Molecular marker for prostatic cancer and application of molecular marker

Abstract

The invention relates to a prostate cancer molecular marker and application thereof, and belongs to the technical field of tumors. The molecular marker for prostatic cancer comprises any one or a combination of the following components: 1) circular RNA circSPIRE1 of which the nucleotide sequence is as shown in SEQ ID NO: 1; and (2) an expression product of the circular RNA circSPIRE1. Differential expression analysis is carried out on circRNAs in metastatic prostate cancer tissue, non-metastatic prostate cancer tissue, prostate cancer tissue and prostate para-carcinoma tissue paired with the prostate cancer tissue, it is found that the expression level of circSPIRE1 in cancer tissue is remarkably higher than that of circSPIRE1 in para-carcinoma tissue, and meanwhile circSPIRE1 encodes a novel protein through a roller translation mechanism. The circular RNA circSPIRE1 and the protein coded by the circular RNA circSPIRE1 have the effect of promoting 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, and its incidence and mortality rate are 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. The study of the proliferation and metastasis mechanism of prostate cancer cells is crucial for the development of effective treatments. Current studies have shown that circular RNA (circRNA) plays an important role in the development of various cancers. Through its unique circular structure, circRNA can escape degradation by nucleases and exist stably in cells. In recent years, multiple circRNAs have been found to regulate gene expression by encoding functional peptides, affecting the growth and metastasis of cancer cells.

[0003] Studies have shown that circRNA can encode specific hidden peptides, which can activate key signaling pathways, immune responses and other functions, thereby affecting cell proliferation and migration. The PI3K / AKT signaling pathway is an important regulatory pathway for cell proliferation, survival and metabolism, and abnormal activation of this pathway exists in many cancers. By regulating the PI3K / AKT signaling pathway, the proliferation and metastasis of prostate cancer cells can be significantly affected.

[0004] Although existing studies have gradually found that circRNA plays a key role in the progression of prostate cancer, the mechanism research is limited to sponge adsorption and protein binding. Although it is recognized that circRNA can mediate translation initiation and encode new polypeptides through ribosome entry sites, m6A, etc., the verification of translation function has not been carried out in depth, and the research on the regulation of prostate cancer by circRNA through its encoded polypeptide is still blank. In previous studies, although it has been confirmed that multiple signaling pathways 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 that there are complex activation mechanisms that have not yet been explored. At present, the proliferation and metastasis mechanism of prostate cancer has not been fully elucidated, and finding new therapeutic targets and strategies has become a hot spot and difficulty in research in this field. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a molecular marker for prostate cancer and its application. The present invention found that in clinical samples of prostate cancer, the expression level of circSPIRE1 in cancer tissue was significantly higher than that in adjacent tissue, and the 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] The present invention differentially expressed circRNAs in metastatic prostate cancer tissues and non-metastatic prostate cancer tissues, prostate cancer tissues and their paired prostate cancer adjacent tissues, and finally obtained circSPIRE1 (i.e., his_circ_0000829). It was found that in clinical prostate cancer samples, the expression level of circSPIRE1 in cancer tissues was significantly higher than that in adjacent tissues. At the same time, it was found that circSPIRE1 encodes a new type of protein through a wheel translation mechanism.

[0012] As a preferred embodiment of the first aspect, the expression product of the circular RNA circSPIRE1 is a polypeptide rtSPIRE1, and its amino acid sequence 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 a drug 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 to increase intracellular Ca 2+ level, activating the PI3K / AKT signaling pathway, and increasing 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 mentioned above, 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 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 immunodetection product of the polypeptide rtSPIRE1, and the immunodetection product of the polypeptide rtSPIRE1 includes an antibody that specifically binds to the polypeptide rtSPIRE1.

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

[0046] The kit can amplify circSPIRE1 through specific primers or detect it through antibodies with affinity to 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 antibodies, it can be determined that the patient has prostate cancer.

