Application of an enhancer RNA FOXA1-eRNA

Abstract

The present application relates to the field of tumor molecular markers, and in particular to an application of enhancer RNA FOXA1-eRNA; the application includes: using the core nucleotide sequence of enhancer RNA FOXA1-eRNA as a molecular marker for prostate cancer; because enhancer RNA has the characteristic of regulating the expression of surrounding protein-coding genes, it can be used as a marker related to prostate cancer, filling the gap in the association between eRNA and prostate cancer.

Description

Technical Field

[0001] The present application relates to the field of tumor molecular markers, and in particular to the application of an enhancer RNA FOXA1-eRNA. Background Art

[0002] Prostate cancer is one of the most common cancers in men worldwide, ranking second in incidence among solid malignant tumors in men. Annually, there are approximately 1.41 million new cases and 375,000 new deaths, with incidence increasing dramatically among men over 70 years old. However, the development of prostate cancer is an extremely complex, multi-step process, involving multiple genes, multiple tumor signaling pathways, and their interactions, the precise mechanisms of which remain largely unknown.

[0003] With the rapid development of next-generation high-throughput sequencing technology, more evidence has confirmed the close relationship between epigenetic regulation and tumor progression. Enhancers, as one of the key components of epigenetic regulation, play a crucial role in tumor progression. Current studies have shown that the more active an enhancer, the more open its chromatin structure, and the more likely it is to transcribe into a new RNA, known as enhancer RNA (eRNA). Multiple regulatory mechanisms exist between eRNA and downstream genes, enabling eRNA to regulate the expression of surrounding protein-coding genes, thereby influencing the development and progression of diseases. This is one of the key biological mechanisms that determine organismal development and the occurrence and progression of diseases. However, there have been no reports of eRNAs being associated with prostate cancer. Therefore, it is important to provide an application of the enhancer RNA FOXA1-eRNA to fill the gap in existing research on the association between eRNA and prostate cancer. Summary of the Invention

[0004] The present application provides an application of enhancer RNA FOXA1-eRNA to fill the gap in the existing technology regarding the association between eRNA and prostate cancer.

[0005] In a first aspect, the present application provides an application of enhancer RNA FOXA1-eRNA in a molecular marker for prostate cancer, the application comprising:

[0006] The core nucleotide sequence of enhancer RNA FOXA1-eRNA was used as a molecular marker for prostate cancer.

[0007] Optionally, the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is shown in SEQ ID NO.1.

[0008] In a second aspect, the present application provides a use of enhancer RNA FOXA1-eRNA in preparing a detection kit for diagnosis and prognosis of prostate cancer, the use comprising:

[0009] The core nucleotide sequence of enhancer RNA FOXA1-eRNA is used to prepare a detection agent in a detection kit, wherein the detection agent is used to prepare a detection kit for diagnosing and prognosing prostate cancer.

[0010] Optionally, the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is shown in SEQ ID NO.1.

[0011] Optionally, the detection agent includes a fluorescent quantitative PCR detection reagent, and the fluorescent quantitative PCR detection reagent includes an amplification primer set to amplify the core nucleotide sequence of the enhancer RNA FOXA1-eRNA.

[0012] Optionally, the amplification primer set includes a first amplification primer, and the nucleotide sequence of the first amplification primer is shown as SEQ ID NO.2.

[0013] Optionally, the amplification primer set further includes a second amplification primer, and the nucleotide sequence of the second amplification primer is shown in SEQ ID NO.3.

[0014] In a third aspect, the present application provides a use of enhancer RNA FOXA1-eRNA in the preparation of a drug for treating prostate cancer, the use comprising:

[0015] The core nucleotide sequence of enhancer RNA FOXA1-eRNA is used to prepare an inhibitor for inhibiting the transcription and / or translation process of enhancer RNA FOXA1-eRNA; wherein the core nucleotide sequence of enhancer RNA FOXA1-eRNA is shown in SEQ ID NO.1.

