KR100861981B1 - Kidney-specific gene HMM03 associated with development of kidney cyst - Google Patents

Abstract

The present invention is involved in the development of kidney cysts (cyst), HMM03 protein specifically expressed in renal tissue, the gene encoding the protein, a recombinant vector comprising the gene, transformants transformed with the recombinant vector The present invention relates to markers and microarrays for diagnosing the development of kidney cysts comprising the genes, antibodies to the protein and microarrays comprising the same, and methods of treating mammalian diseases using the genes.

Kidney Cyst, HMM03 Protein, Marker, Microarray

Description

Kidney-specific gene HMM03 associated with development of kidney cyst}

1 is a schematic of a system for analyzing genes for the development of cysts.

Figure 2 shows the RT-PCR results of the HMM03 gene.

3 shows protein interaction information of the HMM03 gene prepared using STRING.

The present invention is involved in the development of mammalian diseases, particularly kidney cysts, and HMM03 protein specifically expressed in kidney tissue, the gene encoding the protein, a recombinant vector containing the gene, transformed with the recombinant vector Transformants, markers and microarrays for diagnosing the development of renal cysts comprising the gene, antibodies to the protein and microarrays comprising the same, and methods for treating mammalian kidney cysts using the genes will be.

Cyst (cyst) is a pocket-shaped space due to abnormal proliferation of epithelial cells, the body fluid fills up, the cyst spread throughout the organs and loses function, causing a number of serious complications such as end stage renal failure and cardiovascular disease. According to Boletta, A. et al., Mol Cell 6 (5): 1267-73 (2000), the formation of cysts in the kidneys is caused by PKD (Polycystic Kidney Disease). A very common hereditary disease that occurs equally regardless of gender, geographic location, or socioeconomic environment. Cysts form in the proximal and collecting ducts in the nephron of the kidneys, causing abnormal kidney function and end-stage renal failure. It leads.

PKD is a hereditary disorder in which cysts develop on both sides of the kidney, also called polycystic kidney disease or polycystic kidney disease. There are two types, pediatric and adult, which are due to autosomal recessive inheritance, and adult type due to autosomal dominant inheritance. Adult type occurs at a high frequency of about 1 person per 200 to 1,000 people. In addition, about 50% of patients progress to chronic renal failure, with an average death age of about 50 years. The pediatric type is known to have 86% of the patients diagnosed in newborns over 3 months of age, about 50% survive until 10 years old, and 80% of patients diagnosed after 1 year old have a survival rate of 15%. have. There is no specific treatment for both types, and only symptomatic treatment for various symptoms or complications is possible.

PKD's cystogenesis mechanism has been actively studied for the discovery of therapeutic agents. However, the mechanism of cyst formation in PKD has not been fully understood. To date, the cyst formation mechanism has been revealed by the same PKD gene. It is difficult to explain both the two diseases that occur: Autosomal Dominant Polycystic Kidney Disease (ADPKD) and Autosomal Recessive Polycystic Kidney Disease (ARPKD).

Korean Laid-Open Patent Publication No. 2005-0061501 describes a composition, a pharmaceutical preparation, and a method for inhibiting CFTR, which are useful for the study and treatment of cyst fibrosis transmembrane conductance regulator protein (CFTR) -mediated diseases and conditions. There is no description about.

Accordingly, the present inventors have studied the mechanism of formation of cysts, and revealed that HMM03 gene, which is involved in the development of cysts, is specifically expressed in renal tissue, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide HMM03 protein that is specifically expressed in renal tissue.

Another object of the present invention is to provide a gene encoding the protein.

Another object of the present invention is to provide a recombinant vector comprising the gene.

Another object of the present invention is to provide a transformant transformed with the vector.

Another object of the present invention is to provide a marker and a microarray comprising the gene.

Another object of the present invention is to provide an antibody against the protein and a microarray comprising the antibody.

Another object of the present invention to provide a method for treating a mammalian kidney cyst using the gene.

In order to achieve the above object, the present invention provides a HMM03 protein involved in the development of kidney cysts (cysts), consisting of the amino acid sequence represented by SEQ ID NO: 2, which is specifically expressed in kidney tissue.

