Application of inhibiting YAP target gene Hectd1 in inhibiting melanoma metastasis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of inhibiting a YAP target gene Hectd1 in inhibiting melanoma metastasis. The application method comprises the following steps: knocking out the Hectd1 gene, inhibiting the expression of the HECTD1E3 ubiquitin ligase, and inhibiting the migration ability and invasion ability of melanoma cells. A feasible target spot is provided for treatment for inhibiting melanoma metastasis, expression of HECTD1E3 ubiquitin ligase is stopped by knocking out the Hdctd1 gene, and the migration ability and invasion ability of melanoma cells can be effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of inhibiting YAP target gene Hectd1 in inhibiting melanoma metastasis. Background Art

[0002] Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with a global incidence of 0.01‰ to 0.09‰. It originates from melanocytes in the choroid (85%), ciliary body (5-8%), and iris (3-5%), and is highly malignant. Metastasis of UM is an important cause of the high mortality rate of patients, with a total mortality rate of about 31% within 5 years, of which liver metastasis accounts for about 90%, followed by lung metastasis at 30%, and bone metastasis at 23%. Less than 4% of patients with UM can detect signs of metastasis at the time of diagnosis. Many patients have already developed micrometastasis at the time of diagnosis. The one-year mortality rate of patients with UM metastasis is 80%, and the two-year mortality rate is as high as 92%. At present, there is no standard treatment regimen with definite efficacy for metastatic UM. Clinically, systemic chemotherapy, radiotherapy, immunotherapy, and metastatic lesions are mostly used for treatment. However, in recent years, different treatment measures such as chemotherapy and targeted therapy have no significant difference in the survival of patients, and the average median survival time is only about one year.

[0003] The exact mechanism of distant metastasis of UM is still unclear. It is currently believed that the occurrence and metastasis of UM are related to chromosomal abnormalities and gene mutations. The most commonly mutated genes in UM are GNAQ and GNA11. The Q209 site mutation of GNAQ / GNA 11 leads to the loss of GTPase activity, increased binding of GTP to Gα subunits, and continuous activation of multiple downstream cancer-related signaling pathways, among which the YAP (HIPPO) signaling pathway plays an important role in UM. G protein-coupled receptors change the phosphorylation state of YAP through downstream cascade reactions to control its entry and exit of the cell nucleus. As the core transcriptional activator of the HIPPO signaling pathway, YAP plays a very important role in regulating cancer cell proliferation, migration and invasion, escaping contact inhibition, and maintaining cell stemness.

[0004] The YAP pathway regulates the expression of HECTD1 (HECT domain E3 ubiquitin protein ligase 1) downstream. HECTD1 is an E3 ubiquitin ligase that plays an important role in cell cycle progression and tumor development. Studies have shown that HECTD1 promotes the cell cycle by affecting mitosis, interacts with BUB3, and participates in DNA damage response. In addition, studies have found that knocking down HECTD1 in breast cancer cells can reduce the ubiquitination of PIPKIγ90 and inhibit the directionality of tumor cell migration. At the same time, by mutating the ubiquitination site of PIPKIγ90, lung metastasis can be inhibited in vivo. In fibroblasts, HECTD1 affects the diffusion and migration of cells by affecting the dynamic formation of focal adhesions. HECTD1 can ubiquitinate IQGAP1, thereby regulating the FAK / Src signaling pathway to affect the formation of focal adhesions and affect cell migration. The study of HECTD1 has revealed its potential application in cell biology and disease treatment. It is not clear whether targeting HECTD1 has an effect on UM metastasis, and the specific mechanism remains to be explored.

[0005] To this end, we explored whether targeting HECTD1 has an effect on UM and its mechanism of influence, in order to find new targets for UM treatment and provide new references for precision tumor treatment programs. Summary of the invention

[0006] The purpose of the present invention is to provide an application of inhibiting the YAP target gene Hectd1 in inhibiting melanoma metastasis, which can effectively inhibit the migration and invasion ability of melanoma cells by knocking out the Hectd1 gene and preventing the expression of HECTD1 E3 ubiquitin ligase.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides application of Hectd1 gene in inhibiting melanoma metastasis.

