Tetrahedral framework nucleic acid compound for Braf gene silencing as well as preparation method and application of tetrahedral framework nucleic acid compound

Abstract

The invention provides a tetrahedral framework nucleic acid compound for Braf gene silencing as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The tetrahedral framework nucleic acid compound disclosed by the invention is tetrahedral framework nucleic acid loaded with siRNA; the forward sequence of the siRNA is shown as SEQ ID NO.8, and the reverse sequence of the siRNA is shown as SEQ ID NO.7. The compound disclosed by the invention can be enriched at a tumor site, is effectively taken by cancer cells, and shows a good synergistic anti-tumor effect by inhibiting Braf expression, inhibiting proliferation and invasion of cancer cells, promoting cell apoptosis, inhibiting activation of an MEK-ERK signal channel and improving the mitochondrial dysfunction and DNA damage level. Meanwhile, the compound disclosed by the invention has good biological safety and biocompatibility. Therefore, the compound disclosed by the invention is suitable for preparing thyroid cancer related medicines and has a good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a tetrahedral framework nucleic acid complex for Braf gene silencing, a preparation method thereof, and uses thereof. Background Art

[0002] Thyroid cancer is the most common endocrine malignancy. Among them, the most common type is papillary thyroid carcinoma (PTC), accounting for 60%-80% of all thyroid cancers. Although most patients with papillary thyroid carcinoma (PTC) have a good prognosis after receiving appropriate treatment, the recurrence rate is still significant, up to 20%, and a small number of PTCs may develop into more aggressive subtypes and become resistant to conventional treatments. In addition, there is a rare type of thyroid cancer called anaplastic thyroid carcinoma (ATC), accounting for 1%-2% of thyroid cancers. Although it accounts for a small proportion of thyroid cancers, it is a highly aggressive subtype with a disease-specific mortality rate close to 100%, and there is currently no perfect standard treatment plan. Aggressive multimodal treatment for ATC usually only provides short-term efficacy and has limited improvement in long-term survival, with a median overall survival of only 5 months.

[0003] Braf gene mutations are found in approximately 60% of PTCs and 45% of ATCs. This genetic alteration leads to accelerated cell proliferation, enhanced migration ability, increased invasiveness, and resistance to apoptosis. Therefore, the treatment targeting the Braf gene has received extensive attention in recent years. Existing treatment methods targeting the Braf gene have shown significant improvement in response, but are often accompanied by severe adverse reactions and high treatment costs. Therefore, there is an urgent need to explore more effective, safe, and economically feasible treatment options.

[0004] Small interfering RNA (siRNA) initiates the RNA interference mechanism by inducing the silencing of target mRNA with its complementary sequence. Currently, the usage methods of siRNA mainly include transfection of naked siRNA, viral vector delivery, liposome or polymer nanocarrier delivery, etc. However, although using naked siRNA as a therapeutic agent is simple and direct, it faces many limitations, including a short half-life, poor plasma stability, and difficulty in crossing cell membranes. Viral vector delivery is highly efficient, but has immunogenicity and potential safety risks. Liposomes or polymer nanocarriers can improve stability and targeting, but may cause toxic reactions, and the preparation process is complex and costly. There is still room for improvement in terms of efficiency, safety, and clinical application transformation for the above methods.

[0005] Tetrahedral framework nucleic acid (TDN) is a three-dimensional DNA nanomaterial with properties such as self-cell entry, good stability, low immunogenicity, and high editability, and can be used as a drug carrier. However, there is currently no report on using tetrahedral framework nucleic acid to bind siRNA to achieve the silencing of the Braf gene and thus treat thyroid cancer. Summary of the Invention

[0006] To solve the above problems, the present invention provides a tetrahedral framework nucleic acid complex for Braf gene silencing, its preparation method, and uses.

[0007] The present invention provides a tetrahedral framework nucleic acid complex for Braf gene silencing, which is a tetrahedral framework nucleic acid carrying siRNA; the forward sequence of the siRNA is as shown in SEQ ID NO.8, and the reverse sequence is as shown in SEQ ID NO.7.

[0008] Further, the four DNA single-strand sequences of the tetrahedral framework nucleic acid are as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

[0009] Further, the molar ratio of the tetrahedral framework nucleic acid to siRNA is 1:1.

[0010] The present invention also provides a method for preparing the aforementioned tetrahedral framework nucleic acid complex, which includes the following steps:

[0011] (1) Take the four DNA single-strand sequences of the tetrahedral framework nucleic acid, add them to TM buffer, maintain at 90 - 95 °C for 10 - 30 min, quickly cool to 0 - 4 °C and maintain for more than 20 min to obtain a tetrahedral framework nucleic acid with sticky ends;

[0012] (2) Mix the tetrahedral framework nucleic acid with sticky ends and siRNA and incubate to obtain the product.

