Nucleic acid for inhibiting metastasis of thyroid cancer and application thereof

Abstract

The invention provides nucleic acid for inhibiting metastasis of thyroid cancer and application of the nucleic acid. The nucleic acid comprises siRNA (small interfering Ribonucleic Acid) of a Myosin-10 gene, and the siRNA of the Myosin-10 gene has nucleotide sequences as shown in Seq ID NO.1 to 2, Seq ID NO.3 to 4 and Seq ID NO.5 to 6. The siRNA of the Myosin-10 gene inhibits the metastasis of thyroid cancer by inhibiting an FGF / FGFR1 signal axis, CD31 and VEGFA protein expression and inhibiting endothelial angiogenesis. The invention discloses a mechanism for regulating and controlling angiogenesis and metastasis of thyroid cancer cells by Myosin-10, and screening proper siRNA (small interfering Ribonucleic Acid) of Myosin-10, thereby providing a theoretical basis for clinical treatment of proliferation, migration and invasion of thyroid cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nucleic acid for preparing a method for inhibiting the proliferation, migration and invasion of thyroid cancer and its use. Background Art

[0002] Thyroid cancer is a common malignant tumor that seriously affects the quality of life of patients. Although it is less malignant, it is prone to local and distant metastasis, some of which can occur in the early stages. Therefore, clarifying the mechanisms of its proliferation, migration and invasion is the key to curing thyroid cancer.

[0003] RNAi is a new method of silencing gene expression developed in recent years. It uses double-stranded RNA as a medium to degrade RNA with the same sequence. Currently, RNAi is used to specifically inhibit the overexpression of oncogenes, cancer-related genes or mutant genes, keeping these genes in a dormant state. This new method may become one of the effective methods for treating various malignant tumors.

[0004] Angiogenesis refers to the formation of new capillaries by "budding" from existing capillaries. It is the earliest discovered tumor angiogenesis. Angiogenesis is a complex process controlled by multiple signaling pathways and is the main target of anti-tumor angiogenesis therapy. Currently, most anti-tumor vascular drugs target the vascular endothelial growth factor (VEGF) and its receptor VEGFR (vascular endothelialgrowth factors receptors) pathway. In normal vascular endothelial cells, VEGF and VEGFR are expressed at low levels, while they are highly expressed in tumor vascular endothelial cells. Therefore, they are used as targets to inhibit tumor angiogenesis by inhibiting the VEGF pathway, thereby inhibiting tumor development. In addition, FGF (fibroblast growth factor) and FGF receptors, PDGF (platelet-derived growth factor) and PDGF receptors, ANG (angiopoietin) and TIE2 receptor pathways are also involved in the angiogenesis of malignant tumors and can also be used as targets for anti-angiogenic drugs.

[0005] CD31, also known as platelet-endothelial cell adhesion molecule (PECAM-1), is a transmembrane glycoprotein with a molecular weight of 130-140 kDa. CD31 is mainly expressed in vascular endothelial cells, platelets, monocytes / macrophages, granulocytes, lymphocytes (especially marginal zone B cells, peripheral T cells), and is used to identify the differentiation of endothelial cells in tumors and help evaluate the degree of tumor angiogenesis.

[0006] Like other epithelial tumors, the proliferation, migration and invasion of thyroid cancer are the result of imbalanced gene regulation, and its biological characteristics are closely related to a variety of oncogenes and some growth factors. Therefore, it is of great significance to explore the regulatory mechanism of thyroid cancer proliferation, migration and invasion, so as to provide theoretical guidance for the clinical treatment of thyroid cancer. Summary of the invention

[0007] In view of the above problems, the present invention provides a nucleic acid for inhibiting thyroid cancer metastasis and its use. The mechanism of Myosin-10 regulating thyroid cancer metastasis is verified by cell scratch, Western Blot, immunofluorescence and other experimental techniques. The siRNA of Myosin-10 gene obtained by interfering with lentiviral expression vector is used to screen a series of nucleic acid sequences, which can significantly inhibit the FGF / FGFR1 signaling axis, CD31 and VEGFA protein expression and inhibit endothelial angiogenesis, providing a theoretical basis for the clinical treatment of thyroid cancer proliferation, migration and invasion.

