Medicine for treating renal cell carcinoma and application thereof

Abstract

The invention discloses a medicine for treating renal cell carcinoma and application thereof, and belongs to the field of biomedicine, the medicine comprises a reagent capable of ubiquitination degradation of WEE1, FBXO2 overexpression can prevent proliferation and movement of renal cell carcinoma cells, and FBXO2 can reduce abundance of WEE1 through ubiquitination degradation; the interaction and ubiquitination degradation of the WEE1 are mediated by the FBA structural domain of the FBXO2; fBXO2 degrades WEE1 through ubiquitination to inhibit RCC canceration.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a drug for treating renal cell carcinoma and an application thereof. Background Art

[0002] Kidney cancer is a common disease of the urinary system, causing 14,390 deaths and approximately 81,610 new cases in 2024. It accounts for approximately 4% of all cancers. Of these, nearly 90% of cases are renal cell carcinoma, the most common type. As a heterogeneous disease, renal cell carcinoma can be divided into three subtypes: renal clear cell carcinoma, accounting for 70%-80% of all cases; papillary cell carcinoma, accounting for 10%-15% of cases; and renal chromophobia, accounting for 4%-5% of cases. The carcinogenesis of renal cancer is associated with many environmental variables, such as smoking, obesity, and hypertension; however, the exact pathophysiological mechanisms of RCC are still unclear.

[0003] Therefore, exploring the treatment of renal cell carcinoma is a very valuable research. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a drug for treating renal cell carcinoma and its application.

[0005] A drug comprising an agent capable of ubiquitinating and degrading a mutant K292A site of WEE1.

[0006] Preferably, the reagent comprises FBXO2; the sequence of the FBXO2 is SEQ ID NO:1.

[0007] Preferably, the vector of the reagent is pcDNA3.1.

[0008] Preferably, the sequence of the K292A site is SEQ ID NO:5.

[0009] Preferably, the reagent further comprises one or more of FBXO2 shRNA and NC shRNA lentivirus.

[0010] Preferably, the sequence of the FBXO2 shRNA includes SEQ ID NO:2 or SEQ ID NO:3.

[0011] Preferably, the sequence of the NC shRNA is SEQ ID NO:4.

[0012] Use of an agent capable of ubiquitinating and degrading the mutant K292A site of WEE1 in the preparation of a drug for treating renal cell carcinoma.

[0013] Preferably, the sequence of the K292A site is SEQ ID NO:5.

[0014] The beneficial effects of the present invention are as follows:

[0015] The overexpression of FBXO2 of the present invention can prevent the proliferation and movement of RCC cells, and FBXO2 can reduce the abundance of WEE1 through ubiquitination degradation; the FBA domain of FBXO2 mediates the interaction and ubiquitination degradation of WEE1; FBXO2 inhibits RCC carcinogenesis by degrading WEE1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 FBXO2 is downregulated in renal clear cell carcinoma and correlates with a better prognosis; in the TIMER database, the FBXO2 gene is expressed in tumor tissues and matched normal in various tumor types. B Analysis of FBXO2 mRNA expression in KIRC according to the GEPIA website. C. Correlation between overall survival (OS) and FBXO2 expression in the GEPIA database. D FBXO2 expression levels are associated with lymph node metastasis, subtype, patient gender, tumor grade, patient age, patient race, and individual cancer stage in KIRC patients. EF Representative IHC staining and FBXO2 expression scores in 80 KIRC samples and paired adjacent cancer samples. Scale bar: 50 μm. G Kaplan-Meier survival curve of FBXO2 in KIRC patients. H Western blotting analysis of FBXO2 protein in 12 fresh KIRC tissues and adjacent normal tissues. *p<0.05, ***p<0.001.

