Biomarker and application thereof

Abstract

The invention relates to the technical field of molecular biomedicine, and particularly discloses a biomarker and application thereof. Wherein the biomarker comprises a transcription product and / or a translation product generated by jumping of a No.18 exon of the RASAL1 gene. Researches find that the expression of the biomarker in the application is increased in tumors, and the biomarker is positively related to poor prognosis of tumors or cancers, can be used as a biomarker for prognosis risk stratification of tumors or cancers, is helpful for improving clinical management of patients, and also provides powerful support for fundamental research of tumors and drug development.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biomedical technology, in particular to a biomarker and application thereof. BACKGROUND

[0002] In recent years, malignant tumors have become a major public health challenge, and optimizing cancer prevention and treatment strategies has become a top priority. Abnormal activation of the RAS / MAPK signaling pathway is one of the core driving mechanisms of malignant tumor progression. Mutations in BRAF / RAS genes have been identified as molecular markers of poor prognosis in various cancers, which promote tumor proliferation and invasion by activating the RAS / MAPK signaling pathway. Notably, there are still a large number of BRAF / RAS wild-type tumors with persistent activation of the RAS / MAPK pathway. As a key negative regulator of the pathway, RASAL1 catalyzes RAS-GTP hydrolysis through its GTPase-activating protein (GAP) domain, leading to RAS protein inactivation and inhibition of oncogenic RAS / MAPK signaling. Although RASAL1 inactivation has been widely reported in 12 types of solid tumors, its traditional mechanisms of inactivation include gene mutations, deletions, and epigenetic silencing, and therefore there is no biomarker clearly related to RASAL1 that can be used as a biomarker for multi-dimensional risk stratification and prognosis diagnosis system of tumors. SUMMARY

[0003] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide a biomarker and application thereof, aiming to solve the problem of lack of a biomarker clearly related to RASAL1 in the prior art.

[0004] The technical solution of the present application is as follows:

[0005] In a first aspect of the present application, a biomarker is provided, which comprises a transcription product and / or a translation product of skipping of exon 18 of RASAL1 gene.

[0006] Preferably, the skipping of exon 18 of RASAL1 gene occurs at positions 2131-2214 of the nucleotide sequence of exon 18.

[0007] Preferably, the transcription product is a nucleotide sequence as shown in SEQ ID NO. 1.

[0008] Preferably, the translation product is a RASAL1 protein isoform.

[0009] Further preferably, the amino acid sequence of the RASAL1 protein isoform is as shown in SEQ ID NO. 2.

[0010] Preferably, the biomarker further comprises a TERT promoter gene mutant.

[0011] Further preferably, the nucleotide sequence of the TERT promoter gene mutant is shown as SEQ ID NO. 3.

[0012] In a second aspect of the present application, the use of the biomarker of the first aspect of the present application in the preparation of a product for determining whether a subject has a tumor or stratifying the risk of prognosis of a tumor of a subject is provided.

[0013] Preferably, the tumor comprises a malignant tumor; the malignant tumor comprises at least one of adrenocortical carcinoma, urothelial carcinoma of the bladder, squamous cell carcinoma and adenocarcinoma of the cervix, glioblastoma multiforme, squamous cell carcinoma of the head and neck, renal chromophobe carcinoma, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, cutaneous melanoma, thyroid carcinoma, endometrial carcinoma.

[0014] Compared with the prior art, the present application has the following advantages:

[0015] The biomarker in the present application comprises the transcription product and / or translation product of the skipping of exon 18 of the RASAL1 gene. It is found through research that the transcription product of the skipping of exon 18 of the RASAL1 gene is increased in expression in tumors compared with normal tissues, and is positively correlated with the poor prognosis of tumors. In addition, when the inactivation of the RASAL1 gene (the mechanisms include gene mutation, deletion, promoter methylation and translational expression silencing) coexists with the mutation of the TERT promoter, the two have a synergistic effect, strongly promoting tumor invasion, and can be used as a core marker for stratifying the risk of prognosis of tumors. Further, the biomarker in the present application can be used as a biomarker for stratifying the risk of prognosis of tumors or cancers, which helps to improve the clinical management of patients, and also provides strong support for the basic research and drug development of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows.

