CN113318230B - Application of Optineurin in diagnosis and treatment of ocular melanoma - Google Patents
Application of Optineurin in diagnosis and treatment of ocular melanoma Download PDFInfo
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Abstract
The invention provides application of OPTN in eye melanoma diagnosis and treatment. The invention finds that the expression level of OPTN in the melanoma of eyes is higher and is related to poor prognosis. In addition, the malignant progression of the ocular melanoma can be effectively inhibited by specifically knocking out OPTN. Therefore, the OPTN can be used as a molecular biomarker for diagnosing and treating the ocular melanoma, namely as a prognostic index and a potential therapeutic target of the ocular melanoma.
Description
Technical Field
The invention relates to the technical field of molecular biology and medicines, in particular to application of Optineurin in diagnosis and treatment of ocular melanoma.
Background
The ocular melanoma includes Uveal Melanoma (UM) and Conjunctival Melanoma (CM). Among them, UM is the most common intraocular malignancy among adults, with high malignancy, about 50% of UM patients develop liver metastasis, and the median survival time of liver metastasis patients is only 10.2 months. The onset of CM is hidden, the vision is not affected in the early stage, the diffusion and the metastasis are often found, the prognosis is poor, and the life health of the patient is seriously affected. At present, the main treatment means of the eye melanoma is operation and chemoradiotherapy, but the treatment is not sensitive to the conventional chemoradiotherapy and the treatment is troublesome. Therefore, the search for a novel treatment mode which can effectively inhibit the malignant biological characteristics of the eye melanoma so as to improve the treatment effectiveness, prolong the life cycle of a patient and improve the life quality of the patient is an urgent clinical problem to be solved in the field of eye tumor treatment.
Optineurin (OPTN) is a multifunctional autophagy receptor protein and plays an important role in the substrate recognition link of cell selective autophagy. Autophagy is a highly regulated, lysosome-dependent important cellular metabolic process in eukaryotic cells, involved in the degradation of large protein aggregates within cells, senescence-damaged organelles, and pathogens that invade cells. Several studies have shown that tumor cell autophagy is a double-edged sword for the development of tumors. At present, no research on the relationship between OPTN and ocular melanoma exists in the field, and no deep research on the relationship between OPTN and the occurrence and development of ocular melanoma and the potential mechanism of the function of OPTN in ocular melanoma is available.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides an application of Optineurin in the diagnosis and treatment of melanoma on the eye, which solves the problems of the prior art.
In order to achieve the above object, the present invention provides, in a first aspect, a use of an agent for decreasing the expression level of OPTN in the preparation of a medicament for treating or preventing ocular melanoma.
Preferably, the ocular melanoma is Uveal Melanoma (UM) and/or Conjunctival Melanoma (CM).
Further preferably, the substance for reducing the expression level of OPTN is any one or more of the following functions: (1) inhibiting proliferation of melanoma cells of the eye; (2) inhibiting the cloning capacity of melanoma on eyes; (3) inhibiting melanoma autophagy in the eye; (4) reducing the migration capacity of melanoma cells in the eyes.
Further preferably, the substance for reducing the expression level of OPTN comprises an RNA interference molecule or antisense oligonucleotide against OPTN, a small molecule inhibitor, siRNA, and a substance for implementing lentivirus infection or gene knockout.
Further preferably, the substance for implementing gene knockout is an siRNA knockout sequence.
Further preferably, the siRNA knockout sequence is siOPTN-1: sense: GGAAGUUUACUGUUCUGAU, antisense: AUCAGAACAGUAAACUUCC or siOPTN-2: sense: GCGGAAUAUUCCGAUUCAU, antisense: AUGAAUCGGAAUAUUCCGC.
The invention also provides application of the substance for detecting OPTN in preparation of an eye melanoma diagnostic reagent or an auxiliary diagnostic reagent.
The invention also provides another technical scheme, and the application of the substance for detecting OPTN in the preparation of the ocular melanoma prognosis evaluation or auxiliary prognosis evaluation reagent.
Preferably, the prognosis is in particular prediction of survival.
The invention also provides another technical scheme, and the application of the substance for improving the expression level of OPTN in the preparation of products, wherein the functions of the products are any one or more of the following: (1) promoting proliferation of melanoma cells of the eye; (2) promoting the cloning and forming capacity of melanoma cells of eyes; (3) promoting melanoma autophagy of the eye; (4) and (4) preparing an eye melanoma animal model.