[0013] Further, in step (1), the final concentration of each of the four DNA single-strand sequences is 1 μM;

[0014] And / or, in step (1), the pH value of the TM buffer is 8.0.

[0015] Further, in step (2), the molar ratio of the tetrahedral framework nucleic acid with sticky ends to siRNA is 1:1;

[0016] And / or, in step (2), the incubation is at 25 - 37 °C for 20 - 30 min.

[0017] The present invention also provides the use of the aforementioned tetrahedral framework nucleic acid complex in the preparation of a drug for Braf gene silencing.

[0018] The present invention also provides the use of the aforementioned tetrahedral framework nucleic acid complex in the preparation of a drug for preventing and / or treating diseases related to Braf gene silencing;

[0019] Preferably, the disease related to Braf gene silencing is cancer.

[0020] Furthermore, the disease related to Braf gene silencing is thyroid cancer;

[0021] Preferably, the thyroid cancer is papillary thyroid carcinoma and anaplastic thyroid carcinoma.

[0022] The present invention also provides a drug for preventing and / or treating thyroid cancer, which is prepared from the aforementioned tetrahedral framework nucleic acid complex as an active ingredient and pharmaceutically acceptable excipients.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The present invention constructs a novel nano-drug system TDN-siBraf for the first time, in which siBraf binds to TDN through sticky ends, and one siRNA (siBraf) is carried at the vertex of TDN.

[0025] (2) In the TDN-siBraf nano-drug delivery system, TDN can improve the cellular uptake ability and stability of siRNA (siBraf), thereby enhancing the ability of the system to inhibit the proliferation and invasion of cancer cells and promote apoptosis.

[0026] (3) TDN-siBraf treatment can effectively inhibit the activation of the MEK-ERK signaling pathway, thereby leading to increased mitochondrial dysfunction and DNA damage levels.

[0027] (4) TDN-siBraf treatment did not show obvious toxic effects in in vivo experiments.

[0028] (5) TDN-siBraf treatment can effectively inhibit the growth of cancer cells in vivo, and its specific mechanisms include inhibiting the proliferation and invasion of cancer cells and promoting apoptosis.

[0029] In summary, the TDN-siBraf complex of the present invention can be enriched at the tumor site, effectively taken up by cancer cells, inhibit Braf expression, inhibit the proliferation and invasion of cancer cells, promote apoptosis, inhibit the activation of the MEK-ERK signaling pathway, increase the levels of mitochondrial dysfunction and DNA damage, and exhibit a good synergistic anti-tumor effect. At the same time, the TDN-siBraf complex of the present invention has good biosafety and biocompatibility. Therefore, the TDN-siBraf complex of the present invention is suitable for the preparation of drugs related to thyroid cancer and has good prospects for industrial application.

[0030] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0031] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0032] Figure 1 Schematic diagram for the preparation and synthesis of TDN-siBraf.

[0033] Figure 2 Results of 8% non-denaturing PAGE for detecting and verifying the successful synthesis of TDN-siBraf.

[0034] Figure 3 Results of particle size and zeta potential detection of TDN and TDN-siBraf: A is the result of zeta potential detection; B is the result of particle size detection.

[0035] Figure 4 Results of AFM and TEM for detecting the surface morphology of TDN-siBraf.

[0036] Figure 5 Results of fluorescence detection and analysis of the stability of pure siBraf and TDN-siBraf: A is the fluorescence detection graph; B is the statistical analysis result of fluorescence intensity; **p < 0.01.

[0037] Figure 6 Results of confocal microscopy for detecting the uptake effects of pure siBraf and TDN-siBraf by two types of thyroid cancer cells (OCUT-2C cells and KTC-1 cells).

[0038] Figure 7Results of detecting the enrichment effects of simple siBraf and TDN-siBraf on tumors in nude mice