[0008] On the one hand, the present invention provides a nucleic acid for inhibiting thyroid cancer metastasis, including siRNA targeting silencing Myosin-10 gene, wherein the siRNA has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

[0009] The inventor applied for domestic patent CN117310173A in 2023, disclosing a method for regulating the Myosin 10 gene to inhibit the metastasis of thyroid cancer. However, it is still unknown how and where Myosin 10 regulates the metastasis of thyroid cancer. Therefore, the present invention designed a large number of siRNAs based on the Myosin-10 gene, and obtained 3 siRNAs through screening, clarified its regulatory mechanism, and found that interfering with the nucleic acid sequence of Myosin 10 can effectively inhibit the metastasis of thyroid cancer, which has theoretical guiding significance for the clinical treatment of thyroid cancer.

[0010] The nucleic acid for inhibiting thyroid cancer metastasis provided by the present invention can be used to prepare drugs or other forms of products for inhibiting thyroid cancer metastasis, including products in various dosage forms. As long as the nucleic acid is used to inhibit thyroid cancer metastasis, it is within the protection scope of the present invention.

[0011] Furthermore, the siRNA has a nucleotide sequence as shown in Seq ID NO.3-4.

[0012] siRNA, i.e. small interfering RNA, triggers the RNA interference (RNAi) mechanism by precisely complementary pairing with specific mRNA sequences in cells, effectively inhibits the translation of corresponding genes, and realizes temporary and reversible closure of gene functions. siRNA technology is not only suitable for gene function verification and signal pathway analysis in basic research, but also widely used in drug target verification, disease model construction in translational research, and even personalized treatment exploration in clinical research. The present invention obtains 3 siRNAs of the Myosin-10 gene by siRNA synthesis technology screening, and expression verification is performed in thyroid cancer K1 cells and endothelial cells. It is found that after transfection of 3 Myosin-10 interferences in K1 cells and endothelial cells, mRNA and protein have different degrees of expression decrease. Among them, the mRNA and protein of the si-3845 transfection group are significantly reduced, which is the most obvious.

[0013] On the other hand, the present invention provides the use of an agent interfering with Myosin-10 in the preparation of an agent for inhibiting the FGF / FGFR1 signaling axis, wherein the agent interfering with Myosin-10 includes an agent interfering with Myosin-10 gene expression or interfering with Myosin-10 protein production.

[0014] Fibroblast growth factor receptor (FGFR) belongs to receptor tyrosine protein kinase. In recent years, many studies have shown that high expression and mutation of FGFR can lead to abnormal activation of its downstream signaling pathways, promoting cell proliferation, epithelial-mesenchymal transition and angiogenesis, as well as tumor cell invasion, metastasis and tolerance to treatment. The above factors act together on the tumor microenvironment and ultimately trigger a poor prognosis. Therefore, the FGFR family has always been the research focus and hotspot of tumor immunotherapy. The present invention detected the expression levels of FGF3 and FGFR1 proteins after expressing si-Myosin-10 in thyroid cells. The results showed that the expression of FGF3 and FGFR1 was significantly reduced, indicating that interfering with Myosin-10 inhibits thyroid cancer metastasis by inhibiting the FGF / FGFR1 signaling axis.

[0015] Furthermore, the reagent interfering with Myosin-10 includes siRNA interfering with Myosin-10 gene expression, and the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

[0016] Furthermore, the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in Seq ID NO.3-4.

[0017] In another aspect, the present invention provides the use of an agent interfering with Myosin-10 in the preparation of an agent for inhibiting the expression of CD31 and VEGFA proteins, wherein the agent interfering with Myosin-10 includes an agent interfering with Myosin-10 gene expression or interfering with Myosin-10 protein production.

