CN107893119B - Application of ZCCHC12 in osteosarcoma - Google Patents

Abstract

The invention discloses application of ZCCHC12 in osteosarcoma. The invention discloses an application of ZCCHC12 in diagnosis and treatment of osteosarcoma; the invention also discloses a product for diagnosing osteosarcoma, which comprises a reagent for detecting the ZCCHC12 level; the invention also discloses a pharmaceutical composition comprising an inhibitor of ZCCHC 12.

Description

Application of ZCCHC12 in osteosarcoma

Technical Field

The invention belongs to the field of biological medicine, and relates to application of ZCCHC12 in osteosarcoma.

Background

Osteosarcoma (OS) is one of the most common primary malignant bone tumors, occurring in adolescents (Durfee RA, Mohammed M, Luu HH et al, Rheumatotal Heat.2016, 3(2): 221-. Amputation is the standard method for treating osteosarcoma before the 70's of the 20 th century, but the amputation has great influence on the limb functions of patients and brings great psychological damage to the patients. With the rapid development of medical technology, surgical operation, chemotherapy drugs, radiotherapy and chemotherapy adjuvant therapy and other measures are advanced, limb protection therapy gradually becomes the main treatment method of osteosarcoma, and especially the new adjuvant chemotherapy combined limb protection operation has achieved great success in clinical treatment of osteosarcoma, the pain of amputation of patients is avoided, and the survival rate is improved to more than 80% within 5 years [5 ]. After that, molecular targeted therapy, immunotherapy and gene therapy are becoming research hotspots, and new chapters are written for osteosarcoma treatment.

Most osteosarcomas are already diagnosed as high-grade malignant tumors and are highly susceptible to early metastasis. The most common distant metastasis site is the lung, which accounts for about 90% (Osasan S, Zhang M, Shen F et al. anticancer Res. 201636 (9): 4391-4398). Therefore, how to inhibit the metastasis of osteosarcoma is an urgent problem to be solved by researching effective treatment methods and improving the survival rate of patients. Besides the common treatment modes of osteosarcoma such as surgical treatment, radiotherapy, chemotherapy and immunotherapy, a new treatment method for more effective osteosarcoma early diagnosis, early inhibition of osteosarcoma metastasis and reduction of the mortality rate of osteosarcoma patients is researched.

Cytokines and their receptors play an important role in cell signal transduction, proliferation and differentiation of cells, and inevitably cause changes in the expression and distribution of cytokines and their receptors during rapid proliferation of osteosarcoma cells (Saini Vl, Hose CD, Monks A, et al. PLoS one.2012; 7(8): e 41401). Therefore, finding the substance which is characterized in osteosarcoma cells or is produced by osteosarcoma cells abnormally, or the substance produced by host reaction to osteosarcoma stimulation, reflecting the generation and development of tumor, and monitoring the tumor marker of tumor reaction to treatment is the current research focus, for example, patents 201510075917.2, 201510075920.4, 201510075918.7 and 201510075919.1 search for the gene related to the generation and development of osteosarcoma.

With the deep biological research on osteosarcoma molecules, targeted therapy of osteosarcoma is also becoming the key point of research more and more, and represents the future development direction, and a new effective biomarker is searched, which has important significance for early diagnosis and targeted therapy of osteosarcoma.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a product for the diagnosis of osteosarcoma.

The invention also aims to provide a pharmaceutical composition for treating osteosarcoma.

In order to achieve the purpose, the invention adopts the following technical scheme:

a product for the diagnosis of osteosarcoma, said product comprising an agent for detecting the level of ZCCHC 12. Wherein ZCCHC12 is up-regulated in osteosarcoma patients, and the product comprises (but is not limited to) a preparation, a chip and a kit.

Further, the agent is selected from:

a probe that specifically recognizes ZCCHC12 gene; or

Primers for specifically amplifying ZCCHC12 gene; or

An antibody or ligand that specifically binds to a protein encoded by ZCCHC 12.

Furthermore, the primer sequence of the specific amplification ZCCHC12 gene is shown in SEQ ID NO. 1-2.

The present invention provides a pharmaceutical composition comprising an inhibitor of ZCCHC 12.