[0018] Figure 2Western blot study of FBXO2 expression in 786-O and A498 cells transfected with Flag-FBXO2 or empty vector lentivirus. B Effect of fbxo2 overexpression on cell viability was detected by CCK-8. C Cell proliferation of control cells or FBXO2 overexpressing cells was assayed by EdU and colony formation assay. Scale bar, 100 μm. D Quantification of EdU assay and colony formation assay. EF Transwell assay was used to detect the invasion ability of RCC cells with and without FBXO2 overexpression. GH Quantification of apoptosis of RCC cells with and without FBXO2 overexpression by flow cytometry. Sample photos of nude mouse xenograft tumors were shown after subcutaneous injection of 786-O cells infected with vector plasmid (Vec) or FBXO2OE. JK Growth time and tumor weight of RCC xenografts for a total of 35 days. L Western blot analysis of FBXO2 expression in dissected tumor tissues. *p<0.05, **p<0.01, ***p<0.001.

[0019] Figure 3 FBXO2 silencing promotes RCC cell proliferation and motility; A. FBXO2 protein levels in ACHN and Caki-2 cells transfected with FBXO2 shRNA or NC shRNA lentivirus. B. CCK-8 assay was used to determine the viability of ACHN and Caki-2 cells with or without FBXO2 knockout. C. Colony formation assay and EdU assay were used to determine the proliferation of control cells or FBXO2 knockout ACHN and Caki-2 cells. Scale bar, 100 μm. D. Quantification of EdU test and colony formation assay. EF Transwell assay was used to determine the invasion ability of ACHN and Caki-2 cells with or without FBXO2 knockout. GH. Flow cytometry was used to quantify the apoptosis of ACHN and Caki-2 cells with or without FBXO2 knockout. *p<0.05, **p<0.01, ***p<0.001.

[0020] Figure 4FBXO2 binds to and degrades WEE1; A Co-immunoprecipitation technique was used to identify the interaction between endogenous FBXO2 and WEE1 in ACHN and Caki-2 cells. B Interaction of FBXO2 with exogenous WEE1 in 786-O and HEK293T cells. 786-O and HEK 293T cells were transfected with plasmids containing flag-tagged FBXO2 and myc-tagged WEE1. C FBXO2 downregulated endogenous WEE1 protein levels in a dose-dependent manner. HEK293T cells were transfected with increasing amounts of Flag-FBXO2 plasmid in 6-well plates. A pcDNA3.1 control was used to balance the total amount of plasmid. Immunoblotting of cell detachments was performed 48 h after transfection. FBXO2 had no effect on DE WEE1 mRNA levels, but its protein level was negatively regulated. FBXO2 western blots and qRT-PCR were used to detect the expression levels of WEE1 protein and mRNA in 786-O and A498 after overexpression of FBXO2. F-GWestern blots and qRT-PCR were used to detect the expression levels of WEE1 protein and mRNA in ACHN and Caki-2 after knockdown of FBXO2. HWestern blots were used to detect the half-life of WEE1 protein in HEK 293T cells with or without FBXO2 knockdown. 1 Quantification and plotting of WEE1 protein abundance (H). J Proteasome inhibitor MG132 restored the FBXO2-reduced WEE1 protein level. HEK293T cells were transfected with Flag-FBXO2, His-Ub, and Myc-WEE1 plasmids as indicated before collection. They were then treated with 10 μM MG132 for 12 h. Cell lysates and Myc immunoprecipitates were immunoblotted to detect WEE1 ubiquitination. **p<0.01, ***p<0.001.

[0021] Figure 5FBXO2 interacts with the kinase domain of WEE1, and WEE1 interacts with the FBA domain of FBXO2; Schematic representation of the WEE1 deletion mutants used in this study. B Western blots in HEK293T cells transfected with the indicated plasmids. C HEK293T cells were transfected with the indicated plasmids and then treated with MG132 (10 μM) for 12 h before harvesting. Immunoblotting of cell lysates and Myc immunoprecipitates to detect WT and deletion mutant WEE1 ubiquitination. D HEK293T cells were transfected with the indicated plasmids and then treated with MG132 (10 μM, 12 h) before harvesting. Western blots of IPs and wcl. E Schematic representation of the FBXO2 deletion mutants used in this study. F Western blots of HEK293T cells transfected with the indicated plasmids. HEK293T cells were transfected with the indicated plasmids and then treated with MG132 (10 μM, 12 h) before harvesting. His-Ub (WEE1 ubiquitination level) was detected by immunoblotting of cell lysates and immunoprecipitates treated with anti-myc beads. HEK293T cells were transfected with the indicated plasmids and then treated with MG132 (10 μM) for 12 h before collection. Western blots analysis of IPs and WCL. HEK293T cells were co-transfected with His-Ub, Flag-FBXO2, and Myc-WEE1 (WT, K24A, K207A, K219A, K292A, K399A) plasmids to further identify the specific lysine sites of WEE1 ubiquitinated by FBXO2. Cell lysates were immunoprecipitated with Myc antibody.

