CN114480656B - Application of SLC12A3 and/or SLC12A9 as grape membrane melanoma treatment and prognosis detection index - Google Patents

Application of SLC12A3 and/or SLC12A9 as grape membrane melanoma treatment and prognosis detection index Download PDF

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CN114480656B
CN114480656B CN202210215328.XA CN202210215328A CN114480656B CN 114480656 B CN114480656 B CN 114480656B CN 202210215328 A CN202210215328 A CN 202210215328A CN 114480656 B CN114480656 B CN 114480656B
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slc12a3
slc12a9
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周猛
闫聪聪
孙杰
郑钦象
麻晓银
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Eye Hospital of Wenzhou Medical University
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Abstract

The invention discloses application of SLC12A3 and/or SLC12A9 as a treatment and prognosis detection index of uveal melanoma. The invention also discloses a calculation model for predicting the prognosis of the uveal melanoma and a medicament for preventing or treating the uveal melanoma. The invention provides a new method for prognosis monitoring and treatment of patients with uveal melanoma.

Description

Application of SLC12A3 and/or SLC12A9 as grape membrane melanoma treatment and prognosis detection index
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of SLC12A3 and/or SLC12A9 as a grape membrane melanoma treatment and prognosis detection index.
Background
Uveal Melanoma (UVM) is the most common primary malignancy in the eyes of adults, with the age of onset being more than 50-60 years old, and with slightly more male patients. The incidence rate of the foreign uveal melanoma is 0.02%, and the domestic uveal melanoma is the intraocular malignant tumor with the second incidence rate. Uveal melanoma can occur anywhere on the uvea, but is most abundant in the choroid. The disease is usually caused by monocular disease, and the disease caused by binocular disease is rare. The uveal melanoma cells can be transferred to distant sites early along with blood flow, and distant metastasis has begun even before the primary tumor of the eye is found. Uveal melanoma metastasis is primarily the menstrual tract, the most common site of metastasis being the liver, followed by the lungs, bones and skin, and less so the central nervous system. Metastasis occurs in more than 50% of patients with uveal melanoma.
The treatment method of uveal melanoma comprises tumor local excision, eyeball local application treatment, transpupillary thermotherapy and laser photocoagulation, and other treatments include chemotherapy, immunotherapy, gene therapy, cell radiotherapy and the like. Despite improvements in topical treatments, including surgery and radiation, the distant metastasis and mortality of uveal melanoma have remained unchanged for the last decade, indicating that current treatments for primary uveal melanoma do not give a better prognosis. With the research on the mechanism of occurrence, development and metastasis of uveal melanoma, comprehensive treatment is a trend of future treatment. The molecular biological characteristics of the grape membrane melanoma are researched, the pathological mechanism of the tumorigenesis, development and metastasis process is analyzed and described, and valuable specific tumor markers are searched, so that the method has important significance in realizing prognosis evaluation of patients with the grape membrane melanoma and developing new treatment means.
Disclosure of Invention
A first object of the present invention is to provide an agent useful for predicting the prognosis of uveal melanoma;
a second object of the present invention is to provide a method of screening candidate drugs for the treatment of uveal melanoma;
a third object of the invention is to provide a computational model for predicting the prognosis of uveal melanoma;
a fourth object of the present invention is to provide a medicament for preventing or treating uveal melanoma.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a reagent useful for predicting prognosis of uveal melanoma, said reagent being capable of detecting the expression level of a biomarker in a biological sample, said biomarker comprising SLC12A3 and/or SLC12A9, preferably SLC12A3 and SLC12A9.
As a preferred embodiment, the reagent comprises a reagent for measuring the expression level of the biomarker at the mRNA or protein level.
As a preferred embodiment, the reagent for measuring the expression level of the biomarker gene at the mRNA level includes a reagent for detecting the expression level of the biomarker gene by any one of the methods of polymerase chain reaction, real-time fluorescent quantitative reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization method, nucleic acid microarray, RNA blot or DNA chip,
as a more preferred embodiment, the reagent for measuring the expression level of the biomarker gene at the mRNA level comprises a primer pair, a probe or a primer pair and a probe that specifically recognize the whole length of the nucleic acid sequence of the biomarker or a fragment thereof.
As a preferred embodiment, the reagent for measuring the expression level of the biomarker at the protein level comprises a reagent for measuring the expression level of the biomarker gene by multiplex orthostretching analysis, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich analysis, western blot, immunoprecipitation, immunohistochemical staining, fluoroimmunoassay, enzyme substrate color development, antigen-antibody aggregation, fluorescence activated cell sorting, mass spectrometry, multiplex reaction monitoring assay, assay using a set of polyamine-specific stable isotope reagents or protein chip,
as a more preferred embodiment, the reagent for measuring the expression level of the biomarker gene at the protein level comprises an antibody, an antibody fragment, an aptamer, a high affinity polymer or a peptidomimetic which specifically recognizes the full length of the protein of the biomarker or a fragment thereof.