Further, the inhibitor is selected from: nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules.

Further, the inhibitor is a nucleic acid inhibitor siRNA, preferably, the sequence of the siRNA is shown in SEQ ID NO. 9-10.

The invention provides the use of ZCCHC12 as described in any one of the following:

the application of ZCCHC12 in preparing the product for diagnosing osteosarcoma;

the use of ZCCHHC12 in the manufacture of a pharmaceutical composition for the treatment of osteosarcoma;

the use of ZCCHC12 in the manufacture of a pharmaceutical composition for the treatment of osteosarcoma metastases;

use of ZCCHC12 in screening a candidate drug for treating osteosarcoma.

Further, the product described in a comprises reagents for detecting ZCCHC12 using RT-PCR, real-time quantitative PCR, next generation sequencing, in situ hybridization, chip or immunoassay techniques.

Further, the pharmaceutical composition in b or c comprises ZCCHC12 inhibitor, and/or other medicines compatible with the inhibitor and pharmaceutically acceptable carriers and/or auxiliary materials.

Pharmaceutically acceptable carriers and/or adjuvants include, but are not limited to, diluents, excipients, binders, wetting agents, absorption enhancers, surfactants, humectants, adsorbent carriers, lubricants, buffers, stabilizers, bacteriostats, isotonicity agents, chelating agents, pH controlling agents.

The inhibitor is selected from: an interfering molecule that targets ZCCHC12 or its transcript and is capable of inhibiting ZCCHC12 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid; or a binding molecule that specifically binds to a protein encoded by ZCCHC12 (e.g., an antibody or ligand that inhibits the activity of ZCCHC12 protein).

Further, the step of screening for a candidate drug for treating osteosarcoma in d comprises:

treating a system expressing or containing the ZCCHC12 gene or protein encoded thereby with a candidate substance; and

detecting the expression or activity of ZCCHC12 gene or its coded protein in the system.

The invention has the advantages and beneficial effects that:

the invention discovers that the expression level of ZCCHC12 is related to the occurrence and development of osteosarcoma for the first time, and whether a patient has osteosarcoma or is at risk of having osteosarcoma can be judged by detecting the level of ZCCHC 12.

The invention provides a product for diagnosing osteosarcoma, which can diagnose in the early stage of osteosarcoma onset, guide doctors to perform early intervention and improve the survival rate and the life quality of patients.

The invention provides a pharmaceutical composition and a pharmaceutical target for treating osteosarcoma, and the precise treatment can be realized through targeted treatment.

Drawings

FIG. 1 is a graph showing the detection of ZCCHC12 gene expression in osteosarcoma tissue by QPCR;

FIG. 2 is a graph showing the expression of ZCCHC12 protein in osteosarcoma tissues detected by Western blot;

FIG. 3 is a graph of the transfection of ZCCHC12 in osteosarcoma cells; wherein Panel A is a graph of the effect of transfection on ZCCHC12mRNA expression in osteosarcoma cells using QPCR; FIG. B is a graph showing the effect of transfection on ZCCHC12 protein in osteosarcoma cells using Western blot;

FIG. 4 is a graph showing the effect of ZCCHC12 gene on osteosarcoma cell proliferation;

FIG. 5 is a graph showing the effect of ZCCHC12 on osteosarcoma cell migration;

FIG. 6 is a graph showing the effect of ZCCHC12 on osteosarcoma cell invasion.

Detailed Description

The invention is widely and deeply researched, the expression of genes in an osteosarcoma specimen in a tumor tissue and a tissue beside a cancer is detected by a high-throughput sequencing method, a differential expression gene is found, and the relation between the differential expression gene and the occurrence of the osteosarcoma is discussed, so that a better way and a better method are found for the early detection and the targeted treatment of the osteosarcoma. Through screening, the invention discovers that ZCCHC12 is remarkably upregulated in osteosarcoma for the first time. Experiments prove that the growth and invasion of osteosarcoma cells can be effectively inhibited by reducing the expression level of ZCCHC12, the detection of the expression level of ZCCHC12 gene can be one of auxiliary diagnostic indexes for early diagnosis of osteosarcoma, and the interference of ZCCHC12 gene expression can be a new way for preventing or treating osteosarcoma or osteosarcoma transfer.

