FOXP1-196aa protein and application thereof

Abstract

The invention discloses an FOXP1-196aa protein and an application of the FOXP1-196aa protein. The amino acid sequence of the FOXP1-196aa protein is as shown in SEQ ID No. 1. According to the invention, a circRNA with down-regulated expression is found in nasopharyngeal carcinoma through circRNA chip sequencing, the circRNA is derived from 8th to 11th exons of an FOXP1 gene, and the circRNA is named as circFOXP1 (circBase: hsacirc0008234; the genome position is chr3: 71090478 to 71102924, and the genome position is chr3 And the length is 587bp). The invention reveals that circFOXP1 plays a role in inhibiting nasopharyngeal carcinoma tumor by coding a new protein (FOXP1-196aa) with 196 amino acids for the first time, and provides a new strategy for treatment of nasopharyngeal carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a FOXP1-196aa protein and an application thereof. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor that occurs in the nasopharyngeal mucosa. It has a high incidence in South China and Southeast Asia, with distinct ethnic and geographic distribution characteristics. Genetic susceptibility and environmental factors (including Epstein-Barr virus infection) are the main causes of NPC. In recent years, advances in radiotherapy technology and the widespread use of chemotherapy regimens have improved the treatment outcomes for patients with NPC. However, for patients with tumor recurrence or metastasis after radiotherapy, treatment options are still very limited, which remains the main cause of death in these patients.

[0003] In recent years, circular RNAs (circRNAs) have become a research hotspot in the field of noncoding RNA. CircRNAs play a role in multiple important functions, including inhibiting the maturation of miRNAs, regulating alternative splicing, enhancing gene transcription, promoting protein-protein interactions, acting as a scaffold for RNA-binding proteins, and acting as miRNA sponges.

[0004] Like most noncoding RNAs, circRNAs were initially thought to be nontranslatable due to the lack of a well-defined open reading frame (ORF). However, increasing evidence suggests that some circRNAs can be translated through cap-independent translation mechanisms such as internal ribosome entry sites (IRES) and N6-methyladenosine mRNAs. 6 A modification) produces proteins. To date, a small number of known translatable circRNAs, including circZNF609, circE-cadherin, circFBXW7, circGGNBP2, circBUB1B, and circARHGAP35, have been reported to be involved in the IRES-mediated translation process. Under specific conditions, some circRNAs can be expressed through m 6 However, no studies have reported that circRNAs drive the onset and progression of NPC by encoding proteins. Summary of the invention

[0005] The purpose of the present invention is to provide a FOXP1-196aa protein and its application in view of the deficiencies in the prior art.

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

[0007] The first aspect is to provide a FOXP1-196aa protein, wherein the amino acid sequence of the FOXP1-196aa protein is shown in SEQ ID No. 1:

[0008] MMTPQVITPQQMQQILQQQVLSPQQLQVLLQQQQALMLQQQQLQEFYKKQQEQLQLQLLQQQHAGKQPKEQQQVATQQLAFQQQLLQMQQLQQQHLLS LQRQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRERFPCQWL.

[0009] Furthermore, the FOXP1-196aa protein is encoded by circFOXP1, and the circFOXP1 is derived from exons 8-11 of the FOXP1 gene, with a genomic location of chr3:71090478-71102924 and a length of 587 bp.

[0010] The second aspect is to provide the use of the above-mentioned FOXP1-196aa protein in the preparation of products for the treatment of nasopharyngeal carcinoma.

[0011] Furthermore, the FOXP1-196aa protein has an inhibitory effect on nasopharyngeal carcinoma organoids.

[0012] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0013] The present invention discovered a downregulated circRNA in nasopharyngeal carcinoma through circRNA chip sequencing. The circRNA originated from exons 8-11 of the FOXP1 gene and was named circFOXP1 (circBase: hsa_circ_0008234; genomic location chr3: 71090478-71102924; length: 587 bp).

