CN108796083B - Application of CIZ1 as molecular marker for diagnosing and treating tongue hemangioma - Google Patents
Application of CIZ1 as molecular marker for diagnosing and treating tongue hemangioma Download PDFInfo
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Abstract
The invention provides an application of CIZ1 as a molecular marker for diagnosing and treating tongue hemangioma, belonging to the technical field of pharmaceutical preparations. Tests show that the CIZ1 presents a high expression state in human body tongue hemangioma, and quantitative analysis of staining intensity through analyzing average optical density shows that the expression of CIZ1 is up to 16.83 times up-regulated in tongue hemangioma tissues compared with normal tongue tissues, so that the CIZ1 can be used as a molecular marker for diagnosing and treating the tongue hemangioma, and the molecular marker provides a new molecular target for the diagnosis and clinical treatment of the body hemangioma. The research of the application shows that the CIZ1 has the capability of diagnosing and treating human body tongue hemangioma, the application is helpful for better understanding the mechanism with the above results, has important clinical application value in the aspect of diagnosing and treating human body tongue hemangioma, and develops new medicinal application for the CIZ1 and the expression product thereof.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to application of CIZ1 as a molecular marker for diagnosing and treating tongue hemangioma.
Background
Hemangiomas are one of the common benign tumors that occur on the skin and oral mucosa. The Hemangioma of the tongue (Hemangioma of the tongue) often severely affects people's health. Because of the frequent movement of the tongue body, the hemangioma lesion at the position is easy to cause trauma, and complications such as secondary hemorrhage, ulceration, infection and the like are common. The clinical pathological process of hemangioma is generally divided into: a rapid proliferation phase, a regression phase and a regression completion phase. The rapidly proliferating tumor mass exhibits a rapid growth, which microscopic manifestations are due to hyperproliferation of vascular endothelial cells. Under normal conditions, however, vascular endothelial cells generally remain stable. The hyperproliferation of hemangioma endothelial cells is usually regulated by several regulatory factors, such as estrogen (estrogen), Vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bFGF).
CIZ1, a binding protein for p21Cip1/Waf1, plays a crucial role in the assembly of the DNA replication complex and in the initiation of replication. In DNA replication, CIZ1 interacts with cyclin e (cyclin e) and cyclin a (cyclin a) to promote the formation of CDK2/cyclin a complex (cyclin-dependent kinase 2/cyclin a complex). Overexpression of CIZ1 altered the cell cycle distribution by increasing the number of cells in S phase (DNA synthesis phase), whereas knockout of CIZ1 inhibited cells from entering S phase. At present, the expression of CIZ1 is found to be disordered in various types of carcinoma such as breast cancer, hepatocellular carcinoma, colon cancer, gallbladder cancer, prostatic cancer, lung cancer and the like.
However, according to the existing research, the action of CIZ1 in the process of generating and developing tumors varies with different tumors, for example, the research indicates that CIZ1 has high expression in gallbladder cancer, prostate cancer and early non-differentiated liver cancer, shows the effect of promoting the generation and development of tumors, and shows the cancer inhibition effect in leukemia, a vascular malignant tumor. At present, no relation between CIZ1 and tongue hemangioma is reported.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor discovers that CIZ1 presents a high expression state in human body tongue hemangioma through long-term technical and practical exploration, and quantitative analysis of staining intensity through analyzing average optical density shows that the expression of CIZ1 is up to 16.83 times up-regulated in tongue hemangioma tissues compared with normal tongue tissues, so that the CIZ1 is expected to be used as a molecular marker for diagnosing and treating tongue hemangioma, and the molecular marker provides a new molecular target for diagnosing and clinically treating the tongue hemangioma.
One of the purposes of the invention is to provide the application of the CIZ1 gene and the expression product thereof in the preparation of products for diagnosing tongue hemangioma.
The second purpose of the invention is to provide a product for diagnosing tongue hemangioma.
The third purpose of the invention is to provide the application of the CIZ1 gene and the expression product thereof in preparing the medicine for treating tongue hemangioma.
The fourth purpose of the invention is to provide a medicine for treating tongue hemangioma.
In order to achieve the technical purpose, the invention relates to the following technical scheme:
the invention provides the application of CIZ1 gene and its expression product in preparing the product for diagnosing tongue hemangioma;
further, the product can diagnose whether the patient has tongue hemangioma by detecting the expression level of the CIZ1 gene in lung tissue, and the high expression of the CIZ1 gene is related to the occurrence and development of the tongue hemangioma.
