CN114457157A - PDE1A for tumor treatment target and diagnosis biomarker, kit and application - Google Patents

Abstract

The invention discloses a PDE1A for tumor treatment target and diagnosis biomarker, a kit and application, belonging to the technical field of tumor diagnosis and treatment, wherein the PDE1A is detected, differential expression of PDE1A in NSCLC cancer tissues and NSCLC cancer-adjacent tissues is utilized for specific marking of NSCLC, and meanwhile, the target PDE1A can obviously inhibit metastasis of NSCLC and is used for a drug treatment target of tumor anti-metastasis. Therefore, PDE1A is applied to tumor therapy and tumor diagnosis as a tumor therapy target and a labeled molecular marker, and provides a new idea and a new direction for drugs and diagnostic products related to therapy.

Description

A PDE1A and kit and application for tumor therapy target and diagnostic biomarker

technical field

The present application relates to the technical field of tumor diagnosis, and in particular, to a PDE1A, a kit and application for tumor therapy targets and diagnostic biomarkers.

Background technique

The incidence of lung cancer in China and the world ranks first among malignant tumors, and it is also the main reason for the high mortality of all cancers. There are more than 700,000 lung cancer patients diagnosed and 600,000 deaths in China every year, of which nearly 90% of lung cancer patients belong to non-small cell lung cancer patients. In recent years, medical technologies such as surgery, radiotherapy and chemotherapy for lung cancer have been continuously optimized, and treatment methods including molecular targeting and immunotherapy have also made breakthroughs and made new progress. However, the treatment rate of most patients with advanced lung cancer has not The 5-year survival rate is only 16%, which is almost no improvement. Lung cancer has also become one of the most important malignant tumors that endanger people's lives, health and safety. Metastasis is considered to be the main reason for the high mortality rate of lung cancer. A study shows that nearly 80% of lung cancer patients have metastatic stage II, III, and IV disease at the time of diagnosis, and for patients with tumor metastasis, traditional surgery, radiotherapy and chemotherapy and emerging biological therapy bring little benefit. . For lung cancer patients diagnosed earlier, although the treatment has effectively alleviated the disease, the possibility of late recurrence and tumor metastasis is still high. Reducing the incidence of tumor metastasis is the key to reducing the mortality rate of NSCLC patients. Therefore, in-depth research on NSCLC The molecular mechanism of metastasis and the development of reliable prevention and treatment methods are the most urgent problems in lung cancer research.

PDE1A (phosphodiesterase 1A) is a subtype of PDE1, a member of the PDE family, in addition to PDE1B and PDE1C. PDE1A is mainly composed of Ca2+ and calmodulin (CaM), and its function is mainly through the binding of calcium-calmodulin to the CaM domain of PDE1 to relieve the N-terminal auto-inhibition of the catalytic site, thereby promoting enzyme activity. As the earliest discovered isoenzyme, PDE1 can simultaneously hydrolyze cGMP and cAMP. PDE1A is commonly distributed in the vascular smooth muscle system and has a tonic regulation function, while PDE1B is mainly found in the nervous system and is related to memory and immune regulation. PDE1C is distributed in both systems and participates in the biological function of the vascular smooth muscle system.

PDE1A can hydrolyze cAMP and reduce its intracellular expression, thereby regulating various physiological processes in the human body. Reducing the expression of PDE1A can attenuate the proliferation of leukemia cells, affect the cell cycle, and induce apoptosis of leukemia cells. Tehranolide is a natural sesquiterpene lactone with a peroxide group. Noori et al. found that it can inhibit the activity of CaM, further inhibit the activation of PDE1A, lead to the accumulation of intracellular cAMP and activation of PKA, and finally inhibit the proliferation of cancer cells.

Therefore, to explore the specific role and molecular mechanism of PDE1A in the occurrence and development of non-small cell lung cancer, develop new early diagnostic markers and feasible therapeutic targets, and provide a potential clinical diagnosis and treatment background for the diagnosis and treatment of patients with non-small cell lung cancer.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a PDE1A, a kit and application for tumor therapy target and diagnostic biomarker.

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

According to a second aspect of the embodiments of the present invention, there is provided a kit for detecting the expression level of tumor markers in a sample, comprising a molecular probe pair specifically complementary to PDE1A according to claim 1 and/or used for amplification The primer pair of PDE1A as claimed in claim 1.

Further, the primer pair is selected from at least one of the following primer pairs 1-9;

The base sequence of primer pair 1 is shown in SEQ ID NO.2-3; the base sequence of primer pair 2 is shown in SEQ ID NO.4-5; the base sequence of primer pair 3 is shown in SEQ ID NO.6 ～7; the base sequence of primer pair 4 is shown in SEQ ID NO.8～9; the base sequence of primer pair 5 is shown in SEQ ID NO.10～11; the base sequence of primer pair 6 is shown in SEQ ID NO. 12 to 13; the base sequence of primer pair 7 is shown in SEQ ID NO. 14 to 15; the base sequence of primer pair 8 is shown as SEQ ID NO. 16 to 17; the base sequence of primer pair 9 is shown in Shown in SEQ ID NO. 18-19.