In a second aspect the present invention provides a pharmaceutical composition comprising an inhibitor of SLC12A3 and/or an inhibitor of SLC12A9, preferably an inhibitor of SLC12A3 and an inhibitor of SLC12A9,
as a preferred embodiment, the inhibitor comprises a nucleic acid molecule, a carbohydrate, a small molecule compound or an interfering lentivirus.
As a more preferred embodiment, the inhibitor is a nucleic acid molecule, more preferably, the nucleic acid molecule includes, but is not limited to, shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. In a specific embodiment of the invention, the nucleic acid molecule is an siRNA.
As a preferred technical scheme, the inhibitor of SLC12A3 comprises a sequence shown as SEQ ID NO.3, and the inhibitor of SLC12A9 comprises a sequence shown as SEQ ID NO. 2.
In a third aspect, the present invention provides a method of screening for a candidate agent for the treatment of uveal melanoma, said method comprising: treating a system expressing or containing SLC12A3 and/or SLC12A9 genes with a substance to be screened; and detecting the expression of the SLC12A3 and/or SLC12A9 gene in said system; wherein, if the substance to be screened reduces the expression level of SLC12A3 and/or SLC12A9 genes, the substance to be screened is indicated to be a candidate drug for treating uveal melanoma.
The fourth aspect of the invention provides a calculation model for predicting the prognosis of uveal melanoma, which comprises a calculation and analysis module of SLC12A3 and/or SLC12A9, wherein the calculation and analysis module processes the detected data of SLC12A3 and/or SLC12A9 and compares the data with a set predicted value.
A fifth aspect of the invention provides the use of any one of the following:
(1) The use of a reagent according to the first aspect of the invention for the preparation of a product for predicting the prognosis of uveal melanoma;
(2) The application of the biomarker in constructing a calculation model for predicting the prognosis of the uveal melanoma;
(3) The application of the pharmaceutical composition in the second aspect of the invention in preparing medicines for preventing or treating uveal melanoma;
(4) The application of the biomarker in screening candidate medicines for treating uveal melanoma;
the biomarker comprises SLC12A3 and/or SLC12A9, preferably the biomarker is SLC12A3 and SLC12A9.
As a preferred technical scheme, the product comprises a chip, a kit, test paper or a high-throughput sequencing platform.
The high-throughput sequencing platform is a special diagnostic tool, and the product for detecting the expression of the biomarker genes can be applied to the platform to detect the expression of the biomarker genes. With the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes a very convenient task. By comparing the gene expression profiles of the patient with the disease and the normal population, it is easy to analyze which gene abnormality is associated with the disease. Thus, it is within the scope of the present invention to know in high throughput sequencing that abnormalities in the biomarker genes are associated with cancer prognosis as well as the use of the biomarker genes.
Wherein the chip comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises an oligonucleotide probe aiming at the biomarker gene for detecting the transcription level of the biomarker gene; the protein chip comprises a solid phase carrier and a specific antibody of the biomarker protein fixed on the solid phase carrier; the gene chip can be used to detect the expression levels of a plurality of genes including the biomarker genes (e.g., a plurality of genes associated with cancer prognosis). The protein chip can be used to detect the expression levels of a plurality of proteins including the biomarker protein (e.g., a plurality of proteins associated with prognosis of cancer). By detecting a plurality of markers for prognosis of cancer simultaneously, the accuracy of predicting prognosis of cancer can be greatly improved.
Wherein the kit comprises a gene detection kit and a protein immunodetection kit; the gene detection kit comprises reagents for detecting the transcriptional level of the biomarker genes.
