migration ability of NSCLC cells after silencing PDE1A in an embodiment of the present invention; A is a graph showing the siRNA silencing efficiency of PDE1A in A549 and NCI-H1299 cells; B is a graph showing the effect of silencing PDE1A on the migration ability of A549 and NCI-H1299 cells detected by a Transwell assay; C is a graph showing the statistical analysis of the results of experimental B; D is a graph showing the effect of silencing PDE1A on the wound healing ability of A549 and NCI-H1299 cells detected by a scratch assay; and E is a graph showing the statistical analysis of the results of experimental D.

[0031] Figure 9 The present invention provides a graph showing the weakened invasion ability of NSCLC cells after silencing PDE1A; A is a representative graph showing the invasion of A549 and NCI-H1299 cells after silencing PDE1A; B is a statistical analysis graph of the experimental results of A.

[0032] Figure 10 A graph showing the changes in EMT-related proteins in NSCLC cells detected by Western blot after silencing PDE1A according to an embodiment of the present invention;

[0033] Figure 11 A graph showing the changes in mRNA levels of PDE1A, N-cadherin, and Snail detected by qRT-PCR after silencing PDE1A provided in an embodiment of the present invention;

[0034] Figure 12 Figure 1 shows the ability of PDE1A to promote NSCLC cell migration as provided in an embodiment of the present invention; A shows the efficiency of PDE1A overexpression in A549, NCI-H1299, and NCI-H460 cells detected by Western blot; B shows the migration results of three NSCLC cell lines overexpressing PDE1A; C shows the statistical analysis of the experimental results in B.

[0035] Figure 13 The results of the PDE1A overexpression wound healing experiment and the experimental statistical graph provided in the embodiment of the present invention;

[0036] Figure 14 The results of the PDE1A overexpression invasion experiment and the experimental statistical graph provided in the embodiment of the present invention;

[0037] Figure 15The results of Western blot detection of changes in EMT and metastasis-related proteins in A549, NCI-H1299, and NCI-H460 cells after overexpression of PDE1A provided in the embodiments of the present invention are shown;

[0038] Figure 16 Figure 1 is a graph showing the results of overexpression of PDE1A promoting the EMT process of NSCLC cells provided in an embodiment of the present invention; A is a graph showing the results of qRT-PCR detection of changes in the mRNA of PDE1A, N-cadherin, and Slug after overexpression of PDE1A in NCI-H1299 cells; B is a graph showing the results of qRT-PCR detection of changes in the mRNA of PDE1A, N-cadherin, and Slug after overexpression of PDE1A in NCI-H460 cells;

[0039] Figure 17 The results of Western blot detection of PDE1A protein expression level and Transwell assay detection of the effect of PDE1A on the migration and motility of NCI-H1299 cells in vitro provided in the embodiments of the present invention are shown;

[0040] Figure 18 Figures 1 and 2 show the results of PDE1A promoting NCI-H1299 cell metastasis in nude mice as provided in the examples of the present invention. Figure A shows 200×104 NCI-H1299 cells injected into each nude mouse via tail vein. 60 days later, the mice were dissected, their lungs were isolated, and fixed with Bao's fixative. The number of metastatic lesions in each group was observed and counted. Figure B shows H&E staining of lung tissue metastases. Figure C shows the statistical analysis of lung metastases in nude mice in the control group and the PDE1A overexpression group.

[0041] Figure 19 Figure 1 shows the inhibition of NCI-H1299 cell metastasis in nude mice by shPDE1A provided in an embodiment of the present invention. Figure A shows 200×104 NCI-H1299 cells injected into each nude mouse via tail vein injection. 60 days later, the mice were dissected, their lungs were isolated, and fixed with Bao's fixative. The number of metastatic lesions in each group was observed and counted. Figure B shows the statistical analysis results of Figure A.

[0042] Figure 20 The embodiment of the present invention provides a diagram showing the results of H&E staining to detect metastatic lesions in mouse lung tissue;

[0043] Figure 21 A molecular function result diagram of a target protein from Gene Ontology enrichment analysis provided in an embodiment of the present invention;

[0044] Figure 22 A graph showing proteins that bind to PDE1A as detected by silver staining assay according to an embodiment of the present invention;

[0045] Figure 23 A diagram showing the protein interaction between PDE1A and YTHDF2 detected by immunoprecipitation experiments provided in an embodiment of the present invention;