Further, the molecular probe pair is selected from at least one of the following molecular probe pairs 1-7;

The base sequence of molecular probe pair 1 is shown in SEQ ID NO. 20-21; the base sequence of molecular probe pair 2 is shown in SEQ ID NO. 22-23; the base sequence of molecular probe pair 3 is shown in SEQ ID NO. The sequence is shown in SEQ ID NO.24-25; the base sequence of molecular probe pair 4 is shown in SEQ ID NO.26-27; the base sequence of molecular probe pair 5 is shown in SEQ ID NO.28-29 The base sequences of molecular probe pair 6 are shown in SEQ ID NO. 30-31; the base sequences of molecular probe pair 7 are shown in SEQ ID NO. 32-33.

Further, the detection sample includes at least one of non-small cell lung cancer tissue and non-small cell lung cancer adjacent tissue.

According to the third aspect of the embodiments of the present invention, there is provided the use of the PDE1A described in the first aspect in the preparation of a medicament for preventing and/or treating tumors.

Further, the tumor includes lung cancer, preferably, the tumor includes non-small cell lung cancer.

Further, the tumors include primary tumors and metastases.

According to a fourth aspect of the embodiments of the present invention, there is provided an application of a reagent for detecting the expression level of a tumor marker in a sample in preparing a kit for detecting non-small cell lung cancer, the tumor marker PDE1A base The sequence is shown in SEQ ID No.1.

According to a fifth aspect of the embodiments of the present invention, there is provided a medicament for preventing and/or treating tumors, wherein the medicament uses the PDE1A of the first aspect as an active ingredient.

The technical solutions provided by the embodiments of the present application may include the following beneficial effects:

The present invention detects the expression level of PDE1A, a marker, and utilizes the differential expression of this marker in non-small cell lung cancer tissue and its adjacent tissue, which can be used for the diagnosis, prognosis evaluation or evaluation of non-small cell lung cancer. Screen medicine.

It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the present application.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description serve to explain the principles of the application. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. Other related figures are obtained from these figures.

Fig. 1 is a graph showing the effect of PDE1A on the overall survival of NSCLC patients provided in the 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 influence of PDE1A expression on the survival period of NSCLC patients;

Figure 2 is a diagram showing the PDE1 involved in regulating the migration and motility of NSCLC cells and the process of EMT provided by the embodiment of the present invention, A is the result of the Transwell experiment to detect the changes in the migration and motility of NCI-H1299 cells after silencing PDE1A, PDE1B, and PDE1C; B is the result of the A experiment result Statistical analysis chart;

3 is a graph showing the results of the protein change of the transfer inhibitor E-cadherin after A549 cells silenced PDE1 by Western blot detection provided in the embodiment of the present invention;

Figure 4 is a graph of the migration and motility of parental cells T0 and highly metastatic NSCLC cells T3 provided in the example of the present invention; A is a schematic diagram of the method for constructing highly metastatic T3 cells in a Transwell chamber; B is NCI-H1299, A549 cells T0 generation and Representative graph of the migration ability experiment of T3 cells;

Figure 5 is a graph showing the results of Western blot detection of the expression of PDE1A and metastasis-related proteins in NCI-H1299 and A549 parental cells T0 and highly metastatic cells T3 according to the embodiment of the present invention;

Figure 6 is the qRT-PCR detection of the mRNA expression of the transfer-related proteins in parental cells T0 and highly metastatic cells T3 provided in the embodiment of the present invention; mRNA expression of E-cadherin, N-cadherin and PDE1A in T0 and T3 of ANCI-H1299 cells level; B is the mRNA level result of E-cadherin, transcription factor Slug and PDE1A in T0 and T3 of A549 cells;

Figure 7 is a graph showing the expression results of PDE1A in HELF and NSCLC cells provided in the embodiment of the present invention;

Figure 8 is a graph showing the result of the reduced migration ability of NSCLC cells after silencing PDE1A provided by the examples of the present invention; A is the detection of siRNA silencing efficiency of PDE1A in A549, NCI-H1299 cells; B is Transwell assay to detect the effect of silencing PDE1A on the migration of A549, NCI-H1299 cells The graph of the effect of ability; C is the statistical analysis graph of the experimental results of B; D is the graph of the effect of the scratch test to detect the effect of silencing PDE1A on the wound healing ability of A549 and NCI-H1299; E is the statistical analysis graph of the experimental results of D;

Fig. 9 is a graph showing the result of the reduced invasive ability of NSCLC cells after silencing PDE1A in the example of the present invention; A is a representative graph of the invasion of A549 and NCI-H1299 cells after silencing PDE1A; B is a statistical analysis graph of the experimental results of A