(biological) marker

Markers (used alone or in combination with other qualitative terms such as an osteosarcoma marker, an osteosarcoma specific marker, a control marker, an exogenous marker, an endogenous marker) refer to parameters which can be measured, calculated or otherwise obtained, are associated with any molecule or combination of molecules, and can be used as indicators of a biological and/or chemical state. In the present invention, "marker" refers to a parameter associated with one or more biomolecules (i.e., "biomarker"), such as naturally or synthetically produced nucleic acids (i.e., individual genes, as well as coding and non-coding DNA and RNA) and proteins (e.g., peptides, polypeptides). "marker" in the context of the present invention also includes reference to a single parameter which may be calculated or otherwise obtained by taking into account expression data from two or more different markers.

The sarcomas markers refer to specific types of markers that can be used (alone or in combination with other markers) as indicators of osteosarcoma in a subject, and in particular embodiments of the invention, the sarcomas markers can be used to provide markers for clinical assessment of osteosarcoma in a subject (alone or in combination with other markers).

ZCCHC12 gene

ZCCHC12 in the invention comprises wild type, mutant or fragment thereof. A representative ZCCHC12 gene sequence is shown in ZCCHC12 gene (NC-000023.11) in GeneBank of international public nucleic acid database, and the full-length nucleotide sequence of human ZCCHC12 or its fragment can be obtained by PCR amplification, recombination or artificial synthesis.

Detection techniques (methods)

The expression level of the gene of the invention is detected using a variety of detection techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and immunodetection technology.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNAISH can be used to determine the structure of chromosomes. Rnash is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

The present invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip and kit

In the present invention, "chip", "microarray", "array" may be equivalently replaced, including but not limited to: DNA microarrays (e.g., cDNA microarrays and oligonucleotide microarrays), protein microarrays, tissue microarrays, transfection or cell microarrays, chemical compound microarrays, and antibody microarrays. DNA microarrays, often referred to as gene chips, DNA chips or biochips, are collections of microscopic DNA spots attached to a solid surface (e.g., glass, plastic, or silicon chips) that form an array for simultaneous expression profiling or expression level monitoring of thousands of genes. The immobilized DNA fragments, called probes, thousands of which can be used in a single DNA microarray. Microarrays can be used to identify disease genes or transcripts (e.g., ncrnas) by comparing gene expression in disease and normal cells. Microarrays can be fabricated using a variety of techniques, including but not limited to: printing onto a glass slide with a fine-pointed needle, photolithography using a pre-fabricated mask, photolithography using a dynamic micro-mirror device, ink-jet printing, or electrochemical methods on a micro-electrode array.

The kit of the invention can be used for detecting the expression of ZCCHC12, and preferably comprises an effective amount of reagent for detecting ZCCHC12 gene or protein coded by the gene, and one or more substances selected from the following group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent. For example, a solution for suspending or immobilizing cells, a detectable label or label, a solution for facilitating hybridization of nucleic acids, a solution for lysing cells, or a solution for nucleic acid purification.

The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.

Screening for Compounds for prevention or treatment of osteosarcoma

The present invention may utilize the biomarker ZCCHC12 to screen for compounds that prevent or treat osteosarcoma, and in one embodiment of the invention, comprises the steps of: in the test group, adding a test compound in a culture system, and observing the expression amount and/or activity of ZCCHC12 in the cells of the test group; in the control group, no test compound was added to the same culture system, and the expression amount and/or activity of ZCCHC12 in the cells of the control group was observed;

wherein, if the expression level and/or activity of ZCCHC12 of the cells in the test group is lower than that of the control group, the test compound is a candidate drug for treating cancer, which has an inhibitory effect on the expression and/or activity of ZCCHC 12.

As an embodiment of the present invention, the steps further include: the obtained candidate compounds are subjected to further cell experiments and/or animal experiments to further select and determine substances useful for preventing, alleviating or treating osteosarcoma from the candidate compounds.

As an embodiment of the present invention, the system for screening candidate compounds for preventing or treating osteosarcoma is not limited to a cell system, but includes a cell system, a subcellular system, a solution system, a tissue system, an organ system, an animal system, or the like, which is not limited to the above-described forms, as long as the system can detect that a test compound can reduce the expression and/or activity of ASPRV 1.