[0022] K292A-mutant WEE1 base sequence (the uppercase GCT is the mutant base, SEQ ID NO.5):

[0023] atgagcttcctgagccgacagcagccgccgccaccccgccgcgccggggcggcctgcaccttgcggcagaagctgat

[0024] cttctcgccctgcagcgactgtgaggaggaggaagaagaggaggaggaggagggcagcggccacagcaccgggga

[0025] ggactcggcctttcaagagcccgactcgccgctgccgcccgcgcggagccccacggagcccgggcccgagcgccgc

[0026] cgctcgcccgggccggcccccggGagccccggcgagctggaggaggacctgttgctgcccggcgcctgcccgggc

[0027] gcggacgaggcgggcggtggggcggagggcgactcgtgggaggaggagggcttcggctcctcgtcgccggtcaagt

[0028] cgccggcggccccctacttcctgggtagctctttctcgccggtgcgctgcggcggcccaggagatgcgtcgccgcggg

[0029] gttgcggggcgcgccgggcgggcgaaggccgccgctcgccgcggccggaccacccgggcaccccgccacacaag

[0030] accttccgcaagctgcgactcttcgacaccccgcacacgcccaagagtttgctctccaaagctcggggaattgattccagc

[0031] tctgttaaactccggggtagttctctcttcatggatacagaaaaatcaggaaaaagggaatttgatgtgcgacagactcctca

[0032] agtgaatattaatccttttactccggattctttgttgcttcattcctcaggacagtgtcgtcgtagaaagagaacgtattggaatg

[0033] attcctgtggtgaagacatggaagccagtgattatgagcttgaagatgaaacaagacctgctaagagaattacaattactga

[0034] aagcaatatgGCTtcccggtatacaacagaatttcatgagctagagaaaatcggctctggagaatttggttctgtatttaag

[0035] tgtgtgaagaggctggatggatgcatttatgccattaagcgatcaaaaaagccattggcgggctctgttgatgagcagaac

[0036] gctttgagagaagtatatgctcatgcagtgcttggacagcattctcatgtagttcgatatttctctgcgtgggcagaagatgat

[0037] catatgcttatacagaatgaatattgtaatggtggaagtttagctgatgctataagtgaaaactacagaatcatgagttacttta

[0038] aagaagcagagttgaaggatctccttttgcaagttggccgaggcttgaggtatattcattcaatgtctttggttcacatggatat

[0039] aaaacctagtaatattttcatatctcgaacctcaatcccaaatgctgcctctgaagaaggagacgaagatgattgggcatcca

[0040] acaaagttatgtttaaaataggtgatcttgggcatgtaacaaggatctccagtccacaagttgaagagggcgatagtcgtttt

[0041] cttgcaaatgaagttttacaggagaattatacccatctaccaaaagcagatatttttgcgcttgccctcacagtggtatgtgctg

[0042] ctggtgctgaacctcttccgagaaatggagatcaatggcatgaaatcagacagggtagattacctcggataccacaagtgc

[0043] tttcccaagaatttacagagttgctaaaagttatgattcatccagatccagagagaagaccttcagcaatggcactggtaaag

[0044] cattcagtattgctgtccgcttctagaaagagtgcagaacaattacgaatagaattgaatgccgaaaagttcaaaaattcactt