Even if a candidate group of genes involved in the cyst formation mechanism is extracted by a method such as a DNA chip, it is actually a gene involved in the cyst formation mechanism, a housekeeping gene, or an experimental error until the candidate group is knocked out and examined. It is indistinguishable. However, if the candidate group is identified as a tissue-specific gene by performing an Audic-Claverie Test based on the EST (Expressed Sequence Tag) or using an experiment such as RT-PCR, the candidate may be judged as a genetic influence factor. Reliability is increased. The inventors have provided higher reliability for candidate groups of genes involved in cyst development by examining candidate groups of genes involved in cyst development and examining whether they contain tissue specific information.

Cysts are diseases in which empty spaces are formed in tissues due to abnormal proliferation of cells, and fluids are filled and tissues lose their function. In order to determine the cause of cysts in the kidneys, the present inventors investigated the expression status of genes by DNA chip experiments using 6-, 12-, and 18-month-old mice in which cysts developed, and performed functions using UniProt database. In addition, information on protein-protein interactions was extracted from the STRING database. The inventors selected 110 genes as candidate candidates for renal specific genes using Audic-Claverie Test for EST information of genes that were significantly expressed at each expression time point. Among these genes, the HMM03 gene was actually verified by RT-PCR experiment. 1 is a schematic of a system for analyzing genes for the development of cysts. The purpose of this system is to identify whether the hourly significant genes from DNA chip experiments at 6, 12, and 18 months of cyst development are actually tissue-specific genes and to analyze information about protein-protein interactions.

Accordingly, the present invention provides an HMM03 protein involved in the development of renal cysts and specifically expressed in renal tissue. The range of HMM03 protein according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The HMM03 protein according to the invention is preferably derived from, but not limited to, a mouse.

The HMM03 protein according to the present invention can be used as a target of therapeutic drugs for renal cysts. If the drug is designed as a treatment for cyst symptoms, the HMM03 protein may reduce the drug target, allowing for faster treatment at the onset of cysts.

The present invention also provides a gene encoding the HMM03 protein. Genes of the invention include both genomic DNA and cDNA encoding the HMM03 protein. Preferably, the gene of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1.

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 can do. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the gene according to the present invention. The recombinant vector is preferably a recombinant animal expression vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

In the present invention, any promoter known in the art may be used as a promoter of the expression vector. For example, the immediate early promoter of cytomegalovirus, the promoter of SV40 (SV 40 late promoter and SV 40 early promoter), the tk promoter of herpes simplex virus (HSV), and the Adeno 2 major late promoter PAdml ), Adenovirus 2 early promoter (PAdE2), p19 promoter of human parvo virus-associated virus (AAV) and metallothionein promoter of mouse, MT promoter and MMTV LTR promoter, etc. may be used, but are not limited thereto. It is not.

In the present invention, the transcription terminator of the expression vector may include any transcription terminator known in the art. For example, human growth hormone polyadenylation signals, bovine growth hormone polyadenylation signals and SV40 viral polyadenylation signals may be included.

The present invention also provides a transformant transformed with the recombinant vector according to the present invention. The transformant is preferably a mammal. Although preferably human, pets such as dogs or cats, livestock such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like may also be included. In the present invention, a method of transforming a mammal with a recombinant vector includes a micro-injection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 ( 1973)), electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transformation (Wong, TK et al., Gene, 10:87 (1980) ), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568- 9572 (1990)), and the like.

In addition, the present invention is HMM03 according to the present invention Provided are markers for diagnosing renal cyst development, including genes. HMM03 of the present invention Since the gene can be used as a marker for diagnosing or diagnosing a genetic tendency of cyst induction, such information can be used to diagnose and perform treatment before the cyst occurs.

The present invention can also provide a kit for diagnosing the development of kidney cysts comprising the sense and antisense primers of the gene. By performing PCR amplification using the sense and antisense primers of the gene, the onset of cysts can be diagnosed through the generation of desired products. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.

Kits of the invention may also comprise probes complementary to the gene. Hybridization may be performed using a probe complementary to the gene, and the cyst may be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The invention also provides a microarray for diagnosing the development of renal cyst, comprising the HMM03 gene according to the invention. The microarray of the present invention can be easily prepared by a manufacturing method commonly used in the art using the marker of the present invention.