[0018] As an adhesion molecule on vascular endothelial cells, CD31 is currently the only best endothelial differentiation marker. In some pathological conditions, such as tumor growth, CD31 helps identify blood vessels in the tumor area. For example, when CD31 is tested positive, it means that blood vessels in the body are actively expressing this adhesion molecule, which generally indicates tumor infiltration, growth and metastasis. At the same time, VEGFA is secreted by a variety of cells such as endothelial cells and tumors, and was initially shown to be an endothelial growth factor and a regulator of vascular permeability. Since VEGFA is a key regulator of angiogenesis during the growth of solid tumors, treatments targeting VEGFA have become innovative treatments in oncology. After expressing si-Myosin-10, it was found that the positive expression of CD31 and VEGFA was reduced, indicating that the nucleic acid has an inhibitory effect on angiogenesis of endothelial cells.

[0019] Furthermore, the reagent interfering with Myosin-10 includes siRNA interfering with Myosin-10 gene expression, and the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

[0020] In another aspect, the present invention provides the use of an agent interfering with Myosin-10 in the preparation of an agent for inhibiting endothelial angiogenesis, wherein the agent interfering with Myosin-10 includes an agent interfering with Myosin-10 gene expression or Myosin-10 protein production.

[0021] Compared with the control, the length and number of endothelial vascular lumens in the si-Myosin-10 transfection group were significantly reduced, and the positive expression of CD31 and VEGFA decreased, indicating that interfering with the expression of this nucleic acid sequence can significantly inhibit endothelial angiogenesis.

[0022] Furthermore, the reagent interfering with Myosin-10 includes siRNA interfering with Myosin-10 gene expression, and the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

[0023] Furthermore, the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in Seq ID NO.3-4.

[0024] The present invention further proves through animal experiments that interference with the expression of Myosin-10 can significantly inhibit the growth, tissue morphology and endothelial blood vessel formation of thyroid cancer tumor cells.

[0025] The present invention has the following beneficial effects:

[0026] 1. It was found that interfering with Myosin-10 can inhibit the FGF / FGFR1 signaling axis and can be used to prepare preparations that inhibit the FGF / FGFR1 signaling axis;

[0027] 2. It was found that interfering with Myosin-10 can inhibit the expression of CD31 and VEGFA proteins, and can be used to prepare preparations that inhibit the expression of CD31 and VEGFA proteins;

[0028] 3. It was found that interfering with Myosin-10 can inhibit endothelial angiogenesis and can be used to prepare preparations that inhibit endothelial angiogenesis;

[0029] 4. Screen out siRNA that interferes with Myosin-10 gene expression with more significant effects, significantly improving the effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the Myosin-10 mRNA sequence bound by siRNA-3845 in Example 1;

[0031] Figure 2 The results of the verification of the interference efficiency of Myosin-10 in thyroid cancer K1 cells in Example 2;

[0032] Figure 3 The effects of Myosin-10 on the proliferation (A), migration (B) and invasion (C) of thyroid cancer cells in Example 2;

[0033] Figure 4 The effect of Myosin-10 on the FGF3 / FGFR1 pathway in thyroid cancer cells in Example 3;

[0034] Figure 5 The effect of Myosin-10 interference with the conditioned medium of thyroid cancer cells on the expression of CD31 and VEGFA in endothelial cells in Example 4;

[0035] Figure 6 The results of the verification of the interference efficiency of Myosin-10 in vascular endothelial HUVEC cells in Example 5;

[0036] Figure 7 The effects of Myosin-10 interference on endothelial cell tube-forming ability (A) and CD31 (B) and VEGFA (C) expression in Example 5;

[0037] Figure 8 The effect of Myosin-10 interference on tumor volume (A) and body weight (B) of nude mice bearing human thyroid cancer cells in Example 6;

[0038] Fig. 9 HE staining was used to observe the pathological condition of tumor tissue in Example 7 (A) and immunohistochemical staining was used to observe the positive expression of CD31, VEGF and KI67 in tumor tissue. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on it. The reagents used in this example are all known products and were obtained by purchasing commercially available products.