Further, the product for detecting the expression level of the CIZ1 gene comprises: the product for diagnosing the tongue hemangioma is obtained by detecting the expression level of the CIZ1 gene and the expression product thereof through RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization or chip detection.
Wherein, the product for diagnosing the tongue hemangioma by using the chip comprises: protein chips and gene chips; wherein, the protein chip comprises an antibody which is specifically combined with CIZ1 protein, and the gene chip comprises a probe which is hybridized with the nucleic acid sequence of CIZ1 gene.
Further, the gene chip can be used for detecting the expression level of a plurality of genes including the CIZ1 gene (such as a plurality of genes related to tongue hemangioma). The protein chip can be used for detecting the expression level of a plurality of proteins including CIZ1 protein (such as a plurality of proteins related to tongue hemangioma). The accuracy rate of tongue hemangioma diagnosis can be greatly improved by simultaneously detecting a plurality of tongue hemangioma markers.
In a second aspect of the present invention, there is provided a product for diagnosing lingual hemangioma, which is capable of diagnosing lingual hemangioma by detecting the expression level of CIZ1 gene in lung tissue.
Further, the product comprises a chip, or a kit; wherein, the chip comprises a gene chip and a protein chip; wherein, the protein chip comprises an antibody which is specifically combined with CIZ1 protein, and the gene chip comprises a probe which is hybridized with the nucleic acid sequence of CIZ1 gene.
Further, the gene chip can be used for detecting the expression levels of a plurality of genes including the CIZ1 gene (for example, a plurality of genes related to tongue hemangioma). The protein chip can be used for detecting the expression level of a plurality of proteins including CIZ1 protein (such as a plurality of proteins related to tongue hemangioma). The accuracy rate of tongue hemangioma diagnosis can be greatly improved by simultaneously detecting a plurality of tongue hemangioma markers.
In a third aspect of the invention, the application of the CIZ1 gene and its expression product in preparing a medicament for treating tongue hemangioma is provided.
Further, the medicament comprises an inhibitor of the CIZ1 gene and/or its expression product. The inhibitor comprises a substance for inhibiting the expression of a CIZ1 gene, a substance for inhibiting the stability of an expression product of a CIZ1 gene and/or a substance for inhibiting the activity of an expression product of a CIZ1 gene;
further, the drug contains a substance capable of inhibiting the transcription or translation of the CIZ1 gene, or capable of inhibiting the expression or activity of the CIZ1 protein.
Furthermore, the medicine can effectively reduce the expression level of the CIZ1 gene in the hemangioma cells of the tongue body, thereby inhibiting the proliferation, growth, differentiation and/or survival of the tumor cells.
Such drugs include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, polypeptides, proteins, or interfering lentiviruses.
Further, the nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA), or short hairpin RNA (shRNA).
The double-stranded RNA (dsRNA), ribozyme, small interfering RNA (siRNA)), or short hairpin RNA (shRNA) contains a promoter of CIZ1 gene or an information sequence of CIZ1 gene.
The amount of the drug administered is a dose sufficient to reduce transcription or translation of the CIZ1 gene, or to reduce expression or activity of the CIZ1 protein, such that expression of the CIZ1 gene is reduced by at least 10%, 20%, 50%, 80%, 90%, or 99%;
in a fourth aspect of the invention, a medicament for treating tongue hemangioma is provided, which comprises an inhibitor of the CIZ1 gene and/or its expression product. The inhibitor comprises a substance for inhibiting the expression of a CIZ1 gene, a substance for inhibiting the stability of an expression product of a CIZ1 gene and/or a substance for inhibiting the activity of an expression product of a CIZ1 gene;
further, the drug contains a substance capable of inhibiting the transcription or translation of the CIZ1 gene, or capable of inhibiting the expression or activity of the CIZ1 protein.
The medicine can effectively reduce the expression level of CIZ1 gene in tongue hemangioma cells, thereby inhibiting the proliferation, growth, differentiation and/or survival of tumor cells.
Such drugs include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, polypeptides, proteins, or interfering lentiviruses.
Further, the nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA), or short hairpin RNA (shRNA).