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

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

[0049] The present invention reveals the molecular mechanism of prostate cancer proliferation and metastasis, and provides a method for treating prostate cancer by regulating the translation function of circRNA. Through high-throughput sequencing and bioinformatics analysis, the present invention discovered a circRNA named circSPIRE1, which is highly expressed in prostate cancer cells and is regulated by hnRNPA1 to encode a biologically active polypeptide rtSPIRE1 through the IRES mechanism and the wheel translation mechanism.

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

[0051] The present invention clarifies the specific role of circSPIRE1 and its encoded polypeptide rtSPIRE1 in the proliferation and metastasis of prostate cancer cells through experimental means, especially its molecular mechanism of promoting the development of prostate cancer by activating the PI3K / AKT signaling pathway, providing a new perspective for in-depth understanding of the pathogenesis of prostate cancer.

[0052] The present invention verifies the specific biological function of the rtSPIRE1 polypeptide in prostate cancer cells, including its effects on cell proliferation, migration and invasion, through biochemical and cell biological experiments, thus making 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 treating prostate cancer, and the activation level of the PI3K / AKT signaling pathway is regulated by the interaction between rtSPIRE1 and SCN5A, thereby developing an anti-cancer treatment strategy with potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] Figure 2 The 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 expression difference of circSPIRE1 in prostate cancer and adjacent tissues in clinical samples; E is a schematic diagram of the expression difference of circSPIRE1 in prostate cancer cell lines and normal cell lines; F is a schematic diagram of the expression level of circSPIRE1 and the 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 electrophoresis of prostate cancer cell lines; C is a schematic diagram showing the lack of 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 circSPIRE1 fluorescence in situ hybridization experiments; and H is a schematic diagram showing the results of circSPIRE1 nucleocytoplasmic separation experiments;

[0057] Figure 4The schematic diagram of circSPIRE1 promoting the proliferation, metastasis and invasion of prostate cancer, wherein A is a schematic diagram of the expression level of the parent gene SPIRE1 in cancer cells after silencing the expression of circSPIRE1 (si-circSPIRE1#1, si-circSPIRE1#2) in cancer cells; B is a schematic diagram of the expression level of the parent gene SPIRE1 in cancer cells after overexpressing circSPIRE1 (OE-circSPIRE1) in cancer cells; C is a schematic diagram of the proliferation ability of prostate cancer cell lines silencing the expression of circSPIRE1 in cancer cells PC3; D is a schematic diagram of the proliferation ability of prostate cancer cell lines overexpressing circSPIRE1 in cancer cells PC3; E is a schematic diagram of the proliferation ability of prostate cancer cell lines silencing the expression of circSPIRE1 in cancer cells DU145; F is a schematic diagram of the proliferation ability of prostate cancer cell lines overexpressing circSPIRE1 in cancer cells DU145; G is a schematic diagram of the proliferation ability of prostate cancer cell lines showing silencing of circSPIRE1 or overexpression of circSPIRE1 in clone formation experiments;

[0058] Figure 5 Schematic diagram of the results of circSPIRE1 animal experiments in vivo; 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 growth rate and tumor weight of the tumors in 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 verifying the biological function of circSPIRE1; A is a schematic diagram of the circSPIRE1 wheel encoding the new protein; 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 verify the wheel translation mechanism (T1: blank control; T2: inserting 3FLAG tag before ATG of ORF; T3: based on T2, inserting T base after ORF to form a stop codon); D is a schematic diagram of the circular structure of T2 and T3 plasmids; E is a circular circSPIRE1 overexpression vector (T2) constructed with fusion Flag and 3xFlag tag And the schematic diagram of the electrophoresis results of the mutant (T3), the results showed the phenomenon of wheel translation, and the main observable products were 17kDa, 34kDa, and 51kDa, which corresponded to the molecular weights of the 1-circle, 2-circle, and 3-circle encoding products of the circSPIRE1 open reading region, respectively; F is a schematic diagram of protein spectrum analysis that both T2 and T3 plasmids contain specific fragments of circSPIRE1 encoding products 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 5 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 experiment; Figure C is a schematic diagram of the RNA pull-down experiment results; Figure D is a schematic diagram of the mass spectrometry analysis results; Figure E is a schematic diagram of the Western blot analysis to verify the interaction between circSPIRE1 and hnRNPA1; Figure F is a schematic diagram of WB verification of exogenously transfected hnRNPA-HA with different amounts; Figure G is a schematic diagram of dual luciferase activity detection after exogenously transfecting different amounts of hnRNPA-HA and circSPIRE1-IRES activity verification vector R3; Figure H is a schematic diagram of IRES activity detection after transfecting different amounts of hnRNPA1;