[0014] This invention reveals for the first time that circFOXP1 exerts an inhibitory effect on NPC tumors by encoding a new 196-amino acid protein (FOXP1-196aa), providing a new strategy for the treatment of NPC. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Figure 3 shows the expression profile of circRNAs and the characteristics of circFOXP1 in nasopharyngeal carcinoma (NPC); (A) heat map (left) and volcano plot (right) showing 742 differentially expressed circRNAs identified in primary NPC tissues (n=5) and normal nasopharyngeal epithelial tissues (n=5) using Arraystar human circRNA microarray; the cutoff value was fold change>1.5 or <-1.5, P<0.05; (B) Heat map (left) and volcano plot (right) showing 453 differentially expressed circRNAs identified in recurrent NPC tissues (n=5) and primary NPC tissues (n=5); (C) Intersection of circRNA expression differences between the two groups (primary NPC tissue vs. normal tissue, recurrent NPC tissue vs. primary NPC tissue), a total of 2 upregulated and 13 downregulated circRNAs were found; (D) 7 c (E) The expression level of circFOXP1 was detected in normal nasopharyngeal epithelial cells NP69 and 7 NPC cell lines by qRT-PCR; (F) The head-to-tail splicing structure of circFOXP1 in the qRT-PCR product was detected by Sanger sequencing, and its genome size and sequence reported in the circBase database were determined (left); The electrophoresis results of circFOXP1 primers showed that a specific fragment was amplified (right); (G) The circular and linear transcripts of FOXP1 in cDNA and gDNA were detected by divergent primers and polymerized primers; (H) RNase qRT-PCR showed the changes in the expression levels of circFOXP1 and FOXP1 after R enzyme treatment of C666-1 and CNE-1 cells; (I) The localization of circFOXP1 was detected by fluorescence in situ hybridization (FISH); (J) The expression of circFOXP1 in NPC tissue microarray (HNasN110Su01) was detected by circFOXP1 in situ hybridization (ISH) probe, scale bar = 50 μm; (K) The effect of circFOXP1 expression on the overall survival and disease-free survival of NPC patients was evaluated by Kaplan–Meier survival analysis; data are expressed as mean ± SD; P values ​​in D, E, and H were calculated by unpaired two-tailed t test; **P < 0.01, ***P < 0.001.

[0016] Figure 2Figure 3 shows that circFOXP1 encodes a novel protein, FOXP1-196aa; (A) circFOXP1 is 587 nt long and may be translated into a protein (FOXP1-196aa) containing 196 amino acids (about 27 kDa); (B) the putative IRES (internal ribosome entry site) activity of circFOXP1 was detected; the left figure shows five CMV-inducible expression plasmids cloned between the Rluc and Luc reporter genes with independent start (AUG) and stop (UGA) codons (empty vector, EMCVIRES sequence, complete IRES sequence 251–453 of circFOXP1, IRES 251–352 of circFOXP1, IRES 251–3 ... 352–453); the right panel shows the relative luciferase activity of Luc / Rluc in the above plasmids; (C) IRES activity was detected using mCherry and EGFP reporter genes, mCherry is a red fluorescent protein; EGFP is a green fluorescent protein; (D) The positive control (SV40 promoter), negative control (SV40 promoter deletion), IRES251–352 and IRES 352–453, scale bar = 200 μm; (E) 40S, 60S, 80S and polyribosome fractions were extracted from C666-1 and CNE1 cells overexpressing circFOXP1 and circFOXP1-ATG mutant by 5%–50% sucrose gradient ultracentrifugation, and the ribosome distribution between the two groups was plotted; (F) qRT-PCR was performed using junction-specific primers to analyze the translation potential of circFOXP1, and qRT-PCR using linear-specific primers was used as a positive control to determine the translation potential of linear FOXP1; (G) Four plasmids were constructed to detect Flag expression between the four groups (empty plasmid; circFOXP1-Flag plasmid: circFOXP1 with Flag tag 1 sequence was cloned into a CMV-inducible expression vector; circFOXP1-Flag-ATG mutant plasmid: the circFOXP1 sequence with a Flag-tagged start codon mutation (ATG→ATA) was cloned into a CMV-inducible expression vector; FOXP1-196aa: the FOXP1-196aa sequence with a Flag tag was cloned into a CMV-inducible expression vector; FCF and BCF are sequences that can circularize circRNA; (H) After transfection of the empty plasmid group and circFOXP1 plasmid in HEK-293T cells, total proteins were separated by SDS-PAGE; the circFOXP1 band between 25–40 kDa was cut and subjected to mass spectrometry analysis to detect the cyclization site-specific sequence in FOXP1-196aa;(IJ) FOXP1-196aa-Flag was stably transfected into C666-1 and CNE1 cells using lentivirus, and the expression of commercial FOXP1 antibody and FOXP1-196aa-specific antibody was detected by Western blotting to confirm the presence of protein FOXP1-196aa; (K) FOXP1-196aa-specific antibody was used to detect the expression of FOXP1-196aa in NPC tissues and normal specimens; data are expressed as mean ± standard deviation; P values ​​in B, F, and K were calculated by unpaired two-tailed t test; *P < 0.05, ***P < 0.001. ;