[0016] Optionally, the inhibitor includes at least one of nucleic acid molecules, lipids, small molecule chemical drugs, antibody drugs, polypeptides and interfering lentiviruses that can inhibit the normal function of the enhancer RNA FOXA1-eRNA.

[0017] Optionally, the nucleic acid molecule comprises at least one of small interfering RNA, double-stranded short hairpin RNA and antisense oligonucleotide.

[0018] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0019] The present application provides an embodiment of an enhancer RNA FOXA1-eRNA for use as a molecular marker for prostate cancer. By clarifying the transcriptional regulatory mechanism between enhancer RNA and downstream genes, the binding of enhancer RNA and RNA-binding proteins is utilized to enhance the interaction between promoters and enhancers, thereby forming a chromatin loop to regulate the expression levels of surrounding genes and downstream signaling pathways. This allows for clarifying that enhancer RNA can regulate the expression of surrounding protein-coding genes. Utilizing this characteristic, such enhancer RNA can be used as a marker related to prostate cancer, filling the gap in the association between eRNA and prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0022] Figure 1 This is a graph showing the expression of enhancer RNA FOXA1-eRNA in prostate cancer and adjacent tissues provided in the examples of the present application, wherein Adjacent refers to adjacent tissues and Tumor refers to prostate cancer tissues;

[0023] Figure 2 This is a graph showing the expression of the enhancer RNA FOXA1-eRNA in different cell lines provided in the examples of the present application, wherein RWPE-1 is a normal prostate cell line, and PC-3, LNCaP, DU145, 22RV1, and C4-2 are all prostate cancer cell lines;

[0024] Figure 3 Schematic diagram of the experimental results of CCK-8 cell viability assay in which FOXA1-eRNA downregulation inhibits the proliferation of LNCaP prostate cancer cell lines provided in the examples of the present application;

[0025] Figure 4 Schematic diagram of the experimental results of CCK-8 cell viability assay in which FOXA1-eRNA downregulation inhibits the proliferation of 22RV1 prostate cancer cell lines provided in the examples of the present application;

[0026] Figure 5 Figure 2 is a graph showing the results of cell colony formation experiments on different prostate cell lines provided in the examples of this application;

[0027] Figure 6A comparative statistical graph of the results of cell colony formation experiments of different prostate cell lines provided in the examples of this application;

[0028] Figure 7 This is a graph showing the results of EdU fluorescence staining experiments on different prostate cell lines provided in the examples of this application;

[0029] Figure 8 This is a comparative statistical chart of the EdU fluorescence staining experimental results of different prostate cell lines provided in the examples of this application, wherein;

[0030] Figure 9 These are images of tumors of different cell lines in the in vivo experiments in the nude mouse model provided in the examples of this application;

[0031] Figure 10 This is a graph showing the changes in tumor volume over time in different cell lines in an in vivo experiment in a nude mouse model provided in an example of the present application;

[0032] Figure 11 This is a graph showing the change in tumor weight over time of the LNCaP prostate cancer cell line in an in vivo experiment in a nude mouse model provided in an example of the present application;

[0033] Figure 12 This is a graph showing the change in tumor weight over time of the 22RV1 prostate cancer cell line in an in vivo experiment in a nude mouse model provided in an example of the present application;

[0034] Figure 13 This is a data diagram showing the results of the dual luciferase assay provided in the examples of the present application to verify the regulation of FOXA1 transcriptional activity by FOXA1-eRNA;

[0035] Figure 14 This is a graph showing the results of quantitative PCR detection of FOXA1-eRNA and FOXA1 expression in LNCaP prostate cancer cell lines provided in the examples of the present application;

[0036] Figure 15 This is a graph showing the results of quantitative PCR detection of FOXA1-eRNA and FOXA1 expression in the 22RV1 prostate cancer cell line provided in the examples of the present application;

[0037] Figure 16 Schematic diagram of the results of western-blot detection of FOXA1 protein expression provided in the examples of this application;