Figure 10 is a diagram showing the results of Western blot detection of changes in EMT-related proteins in NSCLC cells after silencing PDE1A provided in the embodiment of the present invention;

Figure 11 is a graph showing the results of qRT-PCR detection of the mRNA changes of PDE1A, N-cadherin, and Snail after silencing PDE1A provided in the embodiment of the present invention;

Figure 12 is a graph showing the ability of PDE1A to promote the migration of NSCLC cells provided by the examples of the present invention; A shows the overexpression efficiency of PDE1A in A549, NCI-H1299 and NCI-H460 cells detected by Western blot, and B shows the migration results of PDE1A overexpression in three NSCLC cells; C is the statistical analysis diagram of the experimental results of B;

Fig. 13 is the PDE1A overexpression wound healing experiment result graph and experiment statistical graph provided in the embodiment of the present invention;

FIG. 14 is a result diagram and an experimental statistical diagram of the PDE1A overexpression invasion experiment provided in the embodiment of the present invention;

Figure 15 is a diagram showing the results of Western blot detection of changes in EMT and metastasis-related proteins of A549, NCI-H1299, and NCI-H460 cells after PDE1A overexpression provided in the embodiment of the present invention;

Figure 16 is the result of overexpression of PDE1A to promote the EMT process of NSCLC cells provided in the example of the present invention; A is the result of qRT-PCR detection of the mRNA changes of PDE1A, N-cadherin and Slug after NCI-H1299 cells overexpressed PDE1A; B is qRT-PCR detection of the mRNA changes of PDE1A, N-cadherin and Slug after NCI-H460 cells overexpressed PDE1A;

Figure 17 is the Western blot that provides the embodiment of the present invention to detect the protein expression level of PDE1A, and Transwell experiment detects the effect of PDE1A on NCI-H1299 cell migration and motility in vitro results figure;

Figure 18 is a graph showing the results of PDE1A promoting the transfer of NCI-H1299 cells in nude mice provided in the example of the present invention; A is the injection of 200×104 NCI-H1299 cells into each nude mouse by tail vein injection. After 60 days, The mice were dissected, the lungs of the mice were separated, and fixed with Bauer's fixative, and the number of metastases in each group was observed and counted; B is the map of the metastases in the lung tissue of the mice detected by H&E staining; C is the statistical analysis control group And the results of lung metastases in nude mice overexpressing PDE1A group;

Figure 19 is a graph showing the inhibition of NCI-H1299 cell migration in nude mice by shPDE1A provided in the example of the present invention; A is the injection of 200×104 NCI-H1299 cells into each nude mouse by tail vein injection, and 60 days later, dissection For mice, the lungs of mice were separated and fixed with Bauer's fixative, and the number of metastases in each group was observed and counted. B is the result of statistical analysis of A;

Figure 20 is a graph of the results of H&E staining detection of metastases in mouse lung tissue provided by the embodiment of the present invention;

Fig. 21 is the molecular function result diagram of Gene Ontology enrichment analysis target protein provided in the embodiment of the present invention;

Figure 22 is a map of the protein that is bound to PDE1A detected by the silver staining experiment provided in the embodiment of the present invention;

Figure 23 is an immunoprecipitation experiment provided in the embodiment of the present invention to detect the protein interaction diagram of PDE1A and YTHDF2;

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

Figure 25 is a graph of the results of YTHDF2 reversing the migration of NCI-H1299 cells promoted by PDE1A provided in the example of the present invention; A is a graph of the results of silencing YTHDF2 on PDE1A to promote the migration of NCI-H1299 cells; B is a statistical analysis graph of A; C is a graph of silencing YTHDF2 on PDE1A The graph of the effect of promoting the invasion ability of NCI-H1299 cells; D is the statistical analysis graph of C;

Figure 26 is a graph showing the results of the RNA immunoprecipitation-qPCR assay provided in the embodiment of the present invention to detect the enrichment of SCOS2 mRNA in anti-PDE1A precipitates and anti-YTHDF2 precipitates;

Figure 27 is a graph of the results of overexpressing PDE1A and YTHDF2 after transfecting METTL3 siRNA in NCI-H1299 cells provided in the embodiment of the present invention, collecting mRNA 48 hours after transfection, and detecting the mRNA change of SOCS2 by qRT-PCR;

Figure 28 shows the overexpression of PDE1A/YTHDF2 in NCI-H1299 cells and then transfected siRNA of YTHDF2/PDE1A or the siRNA of YTHDF2 and PDE1A simultaneously transfected in NCI-H1299 cells provided in the example of the present invention, mRNA was collected 48 hours after transfection, and detected by qRT-PCR The result of mRNA change of SOCS2;

Figure 29 shows the results of the degradation of SOCS2 mRNA affected by PDE1A provided in the example of the present invention; A is the analysis of SOCS2 mRNA after silencing PDE1A in A549 cells after treatment with actinomycin D (5 μg/ml) at 0h, 3h and 6h Half-life result graph; B is the result graph of SOCS2 mRNA expression analysis after silencing PDE1A in A549 cells, treated with actinomycin D (5μg/ml) at 0h, 3h and 6h;

Figure 30 shows the results of the degradation of SOCS2 mRNA affected by PDE1A provided in the example of the present invention; A is the analysis of SOCS2 after silencing PDE1A in NCI-H1299 cells at 0h, 3h and 6h with actinomycin D (5μg/ml) treatment The mRNA half-life of ; B is the result of analysis of SOCS2 mRNA expression after silencing PDE1A in NCI-H1299 cells at 0 h, 3 h and 6 h with actinomycin D (5 μg/ml) treatment.