[0017] Figure 3 In reality, FOXP1-196aa inhibits the progression of nasopharyngeal carcinoma (NPC) cells in vivo and in vitro. Empty control, circFOXP1, FOXP1-196aa and circFOXP1-ATG mutants were stably transfected in C666-1 and CNE1 cell lines by lentivirus; (A) Cell viability was detected by CCK-8 assay; (B) Cell replication and self-renewal ability were detected by plate cloning assay; (CD) Cell migration ability was shown by cell scratch assay, scale bar = 200μm; (EF) Cell invasiveness was shown by Transwell assay, scale bar = 200μm; (G) Cell apoptosis ability was evaluated by flow cytometry apoptosis assay; (H) Immunofluorescence results showed the expression level of γ-H2AX, scale bar = 20μm; (I) The expression level of γ-H2AX was shown by Western blotting; (J) 5×10^ 6 Stably transfected C666-1 cells were subcutaneously injected into nude mice. After 30 days, the tumors were removed, photographed and weighed; (K) Tumor volume was measured every 5 days, and the average tumor weight of each group was measured at the end of the experiment (n = 6 per group); (L) Representative H&E stained section images of lung metastasis; (M) The lungs of each nude mouse were divided into four parts, the total area of ​​metastatic lesions in each part was calculated, and the average of the four parts was considered as the average metastatic lung area of ​​each nude mouse. The average tumor area of ​​each group was quantified (n = 6 per group), scale bar = 100 μm (top) and 25 μm (bottom); data are expressed as mean ± SD. P values ​​in AG, K and M were calculated by unpaired two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001.

[0018] Figure 4The expression profile of circRNAs and the characteristics of circFOXP1 in NPC are shown; (A) the left figure shows that on 24 chromosomes, 206 circRNAs are upregulated and 536 circRNAs are downregulated in primary NPC tissues compared with normal tissues; the right figure shows a scatter plot of circRNA expression changes between primary NPC tissues and normal tissues; the values ​​of the X-axis and Y-axis in the scatter plot are the normalized signal values ​​of the samples (in log2 scale) or the average normalized signal values ​​of the sample group; the green line is the foldchange line, and the circRNAs above the top green line and below the bottom green line represent The change between the two groups of compared samples exceeded 1.5-fold; the area between the red line and the top green line or the bottom green line represents the main distribution range of differentially expressed circRNAs; (B) Classification of dysregulated circRNAs between primary NPC tissues and normal tissues; (C) The left figure shows that on 24 chromosomes, 180 circRNAs were upregulated and 273 circRNAs were downregulated in recurrent NPC tissues compared with primary NPC tissues; the right figure shows a scatter plot of circRNA expression changes between recurrent NPC tissues and primary NPC tissues; (D) (E) Detection of circRNA sequences of hsa_circ_0006773, hsa_circ_0003553, hsa_circ_0001280, hsa_circ_0001776, hsa_circ_0007218 and hsa_circ_0003502 by Sanger sequencing; (F) Detection of the expression of circFOXP1 and FOXP1 in C666-1 cells treated with actinomycin D by qRT-PCR; (G) Analysis of circRNAs in the cytoplasm and nucleus of NPC cells by qRT-PCR Changes in the RNA levels of FOXP1, GAPDH, β-actin, and U6; (H) The expression of circFOXP1 in NPC tissue microarray (TFNas0-02) was detected by circFOXP1 in situ hybridization probe, scale bar = 50 μm; (I) The effect of circFOXP1 expression in NPC tissue microarray (TFNas0-02) on the overall survival and disease-free survival of NPC patients was analyzed by Kaplan–Meier survival curve analysis; data are expressed as mean ± SD; P values ​​in G were calculated by unpaired two-tailed t-test; **P < 0.01, ***P < 0.001.