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

[0062] Fig. 9Schematic diagram to verify that rtSPIRE1 activates the PI3K / AKT signaling pathway and promotes metastasis; 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 results of gene function enrichment analysis; Figure I is the expression level of proteins related to the PI3K / AKT signaling pathway detected by Western blot analysis; Figure J is a schematic diagram of the expression level 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 and non-metastatic prostate cancer tissue, prostate cancer tissue and its paired adjacent prostate cancer tissue samples were collected, 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 out 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'-TGAAAACCAACTCCACCACGG-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 and 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 with coding ability were found. 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 clinical prostate cancer 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, the analysis of follow-up information 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 for 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, through the Oligo dT reverse transcription experiment, the Oligo dT primer is used to specifically bind to the Poly-A tail of the mRNA for reverse transcription. If circSPIRE1 is a circRNA (without a Poly-A tail), the reverse transcription efficiency will be low. Next, the RNase R experiment is performed, which degrades linear RNA and retains circular RNA. The changes in RNA before and after treatment are detected by RT-PCR or qPCR. If the circular RNA signal is enhanced and the linear RNA is reduced, it indicates that circSPIRE1 is a circRNA. Finally, the actinomycin D experiment is used to monitor the stability of circSPIRE1 after inhibiting RNA transcription. If the RNA remains stable over time, it further supports that it is a circRNA. These experimental steps can help confirm and repeatedly verify the stable circular structure of circRNA.

[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 RT-qPCR was used to detect the distribution of circSPIRE1 in the nucleus and cytoplasm.

[0086] circSPIRE1 FISH probe:

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

[0088] 2. Results

[0089] The reverse splicing junction sequence of circSPIRE1 determined by Sanger sequencing (Sequence: CGAAAAGTGATGGTTGTGTGCTGCTGCT (SEQ ID NO: 18) was consistent with the predicted result, and 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 and actinomycin D experiments suggest that compared with linear mRNA (Linear SPIRE1), the covalently closed loop structure of circSPIRE1 is highly stable both inside and outside prostate cancer cells ( Figure 3CF). Fluorescence in situ hybridization and nuclear-cytoplasmic separation experiments showed that the circRNA was mainly located in the cytoplasm ( Figure 3 G, H). The above experiments showed that circSPIRE1 was formed by reverse splicing of the parent gene SPIRE1 and was stably expressed in prostate cancer cells.

[0090] Example 3 Biological functions 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 scratch 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 that specifically silenced circSPIRE1 and overexpressed circSPIRE1 ( Figure 4 A silent expression, Figure 4 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 displayed. 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, such as 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 is from Gisai Biotechnology Co., Ltd., specifically the Luc2-IR ES-Report vector, and the vector sequencing primer is 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] By RNA pull-down experiments, proteins binding to circSPIRE1 were enriched from prostate cancer cell lysates using biotin-labeled RNA pull-down probes.

[0120] RNA pulldown probe:

[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 technology were combined to identify and verify the interaction between hnRNPA1 and circSPIRE1.

[0126] 2. Results

[0127] Through the analysis of the TransCirc bioinformatics platform, we found that circSPIRE1 contains a single open reading frame (ORF) that lacks a stop codon. The open reading frame (ORF) sequence is (SEQ ID NO: 19). This feature suggests that circSPIRE1 may encode a new type of protein through a rolling translation mechanism to exert 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 is from Gisai Biotech. Specifically, it is 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 (SEQ ID NO: 3) specifically encoded by ORF was further confirmed by electrophoresis and proteomics analysis ( Figure 6 E, F).