[0017] Figure 1 Mechanism analysis of the skipping event of exon 18 of the RASAL1 gene provided in the embodiments of the present application;

[0018] Figure 2 Analysis of the basis for the skipping event of exon 18.1 and RASAL1-004 as a new broad-spectrum tumor biomarker provided in the embodiments of the present application:

[0019] A is the mechanism analysis of RASAL1 gene 18 exon skipping event; B is the transcriptional expression analysis of RASAL1-004 in tumor cell lines; C, D, E, F, G are the abundance analysis of RASAL1-004 transcriptional expression in different tumor tissues; H, I are the analysis of RASAL1-001 and RASAL1-004 promoting tumor apoptosis ability; J is the analysis of RASAL1-001 and RASAL1-004 inhibiting tumor growth ability; K and L are the analysis of RASAL1-001 and RASAL1-004 inhibiting tumor cell growth and metastasis ability;

[0020] Figure 3 The inactivation mechanism analysis of RASAL1 provided for the embodiments of the application:

[0021] A and B are the mRNA transcription level analysis of wild type RASAL1-001 and RASAL1 splice isomer RASAL1-004; C is the protein expression level analysis of wild type RASAL1-001 and RASAL1 splice isomer RASAL1-004; D and E are the protein synthesis ability analysis of wild type RASAL1-001 and RASAL1-004; F is the ribosome footprint analysis of wild type RASAL1-001 and RASAL1-004 protein translation; G is the splice site analysis of wild type RASAL1-001 and RASAL1 splice isomer RASAL1-004; H and I are the ribosome footprint analysis of wild type RASAL1-001 and RASAL1 splice isomer RASAL1-004 at exon 17-18; J is the RNA structure analysis of wild type RASAL1-001 and RASAL1 splice isomer RASAL1-004 at exon 17-18;

[0022] Figure 4 The feasibility analysis of RASAL1 inactivation and TERT promoter mutation as a clinical prognostic molecular stratification marker for BRAF / RAS wild type tumor provided for the embodiments of the application:

[0023] A is Kaplan-Meier disease-free survival analysis of RASAL1 inactivation with TERT promoter mutation alone or simultaneously; B is pro-apoptotic ability analysis of RASAL1 inactivation alone and RASAL1 inactivation with TERT promoter mutation simultaneously; C is anti-metastasis ability analysis of RASAL1 inactivation alone and RASAL1 inactivation with TERT promoter mutation simultaneously; D is TERT promoter mutation generated using Crispr-Cas9 editing in TERT wild-type tumor cell line WRO; E and F are tumor growth inhibition analysis when RASAL1 is overexpressed in Parental WRO and Crispr / cas9-edited WRO cell lines; G is tumor apoptosis promotion analysis when RASAL1 is overexpressed in Parental WRO and Crispr / cas9-edited WRO cell lines; H is repair of TERT promoter mutation using Crispr-Cas9 editing in TERT promoter mutant tumor cell line TPC-1; I is tumor apoptosis promotion analysis when RASAL1 is overexpressed in Parental TPC-1 and Crispr / cas9-edited TPC-1 cell lines. DETAILED DESCRIPTION

[0024] In order to make the purposes, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0025] It should be noted that if the description of "first", "second" and the like is involved in the embodiments of the present application, the "first", "second" and the like are only for the purpose of description, and cannot be understood as indicating or implying the relative importance and implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can explicitly or implicitly include at least one of the features. In addition, the technical solutions of each embodiment can be combined with each other, but it must be made by those of ordinary skill in the art as the basis, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the protection scope claimed by the present application.

[0026] First, the terms in the embodiments of the present application are explained.

[0027] Exon skipping is a kind of gene splicing variation, which generally refers to that a specific exon is abnormally skipped during RNA processing, so that the generated mRNA is missing the sequence corresponding to the exon.

[0028] Transcription product is various RNA molecules synthesized by RNA polymerase with the DNA of the target gene as a template, and main types include mRNA (messenger RNA) and non-coding RNA (ncRNA). The non-coding RNA can be further divided into miRNA, IncRNA, rRNA and tRNA.

[0029] Translation product is a functional protein formed by folding and modification of a polypeptide chain synthesized by a ribosome with mRNA as a template.

[0030] Gene mutant refers to an individual whose genetic information is changed (mutated) due to changes in gene sequence.

[0031] Tumor prognosis risk stratification is to divide patients into different risk levels according to clinical, pathological and molecular biological characteristics of patients, so as to predict the possibility of disease recurrence, metastasis or death, and to guide individualized treatment and follow-up strategy. Stratification is based on clinical factors, pathological characteristics, molecular markers, biomarkers, imaging and function.