[0019] Figure 5 It shows that CircFOXP1 encodes a new protein FOXP1-196aa; (A) The mRNA of circFOXP1 was predicted using SRAMP software 6The A sequence is located at positions 399 and 555, and then the A base is mutated to a G base. 6 (A) CircFOXP1 with mutation site was transfected into HEK-293T cells, and the expression of FOXP1-196aa was detected; (B) FOXP1-196aa-Flag was stably transfected into C666-1 and CNE1 cells by lentivirus, and the expression of Flag antibody was detected by Western blotting to confirm the presence of FOXP1-196aa; (C) Immunofluorescence experiment was performed in NPC cells using FOXP1-196aa-specific antibody, scale bar = 100 μm; (D) Western blotting was performed to detect the expression of Flag antibody. The expression level of FOXP1-196aa in normal nasopharyngeal epithelial cells NP69 and seven NPC cell lines was detected by blotting; (E) The expression level of FOXP1-196aa in 13 normal tissues and 26 NPC tissues was detected by immunohistochemistry, scale bar = 50 μm; (F) The expression of FOXP1-196aa in NPC tissue microarray (HNasN110Su01) was detected by in situ hybridization (ISH) probe of FOXP1-196aa, scale bar = 100 μm; (G) The effect of FOXP1-196aa expression on overall survival and disease-free survival in NPC patients was evaluated by Kaplan–Meier survival analysis; data are expressed as mean ± SD; P values ​​in C and E were calculated by unpaired two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001.

[0020] Figure 6Figure 1 shows that FOXP1-196aa inhibits the progression of nasopharyngeal carcinoma (NPC) cells and organoids; (A) The expression of FOXP1-196aa was detected by Western blotting using specific antibodies; (B) The levels of γ-H2AX, Ki-67, and DHX9 were detected by immunohistochemistry, scale bar = 50 μm; (C) The percentage of γ-H2AX, Ki-67, and DHX9 positive cells was quantified; (D) H&E staining showed the cell morphology between organoids and their corresponding nasopharyngeal mucosal tissues, scale bar = 200 μm; (E) H&E staining showed the tumor markers of Ki-67 and Vimentin, scale bar = 200 μm; (F) Comparing the copy number variation profiles in the parental tumor and organoids, the organoids retained the chromosomal gains and losses of the parental tumor; (G) SDS-PAGE showed the protein bands of FOXP1-196aa; (H) 4 μg of FOXP1-196aa was added to the NPC organoids. After FOXP1-196aa, the diameter changes of organoids were detected 2 weeks later; (I) MTT assay showed the viability of organoids after FOXP1-196aa treatment for 48 hours; data are expressed as mean ± SD; P values ​​in C, H, and I were calculated by unpaired two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0022] Example 1 Identification and characterization of circFOXP1 in nasopharyngeal carcinoma