Detailed ways

Exemplary embodiments will be described in detail herein, examples of which are illustrated in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings refer to the same or similar elements unless otherwise indicated. The implementations described in the following exemplary examples are not intended to represent all implementations consistent with this application. Rather, they are merely examples of apparatus and methods consistent with some aspects of the present application as recited in the appended claims.

The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to limit the application. As used in this application and the appended claims, the singular forms "a," "the," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will also be understood that the term "and/or" as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

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

The above-mentioned tumor marker PDE1A belongs to RNA tumor markers.

As used herein, "detection" may refer to detection of a sample for the diagnosis, auxiliary diagnosis, or prognostic assessment of non-small cell lung cancer.

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

In some embodiments, the reagents include molecular probes that specifically pair complementary to the PDE1A of the first aspect or a primer pair for amplifying the PDE1A of the first aspect.

Preferably, the reagents include: primer pair 1 for detecting PDE1A.

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

It should be noted that a primer pair usually includes an upstream primer and a downstream primer, and "the base sequence of primer pair 1 is shown in SEQ ID NO. 2-3" herein refers to the base sequence of the upstream primer in the primer pair, as shown in SEQ ID NO. As shown in NO.2, the base sequence of the downstream primer is shown as SEQ ID NO.3.

Preferably, the sample comprises a biological tissue sample.

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

The embodiment of the present invention also provides an application of a reagent for detecting the expression level of a tumor marker in a sample in screening a drug for the treatment of non-small cell lung cancer, where the tumor marker includes PDE1A whose base sequence is shown in SEQID No.1 in sequence , the application is not directly aimed at the diagnosis or treatment of the disease.

The inventors found that the expression of the above-mentioned tumor marker PDE1A in the cancer tissue and paracancerous tissue of patients with non-small cell lung cancer is differentiated. The diagnosis and prognostic assessment of lung cancer play an important role in drug development and prolonged prognosis of non-small cell lung cancer.

The above-mentioned markers can be used for the diagnosis and prognosis evaluation of non-small cell lung cancer. Therefore, through the detection of the above-mentioned markers, the development of specific targeted drugs for patients with non-small cell lung cancer can be accelerated. The PDE1A, the kit and the application for tumor therapy targets and diagnostic biomarkers provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

PDE1A expression is associated with prognosis in lung cancer patients:

First of all, through the search and analysis of the TCGA database in this example, it is found that the risk prognosis of patients with high PDE1A expression in lung cancer patients is higher than that of patients with low PDE1A expression, and the survival time 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 the high expression of PDE1A is closely related to the poor prognosis of lung cancer patients.

Example 2

Migration of PDE1A and NSCLC cells is associated with epithelial-mesenchymal transition:

In this example, siRNA gene silencing experiments were performed on three subtypes of PDE1, PDE1A, PDE1B and PDE1C, respectively, on NSCLC cells NCI-H1299 before the experiment, and then the migration ability of the cells was detected by Transwell method. The results showed that silencing of PDE1 could inhibit the migration and motility of NCI-H1299 cells, among which, after silencing PDE1A, the migration and motility of NCI-H1299 cells was inhibited most significantly (see Figure 2). In addition, this Example also detected the changes of the transfer inhibitor E-cadherin after A549 cells silenced PDE1, and found that the protein of E-cadherin increased most obviously in the silenced PDE1A group (see Figure 3). It is suggested that the expression of PDE1A may be related to the metastasis of NSCLC.

In the early stage of this example, NSCLC/T3 cell line was constructed by 3 times of Transwell chamber enrichment method, and the migration and motility of different metastatic cell lines were studied. Compared with T0), the highly metastatic cells (NCI-H1299/T3, A549/T3) had stronger migratory motility (see Figure 4). In order to further study the correlation between PDE1A and the metastasis of NSCLC cells, this example analyzes cells with different migration abilities from two levels of protein and mRNA. The results showed that the high migration ability cells of A549 and NCI-H1299 had higher protein expressions of the pro-metastasis-related proteins N-cadherin, Vimentin and Snail, but lower expression of the metastasis inhibitory protein E-cadherin. Interestingly, increased levels of phosphorylated proteins of PDE1A and transcription factor smad 3 were also observed in cells with high metastatic potential in this example compared to NSCLC with low metastatic potential (see Figure 5). At the same time, we also further verified this result from the mRNA level (see Figure 6), the above data suggest that PDE1A is likely to have a potential role in promoting metastasis.