Drawings
FIG. 1 is a graph of analysis results of the effect of SLC12 family genes on clinical outcome of uveal melanoma, wherein graph A is a forest map of the effect of SLC12 family gene expression levels on OS and DSS; FIG. B is a graph of the results of Kaplan-Meier analysis of OS and DSS based on SLC12A3 expression levels; FIG. C is a graph of the results of Kaplan-Meier analysis of OS and DSS based on SLC12A9 expression levels; FIG. D is a graph of the results of a multivariate Cox proportional hazards regression analysis of SLC12A3 and SLC12A9 with other clinical features; panel E is a SLC12 family gene correlation heat map; FIG. F is a heat map based on SLC12A3 and SLC12A9 groupings; FIG. G is a Kaplan-Meier survival curve for OS and DSS based on SLC12A3 and SLC12A9 groupings;
FIG. 2 is a graph of the results of analysis of the effect of over-expression of SLC12A3 and/or SLC12A9 on proliferation of uveal melanoma cells, wherein graph A is a negative control graph of immunostaining, graph B is a graph of the results of immunostaining of SLC12A9 of UVM melanoma tissue, graph C is a graph of the results of immunostaining of SLC12A3 of UVM melanoma tissue, graph D is a graph of the results of Western blotting analysis of the protein levels of SLC12A9 in over-expression cells C918 and MUM2C, graph E is a graph of the results of Western blotting analysis of the protein levels of SLC12A3 in over-expression cells C918 and MUM2C, graph F is a graph of C918 after over-expression of SLC12A9, graph G is a graph of C918 after over-expression of SLC12A3, graph H is a graph of MUM2C after over-expression of SLC12A9, graph I is a graph of MUM2C after over-expression of SLC12A3, graph J is a graph of quantitative graph of C918 cell formation of over-expression SLC12A9 and quantitative analysis of SLC 918 cell formation and graph of MUM2C and quantitative analysis of SLC 3 in comparison of quantitative graph of SLC 2C cell formation of SLC12A3 and graph of MUM2C and quantitative analysis of the results of MUM cell formation and the graph;
FIG. 3 is a graph of the results of an analysis of the effect of knockdown of SLC12A3 and SLC12A9 on proliferation of uveal melanoma cells, wherein FIG. A is a graph of the results of an analysis of the knockdown efficiency of specific siRNA on SLC12A3 or SLC12A9 gene, FIG. B is a graph of the growth of C918 cells after knockdown of SLC12A3 and/or SLC12A9, FIG. C is a graph of the growth of MUM2C cells after knockdown of SLC12A3 and/or SLC12A9, and FIG. D is a graph of the results of a colony formation analysis of C918 cells after knockdown of SLC12A3 and/or SLC12A 9; FIG. E is a quantitative analysis chart of clone formation of C918 cells after SLC12A3 and/or SLC12A9 is knocked out, FIG. F is a quantitative analysis chart of clone formation of MUM2C cells after SLC12A3 and/or SLC12A9 is knocked out, FIG. G is a quantitative analysis chart of clone formation of MUM2C cells after SLC12A3 and/or SLC12A9 is knocked out, FIG. H is a quantitative analysis chart of EdU staining of C918 cells after SLC12A3 and/or SLC12A9 is knocked out, FIG. I is a quantitative analysis chart of EdU staining of C918 cells after SLC12A3 and/or SLC12A9 is knocked out, FIG. J is a quantitative analysis chart of EdU staining of MUM2C cells after SLC12A3 and/or SLC12A9 is knocked out;
fig. 4 is a time-dependent ROC analysis result of the prognosis of the uveal melanoma of the SLC12A3 and/or the SLC12A9, wherein fig. a is a time-dependent ROC analysis chart of the SLC12A3, fig. B is a time-dependent ROC analysis chart of the SLC12A9, and fig. C is a time-dependent ROC analysis chart of the SLC12A3 and the SLC12A9.
Detailed Description
The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.
The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).
The term "biomarker" refers to a biomolecule that is present in an individual at different concentrations that can be used to predict the disease state of the individual. Biomarkers can include, but are not limited to, nucleic acids, proteins, and variants and fragments thereof. The biomarker may be DNA comprising all or part of a nucleic acid sequence encoding the biomarker or a complement of such a sequence. Biomarker nucleic acids useful in the present invention are considered to include DNA and RNA comprising all or part of any nucleic acid sequence of interest. In the present invention, biomarkers such as SLC12A3 (solute carrier family 12member 3,gene ID:6559), SLC12A9 (solute carrier family 12member 9,gene ID:56996); including genes and their encoded proteins and their homologs, mutations, and isoforms. The term encompasses full length, unprocessed biomarkers, as well as any form of biomarker derived from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarker. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene.
The term "biological sample" refers to any biological sample from an individual or (control) subject comprising a biomarker of the invention. The biological sample may be a body fluid sample or a tissue sample. For example, biological samples encompassed by the present invention are tissue samples, blood (e.g., whole blood or blood components, such as blood cells/cell components, serum or plasma) samples, urine samples, cerebrospinal fluid (CSF) or samples from other peripheral sources. The biological samples may be mixed or pooled, for example, the sample may be a mixture of a blood sample and a urine sample. The biological sample may be provided by taking the biological sample from an individual or (control) subject, but may also be provided by using a previously isolated sample. For example, a blood sample may be obtained from an individual or (control) subject by conventional blood collection techniques, or a tissue sample may be obtained from an individual or (control) subject by biopsy (biopsy). Biological samples (e.g., urine samples, blood samples, or tissue samples) may be obtained from an individual or (control) subject prior to initiation of therapeutic treatment, during therapeutic treatment, and/or after therapeutic treatment. A biological sample is designated as a "reference biological sample" if it is obtained from at least one (control) subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500 or 1,000 (control) subjects. Preferably, the reference biological sample is from the same source as the biological sample of the individual to be tested, e.g. both are blood samples, urine samples or tissue samples. It is further preferred that both are from the same species, e.g. from a human. It is also preferred (alternatively or additionally) that the measure of the reference biological sample of the (control) subject and the biological sample of the individual to be tested are the same, e.g. both have the same volume. It is particularly preferred that the reference biological sample and the biological sample are from (control) subjects/individuals of the same sex and similar age.