[0023] To determine whether circRNAs regulate the progression of NPC, the present invention performed Arraystar circRNA chip sequencing on normal nasopharyngeal epithelial tissue (n=5), primary NPC tissue (n=5) and recurrent NPC tissue (n=5). Arraystar circRNA chip technology has successfully identified significantly differentially expressed circRNAs in a variety of cancers, such as colorectal cancer, cervical cancer and non-small cell lung cancer. Under the conditions of P<0.05 and Fold change>1.5, it was found that compared with normal tissue, there were 206 significantly upregulated and 536 significantly downregulated circRNAs in primary NPC tissue ( Figure 1 A, Figure 4 A). Compared with primary NPC tissues, there were 180 significantly upregulated and 273 significantly downregulated circRNAs in recurrent NPC tissues ( Figure 1 B, Figure 4C). These circRNAs are mainly located in the exon region ( Figure 4 B, D). The intersection of circRNAs with different expression levels in the two groups (primary NPC tissue and normal tissue, recurrent NPC tissue and primary NPC tissue) showed that there were 2 up-regulated and 13 down-regulated circRNAs ( Figure 1 C). After excluding intron-derived circRNAs (hsa_circ_0000510, hsa_circ_0000552, and hsa_circ_0006783) and exon-intron circRNAs (hsa_circ_007854), seven differentially expressed circRNAs (hsa_circ_0008234, hsa_circ_0006773, hsa_circ_0003553, hsa_circ_0001280, hsa_circ_0001776, hsa_circ_0007218, and hsa_circ_0003502) were further screened, and divergent primers were designed for these circRNAs, and the specific base sequences of the reverse splicing junction sites were detected by Sanger sequencing ( Figure 4 E, Figure 1 F). Real-time quantitative polymerase chain reaction (qRT-PCR) was further used to analyze the expression difference of circFOXP1 in 122 primary NPC samples and 35 normal samples. The results showed that the expression of circFOXP1 in 122 primary NPC samples was significantly lower than that in 35 normal samples, and the expression of circFOXP1 in 38 recurrent NPC samples was also significantly lower than that in primary NPC tissues ( Figure 1 D). In addition, the difference in circFOXP1 expression between the normal nasopharyngeal epithelial cell line NP69 and the nasopharyngeal carcinoma cell line was analyzed by qRT-PCR, and similar results were obtained ( Figure 1 E).

[0024] circFOXP1 is formed by reverse splicing of exons 8, 9, 10, and 11 of the forkhead box protein P1 gene (FOXP1) and is 587 nucleotides long. The bases of the circFOXP1 circularization site identified by Sanger sequencing are consistent with the annotations of the circBase database (http: / / www.circbase.org / , Figure 1 F). In order to characterize circFOXP1, the present invention designed divergent primers and polymerizing primers. First, complementary DNA (cDNA) and genomic DNA (gDNA) extracted from C666-1 and CNE1 cells were used as templates. The gel electrophoresis results showed that circFOXP1 was amplified only from cDNA but not from gDNA ( Figure 1G), indicating that the circular structure of circFOXP1 is generated by back-splicing. circFOXP1, but not linear FOXP1, was found to be resistant to RNase R digestion ( Figure 1 H). Actinomycin D experiments showed that circFOXP1 was more stable than linear FOXP1 mRNA ( Figure 4 F). The localization of circFOXP1 was detected by fluorescence in situ hybridization (FISH) analysis, and the results showed that circFOXP1 was enriched in the cytoplasm of NPC cells ( Figure 1 I). The same conclusion was reached by cytoplasmic and nuclear RNA analysis by qRT-PCR ( Figure 4 G). Finally, according to the RNA in situ hybridization (ISH) staining score, 110 NPC tissue microarray samples HNasN110Su01 were divided into a low expression group (n = 50) and a high expression group (n = 60) ( Figure 1 J). Kaplan–Meier analysis showed that the overall survival and disease-free survival rates of the circFOXP1 low expression group were significantly lower than those of the high expression group ( Figure 1 K). Similar results were also observed in nasopharyngeal carcinoma tissue microarray sample TFNas0-02 ( Figure 4 HI). Taken together, these findings suggest that circFOXP1 is downregulated in NPC and is associated with a good prognosis.

