A molecular marker for prostate cancer and its application

Abstract

The present invention relates to a molecular marker for prostate cancer and its application, belonging to the field of tumor technology. The molecular marker for prostate cancer of the present invention includes any one of the following or a combination thereof: 1) circular RNA circSPIRE1, whose nucleotide sequence is shown in SEQ ID NO: 1; 2) the expression product of circular RNA circSPIRE1. The present invention conducted a differential expression analysis of circRNAs in metastatic prostate cancer tissue and non-metastatic prostate cancer tissue, prostate cancer tissue and its paired adjacent prostate cancer tissue, and found that the expression level of circSPIRE1 in cancer tissue was significantly higher than that in adjacent prostate cancer tissue. At the same time, circSPIRE1 encodes a new protein through a rolling wheel translation mechanism. Circular RNA circSPIRE1 and the protein it encodes have the effect of promoting the proliferation and migration of cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of tumor technology, and in particular to a molecular marker for prostate cancer and application thereof. Background Art

[0002] Prostate cancer is a common and fatal male malignancy, with its incidence and mortality rates increasing year by year worldwide. The treatment of prostate cancer faces many challenges, especially the occurrence of bone metastasis, which greatly reduces the patient's quality of life and life expectancy. Research on the mechanisms of prostate cancer cell proliferation and metastasis is crucial for the development of effective treatments. Current research shows that circular RNA (circRNA) plays an important role in the development of various cancers. Through its unique circular structure, circRNA can evade degradation by nucleases and exist stably in cells. In recent years, multiple circRNAs have been found to regulate gene expression by encoding functional polypeptides, affecting the growth and metastasis of cancer cells.

[0003] Studies have shown that circRNAs can encode specific cryptic peptides that can activate key signaling pathways, immune responses, and other functions, thereby affecting cell proliferation and migration. The PI3K / AKT signaling pathway is a crucial regulatory pathway for cell proliferation, survival, and metabolism, and abnormal activation of this pathway is found in many cancers. Modulating the PI3K / AKT signaling pathway can significantly influence the proliferation and metastasis of prostate cancer cells.

[0004] Although existing research has gradually discovered that circRNA plays a key role in the progression of prostate cancer, mechanistic studies are limited to sponge adsorption and protein binding. Although it is generally recognized that circRNA can mediate translation initiation and encode novel polypeptides through ribosome entry sites and m6A, the verification of translation function has not been carried out in depth, and research on the regulation of prostate cancer by circRNA through its encoded polypeptides is still blank. In previous studies, although multiple signaling pathways have been confirmed to play an important role in the proliferation and metastasis of prostate cancer, the use of signaling pathway inhibitors has failed to effectively improve the prognosis of prostate cancer patients, indicating the existence of complex activation mechanisms that have not yet been explored. At present, the mechanisms of prostate cancer proliferation and metastasis have not been fully elucidated, and the search for new therapeutic targets and strategies has become a hot topic and difficulty in research in this field. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a molecular marker for prostate cancer and its application. The present invention found that in clinical prostate cancer samples, the expression level of circSPIRE1 in cancerous tissue was significantly higher than that in adjacent tissue, and this molecular marker can promote the proliferation and migration of cancer cells.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a molecular marker for prostate cancer, wherein the molecular marker comprises any one or a combination of the following:

[0008] 1) circular RNA circSPIRE1, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0009] TTGTGTGCTGCTCATCTCCCTACTGAATCAGATGCACCAAATCATTATCAGGCAGTATGTCGTGCACTGTTTGCAGAAACAATGGAGCTCCATACATTTCTGACCAAAATTAAGAGTGCGAAAGAGAATCTTAAGAAGATTCAAGAAATGGAAAAGAGCGATGAATCTAGCACAGACTTGGAAGAGC TGAAAAACGCTGACTGGGCACGATTCTGGGTACAGGTGATGAGGGATTTGAGGAATGGGGTAAAACTTAAGAAGGTCCAAGAGCGGCAGTACAACCCTTTGCCCATTGAATATCAGCTCACCCCTTATGAGATGTTAATGGATGACATTCGCTGCAAAAGATACACCTTGCGAAAAGTGATG(SEQID NO: 1);

[0010] 2) Expression products of circular RNA circSPIRE1.

[0011] This study identified circSPIRE1 (his_circ_0000829) by differentially expressing circRNAs in metastatic and non-metastatic prostate cancer tissues, as well as in prostate cancer tissues and their paired adjacent prostate cancer tissues. The study found that circSPIRE1 was expressed at significantly higher levels in prostate cancer tissues than in adjacent prostate cancer tissues in clinical prostate cancer samples. Furthermore, circSPIRE1 was found to encode a novel protein through a rolling translation mechanism.