The body fluid sample may be a urine sample, a blood sample, a sputum sample, a breast milk sample, a cerebrospinal fluid (CSF) sample, a cerumen (cerumen) sample, a gastric fluid sample, a mucus sample, an endolymph sample, an perilymph sample, a peritoneal fluid sample, a pleural fluid sample, a saliva sample, a sebum (skin oil) sample, a semen sample, a sweat sample, a tear sample, a cheek swab, a vaginal secretion sample, a liquid biopsy or vomit sample, including their components or fractions. The term "body fluid sample" also encompasses body fluid components, such as blood components, urine components or sputum components. The body fluid samples may be mixed or pooled. Thus, the body fluid sample may be a mixture of blood and urine samples, or a mixture of blood and cerebrospinal fluid samples.
The term "primer" as used herein refers to a single stranded oligonucleotide capable of binding to a target nucleic acid. Typically, the binding is selective. The exact length of the primer will vary depending on the particular application, but is typically about 15 to about 120 nucleotides. The primer need not reflect the exact sequence of the target nucleic acid template, but must be sufficiently complementary to bind to the template. The term "nucleotide primer" includes "oligonucleotide primer". The oligonucleotides for use as primers can be selected using software known in the art for this purpose. For example, OLIGO4.06 primer analysis software (available from National Biosciences, plymouth, MN) can be used to select primers each up to 30-100 nucleotides, and to analyze larger polynucleotides up to 5,000 nucleotides from an input polynucleotide sequence up to 32 kilobases. Similar primer selection procedures have incorporated additional features to extend performance. For example, the Primou primer selection program (publicly available from Genome Center at University of Texas South West Medical Center (Dallas TX)) is capable of selecting specific primers from megabase sequences and thus can be used to design primers in a Genome-wide manner. Primer3 Primer selection procedure (publicly available from Whitehead Institute/MITCenter for Genome 5Research,Cambridge MA) allows the user to input "missprimingligary", wherein the sequence to be avoided as the Primer binding site is user-specified. Primer3 is particularly useful for selecting nucleotides for microarrays. (the source codes of the latter two primer selection procedures may also be obtained from their respective sources and modified to suit the specific needs of the user). The PrimeGen program (publicly available from UK Human Genome MappingProjectResource Centre, cambridge UK) designs primers based on multiple sequence alignments, allowing the selection of primers that bind or hybridize to the most conserved or least conserved regions of aligned nucleic acid sequences. Thus, this procedure can be used to identify unique and conserved nucleotides as well as polynucleotide fragments.
The term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can confirm the presence of the target substance in the sample by the above-mentioned binding specificity. The type of probe is not limited and may be peptide nucleic acid (PNA, peptide nucleic acid), locked nucleic acid (LNA, locked nucleic acid), peptide, polypeptide, protein, ribonucleic acid or deoxyribonucleic acid, and most preferably peptide nucleic acid, which are commonly used in the art. More specifically, the above-mentioned probes are biomass including those derived from or similar to biological materials or produced in vitro, and may include, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, deoxyribonucleic acids including complementary deoxyribonucleic acids (cdnas), genomic deoxyribonucleic acids, oligonucleotides, ribonucleic acids including genomic ribonucleic acids, messenger ribonucleic acids, oligonucleotides, and the like, as examples of proteins including antibodies, antigens, enzymes, peptides, and the like.
The term "antisense" as used herein refers to an oligomer that hybridizes to a target sequence in ribonucleic acid by base-pairing, typically allowing formation of messenger ribonucleic acid and ribonucleic acid in the target sequence, an oligomeric heteroduplex, a sequence of nucleotide bases and an intersubunit backbone. The oligomer may have precise sequence complementarity or near complementarity to the target sequence.
The term "antibody" refers to a substance that specifically binds to an antigen to elicit an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a biomarker of the present invention for predicting the prognosis of uveal melanoma. Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibodies described above can be readily prepared using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art including the process of injecting an animal with a biomarker protein antigen described above that predicts the prognosis of uveal melanoma and collecting blood from the animal to obtain serum containing the antibodies. Such polyclonal antibodies may be prepared by any animal such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, mice, etc. Also, monoclonal antibodies can be prepared using hybridoma methods or phage antibody library techniques well known in the art. The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, etc. Furthermore, the antibodies of the invention include not only intact forms having 2 full length light chains and 2 full length heavy chains, but also functional fragments of the antibody molecules. Functional fragments of antibody molecules refer to fragments having at least antigen binding function, and include Fab, F (ab') 2, fv, and the like. Furthermore, the antibodies of the invention are commercially available.
Any method available in the art for detecting expression of a molecular marker is encompassed herein. Expression of the molecular markers of the invention may be detected at the nucleic acid level (e.g., RNA transcripts) or at the protein level. By "detecting expression" is intended determining the amount or presence of an expression product of an RNA transcript or a molecular marker gene thereof. Thus, "detecting expression" includes instances where a molecular marker is determined to be not expressed, not to be detected for expression, expressed at a low level, expressed at a normal level, or over-expressed. To determine low expression, the body sample being examined can be compared to a corresponding body sample from a control.