[0046] Figure 24 Graph showing the YTHDF2 silencing efficiency results provided in an embodiment of the present invention; A is a graph showing the siRNA silencing efficiency of YTHDF2 in A549 and NCI-H1299 cells detected by Western blot; B is a graph showing the silencing efficiency results of siYTHDF2 detected by qRT-PCR;

[0047] Figure 25 A graph showing the effect of YTHDF2 on the reversal of PDE1A-promoted NCI-H1299 cell migration by reversing PDE1A provided in an embodiment of the present invention; A is a graph showing the effect of silencing YTHDF2 on the effect of PDE1A on the migration of NCI-H1299 cells; B is a statistical analysis graph of A; C is a graph showing the effect of silencing YTHDF2 on the effect of PDE1A on the invasion of NCI-H1299 cells; D is a statistical analysis graph of C;

[0048] Figure 26 Figure 2 is a diagram showing the enrichment results of SCOS2 mRNA in anti-PDE1A precipitates and anti-YTHDF2 precipitates detected by RNA immunoprecipitation-qPCR experiments provided in an embodiment of the present invention;

[0049] Figure 27 Figure 1 shows the results of an embodiment of the present invention in which NCI-H1299 cells were transfected with METTL3 siRNA to overexpress PDE1A and YTHDF2, mRNA was collected 48 hours after transfection, and the mRNA changes of SOCS2 were detected by qRT-PCR;

[0050] Figure 28 Figure 1 shows the results of qRT-PCR detection of SOCS2 mRNA changes after overexpressing PDE1A / YTHDF2 in NCI-H1299 cells, followed by transfection with YTHDF2 / PDE1A siRNA or simultaneous transfection with YTHDF2 and PDE1A siRNA, and collecting mRNA 48 hours after transfection.

[0051] Figure 29 Figure 1 shows the effect of PDE1A on SOCS2 mRNA degradation in an embodiment of the present invention; A shows the results of analyzing SOCS2 mRNA half-life after silencing PDE1A in A549 cells and treating with actinomycin D (5 μg / ml) at 0, 3, and 6 hours; B shows the results of analyzing SOCS2 mRNA expression after silencing PDE1A in A549 cells and treating with actinomycin D (5 μg / ml) at 0, 3, and 6 hours;

[0052] Figure 30 Figure 1 is a graph showing the effect of PDE1A on SOCS2 mRNA degradation in an embodiment of the present invention; A is a graph showing the analysis of SOCS2 mRNA half-life after silencing PDE1A in NCI-H1299 cells and treating them with actinomycin D (5 μg / ml) at 0 h, 3 h, and 6 h; B is a graph showing the analysis of SOCS2 mRNA expression after silencing PDE1A in NCI-H1299 cells and treating them with actinomycin D (5 μg / ml) at 0 h, 3 h, and 6 h. DETAILED DESCRIPTION

[0053] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. In the following description, when referring to the drawings, identical numerals in different figures represent identical or similar elements, unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.

[0054] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. As used in this application and the appended claims, the singular forms "a," "an," "the," and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0055] The present invention provides use of a reagent for detecting the expression level of PDE1A in a sample in preparing a kit for detecting non-small cell lung cancer.

[0056] The above-mentioned tumor marker PDE1A is an RNA tumor marker.

[0057] The term "detection" herein may refer to detection of a sample for diagnosis, auxiliary diagnosis or prognosis assessment of non-small cell lung cancer.

[0058] Specifically, the base sequence of PDE1A is shown in SEQ ID NO.1.

[0059] In some embodiments, the reagent comprises a molecular probe specifically complementary to the PDE1A of the first aspect or a primer pair for amplifying the PDE1A of the first aspect.

[0060] Preferably, the reagent comprises: a primer pair 1 for detecting PDE1A.

[0061] The base sequences of primer pair 1 are shown in SEQ ID NOs. 2-3.

[0062] It should be noted that a primer pair generally includes an upstream primer and a downstream primer. In this article, "the base sequence of primer pair 1 is shown in SEQ ID NO.2-3" means that the base sequence of the upstream primer in the primer pair is shown in SEQ ID NO.2, and the base sequence of the downstream primer is shown in SEQ ID NO.3.

[0063] Preferably, the sample comprises a biological tissue sample.

[0064] Preferably, the biological tissue sample includes at least one of non-small cell lung cancer tissue and non-small cell lung cancer adjacent tissue.

[0065] The present invention also provides an embodiment of the present invention for use of a reagent for detecting the expression level of a tumor marker in a sample in screening for drugs for treating non-small cell lung cancer. The tumor marker includes PDE1A having a base sequence as shown in SEQ ID No. 1. The application is not directly intended for diagnosis or treatment of a disease.

