Application of OLFM3 as a drug target for neuroblastoma

Abstract

The present invention belongs to the field of gene diagnosis and treatment, and specifically relates to the application of OLFM3 as a drug target for neuroblastoma. The present invention provides a reagent for inhibiting the expression of OLFM3 gene in the preparation of a therapeutic agent. MYCN The invention discloses that OLFM3 is involved in the treatment of neuroblastoma. MYCN Amplified neuroblastoma cells specifically expressed MYCN Very low expression in non-amplified neuroblastoma cells; MYCN In amplified neuroblastoma cells, knockdown of OLFM3 attenuated the proliferation and clone formation ability of neuroblastoma cells. OLFM3 promotes MYCN Amplification of malignant proliferation of neuroblastoma cells is MYCN Amplification of a new driver gene for malignant proliferation of neuroblastoma cells suggests that it is MYCN Amplification of novel therapeutic targets for neuroblastoma.

Description

Technical Field

[0001] The invention belongs to the field of gene diagnosis and treatment, and specifically relates to the application of OLFM3 as a drug target for neuroblastoma. Background Art

[0002] Neuroblastoma (NB) originates from the abnormal neural crest of the sympathetic nervous system and is the most common extracranial solid tumor in children, with an incidence of approximately 10.2 cases / 1 million / children under 15 years old, accounting for about 15% of all childhood cancer deaths. Although the clinical precision treatment methods for NB have been increasingly improved in recent years, and the application of multidisciplinary joint diagnosis and treatment models such as high-dose chemotherapy, surgical resection and targeted therapy has gradually matured, about 50% of high-risk NB children still experience progression or recurrence during treatment, and the 5-year survival rate still hovers around 30-40%, with extremely poor long-term prognosis. It can be seen that the existing treatment methods and systems are difficult to meet the needs of precision treatment, and the clinical diagnosis and treatment of NB children faces huge challenges. Therefore, establishing more accurate targeted diagnosis and treatment strategies to improve the outcome of NB with poor prognosis is a huge challenge and research hotspot in the current clinical diagnosis and treatment of NB.

[0003] However, the current international research on the precise molecular characteristics of NB is limited to a few important cancer genes, such as MYCN , ALK But MYCN There is currently a lack of targeted drugs. ALK The mutation rate in NB is relatively small, and it is difficult to meet the needs of accurate, efficient and widely used treatment for NB. The only drug targeting neuroblastoma (NB) on the market is anti-GD2 antibody, such as Dinutuximab, which has been approved by the US FDA and included in the first-line treatment of NB, but has side effects such as severe pain, fever, and low platelet count. Therefore, discovering molecules that are specifically expressed in NB cells and developing targeted therapeutic drugs for these molecules is a new idea for treating NB.

[0004] Studies have shown that NB has multiple potential therapeutic targets, such as the histone modification enzyme EZH2, which promotes NB cell proliferation, and EZH2 inhibitors combined with other drugs can inhibit NB growth in vitro; ALK Some NB tumors have mutations, and ALK inhibitors such as crizotinib and lorlatinib can target ALKMutants inhibit NB development; Nutlins, SAR405838 (MI-77301), RG7388 (RO5503781 or idasanutlin), inhibitors targeting MDM2, inhibit p53-MDM2 interaction in NB and activate p53-induced apoptotic signaling; AuroraA kinase (AURKA) inhibitor MLN8237 (alisertib) promotes MYCN Degradation can inhibit NB growth in xenograft mouse models. However, the above target molecules are located in the cell membrane and cannot be treated by antibody therapy. In recent years, studies have found that the transmembrane protein GPC2 is a new potential NB cell surface target molecule, but targeted GPC2 therapy still lacks clinical evidence related to safety and effectiveness.

[0005] Therefore, the molecular mechanism of the occurrence and development of NB needs further in-depth study. Summary of the invention

[0006] The present invention provides a transmembrane protein OLFM3 gene and its encoding product in MYCN Amplification of neuroblastoma can be used as MYCN Amplification of therapeutic and drug screening targets for neuroblastoma MYCN Amplification provides new ideas for neuroblastoma treatment and drug screening.