As used herein, the inhibitor is selected from: an interfering molecule targeting the biomarker gene or a transcript thereof and capable of inhibiting expression of the biomarker gene or transcription of the gene, comprising: nucleic acid molecules, carbohydrates, small molecule compounds or interfering lentiviruses. Wherein the nucleic acid molecule includes, but is not limited to, an shRNA (small hairpin RNA), a small interfering RNA (siRNA), a dsRNA, a microrna, an antisense nucleic acid, or a construct capable of expressing or forming the shRNA, the small interfering RNA, the dsRNA, the microrna, the antisense nucleic acid. In a specific embodiment of the invention, the nucleic acid molecule is an siRNA.
The term "siRNA" as used herein refers to preventing over-expression of a target gene. Standard techniques for introducing siRNA into cells are used, including those in which RNA is transcribed using DNA as a template. The siRNA includes a sense nucleic acid sequence (also referred to as the "sense strand"), an antisense nucleic acid sequence (also referred to as the "antisense strand"), or both. The siRNA can be constructed such that a single transcript has an antisense nucleic acid sequence complementary thereto to the sense nucleic acid sequence of the target gene, e.g., a hairpin structure. The siRNA may be dsRNA or shRNA.
The term "dsRNA" as used herein refers to a construct comprising two RNA molecules of mutually complementary sequences through which the two RNA molecules anneal to form a double stranded RNA molecule.
In the present invention, the term "shRNA" refers to: siRNA having a stem-loop structure comprising a first region and a second region (i.e., a sense strand and an antisense strand) complementary to each other. The degree and orientation of complementarity of the two regions is sufficient to allow base pairing between the two regions, the first and second regions being joined together by a loop region formed by the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of the shRNA is a single stranded region between the sense and antisense strands, and may also be referred to as an "intervening single strand" (intervening single-strand) ".
In the present invention, "drug", "pharmaceutical composition" may be used in general. The pharmaceutical compositions of the invention are characterized as being at least sterile and pyrogen-free. In preparing these pharmaceutical compositions, the active ingredient is typically admixed with or diluted with an excipient or enclosed in a carrier which may be in the form of a capsule or sachet. When the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water and the like. The formulation may further comprise wetting agents, emulsifying agents, preserving agents (e.g., methyl and propyl hydroxybenzoates), sweetening agents, and the like.
The use of the pharmaceutical composition provides a method for the treatment of a tumor, in particular a method for the prevention or treatment of a tumor in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition.
For use in preventing or treating a tumor in a subject, an effective amount of the pharmaceutical composition is administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the portion of the tumor that grows, proliferates, recurs and/or metastasizes is inhibited.
The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Experimental method
1. UVM patient cohort acquisition
Tumor genomic profile (TCGA) transcript (mRNA, miRNA, lncRNA) and clinical data were obtained from the UCSC Xena database (https:// Xena. UCSC. Edu /) for 80 UVM patients. The study excludes 3 UVM samples of Formalin Fixed Paraffin Embedded (FFPE). Two independent UVM queues with microarray expression profiles and clinical data were downloaded from GEO databases with accession numbers GSE84976 (n=28) and GSE22138 (n=63).
2. Differential Expression Gene (DEG) analysis
Differential expression between the different groups was screened for mRNAs (DEmRNAs), miRNAs (DEmiRNAs) and LncRNAs (DElncRNAs) using a two-tailed unpaired Mann-Whitney U assay. Only mRNAs expressed in at least 80% of the samples remain.
DEmiRNAs and DElncRNAs selection criteria: log2 (fold change) >1 (up-regulated) or < -1 (down-regulated), FDR <0.05.
DEmRNAs screening criteria: log2 (fold change) >2 (up-regulated) or < -2 (down-regulated), FDR <0.05.
3. Functional enrichment analysis
GO and KEGG pathway enrichment analysis was performed using R package cluster profile (18) to determine significantly enriched GO Biological Processes (BP) terms and KEGG pathways. The z-score is then calculated using the R-packet GOplot to determine whether the rich terms or paths are more likely to decrease (negative values) or increase (positive values). The gene sets enriched or overexpressed in the different subtypes were analyzed using gene set enrichment analysis software (http:// software.broadenstitute.org/gsea/index.jsp).
4. Calculation and quantification of tumor immune microenvironment
Gene profiles associated with 16 immune cell subsets were obtained from a study of Charoentong (Charoentong P, finotello F, angelova M, mayer C, efrenova M, rieder D, et al Pan-cancer Immunogenomic Analyses Reveal Genotype-Immunophenotype Relationships and Predictors of Response to Checkpoint Block ade. Cell Rep.2017;18 (1): 248-62.). The relative abundance of 16 immune cell subsets in the tumor immune microenvironment was quantified per patient using a single sample gene set enrichment analysis (single-sample gene set enrichment Analysis, ssGSEA) with a specific gene set of R-packets.