[0066] The inventors discovered that the tumor marker PDE1A is differentially expressed in the cancerous tissues and adjacent tissues of patients with non-small cell lung cancer. By detecting the marker in the cancerous tissues and adjacent tissues of patients, non-small cell lung cancer can be diagnosed and the prognosis evaluated, which plays an important role in the development of drugs for non-small cell lung cancer and prolonging the prognosis.

[0067] These markers can be used for the diagnosis and prognosis of non-small cell lung cancer. Therefore, detection of these markers can accelerate the development of targeted drugs specific for non-small cell lung cancer patients. The following, in conjunction with the examples, describes in detail PDE1A, a tumor therapeutic target and diagnostic biomarker, as well as kits and applications provided by the present invention. However, these examples should not be construed as limiting the scope of the present invention.

[0068] Example 1

[0069] PDE1A expression is associated with the prognosis of lung cancer patients:

[0070] First, this example found through a search and analysis of the TCGA database that lung cancer patients with high PDE1A expression have a higher risk prognosis than those with low PDE1A expression, and the survival of lung cancer patients with high PDE1A expression is significantly shortened (see Figure 1 ). It can be seen that this example successfully predicts that PDE1A may be involved in the occurrence and development of lung cancer, and high expression of PDE1A is closely related to the poor prognosis of lung cancer patients.

[0071] Example 2

[0072] PDE1A is associated with the migration and epithelial-mesenchymal transition of NSCLC cells:

[0073] Before the experiment, this example performed siRNA gene silencing experiments on the three isoforms of PDE1, PDE1A, PDE1B, and PDE1C, in NSCLC cells NCI-H1299. The cell migration ability was then tested using the Transwell method. The results showed that silencing PDE1 inhibited the migration ability of NCI-H1299 cells. Among them, silencing PDE1A inhibited the migration ability of NCI-H1299 cells most significantly (see Figure 2 In addition, this example also examined the changes in the metastasis suppressor protein E-cadherin after silencing PDE1 in A549 cells, and found that the E-cadherin protein increased most significantly in the PDE1A silencing group (see Figure 3 ). These results suggest that PDE1A expression may be associated with the metastasis of NSCLC.

[0074] In the early stage of this example, NSCLC / T3 cell lines were constructed by three-fold Transwell enrichment to study the migration and motility of different metastatic cell lines. As shown in the figure, compared with the parental cells (NCI-H1299 / T0, A549 / T0), highly metastatic cells (NCI-H1299 / T3, A549 / T3) have stronger migration and motility (see Figure 4 ). To further investigate the correlation between PDE1A and NSCLC cell metastasis, this example analyzed cells with different migration abilities at both the protein and mRNA levels. The results showed that A549 and NCI-H1299 cells with high migration abilities expressed higher levels of the metastasis-promoting proteins N-cadherin, Vimentin, and Snail, while expressing lower levels of the metastasis-suppressing protein E-cadherin. Interestingly, compared with NSCLC cells with low metastasis abilities, this example also observed increased levels of phosphorylated proteins of PDE1A and transcription factor smad 3 in cells with high metastasis abilities (see Figure 5 At the same time, we further verified this result at the mRNA level (see Figure 6 ), the above data suggest that PDE1A may have a potential role in promoting metastasis.

[0075] Example 3

[0076] PDE1A expression differences between HELF and NSCLC cells: To evaluate the expression of PDE1A in NSCLC cells, normal human lung fibroblasts and different NSCLC cell lines were selected for study. Western blot results showed that compared with normal human lung fibroblasts (HELF), PDE1A expression was higher in NSCLC cells A549, moderate in NCI-H1299, and low in NCI-H460, while almost absent in HELF (see Figure 7 This suggests that the expression level of PDE1A protein is associated with the malignancy of NSCLC cells.

[0077] Example 4

[0078] PDE1A promotes the migration and invasion ability of NSCLC cells:

[0079] First, the Transwell assay was used to examine the changes in the migration ability of NSCLC cells before and after silencing PDE1A. It was found that after silencing PDE1A, the number of migrating cells in A549 and NCI-H1299 cells was significantly less than that in the negative control group (see Figure 8 ); At the same time, the effect of silencing PDE1A on the wound healing ability of NSCLC cells was detected by scratch assay. The results showed that silencing PDE1A could reduce the wound healing ability of A549 and NCI-H1299 cells (see Figure 9 Invasion assays using Matrigel-based chambers revealed that after silencing PDE1A, the number of cells penetrating Matrigel was significantly lower than that in the negative control group (see Figure 10 ), the above results indicate that silencing PDE1A can inhibit the migration and invasion ability of NSCLC cells.