[0012] As a preferred embodiment of the first aspect, the expression product of the circular RNA circSPIRE1 is the polypeptide rtSPIRE1, the amino acid sequence of which is shown in SEQ ID NO: 3:

[0013] MELHTFLTKIKSAKENLKKIQEMEKSDESSTDLEELKNADWARFWVQVMRDLRNGVKLKKVQERQYNPLPIEYQLTPYEMLMDDIRCKRYTLRKVMLCAAHLPTESDAPNHYQAVCRALFAETMELHTFLTKIKSAKENLKKIQEMEKSDESSTDLEELKNADWARFWVQVMRDLRNGVKLKKVQERQYNPLPIEYQLTPYEMLMDDIRCKRYTLRKVMLCAAHLPTESDAPNHYQAVCRALFAETMELHTFLTKIKSAKENLKKIQEMEKSDESSTDLEELKNADWARFWVQVMRDLRNGVKLKKVQERQYNPLPIEYQLTPYEMLMDDIRCKRYTLRKVMLCAAHLPTESDAPNHYQAVCRALFAET(SEQ ID NO: 3).

[0014] As a preferred embodiment of the first aspect, the nucleic acid sequence encoding the polypeptide rtSPIRE1 is as shown in SEQ ID NO: 19.

[0015] ATGGAGCTCCATACATTTCTGACCAAAATTAAGAGTGCGAAAGAGAA

[0016] TCTTAAGAAGATTCAAGAAATGGAAAAGAGCGATGAATCTAGCACAGAC

[0017] TTGGAAGAGCTGAAAAACGCTGACTGGGCACGATTCTGGGTACAGGTGAT

[0018] GAGGGATTTGAGGAATGGGGTAAAACTTAAGAAGGTCCAAGAGCGGCAG

[0019] TACAACCCTTTGCCCATTGAATATCAGCTCACCCCTTATGAGATGTTAATG

[0020] GATGACATTCGCTGCAAAAGATACACCTTGCGAAAAGTGATGTTGTGTGC

[0021] TGCTCATCTCCCTACTGAATCAGATGCACCAAATCATTATCAGGCAGTATG

[0022] TCGTGCACTGTTTGCAGAAACAATGGAGCTCCATACATTTCTGACCAAAA

[0023] TTAAGAGTGCGAAAGAGAATCTTAAGAAGATTCAAGAAATGGAAAAGAG

[0024] CGATGAATCTAGCACAGACTTGGAAGAGCTGAAAAACGCTGACTGGGCA

[0025] CGATTCTGGGTACAGGTGATGAGGGATTTGAGGAATGGGGTAAAACTTAA

[0026] GAAGGTCCAAGAGCGGCAGTACAACCCTTTGCCCATTGAATATCAGCTCA

[0027] CCCCTTATGAGATGTTAATGGATGACATTCGCTGCAAAAGATACACCTTG

[0028] CGAAAAGTGATGTTGTGTGCTGCTCATCTCCCTACTGAATCAGATGCACCA

[0029] AATCATTATCAGGCAGTATGTCGTGCACTGTTTGCAGAAACAATGGAGCT

[0030] CCATACATTTCTGACCAAAATTAAGAGTGCGAAAGAGAATCTTAAGAAGA

[0031] TTCAAGAAATGGAAAAGAGCGATGAATCTAGCACAGACTTGGAAGAGCT

[0032] GAAAAACGCTGACTGGGCACGATTCTGGGTACAGGTGATGAGGGATTTGA

[0033] GGAATGGGGTAAAACTTAAGAAGGTCCAAGAGCGGCAGTACAACCCTTT

[0034] GCCCATTGAATATCAGCTCACCCCTTATGAGATGTTAATGGATGACATTCG

[0035] CTGCAAAAGATACACCTTGCGAAAAGTGATGTTGTGTGCTGCTCATCTCCC

[0036] TACTGAATCAGATGCACCAAATCATTTATCAGGCAGTATGTCGTGCACTGTTTGCAGAAACA (SEQ IDNO: 19)

[0037] In a second aspect, the present invention provides the use of the molecular marker described in the first aspect in the preparation of a preparation for screening drugs for treating prostate cancer. Experiments have shown that the circular RNA circSPIRE1 of the present invention promotes the development of prostate cancer by activating the PI3K / AKT signaling pathway, in particular, the polypeptide rtSPIRE1 interacts with SCN5A, increasing intracellular Ca 2+ level, activates the PI3K / AKT signaling pathway, and can increase the proliferation and migration speed of cancer cells.

[0038] In a third aspect, the present invention provides a method for screening candidate drugs for treating prostate cancer, the method comprising: detecting the expression level of the prostate cancer molecular marker described in the first aspect in prostate cancer cells after treatment with the drug to be screened, if the drug to be screened can reduce the expression level of the prostate cancer molecular marker, it indicates that the drug to be screened is a candidate drug for treating prostate cancer.