[0045] ttacaaaaagaactcaagaaagcacagatggcaaaagctgcagctgaggaaagagcactcttcactgaccggatggcca

[0046] ctaggtccaccaccaccagagtaatagaacatctcgacttattggaaagaaaatgaaccgctctgtcagccttactatatac

[0047]

[0048] Figure 6 WEE1 inhibits RCC cell proliferation and motility; A Protein levels of WEE1 in ACHN and Caki-2 cells transfected with WEE1 shRNA or NC shRNA lentivirus. B Viability of ACHN and Caki-2 cells with or without WEE1 knockdown was determined by CCK-8 assay. C Colony formation assay and EdU assay were used to determine proliferation of control cells and wee1 knockdown cells. Scale bar, 100 μm. D Quantification of colony formation assay and EdU assay. EF Transwell assay was used to detect invasion ability of ACHN and Caki-2 cells with or without WEE1 knockdown. GH Apoptosis of ACHN and Caki-2 cells with or without WEE1 knockdown was analyzed by flow cytometry. *p<0.05, **p<0.01, ***p<0.001.

[0049] Figure 7 In vitro, knockdown of WEE1 partially abolished the promotion of RCC cell proliferation and motility caused by FBXO2 silencing. Western blotting was used to analyze the expression of FBXO2 and WEE1 in ACHN and Caki-2 cells treated with NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1. CCK-8 and colony formation assays were used to detect BC cell proliferation in ACHN and Caki-2 cells treated with NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1. Scale bar, 100 μm. DTranswell assay was used to detect the invasion ability of NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1 on ACHN and Caki-2 cells. EFlow cytometry was used to detect the apoptosis level of ACHN and Caki-2 cells treated with NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1. Quantification of FH colony formation assay (C), transwell assay (D) and cell analysis (E). *shFBXO2 or shWEE1 treated group vs NC shRNA group, *p<0.05, **p<0.01, **p<0.001. #shFBXO2+shWEE1 vs shFBXO2 or shWEE1 group, #p<0.05, ##p<0.01, ##p<0.001.

[0050] Figure 8In vivo, knockdown of WEE1 partially abolished the cell proliferation-promoting effect caused by FBXO2 silencing in RCC. Representative images of xenograft tumors were shown after subcutaneous injection of ACHN cells infected with NCshRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1 into nude mice. BC Tumor weights were followed during the growth of RCC xenografts for a total of 25 days. D Representative images of immunohistochemical staining showing the expression of FBXO2, WEE1, and Ki67 in NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1 xenograft mice. Scale bar, 20 μm. E Representative images of IHC staining for FBXO2, WEE1, and Ki67 in NC shRNA, shFBXO2, shFBXO2, and shFBXO2+shWEE1 xenograft mice. *shFBXO2 or shWEE1 treated group vs NCshRNA group, *p<0.05, **p<0.01, **p<0.001, ****p<0.0001. #shFBXO2+shWEE1 vs shFBXO2 or shWEE1 group, ##p<0.01, ####p<0.0001.

[0051] Fig. 9 Immunohistochemical (IHC) staining of FBXO2 in KIRC samples.

[0052] Fig.10 Overexpression of FBXO2 inhibits RCC cell migration. A Western blotting was used to detect the expression of FBXO2 protein in the human renal tubular cell line HK-2 and different RCC cell lines. B FBXO2 mRNA expression was significantly increased in RCC cells compared with HK-2 cells. In 786-O and A498 cells with or without FBXO2 overexpression, the migration ability was evaluated using a transwell assay without matrix. Cell motility was detected by the EF wound healing assay in 786-O and A498 cells with or without FBXO2 overexpression, scale bar: 400 μm, *p<0.05, **p<0.01, ***p<0.001.

[0053] Fig.11 FBXO2 knockdown promotes RCC cell migration. The migration of RCC cells treated with FBXO2 shRNA or Scramble shRNA was detected by AB Transwell assay. Wound healing was observed by wound healing assay using C-DCaki-2 and ACHN cells. Scale bar: 400 μm. *p<0.05, **p<0.01, ***p<0.001.