[0025] Example 2 circFOXP1 can encode a new protein FOXP1-196aa with 196 amino acids

[0026] In order to explore whether circFOXP1 can encode a new protein, the present invention predicted based on the circRNADb database (http: / / reprod.njmu.edu.cn / cgi-bin / circrnadb / circRNADb.php). The results showed that the circFOXP1 sequence contained an open reading frame (ORF) with a start codon ATG and an internal ribosome entry site (IRES) from position 251 to 453, which may encode a protein of 196 amino acids (FOXP1-196aa; Figure 2 A). The activity of IRES was evaluated using a luciferase assay. The full-length or truncated circFOXP1 putative IRES sequence was cloned into the plasmid, and the results of the luciferase assay showed that the activity of IRES251-352 and IRES 352-453 was significantly higher than that of the control group ( Figure 2B). To further confirm the activity of IRES, mCherry and EGFP reporter gene experiments were performed. In this experiment, when translation is initiated by a putative IRES, red fluorescent proteins mCherry and EGFP will be expressed. In contrast, if IRES fails to recruit ribosomes, only weak EGFP expression will be detected ( Figure 2 C). The results showed that the EGFP expression in the IRES251-352 group was significantly higher than that in the negative control group, while the EGFP expression in the IRES 352-453 group and the negative control group was similar. Therefore, IRES251-352 may drive the translation of circFOXP1 ( Figure 2 D).

[0027] In addition, the translation of circRNA may be regulated by N6-methyladenosine (m 6 A) Modification drive, it has also been reported that m 6 A modification does not promote the translation of exogenous circRNAs, which indicates that m 6 A modification may improve the translation efficiency of some circRNAs, but it is not necessarily required for all translatable circRNAs. The present invention used SRAMP software (http: / / www.cuilab.cn / sramp) to predict the m 6 The A sequence was present at positions 399 and 555, and the A base was mutated to a G base. The results showed that there was no significant change in the protein level of circFOXP1 translation ( Figure 5 A). To determine the translation of circFOXP1, polysome profiling was performed using sucrose density gradient centrifugation. C666-1 and CNE1 cells were transfected with Lv-circFOXP1 or Lv-circFOXP1 ATG mutant (non-ATG) plasmids, and the ribosome profiles between the two groups showed differences in the 40S, 60S, 80S, and polysome fractions, especially in the 80S fraction ( Figure 2 E). qRT-PCR detected circFOXP1 in these fractions, but no circFOXP1 was detected in the circFOXP1-ATG mutant group ( Figure 2 F). As a positive control, linear FOXP1 was mainly detected in the 80S and polysome fractions and was not affected by circFOXP1 transfection ( Figure 2 F).