[0039] The circular RNA circSPIRE1 of the present invention can increase the proliferation and migration speed of cancer cells and can be used to screen drugs for treating prostate cancer. If the candidate drug can reduce the expression level of circSPIRE1 in cells, it indicates that it has the efficacy of treating prostate cancer.

[0040] In a fourth aspect, the present invention provides a detection kit for diagnosing prostate cancer, comprising a reagent for detecting the molecular marker described in the first aspect. As previously described, circSPIRE1 of the present invention is used as a molecular marker for prostate cancer. Detecting a high expression level of circSPIRE1 in a sample indicates prostate cancer.

[0041] As a preferred embodiment of the fourth aspect, it comprises a forward primer and a reverse primer that can amplify the molecular marker described in the first aspect, the sequence of the forward primer is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO: 5.

[0042] Sequence of forward primer: TCGCTGCAAAAGATACACCT (SEQ ID NO: 4)

[0043] Sequence of the reverse primer: GAGATGAGCAGCACACAACAT (SEQ ID NO: 5).

[0044] As a preferred embodiment of the fourth aspect, it also includes an immunoassay product for the polypeptide rtSPIRE1, wherein the immunoassay product for the polypeptide rtSPIRE1 includes an antibody that specifically binds to the polypeptide rtSPIRE1.

[0045] As a preferred embodiment of the fourth aspect, a positive control and a negative control are further included, wherein the positive control is a prostate cancer cell line positive for circular RNA circSPIRE1; and the negative control is a prostate cell line negative for circular RNA circSPIRE1.

[0046] The kit can amplify circSPIRE1 through specific primers or detect it through antibodies with affinity for the polypeptide rtSPIRE1. The test results are compared with positive and negative results. If the same fragment of circSPIRE1 is amplified or the sample is detected to contain the molecular marker of the present invention through antibody detection, it can be determined that the patient has prostate cancer.

[0047] In a fifth aspect, the present invention provides a kit for the diagnosis and prognosis of prostate cancer, which is composed of reagents for specifically detecting the gene expression levels of the molecular markers described in the first aspect.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] This study reveals the molecular mechanisms of prostate cancer proliferation and metastasis and provides a method for treating prostate cancer by regulating circRNA translation. Through high-throughput sequencing and bioinformatics analysis, the researchers discovered a circRNA, circSPIRE1, which is highly expressed in prostate cancer cells and regulated by hnRNPA1 through an IRES mechanism, encoding the biologically active polypeptide rtSPIRE1 via rolling wheel translation.

[0050] The present invention uses circSPIRE1 and its encoded polypeptide rtSPIRE1 as molecular markers for the diagnosis and prognosis evaluation of prostate cancer, which makes up for the lack of effective molecular markers in the existing technology and helps to improve the accuracy of diagnosis and the reliability of prognosis evaluation.

[0051] The present invention uses experimental methods to clarify the specific role of circSPIRE1 and its encoded polypeptide rtSPIRE1 in the proliferation and metastasis of prostate cancer cells, especially the molecular mechanism by which it promotes the development of prostate cancer by activating the PI3K / AKT signaling pathway, providing a new perspective for a deeper understanding of the pathogenesis of prostate cancer.

[0052] The present invention verifies the specific biological function of the rtSPIRE1 polypeptide in prostate cancer cells through biochemical and cell biological experiments, including its effects on cell proliferation, migration and invasion, which makes up for the problem of insufficient identification and verification of functional polypeptides in the prior art.

[0053] The present invention confirms that circSPIRE1 and its encoded polypeptide rtSPIRE1 can be used as new sites for the treatment of prostate cancer. By regulating the activation level of the PI3K / AKT signaling pathway through the interaction between rtSPIRE1 and SCN5A, an anti-cancer treatment strategy with potential clinical application value can be developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of circSPIRE1 translation and regulation;

[0055] Figure 2 Schematic diagram of the screening of circSPIRE1 in the present invention, wherein A is a schematic diagram of the screening process of circSPIRE1; B is a schematic diagram of the secondary structure of circSPIRE1; C is a schematic diagram of the coding open reading region; D is a schematic diagram of the differential expression of circSPIRE1 in prostate cancer and adjacent tissues in clinical samples; E is a schematic diagram of the differential expression of circSPIRE1 in prostate cancer cell lines and normal cell lines; F is a schematic diagram of the relationship between circSPIRE1 expression levels and biochemical recurrence-free survival of prostate cancer patients;

[0056] Figure 3 Figure 1 is a schematic diagram of the structure and cellular localization of circSPIRE1, where A is a schematic diagram showing that circSPIRE1 is formed by the circularization of exons 4-6 of SPIRE1 mRNA; B is a schematic diagram showing the results of agarose gel electrophoresis of a prostate cancer cell line; C is a schematic diagram showing the lack of a 3'Poly(A) structure in circSPIRE1 confirmed by reverse transcription using oligo dT primers and random primers; D, E, and F are schematic diagrams showing the ability of circSPIRE1 and linear SPIRE1 to resist RNase R digestion; G is a schematic diagram showing the results of fluorescence in situ hybridization experiments on circSPIRE1; and H is a schematic diagram showing the results of nuclear-cytoplasmic fractionation experiments on circSPIRE1.