[0054] Fig.12WEE1 specifically interacts with FBXO2.

[0055] Fig.13 FBXO2 interacts with the WEE1 kinase domain; Western blots were performed on 786-O cells transfected with the indicated plasmids. Figure 5 Quantification of WEE1 band intensity in CD4 T cells. CD Western blots were used to detect the half-life of WEE1 protein in 786-O cells transfected with Vec, FBXO2-WT or FBXO2-N terminus.

[0056] Fig.14 In RCC, knockdown of WEE1 partially abolished the promotion of cell migration caused by FBXO2 silencing. AB Transwell assay was used to measure the cell motility of ACHN and Caki-2 cells treated with NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1. Wound healing assay was used to measure the CD cell motility of ACHN and Caki-2 cells treated with NC shRNA, shFBXO2, shWEE1, and shFBXO2+shWEE1. *shFBXO2 or shWEE1 treatment group vs NC shRNA group, *p<0.05, **p<0.01, **p<0.001. #shFBXO2+shWEE1 group vs shFBXO2 or shWEE1 group, scale bar: 400μm. #p<0.05, ##p<0.01, ##p<0.001. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] The sequence of FBXO2 is SEQ ID NO: 1:

[0059] MDGDGDPESVGQPEEASPEEQPEEASAEEERPEDQQEEEAAAAAAYLDELPEPLLLRVLAALPAAELVQACRLVCLRWKELVDGAPLWLLKCQQEGLVPEGGVEEERDHWQQFYFLSKRRRNLLRNPCGEEDLEGWCDVEHGGDGWRV EELPGDSGVEFTHDESVKKYFASSFEWCRKAQVIDLQAEGYWEELLDTTQPAIVVKDWYSGRSDAGCLYELTVKLLSEHENVLAEFSSGQVAVPQDSDGGGWMEISHTFTDYGPGVRFVRFEHGGQDSVYWKGWFGARVTNSSVWVEP.

[0060] The sequence of FBXO2 shRNA is SEQ ID NO: 2:

[0061] 5′-CACCGTTAAGCTACTGTCCGA-3′

[0062] Or SEQ ID NO: 3: 5'-TCGTGGTGAAGGACTGGTACT-3'.

[0063] The sequence of NC shRNA is SEQ ID NO:4: 5′-TCTCGCTTGGGCGAGAGTAAG-3′.

[0064] Example 1 Increased expression of FBXO2 is associated with a favorable prognosis in renal clear cell carcinoma (KIRC)

[0065] According to the GEPIA database research, GEPIA is an online bioinformatics analysis tool with zero-code operation. GEPIA organizes the expression values ​​of each searchable gene in different tumor samples, and can calculate the expression level of a gene in a certain tumor. It can also analyze the relationship between genes and tumor prognosis, co-expression between genes, etc. Website: http: / / gepia.cancer-pku.cn / . Enter the gene FBXO2 in the search box to query its gene expression level in a certain cancer type or certain cancers. Survival analysis can also be performed. We found that FBXO2 was positively correlated with the overall survival rate of KIRC patients ( Figure 1 C in Figure ). Results from the UALCAN database showed that FBXO2 expression was correlated with various clinical characteristics, such as lymph node metastasis, subtype, patient sex, tumor grade, patient age, patient race, and individual cancer stage ( Figure 1In addition, immunohistochemistry (IHC) staining showed that FBXO2 expression was significantly higher in tumor tissues of 80 KIRC patients ( Fig. 9 A) is significantly lower than the paired normal tissue ( Figure 1 E, F). Kaplan-Meier survival curve analysis showed that increased FBXO2 expression was associated with a better prognosis in KIRC patients ( Figure 1 Strikingly, western blotting analysis of freshly collected protein samples from KIRC patients revealed that FBXO2 was downregulated in KIRC tissues compared with individual paired controls ( Figure 1 In addition, we found that FBXO2 expression was downregulated in five human renal cancer cell lines compared with the human renal tubular cell line HK-2. Fig.10 In summary, the above data indicate that FBXO2 is downregulated in KIRC and is associated with a better prognosis.