[0028] In order to verify the existence of FOXP1-196aa, the present invention constructed four plasmids with Flag tags (empty vector, circFOXP1-Flag: circFOXP1 sequence with Flag tag cloned into a CMV-induced expression vector, FOXP1-196aaFlag: FOXP1-196aa sequence with Flag tag cloned into a CMV-induced expression vector, circFOXP1-Flag-mut: circFOXP1 sequence with start codon mutation (ATG→ATA) cloned into a CMV-induced expression vector). Western blot analysis showed that after circFOXP1 or FOXP1-196aa transfection, the Flag-tagged FOXP1-196aa protein of about 27 kDa was detected ( Figure 2 G). In HEK-293T cells, total proteins were separated by SDS-PAGE after pull-down experiments after transfection with empty vector group and circFOXP1 plasmid. The circFOXP1 band was cut between 25–40 kDa and subjected to liquid chromatography-mass spectrometry (LC-MS) / MS analysis ( Figure 5 B). LC-MS / MS successfully detected the partial amino acid sequence and cyclization site-specific sequence (RFPCQWL) of FOXP1-196aa ( Figure 2 H). Using a specific antibody with the cyclization site (RFPCQWL) of the FOXP1-196aa-specific sequence, as well as a Flag antibody and a FOXP1 antibody (recognizing the same amino acid sequence as FOXP1-196aa), FOXP1-196aa-Flag was overexpressed in two nasopharyngeal carcinoma cell lines, C666-1 and CNE1. The results showed that these antibodies overexpressed the FOXP1-196aa band ( Figure 2 I, J, Figure 5 C). In addition, no significant difference in the expression of the maternal gene FOXP1 was observed ( Figure 2 I). After nasopharyngeal carcinoma cells were transfected with Lv-vector, Lv-circFOXP1, Lv-FOXP1-196aa, and Lv-circFOXP1-ATG mut, immunofluorescence experiments showed that the fluorescence intensity of FOXP1-196aa in the Lv-circFOXP1 and Lv-FOXP1-196aa groups was higher than that in the control group, while the expression of FOXP1-196aa in the circFOXP1-ATG mutant group was significantly lower than that in the Lv-circFOXP1 group ( Figure 5D). In addition, FOXP1-196aa expression in 20 NPC tissues and 20 normal NPC tissues was detected using FOXP1-196aa specific antibody. The results showed that FOXP1-196aa expression in NPC tissues was significantly downregulated ( Figure 2 K). Similarly, it was found that the expression of FOXP1-196aa in the normal cell line NP69 was significantly higher than that in the nasopharyngeal carcinoma cell line ( Figure 5 E). Immunohistochemistry results showed that the expression level of FOXP1-196aa in 26 NPC tissue samples was significantly lower than that in 13 normal control groups ( Figure 5 F). Finally, according to the ISH staining score ( Figure 5 G), 110 nasopharyngeal carcinoma tissue microarray samples HNasN110Su01 were divided into low expression group (n = 53) and high expression group (n = 57). Kaplan–Meier analysis showed that the overall survival rate and disease-free survival rate of the FOXP1-196aa low expression group were significantly lower than those of the high expression group ( Figure 5 H). Overall, these findings suggest that circFOXP1 encodes a novel 196-amino acid protein, FOXP1-196aa, in NPC and its expression is associated with a favorable prognosis.

[0029] Example 3 Verification of the inhibitory effect of FOXP1-196aa on nasopharyngeal carcinoma

[0030] In order to explore the biological function of FOXP1-196aa, the present invention transfected four Lv vectors (empty vector, circFOXP1, FOXP1-196aa and circFOXP1-ATG mutant) into nasopharyngeal carcinoma cell lines. Western blot experiments showed that FOXP1-196aa increased significantly in the circFOXP1 and FOXP1-196aa groups, while no significant difference was observed in the circFOXP1-ATG mutant group ( Figure 6 A). Functional experiments showed that the proliferation, cloning, metastasis and invasion of NPC cells were inhibited after transfection with Lv-circFOXP1 or Lv-FOXP1-196aa. In addition, the tumorigenicity of the Lv-circFOXP1-ATG mutant group was significantly greater than that of the Lv-circFOXP1 group, but lower than that of the Lv empty vector group ( Figure 3A–F). Further, the apoptosis rate was detected by flow cytometry, and the γ-H2AX level was detected by immunofluorescence and Western blot, and it was found that Lv-circFOXP1 and Lv-FOXP1-196aa significantly promoted apoptosis and upregulation of γ-H2AX. Similarly, the apoptosis to DNA damage ratio of the Lv-circFOXP1-ATG mutant group was significantly higher than that of the normal group, but lower than that of the Lv-circFOXP1 group ( Figure 3 G–I).