Example 3

Differences in the expression of PDE1A in HELF and NSCLC cells: In order to evaluate the expression of PDE1A in NSCLC cells, normal human lung fibroblasts and different NSCLC cell lines were selected for research in this example. Western blot experiments showed that compared with normal human lung fibroblasts HELF, PDE1A was highly expressed in NSCLC cells A549, moderately expressed in NCI-H1299, lower in NCI-H460, and almost not expressed in HELF ( See Figure 7). This suggests that the expression level of PDE1A protein is related to the malignancy of NSCLC cells.

Example 4

PDE1A promotes the migration, invasion ability of NSCLC cells:

First, the migration ability of NSCLC cells before and after silencing PDE1A was detected by Transwell assay, and 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); Effects of silencing PDE1A on the wound healing ability of NSCLC cells. The results showed that silencing of PDE1A reduced the wound healing ability of A549, NCI-H1299 cells (see Figure 9). The Matrigel matrigel-coated chamber was used for the invasion assay, and it was found that after silencing PDE1A, the number of cells passing through Matrigel was significantly lower than that of the negative control group (see Figure 10). The above results indicate that silencing PDE1A can inhibit the migration and invasion of NSCLC cells.

During the process of tumor metastasis, the cytoskeleton or movement-related proteins of tumor cells will change. An important process in tumor metastasis is epithelial-mesenchymal transition (EMT), which is accompanied by changes in related proteins, such as metastasis suppressor protein E-cadherin and pro-metastatic proteins Fibronectin, N-cadherin, etc. Therefore, we further studied the silencing of Effects of PDE1A on these proteins. The results showed that after silencing PDE1A, the protein levels of the transcription factors Snail and Slug decreased, and 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 detected. The results of qRT-PCR experiments showed that silencing PDE1A inhibited the expression of the transcription factor Snail and the mRNA expression of the pro-transfer protein N-cadherin (see Figure 12). The above protein expression and mRNA expression levels were the same as those in this example. The transfer motion phenomenon observed earlier is consistent.

In order to further study the effect of PDE1A on the ability of metastasis and invasion of NSCLC cells, in this example, PDE1A was overexpressed in A549, NCI-H1299 and NCI-H460 cells, and the migration and invasion abilities of the cells were detected. As shown in the figure, overexpression of PDE1A significantly increased the number and distance of cell migration, that is, the cell migration ability was enhanced (see Figure 13-14). Matrigel Matrigel-coated chamber Transwell experiments further showed that overexpression of PDE1A could significantly enhance cell migration. The invasion ability of NSCLC cells (Figure 10 (see Figure 15). The above results show that PDE1A can promote the migration and invasion ability of NSCLC cells.

Next, in this example, the changes of transfer-related proteins after overexpression of PDE1A were detected by Western blot. The results showed that the overexpression of PDE1A would increase the phosphorylation levels of transcription factors STAT3 and smad3, decrease the expression of the metastasis suppressor protein E-cadherin, and increase the expression of the transfer-promoting protein N-cadherin (see Figure 16). In addition, the PDE1A background was selected. In NCI-H1299 and NCI-H460 cells with low protein expression, PDE1A overexpression experiment was performed, and then the mRNA changes of PDE1A and EMT-related proteins and transcription factors were detected. The results showed that overexpression of PDE1A can upregulate the mRNA of N-cadherin and Slug (See Figure 17). The above suggests that PDE1A is involved in the EMT process of NSCLC cells and plays an important role in its metastasis.

Example 5

The effect of PDE1A on the metastasis of NCI-H1299 cells in nude mice:

Through the previous studies, this example found that PDE1A has a significant promoting effect on the migration and invasion ability of NSCLC cells. However, the occurrence and development of tumors are determined by the tumor cells themselves and the tumor microenvironment. In order to further study the effect of PDE1A on the metastasis and colonization ability of NSCLC cells in vivo, the NCI-H1299 cell line (PDE1A stably overexpressing PDE1A) was constructed in this example. : pLenti-CMV-PDE1A-Flag) and control (vect: pLenti-CMV-PCIG3-Flag). The results showed that the NCI-H1299 cells stably overexpressing PDE1A had stronger migration ability (see Figure 18), that is, the stable overexpression model was successfully constructed. In addition, a nude mouse model was constructed by means of tail vein injection, which further confirmed the pro-metastatic effect of PDE1A in vivo. After dissecting the various organs of the mice, the lungs of nude mice were stained with encapsulated liquid, and it was found that the number of foci in the lungs of nude mice with stable overexpression of PDE1A cells was more than that of the control group. The results showed that there were obvious metastases in the lungs of the nude mice in the PDE1A overexpression group (see Figure 19). Therefore, PDE1A can promote NSCLC cell metastasis in nude mice.