[0066] Example 2 FBXO2 overexpression inhibits RCC cell proliferation and motility in vitro and in vivo

[0067] A cell model of FBXO2 overexpression was established in 786-O and A498 cells using FBXO2-flag lentivirus. Figure 2 CCK8, EdU and cell cloning experiments confirmed that overexpression of FBXO2 significantly attenuated the proliferation of RCC cells ( Figure 2 Transwell and wound healing experiments showed that cell migration and invasion were significantly reduced in the FBXO2 overexpression cell model ( Figure 2 e, F and Fig.10 In contrast, our flow cytometry results showed that FBXO2 has the potential to induce apoptosis in RCC cells ( Figure 2 G, H in Figure 3). We established a xenograft mouse model to further explore whether FBXO2 can also inhibit the development of RCC cells in vivo. The results showed that the tumor size and weight of the FBXO2 overexpression group were smaller than those of the control group ( Figure 2 k). The FBXO2 protein level in animals injected with cells overexpressing FBXO2 was significantly increased, as shown in Figure 2 These results demonstrate that FBXO2 overexpression can block RCC cell proliferation and motility both in vitro and in vivo.

[0068] Example 3 Loss of FBXO2 promotes proliferation and motility of RCC cells

[0069] Two different short hairpin RNAs (shRNAs) were used to construct a cell model of FBXO2 deficiency ( Figure 3 CCK8, EdU and cell cloning experiments confirmed that inhibition of FBXO2 significantly increased the proliferation of RCC cells ( Figure 3 B, C, and D in Figure 3). Transwell and wound healing experiments confirmed that FBXO2 silencing significantly increased cell invasion and migration ( Figure 3 e, F and Fig.11 A, B, C, D in Figure ). Flow cytometry results showed that the loss of FBXO2 significantly reduced the apoptosis of RCC cells ( Figure 3 G, H in.

[0070] Example 4 FBXO2 negatively regulates the stability of WEE1 by ubiquitination and destruction of WEE1

[0071] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used. Briefly, we transiently transfected human HEK293T cells with Flag-FBXO2, isolated the FBXO2 immune complexes, and detected them using LC-MS / MS. Fig.12 As shown in Figure A, LC-MS / MS analysis identified the peptide corresponding to WEE1. We further detected the binding of FBXO2 to WEE1 by immunoprecipitation and western blot. Figure 4 As shown in A and B, both endogenous and exogenous FBXO2 were able to interact with WEE1 in two different RCC cell lines. We found that ectopic overexpression of FBXO2 significantly reduced WEE1 protein levels in HEK293T, A498, and 786-O cells, but WEE1 mRNA levels were not affected ( Figure 4 Consistent with this, our immunoblotting experiments also confirmed that FBXO2 depletion accelerated the protein level of WEE1 but barely altered the WEE1 mRNA level ( Figure 4 In addition, cycloheximide (CHX) treatment showed that depletion of endogenous FBXO2 significantly prolonged the half-life of WEE1 ( Figure 4 To confirm this conclusion, we used the MG132 assay to quantify the effect of FBXO2 on WEE1 stability. Figure 4 As shown in J, MG132 (a proteasome inhibitor) reduced FBXO2-mediated downregulation of WEE1. In addition, in vivo ubiquitination assays showed that FBXO2 promoted the ubiquitination of WEE1 ( Figure 4 K in Figure ). Taken together, these data suggest that FBXO2 can reduce the abundance of WEE1 through ubiquitination and degradation.