The invention has the beneficial effects that the invention provides the application of OPTN in eye melanoma diagnosis and treatment. The invention discovers that the expression level of OPTN in the melanoma of eyes is higher, and the OPTN is related to poor prognosis. In addition, the malignant progression of the ocular melanoma can be effectively inhibited by specifically knocking out OPTN. Therefore, the OPTN can be used as a molecular biomarker for diagnosing and treating the ocular melanoma, namely as a prognostic index and a potential therapeutic target of the ocular melanoma. The invention discovers for the first time that the expression quantity of OPTN is closely related to the poor prognosis of the ocular melanoma, and the malignant progression of the ocular melanoma can be effectively inhibited by specifically knocking out the OPTN. Therefore, the OPTN can be used as a prognostic index and a potential therapeutic target of the ocular melanoma and has good clinical application value.
Description of the drawings:
FIG. 1 is a graph showing the expression level and prognosis of OPTN in ocular melanoma in the examples of the present invention. A is a representative picture of the expression condition of OPTN in ocular melanoma and normal melanoma by immunofluorescence staining detection, and the high expression of OPTN in ocular melanoma tissues can be seen. B is a statistical plot of the relative expression of OPTN in 81 ocular melanomas and 26 normal lentigines, showing significantly high OPTN expression in ocular melanomas (. p < 0.05). C, counting the relation between the OPTN expression amount and the recurrence of the eye melanoma patients, and showing that the recurrence-free survival rate of the patients with high OPTN expression is obviously reduced compared with the patients with low OPTN expression, and the logrank test p is less than 0.01.
FIG. 2 qPCR results plot for the identification of knock-out effect in example 2.
FIG. 3 is a Western blot result chart for identifying the knockout effect in example 2.
FIG. 4 is a graph showing the results of plate cloning for reducing the proliferation ability of ocular melanoma cells by knocking out OPTN in the present invention. A is a representative graph showing that tumor cells were significantly reduced in proliferative capacity after knocking out OPTN with siOPTN-1 and siOPTN-2 in MUM2B and crm 1 cells, and B is a statistical graph of the relative number of colonies formed, # p <0.01, # p <0.001, # p < 0.0001.
FIG. 5 is a diagram showing the results of Transwell that the deletion of OPTN reduces the migration ability of ocular melanoma cells in the present invention. A is a representative graph showing that tumor cells had significantly reduced migratory capacity after knocking out OPTN with siOPTN-1 and siOPTN-2 in MUM2B and CRMM1 cells, and B is a statistical graph of the relative number of migrating cells, p < 0.0001.
Detailed Description
The invention will be further understood by reference to the following examples.
EXAMPLE 1 tissue sections immunofluorescent staining
Experimental materials: in this example, the tissue samples were obtained from ocular melanoma samples and ocular melanoma samples obtained from ophthalmology in the ninth national hospital affiliated to Shanghai university of transportation medical school. The study protocol was approved by the hospital ethics committee and with written informed consent from the patients.
The experimental steps are as follows: human ocular melanoma tissue or melanotic nevus tissue samples were fixed with 4% paraformaldehyde. Paraffin-embedded tissue sections were 5 μm thick and mounted on glass slides and 1mM EDTA was used for antigen retrieval. Sections were incubated in methanol containing 3% hydrogen peroxide to inactivate endogenous peroxidase activity, and then sections were rinsed in PBS for 6 min. Sections were blocked in goat serum for 2h at room temperature and incubated overnight with primary antibody at 4 ℃. The sections were washed with PBS and incubated with fluorescent secondary antibody at room temperature for 1-2h in the dark. Then, the cells were observed and photographed under a fluorescent microscope (A in FIG. 1), and the relative expression amount of OPTN was measured by Photoshop software, recorded and counted (B in FIG. 1). Further, the expression level of OPTN was analyzed in association with the recurrence of the patients, and a statistical chart was plotted (C in FIG. 1).
Example 2 knock-out and plate clone formation experiments for OPTN
Experimental materials: human uveal melanoma cell line MUM2B, human conjunctival melanoma cell line CRMM 1. Lipofectamine 2000 was purchased from Thermo Fisher (USA), and 4% paraformaldehyde, crystal violet dye were purchased from Biochemical company (China).
The experimental steps are as follows: (1) MUM2B, MEL290, CRMM1, CM2005.1, PIG1 cells were routinely cultured at 37 ℃, 5% CO2 in an incubator. MUM2B and MEL290 were cultured in 10% FBS-containing DMEM, and CRMM1, CM2005.1 and PIG1 were cultured in 10% FBS-containing F12-K.
(2) Digesting and centrifuging MUM2B and CRMM1 cells, re-suspending with complete culture medium, counting and taking 300,000 cells/hole, paving on a six-hole plate, and replacing with serum-free culture medium when the cells are attached to the wall and the growth density is 50% -60%.