[0032] The embodiment of the present application provides a biomarker, which comprises a transcription product and / or a translation product generated by skipping of the 18th exon of a RASAL1 gene.

[0033] As a negative regulatory factor related to the RAS signal pathway, RASAL1 gene is considered as an important tumor suppressor gene. RASAL1 contains a highly conserved GAP domain (about 300 amino acids), and its core function is to induce conformational changes of RAS protein through the SwitchII region of RAS protein, thereby activating the activity of endogenous GTPase of RAS, increasing the affinity of GDP, and then promoting the inactivation of RAS protein to inhibit RAS-related oncogenic signals. Inactivation of RASAL1 has been widely reported in 12 kinds of solid tumors, and RASAL1 deletion promotes cell proliferation, migration and invasion, and is related to tumor size, differentiation degree, infiltration depth, lymph node metastasis and TNM stage. The embodiment of the present application combines omics and molecular biology analysis, and finds that compared with normal tissues, the transcription product generated by skipping of the 18th exon of the RASAL1 gene is increased in tumors, and is positively correlated with poor prognosis of tumors, thereby indicating that the transcription product generated by skipping of the 18th exon of the RASAL1 gene can be used as a biomarker related to tumors.

[0034] Further, the skipping of the 18th exon of the RASAL1 gene occurs at positions 2131-2214 of the 18th exon.

[0035] In some embodiments, the nucleotide sequence of the transcription product is as shown in SEQ ID NO. 1.

[0036] In some embodiments, the translation product is a RASAL1 protein isoform. Further, the amino acid sequence of the RASAL1 protein isoform is as shown in SEQ ID NO. 2.

[0037] The present application provides evidence that RASAL1 inactivation (such as gene mutation, deletion, promoter methylation and transcriptional silencing) and TERT promoter mutation synergize as a clinical prognostic molecular stratification marker for BRAF / RAS wild-type tumors. According to Kaplan-Meier survival analysis and Cox regression model, the survival rate of patients carrying RASAL1 inactivation and TERT promoter mutation significantly decreased, and the risk of clinical disease progression was significantly higher than that of other groups. The TERT promoter mutation generated by CRISPR / Cas9 technology promotes the anti-tumor effect of inhibiting tumor cell growth and promoting apoptosis mediated by RASAL1 overexpression. Repairing the TERT promoter mutation to wild type by CRISPR / Cas9 technology eliminates the anti-tumor effect of inhibiting tumor cell growth and promoting apoptosis mediated by RASAL1 overexpression. Figure 1 The present application provides evidence that RASAL1 inactivation (such as gene mutation, deletion, promoter methylation and transcriptional silencing) and TERT promoter mutation synergize as a clinical prognostic molecular stratification marker for BRAF / RAS wild-type tumors. According to Kaplan-Meier survival analysis and Cox regression model, the survival rate of patients carrying RASAL1 inactivation and TERT promoter mutation significantly decreased, and the risk of clinical disease progression was significantly higher than that of other groups. The TERT promoter mutation generated by CRISPR / Cas9 technology promotes the anti-tumor effect of inhibiting tumor cell growth and promoting apoptosis mediated by RASAL1 overexpression. Repairing the TERT promoter mutation to wild type by CRISPR / Cas9 technology eliminates the anti-tumor effect of inhibiting tumor cell growth and promoting apoptosis mediated by RASAL1 overexpression.

[0038] In some embodiments, the nucleotide sequence of the TERT promoter gene mutant is as shown in SEQ ID NO. 3.

[0039] The present application provides evidence that RASAL1 inactivation (such as gene mutation, deletion, promoter methylation and transcriptional silencing) and TERT promoter mutation synergize as a clinical prognostic molecular stratification marker for BRAF / RAS wild-type tumors. According to Kaplan-Meier survival analysis and Cox regression model, the survival rate of patients carrying RASAL1 inactivation and TERT promoter mutation significantly decreased, and the risk of clinical disease progression was significantly higher than that of other groups. The TERT promoter mutation generated by CRISPR / Cas9 technology promotes the anti-tumor effect of inhibiting tumor cell growth and promoting apoptosis mediated by RASAL1 overexpression. Repairing the TERT promoter mutation to wild type by CRISPR / Cas9 technology eliminates the anti-tumor effect of inhibiting tumor cell growth and promoting apoptosis mediated by RASAL1 overexpression.