In order to further study the effect of PDE1A on the transfer of NCI-H1299 cells in nude mice, the shPDE1A model of silencing PDE1A was also constructed in this example, and the NCI-H1299 cell lines stably transfected with shPDE1A and control plko. inoculated into nude mice. The results showed that shPDE1A inhibited the metastasis of NCI-H1299 cells in nude mice. The number of metastases in the lungs of nude mice in the shPDE1A group was significantly less than that in the control plko.1 group, and the results of H&E staining showed that there were no obvious metastases in the shPDE1A group (see Figure 20-21). The above results indicated that shPDE1A inhibited the metastasis of NCI-H1299 cells in nude mice.

Example 6

PDE1A binds to YTHDF2 and reverses PDE1A to promote NSCLC cell migration and invasion:

In order to further search for the downstream target molecules involved in regulating PDE1A to promote the metastasis of non-small cell lung cancer cells, in this example, after plasmid transfection of A549 cells to overexpress PDE1A, samples were collected for immunoprecipitation experiments. The samples were stained with Maas Brilliant Blue and sent for protein profiling. According to the Score in the mass spectrometry results (see Table 1) and the Gene Ontology enrichment analysis (see Figures 22-23), we found that the m6A-binding protein (GO: 1990247) had a larger enrichment fold, and the binding protein had the most significant enrichment. , comprehensive silver staining results, and finally selected YTHDF2 as the next research object.

Table 1 Protein profiling analysis is highly correlated with PDE1A expression

PDE1A is mainly distributed in the cytoplasm of cells. Generally, the proteins in the cytoplasm often realize their biological functions by combining with proteins. Therefore, immunoprecipitation experiments were used to detect whether PDE1A has protein binding effect with YTHDF2. As a result, it was found that PDE1A was able to interact with YTHDF2 (see Fig. 24). Then we examined the effect of silencing YTHDF2 on PDE1A-promoted NSCLC cell migration and invasion, and found that YTHDF2 could reverse PDE1A-promoted NSCLC cell migration and enhanced invasion (see Figures 25-26).

YTHDF2 is one of the currently known readers during m6A modification. YTHDF2 recognizes m6A modifications on mRNAs that direct the CCR4-NOT protein complex to degrade the recognized mRNAs. Some literatures show that SOCS2, as a downstream target of YTHDF2, can be degraded by YTHDF2 for its mRNA. Therefore, RNA immunoprecipitation-qPCR technology was used to verify whether there is RNA binding between PDE1A and YTHDF2 and SOCS2. The SOCS2 mRNA enriched by PDE1A antibody and YTHDF2 antibody was significantly increased, which indicated that PDE1A and YTHDF2 were combined with SOCS2 mRNA (see Figure 27). In this example, fluorescence quantitative PCR technology was used to detect changes in the expression levels of SOCS2 caused by changes in the expressions of METTL3, YTHDF2 and PDE1A, and it was found that affecting the expressions of METTL3, YTHDF2 and PDE1A could cause changes in the mRNA levels of SOCS2 (see Figure 28 ). -29). In addition, mRNA stability assays showed that SOCS2 mRNA expression was elevated and SOCS2 mRNA half-life continued to be prolonged by PDE1A silencing in A549 and NCI-H1299 cells (see Figure 30).

The primer pair sequences and probe base pair sequences described in the above embodiments are shown in Table 2 in detail.

Table 2:

experimental method:

1. Cell Culture

Human non-small cell lung cancer cells A549, NCI-H1299, NCI-H460 and human umbilical vein endothelial cells HUVEC were cultured in medium with 10% FBS and 90% RPMI 1640. All cells were cultured in a cell incubator at 37°C with 5% CO ₂ .

2. Transwell experiment

(1) Cell Migration Experiment

Lung cancer cells with high and low expression of circPOLK were serum-starved overnight, digested and counted, and inoculated into transwell chambers at a density of 2×10 ⁴ /well, with 200 μl of serum-free culture medium per well. 600 μl of culture medium containing 20% serum was added to the corresponding 24-well plate. After the cells were incubated for 24 hours, the chamber was stained with 1% crystal violet for 30 min in the dark, then the cells inside the chamber were washed with 1×PBS, and the cells that migrated to the lower surface of the chamber were photographed and counted under a microscope.

(2) Cell invasion assay

Matrigel was thawed at 4°C in advance, and the chamber, 24-well plate and pipette tips used were pre-cooled at -20°C. Dilute Matrigel 1:20 with cell culture medium, add 100 μl of Matrigel to each chamber, place the chamber in a 24-well plate and place it in a 37°C incubator for 30 minutes, take out the 24-well plate, and discard the uncoagulated Matrigel in the chamber. , and 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 inoculated into a transwell chamber with Matrigel gel at a density of 3×10 ⁴ /well, and each well of serum-free medium was 200 μl. 600 μl of culture medium containing 20% serum was added to the corresponding 24-well plate. After the cells were incubated for 24 hours, the chamber was stained with 1% crystal violet for 30 min in the dark, and then the cells inside the chamber were washed with 1×PBS, and the cells that invaded the lower surface of the chamber were photographed and counted with a microscope.