[0039] Figure 8 Results of detecting the knockout effects of simple siBraf and TDN-siBraf on Braf by Western Blot and immunofluorescence: A is the result of Western Blot; B is the result of immunofluorescence staining; C is the statistical analysis result of Western Blot; D is the statistical analysis result of immunofluorescence; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0040] Figure 9 Results of detecting the inhibitory effects of simple siBraf and TDN-siBraf on the proliferation and migration of thyroid cancer cells and the promoting effect on apoptosis by Transwell, CCK8, and flow cytometry: A is the staining result of Giemsa staining solution; B is the statistical result of the number of migrated cells in each group; C and D are the CCK8 proliferation curves of thyroid cancer cells after treatment in each group, C is OCUT-2C cells, D is KTC-1 cells; E is the result of flow cytometry detection and analysis of apoptosis of thyroid cancer cells after treatment in each group; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0041] Figure 10 Results of mitochondrial membrane potential staining analysis of the effects of simple siBraf and TDN-siBraf on the membrane potential of thyroid cancer cells and semi-quantitative analysis: A is the mitochondrial membrane potential staining, red represents complex JC-1, and green represents monomeric JC-1; B-C are the statistical analyses of each group, the ratio of complex / monomer, B is OCUT-2C cells, C is KTC-1 cells; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0042] Figure 11To detect the effects of simple siBraf and TDN-siBraf on the mitochondrial morphology of thyroid cancer cells by TEM and semi-quantitative analysis: A shows the mitochondrial morphology photographed by TEM; B-C show the statistical analysis of mitochondrial quantity, B for OCUT-2C cells, C for KTC-1 cells; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0043] Figure 12 To detect the effects of simple siBraf and TDN-siBraf on DNA damage of thyroid cancer cells by immunofluorescence staining and semi-quantitative analysis: A shows γH2AX fluorescence staining; B-C show the statistical analysis of fluorescence intensity, B for OCUT-2C cells, C for KTC-1 cells; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0044] Figure 13 To show the changes in body weight and tumor size of mice after treatment with simple siBraf, TDN and TDN-siBraf in subcutaneous thyroid cancer tumors: A shows the results of body weight changes; B shows the results of tumor size changes; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0045] Figure 14 To show the tumor volume size on the day of sample collection after treatment with simple siBraf, TDN and TDN-siBraf in subcutaneous thyroid cancer tumors: A shows the tumor entity diagram; B shows the statistical chart of tumor mass on the day of sample collection; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0046] Figure 15 To show the H&E staining results of each tissue during the biosafety detection of siBraf, TDN and TDN-siBraf.

[0047] Figure 16 To show the hematological changes in mice after treatment with simple siBraf, TDN and TDN-siBraf: A shows the main indicators of blood detection; B shows the detection results of white blood cell count and classification; ns indicates no significant difference among groups.

[0048] Figure 17For the detection of apoptosis in thyroid cancer cells by TUNEL staining, the expression and semi-quantitative analysis of Braf, Ki-67 and MMP9; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0049] Figure 18 For RNA sequencing of tumor tissues treated with simple siBraf and TDN-siBraf, and comparison of gene expression changes by KEGG and GO analysis: A-B shows the up-regulated and down-regulated genes in the comparison of TDN-siBraf and siBraf by enriched KEGG pathway terms, A is the up-regulated genes, B is the down-regulated genes; C-D shows the GO and KEGG enrichment of down-regulated terms in the comparison of TDN-siBraf and siBraf, C is GO enrichment, D is KEGG enrichment.

[0050] Figure 19 For immunofluorescence staining to detect the inhibition of the pathway after treatment with siBraf and TDN-siBraf and semi-quantitative analysis of the results; A shows the immunofluorescence staining results of p-MEK in each group of cells; B shows the statistical results of the fluorescence intensity of p-MEK in OCUT-2C cells; C shows the statistical results of the fluorescence intensity of p-MEK in KTC-1 cells; D shows the immunofluorescence staining results of p-ERK in each group of cells; E shows the statistical results of the fluorescence intensity of p-ERK in OCUT-2C cells; F shows the statistical results of the fluorescence intensity of p-ERK in KTC-1 cells; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05.

[0051] Figure 20 For WB to detect the inhibition of the pathway after treatment with siBraf and TDN-siBraf and semi-quantitative analysis of the results; A shows the WB bands of p-MEK and p-ERK in each group of cells; B shows the WB quantitative analysis of OCUT-2C cells; C shows the WB quantitative analysis of KTC-1 cells; *: TDN-siBraf vs Con p<0.05; #: TDN-siBraf vs siBraf p<0.05; ^: TDN-siBraf vs TDN p<0.05. Specific embodiments

[0052] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0053] The nucleotide sequences involved in the present invention are shown in Table 1.

[0054] Table 1. Nucleotide sequences involved in the present invention

[0055]

[0056]

[0057] Example 1. Synthesis of the tetrahedral framework nucleic acid complex (TDN-siBraf) for Braf gene silencing of the present invention

[0058] The schematic diagram of the preparation and synthesis of TDN-siBraf is shown in Figure 1 shown.

[0059] 1. Preparation method of TDN with sticky ends

[0060] The four DNA single strands (S1, S2', S3, S4) in Table 1 were added to 96 μL of TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) in an equimolar ratio. The final concentration of the four DNA single strands was 1 μM. The mixture was fully mixed, quickly heated to 95°C and maintained for 10 minutes, and then quickly cooled to 4°C and maintained for 20 minutes to obtain tetrahedral framework nucleic acids with sticky ends, referred to as TDN-sticky ends.