[0072] Example 5

[0073] Generate wild-type or a series of truncated forms of WEE1 ( Figure 5 A and Fig.13 A in Figure 5 As shown in B and C, the N-terminal domain blocked FBXO2-mediated WEE1 downregulation and destruction. In addition, all deletion mutants except the N-terminal domain interacted with FBXO2 ( Figure 5 D in ). Therefore, the above data indicate that FBXO2 binds to the kinase domain of WEE1. Since FBXO2 contains two conserved domains, including the F-box associated (FBA) domain that interacts with glycoprotein function, the F-box domain directly interacts with Skp1. To determine whether the FBA domain promotes the interaction between FBXO2 and WEE1, we synthesized three different versions of FBXO2: FL, N-terminal (F-box domain), and C-terminal (FBA domain) ( Figure 5 As shown in Figure 5F, the N-terminus and C-terminus, especially the N-terminus, significantly weakened the downregulation and degradation of WEE1 mediated by FBXO2 ( Figure 5 In F, G). Compared with the C-terminus of WEE1 and WT, the N-terminus largely blocked the interaction with FBXO2 ( Figure 5 H in ). Since ubiquitination often occurs on lysine residues, we constructed five WEE1 mutants (K24A, K207A, K219A, K292A, K399A) based on the GPSUber website that predicts ubiquitination sites. Ubiquitination experiments showed that the K292A mutation significantly reduced FBXO2-mediated ubiquitination of WEE1 ( Figure 5 In addition, HEK293T and 786-O cells transfected with the N-terminal mutant showed a prolonged WEE1 half-life compared with those transfected with FBXO2WT ( Fig.13 Therefore, the above data indicate that the FBA domain of FBXO2 mediates the interaction and ubiquitination degradation of WEE1.

[0074] Example 6

[0075] FBXO2 and WEE1-deficient cells were constructed using shRNA for rescue experiments. Western blot results showed that WEE1 silencing could offset the increase in WEE1 protein levels caused by FBXO2 depletion ( Figure 7 According to CCK-8, colony formation and invasion assays ( Figure 7 B, C, D, F, G), after WEE1 knockdown, FBXO2 depletion-induced cell proliferation and invasion were significantly restored. In addition, when WEE1 was absent, the reduction in apoptosis caused by FBXO2 silencing was restored ( Figure 7In addition, we investigated the impact of the FBXO2-WEE1 axis in RCC tumorigenesis in vivo. As expected, the increase in weight and volume of xenograft tumors when FBXO2 was depleted was largely counteracted by the depletion of WEE1, as demonstrated by xenografts of ACHN cells ( Figure 8 IHC staining confirmed the upregulation of WEE1 and Ki67 in the ACHN-shFBXO2 group, and when WEE1 was silenced, the upregulation of WEE1 and Ki67 was restored ( Figure 8 These results suggest that FBXO2 inhibits RCC carcinogenesis by degrading WEE1.

[0076] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A drug, characterized in that Included are reagents capable of ubiquitinating and degrading the mutant K292A site of WEE1.

2. The drug according to claim 1, characterized in that The reagent includes FBXO2; the sequence of the FBXO2 is SEQ ID NO:

1.

3. The drug according to claim 1, characterized in that The vector of the reagent is pcDNA3.

1.

4. The drug according to claim 1, characterized in that The sequence of the K292A site is SEQ ID NO:

5.

5. The drug according to claim 1, characterized in that The reagents also include one or more of FBXO2 shRNA and NCshRNA lentivirus.

6. The drug according to claim 5, characterized in that The sequence of the FBXO2 shRNA includes SEQ ID NO:2 or SEQ ID NO:

3.

7. The drug according to claim 5, characterized in that The sequence of the NC shRNA is SEQ ID NO:

4.

8. Use of an agent capable of ubiquitinating and degrading the mutant K292A site of WEE1 in the preparation of a drug for treating renal cell carcinoma.

9. Use of the reagent capable of ubiquitinating and degrading the mutant K292A site of WEE1 according to claim 5 in the preparation of a drug for treating renal cell carcinoma, characterized in that: The vector of the reagent is pcDNA3.

1.

10. Use of the reagent capable of ubiquitinating and degrading the mutant K292A site of WEE1 according to claim 5 in the preparation of a drug for treating renal cell carcinoma, characterized in that: The sequence of the K292A site is SEQ ID NO:5.

Images