3. Wound Healing Experiment

The cells were seeded in a six-well plate at a density of 2×10 ⁵ /well, and after the cells adhered, the desired plasmids or siRNA were transfected. When the cell confluence reaches 80-100%, scratch the cell surface with a pipette, wash off the cells dropped on the surface with PBS, replace with a new medium, and take a microscope to take a picture of the wound distance for 0h. After 24h, use PBS The six-well plate was washed to remove non-adherent cells, and the wound distance for 24 hours was photographed with a microscope to calculate the wound healing.

4. Western blot method

(1) Extraction and quantification of protein samples

①Extraction: Collect the pretreated cells, centrifuge at 2000rpm for 4min, discard the supernatant, transfer the pellet to an EP tube, centrifuge at 2000rpm for 4min, and discard the supernatant. Add whole cell lysate according to the number of cells, vortex every 10min,

Perform 3 times, fully mix the cells and the lysate, centrifuge at 12,000 rpm and 4° C. for 30 min, and collect the supernatant, which is the protein sample.

②Quantitative: BSA (2mg/ml) was diluted by half and half with deionized water as the standard curve, and the sample protein was diluted 10 times with deionized water. Take 5 μl of BSA solution and sample protein with different concentrations respectively, add 200 μl of BCA reagent mixture (solution A: solution B = 50:1), mix well and place at 37°C for 30 min, read with a microplate reader Absorbance at wavelength 562 nm. According to the formula of the obtained standard curve, the concentration of the sample protein was calculated, and the protein was calibrated according to a certain amount.

③Direct loading method

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

(2) Western blot

①Gel electrophoresis: Prepare 500ml of 1×Running Buffer, and load protein samples in a certain order and in a certain amount. First adjust the voltage to 70V. When the sample runs to the separation gel, increase the voltage to 110V. When the sample is 1cm away from the bottom of the gel, stop electrophoresis.

②Membrane transfer: Prepare 900ml of 1×Transfer Buffer, activate the PVDF membrane of suitable size with 100ml of absolute ethanol for 1min, and add the prepared Transfer Buffer. According to the protein size required for the experiment, cut the glue into the corresponding size, transfer the membrane in the way of black glue and white film, 330mA horizontal current, and transfer the membrane in an ice-water bath for 90min.

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

④Incubate the secondary antibody: Recover the primary antibody and wash the strip with T-PBS 3 times for 10 min each time. The horseradish peroxidase-labeled secondary antibody corresponding to the primary antibody was added, incubated at room temperature for 90 min on a shaker, discarded the secondary antibody, and washed 3 times with T-PBS.

⑤Exposure: Prepare the exposed things in advance, incubate the strips with ECL chromogenic solution (1:1 solution A and solution B in the ECL kit) in the dark for 1 min, transfer to the exposure box, and press X-ray film in the darkroom , a short time of 5-20s (depending on the fluorescence intensity of the strip), a long time of 30min, the film is taken out, placed in the developer solution for 1.5min, rinsed with tap water, placed in the fixer solution for 1.5min, and imaged.

5. RNA extraction and reverse transcription

Discard the supernatant from the pre-treated six-well plate cells, rinse twice with pre-cooled PBS, add 1 ml of Trizol to each well, and repeatedly pipet until the solution is transparent. The cells were transferred to a 1.5 ml RNase-free EP tube, 200 μl of chloroform was added, vigorously shaken for 30 s, stood at 4° C. for 2 min, and centrifuged at 12,000 rpm and 4° C. for 10 min. Pipette 450 μl of the supernatant into a new EP tube, add an equal amount of isopropanol, gently invert 5 times, stand on ice for 10 min, centrifuge at 12000 rpm for 15 min at 4°C. The supernatant was discarded, the pellet was gently washed with 500 μl of pre-chilled 75% ethanol, and centrifuged at 8000 rpm for 5 min at 4°C. Repeat the washing, place in a ventilated place, and after the ethanol evaporates, add 20 μl of DEPC water to dissolve the mRNA, and measure the mRNA concentration.

The mRNA was reverse transcribed to complementary DNA (cDNA) using the TransStart Top Green qPCR SuperMix (Lot#40426) kit in full gold.

6. qRT-PCR

The qRT-PCR system was 10 μl, 5 μl of 2×QuantiTect SYBR Green PCR Master Mix, 1 μl of template cDNA, 0.6 μl of target forward and reverse primers, and 2.8 μl of DEPC water. The reaction conditions were pre-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, 39 cycles.