[0080] During the metastasis process, the cytoskeleton or motility-related proteins of tumor cells change. An important process in tumor metastasis is epithelial-mesenchymal transition (EMT), which is accompanied by changes in related proteins, such as the metastasis suppressor protein E-cadherin and the metastasis-promoting proteins Fibronectin and N-cadherin. Therefore, we further studied the effect of silencing PDE1A on these proteins. The results showed that after silencing PDE1A, the protein levels of transcription factors Snail and Slug decreased. The transcription factors inhibited the promoter activity of E-cadherin, activated its protein transcription, and reduced the expression of N-cadherin protein, thereby inhibiting tumor metastasis (see Figure 11 In addition, the mRNA level was tested, and the results of qRT-PCR experiments showed that silencing PDE1A inhibited the expression of the transcription factor Snail and the mRNA expression of the metastasis-promoting protein N-cadherin (see Figure 12 ), the above protein expression and mRNA expression levels are consistent with the metastatic movement phenomenon observed in the previous example.

[0081] To further investigate the effect of PDE1A on the metastatic and invasive motility of NSCLC cells, this example overexpressed PDE1A in A549, NCI-H1299, and NCI-H460 cells and tested their migration and invasion abilities. As shown in the figure, overexpression of PDE1A significantly increased the number and distance of cell migration, indicating enhanced cell migration ability (see Figure 13-14 Matrigel matrix gel-coated transwell assay further demonstrated that overexpression of PDE1A could significantly enhance the invasive ability of cells (Figure 10 (see Figure 15 The above results indicate that PDE1A can promote the migration and invasion of NSCLC cells.

[0082] Next, this example used Western blot to detect changes in metastasis-related proteins after overexpression of PDE1A. The results showed that overexpression of PDE1A increased the phosphorylation levels of transcription factors STAT3 and smad3, decreased the expression of metastasis suppressor protein E-cadherin, and increased the expression of metastasis-promoting protein N-cadherin (see Figure 16 ); In addition, NCI-H1299 and NCI-H460 cells with low background expression of PDE1A were selected for PDE1A overexpression experiments. The mRNA changes of PDE1A and EMT-related proteins and transcription factors were then detected. The results showed that overexpression of PDE1A could upregulate the mRNA of N-cadherin and Slug (see Figure 17 These results suggest that PDE1A participates in the EMT process of NSCLC cells and plays an important role in their metastasis.

[0083] Example 5

[0084] Effect of PDE1A on NCI-H1299 cell metastasis in nude mice:

[0085] Through previous studies, this example found that PDE1A significantly promotes the migration and invasion of NSCLC cells. However, the occurrence and development of tumors are determined by both the tumor cells themselves and the tumor microenvironment. To further study the effect of PDE1A on the metastasis and colonization of NSCLC cells in vivo, this example constructed an NCI-H1299 cell line stably overexpressing PDE1A (PDE1A: pLenti-CMV-PDE1A-Flag) and a control (vect: pLenti-CMV-PCIG3-Flag). The results showed that NCI-H1299 cells stably overexpressing PDE1A had a stronger migration ability (see Figure 18 ), indicating that a stable overexpression model was successfully established. Furthermore, a nude mouse model was established by tail vein injection, further confirming the metastatic effect of PDE1A in vivo. After dissecting the various organs of the mice, the lungs of the nude mice were stained with Parfum, revealing that the number of lung foci in the nude mice with stable PDE1A overexpression group was greater than that in the control group. Paraffin-embedded lung tissue of the nude mice was subjected to H&E staining, which revealed that the lungs of the nude mice with PDE1A overexpression group had obvious metastatic foci (see Figure 19 ). Therefore, PDE1A can promote NSCLC cell metastasis in nude mice.

[0086] To further investigate the effect of PDE1A on NCI-H1299 cell metastasis in nude mice, this example also constructed a PDE1A-silencing shPDE1A model. NCI-H1299 cells stably transfected with shPDE1A and the control plko.1 were inoculated into nude mice via tail vein injection. The results showed that shPDE1A inhibited NCI-H1299 cell metastasis in nude mice. The number of lung metastases in the shPDE1A group was significantly lower than that in the control plko.1 group, and H&E staining revealed no obvious metastatic lesions in the shPDE1A group (see ). Figure 20-21 The above results indicate that shPDE1A inhibits the metastasis of NCI-H1299 cells in nude mice.