[0057] Figure 4Figure 1 is a schematic diagram showing how circSPIRE1 promotes the proliferation, metastasis and invasion of prostate cancer. Figure 1 is a schematic diagram showing the expression level of the parent gene SPIRE1 in cancer cells after silencing circSPIRE1 expression (si-circSPIRE1#1, si-circSPIRE1#2); Figure 1 is a schematic diagram showing the expression level of the parent gene SPIRE1 in cancer cells after overexpressing circSPIRE1 (OE-circSPIRE1); Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with silencing circSPIRE1 expression in PC3 cancer cells; Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with overexpression of circSPIRE1 in PC3 cancer cells; Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with silencing circSPIRE1 expression in DU145 cancer cells; Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with overexpression of circSPIRE1 in DU145 cancer cells; Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with silencing circSPIRE1 expression in DU145 cancer cells; Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with overexpression of circSPIRE1 in DU145 cancer cells; Figure 1 is a schematic diagram showing the proliferation ability of prostate cancer cell lines with silencing circSPIRE1 or overexpressing circSPIRE1 in clone formation experiments.

[0058] Figure 5 Schematic diagram of the results of circSPIRE1 in vivo experiments; A is a schematic diagram of the tumor growth rate of nude mice overexpressing circSPIRE1 (OE-circSPIRE1); B is a schematic diagram of the tumor volume of nude mice overexpressing circSPIRE1; C is a schematic diagram of the tumor growth rate and tumor weight of the circSPIRE1 overexpression group and the control group (Empty Vector); D is a schematic diagram of the HE staining and Ki-67 immunohistochemical staining results of the circSPIRE1 overexpression group;

[0059] Figure 6Schematic diagram of the results of validating the biological function of circSPIRE1; A is a schematic diagram of the nascent protein encoded by the circSPIRE1 roller; B is a schematic diagram of the full-length sequence of circSPIRE1 (i.e., hsa_circ_0000829); C is a schematic diagram of the plasmid structure constructed to validate the roller translation mechanism (T1: blank control; T2: inserting a 3FLAG tag before the ATG of the ORF; T3: based on T2, inserting a T base after the ORF to form a stop codon); D is a schematic diagram of the circular structure of T2 and T3 plasmids; E is a schematic diagram of the construction of a circular circSPIRE1 overexpression vector (T2) fused with Flag and tagged with 3xFlag And a schematic diagram of the electrophoresis results of the mutant (T3), which showed a rolling wheel translation phenomenon. The main observable products were 17kDa, 34kDa, and 51kDa, corresponding to the molecular weights of the 1st, 2nd, and 3rd circle encoding products of the circSPIRE1 open reading region, respectively; F is a schematic diagram of protein spectrum analysis showing that both T2 and T3 plasmids contain specific fragments of the circSPIRE1 encoding product and their amino acid sequences; G, H, I, J are schematic diagrams of the effects of T1, T2, and T3 overexpression of circSPIRE1 on the proliferation of prostate cancer cell lines; K is a schematic diagram of the effects of T1, T2, and T3 overexpression of circSPIRE1 on the invasion and metastasis of prostate cancer cell lines;

[0060] Figure 7 Schematic diagram to verify that circSPIRE1 is translated by IRES and that the translation process is regulated by hnRNPA1; Figure A is a schematic diagram of the structures of five verification vectors constructed based on the IRES active fragment of circSPIRE1; Figure B is a schematic diagram of the IRES activity in the dual luciferin reporter assay; Figure C is a schematic diagram of the RNA pull-down assay results; Figure D is a schematic diagram of the mass spectrometry analysis results; Figure E is a schematic diagram of the Western blot analysis results verifying the interaction between circSPIRE1 and hnRNPA1; Figure F is a schematic diagram of the Western blot verification of exogenously transfected hnRNPA-HA at different amounts; Figure G is a schematic diagram of the dual luciferase activity assay after simultaneous exogenous transfection of different amounts of hnRNPA-HA and the circSPIRE1-IRES activity verification vector R3; Figure H is a schematic diagram of the IRES activity assay after transfection of different amounts of hnRNPA1;