In the microarray of the present invention, "microarray" refers to a microarray in which a group of polynucleotides is immobilized to a high density on a substrate, and each of the polynucleotide groups is immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The term “substrate” refers to any substrate to which an oligonucleotide probe can be coupled under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose) or microspheres (beads) or chips. Prior to application or immobilization on the membrane, nucleic acid probes can be modified to promote immobilization or to improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

The invention also provides an antibody against the HMM03 protein according to the invention. The antibody of the present invention can be produced by a conventional method from the obtained protein by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the HMM03 gene. This includes partial peptides that can be made from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

The invention also provides a microarray for diagnosing the development of kidney cysts, comprising an antibody of the HMM03 protein according to the invention. The microarray of the present invention may refer to the aforementioned microarray except that the antibody of the present invention is immobilized in place of the polynucleotide in the microarray.

The invention also provides a method of treating a mammalian kidney cyst by inhibiting the expression of the HMM03 gene according to the invention in a mammal. Preferably, the mammal is a human, but may also include non-human animals such as pets such as dogs and cats, livestock such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like.

As a method of inhibiting the expression of the HMM03 gene in a mammal, various methods known in the art may be used. Said "inhibition of gene expression" includes inhibition of gene transcription and translation into protein. Also included are not only complete stops of gene expression, but reduced expression.

Antisense molecules are most commonly used to inhibit the expression of certain endogenous genes in mammals. Antisense molecules inhibit the expression of target genes by inhibiting transcriptional initiation by triple chain formation, transcriptional inhibition by hybridization at sites where local open loop structure is formed by RNA polymerase, and in RNA where synthesis is in progress. Inhibition of transcription by hybrid formation, inhibition of splicing by hybridization at the junction of introns and exons, inhibition of splicing by hybridization at the site of splicosomal formation, transition from nucleus to cytoplasm by hybridization with mRNA Inhibition of translation initiation by hybridization at the site of translation initiation factor binding. They inhibit the expression of target genes by inhibiting transcription, splicing or translation processes.

The antisense molecule used in the present invention may inhibit the expression of the target gene by any of the above actions. Representative antisense molecules include triplets, ribozymes, RNAi, or antisense nucleic acids. The triple agent allows the initiation of transcription to be suppressed by winding around double helix DNA to form a three-stranded helix (Maher et al., Antisense Res. And Dev., 1 (3): 227, 1991; Helene, C., Anticancer Drug Design, 6 (6): 569, 1991). Ribozymes are RNA molecules that possess the ability to specifically cleave single stranded RNA. Ribozymes can be engineered to recognize and cleave site specific nucleotide sequences in RNA molecules (Cech, J. Amer. Med. Assn., 260: 3030, 1998). Thus, the main advantage of this method is that only mRNAs with specific sequences are inactivated because they are sequence specific. RNAi is a method of inhibiting gene expression at the transcriptional level or post-transcriptional level by using small-molecule RNA in the form of a hairpin that is specifically sequenced (Mette et al., EMBO J., 19: 5194-5201, 2000). . Antisense nucleic acid refers to a DNA or RNA molecule that is at least partially complementary to a particular mRNA molecule (Weintraub, Scientific American, 262: 40, 1990). In cells, antisense nucleic acids hybridize with the corresponding mRNAs to form double stranded molecules. This inhibits the translation of mRNA (Marcus-Sakura, Anal. Biochem., 172: 289, 1988).

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example

Example  One: DNA  Chip experiment

According to Zoja, C. Cytogenet Genome Res 105 (2-4): 479-8 (2004), in 18-month-old mice, cysts develop in the kidneys of 6-month-old mice, and their cysts increase over time. The cysts were very large and several water clots were observed. DNA chip experiments were performed using CodeLink Bioarray: UniSet Mouse 20K I Bioarray (GE Healthcare; hppt: //www.gehealthcare.com), and Quintet (Choe, JK LNCS 3642: 392). (2005)) was used to extract a list of significant genes in the 6, 12, and 18 months following cyst changes.

Table 1 below shows the number of significant genes over time as a result of DNA chip experiments.

time Significant gene (TG / WT) Kidney Specific Related Significant Genes 6 months 1521 (845/676) 67 12 months 1220 (639/581) 51 18 months 1533 (720/813) 45

In Table 1, TG is a mouse with a cyst, and WT represents a mouse without a cyst. The HMM03 gene is not specifically expressed in TG, and only 12 months have been shown to down-regulated TG / WT ratio to 0.487.