[0007] Olfactory domain family protein 3 (Olfactomedin-3, OLFM3) belongs to the Olfactomedin domain family protein. The present invention found that OLFM3 (GENE ID: 118427) MYCN Amplify the specific expression in NB. In-depth functional studies have found that knocking down the OLFM3 gene significantly inhibits MYCN The above results show that OLFM3 promotes the proliferation of NB cells. MYCN Amplification of NB proliferation and tumorigenesis may serve as a new therapeutic target for NB.

[0008] The present invention provides OLFM3 gene as MYCN Application of amplified biomarkers of neuroblastoma in the preparation of diagnostic preparations for neuroblastoma, wherein the OLFM3 gene is expressed in MYCN The expression level was significantly increased in amplified neuroblastoma.

[0009] The use of the diagnostic preparation includes: detecting the expression of OLFM3 gene in the test cells of the subject; comparing the detected value with the reference value, if the OLFM3 gene is significantly higher than the reference value, it indicates that the subject suffers from neuroblastoma. The reference value level is the expression level of OLFM3 gene in normal cells, i.e., normal human nerve tissue cells, the normal cells are of the same strain as the test cells and are not cancerous. The test cells are known or suspected to contain tumor cells.

[0010] The present invention provides a reagent for inhibiting OLFM3 gene expression for preparing a therapeutic MYCN Expanded application of drugs in neuroblastoma.

[0011] According to a specific embodiment of the present invention, the use of an agent for inhibiting the expression of OLFM3 gene comprises shRNA or siRNA specifically targeting OLFM3 gene.

[0012] Preferably, the nucleotide sequence of the siRNA is as follows:

[0013] SEQ ID NO.1: 5'-GTATGCAACTAACCAGAAT-3',

[0014] SEQ ID NO.2: 5'-GTCACCAACTCCCACTTAA-3', or,

[0015] SEQ ID NO. 3: 5'-GCTGAATCAAGGACATACA-3'.

[0016] Preparation of siRNA targeting OLFM3 gene MYCN The application of the siRNA in amplifying neuroblastoma therapeutic drugs, the nucleotide sequence of the siRNA is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0017] According to a specific embodiment of the present invention, a method for MYCN A product for clinical diagnosis of amplified neuroblastoma, the product comprising a reagent for detecting the expression level of a biomarker, wherein the biomarker is the OLFM3 gene.

[0018] The present invention provides diagnostic MYCN A kit for amplifying neuroblastoma, the kit comprising the detection reagent for the expression level of the OLFM3 gene.

[0019] Wherein, the kit also includes a negative control, a positive control, a primer, a probe or an antibody.

[0020] As used herein, the term "primer" refers to a single-stranded polynucleotide that is capable of hybridizing to a nucleic acid and allowing polymerization of a complementary nucleic acid (generally by providing a free 3'-OH group).

[0021] Preferably, the primer sequences are as follows:

[0022] SEQ ID NO.4: 5'-CAGGCAACTTCGCCAACTACT-3';

[0023] SEQ ID NO. 5: 5'-CCAGCACGGGGATCAAAGG-3'.

[0024] The present invention also provides an inhibitor of OLFM3 in the preparation MYCN Expanded application of drugs in the treatment of neuroblastoma.

[0025] According to the use of the inhibitor of OLFM3 phosphatase in a specific embodiment of the present invention, the reagent for inhibiting the expression of OLFM3 gene includes shRNA or siRNA specifically targeting OLFM3 gene.

[0026] The present invention also provides a reagent for detecting the OLFM3 gene in the preparation of the detection MYCN Application of reagents to amplify neuroblastoma.

[0027] Treatment according to a specific embodiment of the present invention MYCN A drug for amplifying neuroblastoma containing any one or more of the following:

[0028] (1) containing the OLFM3 gene or a biological material containing the OLFM3 gene, wherein the biological material is an expression cassette, an expression vector, a plasmid, a host bacterium, or a host cell;

[0029] (2) containing an agent that downregulates the expression of the OLFM3 gene in the body;

[0030] (3) An agent that inhibits or blocks the expression of the OLFM3 gene in the body.

[0031] Treatment according to a specific embodiment of the present invention MYCN The drug for amplifying neuroblastoma is characterized by comprising shRNA or siRNA specifically targeting the OLFM3 gene.

[0032] In the present invention, this is achieved by inhibiting or reducing the expression of the gene encoding the active protein to be inhibited, specifically, by gene knockout or gene silencing.