[0061] Figure 8 Schematic diagram to verify that the newly synthesized protein circSPIRE1 targets SCN5A and activates Wnt / β-catenin; Figure A is a schematic diagram of the immunoprecipitation (IP) experiment results; Figure B is a schematic diagram of the mass spectrometry analysis results; Figure C is a schematic diagram of the Western blot analysis results;

[0062] Figure 9Schematic diagram to verify that rtSPIRE1 activates the PI3K / AKT signaling pathway and promotes metastasis; among them, A, B, C, and D are schematic diagrams of the gene expression changes of circSPIRE1-flag and Vector, and circSPIRE1-flag and circSPIRE1-flag-mut; Figures E, F, G, and H are schematic diagrams of the gene function enrichment analysis results; Figure I is the expression levels of PI3K / AKT signaling pathway-related proteins detected by Western blot analysis; Figure J is a schematic diagram of the expression levels of epithelial-mesenchymal transition (EMT) markers detected by Western blot analysis. DETAILED DESCRIPTION

[0063] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0064] Example 1 Screening and identification of circSPIRE1

[0065] 1. Methods

[0066] 1.1 Sample collection and processing:

[0067] Metastatic prostate cancer tissue, non-metastatic prostate cancer tissue, prostate cancer tissue and its paired adjacent prostate cancer tissue samples were collected, and total RNA was extracted. rRNA was removed using the Ribo-Zero rRNA Removal Kit, and the resulting RNA was purified.

[0068] 1.2 High-throughput sequencing:

[0069] The obtained sample RNA was sequenced using the Illumina HiSeq platform to obtain sequence data, and differentially expressed circRNAs were screened through bioinformatics analysis.

[0070] 1.3 Cell experiments to verify circSPIRE1 expression:

[0071] Specific primers were designed, and RT-qPCR technology was used to verify the expression level of circSPIRE1 in prostate cancer cell lines (PC3, DU145, C4-2, LNCaP, and 22Rv1).

[0072] Specific primers:

[0073] Forward primer: 5'-TGAAACCAACTCCACCACGG-3' (SEQ ID NO: 6)

[0074] Reverse primer: 5'-GTGGCCGCATTGCTAATCTG-3' (SEQ ID NO: 7).

[0075] 2. Results

[0076] Differential expression analysis of circRNAs in metastatic prostate cancer tissues, non-metastatic prostate cancer tissues, prostate cancer tissues and their paired adjacent prostate cancer tissues was performed, and 59 differentially expressed circRNAs were successfully identified. After coding potential prediction, 4 circRNAs were found to have coding ability. After screening by expression abundance identification of tissue samples, circSPIRE1 (his_circ_0000829) was finally obtained. The secondary structure and coding open reading region of circSPIRE1 are shown in Figure 2. Figure 2 As shown in B. In prostate cancer clinical samples, the expression level of circSPIRE1 in cancer tissues is generally higher than that in adjacent tissues ( Figure 2 D). To further verify this finding, we measured the relative expression of circSPIRE1 in the normal prostate cell line RWPE1 and prostate cancer cell lines PC3, DU145, C4-2, LNCaP, and 22Rv1. The results showed that the expression abundance of circSPIRE1 in prostate cancer cell lines was significantly increased compared with normal cell lines ( Figure 2 E), more notably, follow-up analysis of a larger cohort of prostate cancer patients suggested that high expression of circSPIRE1 was positively correlated with poor prognosis of prostate cancer ( Figure 2 F).

[0077] Example 2 Structural characteristics and subcellular localization of circSPIRE1

[0078] 1. Methods

[0079] 1.1 Structural Verification: Specific primers targeting the reverse splicing junction of circSPIRE1 were designed and amplified by RT-PCR. The reverse splicing junction sequence of circSPIRE1 was confirmed by Sanger sequencing.

[0080] Specific primers:

[0081] Forward primer: 5'-TGAAAACCAACTCCACCACGG-3' (SEQ ID NO: 8), Reverse primer: 5'-GTGGCCGCATTGCTAATCTG-3' (SEQ ID NO: 9).

[0082] 1.2 Covalent closed ring structure stability experiment:

[0083] The stability of the covalently closed ring structure was verified by Oligo dT reverse transcription, RNase R assay, and actinomycin D assay.

[0084] First, an oligo dT reverse transcription assay is performed, using an oligo dT primer that specifically binds to the Poly-A tail of mRNA for reverse transcription. If circSPIRE1 is a circRNA (without a Poly-A tail), the reverse transcription efficiency will be low. Next, an RNase R assay is performed, which degrades linear RNA while retaining circular RNA. RT-PCR or qPCR is used to detect changes in RNA before and after treatment. If the circular RNA signal increases and the linear RNA signal decreases, it indicates that circSPIRE1 is a circRNA. Finally, an actinomycin D assay is performed to monitor the stability of circSPIRE1 after inhibiting RNA transcription. If the RNA remains stable over time, it further supports its identity as a circRNA. These experimental steps can help confirm and repeatedly verify the stable circular structure of circRNAs.