[0038] Among them, shControl is the FOXA1-eRNA knockdown control plasmid, shFOXA1-eRNA-1 and shFOXA1-eRNA-2 are FOXA1-eRNA knockdown plasmids, and LNCaP and 22RV1 are prostate cancer cell lines. DETAILED DESCRIPTION

[0039] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0040] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0041] The creative thinking of this application is:

[0042] Current research indicates that the more active an enhancer, the more open its chromatin structure, and the more likely it is to transcribe a new RNA, known as enhancer RNA (eRNA). Multiple regulatory mechanisms exist between eRNA and downstream genes, and depending on their distance from the target gene, these regulatory modes are primarily categorized as cis- and trans-regulation. Generally, eRNA binds to RNA-binding proteins, enhancing promoter-enhancer interactions and forming chromatin loops to regulate the expression levels of surrounding genes and downstream signaling pathways.

[0043] Based on the above characteristics, since the driver transcription factor FOXA1 (pioneer TF) is a regulatory factor closely related to the proliferation and differentiation of prostate cancer cells, it is reasonable to speculate that enhancer RNA related to the driver transcription factor FOXA1 can serve as an associated biological marker for prostate cancer.

[0044] The present application provides an application of enhancer RNA FOXA1-eRNA in a prostate cancer molecular marker, the application comprising:

[0045] The core nucleotide sequence of enhancer RNA FOXA1-eRNA was used as a molecular marker for prostate cancer.

[0046] In some optional embodiments, the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is shown as SEQ ID NO.1.

[0047] The examples of the present application provide the specific sequence structure of the core nucleotide sequence that controls the enhancer RNA FOXA1-eRNA, thereby clarifying that the core of the existing enhancer RNA FOXA1-eRNA can also meet the requirements of being a molecular marker for prostate cancer.

[0048] Based on a general inventive concept, the present application also provides an embodiment of a method for preparing a detection kit for diagnosing and prognosing prostate cancer using an enhancer RNA FOXA1-eRNA. The method comprises:

[0049] The core nucleotide sequence of enhancer RNA FOXA1-eRNA is used to prepare a detection agent in a detection kit, wherein the detection agent is used to prepare a detection kit for diagnosing and prognosing prostate cancer.

[0050] This application is based on the application of the above-mentioned enhancer RNA FOXA1-eRNA in a molecular marker for prostate cancer. The specific principles of this application can be referred to the above-mentioned embodiments. Since this application adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0051] In the examples of the present application, by limiting the specific application scenario of the enhancer RNA FOXA1-eRNA in the detection kit to a detection agent, the principle that the enhancer RNA FOXA1-eRNA can be a prostate molecular marker can be clearly utilized, thereby ensuring the detection accuracy of the detection agent.

[0052] In some optional embodiments, the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is shown as SEQ ID NO.1.

[0053] In the examples of the present application, by defining the specific nucleotide sequence of the core of the enhancer RNA FOXA1-eRNA used for the detection agent, the feasibility of the specific nucleotide sequence as a detection agent can be clarified, thereby improving the accuracy of the detection.

[0054] In some optional embodiments, the detection agent includes a fluorescent quantitative PCR detection reagent, and the fluorescent quantitative PCR detection reagent includes an amplification primer set to amplify the core nucleotide sequence of the enhancer RNA FOXA1-eRNA.

[0055] In the examples of the present application, by limiting the specific form of the detection agent, a fluorescent quantitative PCR detection reagent including an amplification primer set is used to amplify the core nucleotide sequence of the enhancer RNA FOXA1-eRNA to ensure sufficient capacity of the detection sample, thereby improving the accuracy of the detection.

[0056] In some optional embodiments, the amplification primer set includes a first amplification primer, and the nucleotide sequence of the first amplification primer is shown as SEQ ID NO.2.

[0057] In the embodiment of the present application, by controlling the specific composition of the first amplification primer in the amplification primer set, it is possible to ensure that the amplification product of the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is sufficient and accurately amplified, thereby ensuring the accuracy of the detection kit.