Inhibitors and pharmaceutical compositions

Based on the discovery of the inventor, the invention provides application of an inhibitor of ZCCHC12 in preparing a pharmaceutical composition for inhibiting osteosarcoma. As used herein, the inhibitors of ZCCHC12 include, but are not limited to, inhibitors, antagonists, blockers, nucleic acid inhibitors, and the like.

The ZCCHC12 gene or protein inhibitor is any substance capable of reducing the activity of ZCCHC12 protein, reducing the stability of ZCCHC12 gene or protein, reducing the expression of ZCCHC12 protein, reducing the effective action time of ZCCHC12 protein, or inhibiting the transcription and translation of ZCCHC12 gene, and the substances can be used for the invention, and can be used for reducing ZCCHC12, thereby being used for preventing or treating osteosarcoma. For example, the inhibitor is: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, proteolytic enzymes, protein binding molecules, as long as they are capable of down-regulating the expression of ZCCHC12 protein or its encoding gene at the protein or gene level.

In an alternative form of the invention, the inhibitor of ZCCHC12 is an antibody that specifically binds ZCCHC 12. The specific antibody comprises a monoclonal antibody and a polyclonal antibody; the invention encompasses not only intact antibody molecules, but also any fragment or modification of an antibody, e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc. As long as the fragment retains the ability to bind to ZCCHC12 protein. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies.

In a preferred mode of the invention, the inhibitor of ZCCHC12 is a small interfering RNA molecule specific to ZCCHC 12. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, a process known as RNA interference (RNAInterferce). Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by transfecting an osteosarcoma cell line with a transfection reagent respectively, selects siRNA with the best interference effect, has the sequences shown in SEQ ID NO.9 and SEQ ID NO.10 respectively, further performs experiments at a cell level, and proves that the inhibition efficiency is very high for cell experiments.

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

In an alternative embodiment of the present invention, the ZCCHC12 inhibitor may be a Small hairpin RNA (shRNA) that is a non-coding Small RNA molecule capable of forming a hairpin structure, wherein the Small hairpin RNA is capable of inhibiting gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. Under the action of DICER enzyme in eukaryotic cells, shRN can be cut into small interfering RNA molecules, thereby entering an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

The invention also provides a pharmaceutical composition which contains an effective amount of the ZCCHC12 inhibitor and a pharmaceutically acceptable carrier. The composition can be used for inhibiting osteosarcoma. Any of the foregoing inhibitors of zchc 12 may be used in the preparation of the compositions. Such carriers include, but are not limited to, diluents, excipients, binders, disintegrants, absorption enhancers, surfactants, humectants, adsorptive carriers, lubricants, buffers, stabilizers, bacteriostats, isotonicity agents, chelating agents, pH control agents.

As used herein, the "effective amount" refers to an amount that is functional or active in and acceptable to humans and/or animals. The effective amount of the inhibitor may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the ZCCHC12 gene inhibitor such as bioavailability, metabolism, half-life and the like; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical composition of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The pharmaceutical compositions of the invention may also be combined with other agents for the treatment of osteosarcoma, and the other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient.

Preferably, it can be carried out by means of gene therapy. For example, an inhibitor of ZCCHC12 may be administered directly to a subject by a method such as injection; alternatively, expression units carrying an inhibitor of zchc 12 (e.g. expression vectors or viruses, etc., or siRNA or shRNA) can be delivered to the target and allowed to express the active zchc 12 inhibitor by a route, depending on the type of inhibitor, as is well known to those skilled in the art.

The experiments in the invention are all completed according to at least 3 times of repetition, the result data are all expressed in a mode of mean value plus or minus standard deviation, statistical analysis is carried out by SPSS18.0 statistical software, pairing comparison of cancer and paracarcinoma tissues adopts t test, and the statistical significance is considered when P is less than 0.05.

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of genetic markers associated with osteosarcoma

1. Sample collection

6 osteosarcoma and paracarcinoma tissue samples were collected, and obtained with informed consent from the patients and with consent from the tissue ethics committee.