The double-stranded RNA (dsRNA), ribozyme, small interfering RNA (siRNA)), or short hairpin RNA (shRNA) contains a promoter of CIZ1 gene or an information sequence of CIZ1 gene.
The amount of the drug administered is a dose sufficient to reduce transcription or translation of the CIZ1 gene, or to reduce expression or activity of the CIZ1 protein, such that expression of the CIZ1 gene is reduced by at least 10%, 20%, 50%, 80%, 90%, or 99%;
further, the medicine also comprises one or more pharmaceutically or dietetically acceptable auxiliary materials. The adjuvants can be solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, pills and suppositories. Powders and tablets may contain from about 0.1% to about 99.9% of the active ingredient. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid form preparations include solutions, suspensions and emulsions, examples of which are aqueous parenteral solutions or water-propylene glycol solutions, or oral solutions with the addition of sweeteners and contrast agents. In addition, it can also be made into small water injection for injection, lyophilized powder for injection, large infusion solution or small infusion solution;
preferably, the medicament is a solid oral preparation, a liquid oral preparation or an injection;
further preferably, the medicament is a tablet, a dispersible tablet, an enteric-coated tablet, a chewable tablet, an orally disintegrating tablet, a capsule, a sugar-coated agent, a granule, a dry powder, an oral solution, a small water injection for injection, a freeze-dried powder injection for injection, a large infusion solution or a small infusion solution.
When the medicament is used for treating human body tongue hemangioma, an effective dose of the medicament can be applied to a human body.
The term "effective dose" as used herein means the amount of therapeutic agent required to treat, ameliorate the targeted disease or condition, or to exhibit a detectable therapeutic effect.
Further, the dosage of the drug may be decided by a doctor according to the relevant circumstances. These include: the physical condition of the subject, the route of administration, the age, body weight, individual response to the drug, severity of the symptoms, etc., are within the skill of the skilled practitioner.
The invention has the beneficial effects that: the invention provides a marker for diagnosing and treating tongue hemangioma. Tests show that the CIZ1 presents a high expression state in human body tongue hemangioma, and quantitative analysis of staining intensity through analyzing average optical density shows that the expression of CIZ1 is up to 16.83 times up-regulated in tongue hemangioma tissues compared with normal tongue tissues, so that the CIZ1 can be used as a molecular marker for diagnosing and treating the tongue hemangioma, and the molecular marker provides a new molecular target for the diagnosis and clinical treatment of the body hemangioma.
Our research shows that the CIZ1 has the capability of diagnosing and treating human body tongue hemangioma, and the research is helpful for better understanding the mechanism of the above results, has important clinical application value in the aspect of diagnosing and treating human body tongue hemangioma, and opens up new medicinal application for the CIZ1 and the expression product thereof.
Drawings
FIG. 1(a) is a photograph showing the expression of CIZ1 in normal tongue body tissues and tongue body hemangioma tissues by immunohistochemical staining (magnification: 400X); fig. 1(b) is a histogram of CIZ1 immunostaining activity expression in normal tongue tissue and tongue hemangioma tissue (P < 0.01);
FIG. 2(a) is a graph showing the expression of CIZ1 and GAPDH in Human Umbilical Vein Endothelial Cells (HUVECs) transfected with CIZ1-shRNA (i.e., shCIZ1 group) and in human umbilical vein endothelial cells not transfected with CIZ1-shRNA (i.e., shNC group); fig. 2(b) is a graph showing the relative cell numbers of the shCIZ1 group and the shNC group measured by MTT (n-4, P < 0.01); fig. 2(c) is a representative flow cytogram of shCIZ1 and shNC groups detected by flow cytometry after EdU staining; fig. 2(d) is a histogram of positive cells in shCIZ1 and shNC groups after EdU staining (n-4, P < 0.05); fig. 2(e) is a graph of typical scratches at 12h and 24h after the shCIZ1 group and the shNC group were scratched; fig. 2(f) is a bar graph of shCIZ1 group and shNC group at 12h and 24h after scratch treatment (n is 3, P is 0.05).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
In a typical embodiment of the invention, the application of the CIZ1 gene and its expression product in preparing a product for diagnosing tongue hemangioma is provided;
in still another embodiment of the invention, the product can diagnose whether the patient has tongue hemangioma by detecting the expression level of CIZ1 gene in lung tissue, and the high expression of CIZ1 gene is related to the occurrence and development of tongue hemangioma.