[0085] 1.3 Subcellular localization: Fluorescence in situ hybridization (FISH) was used to detect the localization of circSPIRE1 in prostate cancer cells. Nuclear and cytoplasmic separation experiments were performed, and the distribution of circSPIRE1 in the nucleus and cytoplasm was detected by RT-qPCR.

[0086] circSPIRE1 FISH probe:

[0087] GGGAGATGAGCAGCACACAAACATCACTTTTCGCAAGGTG T(5'Cy3) (SEQ ID NO: 10).

[0088] 2. Results

[0089] The reverse splicing junction sequence of circSPIRE1 (Sequence: CGAAAAGTGATGGTTGTGTGCTGCTGCT (SEQ ID NO: 18) was determined by Sanger sequencing and was consistent with the predicted result. It was confirmed that circSPIRE1 was formed by the circularization of exons 4-6 of SPIRE1 mRNA ( Figure 3 A), and circSPIRE1 was detected in prostate cancer cell lines (PC3, DU145) by agarose electrophoresis ( Figure 3 B). Oligo dT reverse transcription, RNase R assay, and actinomycin D assay indicated that, compared with linear mRNA (Linear SPIRE1), the covalently closed loop structure of circSPIRE1 was highly stable both inside and outside prostate cancer cells ( Figure 3CF). Fluorescence in situ hybridization and nuclear cytoplasm fractionation experiments showed that the circRNA was mainly located in the cytoplasm ( Figure 3 G, H). The above experiments show that circSPIRE1 is formed by reverse splicing of the parent gene SPIRE1 and is stably expressed in prostate cancer cells.

[0090] Example 3 Biological Function of circSPIRE1

[0091] 1. Methods

[0092] 1.1 Construction of circSPIRE1 overexpression and silencing vectors:

[0093] Construction of an overexpression vector carrying the full-length sequence of circSPIRE1 (circSPIRE1) and a shRNA silencing vector targeting circSPIRE1

[0094] si-circSPIRE1#1:

[0095] Sense strand:UUGCGAAAAGUGAUGUUGUTT (SEQ ID NO: 11)

[0096] Antisense strand:ACAACAUCACUUUUCGCAATT (SEQ ID NO: 12)

[0097] si-circSPIRE1#2:

[0098] Sense strand:GUGAUGUUGUGUGCUGCUCTT (SEQ ID NO: 13)

[0099] Antisense strand: GAGCAGCACACAACAUCACTT (SEQ ID NO: 14).

[0100] The vectors were transfected into prostate cancer cell lines PC-3 and DU145, respectively.

[0101] 1.2 Functional Verification:

[0102] Cell proliferation assay (CCK-8 method), colony formation assay, Transwell migration assay and wound wound healing assay were performed to evaluate the effect of circSPIRE1 on the proliferation and migration of prostate cancer cells.

[0103] 2. Results

[0104] By designing siRNAs to construct circSPIRE1 silencing expression vectors and constructing circSPIRE1 overexpression vectors, the vectors were packaged with lentivirus and infected with prostate cancer PC3 and DU145 cell lines to obtain cell lines with specific circSPIRE1 silencing and circSPIRE1 overexpression ( Figure 4 A silent expression, Figure 4 B overexpression). In vitro experiments showed that silencing circSPIRE1 inhibited the proliferation of prostate cancer cells, while upregulating circSPIRE1 enhanced the proliferation and migration of prostate cancer cells ( Figure 4 CG).

[0105] We further verified the role of circSPIRE1 in prostate cancer proliferation. By constructing a cell line stably overexpressing circSPIRE1 (OE-circSPIRE1) and a corresponding control cell line (Empty vector), we established a subcutaneous tumor model in nude mice. The results showed that compared with the control group (vector group), the tumor growth rate of nude mice overexpressing circSPIRE1 was significantly accelerated. This finding was confirmed by Figure 5 A, 5B, and 5C were intuitively demonstrated. In addition, through E staining and Ki-67 immunohistochemical staining techniques, we further observed that the positive expression rates of Ki-67 and EMT in tumor cells in the circSPIRE1 overexpression group were significantly higher than those in the control group. Figure 5 D. These preliminary animal experimental results strongly support the potential role of circSPIRE1 in promoting prostate cancer proliferation.

[0106] Example 4 Translation mechanism of circSPIRE1

[0107] 1. Methods

[0108] 1.1 IRES activity verification:

[0109] A dual fluorescence reporter gene vector containing the circSPIRE1 IRES sequence was constructed.