2. Preparation of RNA samples

The tissue RNA extraction kit from Invitrogen corporation was used to extract RNA, and the detailed procedures were as described in the manual.

3. Mass analysis of RNA samples

The concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The concentration is more than or equal to 200 ng/mul, and the OD260/280 is between 1.8 and 2.2.

4. Removal of rRNA

Ribosomal RNA was removed from total RNA using Ribo-Zero kit.

5. Construction of cDNA library

The construction of cDNA library was carried out using the Truseq RNAscope Prep Kit from Illumina, the detailed procedures were as described in the specification.

6. Sequencing on machine

The cDNA library was sequenced using the Hiseq4000 sequencing platform, the specific procedures were as described in the specification.

7. High throughput transcriptome sequencing data analysis

And (3) performing bioinformatics analysis and processing on the sequencing result, wherein the screening standard of the differential genes is that fdr is less than 0.05, and the difference between the fpkm average values of the two groups is more than 5.

8. Results

The RNA-seq results showed significant upregulation of ZCCHC12 expression levels in osteosarcoma patients, with differences of statistical significance (P < 0.05).

Example 2QPCR sequencing validation of differential expression of ZCCHC12 Gene

1. Large sample QPCR validation was performed on differential ZCCHC12 gene expression. The paracarcinoma tissues and osteosarcoma tissues of osteosarcoma patients were selected in 50 cases according to the sample collection method in example 1.

2. The specific procedure for RNA extraction was as described in example 1.

3. Reverse transcription

mRNA reverse transcription was performed using the FastQuant cDNA first strand synthesis kit (cat # KR 106). The method comprises the following specific steps:

(1) 5 × gDNABuffer 2.0. mu.l, total RNA 1. mu.g, RNase Free ddH₂O, heating to 42 ℃ in a water bath for 3min until the total volume is 10 mu l;

(2) a20. mu.l reaction system was constructed of 10 × Fast RT Buffer 2.0. mu.l, RT Enzyme Mix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂Adding O5.0 mul into the mixed solution in the step (1) after mixing, and mixing uniformly;

(3) heating in water bath at 42 deg.C for 15min, heating at 95 deg.C for 3min, and storing at-20 deg.C for use.

4. QPCR amplification

(1) Primer design

QPCR amplification primers were designed based on the coding sequences of ZCCH 12 gene and housekeeping GAPDH gene in Genebank and synthesized by Bomaide.

Primer sequence of ZCCHC12 gene:

the forward primer sequence is 5'-GTTAATCAGAATGGTAAG-3' (SEQ ID NO. 1);

the reverse primer sequence is 5'-CATCTATCTCTATCACAT-3' (SEQ ID NO. 2).

Primer sequence of GAPDH gene:

the forward primer sequence is 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO. 3);

the reverse primer sequence is 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO. 4).

(2) PCR reaction system including 0.6. mu.l each of forward and reverse primers, 10. mu.l of 2 × SuperReal Premix Plus, 2. mu.l of DNA template, ddH₂O 7.4μl，50×ROX Reference Dye ^△2. mu.l of sterile distilled water, 4.8. mu.l.

(3) And (3) PCR reaction conditions: 95 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 32s) x 40 cycles, 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15 s. PCR reaction is carried out on an ABI 7300 type fluorescence quantitative PCR instrument, a target band is determined by melting curve analysis and electrophoresis, and relative quantification is carried out by a delta CT method.

5. Results

Results as shown in figure 1, ZCCHC12 was up-regulated in osteosarcoma tissue compared to para-carcinoma tissue, with statistical differences (P <0.05), consistent with high throughput sequencing results.

Example 3 detection of differential expression of ZCCHC12 protein by Western blotting assay

1. Extraction of total tissue protein

Shearing tissue with scissors, placing into a glass homogenizer in ice, mixing RIPA lysate and PMSF at a ratio of 100:1, adding RIPA lysate of corresponding amount into tissue specimen of 20mg per 100 μ l lysate, grinding tissue with glass homogenizer until it is fully lysed, sucking the lysed liquid into EP tube, centrifuging at 14000rpm at 4 deg.C for 5min, and collecting supernatant.