[0040] The present application also provides a use of the biomarker as described above in the preparation of a product for determining whether a subject has a tumor or stratifying the risk of tumor prognosis of a subject.

[0041] The product of tumor prognosis risk stratification can confirm the alternative splicing (RASAL1 gene exon 18 skipping) variant state by RNA transcriptome sequencing of the biopsy sample. Then the alternative splicing event in the RASAL1 transcription process is quantitatively analyzed: the relative frequency of an exon in all splicable transcripts is measured using PSI. The PSI is calculated using the SplAdder method, and the abundance of each splicing event in each sample is calculated according to the RNA sequencing data. Finally, the RASAL1 splicing isomer is quantitatively analyzed: the transcriptional expression data of the four protein subtypes of the RASAL1 gene in normal and tumor tissues are calculated.

[0042] In some embodiments, the tumor comprises at least one of adrenocortical carcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma and adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, cutaneous melanoma, thyroid carcinoma, endometrial carcinoma.

[0043] Further illustrated below are specific examples. Unless otherwise specified, the reagents, consumables and experimental methods used in the examples are conventional technical means in the art.

[0044] Example 1

[0045] This example verifies that the exon 18.1 skipping event and RASAL1-004 can be used as a tumor biomarker. The specific steps are as follows:

[0046] RNA transcriptome sequencing is performed on biopsy samples, including one of the following malignant tumors derived from the adrenal cortex, bladder urothelial tissue, cervical tissue, brain glial tissue, head and neck tissue, kidney tissue, colon tissue, breast tissue, pancreatic tissue, ovarian tissue, liver tissue, lung tissue, prostate tissue, skin tissue, thyroid tissue and uterine tissue. The following alternative splicing variant states are confirmed. The RASAL1 alternative splicing event is quantitatively analyzed: the relative frequency of an exon in all splicable transcripts is measured using PSI. The PSI is calculated using the SplAdder method, and the abundance of each splicing event in each sample is calculated according to the RNA sequencing data. The RASAL1 splicing isomer is quantitatively analyzed: the transcriptional expression data of the four protein subtypes of the RASAL1 gene in normal and tumor tissues are calculated.

[0047] The abundance of the splicing event in each sample is calculated as shown in C, D, E, F and G in Figure 2 Figure 2 ​As shown in C, D, and E, the proportion of exon 18.1 skipping events decreased in six types of tumor tissues (46%, 6 / 13), including adrenocortical carcinoma (ACC), poorly differentiated glioma (LGG), glioblastoma multiforme (GBM), thyroid cancer (THCA), colon cancer (COAD), and breast cancer (BRCA). Figure 2 A lower proportion of exon 18.1 skipping events is associated with shorter overall survival in cancer patients, including those with adrenocortical carcinoma (ACC), clear cell renal cell carcinoma (KIRC), endometrial carcinoma (UCEC), and poorly differentiated glioma (LGG). Figure 2 (D). Delete exon 18.1, which is associated with shorter progression-free survival (PFI) in cancer patients, including those with rectal cancer (READ), thyroid cancer (THCA), bladder cancer (BLCA), cervical cancer (CESC), head and neck squamous cell carcinoma (HNSC), papillary renal cell carcinoma (KIRP), clear cell renal cell carcinoma (KIRC), and poorly differentiated glioma (LGG). Figure 2 E).

[0048] Furthermore, analysis of the transcriptional expression of RASAL1-004 in tumor cell lines, as shown in Figure B, revealed that the transcriptional expression of RASAL1-004 was increased in tumor cell lines compared to normal cells Nthy-ori3-1.

[0049] Furthermore, from Figure 2 The results show that in nearly half (48%, 15 / 31) of tumor types, the transcriptional level of RASAL1-004 was significantly increased, including cervical cancer (CESC), colon cancer (COAD), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian cancer (OV), pancreatic cancer (PAAD), rectal adenocarcinoma (READ), thyroid cancer (THCA), and endometrial cancer (UCEC) screened out in the alternative splicing analysis. Figure 2 As can be seen from the data, high expression of RASAL1-004 is associated with shorter survival in bladder cancer (BLCA), lung adenocarcinoma (LUAD), pancreatic cancer (PAAD), and ovarian cancer (OV).