[0110] hsa_circ_0000829 full-length sequence:

[0111] TTGTGTGCTGCTCATCTCCCTACTGAATCAGATGCACCAAATCATTATCAGGCAGTA TGTCGTGCACT GTTTGCAGAAACAATGGAGCTCCATACATTTCTGACCAAAATTAAGAGTGCGAAAGAGAATCTTAAGAAGATTCAA GAAATGGAAAAGAGCGATGAATCTAGCACAGACTTGGAAGAGCTGAAAAACGCTGACTGGGCACGATTCTGGGTACAGGTGATGAGGGATTTGAGGAATGGGGTAAAACTTAAGAAGGTCCAAGAGCGGCAGTACAACCCTTTGCCCATTGAATATCAGCTCACCCCTTATGAGATGTTAATGGATGACATTCGCTGCAAAAGATACACCTTGCGAAAAGTGATG(SEQID NO: 1) Among them, IRES active sequence: 58-147

[0112] TGTCGTGCACTGTTTGCAGAAACAATGGAGCTCCATACATT TCTGACCAAAATTAAGAGTGCGAAAGAGAATCTTAAGAAGATT CAAGAA (SEQ ID NO: 2)

[0113] The R1-R5 reporter plasmid vectors were constructed in sequence to verify the IRES activity. The constructed plasmid vectors are shown in Table 1:

[0114] Table 1: Constructed plasmid vector information

[0115]

[0116] The plasmid vector constructed in this example was from Gisai Biotechnology Co., Ltd., specifically the Luc2-IR ES-Report vector, and the vector sequencing primer was Luc-seqF: CAGGAGGACGCTCCA GATGA (SEQ ID NO: 15).

[0117] Vectors R1-R5 were transfected into prostate cancer cell lines, and the fluorescence intensity was detected.

[0118] 1.2 Verification of the interaction between hnRNPA1 and circSPIRE1:

[0119] RNA pull-down experiments were performed using biotin-labeled RNA pull-down probes to enrich proteins binding to circSPIRE1 from prostate cancer cell lysates.

[0120] RNA pulldown probes:

[0121] NC Sense S strand:

[0122] TCGAAAAG+TGATGT+TGTGTGC+TGCCTCATC (SEQ ID NO: 16)

[0123] circSPIRE1 Sense S strand:

[0124] GATGAGCAGCACACAACA+TCACTTT+TCGCAA (SEQ ID NO: 17),

[0125] Mass spectrometry analysis and Western blot were combined to identify and verify the interaction between hnRNPA1 and circSPIRE1.

[0126] 2. Results

[0127] Through analysis using the TransCirc bioinformatics platform, we found that circSPIRE1 contains a single open reading frame (ORF) that lacks a stop codon. The ORF sequence is (SEQ ID NO: 19). This feature suggests that circSPIRE1 may encode a novel protein through a rolling translation mechanism, thereby exerting its biological function ( Figure 6 A, B). To verify the coding potential of circSPIRE1, we designed and constructed three different plasmids: 1) blank control plasmid; 2) inserting a 3FLAG tag before the start ATG of the ORF; 3) based on 2), inserting a T base after the ORF to form a stop codon ( Figure 6 C, D). The plasmid vector constructed in this example was obtained from Gisai Biotechnology Co., Ltd. Specifically, GS0108: lentiviral overexpression vector pLC5-ciR (GFP) vector. These plasmids were then transfected into HEK 293T cells, and protein expression was verified by Western Blot. The amino acid sequence specifically encoded by the ORF (SEQ ID NO: 3) was further confirmed by electrophoresis and proteomic analysis ( Figure 6 E, F).

[0128] To further verify that circSPIRE1 plays a functional role in prostate cancer cells through its encoded protein, we conducted in vitro experiments. By knocking down circSPIRE1 and performing rescue experiments, we compared cells transfected with circSPIRE1 (T2) and its mutant (T3). The results showed that compared with the mutant, cells re-expressing circSPIRE1 exhibited stronger prostate cancer proliferation and metastasis capabilities ( Figure 6 Based on the above experimental data, we speculate that circSPIRE1 may promote prostate cancer progression by encoding the newly expressed protein rtSPIRE1 through a wheel translation mechanism.

[0129] It is predicted that circSPIRE1 contains a sequence with IRES (internal ribosome entry site) activity. To verify this prediction, we constructed the five different vectors mentioned above ( Figure 7 A: plasmids R1 to R5), and the IRES activity of these sequences was tested by dual fluorescence reporter gene assay ( Figure 7 B) The experimental results show that this specific fragment does have IRES activity, and the main activity is concentrated in the first half of the fragment. This finding indicates that circSPIRE1 is translated in a cap-independent mechanism.