[0009] Furthermore, the application method is:

[0010] By knocking out the Hectd1 gene, the migration and invasion abilities of melanoma cells are inhibited.

[0011] The present invention provides application of HECTD1 E3 ubiquitin ligase in inhibiting melanoma metastasis.

[0012] Furthermore, the application method is:

[0013] By inhibiting the expression of HECTD1 E3 ubiquitin ligase, the migration and invasion abilities of melanoma cells are suppressed.

[0014] The present invention has at least the following beneficial effects:

[0015] The present invention provides a feasible target for the treatment of inhibiting melanoma metastasis, and can effectively inhibit the migration and invasion abilities of melanoma cells by knocking out the Hectd1 gene and preventing the expression of HECTD1 E3 ubiquitin ligase. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0017] Figure 1 Screening diagram of new downstream genes of YAP (a) and correlation analysis diagram of YAP and HECTD1 (b);

[0018] Figure 2 This is a picture of the scratch experiment of Hectd1 knockout cells;

[0019] Figure 3 Detection of migration ability of Hectd1 knockout cells. Note: Compared with the control group, ****P<0.0001.

[0020] Figure 4 Detection of the invasion ability of Hectd1 knockout cells. Note: Compared with the control group, ***P<0.001; ****P<0.0001. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] The sequence listing of Hectd1 gene is shown in SEQ ID NO:1.

[0023] The main purpose of the present invention is to provide the effects of targeting HECTD1 on UM metastasis, as follows:

[0024] First, ChIP-Seq was used to screen new downstream genes regulated by YAP in UM cells, and HECTD1 ( Figure 1 (a); Gepi a2 database analysis showed that in UM patient samples, the expression levels of YAP1 and HECTD1 mRNA transcripts were positively correlated. ( Figure 1 (b)

[0025] The CRISPR cas9 system was used to knock out the Hectd1 gene in the mouse melanoma cell line B16-F10 (hereinafter referred to as B16), and two sgRNA knockout plasmids were designed and constructed. The sgRNA sequences were:

[0026] sgHectd1-1#:5'-GGAGGACCAAGAATACGAGG-3'

[0027] sgHectd1-2#:5'-AGATACATTACACTCTGCGA-3'

[0028] The knockout plasmid and control empty plasmid were transferred into B16 cells by transfection, and then monoclonal screening was performed to obtain Hectd1 knockout and control cell lines, and the protein knockout efficiency was verified by Western Blot.

[0029] The cells were subjected to scratch assay, migration assay and invasion assay to verify the effect of Hectd1 knockout on the migration and invasion ability of B16 cells in vitro.

[0030] 1. Scratch test:

[0031] 1. Plate cells at a density of 250,000 cells per well in a 24-well plate, with 3 replicate wells for each cell type;

[0032] 2. Observe overnight. When the confluence reaches 100%, use a 10μl white pipette tip and a ruler to draw a cross in the well, discard the culture medium, and gently rinse with PBS 1-2 times to remove floating cells. Then add 500μl serum-free 1640 culture medium to each well.

[0033] 3. Place the cell in the real-time living cell imaging analyzer Incucyte for imaging. Set the full-well imaging mode and start imaging from 0h, then every 3h for a total of 24h.

[0034] 4. Export the image to observe the width of the scratch;

[0035] 5. The experiment was repeated three times.

[0036] See also Figure 2 It can be seen that the degree of scratch healing in the Hectd1 knockout group was lower than that in the control group, indicating that knocking out Hectd1 can inhibit the migration ability of B16 cells.

[0037] 2. Migration Experiment

[0038] 1. Preparation before the experiment:

[0039] Experimental cells were starved for 12 h using serum-free 1640 medium;

[0040] 2. Collect the cells, centrifuge to remove the supernatant, resuspend the cells in serum-free medium and count. Preparation of each group of cells: 1 well per group. Take 200,000 cells / 300μl per well as an example to prepare 2 replicate wells (1 more well), that is, 400,000 cells / 600μl;

[0041] 3. Add 700 μl of complete 1640 medium containing 10% fetal bovine serum to the lower chamber of the Transwell, and add 300 μl of cells prepared in the previous step to the upper chamber;

[0042] 4. Culture in a cell culture incubator at 37°C, 5% CO2 for 24 hours;

[0043] 5. Fixation. Soak in 4% paraformaldehyde at room temperature for 30 minutes, discard the solution and rinse once with PBS, stain with 1% crystal violet solution at room temperature for 2 hours, gently wipe the cells in the upper chamber with a cotton swab, wash off the stain with double distilled water, air-dry appropriately, take pictures, count, and perform statistical analysis.