[0058] In some optional embodiments, the amplification primer set includes a second amplification primer, and the nucleotide sequence of the second amplification primer is shown as SEQ ID NO.3.

[0059] In the embodiment of the present application, by controlling the specific composition of the second amplification primer in the amplification primer set, it is possible to ensure that the amplification product of the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is sufficient and accurately amplified, thereby ensuring the accuracy of the detection kit.

[0060] In some optional embodiments, the amplification primers further include a third amplification primer and a fourth amplification primer, the nucleotide sequence of the third amplification primer is shown as SEQ ID NO.4, and the nucleotide sequence of the fourth amplification primer is shown as SEQ ID NO.5.

[0061] In the embodiment of the present application, the third amplification primer and the fourth amplification primer are introduced to amplify the converter-driving factor FOXA1, thereby ensuring sufficient FOXA1 production.

[0062] Based on a general inventive concept, the present application provides a use of enhancer RNA FOXA1-eRNA in the preparation of a drug for treating prostate cancer, the application comprising:

[0063] The core nucleotide sequence of enhancer RNA FOXA1-eRNA is used to prepare an inhibitor for inhibiting the transcription and / or translation process of enhancer RNA FOXA1-eRNA; wherein the core nucleotide sequence of enhancer RNA FOXA1-eRNA is shown in SEQ ID NO.1.

[0064] This application is based on the application of the above-mentioned enhancer RNA FOXA1-eRNA in a molecular marker for prostate cancer. The specific principles of this application can be referred to the above-mentioned embodiments. Since this application adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0065] In the examples of the present application, the characteristics of the enhancer RNA FOXA1-eRNA as a molecular marker for prostate cancer are utilized, and the specific composition of the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is defined. Thus, through the specific working principle of the enhancer RNA FOXA1-eRNA, the transcription and translation of the enhancer RNA FOXA1-eRNA can be inhibited, thereby ensuring the inhibition of prostate cancer cell proliferation and differentiation, embodying the use of the inhibitor in drugs for the treatment of prostate cancer.

[0066] In some optional embodiments, the inhibitor includes at least one of nucleic acid molecules, lipids, small molecule chemical drugs, antibody drugs, polypeptides and interfering lentiviruses that can inhibit the normal function of enhancer RNA FOXA1-eRNA.

[0067] In the examples of the present application, controlling the specific composition of the inhibitor can ensure that the normal physiological function of the enhancer RNA FOXA1-eRNA is inhibited, thereby ensuring the therapeutic effect of the therapeutic drug.

[0068] In some optional embodiments, the nucleic acid molecule includes at least one of small interfering RNA, double-stranded short hairpin RNA and antisense oligonucleotide.

[0069] In the embodiments of the present application, the specific composition of the nucleic acid molecule is controlled to encompass all stages of the normal function of the enhancer RNA FOXA1-eRNA, thereby ensuring that the inhibitor inhibits the enhancer RNA FOXA1-eRNA.

[0070] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples where specific conditions are not specified are usually measured in accordance with national standards. If there are no corresponding national standards, then the methods are carried out in accordance with general international standards, conventional conditions, or according to the conditions recommended by the manufacturer.

[0071] Example 1

[0072] Real-time quantitative PCR was used to detect the expression levels of FOXA1-eRNA and FOXA1 in prostate cancer tissues and different cell lines. The specific steps are as follows:

[0073] 1. Primer Design: The core 2000bp sequence of FOXA1-eRNA was found in the NCBI gene library as shown in SEQ ID NO. 1; the FOXA1 gene ID is 3169, and the transcript is NM_004496. Primers were designed using Primer software as follows:

[0074] The primers for FOXA1-eRNA are as follows:

[0075] First amplification primer: 5'-CGGGAGGGAGGAGAGTAAATAC-3' (SEQ ID NO. 2);