[0040] Example 1. Design and synthesis of siRNA targeting Myosin-10 gene

[0041] In order to select the best siRNA for targeting and silencing the Myosin-10 gene, this example designed and synthesized siRNA and empty control siRNA-NC according to the CDS region sequence of Myosin-10 (NM_005964.5, Gene ID: 4628) published by NCBI, referring to the method of designing siRNA website (http: / / siDirect2.rnai.jp). siRNA design was carried out, combined with bioinformatics and gene data analysis, to reduce the possibility of other genes targeting thyroid cancer metastasis and proliferation, and obtain siRNA.

[0042] The obtained siRNA sequence and empty lentiviral vector were synthesized by homologous recombination method, and further used a four-plasmid lentiviral system for virus packaging process, which includes the following plasmids: (1) lentiviral transfer plasmid carrying the target fragment; (2) virus packaging auxiliary plasmid LV003-VSVG; (3) virus packaging auxiliary plasmid LV003-REV; (4) virus packaging auxiliary plasmid LV003-GP. After high-purity endotoxin-free extraction of the four plasmid vectors, the four plasmids were co-transfected into 293T cells using Easyfectin transfection reagent (purchased from Invitrogen). The virus supernatant was collected at 48h and 72h after transfection. The high-titer lentiviral storage solution was obtained by ultracentrifugation concentration and purification, and finally the various indicators of the lentivirus were determined according to strict quality standards.

[0043] Three pairs of siRNAs were further screened through in vitro cell transfection experiments and gene expression analysis methods. Specifically, the candidate siRNAs were first transfected into cell lines expressing the Myosin-10 gene, and then the expression level of the Myosin-10 gene was detected using fluorescent quantitative PCR or Western blot and other technologies to evaluate the silencing efficiency of each candidate siRNA. By comparison, the three pairs of siRNAs with the most significant silencing effects were selected, namely siRNA-1024, siRNA-3845, and siRNA-2190 (see the sequence table for specific sequences), while the remaining siRNAs had no effect. The Myosin-10 mRNA sequence bound by siRNA-3845 is as follows: Figure 1 These three pairs of siRNA not only have high specificity and affinity in the target area, but also can effectively reduce the expression of Myosin-10 gene, which will help the subsequent study of its role and mechanism in the metastasis and proliferation of thyroid cancer.

[0044] Example 2: Effect of Myosin-10 interference on proliferation, migration and invasion of thyroid cancer cells

[0045] To investigate the effect of Myosin-10 gene on thyroid cancer, human thyroid cancer cell K1 cells (iCell-h394, Cybio) were first cultured in F12K medium (iCell-0007, Cybio) in a 37°C 5% CO2 incubator, and then Myosin-10 siRNA and its empty vector siRNA-NC (specific sequence see sequence table) were transfected into the cells using Lipofectamine transfection reagent (purchased from Invitrogen). After the transfection was verified, subsequent detection was performed.

[0046] (I) Verification of Myosin-10 interference in thyroid cancer cells

[0047] In order to verify the interference effect of Myosin-10 in thyroid cancer cells, the expression level of Myosin-10 siRNA in thyroid cancer K1 cells was analyzed by qPCR technology. Total RNA from K1 cells was extracted using Trizon reagent; total RNA was extracted using RNA ultrapure extraction kit (CW0581M, CWBIO); and the concentration and purity of RNA (OD 260 / OD 280 ) ; cDNA was synthesized by RNA reverse transcription kit; fluorescent PCR instrument (CFX Connect TMReal-time fluorescence quantitative PCR was performed by Bio-Rad Biomedical Products (Shanghai) Co., Ltd. The reaction steps were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s; annealing at 58°C for 30 s; extension at 72°C for 30 s; 40 cycles. β-actin was used as an internal reference, and the relative expression of the gene was calculated based on 2 -△△Ct The primer sequences are shown in Table 1:

[0048] Table 1 Primer sequences

[0049]

[0050] like Figure 2 The results showed that compared with the si-NC group, the expression levels of mRNA and protein of K1 cells transfected with three Myosin-10 siRNAs decreased to varying degrees, among which si-3845 decreased most significantly. si-3845 was preferred, and si-Myosin-10 was used to represent si-3845 in the subsequent functional verification test results.