Example  2: TISA  Set Up Renal Specific Gene Candidates Using Database

Noh, S.J. DNA Res 13 (5): 229-43 (2006) published a Tissue-specific Alternative Splicing (TISA) database that contains information on genes, transcript structure, alternative splicing events and tissue specificity. In order to check whether there are significant genes extracted from the TISA experiments in the TISA, check whether the Refseq information of the TISA genes is the same as the Refseq corresponding to the probe on the DNA chip, and connect the TISA database with the DNA chip. If any of the kidney specific genes predicted in the gene was significantly expressed on the DNA chip, it was extracted as a candidate group of the kidney specific genes.

Example  3: renal specific gene candidate group RT - PCR  Experiment

Mice were male and female of 9-10 weeks old B6 strain. There are 20 types of tissues that can be extracted: virgin breast, breast during lactation, breast after breast feeding, colon, ear, pancreas, kidney, liver, lung, cerebral, cerebellum, ovary, uterus, testes, thymus, stomach, spleen, Skin, heart and duodenum. Taking into account the characteristics of each tissue, the lipid, breast, cerebral, and cerebellum are crushed about 100 mg with TissueLyser (Qiagen, Valencia, CA, USA), and using the RNeasy lipid tissue mini kit (Qiagen, Valencia, CA, USA). RNA was extracted. The remaining tissue was ground to about 30 mg with the same homogenizer and RNA was obtained with the RNeasy mini kit (Qiagen, Valencia, CA, USA). From the extracted RNA 1ug, RT-PCR was performed using Standard Two-step RT-PCR- & GO ™ (Q-biogene, Cat # RT2ST100) to obtain about 100ul of cDNA. Primers for these genes (forward primers: 5'-CAAGGTCCTGGTGATCACTGTC-3 '(SEQ ID NO: 3) and reverse primers: 5'-CTTAGAGATGCCTGGCACAGC-3' (SEQ ID NO: 4)) to identify kidney specific genes (PCR product size 536 bp) and PCR was performed using cDNA for the 20 tissues obtained above. PCR conditions were last extended for 30 min of predenatured 95 ° C. 5 min, followed by 30 cycles of 95 ° C. 1 min, annealing 58 ° C. 1 min, extension 72 ° C. 1 min, and 72 ° C. 5 min. 1% agarose gel electrophoresis was performed to confirm the PCR product.

Figure 2 shows the RT-PCR results of the HMM03 gene. Description of each lane of Figure 2 is as follows:

Br1: virgin breast Ce2: cerebral Th: thymus Br2: breast during lactation Pa: Pancreas Ov: Ovary Br3: breast after lactation Ki: height Li: liver Co: Colon Sk: skin Ut: Uterus Ea: Ear He: heart Sp: Spleen Du: duodenum Te: Testicle St: above Ce1: Cerebellum Lu: lung

As can be seen in Figure 2, it was found that the HMM03 gene is rarely expressed in other tissues, but specifically expressed in the kidney.

Example  4: UniProt Reference of Kidney Specific Gene Candidates

UniProt (http://www.ebi.uniprot.org) was developed in 2002 as an EBI / SIB Swiss-Prot, TrEMBL database PIR-Protein Sequence Database (PIR-PSD). It is a way to combine the contents into a UniProt consortium and provide them with mutual cooperation. The consortium provides comprehensive, accurate information about protein sequences, and maintains an accessible interface despite providing many references.

To identify which protein candidates are specific to kidneys and which functions are linked to UniProt, the sequence data of kidney-specific gene candidates was applied to BLAST alignment provided by UniProt's website and identified for accuracy. Only data with values greater than 95% were adopted.

Example  5: STRING Analysis of Protein Interaction in Renal Specific Gene Candidates

STRING (Search Tool for the Retrieval of Interacting Proteins; http://string.embl.de) is a database that provides comprehensive information on protein interactions.In 2000, Snel B 2000 Nucleic Acids Res 28 (18) 3442 Version 7 was released in 2007 as a continual upgrade since its release by -4.

In order to confirm the protein interaction information of the kidney-specific gene candidate group, the mouse sequence in STRING was determined by using the candidate data and BLAST alignment (identity> = 84% and coverage> = 60%), Corresponding protein interaction information was extracted.