[0076] Second amplification primer: 5'-TTCGTTTCTCCCTTCGCAAC-3' (SEQ ID NO. 3);

[0077] The primers for FOXA1 are as follows:

[0078] The third amplification primer: 5'-GCAATACTCGCCTTACGGCT-3' (SEQ ID NO. 4);

[0079] Fourth amplification primer: 5'-TACACACCTTGGTAGTACGCC-3' (SEQ ID NO. 5);

[0080] 2. Clinical sample collection: The sample collection process for this application was reviewed and approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

[0081] A total of 66 patients with prostate cancer diagnosed at Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, between January 2018 and January 2022 were included in this analysis. All patients underwent laparoscopic radical prostatectomy for prostate cancer, and the corresponding prostate cancer and adjacent tissues were preserved in liquid nitrogen within 30 minutes after surgery. The diagnosis of prostate cancer was confirmed by clinical presentation, PSA level, magnetic resonance imaging (MRI) results, and histopathological analysis and immunohistochemical staining of all prostate cancers.

[0082] 3. Real-time quantitative PCR: Total RNA from cells or tissues was extracted using Trizol reagent according to the product instructions. RNA purity and concentration were determined using a Nano-Drop 1000. 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit. Real-time quantitative PCR was performed using SYBR Green Mix reagent according to the manufacturer's instructions. The PCR amplification system is shown in Table 1.

[0083] Table 1

[0084] Components Volume (μL) SYBR Green Mix 10 Forward primer 0.5 Reverse primer 0.5 cDNA template 2 Enzyme-free water 7

[0085] Meanwhile, the PCR amplification conditions were 50°C for 2 min, 95°C for 2 min, 95°C for 15 s, and 60°C for 30 s for 40 cycles.

[0086] 4. Test results: Figures 1 to 2As shown in the figure, compared with adjacent prostate cancer tissues and normal prostate cells, the expression of FOXA1-eRNA in prostate cancer tissues and prostate cancer cells was significantly increased.

[0087] Example 2

[0088] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is:

[0089] The effect of FOXA1-eRNA on the proliferation of LNCaP and 22RV1 prostate cancer cells was detected using the following steps:

[0090] 1. Cell Culture: LNCaP and 22RV1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When the cell density reached 80%-90%, the cells were dissociated with 0.25% trypsin and subcultured at a 1:3 ratio.

[0091] 2. Cell transfection:

[0092] Place approximately 6 x 10 5 293T cells were plated into six-well plates and the lentiviral packaging operation was started after the density reached about 80%.

[0093] Mix 7 μL of transfection reagent Lipo3000 with 200 μL of reduced serum medium Opti-MEM, then add 2250 ng of target plasmids (shControl, shFOXA1-eRNA-1, and shFOXA1-eRNA-2), 800 ng of pHelp1.0, and 800 ng of pHelp2.0 helper plasmids in sequence. Incubate at room temperature for 10 to 15 minutes (during which time the 293T cells are replaced with a medium), then rotate and add dropwise to the DMEM medium of the 293T cells, and gently shake to mix the added liquid and the medium evenly.

[0094] After 6 to 8 hours, the medium of 293T cells was changed.

[0095] After 48 hours, the 293T cells were observed under a fluorescence microscope to observe the intensity of green fluorescence.

[0096] The supernatant was collected and filtered through a 0.2 μM pore size filter, then added dropwise to a six-well plate containing the target cells (LNCaP and 22RV1) prepared in advance. After 48 hours, the viral fluid was aspirated, washed 2-3 times with PBS, and replaced with fresh complete medium. One thousandth of puromycin was added to the medium for selection until a cell line stably overexpressing or interfering with the target gene was identified and stored for subsequent experiments.

[0097] 3.CCK8 experiment:

[0098] When the target cell density reaches 60% to 70%, the CCK-8 proliferation assay can be performed. Count the target cells using a Bio-Rad cell counter and plate 2,000 target cells per well in five 96-well plates, with five replicate wells in each 96-well plate.