(3) A knockout group (siOPTN) and a control group (siNC) were set.
siOPTN-1:
sense:GGAAGUUUACUGUUCUGAU
antisense:AUCAGAACAGUAAACUUCC
siOPTN-2:
sense:GCGGAAUAUUCCGAUUCAU
antisense:AUGAAUCGGAAUAUUCCGC
MUM2B and CRMM1 cells were transfected with Lipofectamine 2000, placed in a 37 ℃ incubator for starvation culture for 6h and then replaced with 10% FBS complete medium. And (3) digesting the cells after 72h to collect RNA and protein, and identifying by qPCR and Western blot to show that the knockout is successful, wherein the specific steps are shown in figures 2 and 3.
(4) The remaining cells were counted and 1000 cells/well plated in six well plates containing 2ml of complete medium per well. Periodically changing the culture solution, discarding the culture solution after 2 weeks, fixing with 4% paraformaldehyde, staining with crystal violet, washing with PBS 2 times, counting cell colonies by photographing (A in FIG. 4), and performing statistical analysis (B in FIG. 4). The result shows that the OPTN knockout obviously inhibits the proliferation capacity of the ocular melanoma cells.
Example 3 knock-out of OPTN and Transwell cell migration experiments
Experimental materials: human uveal melanoma cell line MUM2B, human conjunctival melanoma cell line CRMM 1. Lipofectamine 2000 was purchased from Thermo Fisher (USA), 8 μm 24-well plate Transwell cell from Millipore (USA), and 4% paraformaldehyde, crystal violet dye from Bio Inc. (China).
The experimental steps are as follows: (1) (2) and (3) the steps are the same as the steps.
(4) After identification of the knock-out effect, the remaining cells were counted. A24-well plate was loaded with 900. mu.l of 10% FBS medium per well, and 8 μm Transwell chambers were suspended in the well, and 20000 cells were seeded in 250. mu.l of 2% FBS medium per chamber. After culturing for 48-72 h at 37 ℃ in a 5% CO2 incubator, sucking out culture solution in the holes and the chambers, carefully washing twice with PBS, fixing with 4% paraformaldehyde for 30min, dyeing with crystal violet for 30min, and carefully washing off excessive dye with PBS. The cells in the chamber were all wiped clean, and the cells outside the chamber were photographed by observation under a microscope (A in FIG. 5). Cells outside the chamber were counted and statistically analyzed using ImageJ software (B in fig. 5), and the results showed that knockout of OPTN significantly inhibited ocular melanoma cell migration.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
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Application of Optineurin in diagnosis and treatment of ocular melanoma
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Claims (10)
1. The application of the substance for reducing the expression level of OPTN in the preparation of the medicine for treating or preventing the ocular melanoma.
2. The use according to claim 1, wherein the substance that reduces the expression level of OPTN is functional as any one or more of: (1) inhibiting proliferation of melanoma cells of the eye; (2) inhibiting the cloning capacity of melanoma on eyes; (3) inhibiting melanoma autophagy in the eye; (4) reducing the migration capacity of melanoma cells in the eyes.
3. The use according to claim 1, wherein the ocular melanoma is uveal and/or conjunctival melanoma.
4. Use according to claim 1, wherein the substance that reduces the expression level of OPTN comprises RNA interfering molecules or antisense oligonucleotides directed against OPTN, small molecule inhibitors, siRNA, and substances that effect lentiviral infections or gene knock-outs.
5. The use of claim 4, wherein the agent that effects gene knock-out is an siRNA knock-out sequence.
6. The use of claim 5, wherein the siRNA knockdown sequence is siOPTN-1: sense: GGAAGUUUACUGUUCUGAU, antisense: AUCAGAACAGUAAACUUCC, or
siOPTN-2:sense:GCGGAAUAUUCCGAUUCAU，
antisense:AUGAAUCGGAAUAUUCCGC。
7. The application of the substance for detecting OPTN in the preparation of eye melanoma diagnostic reagents or auxiliary diagnostic reagents.
8. The application of the substance for detecting OPTN in the preparation of the ocular melanoma prognosis evaluation or auxiliary prognosis evaluation reagent.
9. The use according to claim 8, wherein the prognosis is in particular prediction of survival.
10. The application of the substance for improving the expression level of OPTN in the preparation of products, wherein the functions of the products are any one or more of the following: (1) promoting proliferation of melanoma cells of the eye; (2) promoting the cloning and forming capacity of melanoma cells of eyes; (3) promoting melanoma autophagy of the eye; (4) and (4) preparing an eye melanoma animal model.
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