[0051] (II) Effects of myosin-10 on the proliferation, migration and invasion of thyroid cancer cells

[0052] In order to further explore the effect of Myosin-10 on the proliferation, migration and invasion of thyroid cancer cells, the proliferation, migration and invasion abilities of thyroid cancer cells were analyzed by CCK8 assay, cell scratch assay and Transwell assay.

[0053] CCK8 experimental steps:

[0054] 1. Take K1 cells in good condition, discard the culture supernatant, wash twice with 1×PBS, and add 0.25% trypsin (containing 0.02% EDTA) for digestion;

[0055] 2. When the cells become round, add culture medium to terminate digestion and collect the cell suspension into a 10 ml centrifuge tube. Centrifuge at 1000 rpm for 3 min, discard the supernatant and add culture medium to resuspend the cells.

[0056] 3. Inoculate the cells into a 96-well plate, 7×103 cells / 100ul per well, and conduct the experiment after the cells are completely attached;

[0057] 4. CCK8 detection method: add 10 μl CCK8 reagent (KGA317, Keygene Biotechnology) to each well and incubate at 37°C for 2 hours; use an ELISA reader to detect the OD value of each well at a wavelength of 450 nm to calculate the survival rate.

[0058] Cell viability (%) = [A (experimental group) - A (blank culture medium)] / [A (control group) - A (blank culture medium)] × 100%. In the results, control represents blank culture medium, and si-NC represents control group.

[0059] Cell scratch assay steps:

[0060] 1. After the cell density of each group reaches 90% or more, streak each well with a 200 μl pipette tip, discard the culture medium, wash with PBS three times, replace with serum-free culture medium, and take pictures of each well;

[0061] 2. Place the cells in an incubator and take photos of the scratches in each well again after 24 hours. Use the 24-hour scratch data and the 0-hour scratch data to calculate the corresponding scratch area and then calculate the degree of cell healing.

[0062] Healing degree = 100% × (0 h scratch area - 0 h scratch area) / 0 h scratch area.

[0063] Transwell Experiment Steps:

[0064] 1. Take cells in good condition, digest them, centrifuge them, resuspend them in serum-free medium, and count them. The number of cells in each chamber is: 3% × 104;

[0065] 2. Add 500ul of complete culture medium to the lower chamber of the invasion chamber, and the total volume of cells and serum-free drug-containing culture medium in the upper chamber is about 300ul. After the corresponding treatment time, crystal violet staining is performed;

[0066] 3. Remove the culture medium in the wells, then add PBS to wash for 5 minutes, then add the prepared 0.1% crystal violet and let it stand for staining for 1 hour. After staining, wipe the cells in the inner chamber of the chamber with a cotton swab, turn the chamber upside down on a slide and take a picture. After taking the picture, remove the staining solution in the wells, add 1 ml of prepared 33% acetic acid to each well to dissolve the staining solution in the cells, mix well and let it stand, then aspirate 200ul of each well and place it in a 96-well plate, and measure it with a multifunctional microplate reader at a wavelength of 562nm. Use 200ul of 33% acetic acid in each well as a control to measure the absorbance of the solution in each well.

[0067] Cell invasion ability = (absorbance of experimental group - absorbance of control group) / (absorbance of control group) × 100%.

[0068] like Figure 3The results showed that compared with the Control and si-NC groups, the cell proliferation rate, 24h migration level, and 24h invasion ability of the si-Myosin-10 group were significantly reduced; the cell proliferation rate, migration level, and invasion ability of the si-1024 and si-2190 groups were reduced, but the effects were not as good as those of the si-Myosin-10 group. Therefore, si-Myosin-10 was preferred.