[0099] 200 μl of fresh 1640 medium containing 10% FBS was added to each well, and 200 μl of PBS buffer was added to each well in the outermost circle of the 96-well plate to reduce the influence of edge effect on the results.

[0100] Before measuring the absorbance, prepare a 10% CCK-8 solution (9 parts serum-free culture medium plus 1 part CCK-8 active reagent) in a biosafety cabinet, aspirate the original culture medium, add 100 μl of 10% CCK-8 reagent mixed solution to each well, and incubate in a 37°C constant temperature cell culture incubator for 90 min. Then take out the 96-well plate and place it in a microplate reader to measure the absorbance at 450 nm.

[0101] The test was conducted every 24 hours, and the five absorbances were used as the values ​​of 0h, 24h, 48h, 72h, and 96h, respectively. A line graph was drawn and statistical analysis was performed. The results are shown in the figure below. Figures 3 and 4 shown.

[0102] 4.Cell colony formation assay:

[0103] Use a cell counter to count the target cells.

[0104] After counting, the target cells were plated into six-well plates (1500 cells per well for LNCaP and 1000 cells per well for 22RV1).

[0105] After the cells were evenly spread, the 6-well plate was placed in a cell culture incubator and cultured for 14 days, with the cell medium being changed every 2-3 days.

[0106] After 14 days, the colony size was observed under a microscope, the culture medium was removed, 1 mL of methanol was added for fixation for 30 min, the methanol was removed, and 1 mL of 5% crystal violet dye was added for staining for 30 min.

[0107] After staining, absorb the crystal violet dye, carefully rinse off the remaining dye with running water, dry overnight, and take pictures. Figure 5 The pictures shown in the figure are counted and statistically analyzed. The results are as follows Figure 6 shown.

[0108] 5. EdU proliferation assay: Use a cell counter to count the target cells.

[0109] After the counting is completed, the target cells are plated into a 12-well plate, two replica wells are set up, 100,000 target cells are plated in each well, and placed in a cell culture incubator overnight to allow them to adhere to the wall.

[0110] The EdU kit of Guangzhou Ruibo Company was used and the steps in the instructions were followed. The target cells were fixed and stained with 4% paraformaldehyde. The nuclei were stained red and the cytoplasm was stained blue. The cells were photographed under an inverted fluorescence microscope. The results are as follows. Figure 7 The analysis results are shown in Figure 8 shown.

[0111] The experimental results showed that FOXA1-eRNA was significantly correlated with the proliferation ability of prostate cancer.

[0112] Example 3

[0113] Comparing Example 3 with Example 2, the difference between Example 3 and Example 2 is:

[0114] To monitor the effects of FOXA1-eRNA interference on tumor volume and weight in an in vivo mouse model, follow these steps:

[0115] 1. Nude mouse subcutaneous tumor formation experiment: Logarithmically growing prostate cancer cells were taken and digested with 0.25% trypsin.

[0116] After washing in serum-free medium, the cells were collected and counted. The cell concentration of the target cells was adjusted to 5 × 10 6 / mL. Each nude mouse was inoculated into the soft skin of the right forelimb at a dose of 0.1mL / mouse. Figure 9 As shown, the major diameter (mm) and minor diameter (mm) were measured with a vernier caliper every 7 days.

[0117] Four weeks later, the nude mice were sacrificed and the long and short diameters of the tumor nodules were measured with a vernier caliper. The volume curve of the subcutaneous transplanted tumor in nude mice was drawn. The results are shown in Figure 2. Figure 10 The weight of the subcutaneous transplanted tumor in nude mice was measured. Figure 11 and Figure 12 shown.

[0118] 2. Experimental results:

[0119] like Figures 10 to 12 As shown, compared with the control group, the tumor volume and weight of mice in the shFOXA1-eRNA-1 and shFOXA1-eRNA-2 groups were significantly decreased.