2. Total protein concentration determination

The protein concentration was determined according to the instructions of the BCA protein concentration determination kit.

3. SDS-PAGE electrophoresis

8% of separation gel and 5% of concentrated gel were prepared and electrophoresed according to the instruction of SDS-PAGE gel preparation kit.

4. Western detection

1) Electrotransfer

And (3) putting the PVDF membrane into a methanol solution for activating for 5min, and putting the PVDF membrane into a membrane transferring buffer solution for balancing for 20 min. Taking out the PAGE gel, putting the PAGE gel into a membrane transferring buffer solution, cutting off the corresponding PAGE gel, putting the PAGE gel, the filter paper, the PVDF membrane, the PAGE gel and the filter paper in sequence from bottom to top into a semi-dry membrane transferring instrument, and transferring the membrane for 1.5h at constant pressure of 25V;

2) immunological hybridization

Taking out the PVDF membrane, washing the PVDF membrane by PBS, placing the washed PVDF membrane in a 5% BSA solution, shaking and sealing the PVDF membrane for 2 hours at room temperature, placing the PVDF membrane in a hybridization bag, adding a primary antibody for overnight, washing the PVDF membrane by a TBST buffer solution, adding a corresponding secondary antibody, incubating the PVDF membrane for 2 hours at room temperature, and washing the PVDF membrane by the TBST buffer solution.

3) DAB color development

And (3) dropwise adding a freshly prepared DAB color development solution after the PVDF membrane is slightly dried, and scanning and recording after the PVDF membrane develops color. And (3) taking beta-actin as an internal reference, performing semi-quantitative gray scale analysis on the strip by adopting a Quantity One gel imaging analysis system, repeating the experiment for 3 times, and taking an average gray scale value as a result.

5. Results

The results are shown in fig. 2, and the expression level of ZCCHC12 protein in osteosarcoma tissues is obviously higher than that in paracarcinoma tissues.

Example 4 silencing of ZCCHC12 Gene

1. Cell culture

Human osteosarcoma cell line U-2OS is cultured in DMEM medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. The liquid is changed for 1 time in 2-3 days, the cells grow well and grow in a monolayer adherent manner. Passage was routinely digested with 0.25% EDTA-containing trypsin.

2. Transfection

1) Treatment of cells prior to transfection

One day before transfection, 3-5 × 10 are planted on 6-hole culture plates⁵And (3) culturing each cell/hole in an antibiotic-free culture medium for one day, wherein the cell density is 30-50% during transfection, and the cell/hole is replaced by a serum-free culture medium before transfection.

2) Design of siRNA

Negative control siRNA sequence (siRNA-NC):

the sense strand is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.5)

The antisense strand is 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.6)

siRNA1：

The sense strand is 5'-ACAACUUCAUGUUUCUGUCUG-3' (SEQ ID NO.7)

The antisense strand is 5'-GACAGAAACAUGAAGUUGUUC-3' (SEQ ID NO.8)

siRNA2：

The sense strand is 5'-UCCGUUUAAUAAAAGCAUCAU-3' (SEQ ID NO.9)

The antisense strand is 5'-GAUGCUUUUAUUAAACGGAAG-3' (SEQ ID NO.10)

siRNA3：

The sense strand is 5'-AUCUAUCUCUAUCACAUUGCA-3' (SEQ ID NO.11)

The antisense strand is 5'-CAAUGUGAUAGAGAUAGAUGA-3' (SEQ ID NO.12)

The experiment was divided into three groups: a control group (U-2OS), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2, siRNA3), wherein the siRNA of the negative control group has no homology with the sequence of ZCCHC12 gene.

3) Transfection

Liposome Lipofectamine 3000 from Invitrogen was used for transfection, and the procedures were performed as described

5. QPCR detection of transcriptional level of ZCCHC12 Gene

5.1 extraction of Total RNA from cells

The RNA in the cells was extracted using Qiagen's cell RNA extraction kit, and the experimental procedures were performed according to the instructions.

5.2 reverse transcription procedure as in example 2.

5.3QPCR amplification procedure as in example 2.