5. Network analysis
The co-expression relationship between genes was measured using Pearson correlation coefficients, and gene pairs of Pearson' s|r| >0.2 and p <0.05 were used to construct a gene co-expression network, and repeated edges were removed and visualized after self-loop. The highly linked gene modules were generated using the molecular complex detection (molecular complex detection, MCODE) algorithm, node score cutoff at 0.1 and other parameters set to default values.
Deregulated competitive endogenous RNA (competing endogenous RNA, ceRNA) networks were constructed based on the ceRNA hypothesis as follows:
1) Retrieving human miRNA-SLC12A3/A9 and miRNA-lncRNAs interactions from ENCORI, targetScan (release 8.0) and mirDIP;
2) The expression correlation of DEmiRNAs with SLC12A3/A9, and DEmiRNAs with DElncRNAs, DElncRNAs with SLC12A3/A9 is measured by using a Pearson correlation coefficient;
3) The SLC12A3/A9-DEmiRNA-DElncRNAs triplets were selected as the ceRNA triplets that were positively correlated with DElncRNA-SLC12A3/A9 (Pearson's r > 0.2) and down-regulated by the same DEmiRNAs (Pearson's r < -0.2). These ceRNA triplets are integrated into a ceRNA network.
6. Cell culture and gene knockout
Grape membrane melanoma cell lines C918 and MUM2C were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum and antibiotics, and placed in 5% CO 2 Culturing at 37deg.C. In siRNA studies, cells were cultured in 12-well plates to about 50% confluence, then with LipoJet TM 40pmol of siRNA was transfected with reagent (SignagEN). siRNA was designed and synthesized by Shanghai gene pharmaceutical Co., ltd. In China, the sequence is as follows:
si-NC:5’-UUCUCCGAACGUGUCACGUTT(SEQ ID NO.1);
si-SLC12A9:5’-GCATTGGGCTCATGTTCTA(SEQ ID NO.2);
si-SLC12A3:5‘-GCCCACAUAUGAGCACUAUTT(SEQ ID NO.3)。
7. gene overexpression using lentiviral infection
SLC12A3 over-expressed lentivirus (nm_ 001126108) was purchased from geiki gene limited (Shanghai, china) while SLC12A9 over-expressed lentivirus (nm_ 020246) was purchased from Yijin biotechnology limited (Guangzhou, china). 1 day prior to infection, C918 cells were cultured in 12-well plates to about 50% confluency. Then 10. Mu.L of SLC12A3 or SLC12A9 over-expressed lentivirus (2X 10 8 TU/ml) was added to FBS-free DMEM for about 6 hours, cellsCulturing in complete medium. The SLC12A3 or SLC12A9 protein was analyzed by Western blotting 72 hours after infection.
8、Western blotting
UVM cells were washed with PBS and lysed with RIPA (Biyuntian, china) on ice for 30 min. An equal amount of protein lysate was separated by SDS-PAGE and transferred onto PVDF membrane, blocked with 5% skimmed milk for 2 hours at room temperature, then incubated overnight at 4 ℃. The antibody comprises: anti-SLC 12A9 antibody (1:1000,HuaBio CHINA), anti-SLC 12A3 antibody (1:1000, abcam, ab23401), or anti-GAPDH antibody (1:4000,Cell Signaling Technology,5174S). Primary antibodies were identified with fluorescein-conjugated secondary antibodies (LI-COR) at room temperature over 2 hours, and blots were analyzed using the Physsey CLx System (LI-COR) and quantitative densitometry of the bands was performed using imageJ software.
7. Analysis of cell growth curve
Cell viability was analyzed using a Cell Counting Kit-8 kit (CCK-8,Beyotime Biotechnology Co, ltd). 1000 melanoma cells were cultured in 96-well plates, and after adding 10uL of CCK-8 working solution for 2 hours at 0h, 24h, 48h, 78h and 96h, the optical density at 450nm was measured.
9. EdU incorporation method
BeyoClick with Alexa Fluor 594 TM The EdU cell proliferation kit was purchased from the yunnan biotechnology company (Shanghai, china). Cells were incubated in 6-well plates and incubated in 10. Mu. Mol/L EdU working solution for 2 hours at 37 ℃. Cells were then fixed in 4% polyoxymethylene for 15 min at room temperature and incubated with 0.3% triton X-100 for 15 min in the dark. After staining nuclei with DAPI for 5 minutes at room temperature, the cells were observed under an inverted fluorescence microscope.
10. Immunohistochemical (IHC) staining
The tissue specimens were subjected to immunohistochemical analysis, and the slice thickness was 5. Mu.m. Dewaxing with xylene, washing the hydration with a series of graded alcohols, thermal antigen retrieval using EDTA at 100 ℃ for 20min and rinsing in PBS. The antibody comprises: anti-SLC 12A9 antibody (1:200, huaBio, CHINA), anti-SLC 12A3 antibody (1:200, ab23401, abcam, USA). Negative controls were processed in parallel using the same protocol, but omitting primary antibodies, and images were captured using the Leica DM750 automated microscope system.