[0050] In addition, the ability of RASAL1-001 and RASAL1-004 to inhibit tumor apoptosis was analyzed, and the results were as follows: Figure 2 As shown in H and I, from Figure 2 As can be seen from H and I, RASAL1-004's ability to inhibit tumor apoptosis is impaired compared to wild-type RASAL1-001. Similarly, compared to normally functioning RASAL1-001, RASAL1-004's ability to inhibit tumor cell growth and metastasis is also impaired.Figure 2 K and 1L).

[0051] The above results provide evidence for the RASAL1 gene exon 18.1 skipping event and RASAL1-004 as a novel broad-spectrum tumor biomarker.

[0052] Example 2

[0053] This embodiment provides the inactivation mechanism of RASAL1: RNA secondary structure-ribosome arrest. The study revealed that the exon 18.1 skipping RNA molecule has a more complex folding structure at the junction of exon 17 and exon 18, causing ribosome arrest, hindering protein translation, and thus leading to a significant decrease in RASAL1-004 protein expression levels. The specific steps are as follows:

[0054] (1) mRNA transcription and protein expression levels were analyzed by overexpressing wild-type RASAL1-001 and the RASAL1 splice isoform RASAL1-004 in the WRO cell line. The results are as follows: Figure 3 As shown in A, B, and C. From... Figure 3 As can be seen from A and B, the wild-type RASAL1-001 and the RASAL1 splice isoform RASAL1-004 are similar at the mRNA transcription level. However, from... Figure 3 As can be seen from the data in Figure C, the protein level of RASAL1-004 is significantly lower than that of RASAL1-001.

[0055] (2) The protein synthesis capabilities of wild-type RASAL1-001 and the RASAL1 splice isoform RASAL1-004 were analyzed, and the results are as follows: Figure 3 As shown in D and E. From Figure 3 As can be seen from D and E, RASAL1-001 has a stronger protein synthesis ability than RASAL1-004; based on this, further analysis of the protein translation process of both showed the following results. Figure 3 As shown in F and H. From Figure 3 As shown in F and H, the number of ribosomal footprints at exon 18 in RASAL1-004 is greater than that in RASAL1-001. Based on these results, the RNA structures of wild-type RASAL1-001 and the RASAL1 splice isoform RASAL1-004 during transcription were compared, and the results are as follows: Figure 3 As shown in J. From Figure 3 As can be seen from the data, the RNA structure of exon 18.1 in RASAL1-004 is more complex than that in RASAL1-001, suggesting that the deletion of exon 18.1 alters the RNA structure of RASAL1-004 and promotes ribosomal translation arrest at that location.

[0056] From the results of the present embodiment, it can be concluded that the inactivation mechanism of RASAL1 is through RNA secondary structure-ribosome stalling.

[0057] Example 3

[0058] This example verifies the synergistic effect of RASAL1 inactivation (including genetic mutation, deletion, promoter methylation, and transcriptional silencing) and TERT promoter mutant. The specific steps are as follows:

[0059] (1) Confirm the diagnosis of 13 types of cancer, including adrenocortical carcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma and adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, cutaneous melanoma, thyroid carcinoma, and endometrial carcinoma. Pathological examination was performed according to the World Health Organization (WHO) standard. Biopsy samples were obtained from patients for subsequent molecular biology detection. Pan-cancer tumor datasets TCGA and TCPA were used. A total of 968 tumor patients with complete clinical follow-up and genetic information carrying wild-type BRAF / RAS were collected from the TCGA cohort, of which 246 (88 females, 154 males, 4 unknown gender) carried a median age (IQR) of 59.00 (38.00, 67.25) years.

[0060] (2) Genetic testing of biopsy samples to confirm the following genetic variation status.

[0061] Detection of RASAL1 inactivation: PCR and sequencing techniques were used to detect genetic changes in the RASAL1 gene (including mutations, deletions), the presence of gene promoter region methylation, and mRNA expression. Missense mutations of RASAL1 are defined as deleterious mutations; the average beta value of all CpG islands in the RASAL1 promoter region is used to define the methylation status, and when the average beta value is greater than 0.2, it is defined as high methylation; RASAL1 mRNA levels below the mean are defined as RASAL1 transcriptional silencing.