In another embodiment of the present invention, the product for detecting the expression level of CIZ1 gene comprises: the product for diagnosing the tongue hemangioma is obtained by detecting the expression level of the CIZ1 gene and the expression product thereof through RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization or chip detection.
Wherein, the product for diagnosing the tongue hemangioma by using the chip comprises: protein chips and gene chips; wherein, the protein chip comprises an antibody which is specifically combined with CIZ1 protein, and the gene chip comprises a probe which is hybridized with the nucleic acid sequence of CIZ1 gene.
In another embodiment of the present invention, the gene chip can be used to detect the expression levels of a plurality of genes including the CIZ1 gene (e.g., a plurality of genes associated with tongue hemangioma). The protein chip can be used for detecting the expression level of a plurality of proteins including CIZ1 protein (such as a plurality of proteins related to tongue hemangioma). The accuracy rate of tongue hemangioma diagnosis can be greatly improved by simultaneously detecting a plurality of tongue hemangioma markers.
In still another embodiment of the present invention, there is provided a product for diagnosing lingual hemangioma, which is capable of diagnosing lingual hemangioma by detecting the expression level of CIZ1 gene in lung tissue.
In yet another embodiment of the invention, the product comprises a chip, or a kit; wherein, the chip comprises a gene chip and a protein chip; wherein, the protein chip comprises an antibody which is specifically combined with CIZ1 protein, and the gene chip comprises a probe which is hybridized with the nucleic acid sequence of CIZ1 gene.
In still another embodiment of the present invention, the gene chip can be used to detect the expression levels of a plurality of genes including the CIZ1 gene (e.g., a plurality of genes associated with tongue hemangioma). The protein chip can be used for detecting the expression level of a plurality of proteins including CIZ1 protein (such as a plurality of proteins related to tongue hemangioma). The accuracy rate of tongue hemangioma diagnosis can be greatly improved by simultaneously detecting a plurality of tongue hemangioma markers.
In another embodiment of the invention, the application of the CIZ1 gene and its expression product in preparing a medicament for treating tongue hemangioma is provided.
In yet another embodiment of the present invention, the medicament comprises an inhibitor of the CIZ1 gene and/or its expression product. The inhibitor comprises a substance for inhibiting the expression of a CIZ1 gene, a substance for inhibiting the stability of an expression product of a CIZ1 gene and/or a substance for inhibiting the activity of an expression product of a CIZ1 gene;
in still another embodiment of the present invention, the medicament comprises a substance capable of inhibiting the transcription or translation of the CIZ1 gene, or the expression or activity of the CIZ1 protein.
The medicine can effectively reduce the expression level of CIZ1 gene in tongue hemangioma cells, thereby inhibiting the proliferation, growth, differentiation and/or survival of tumor cells.
Such drugs include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, polypeptides, proteins, or interfering lentiviruses.
In yet another embodiment of the present invention, the nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA), or short hairpin RNA (shRNA).
The double-stranded RNA (dsRNA), ribozyme, small interfering RNA (siRNA)), or short hairpin RNA (shRNA) contains a promoter of CIZ1 gene or an information sequence of CIZ1 gene.
The amount of the drug administered is a dose sufficient to reduce transcription or translation of the CIZ1 gene, or to reduce expression or activity of the CIZ1 protein, such that expression of the CIZ1 gene is reduced by at least 10%, 20%, 50%, 80%, 90%, or 99%;
in still another embodiment of the present invention, there is provided a medicament for treating tongue hemangioma, which comprises an inhibitor of CIZ1 gene and/or its expression product. The inhibitor comprises a substance for inhibiting the expression of a CIZ1 gene, a substance for inhibiting the stability of an expression product of a CIZ1 gene and/or a substance for inhibiting the activity of an expression product of a CIZ1 gene;
in still another embodiment of the present invention, the medicament comprises a substance capable of inhibiting the transcription or translation of the CIZ1 gene, or the expression or activity of the CIZ1 protein.
In yet another embodiment of the present invention, the medicament is effective to decrease the expression level of CIZ1 gene in tongue hemangioma cells, thereby inhibiting proliferation, growth, differentiation and/or survival of tumor cells.
Such drugs include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, polypeptides, proteins, or interfering lentiviruses.
In yet another embodiment of the present invention, the nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA), or short hairpin RNA (shRNA).