11. Statistical analysis
All analyses were performed in R version 3.6. Statistical differences in continuous variables between the two groups were determined by using a two-tailed Wilcoxon rank sum test. Each experiment was repeated 3 times and the results are expressed as mean ± Standard Deviation (SD). Statistical significance between experimental and control groups was assessed using Student's t test. And (3) establishing a single-factor and multi-factor Cox proportion risk regression model by adopting a coxph function of R-pack survival, and checking the influence of specific factors and clinical variables on the survival of the patient. The risk ratio (HR) and 95% Confidence Interval (CI) were calculated. The differences in survival between groups were assessed using the Kaplan-Meier method and the log-rank test. And carrying out correlation analysis on the R package 'H misc', and carrying out correlation matrix visualization by using the R package 'corrplot'. Time-dependent subject work characteristics (time-dependent receiver operating characteristic, timeROC) were plotted to evaluate the performance of the prognostic model by R-package "timeROC" and calculate the area under the curve (AUC). p <0.05 is considered statistically significant.
EXAMPLE 1 correlation of SLC12A3 and SLC12A9 expression levels with prognosis of UVM patients
To evaluate the relationship between SLC12s and prognosis in UVM patients, the present invention performed univariate Cox regression analysis in the TCGA-UVM cohort. The results showed that high expression of SLC12A3 and SLC12A9 correlated significantly with poor Overall Survival (OS) (SLC 12A3: hr=1.16, 95% ci=1.03-1.29, p=0.01; SLC12A9: hr=2.36, 95% ci=1.14-4.88, p=0.02), and disease-specific survival (disease-specific survival, DSS) (SLC 12A3: hr=1.16, 95% ci=1.03-1.31, p=0.01; SLC12A9: hr=2.55, 95% ci=1.20-5.45, p=0.02), whereas other SLC12 family members did not exhibit significant correlation (as shown in fig. 1A).
Survival analysis also showed that 77 UVM patients could be divided into high-risk and low-risk groups (Log-Rank P < 0.05) with significant differences in OS and DSS based on SLC12A3 and SLC12A9 expression levels (as shown in figures 1B and 1C). In gender, age, stage, etc. multifactorial Cox regression analysis, SLC12A3, SLC12A9 remained significantly correlated with OS (SLC 12A3: hr=1.20, 95% ci=1.1-1.4, p=0.005; SLC12A9: hr=2.44, 95% ci=1.10-5.70, p=0.038), and DSS remained significantly correlated (SLC 12A3: hr=1.20, 95% ci=1.04-1.40, p=0.009; SLC12A9: hr=2.70, 95% ci=1.10-6.50, p=0.031) (as shown in fig. 1D), indicating that SLC12A3 and SLC12A9 are independent prognostic factors affecting clinical outcome. The invention compares the predictive ability of patients in 3 years using a time dependent ROC analysis, as shown in fig. 4, with A3 year AUC of SLC12A3 of 0.68; the 3-year AUC for SLC12A9 was 0.67, and the 3-year AUC for the combined predicted prognosis for SLC12a3 and SLC12A9 was 0.74. The above results indicate that SLC12A3 and SLC12A9 have better prognostic predictive power in UVM patients.
The present invention measures the expression correlation between members of the SLC12 family and finds no significant correlation between SLC12A3 and SLC12A9 (as shown in fig. 1E). It was further investigated whether the combination of SLC12A3 and SLC12A9 has a synergistic effect on the prognosis of the patient. All patients were divided into 4 risk groups based on the expression levels of SLC12A3 and SLC12A9. As shown in fig. 1G, there is a significant difference in OS and DSS between the four hazard groups (OS: log-rank p=0.015; DSS: log-rank p=0.016). Patients with high expression of SLC12A3 and SLC12A9 (designated A3 H A9 H Group) OS and DSS were worst, whereas patients with low expression of SLC12A3 and SLC12A9 (termed A3 L A9 L Group) OS and DSS are longest. These findings indicate that the effect of predicting prognosis of the combination of SLC12A3 and SLC12A9 is superior to that of the single marker.
Example 2 Effect of over-expression of SLC12A3 and/or SLC12A9 on uveal melanoma cell proliferation
The invention adopts an immunohistochemical method to analyze the expression of SLC12A3 and SLC12A9 in the uveal melanoma tissues. Immunohistochemical staining showed low expression of SLC12A3 and SLC12A9 in normal pararetinal cancer tissues and high expression in uveal melanoma tissues (as shown in fig. 2A-C). To investigate the functional role of SLC12A3 and SLC12A9 in uveal melanoma, the present invention utilized lentiviral infection to overexpress SLC12A3 and SLC12A9 in uveal melanoma cell lines C918 and MUM 2C. Western blotting results showed that SLC12A3 and SLC12A9 protein levels were elevated in the overexpressed cell lines, respectively (as shown in FIG. 2D and FIG. 2E). Cell growth curves showed that overexpression of both SLC12A3 and SLC12A9 increased cell proliferation activity compared to the EGFP control group (as shown in FIGS. 2F-I). In addition, overexpression of SLC12A3 and SLC12A9 also accelerated colony formation of uveal melanoma cells (as shown in FIGS. 2J-M). These results clearly demonstrate that both SLC12A3 and SLC12A9 are highly expressed in uveal melanoma and promote uveal melanoma cell proliferation.