[0120] Example 4

[0121] Comparing Example 4 with Example 3, the difference between Example 4 and Example 3 is:

[0122] To detect the effect of FOXA1-eRNA on the transcriptional regulation of the target gene FOXA1, the specific steps are as follows:

[0123] 1. Dual luciferase assay: The FOXA1 promoter and enhancer regions were inserted into the PGL3-basic vector by PCR cloning, and the constructed luciferase plasmid was transformed into the stably transfected target cell line. 100 μL PBL was added 48 hours after transfection. After shaking at room temperature for 15 minutes, 20 μL of the product was added to an equal volume of LAR II solution to measure the fluorescence intensity. After completion, 20 μL of stop solution was added. The fluorescence intensity was measured again, and the relative fluorescence intensity was calculated. The results are shown in Figure 2. Figure 13 shown.

[0124] 2. Quantitative PCR and western blot detection of FOXA1 expression level: RNA and protein were extracted from target cells transfected with interference control plasmid (shControl) and interference plasmids (shFOXA1-eRNA-1 and shFOXA1-eRNA-2), and the expression level of target gene FOXA1 was detected by quantitative PCR and western blot. The results are as follows: Figures 14 to 16 shown.

[0125] 3. Experimental results: Figures 13 to 16 As shown in the results, FOXA1-eRNA can significantly enhance the transcriptional activity of FOXA1 and increase the expression level of the target gene FOXA1.

[0126] One or more technical solutions in the embodiments of the present application may have at least the following technical effects or advantages:

[0127] (1) The present application provides an embodiment of an enhancer RNA FOXA1-eRNA for use as a molecular marker for prostate cancer. By clarifying that enhancer RNA can regulate the expression of surrounding protein-coding genes, this property is utilized to ensure that this type of enhancer RNA can be used as a marker related to prostate cancer, thus filling the gap in the association between eRNA and prostate cancer.

[0128] (2) The examples of this application provide an application of an enhancer RNA FOXA1-eRNA in a molecular marker for prostate cancer. For the first time, an enhancer RNA expression library based on the general population was established, and for the first time, the enhancer RNA FOXA1-eRNA was found to be highly expressed in prostate cancer tissue.

[0129] (3) The use of an enhancer RNA FOXA1-eRNA provided in the examples of the present application in the preparation of a detection kit for the diagnosis and prognosis of prostate cancer provides a new therapeutic target for prostate cancer. The multidisciplinary technical means such as bioinformatics, genomics of clinical multi-stage tissue samples, and molecular biology were used to confirm the mechanism by which FOXA1-eRNA promotes the progression of prostate cancer.

[0130] (4) The use of an enhancer RNA FOXA1-eRNA provided in the examples of this application in the preparation of drugs for the treatment of prostate cancer provides new ideas and strategies for studying the upstream expression regulation mechanism of FOXA1, an important signaling pathway related to prostate cancer, and also provides clues and scientific basis for screening potential drug treatment targets for prostate cancer.

[0131] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a hard limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numbers within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0132] In this application, unless otherwise specified, the directional words used, such as "upper" and "lower", refer specifically to the directions of the drawings in the accompanying drawings. In addition, in the description of the present application specification, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" refers to one or more, and "plurality" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple.

[0133] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. Use of a reagent for detecting the expression level of enhancer RNA FOXA1-eRNA in the preparation of a detection kit for diagnosing and prognosing prostate cancer, wherein the core nucleotide sequence of the enhancer RNA FOXA1-eRNA is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The detection agent includes a fluorescent quantitative PCR detection reagent, and the fluorescent quantitative PCR detection reagent includes an amplification primer set to amplify the core nucleotide sequence of the enhancer RNA FOXA1-eRNA.

3. The use according to claim 2, characterized in that The amplification primer set includes a first amplification primer, and the nucleotide sequence of the first amplification primer is shown as SEQ ID NO.

2.

4. The use according to claim 2, characterized in that The amplification primer set further includes a second amplification primer, and the nucleotide sequence of the second amplification primer is shown in SEQ ID NO.3.

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