[0069] Example 3: Effect of Myosin-10 interference on FGF3 / FGFR1 pathway in thyroid cancer cells

[0070] Studies have found that high expression or mutation of FGFR can lead to abnormal activation of its downstream signaling pathways, promoting cell proliferation, cancer cell invasion, migration, etc. In order to explore the effect of Myosin-10 interference on the FGF3 / FGFR1 pathway in thyroid cancer cells, Western blot technology was used to detect the expression levels of FGF3 and FGFR1 proteins. The experimental steps are as follows:

[0071] 1. Take the cells, discard the culture medium, extract the total protein with RIPA lysis buffer, centrifuge at 12000rpm / min at 4℃ for 10min, take the supernatant, and quantify the total protein with BCA protein quantification kit;

[0072] 2. After the protein samples were denatured, sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) was performed for 1.5 hours, and then the membrane was transferred at a constant current of 300 mA. After the PVDF membrane (millipore) was blocked with skim milk powder, the primary antibodies Myosin-10 (DF13164, Affinity, 1 / 1000), FGF3 (A16850, ABclonal, 1 / 1000), and FGFR1 (A21219, ABclonal, 1 / 5000) were incubated at 4°C overnight. The next day, the PVDF membrane was incubated with the secondary antibody IgG (H+L) (GB23303, Servicebio, 1 / 2000) at room temperature for 2 hours, and the PVDF membrane was soaked with luminescent liquid and placed in an ultra-high sensitivity chemiluminescence imaging system for development.

[0073] like Figure 4The results showed that compared with the control, the expression level of FGF3 protein in the si-Myosin-10 group cells was significantly reduced, and the expression of FGFR1 protein did not change significantly; compared with the si-NC group, the expression of FGF3 and FGFR1 proteins in the si-Myosin-10 group cells was significantly reduced; the expression levels of FGF3 and FGFR1 proteins in the si-1024 group and the si-2190 group were reduced compared with the control and si-NC groups, but the reduction trend was not as good as that in the si-Myosin-10 group. Therefore, si-Myosin-10 is preferred in inhibiting the FGF3 / FGFR1 pathway.

[0074] Example 4: Effect of Myosin-10 on thyroid cancer cell conditioned medium on endothelial cell angiogenesis

[0075] In order to explore the effect of Myosin-10 interference with thyroid cancer cell conditioned medium on endothelial cell tube-forming ability, HUVEC cells were cultured, and according to the experimental grouping information, the conditioned medium of K1 cells in the corresponding treatment group was added to the culture medium at a ratio of 1:1 and incubated for 6 hours. The cell tube-forming experiment was performed, and the number and length of the tube lumens were calculated using Image J analysis software; the conditioned medium of K1 cells in the corresponding treatment group was added to the culture medium at a ratio of 1:1 and incubated for 6 hours before subsequent immunofluorescence detection experiments were performed.

[0076] The steps of immunofluorescence detection are as follows:

[0077] 1. Cell climbing: Wash the culture dish with cells climbed on the culture plate with PBS for 3 times, 5 minutes each time, and 3 times with PBS, 5 minutes each time. Drain the PBS with a pipette, add 5% BSA to the culture dish, and block at 37 degrees for 30 minutes.

[0078] 2. Remove the blocking solution with a pipette, do not wash, add a sufficient amount of diluted primary antibody to the culture dish: VEGFA (bs-1313R, Bioss, 1 / 200), CD31 (GB11063-2, Servicebio, 1 / 200), put it in a wet box, and incubate it at 4°C overnight;

[0079] 3. Wash the dish with PBS 3 times, 5 minutes each time, drain the excess liquid in the dish with a pipette, then add the diluted fluorescent secondary antibody Cy3 (1:200), incubate at 37℃ for 30 minutes, wash the dish with PBS 3 times, 5 minutes each time; from the addition of fluorescent secondary antibody, all subsequent steps should be performed in a dark place as much as possible. Add DAPI and incubate in the dark for 5 minutes, stain the nucleus of the specimen, and rinse the excess DAPI with running water; seal the dish with 50% glycerol, and then observe and collect images under a fluorescence microscope.