[0044] 6. The experiment was repeated three times.

[0045] See also Figure 3 The results of Transwell chamber migration showed that after knocking out Hectd1, the number of B16 cells migrating under serum chemotactic conditions was significantly reduced, and the migration ability was decreased, which was statistically significant (p < 0.0001).

[0046] III. Invasion Assay

[0047] 1. Preparation before the experiment:

[0048] (1) Experimental cells were starved for 12 h using serum-free 1640 medium;

[0049] (2) Thaw Matrigel overnight at 4°C and transfer to an ice box before the experiment;

[0050] (3) Precool the pipette tip, EP tube, 24-well plate with Transwell chamber, and serum-free 1640 culture medium to 4°C and transfer them to an ice box before the experiment;

[0051] 2. Matrigel plating:

[0052] (1) Dilute Matrigel: First, add 8 μl of Matrigel to 64 μl of pre-cooled serum-free medium (1:8 dilution ratio), and then mix thoroughly with a pipette tip. Note that the whole process should be carried out in an ice box or on an ice plate at low temperature.

[0053] (2) Evenly spread the gel: Pipette 60 μl of diluted Matrigel matrix gel, vertically add it to the upper chamber of the Transwell, and evenly spread it on the bottom. Do not generate bubbles during the spreading process;

[0054] (3) Gel. The Transwell chamber covered with Matrigel was placed in an incubator (37°C, 5% CO2) and incubated for 1 to 3 hours to polymerize into a thin film.

[0055] (4) Hydrate the basement membrane, remove excess liquid from the upper chamber, add 100 μl of serum-free culture medium to each well, and place in an incubator for 30 min;

[0056] 3. Collect the cells, centrifuge to remove the supernatant, resuspend the cells in serum-free medium and count them. Preparation of each group of cells: 1 well per group. Take 200,000 cells / 200μl per well as an example to prepare 2 replicate wells (1 more well), that is, 400,000 cells / 400μl;

[0057] 4. Add 700 μl of complete culture medium containing 10% fetal bovine serum to the lower chamber of the Transwell, and add 200 μl of cells prepared in the previous step to the upper chamber;

[0058] 5. Culture in a cell culture incubator for 24 hours;

[0059] 6. Fixation: Add 4% paraformaldehyde to the cells at room temperature for 30 minutes, discard the solution, rinse once with PBS, stain with 1% crystal violet solution at room temperature for 2 hours, gently wipe the cells in the upper chamber with a cotton swab, wash off the stain with double distilled water, air-dry appropriately, take pictures, count, and perform statistical analysis.

[0060] 7. The experiment was repeated three times.

[0061] like Figure 4 The results of Transwell invasion showed that after knocking out Hectd1, the number of B16 cells that invaded under serum chemotaxis conditions was significantly reduced, and the invasion ability was decreased, which was statistically significant (p < 0.001).

[0062] Based on the above, it can be seen that the present invention provides a feasible target for the treatment of inhibiting melanoma metastasis. By knocking out the Hectd1 gene and preventing the expression of HECTD1 E3 ubiquitin ligase, the migration and invasion abilities of melanoma cells can be effectively inhibited.

[0063] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of Hectd1 gene in inhibiting melanoma metastasis.

2. The use according to claim 1, characterized in that: The application method is: By knocking out the Hectd1 gene, the migration and invasion abilities of melanoma cells are inhibited.

3. Application of HECTD1 E3 ubiquitin ligase in inhibiting melanoma metastasis.

4. The use according to claim 3, characterized in that: The application method is: By inhibiting the expression of HECTD1 E3 ubiquitin ligase, the migration and invasion abilities of melanoma cells are suppressed.

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