[0062] Detection of BRAF / RAS mutations and RET rearrangements: Sequencing techniques are used to detect the mutational status of the BRAF and RAS genes. BRAF driver mutations include: D594G, D594N, G466E, G469V, G596R, K601E, K601E, L245F, K601E, P731S, L613F, N581S, P367S, L597Q, R462K, V600E, V600K, V600R. KRAS driver mutations include: A146T, G12A, G12C, G12D, G12V, G13C, G13D, K117N, K5E, Q61H, Q61K, Q61L, Q61R, Q61R, E62K. NRAS driver mutations include: G12A, G12R, G13R, Q61H, Q61K, Q61K, Q61R, Q61L, Q61R. HRAS driver mutations include: A59T, G12S, G13D, G13S, G13R, Q61K, Q61L, Q61R. FISH techniques are used to detect the presence of RET fusion genes, such as RET fusions with YAKAP13, CCDC6, MRLN, YERC1, FKBP15, NCOA4, TRIM33, and SPECC1L genes.

[0063] Detection of TERT promoter mutations: PCR and sequencing are used to detect mutations in the TERT promoter region, such as TERT C228T, TERT C228A, TERT C250T, TERT C242T, TERT C243T, and TERT C169T.

[0064] Detection of tumor growth phenotype status in tumor patients: A tumor growth phenotype composite score is calculated, including pro-apoptotic marker proteins (CASPASE 3, CASPASE 7 CLEAVED D198, CASPASE 8, BIM, P53, SMAC, BID, BAX, BAK) and metastatic marker proteins (ECADHERIN, NCADHERIN, PCADHERIN, CAVEOLIN1, PAXILLIN, FIBRONECTIN).

[0065] (3) Overall analysis of the TCGA dataset showed an antagonistic relationship between BRAF mutations, RAS mutations, and RET rearrangements and RASAL1 inactivation in the MAPK pathway. Given that both BRAF / RAS mutations and RET rearrangements can coexist with TERT promoter mutations, patients with RET rearrangements were excluded from the analysis in both thyroid cancer and non-small cell lung cancer. Kaplan-Meier analysis of overall survival was performed on 968 patients with wild-type BRAF / RAS for the four genotype groups shown. RASAL1 Normal TERTp Wt (patients with both normal RASAL1 function and wild-type TERT); RASAL1 Normal TERTp Mt (patients with both normal RASAL1 function and TERT promoter mutation); RASAL1 Loss-TERTp Wt (patients with both RASAL1 inactivation and wild-type TERT); RASAL1 Loss-TERTp Mt (patients with both RASAL1 inactivation and TERT promoter mutation). The results are shown in Table 1. Among the cohort of patients with wild-type BRAF / RAS, 246 / 968 (25.73%) had both RASAL1 inactivation and TERT promoter mutation. The co-occurrence test for the interaction of RASAL1 inactivation and TERT promoter mutation showed a co-occurrence index of 1.10 (95% CI 1.03-1.18, p=0.009), indicating a significant correlation between the two. In Table 1, Co-occurrence index = [n(RET / PTC mt, TERTp mt) / n(TERTp, mt)] / [n(RET / PTC mt, TERTp wt) / n(TERTp, wt)]; P value for chi-square test; BRAF / RAS wt, wild-type, i.e., no BRAF / RAS mutation.

[0066] Table 1 Co-occurrence test for the interaction of RASAL1 inactivation and TERT promoter mutation

[0067]

[0068]

[0069] (4) As shown in Table 2, wild-type BRAF / RAS patients were divided into four genotype groups, and it was found that the mortality rate of patients without double gene alterations was 35 / 164 (21.34%), the mortality rate of patients carrying TERT promoter mutation alone was 21 / 51 (41.18%), the mortality rate of patients carrying RASAL1 inactivation alone was 147 / 495 (29.70%), and the mortality rate of patients with both of these two gene alterations was 107 / 246 (43.50%). These results show that the coexistence of RASAL1 inactivation and TERT promoter mutation gene duet is strongly associated with the highest mortality rate.