[0080] like Figure 5 As shown in the results, compared with the Control and si-NC groups, the positive expression of CD31 and VEGFA in HUVEC cells was reduced after treatment with K1 cell conditioned medium in the si-Myosin-10 group, and the positive expression levels of CD31 and VEGFA in the si-1024 group and si-2190 group were also reduced after treatment, but the effect was not as good as that in the si-Myosin-10 group. Therefore, si-Myosin-10 is preferred in this example.

[0081] Example 5: Effect of Myosin-10 Interference on Endothelial Cell Angiogenesis

[0082] To investigate the effect of Myosin-10 interference on the cell tube-forming ability in endothelial cells, Myosin-10 siRNA and its empty vector were transfected into vascular endothelial HUVEC cells using Lipofectamine transfection reagent (purchased from Invitrogen), and the expression level of Myosin-10 siRNA in vascular endothelial HUVEC cells was analyzed using qPCR technology. The experimental method was the same as Example 2.

[0083] according to Figure 6 The results showed that compared with the si-NC group, the expression of Myosin-10 mRNA and protein in vascular endothelial HUVEC cells decreased to varying degrees after transfection with Myosin-10 siRNA. Among them, the mRNA and protein in the si-3845 transfection group decreased most significantly, and si-3485 was preferred. In the subsequent functional verification test results, si-Myosin-10 represented si-3845.

[0084] Then, a cell tube formation experiment was performed to calculate the number and length of tube lumens, and a subsequent immunofluorescence test was performed. The experimental method and steps were the same as those in Example 4.

[0085] like Figure 7 As shown in the results of A, compared with the si-NC group, the lumen length and number of endothelial cell tube formation experiments in the si-Myosin-10 group were significantly reduced, while the reduction effects of lumen length and number of lumens in the si-1024 group and the si-2190 group were not as good as those in the si-Myosin-10 group; Figure 7 As shown in the results of B and C, compared with the si-NC group, the positive expression of CD31 and VEGFA in endothelial cells in the si-Myosin-10 group was reduced, indicating that inhibiting the expression of Myosin-10 in endothelial cells can inhibit their angiogenesis, and si-Myosin-10 is preferred.

[0086] Example 6: Effect of Myosin-10 Interference on Thyroid Cancer Cell Tumor Growth

[0087] In order to verify the effect of Myosin-10 interference on the growth of thyroid cancer cells, this example injected si-Myosin-10 and empty vector into the tumor, and the tumor growth results were Figure 8 As shown. Starting from the 12th day after cell inoculation, the tumor volume of nude mice in the si-Myosin-10 group was smaller than that in the empty group; on the 21st day, the tumor volume of nude mice in the si-Myosin-10 group was significantly smaller than that in the empty group, and the difference in tumor volume became larger and larger. There was no significant difference in the body weight of the two groups of mice, indicating that Myosin-10 interference inhibited the tumorigenicity of thyroid cancer K1 cells.

[0088] Example 7: Effects of Myosin-10 interference on thyroid cancer cell tumor tissue morphology, cell angiogenesis and proliferation

[0089] In order to further verify the effect of Myosin-10 interference on the tissue morphology, cell angiogenesis, and proliferation of thyroid cancer tumor cells, this example used HE staining to observe the pathological conditions of tumor tissues, and used immunohistochemistry to detect the expression of KI67, VEGF, and CD31 in tumors.