[0128] To further verify that circSPIRE1 plays a functional role in prostate cancer cells through the 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 showed stronger prostate cancer proliferation and metastasis ability ( Figure 6 Based on the above experimental data, we speculate that circSPIRE1 may promote prostate cancer progression by encoding the new protein rtSPIRE1 through the 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 above five different vectors ( Figure 7 A: Plasmids R1-5), 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, which indicates that circSPIRE1 is translated by a cap-independent mechanism.

[0130] In order to further explore the factors regulating the translation 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 translation function of circSPIRE1. Then, hnRNPA1-HA and circSPIRE1 were co-transfected into HEK-293T cells, and double fluorescein experiments were performed, and 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 and 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 PI3K / AKT signaling pathway was detected.

[0138] 2. Results

[0139] Immunoprecipitation (IP) experiments used Flag antibodies to immunoprecipitate rtSPIRE1, IgG was used as a control, and the binding of proteins in different samples was detected. Marker was used to indicate the size of the protein. Immunoprecipitation was used to find the downstream mechanism of rtSPIRE1 function. Combined with mass spectrometry and WB, it was found that rtSPIRE1 can interact with the ion channel SCN5A ( Figure 8 A). Mass spectrometry analysis results show the mass spectra of SCN5A in the immunoprecipitated samples, 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 reference) after co-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 changes of SCN5A may affect the key components of the Wnt / β-catenin signaling pathway directly or indirectly. Fig. 9 ), such as the stability and nuclear translocation of β-catenin. Fig. 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. Fig. 9 EH shows the results of gene function enrichment analysis, in which the gene ontology (GO) enrichment analysis diagram shows the significantly enriched GO entries, and the gene set enrichment analysis (GSEA) diagram shows the significant enrichment of the PI3K-Akt signaling pathway. Fig. 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. Fig. 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 SCN5A ligase; overexpression of rtSPIRE1 stabilized SCN5A, reduced its degradation, promoted the activation of sodium-calcium transporters, 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 mentioned above, 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 comprises a forward primer and a reverse primer for amplifying circSPIRE1, the sequence of the forward primer is shown as SEQ ID NO: 4, and the sequence of the reverse primer is shown as 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 solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A molecular marker for prostate cancer, characterized in that: The molecular markers include any one or a combination of the following: 1) circular RNA circSPIRE1, the nucleotide sequence of which is shown in SEQ ID NO: 1; 2) Expression products of circular RNA circSPIRE1.

2. The molecular marker according to claim 1, characterized in that The expression product of the circular RNA circSPIRE1 is the polypeptide rtSPIRE1, and its amino acid sequence is shown in SEQ ID NO:

3.

3. The molecular marker according to claim 1, characterized in that The nucleic acid sequence encoding the polypeptide rtSPIRE1 is shown in SEQ ID NO:

19.

4. Use of the molecular marker as claimed in claim 1 or 2 in the preparation of a preparation for screening drugs for treating prostate cancer.

5. A method for screening candidate drugs for treating prostate cancer, characterized in that: The method comprises: detecting the expression level of the prostate cancer molecular marker according to claim 1 or 2 in prostate cancer cells after being treated 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.

6. A detection kit for diagnosing prostate cancer, characterized in that: Comprising a reagent for detecting the molecular marker according to claim 1 or 2.

7. The detection kit according to claim 6, characterized in that It comprises a forward primer and a reverse primer that can amplify the molecular marker according to claim 1, wherein the sequence of the forward primer is shown as SEQ ID NO: 4, and the sequence of the reverse primer is shown as SEQ ID NO:

5.

8. The detection kit according to claim 6, characterized in that Also included is an immunodetection product of polypeptide rtSPIRE1, wherein the immunodetection product of polypeptide rtSPIRE1 includes an antibody that specifically binds to polypeptide rtSPIRE1.

9. The detection kit according to claim 6, characterized in that A positive control and a negative control are also included. The positive control is a prostate cancer cell line that is positive for circular RNA circSPIRE1; and the negative control is a prostate cell line that is negative for circular RNA circSPIRE1.

10. A kit for diagnosis and prognosis of prostate cancer, characterized in that: The invention is composed of reagents for specifically detecting the gene expression level of the molecular markers according to claim 1 or 2.

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