[0087] Example 6

[0088] PDE1A binds to YTHDF2 and reverses PDE1A-induced migration and invasion of NSCLC cells:

[0089] To further explore downstream target molecules involved in regulating PDE1A to promote metastasis of non-small cell lung cancer cells, this example used A549 cells to overexpress PDE1A by plasmid transfection. Samples were collected for immunoprecipitation experiments. After Western blot running, the gel was stained with Coomassie Brilliant Blue and sent for protein spectrum analysis. According to the score results of mass spectrometry analysis (see Table 1) and Gene Ontology enrichment analysis (see Figure 22-23 ), we found that the m6A binding protein (GO:1990247) was enriched at a large multiple, and the RNA binding protein was most significantly enriched. Based on the silver staining results, YTHDF2 was finally selected as the next research object.

[0090]

[0091] PDE1A is mainly distributed in the cell cytoplasm. Generally, proteins in the cytoplasm often achieve their biological functions by binding to proteins. Therefore, immunoprecipitation experiments were used to detect whether PDE1A and YTHDF2 have protein binding. The results showed that PDE1A can bind to YTHDF2 (see Figure 24 We then examined the effect of silencing YTHDF2 on the migration and invasion of NSCLC cells promoted by PDE1A. We found that YTHDF2 could reverse the effects of PDE1A on the migration and invasion of NSCLC cells (see Figures 25-26 ).

[0092] YTHDF2 is one of the currently known reader proteins in the m6A modification process. YTHDF2 recognizes the m6A modification on mRNA and guides the CCR4-NOT protein complex to degrade the recognized mRNA. Literature has shown that SOCS2, as a downstream target of YTHDF2, can be degraded by YTHDF2. Therefore, RNA immunoprecipitation-qPCR technology was first used to verify whether PDE1A and YTHDF2 bind to SOCS2 RNA. The experimental results showed that compared with the IgG group, the SOCS2 mRNA enriched by PDE1A antibodies and YTHDF2 antibodies respectively increased significantly. This result indicates that PDE1A and YTHDF2 bind to SOCS2 mRNA (see Figure 27 This example also uses fluorescent quantitative PCR technology to detect changes in SOCS2 expression levels caused by changes in the expression of METTL3, YTHDF2, and PDE1A. The results show that affecting the expression of METTL3, YTHDF2, and PDE1A can cause changes in the mRNA level of SOCS2 (see Figures 28-29Furthermore, mRNA stability assays showed that SOCS2 mRNA expression was elevated and the mRNA half-life of SOCS2 was prolonged by PDE1A silencing in A549 and NCI-H1299 cells (see Figure 30 ).

[0093] The primer pair sequences and probe base pair sequences described in the above examples are specifically shown in Table 2.

[0094] Table 2:

[0095]

[0096] 1. Cell Culture

[0097] Human non-small cell lung cancer cells A549, NCI-H1299, NCI-H460 and human umbilical vein endothelial cells HUVEC were cultured in a medium containing 10% FBS and 90% RPMI 1640. All cells were cultured in a cell culture incubator at 5% CO2 and 37°C.

[0098] 2. Transwell assay

[0099] (1) Cell migration assay

[0100] Lung cancer cells with high and low circPOLK expression were serum starved overnight, digested and counted, and seeded into transwell chambers at a density of 2×10 4 200 µl of serum-free culture medium was added to each well of the corresponding 24-well plate. 600 µl of culture medium containing 20% ​​serum was added to the corresponding 24-well plate. After 24 hours of incubation, the chambers were stained with 1% crystal violet for 30 minutes in the dark. The cells inside the chambers were then washed with 1× PBS. Cells that migrated to the lower surface of the chambers were then photographed and counted using a microscope.

[0101] (2) Cell invasion assay

[0102] Matrigel was thawed at 4°C in advance, and the chamber, 24-well plate, and pipette tips were pre-cooled at -20°C. Matrigel was diluted 1:20 with cell culture medium, 100 μl of Matrigel was added to each chamber, and the chamber was placed in a 24-well plate and incubated at 37°C for 30 minutes. The 24-well plate was removed, the unsolidified Matrigel in the chamber was discarded, and the cells were rinsed with culture medium. At the same time, non-small cell lung cancer cells with high and low expression of circPOLK were serum starved overnight, digested and counted, and seeded into transwell chambers with Matrigel at a density of 3×10 4200 µl of serum-free culture medium was added to each well of the corresponding 24-well plate. 600 µl of culture medium containing 20% ​​serum was added to the corresponding 24-well plate. After incubation for 24 hours, the chambers were stained with 1% crystal violet for 30 minutes in the dark. The cells inside the chambers were then washed with 1× PBS. Cells invading the lower surface of the chambers were then photographed and counted using a microscope.