6. Western detection of expression level of ZCCHC12 protein

The cells of different treatment groups in log phase were collected, the cells were washed with pre-cooled PBS, RIPA lysate was added, placed on ice for 30min, lysed cells were scraped off using a cell scraper, lysed liquid was pipetted into an EP tube using a pipettor, centrifuged at 14000rpm for 5min at 4 ℃, the centrifuged supernatant was collected, and the remaining steps were the same as in example 3.

7. Results

The results are shown in fig. 3, wherein the reduction of the expression level of siRNA2 is most significant in the experimental group compared to the non-transfected group and the transfected siRNA-NC group, and the difference is statistically significant (P <0.05), and siRNA2 was selected for subsequent experiments.

Example 5 Effect of ZCCHC12 Gene on osteosarcoma cell proliferation

MTT (methyl thiazolyl tetrazolium) experiment is adopted to detect influence of ZCCHC12 gene on proliferation capacity of osteosarcoma cells

1. Taking cells with good growth conditions, conventionally digesting the cells into single cell suspension, counting the cells, and diluting the cells into cell suspension with proper concentration.

2. Inoculating the diluted cells of different treatment groups into 2000 cells per well in 96-well culture plate, setting at least 3 parallel wells and no-cell culture medium control, 37 deg.C, 5% CO₂And culturing for 24 h.

3. Cells from 3 wells were sampled 1, 2, 3, 4, and 5 days after inoculation and their OD values at 490nm were measured by MTT method, counted, and the average value was calculated.

4. The supernatant was discarded before detection, the culture medium was washed 3 times, 10. mu.l of MTT serum-free medium solution (5mg/ml) was added to each well, and the culture was continued for 4 hours in an incubator at 37 ℃ to terminate the culture.

5. Add 100. mu.l of Formanzan's solution to each well and shake the shaker slowly for 1 min. The Optical Density (OD) was measured at 490nm on a microplate reader, and the cell growth curve was plotted with time as the horizontal axis and the optical density as the vertical axis.

6. Results

The results are shown in fig. 4, compared with the control, the experimental group has obviously inhibited cell proliferation after being transfected with siRNA2, and the difference has statistical significance (P <0.05), which indicates that ZCCHC12 is closely related to the proliferation of osteosarcoma cells.

Example 6 Effect of ZCCHC12 Gene on osteosarcoma apoptosis

The effect of ZCCHC12 gene on apoptosis was examined using a flow cytometer.

1. The cell culture and transfection procedures were the same as in example 3.

2. Flow cytometry detection

1) Cells from different treatment groups in the logarithmic growth phase were trypsinized and blown into cell suspensions and counted. Get 10⁶Centrifuging the cell suspension at 1000rpm for 5 min;

2) discarding the supernatant, adding 195. mu.l Annexin V-FITC binding solution to gently resuspend the cells;

3) adding 5 μ l Annexin V-FITC, mixing, and incubating at room temperature in dark for 10 min;

4) centrifuging at 1000rpm for 5min, discarding the supernatant, and adding 190 μ l Annexin V-FITC binding solution to gently resuspend the cells;

5) and adding 10 mu l of Propidium Iodide (PI) staining solution, mixing gently, placing in ice bath and in dark, detecting the apoptosis condition by using a flow cytometer, repeating all experiments for 3 times, and taking an average value of results.

3. As a result:

the results show that there was no significant change in the apoptosis rate of the cells in the experimental group compared to the control group, indicating that ZCCHC12 had no significant effect on apoptosis of osteosarcoma.

Example 7 Effect of ZCCHC12 on cell migration

1. 1ml of 50. mu.g/ml fibronectin per well was added to 6-well plates and placed in a refrigerator at 4 ℃ overnight.

2. Discarding the rest fibronectin solution, washing with serum-free medium, subjecting the cells of different groups in logarithmic growth phase to trypsinization and resuspension, inoculating into 6-well plate paved with fibronectin, wherein each group of cells has 2 multiple wells, each well has 5 × 10⁵Individual cell, 37 ℃ and 5% CO₂The culture was carried out overnight in an incubator.

3. When the cells grow to about 90% confluence, a 10 μ l Tip is used to scratch a cell-free scratch, PBS solution is used to wash off the detached cells, and serum-free medium is added for continuous culture.