7. Statistical analysis

Statistical analysis was tested using t-test and p<0.05 was considered to be significantly different (*P<0.05, **P<0.01 and ***P<0.001). Each experiment was repeated at least three times.

Other embodiments of the present application will readily occur to those skilled in the art upon consideration of the specification and practice of what is disclosed herein. This application is intended to cover any variations, uses or adaptations of this application that follow the general principles of this application and include common knowledge or conventional techniques in the technical field not disclosed in this application . The specification and examples are to be regarded as exemplary only, with the true scope and spirit of the application being indicated by the claims.

It is to be understood that the present application is not limited to the precise structures described above and illustrated in the accompanying drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.

sequence listing

<110> Zhejiang University City College

<120> A PDE1A and kit and application for tumor therapy target and diagnostic biomarker

<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 aaatttcgga 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 gtttcttgcc 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 atttatccaa 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 caaccatggt ggcccagtca caaataggtt tcatcgattt catagtagag 1440

ccaacatttt ctcttctgac agactcaaca gagaaaattg ttattcctct tatagaggaa 1500

gcctcaaaag ccgaaacttc ttcctatgtg gcaagcagct caaccaccat tgtggggtta 1560

ccattgctg 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> 23

<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> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 20

ccacgugagu gcagcuuaut t 21

<210> 21

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 21

auaagcugca cucacguggt t 21

<210> 22

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 22

ccagcagcug ucaucguaat t 21

<210> 23

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 23

uuacgaugac agcugcuggt t 21

<210> 24

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 24

gcuggaagcc gucuacauat t 21

<210> 25

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 25

uauguagacg gcuuccagct t 21

<210> 26

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 26

gcugcagcua uccagauut t 21

<210> 27

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 27

aaucauggau agcugcagct t 21

<210> 28

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 28

gcuucagugg uagaucuuat t 21

<210> 29

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 29

uaagaucuac cacugaagct t 21

<210> 30

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 30

ccagcuguua uugaggcaut t 21

<210> 31

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 31

augccucaau aacagcuggt t 21

<210> 32

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 32

aaggacguuc ccaauagcca a 21

<210> 33

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 33

uuggcuauug ggaacguccu u 21

Claims (10)

1. PDE1A for use as a tumor therapy target and a diagnostic biomarker, wherein the base sequence of PDE1A is as shown in SEQ ID No. 1.

2. A kit for detecting the expression level of a tumor marker in a sample, comprising a pair of molecular probes specifically complementary-paired to PDE1A of claim 1 and/or a pair of primers for amplifying PDE1A of claim 1.

3. The kit according to claim 2, wherein the primer pair is at least one selected from the group consisting of the following primer pairs 1 to 9;

wherein the base sequence of the primer pair 1 is shown as SEQ ID NO. 2-3; the base sequence of the primer pair 2 is shown as SEQ ID NO. 4-5; the base sequence of the primer pair 3 is shown as SEQ ID NO. 6-7; the base sequence of the primer pair 4 is shown as SEQ ID NO. 8-9; the base sequence of the primer pair 5 is shown as SEQ ID NO. 10-11; the base sequence of the primer pair 6 is shown as SEQ ID NO. 12-13; the base sequence of the primer pair 7 is shown as SEQ ID NO. 14-15; the base sequence of the primer pair 8 is shown as SEQ ID NO. 16-17; the base sequence of the primer pair 9 is shown in SEQ ID NO. 18-19.

4. The reagent according to claim 2, wherein the molecular probe pair is at least one selected from the group consisting of the following molecular probe pairs 1 to 7;

wherein the base sequence of the molecular probe pair 1 is shown as SEQ ID NO. 20-21; the base sequence of the molecular probe pair 2 is shown as SEQ ID NO. 22-23; the base sequence of the molecular probe pair 3 is shown as SEQ ID NO. 24-25; the base sequence of the molecular probe pair 4 is shown as SEQ ID NO. 26-27; the base sequence of the molecular probe pair 5 is shown in SEQ ID NO. 28-29; the base sequence of the molecular probe pair 6 is shown in SEQ ID NO. 30-31; the base sequence of the molecular probe pair 7 is shown in SEQ ID NO. 32-33.

5. The kit of claim 2, wherein the test sample comprises at least one of non-small cell lung cancer tissue and non-small cell lung cancer paracancerous tissue.

6. Use of PDE1A according to claim 1 for the preparation of a medicament for the prophylaxis and/or treatment of tumors.

7. The use of claim 6, wherein the tumor comprises lung cancer, preferably, the tumor comprises non-small cell lung cancer.

8. The use of claim 6, wherein the tumor comprises primary and metastatic tumors.

9. An application of a reagent for detecting the expression level of a tumor marker in a sample in preparing a kit for detecting non-small cell lung cancer, wherein the base sequence of the tumor marker PDE1A is shown as SEQ ID No. 1.

10. A pharmaceutical agent for preventing and/or treating tumor, which comprises the PDE1A according to claim 1 as an active ingredient.

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