[0103] 3. Wound Healing Experiment

[0104] The cells were plated at 2 × 10 5 Cells were seeded at a density of 100 μg / well in a six-well plate. After cells adhered, the desired plasmid or siRNA was transfected. When cell confluence reached 80-100%, the cell surface was scratched with a pipette. Removed cells were washed with PBS and replaced with fresh culture medium. The wound distance at 0 h was photographed microscopically. After 24 h, the six-well plate was washed with PBS to remove non-adherent cells. The wound distance at 24 h was photographed microscopically to calculate wound healing.

[0105] 4. Western blot method

[0106] (1) Protein sample extraction and quantification

[0107] ① Extraction: Collect the pre-treated cells, centrifuge at 2000 rpm for 4 min, discard the supernatant, transfer the precipitate to an EP tube, centrifuge at 2000 rpm for 4 min, discard the supernatant. Add whole cell lysis buffer according to the number of cells, vortex once every 10 min,

[0108] Perform this step three times to thoroughly mix the cells and lysate, centrifuge at 12,000 rpm and 4°C for 30 min, and collect the supernatant, which is the protein sample.

[0109] ② Quantification: Prepare a standard curve using a 1 / 2-fold dilution of BSA (2 mg / ml) with deionized water. Dilute the sample protein 10-fold with deionized water. Add 5 μl of each BSA solution and sample protein to 200 μl of freshly prepared BCA reagent mixture (Solution A:Solution B = 50:1) and mix thoroughly. Incubate at 37°C for 30 minutes and read the absorbance at 562 nm using a microplate reader. Calculate the sample protein concentration using the formula from the resulting standard curve and calibrate the protein at a specific amount.

[0110] ③ Direct loading method

[0111] The supernatant of the pretreated cells was discarded, and the cells were washed twice with PBS. According to the cell density, the corresponding amount of 2.5× loading was added and stirred to directly harvest the cells. The cells were centrifuged, boiled in boiling water for 10 min to inactivate the protein, cooled and centrifuged, and stored at -20°C.

[0112] (2) Protein immunoblotting

[0113] ① Gel electrophoresis: Prepare 500 ml of 1× Running Buffer and load the protein samples in the specified order and amount. Initially, adjust the voltage to 70 V. Once the samples reach the separating gel, increase the voltage to 110 V. Stop electrophoresis when the samples are 1 cm from the bottom of the gel.

[0114] ② Transfer: Prepare 900 ml of 1× Transfer Buffer. Activate an appropriately sized PVDF membrane with 100 ml of anhydrous ethanol for 1 minute. Add the prepared Transfer Buffer. Cut the gel into appropriate sizes based on the desired protein size. Transfer the membrane using the black gel and white membrane method. Use a constant current of 330 mA and transfer in an ice-water bath for 90 minutes.

[0115] ③ Milk blocking and incubation with primary antibody: After the transfer time is up, block the PVDF membrane in 5% skim milk (prepared in T-PBS) at room temperature for 1 hour. Discard the milk, wash the PVDF membrane three times with T-PBS, add the desired primary antibody, and incubate overnight at 4°C on a shaker.

[0116] ④ Incubate with secondary antibody: Recover the primary antibody and wash the strips three times with T-PBS for 10 minutes each. Add the horseradish peroxidase-conjugated secondary antibody corresponding to the primary antibody and incubate on a shaker at room temperature for 90 minutes. Discard the secondary antibody and wash the strips three times with T-PBS.

[0117] ⑤ Exposure: Prepare the exposure materials in advance, incubate the strips with ECL developer (1:1 solution A and solution B in the ECL kit) in the dark for 1 min, transfer them to the exposure box, and press them with X-ray film in a darkroom. The short exposure time is 5-20 s (depending on the fluorescence intensity of the strips) and the long exposure time is 30 min. Take out the film, place it in the developer for 1.5 min, rinse with tap water, place it in the fixer for 1.5 min, and image it.