[0130] To further explore the factors that regulate the translational activity of circSPIRE1, we used RNA pull-down technology combined with mass spectrometry analysis and Western Blot (WB) experiments to identify proteins that interact with circSPIRE1. RNA pull-down experiments suggested that circSPIRE1 can bind to hnRNPA1, and hnRNP A1 protein plays a key role in regulating the translational function of circSPIRE1. hnRNPA1-HA and circSPIRE1 were co-transfected into HEK-293T cells, and a double fluorescein experiment was performed. It was found that the level of hnRNPA1 was positively correlated with the activity of IRES ( Figure 7 CH).

[0131] Example 5 Function and mechanism of action of rtSPIRE1

[0132] 1. Methods

[0133] 1.1 Interaction between rtSPIRE1 and SCN5A:

[0134] The interaction between rtSPIRE1 and SCN5A was verified by co-immunoprecipitation experiments combined with mass spectrometry analysis.

[0135] Western blot was used to detect the interaction between rtSPIRE1 and SCN5A in prostate cancer cells.

[0136] 1.2 Effect of rtSPIRE1 on PI3K / AKT signaling pathway activation:

[0137] Prostate cancer cells and HEK-293T cells were transfected with rtSPIRE1 overexpression or mutation vectors, and the activation level of the PI3K / AKT signaling pathway was detected.

[0138] 2. Results

[0139] Immunoprecipitation (IP) experiments used Flag antibodies to immunoprecipitate rtSPIRE1, with IgG as a control, to detect protein binding in different samples. Markers were used to indicate protein size. Through immunoprecipitation, the downstream mechanism of rtSPIRE1 function was explored. Combined with mass spectrometry and western blot, it was found that rtSPIRE1 can interact with the ion channel SCN5A ( Figure 8 A). Mass spectrometry analysis results show the mass spectrum of SCN5A in the immunoprecipitated sample, confirming the binding of rtSPIRE1 to SCN5A ( Figure 8 B). Western blot was used to detect the expression levels of SCN5A and GADPH (as an internal control) after immunoprecipitation to verify the specific binding of rtSPIRE1 to SCN5A. The experimental groups included Input (input sample), Flag (rtSPIRE1 precipitation) and IgG (control group) ( Figure 8 C).

[0140] The results of detecting the activation level of the PI3K / AKT signaling pathway showed that the activity change of SCN5A may affect the key components of the Wnt / β-catenin signaling pathway directly or indirectly. Figure 9 ), such as the stability and nuclear translocation of β-catenin. Figure 9 AD shows the gene expression changes of circSPIRE1-flag and Vector, circSPIRE1-flag and circSPIRE1-flag-mut, among which the volcano plot shows the significantly differentially expressed genes, and the heat map shows the expression of significantly differentially expressed genes in different samples. Figure 9 EH shows the results of gene function enrichment analysis, where the gene ontology (GO) enrichment analysis diagram shows significantly enriched GO terms, and the gene set enrichment analysis (GSEA) diagram shows the significant enrichment of the PI3K-Akt signaling pathway. Figure 9 I shows the expression levels of proteins related to the PI3K / AKT signaling pathway detected by Western blot analysis, in which the expression of mTOR, p-mTOR (Ser2448), pan-AKT, p-AKT (Thr308), p-AKT (Ser473) and Gadph in PC3 cells were detected. Figure 9 J showed the detection of epithelial-mesenchymal transition (EMT) marker expression levels by Western blot analysis, in which the expression of E-Cadherin (E-Cad), N-Cadherin (N-Cad), Vimentin and Gadph in PC3 cells was detected.

[0141] The results of the above-mentioned co-immunoprecipitation, mass spectrometry analysis, Western blot, and molecular docking experiments showed that rtSPIRE1 closely interacted with the SCN5A ligase; overexpression of rtSPIRE1 stabilized SCN5A, reduced its degradation, promoted the activation of the sodium-calcium transporter, increased the intracellular sodium ion level, and thereby activated the PI3K / AKT signaling pathway.

[0142] Example 6

[0143] This embodiment provides a detection kit for diagnosing prostate cancer, including a reagent for detecting circSPIRE1. As previously mentioned, circSPIRE1 of the present invention is used as a molecular marker for prostate cancer. If a high expression level of circSPIRE1 in a sample is detected, it can indicate prostate cancer.

[0144] The kit includes a forward primer and a reverse primer for amplifying circSPIRE1, the sequence of the forward primer is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO: 5.

[0145] Sequence of forward primer: TCGCTGCAAAAGATACACCT (SEQ ID NO: 4)

[0146] Sequence of the reverse primer: GAGATGAGCAGCACACAACAT (SEQ ID NO: 5).

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a reagent for detecting circular RNA circSPIRE1 in the preparation of a preparation for diagnosing prostate cancer, characterized in that: The nucleotide sequence of the circular RNA circSPIRE1 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The reagent for detecting circular RNA circSPIRE1 is an amplification primer; The amplification primers include a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO: 5.

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