4. The healing condition of the cell scratch is observed at 0h and 48h after scratching respectively and photographed. The experiment was repeated 3 times and the results averaged.

5. Results

The results are shown in fig. 5, compared with the control group, the migration distance of the cells after transfection of siRNA2 is obviously reduced, and no significant difference exists between the control groups, which indicates that ZCCHC12 overexpression can promote migration of osteosarcoma cells.

Example 8 Effect of ZCCHC12 on cell invasion

1. Transwell cell preparation

1) Diluting 50mg/L Matrigel with a serum-free culture medium pre-cooled at 4 ℃ in a ratio of 1:8, and mixing uniformly;

2) mu.l to 80. mu.l of diluted Matrigel gel (3.9. mu.g/. mu.l) was placed on a polycarbonate membrane in a Transwell upper chamber having a pore size of 8 μm so that all micropores on the membrane were covered with Matrigel, and the membrane was allowed to polymerize into a gel at 37 ℃ for 30 min.

2. Preparing a cell suspension

The cells of different treatment groups in logarithmic growth phase were trypsinized and resuspended in serum-free medium, and the cell concentration was adjusted to 5 × 10⁴One per ml.

3. Cell seeding

2ml of cell suspension was added to the upper chamber of the Transwell, 1ml of complete medium containing 10% fetal bovine serum was added to the lower chamber, and the mixture was placed in a matched 6-well plate and incubated at 37 ℃ with 5% CO₂Culturing for 20-24h under the condition; the Transwell chamber was removed and the cotton swab wiped to remove Matrigel and non-membrane-penetrating cells from the upper chamber.

4. Dyeing process

After the cell culture is finished, taking out the Transwell chamber, wiping off Matrigel glue on the upper chamber surface and cells which do not penetrate through the membrane with a cotton swab, fixing the lower chamber surface with 95% alcohol for 15min, staining with hematoxylin for 2min, and randomly taking 5 high-power lenses under an inverted microscope for visual field observation, counting and photographing. Counting the number of cells on the lower surface of the chamber, namely the number of cells penetrating the Matrigel gel, taking the average number as an experimental result, representing the invasiveness of the tumor cells by the number of the cells, repeating the experiment for 3 times, and arranging 3 compound holes in each group of the cells.

5. Results

As shown in FIG. 6, the number of cells passing through polycarbonate membrane of Transwell chamber was significantly reduced in the experimental group transfected with siRNA2 compared to the control group, while there was no significant difference between the control groups, indicating that interference with the expression of ZCCHC12 decreased the invasion of osteosarcoma cells.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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Claims (9)

1. Application of a reagent for detecting ZCCHC12 gene or protein expression level in preparing a product for diagnosing osteosarcoma.

2. The use according to claim 1, wherein the product comprises reagents for detecting ZCCHC12 using RT-PCR, real-time quantitative PCR, next generation sequencing, in situ hybridization, chip or immunoassay techniques.

3. Use according to claim 2, wherein said agent is selected from:

a probe that specifically recognizes ZCCHC12 gene; or

Primers for specifically amplifying ZCCHC12 gene; or

An antibody or ligand that specifically binds to a protein encoded by ZCCHC 12.

4. Use of an agent that inhibits the expression level of ZCCHC12 gene or protein in the preparation of a pharmaceutical composition for the treatment of osteosarcoma.

5. The use according to claim 4, wherein the pharmaceutical composition comprises an inhibitor of the expression level of ZCCHC12 gene or protein, and/or other drugs compatible with the inhibitor and pharmaceutically acceptable or auxiliary materials.

6. The use of claim 5, wherein the inhibitor is a nucleic acid inhibitor siRNA.

7. The use of claim 6, wherein the siRNA has the sequence shown in SEQ ID No. 9-10.

8. Application of the reagent for detecting the expression level of ZCCHC12 gene or protein in screening candidate drugs for treating osteosarcoma.

9. The use of claim 8, wherein the step of screening for a candidate agent for the treatment of osteosarcoma comprises:

treating a system expressing or containing the ZCCHC12 gene or protein encoded thereby with a candidate substance; and

detecting the expression level of ZCCHC12 gene or protein coded by the same in the system.

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