[0033] The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0034] Gene silencing refers to the phenomenon of making a gene non-expressed or low-expressed without damaging the original DNA. Gene silencing can occur at two levels: one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization, and position effects, and the other is post-transcriptional gene silencing, which is to inactivate the gene by specifically inhibiting the target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi), and microRNA (miRNA)-mediated translation inhibition.

[0035] Beneficial effects of the present invention:

[0036] The present invention found that OLFM3 MYCN High expression in MYCN-amplified NB cells, but very low expression in MYCN-non-amplified NB cells; MYCN In the expanded NB cells, knockdown of OLFM3 weakened the proliferation and clone-forming ability of NB cells.

[0037] OLFM3 promotes MYCN Amplify the malignant proliferation of NB cells, MYCN Amplification of new driver genes for malignant proliferation of NB cells, suggesting that it is MYCN Amplified novel therapeutic targets for NB. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 Shows the expression of OLFM3 in different NB cell lines;

[0040] Figure 2 Knockdown of OLFM3 has been shown to MYCN The effect of amplification on the proliferation of the NB cell line SK-N-BE(2);

[0041] Figure 3 Knockdown of OLFM3 has been shown to MYCN The effect of not amplifying the proliferation capacity of the NB cell line SK-N-AS;

[0042] Figure 4 Knockdown of OLFM3 has been shown to MYCN The effect of not amplifying the proliferation capacity of the NB cell line SH-SY5Y;

[0043] Figure 5 Knockdown of OLFM3 has been shown to MYCN The effect of amplification on the proliferation rate of the NB cell line SK-N-BE(2);

[0044] Figure 6 Knockdown of OLFM3 has been shown to MYCN The effect of expanded NB cells on the number of clones formed. DETAILED DESCRIPTION

[0045] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0046] MYCN Amplified neuroblastoma is a type of neuroblastoma in which the number of copies of the MYCN gene is 4 or more.

[0047] MYCN Non-amplified NB cell lines are neuroblastoma cell lines with less than 4 copies of the MYCN gene.

[0048] In the present invention, MYCN Non-amplified NB cell lines include SK-N-SH, SK-N-AS, SH-SY5Y,

[0049] MYCN Expanded NB cell lines include SK-N-BE(2), IMR32, and BE(2)-C.

[0050] SK-N-SH: transferred from the bone marrow MYCN Tumor cell lines isolated from non-amplifying human neuroblastoma express higher levels of dopamine-β-hydroxylase.

[0051] SK-N-AS: transferred from the bone marrow MYCN A tumor cell line isolated and established from a non-proliferating human neuroblastoma with moderate levels of dopamine β-hydroxylase activity.

[0052] SH-SY5Y: transferred from the bone marrow MYCN A tumor cell line isolated and established from a non-proliferating human neuroblastoma with moderate levels of dopamine β-hydroxylase activity.

[0053] SK-N-BE(2): Transferred from bone marrow MYCN An established tumor cell line isolated from a proliferated human neuroblastoma that displays moderate levels of dopamine-β-hydroxylase activity.

[0054] IMR32: From the primary lesion in the lower abdomen MYCN An established tumor cell line isolated from a proliferated human neuroblastoma that displays moderate levels of dopamine-β-hydroxylase activity.

[0055] BE(2)-C: metastasis from bone marrow MYCN An established tumor cell line isolated from a proliferated human neuroblastoma that displays moderate levels of dopamine-β-hydroxylase activity.

[0056] The above cell lines were purchased.

[0057] Example 1

[0058] Choose 3 MYCN Non-expandable NB cell lines (SK-N-SH, SK-N-AS, SH-SY5Y) and three MYCN The RNA levels of OLFM3 were detected in the expanded NB cell lines (SK-N-BE(2), IMR32, and BE(2)-C).

[0059] RNA extraction, reverse transcription and real-time quantitative PCR (qPCR): Total cellular RNA was extracted by Trizol method, and the concentration was determined by Nanodrop 8000 spectrophotometer. Reverse transcription kit produced by TaKaRa was used to obtain cDNA with 1 μg RNA as template.

[0060] The target gene was amplified using the SYBR Green PCR Master Mix kit and the Applied Biosystems ABI Viia7 detection system under standard cycling conditions (primers are shown in Table 1), with GAPDH as the internal reference and 2 -△△Ct To calculate the changes in mRNA expression levels.