[0031] To further evaluate the tumor suppressor effect of FOXP1-196aa encoded by circFOXP1 in vivo, 5×10^ 6 C666-1 cells (Lv empty vector, Lv-circFOXP1, Lv-FOXP1-196aa, or Lv-circFOXP1-ATG mutant group). After transplantation, the tumor size of mice was monitored every five days, and the tumor volume was calculated using the modified ellipsoid formula (volume = length × width2 / 2). After 30 days, the mice were sacrificed and the final tumor weight was measured. The tumors were fixed and prepared for histological examination ( Figure 6 B). The results showed that the tumor growth rate and final weight of the Lv-circFOXP1 and Lv-FOXP1-196aa groups were significantly lower than those of the empty vector control group. However, the growth rate and tumor weight of the Lv-circFOXP1-ATG mutant group were faster and heavier than those of the Lv-circFOXP1 group, but significantly lower than those of the empty vector control group ( Figure 3 JK). In addition, compared with the Lv empty vector and Lv-circFOXP1-ATG mutant groups, the proliferation marker Ki67 was downregulated in the Lv-circFOXP1 and Lv-FOXP1-196aa groups, while the expression level of γ-H2AX was upregulated in the Lv-circFOXP1 and Lv-FOXP1-196aa groups ( Figure 6 CD). In order to evaluate the metastasis in vivo, 2×10^ 6 C666-1 cells (transfected with Lv empty vector, Lv-circFOXP1, Lv-FOXP1-196aa, or Lv-circFOXP1-ATG mutant) were injected into nude mice via the tail vein. Approximately two months later, the mice were sacrificed, and the lung tissues were fixed, paraffin-embedded, and sectioned for histopathological examination ( Figure 6 B). Figure 3LM shows H&E staining of lung metastasis and the average tumor area of ​​lung metastasis in each group. The average tumor area of ​​lung metastasis was lower in the Lv-circFOXP1 and Lv-FOXP1-196aa groups, while the invasion and metastasis ability of the Lv-circFOXP1-ATG mutant group was stronger than that of the Lv-circFOXP1 group, but significantly lower than that of the empty vector control group. Taken together, these results suggest that circFOXP1 may exert an inhibitory effect through the encoded FOXP1-196aa protein. In addition, given that the proliferation and migration abilities of the Lv-circFOXP1-ATG mutant group were weaker than those of the Lv empty vector group, while the proliferation and migration abilities of the Lv-circFOXP1-ATG mutant group were stronger than those of the Lv-circFOXP1 group in both in vivo and in vitro experiments, this suggests that circFOXP1 may inhibit tumor progression through other functional mechanisms, such as binding to proteins or miRNA sponging.

[0032] Subsequently, the present invention cultured organoids from tissues collected from NPC patients and stained them with H&E. The results showed that the cell morphology of the organoids was consistent with that of the primary nasopharyngeal mucosal tissue ( Figure 6 E). In addition, the organoids and their primary NPC tissues were positive for Ki67 and Vimentin ( Figure 6 F). Copy number variation analysis by exome sequencing showed that chromosomal gains and losses from primary tumors were well preserved in NPC organoids ( Figure 6 G). Overall, we found that organoids retained the characteristics of NPC tumors. Subsequently, FOXP1-196aa protein solution was synthesized at Novoprotein and confirmed by SDS-PAGE that the protein band was visible at approximately 27 kDa ( Figure 6 H). After NPC organoids were treated with FOXP1-196aa, the growth rate of organoids was significantly slowed down ( Figure 6 I). MTT experiments also showed that FOXP1-196aa inhibited the survival rate of organoids ( Figure 6 J). Therefore, FOXP1-196aa also has an inhibitory effect on NPC organoids.

[0033] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A FOXP1-196aa protein, characterized in that The amino acid sequence of the FOXP1-196aa protein is shown in SEQ ID No. 1: MMTPQVITPQQMQQILQQQVLSPQQLQVLLQQQQALMLQQQQLQEFYKKQQEQLQLQLLQQQHAGKQPKEQQQVATQQLAFQQQLLQMQQLQQQHLLS LQRQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRERFPCQWL.

2. The FOXP1-196aa protein according to claim 1, characterized in that The FOXP1-196aa protein is encoded by circFOXP1, and the circFOXP1 is derived from exons 8-11 of the FOXP1 gene, with a genomic location of chr3:71090478-71102924 and a length of 587 bp.

3. Use of the FOXP1-196aa protein as claimed in claim 1 in the preparation of a product for treating nasopharyngeal carcinoma.

4. The use according to claim 3, characterized in that: The FOXP1-196aa protein has an inhibitory effect on nasopharyngeal carcinoma organoids.

Images