Example 3 effects of knock-down of SLC12A3 and SLC12A9 on uveal melanoma cell proliferation
According to the invention, SLC12A3 or SLC12A9 in the uveal melanoma cell line is knocked down, or SLC12A3 and SLC12A9 are knocked down simultaneously, and both si-SLC12A3 (abbreviated as si-A3) and si-SLC12A9 (abbreviated as si-A9) are found to be capable of effectively knocking down SLC12A3 and SLC12A9 respectively (shown in figure 3A). Cell growth curves showed that knockdown of SLC12A3 and SLC12A9 inhibited proliferation of C918 cells (as shown in fig. 3B). Simultaneously knocking down SLC12A3 and SLC12A9 in C918 cells has stronger inhibition effect on cell proliferation (as shown in fig. 3B). To further confirm this result in other uveal melanoma cell lines, the present invention also knockdown SLC12A3 and SLC12A9 in the MUM2C cells, and the cell growth curve also showed that proliferation of SLC12A3 and SLC12A9 knockdown cells was inhibited (fig. 3C). Consistent with cell proliferation data, knockdown of SLC12A3 and SLC12A9 reduced colony formation of C918 and MUM2C cells, while knockdown of SLC12A3 and SLC12A9 had a synergistic effect (fig. 3D-G). In addition, in C918 (as shown in fig. 3H and 3I) and in MUM2C cells (fig. 3J and K), the proportion of cells that were positive for EdU staining in the cells decreased after the SLC12A3 or SLC12A9 gene knockdown (EdU marks s-phase proliferating cells) compared to NC cells.
The preferred embodiments of the present application have been described in detail above with reference to the accompanying drawings, but the present application is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in detail.
Moreover, any combination of the various embodiments of the present application may be made without departing from the spirit of the present application, which should also be considered as disclosed herein.
Sequence listing
<110> Ocular eye sight hospital attached to university of medical science in Wenzhou
<120> use of SLC12A3 and/or SLC12A9 as an index for the treatment and prognostic assay of uveal melanoma
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Claims (10)

1. The use of any one of the following:
(1) Use of a reagent for detecting the expression level of a biomarker in a biological sample in the preparation of a product for predicting the prognosis of uveal melanoma;
(2) Application of biomarker in preparing calculation model for predicting grape membrane melanoma prognosis;
(3) Use of a pharmaceutical composition comprising an inhibitor of SLC12A3 and/or an inhibitor of SLC12A9 in the manufacture of a medicament for the treatment of uveal melanoma;
the biomarker is SLC12A3 and/or SLC12A9, the inhibitor of SLC12A3 is a sequence shown as SEQ ID NO.3, and the inhibitor of SLC12A9 is a sequence shown as SEQ ID NO. 2.
2. The use of claim 1, wherein the biomarkers are SLC12A3 and SLC12A9.
3. The use of claim 1, wherein the pharmaceutical composition comprises an inhibitor of SLC12A3 and an inhibitor of SLC12A9.
4. The use of claim 1, wherein the product is a chip, a kit, a test strip or a high throughput sequencing platform.
5. The use according to claim 1, wherein the reagent of (1) comprises a reagent for measuring the expression level of the biomarker at the mRNA or protein level.
6. The use according to claim 5, wherein the reagent for measuring the expression level of the biomarker gene at the mRNA level comprises a reagent for detecting the expression level of the biomarker gene by any one of polymerase chain reaction, nuclease protection analysis, in situ hybridization method, nucleic acid microarray, northern blotting or DNA chip method.
7. The use according to claim 5, wherein the reagent for measuring the expression level of the biomarker gene at the mRNA level comprises a primer pair, a probe or a primer pair and a probe that specifically recognizes the full length of the nucleic acid sequence of the biomarker or a fragment thereof.
8. The use according to claim 5, wherein the reagent for measuring the expression level of the biomarker at the protein level comprises a reagent for measuring the expression level of the biomarker gene by multiplex orthostretching analysis, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, western blot, immunoprecipitation, immunohistochemical staining, fluorescent immunoassay, enzyme substrate color development, antigen-antibody aggregation, fluorescence activated cell sorting, mass spectrometry, multiplex reaction monitoring assay, assay using a set of polyamine-specific stable isotope reagents or protein chip.
9. The use according to claim 5, wherein the reagent for measuring the expression level of the biomarker gene at the protein level comprises an antibody, an antibody fragment, an aptamer, a high affinity polymer or a peptidomimetic which specifically recognizes the full length of the protein of the biomarker or a fragment thereof.
10. The use of claim 1, wherein the computational model of (2) comprises a computational analysis module of SLC12A3 and/or SLC12A9, the computational analysis module processing the detected data of SLC12A3 and/or SLC12A9 and comparing with a set prediction value.
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