3 shows protein interaction information of the HMM03 gene prepared using STRING. In FIG. 3, red dots represent proteins aligned with 100% identity and 100% coverage on HMM03 and BLAST alignment. Proteins that interact with the HMM03 protein are proteins that act as transporters of amino acids, which is an important function of the kidney.

To determine the cause of the cyst's development, it is important to identify information about the protein-protein interactions, pathways, or regulatory networks involved in the cyst's development. However, analyzing and generating individual genes to reveal this information takes a very long time to identify the genes actually involved in the development of cysts.

The present inventors provide the fact that the HMM03 gene, which is involved in the development of cysts, is specifically expressed in renal tissue, allowing researchers to easily access information such as protein-protein networks or pathways for the development of cysts. The fact that it is a kidney-specific gene can reduce false positives in studies of cyst symptoms. In addition, the HMM03 gene can be used as a marker for the diagnosis of cysts, making it easier to diagnose. When designing a drug as a therapeutic agent for cyst symptoms, the drug target for HMM03 can be reduced. Faster treatment is possible early in the onset of cysts.

<110> Sookmyung Womens University Industry Academic Cooperation Foundation <120> Kidney-specific gene HMM03 associated with development of kidney          cyst <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 696 <212> DNA <213> Mouse C57 BL / 6 <400> 1 ctccagccaa ctgtggaaaa ccaccttcag ggacagagta caggatgtat aaaagcccag 60 gaggactctt tttggactaa tttctttctc ctgttcactg gttgccttaa ccctactaaa 120 gcatgatgct tttcaaggtc ctggtgatca ctgtcttctg tggtctgact gtggctttcc 180 ccctgtcaga attagtttca atcaataaag aactacagaa ttcaatcatt gacctactaa 240 actcagtctt tgaccaactg ggatcataca gagggacaaa agctcctcta gaggattata 300 cagatgatga tttaagcact gactctgagc agatcatgga cttcacgcca gccgcaaaca 360 aacagaattc tgagttctct actgatgttg agacagtctc ctccggcttt ctggaagaat 420 tcactgagaa cacagacatc acagtgaaaa ttccattagc tgggaatcca gtctccccta 480 cttcctgaga agactccaac tttgaaataa acctgcgact gccattcagg atattgtctt 540 tgtagaaaat gatataaatc taagatgctc ccagcaaaga agaaagaacg aggaagtgat 600 cctggatgag aaatgctgtg ccaggcatct ctaagctctg cccttactct gtgtgggttt 660 aagaaataaa cgttcatttg aattgcatta tgagag 696 <210> 2 <211> 121 <212> PRT <213> Mouse C57 BL / 6 <400> 2 Met Met Leu Phe Lys Val Leu Val Ile Thr Val Phe Cys Gly Leu Thr   1 5 10 15 Val Ala Phe Pro Leu Ser Glu Leu Val Ser Ile Asn Lys Glu Leu Gln              20 25 30 Asn Ser Ile Ile Asp Leu Leu Asn Ser Val Phe Asp Gln Leu Gly Ser          35 40 45 Tyr Arg Gly Thr Lys Ala Pro Leu Glu Asp Tyr Thr Asp Asp Asp Leu      50 55 60 Ser Thr Asp Ser Glu Gln Ile Met Asp Phe Thr Pro Ala Ala Asn Lys  65 70 75 80 Gln Asn Ser Glu Phe Ser Thr Asp Val Glu Thr Val Ser Ser Gly Phe                  85 90 95 Leu Glu Glu Phe Thr Glu Asn Thr Asp Ile Thr Val Lys Ile Pro Leu             100 105 110 Ala Gly Asn Pro Val Ser Pro Thr Ser         115 120 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caaggtcctg gtgatcactg tc 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cttagagatg cctggcacag c 21  

Claims (13)

delete delete delete delete delete delete delete delete Microarray for diagnosing the development of kidney cyst, comprising the nucleotide sequence represented by SEQ ID NO: 1. delete A microarray for diagnosing the development of renal cyst, comprising an antibody against HMM03 protein involved in the development of renal cyst (cyst), consisting of the amino acid sequence represented by SEQ ID NO: 2, and which is superior in renal tissue only. delete delete

Images