The double-stranded RNA (dsRNA), ribozyme, small interfering RNA (siRNA)), or short hairpin RNA (shRNA) contains a promoter of CIZ1 gene or an information sequence of CIZ1 gene.
The amount of the drug administered is a dose sufficient to reduce transcription or translation of the CIZ1 gene, or sufficient to reduce expression or activity of CIZ1 protein. Such that the expression of the CIZ1 gene is reduced by at least 10%, 20%, 50%, 80%, 90% or 99%;
in another embodiment of the present invention, the medicament further comprises one or more pharmaceutically or dietetically acceptable excipients. The adjuvants can be solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, pills and suppositories. Powders and tablets may contain from about 0.1% to about 99.9% of the active ingredient. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid form preparations include solutions, suspensions and emulsions, examples of which are aqueous parenteral solutions or water-propylene glycol solutions, or oral solutions with the addition of sweeteners and contrast agents. In addition, it can also be made into small water injection for injection, lyophilized powder for injection, large infusion solution or small infusion solution;
in still another embodiment of the present invention, the medicament is a solid oral preparation, a liquid oral preparation or an injection;
in another embodiment of the present invention, the drug is a tablet, a dispersible tablet, an enteric coated tablet, a chewable tablet, an orally disintegrating tablet, a capsule, a sugar coated agent, a granule, a dry powder, an oral solution, a small water injection for injection, a freeze-dried powder injection for injection, a large infusion solution or a small infusion solution.
When the medicament is used for treating human body tongue hemangioma, an effective dose of the medicament can be applied to a human body.
The term "effective dose" as used herein means the amount of therapeutic agent required to treat, ameliorate the targeted disease or condition, or to exhibit a detectable therapeutic effect.
In still another embodiment of the present invention, the dosage of the drug may be decided by a physician according to the relevant circumstances. These include: the physical condition of the subject, the route of administration, the age, body weight, individual response to the drug, severity of the symptoms, etc., are within the skill of the skilled practitioner.
The operation of the present invention will be described in further detail below with reference to examples.
Examples
1. Materials and methods
1.1 immunohistochemistry
The study complies with relevant regulations of the ethical committee of the university of Shandong: 16 tongue hemangioma tissues and 3 normal tongue tissues were collected during the period from 2 months 2012 to 2 months 2017 in the pathology department of the Qilu hospital, Shandong university. All samples were formalin fixed and paraffin embedded. All specimens taken received no other treatment prior to surgical resection. These wax masses were cut into 6 μm thick sections. These sections were then dewaxed for 20min using xylene and then rehydrated using a gradient of ethanol (90%, 80% and 70%). The sections were boiled in 0.01M citrate buffer for 2min and then cooled naturally for antigen retrieval. The endogenous peroxidase was eliminated by incubation with a 3% H2O2 solution for 10min at room temperature. After three PBS washes, rabbit polyclonal CIZ1 antibody (1: 500; ab 102013; Abcam, Cambridge, MA, USA) was incubated overnight at 4 ℃. PBS was washed three times and a secondary antibody was incubated at room temperature for 20min (KIT-5020; MaxVision, Fuzhou, China). DAB (3, 3-diaminobenzidine tetra-hydrochloric acid) color development, hematoxylin counterstain, and pictures taken by an inverted microscope. Image Pro Plus5.0(Media Cybernetics, Silver Spring, MD) was used to analyze the immunoreactivity of CIZ 1. For each sample, 6 sections were counted, 6 fields were taken for each section, and then the mean optical density (IOD) and Mean Stained Area (MSA) of 6 × 6 were calculated.
1.2 cell culture
HUVECS were placed in DMEM double-antibody medium (penicillin 100U/ml, streptomycin 0.1mg/ml) containing 10% FBS in 5% CO2Culturing in a constant-temperature incubator.
1.3ShRNA transfection
Establishment of a CIZ1 knockout stably transfected cell line: HUVECs were transfected with RNAi-bearing (ShNC and ShCIZ1) lentiviral particles containing a puromycin resistance gene for 24h, fresh medium was substituted for the lentiviral particle-containing medium and incubation continued for 2 d. Stable transfected cell lines were selected using 1. mu.g/ml puromycin (Sigma, St. Louis, MO, USA). After obtaining a stable transfected cell line, the culture was maintained with a medium containing 0.1. mu.g/ml puromycin.