[0118] 5. RNA Extraction and Reverse Transcription

[0119] Discard the supernatant from the pre-treated six-well plate, rinse twice with ice-cold PBS, add 1 ml of Trizol to each well, and repeatedly pipette until the solution becomes transparent. Transfer the cells to a 1.5 ml RNase-free EP tube, add 200 µl of chloroform, shake vigorously for 30 seconds, incubate at 4°C for 2 minutes, and centrifuge at 12,000 rpm for 10 minutes. Pipette 450 µl of the supernatant into a new EP tube, add an equal amount of isopropanol, gently invert five times, incubate on ice for 10 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. Discard the supernatant, and gently rinse the pellet with 500 µl of ice-cold 75% ethanol and centrifuge at 8,000 rpm for 5 minutes at 4°C. Repeat this wash cycle, place in a well-ventilated area, and allow the ethanol to evaporate. Add 20 µl of DEPC-free water to dissolve the mRNA and measure its concentration.

[0120] mRNA was reverse transcribed into complementary DNA (cDNA) using the TransStart Top Green qPCR SuperMix kit (Lot#40426) from Total Gold.

[0121] 6.qRT-PCR

[0122] The qRT-PCR system consisted of 10 µl of 2× QuantiTect SYBR Green PCR MasterMix, 1 µl of template cDNA, 0.6 µl each of the forward and reverse primers of interest, and 2.8 µl of DEPC water. The reaction conditions were 39 cycles of initial denaturation at 95°C for 15 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s, and extension at 72°C for 30 s.

[0123] 7. Statistical Analysis

[0124] Statistical analysis was performed using the t-test, and p < 0.05 was considered significant (* P < 0.05, ** P < 0.01, and *** P < 0.001). Each experiment was repeated at least three times.

[0125] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present application that follow the general principles of the present application and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered merely as exemplary, and the true scope and spirit of the present application are indicated by the claims.