[0070] Table 2 Demographic and genetic characteristics of wild-type BRAF / RAS patient cohort

[0071]

[0072] Further, as can be seen from Figure 4 Figure 2, in the Kaplan-Meier disease-free survival analysis of wild-type BRAF / RAS patients, the survival curve of patients without double gene alterations is flat, the survival curve of patients carrying TERT promoter mutation alone is slightly decreased, the survival curve of patients carrying RASAL1 inactivation alone is decreased, and the disease-free survival rate of patients with both of these two gene alterations is sharply decreased. By Cox regression analysis (as shown in Table 3), it was found that compared with patients without gene alterations, the survival HR (95% CI) of the TERT promoter mutation alone group was 1.27 (0.73-2.19), the survival HR of the RASAL1 inactivation alone group was 1.32 (0.39-4.50), and the survival HR of the group with both was 3.34 (1.86-5.99); the former two were not significant, and the latter was significant. After adjusting the patient's gender, age and AJCC Tumor stage, these HRs remained basically unchanged, indicating that the two gene alterations have a strong synergistic effect on the survival of patients with poor prognosis.

[0073] Table 3 Effect of RASAL1 inactivation and TERT promoter mutation on disease-free survival of BRAF / RAS wild-type tumor patients in Cox regression model

[0074]

[0075]

[0076] (5) The TCPA data was analyzed for tumor growth phenotypes such as apoptosis and expression levels of metastasis-related marker proteins. As Figure 4B and C show that, compared with RASAL1 inactivation alone, RASAL1 inactivation combined with TERT promoter mutation reduces the expression of pro-apoptotic and anti-metastatic marker proteins in tumor tissues, and reduces the pro-apoptotic and anti-metastatic ability of tumors.

[0077] Further, a cell line edited by CRISPR-Cas9 to repair the TERT promoter mutation is constructed, as shown in Figure 4 D, TERT promoter mutation is generated in TERT wild-type thyroid tumor WRO cell line using CRISPR-Cas9 technology. Overexpression of RASAL1 in Parental WRO and Crispr / cas9-edited WRO cell lines, and the results are as shown in Figure 4 E and F show that, compared with TERT wild-type cells, RASAL1 has stronger ability to inhibit tumor growth in TERT promoter mutant cells. Similarly, compared with TERT wild-type cells, RASAL1 has stronger ability to promote tumor apoptosis in TERT promoter mutant cells Figure 4 G).

[0078] Further, a cell line edited by CRISPR-Cas9 to repair the TERT promoter mutation is constructed, as shown in Figure 4 H, TERT promoter mutation is repaired in TERT promoter mutant tumor cell line TPC-1 using Crispr-Cas9 editing. Overexpression of RASAL1 in Parental TPC-1 and Crispr / cas9-edited TPC-1 cell lines, and the results are as shown in Figure 4 I show that, compared with TERT promoter mutant cells, RASAL1 has weaker ability to promote tumor apoptosis in TERT wild-type cells.

[0079] In summary, the biomarkers in the present application can be used as biomarkers for tumor or cancer prognosis risk stratification, which helps to improve the clinical management of patients, and also provides strong support for the basic research and drug development of tumors.

[0080] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the claims attached to the present application.

Claims

1. A biomarker characterized in that, The biomarker comprises a transcription product and / or a translation product of skipping of exon 18 of a RASAL1 gene.

2. The biomarker of claim 1, wherein, The skipping of exon 18 of the RASAL1 gene occurs at positions 2131-2214 of the nucleotide sequence of exon 18.

3. The biomarker according to claim 1 or 2, characterized in that, The nucleotide sequence of the transcription product is shown as SEQ ID NO.

1.

4. The biomarker according to claim 1 or 2, characterized in that, The translation product is a RASAL1 protein isoform.

5. The biomarker of claim 4, wherein, The amino acid sequence of the RASAL1 protein isoform is shown as SEQ ID NO.

2.

6. The biomarker of claim 1, wherein, The biomarker further comprises: a TERT promoter gene mutant.

7. The biomarker of claim 6, wherein, The nucleotide sequence of the TERT promoter gene mutant is shown as SEQ ID NO.

3.

8. Use of the biomarker of any one of claims 1 to 7 in the preparation of a product for determining whether a subject has a tumor or stratifying a tumor prognosis risk of a subject.

9. Use according to claim 8, characterized in that, The tumor comprises a malignant tumor; The malignant tumor comprises at least one of an adrenocortical carcinoma, a bladder urothelial carcinoma, a cervical squamous cell carcinoma and adenocarcinoma, a glioblastoma multiforme, a head and neck squamous cell carcinoma, a kidney chromophobe carcinoma, a low-grade glioma, a hepatocellular carcinoma, a lung adenocarcinoma, a prostate adenocarcinoma, a cutaneous melanoma, a thyroid carcinoma, an endometrial carcinoma.

Images