[0090] The specific experimental steps of HE staining are as follows: take out the tumor tissue fixed in 4% paraformaldehyde and rinse with running water for several hours, dehydrate with 70%, 80%, and 90% ethanol solutions at different levels, and then dehydrate with equal amounts of pure alcohol and xylene for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes until transparent. Put it into a mixture of half xylene and half paraffin for 15 minutes, then put it into paraffin I for 50-60 minutes, paraffin II for 50-60 minutes, and then embed it in paraffin and slice it. The paraffin after slicing is baked, dewaxed, and hydrated. After washing with distilled water for 5 seconds, put the slices into a hematoxylin aqueous solution for staining for 3 minutes, differentiate them with hydrochloric acid ethanol differentiation solution for 15 seconds, and wash them with water; turn blue with blue solution for 15 seconds, rinse with running water; stain with eosin for 3 minutes, rinse with running water; dehydrate, make it transparent, seal the slices, and examine them under a microscope. The cell nucleus is blue-purple and the cytoplasm is red.

[0091] Immunohistochemistry: Mouse tumor tissue sections, baked, dewaxed, and hydrated, antigen retrieval was performed with citric acid buffer. After 5% BSA antigen blocking, primary antibody incubation was performed with primary anti-VEGFA antibody (bs-1313R, Bioss, 1 / 200), CD31 antibody (GB11063-2, Servicebio, 1 / 200), and Ki67 antibody (HA721115, Huaan Bio, 1 / 200); after overnight at 4°C, horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:100) was incubated, color was developed with DAB, counterstained with hematoxylin, dehydrated and transparent, and then mounted and observed under a microscope (BX43, Olympus).

[0092] like Fig. 9 As shown in A, the cells in the empty group were closely arranged, numerous, filled with cells, rich cytoplasm, clearly visible nucleoli, and moderate staining. In the si-Myosin-10 group, the tumor tissue cells were disorderly arranged, loose, and irregular in shape. The tumor cells were significantly reduced in size, with pyknosis of the nucleus, unclear boundaries between the cytoplasm and the nucleus, and necrosis. The staining was deepened and uneven (red arrow).

[0093] Immunohistochemical staining results Fig. 9 As shown in B, compared with the empty group, the positive expressions of CD31, VEGF, and KI67 in tumor tissues of the si-Myosin-10 group were significantly reduced, indicating that Myosin-10 interference can inhibit angiogenesis in tumor tissues and inhibit tumor cell proliferation.

[0094] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

[0095]

[0096]

Claims

1. A nucleic acid for inhibiting thyroid cancer metastasis, characterized in that: The invention comprises siRNA targeting silencing Myosin-10 gene, wherein the siRNA has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

2. The nucleic acid according to claim 1, characterized in that The siRNA has the nucleotide sequence shown in Seq ID NO.3-4.

3. Use of an agent interfering with Myosin-10 in the preparation of an agent for inhibiting the FGF / FGFR1 signaling axis, characterized in that: The reagents interfering with Myosin-10 include reagents interfering with Myosin-10 gene expression or reagents interfering with Myosin-10 protein production.

4. The use according to claim 3, characterized in that The reagent interfering with Myosin-10 includes siRNA interfering with Myosin-10 gene expression, and the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

5. The use according to claim 4, characterized in that The siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in Seq ID NO.3-4.

6. Use of an agent interfering with Myosin-10 in the preparation of an agent for inhibiting the expression of CD31 and VEGFA proteins, characterized in that: The reagents interfering with Myosin-10 include reagents interfering with Myosin-10 gene expression or reagents interfering with Myosin-10 protein production.

7. The use according to claim 6, characterized in that The reagent interfering with Myosin-10 includes siRNA interfering with Myosin-10 gene expression, and the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

8. Use of an agent interfering with Myosin-10 in the preparation of an agent for inhibiting endothelial angiogenesis, characterized in that: The reagents interfering with Myosin-10 include reagents interfering with Myosin-10 gene expression or reagents interfering with Myosin-10 protein production.

9. The use according to claim 8, characterized in that The reagent interfering with Myosin-10 includes siRNA interfering with Myosin-10 gene expression, and the siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in any one or more of Seq ID NO.1-2, Seq ID NO.3-4, and Seq ID NO.5-6.

10. The use according to claim 9, characterized in that The siRNA interfering with Myosin-10 gene expression has a nucleotide sequence as shown in Seq ID NO.3-4.

Images