[0126] It should be understood that the present application is not limited to the exact structures described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims. Sequence Listing <110> Zhejiang University City College <120> PDE1A, a tumor treatment target and diagnostic biomarker, and a kit and application thereof <160> 33 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2136 <212> DNA <213> Artificial Sequence <400> 1 agagaggaat tcagcttctt ctggagcgcg aaagtcattc acgtttctct tgtgcataat 60 agagctcgta aactgtagga attctgatgt gcttcagtgc acagaacagt aacagatgag 120 ctgcttttgg ggagagcttg agtactcagt cggagcatca tcatggggtc tagtgccaca 180 gagattgaag aattggaaaa caccactttt aagtatctta caggagaaca gactgaaaaa 240 atgtggcagc gcctgaaagg aatactaaga tgcttggtga agcagctgga aagaggtgat 300 gttaacgtcg tcgacttaaa gaagaatatt gaatatgcgg catctgtgct ggaagcagtt 360 tatatcgatg aaacaagaag acttctggat actgaagatg agctcagtga cattcagact 420 gactcagtcc catctgaagt ccgggactgg ttggcttcta cctttacacg gaaaatgggg 480 atgacaaaaa agaaacctga ggaaaaacca aaattcgga gcattgtgca tgctgttcaa 540 gctggaattt ttgtggaaag aatgtaccga aaaacatatc atatggttgg tttggcatat 600 ccagcagctg tcatcgtaac attaaaggat gttgataaat ggtctttcga tgtatttgcc 660 ctaaatgaag caagtggaga gcatagtctg aagtttatga tttatgaact gtttaccaga 720 tatgatctta tcaaccgttt caagattcct gttctctgcc taatcacctt tgcagaagct 780 ttagaagttg gttacagcaa gtacaaaaat ccatatcaca atttgattca tgcagctgat 840 gtcactcaaa ctgtgcatta cataatgctt catacaggta tcatgcactg gctcactgaa 900 ctggaaattt tagcaatggt ctttgctgct gccattcatg attatgagca tacagggaca 960 acaaacaact ttcacattca gacaaggtca gatgttgcca ttttgtataa tgatcgctct 1020 gtccttgaga atcaccacgt gagtgcagct tatcgactta tgcaagaaga agaaatgaat 1080 atcttgataa attatccaa agatgactgg agggatcttc ggaacctagt gattgaaatg 1140 gttttatcta cagacatgtc aggtcacttc cagcaaatta aaaatataag aaacagtttg 1200 cagcagcctg aagggattga cagagccaaa accatgtccc tgattctcca cgcagcagac 1260 atcagccacc cagccaaatc ctggaagctg cattatcggt ggaccatggc cctaatggag 1320 gagtttttcc tgcagggaga taaagaagct gaattagggc ttccattttc cccactttgt 1380 gatcggaagt caccatggt ggcccagtca caaataggtt tcatcgattt catagtagag 1440 ccaacatttt ctcttctgac agactcaaca gagaaaattg ttattcctct tatagaggaa 1500 gcctcaaaag ccgaaacttc ttcctatgtg gcaagcagct caaccaccat tgtggggtta 1560 cacattgctg atgcactaag acgatcaaat acaaaaggct ccatgagtga tgggtcctat 1620 tccccagact actcccttgc agcagtggac ctgaagagtt tcaagaacaa cctggtggac 1680 atcattcagc agaacaaaga gaggtggaaa gagttagctg cacaagaagc aagaaccagt 1740 tcacagaagt gtgagtttat tcatcagtaa acacctttaa gtaaaacctc gtgcatggtg 1800 gcagctctaa tttgaccaaa agacttggag attttgatta tgcttgctgg aaatctaccc 1860 tgtcctgtgt gagacaggaa atctattttt gcagattgct caataagcat catgagccac 1920 ataaataaca gctgtaaact ccttaattca ccgggctcaa ctgctaccga acagattcat 1980 ctagtggcta catcagcacc ttgtgctttc agatatctgt ttcaatggca ttttgtggca 2040 tttgtcttta ccgagtgcca ataaattttc tttgagcagc taattgctaa ttttgtcatt 2100 tctacaataa agcttggtcc acctgttttc acctta 2136 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 cagtaacaga tgagctgc 18 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <400> 3 gtattccttt caggcg 16 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tagccaactg cgacacattc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 cacgaccttg acgttccttt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tgcaaggata agcggacagg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 cagagatggt gctgacgtgt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 gaacgcattg ccacatacac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 gaattcgggc ttgttgtcat 20 <210> 10 <211> twenty three <212> DNA <213> Artificial Sequence <400> 10 ctcctatgag tggaacagga acg 23 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <400> 11 ttggatcaat gtcatattca agtgctgta 29 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 cgtttttcca gaccctggtt 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <400> 13 ctgcagatga gccctcag 18 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 cgtttttcca gaccctggtt 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <400> 15 ctgcagatga gccctcaga 19 <210> 16 <211> 58 <212> DNA <213> Artificial Sequence <400> 16 ccgggatgca ctaagacgat caaatctcga gatttgatcg tcttagtgca tctttttg 58 <210> 17 <211> 58 <212> DNA <213> Artificial Sequence <400> 17 aattcaaaaa gatgcactaa gacgatcaaa tctcgagatt tgatcgtctt agtgcatc 58 <210> 18 <211> 58 <212> DNA <213> Artificial Sequence <400> 18 ccgggcctga aaggaatact aagatctcga gatcttagta ttcctttcag gctttttg 58 <210> 19 <211> 58 <212> DNA <213> Artificial Sequence <400> 19 aattcaaaaa gcctgaaagg aatactaaga tctcgagatc ttagtattcc tttcaggc 58 <210> 20 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 20 ccacgugagu gcagcuuaut t 21 <210> twenty one <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty one auaagcugca cucacguggt t 21 <210> twenty two <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty two ccagcagcug ucaucguaat t 21 <210> twenty three <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty three uuacgaugac agcugcuggt t 21 <210> twenty four <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty four gcuggaagcc gucuacauat t 21 <210> 25 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 25 uauguagacg gcuuccagct t 21 <210> 26 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 26 gcugcagcua uccaugauut t 21 <210> 27 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 27 aaucauggau agcugcagct t 21 <210> 28 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 28 gcuucagugg uagaucuuat t 21 <210> 29 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 29 uaagaucuac cacugaagct t 21 <210> 30 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 30 ccagcuguua uugaggcaut t 21 <210> 31 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 31 augccucaau aacagcuggt t 21 <210> 32 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 32 aaggacguuc ccaauagcca a 21 <210> 33 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 33 uuggcuauug ggaacguccu u 21

Claims

1. Use of siPDE1A in the preparation of a drug for inhibiting tumor metastasis, wherein the tumor is non-small cell lung cancer; The siPDE1A is siPDE1A#1 or siPDE1A#2, the base sequence of the siPDE1A#1 is shown in SEQ ID NOs. 20-21, and the base sequence of the siPDE1A#2 is shown in SEQ ID NOs. 22-23.

2. A drug for inhibiting tumor metastasis, comprising the siPDE1A according to claim 1 as an active ingredient, and the tumor is non-small cell lung cancer.

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