1.4 expression of Western blot detection protein
Removing the cell culture solution, washing with PBS for three times, lysing the cells with RIPA lysate at 4 deg.C for 15min, mixing the cell lysate with the sample buffer solution, and boiling for 5min to obtain the protein sample. After the protein sample was separated by 10% SDS-PAGE, it was transferred to a PVDF membrane, and after blocking with TBST containing 5% skim milk at room temperature for 1 hour, CIZ1 antibody (dilution ratio 1: 1500; cat # ab 102013; brand: Abcam) and GAPDH antibody (dilution ratio 1: 1500; cat # 14C 10; brand: CellSignaling, Danvers, MA, USA) were added, incubated overnight at 4 ℃, and after washing the membrane three times on the next day, horseradish peroxidase-conjugated secondary antibody was added, and incubated at room temperature for 1.5 hours. After the TBST is washed again, ECL developer is added and developed in the VILBER FUSION FX7 imaging system.
1.5 cell proliferation assay
The present study used the MTT assay and the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay to detect cell proliferation. The cellular absorbance was measured at a wavelength of 570 and 670nm using MTT reagent (Solarbio, Beijing, China) and on SpectraMax M2(Devices, Sunnyvale, Calif., USA). EdU incorporation assay ShCIZ1 and ShNC cells were fluorescently stained according to the fluorescent staining method of EdU reagent (lebo, guangzhou, china), respectively, and flow cytometric detection was performed on an up-flow cytometer (BD Biosciences, San Jose, CA, USA) with a detection channel of 550.
1.6 scratch test
Cell migration ability was evaluated by scratch test. ShNC and ShCIZ1 cells were seeded in 6-well plates at approximately 1X 10 per well6. When the cells were substantially full, the two groups of cells were lined with 100 μ L autoclaved tips, gently washed 2 times with PBS, added 2mL of low serum medium (containing 1% PBS) and photographed under a microscope to record 0hr scratch area, and cultured, and the same scratch area was photographed at 12hr and 24hr, respectively. Relative migration distance between cells was analyzed using ImageJ software, and the scratch healing rate was (0hr scratch width-24 hr scratch width)/0 h scratch width × 100%. The experiment was repeated three times.
1.7 statistical analysis
All data were analyzed using GraphPad Prism 5.0(GraphPad), data are reported as mean ± standard deviation, and differences between groups were analyzed by t-test. P <0.05 had statistical differences.
2 results of the test
2.1 increased expression of CIZ1 in human tongue hemangioma tissues
16 human tongue hemangioma tissues and 3 normal tongue tissues were collected in this study, and the expression of CIZ1 was confirmed by immunohistochemical staining. The results showed that CIZ1 was almost not expressed in normal tongue tissue (FIG. 1a), and was highly expressed in 16 tongue hemangioma endothelial cells (FIG. 1 a). And the site of positive staining was located in the nucleus, which is consistent with previous findings. Quantitative analysis of staining intensity by analysis of mean optical density indicated that expression of CIZ1 was 16.83-fold (P <0.01) up-regulated in tongue hemangioma tissues compared to normal tongue tissues (fig. 1 b). Thus, our results demonstrate that expression of CIZ1 is significantly elevated in lingual hemangioma tissues.
2.2 knockdown of CIZ1 inhibits the proliferation and migration of HUVECs
According to the research, the expression of CIZ1 in HUVECs is knocked out by using lentivirus particles, and the protein expression is detected by Western blot to verify the knocking-out efficiency. FIG. 2a shows that CIZ1 was successfully knocked out. MTT experiments showed that the proliferation rate of HUVECs after CIZ1 knockout was 39.6% lower than that of the control group (FIG. 2 b). In addition, the EdU assay was used to detect cells in proliferative phase and demonstrated that the proliferation rate of HUVECs decreased by 18.96% (P <0.05) after CIZ1 knockout (fig. 2c, d). In view of the essential role of endothelial cell migration in the development of hemangiomas, this study validated the migration efficiency of HUVECs following CIZ1 knockdown using a scratch test. Fig. 2e, f shows that scratch healing area was significantly reduced at 12hr (P <0.05) and 24hr (P <0.05) after CIZ1 knockout. These results indicate that CIZ1 may be involved in the development of hemangiomas by promoting the proliferation and migration of vascular endothelial cells.