[0061]

[0062] The results are as follows Figure 1 As shown, MYCN The expression level of OLFM3 in the amplified cell lines IMR32, SK-N-BE(2), and BE(2)-C was significantly higher than MYCN The non-amplified cell lines SK-N-SH, SH-SY5Y, and SK-N-AS showed that OLFM3 MYCN The amplified NB cell lines expressed high levels of leukemia.

[0063] Example 2

[0064] Three siRNAs with different sequences (siOLFM3-1#, siOLFM3-2#, siOLFM3-3#) were used to knock down OLFM3 in NB cell lines, inhibit OLFM3 expression, and investigate the effects on the proliferation ability of the cell lines.

[0065] The cells were cultured at 1.2 × 10 5 The cells were inoculated into 6-well plates at a density of 100 cells / ml, with 2 ml of cell suspension per well. After the cells adhered to the wall and the confluence reached 20%-30%, siRNA transfection was performed according to the Lipofectamine RNAi MAX kit, with a final siRNA concentration of 60 nmol / ml. A total of 4 groups were set up, with the control group targeting the luciferase reporter gene siControl ( siCtrl ) and experimental group siOLFM3-1# , siOLFM3-2# , siOLFM3-3# The target sequence is as follows:

[0066] siOLFM3-1 #:5'-GTATGCAACTAACCAGAAT-3',

[0067] siOLFM3-2 #:5'-GTCACCAACTCCCACTTAA-3',

[0068] siOLFM3-3 #:5'-GCTGAATCAAGGACATACA-3'.

[0069] use siOLFM3-1#, siOLFM3-2#, siOLFM3-3 #SK-N-BE(2) cells were transfected with three siRNAs respectively. 48 hours after transfection, RNA was extracted from the cells, reverse transcribed, and real-time quantitative PCR (qPCR) was performed.

[0070] The results are as follows Figure 2 As shown in the figure, compared with the control group siCtrl, the three siRNAs targeting OLFM3 can reduce the mRNA level of OLFM3 in cells and inhibit the expression of OLFM3, indicating that knocking down OLFM3 can significantly inhibit MYCN Amplify the proliferation capacity of NB cell line SK-N-BE(2).

[0071] The results are as follows Figure 3 , 4 As shown, knockdown of OLFM3 significantly MYCN The proliferation capacity of NB cell lines SK-N-AS and SH-SY5Y was slightly affected by non-amplification.

[0072] Therefore, reducing the expression of OLFM3 has an important effect on MYCN The viability of amplified NB cells was more significantly inhibited, and OLFM3 specifically promotedMYCN Amplify the viability of high-risk NB tumor cells.

[0073] Example 3

[0074] Three siRNAs with different sequences were used to knock down OLFM3 in NB cell lines to inhibit OLFM3 expression and investigate the effect on the proliferation rate of the cell lines.

[0075] Real-time xCELLigence analysis system (RTCA): 24 h after cells were transfected with siRNA, the cells were cultured at a density of 2×10 4 Cells were inoculated into RTCA special detection wells with 100 µl of cell suspension per well. The number of cells was detected in real time and the proliferation of cells was recorded within 120 h.

[0076] The target sequence is the same as in Example 2.

[0077] The real-time label-free cell analysis system showed that siCtrl In comparison, the results Figure 5 As shown, the knockdown group siOLFM3-1# , siOLFM3-2# and siOLFM3-3# The cell growth rate was significantly slowed down, and knockdown of OLFM3 significantly inhibited MYCN The proliferation rate of the NB cell line SK-N-BE(2) was amplified.

[0078] like Figure 6 Compared with the control group siCtrl Compared with the knockdown group siOLFM3-1# , siOLFM3-2# and siOLFM3-3# The number of cell clones was significantly reduced. Thus, knockdown of OLFM3 significantly weakened the clone-forming ability of the MYCN-amplified NB cell line SK-N-BE(2).

[0079] It can be seen that in MYCN After knockdown of OLFM3 in expanded NB cells, cell proliferation and clone formation abilities were reduced.

[0080] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of an agent for inhibiting OLFM3 gene expression in the preparation of a drug for treating MYCN amplified neuroblastoma, characterized in that: The reagent for inhibiting the expression of OLFM3 gene is siRNA specifically targeting OLFM3 gene, and the nucleotide sequence of siRNA is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

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