3. Discussion and conclusions
Hemangiomas are a typical proliferative disease caused by hyperproliferation of endothelial cells. The research firstly proves that CIZ1 presents a high expression state in the hemangioma tissues of tongue bodies and has the function in the proliferation and migration processes of vascular endothelial cells. This, in part, reveals a role for CIZ1 in the development of tongue hemangiomas.
Immunohistochemical results in this study showed that positive staining of CIZ1 occurred in the nuclear matrix of endothelial cells, and as a nuclear matrix protein, CIZ1 promoted the formation of DNA replication precursors and replication particles by interacting with a range of cell cycle regulators such as p21, cyclin a, cyclin E, CDK2, CDC6 and PCNA to promote DNA replication. Our findings demonstrate that knockout of CIZ1 significantly inhibits endothelial cell proliferation and migration, and thus CIZ1 may promote the development of hemangiomas by modulating endothelial cell function.
At present, the specific role of CIZ1 in the pathogenesis of hemangioma, particularly tongue hemangioma is still unclear, and we prove for the first time that the expression of CIZ1 in tongue hemangioma tissues is increased, and the in vitro knockout of CIZ1 can inhibit the proliferation and migration of HUVECs. The research shows that the CIZ1 is involved in the pathogenesis of tongue hemangioma and is possibly a new target point for hemangioma treatment.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (9)
- Application of CIZ1 gene and its expression product in preparing product for diagnosing tongue hemangioma.
- 2. The use of claim 1, wherein the product is used for diagnosing whether a patient has a tongue hemangioma by detecting the expression level of CIZ1 gene in tongue tissue.
- 3. The use of claim 2, wherein the product for detecting the expression level of CIZ1 gene comprises: by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection.
- 4. The use of claim 3, wherein the product for diagnosing lingual hemangioma with a chip comprises: protein chips and gene chips; wherein, the protein chip comprises an antibody which is specifically combined with CIZ1 protein, and the gene chip comprises a probe which is hybridized with the nucleic acid sequence of CIZ1 gene.
- The application of the CIZ1 gene or the expression product thereof in preparing the medicine for inhibiting the proliferation and migration of tongue hemangioma endothelial cells, which is characterized in that the medicine comprises an inhibitor of the CIZ1 gene and/or the expression product thereof.
- 6. The use of claim 5, wherein the inhibitor comprises a substance inhibiting the expression of CIZ1 gene, a substance inhibiting the stability of the expression product of CIZ1 gene, a substance inhibiting the activity of the expression product of CIZ1 gene.
- 7. The use of claim 5, wherein the medicament comprises a substance capable of inhibiting transcription or translation of the CIZ1 gene, or capable of inhibiting expression or activity of CIZ1 protein.
- 8. The use of claim 5, wherein the medicament comprises: a nucleic acid molecule, polypeptide, protein or interfering lentivirus.
- 9. The use of claim 8, wherein the nucleic acid molecule comprises: antisense oligonucleotides, double-stranded RNA, ribozymes, small interfering RNAs, or short hairpin RNAs.
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| CN1720442A (en) * | 2002-12-05 | 2006-01-11 | 约克舍癌病研究所 | CIZI replication protein |
| WO2010089559A1 (en) * | 2009-02-05 | 2010-08-12 | Cizzle Biotechnology Limited | Cancer diagnosis and treatment |
| CN103328500A (en) * | 2010-08-04 | 2013-09-25 | 西兹尔生物技术有限公司 | Methods and compounds for the diagnosis and treatment |
| CN103421886A (en) * | 2012-05-21 | 2013-12-04 | 上海吉凯基因化学技术有限公司 | Applications of CIZI gene and relevant medicines |
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| CN1720442A (en) * | 2002-12-05 | 2006-01-11 | 约克舍癌病研究所 | CIZI replication protein |
| WO2010089559A1 (en) * | 2009-02-05 | 2010-08-12 | Cizzle Biotechnology Limited | Cancer diagnosis and treatment |
| CN103328500A (en) * | 2010-08-04 | 2013-09-25 | 西兹尔生物技术有限公司 | Methods and compounds for the diagnosis and treatment |
| CN103421886A (en) * | 2012-05-21 | 2013-12-04 | 上海吉凯基因化学技术有限公司 | Applications of CIZI gene and relevant medicines |
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