[0061] 2. Preparation of the TDN-siBraf complex

[0062] The prepared TDN-sticky ends and the siBraf (siBraf-sticky ends) obtained by annealing the complementary sequence with sticky ends were mixed at a molar ratio of 1:1 and incubated at room temperature for 20 minutes to finally obtain the complex TDN-siBraf. The forward sequence of siBraf is shown in SEQ ID NO.8, and the reverse sequence is shown in SEQ ID NO.7.

[0063] Comparative Example 1: Preparation of Tetrahedral Framework Nucleic Acid (TDN)

[0064] The four DNA single strands (S1, S2, S3, S4) in Table 1 were added to 96 μL of TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) in an equimolar ratio, and the final concentration of the four DNA single strands was 1 μM. They were fully mixed, quickly heated to 95°C and maintained for 10 minutes, and then quickly cooled to 4°C and maintained for 20 minutes to obtain tetrahedral framework nucleic acid, referred to as TDN.

[0065] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0066] Unless otherwise specified, the samples involved in the test examples were prepared according to the methods described in Example 1 and Comparative Example 1.

[0067] Test Example 1. Identification of TDN-siBraf of the present invention

[0068] 1. Identification method

[0069] Polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to verify the successful synthesis of TDN-siBraf and observe its size and shape. Dynamic light scattering DLS (Nano ZS, Malvern, UK) was used to measure the particle size and Zeta potential of TDN-siBraf. The PerkinElmer IVIS fluorescence system was used to detect the degradation of TDN-siBraf.

[0070] (1) PAGE: The successfully synthesized TDN-siBraf was first characterized by 8% polyacrylamide gel electrophoresis. The sample was loaded and run on the gel at a constant voltage of 100 V for 80 min, and then stained and exposed with 1:50 Goldview for observation. S1, S2-stickyends, S3, S4, siBraf-sticky ends, TDN, and TDN-sticky ends were used as controls.

[0071] (2) DLS: The synthesized TDN and TDN-siBraf were diluted to 250 nM with secondary distilled water, and the particle size and potential of TDN and TDN-siBraf were measured using a Zetasizer Nano ZS90 (Malvern Instruments Ltd, UK).

[0072] (3) AFM: The surface morphology of TDN-siBraf nanoparticles was characterized by an atomic force microscope, which was completed by a Shimadzu SPM-9700 atomic force microscope in the tapping scan mode. TDN-siBraf was diluted to 20 nM with TM buffer solution, and then 10 μL of this solution was treated for about 15 min and then observed. TDN was used as a control.

[0073] (4) TEM: The microstructure of TDN-siBraf was observed by transmission electron microscopy, showing that the TDN-siBraf nanomaterial was small particles with a uniform particle size of 10 nm. TDN was used as a control.

[0074] (5) Fluorescence determination: FAM-siBraf and TDN-siBraf were placed at room temperature, and then the fluorescence intensity was detected using a PerkinElmer IVIS Spectrum at 0.5, 1, 3, 6, 12, 24, 36, and 48 hours. The signal of pure FAM-siBraf was significantly weakened after 6 hours of incubation, indicating its degradation, and no signal could be detected after 24 hours. In contrast, in the TDN-siBraf group, FAM-siBraf dissociated from TDN still showed a significant signal after 24 hours. FAM-siBraf refers to siBraf with a fluorescence label (FAM). The forward sequence of siBraf is shown in SEQ ID NO.6, and the reverse sequence is shown in SEQ ID NO.7, which are obtained by annealing the forward and reverse sequences. The TDN-siBraf used here was obtained by incubating siBraf with a fluorescence label (FAM) according to the method described in Example 1 with TDN.

[0075] 2. Identification results

[0076] PAGE showed that the mobilities of TDN, TDN-sticky ends, and TDN-siBraf were consistent with expectations, indicating that all three nanoparticles were successfully synthesized ( Figure 2 ). Dynamic light scattering analysis showed that the particle sizes of TDN and TDN-siBraf were approximately 11 and 13 nm, respectively, and charge determination proved that both TDN and TDN-siBraf were negatively charged ( Figure 3 ). Atomic force microscopy and transmission electron microscopy detection results showed that TDN-siBraf was evenly distributed and approximately 10 nm in size ( Figure 4 ). From the above results, it can be seen that the synthesis of TDN-siBraf was successful. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to further observe the nanostructure of the particles. The results showed that the TDN core was roughly pyramid-shaped, and a "tail" structure could be observed at the vertex of TDN-siBraf, which was consistent with the expected design ( Figure 4 ). Fluorescence detection results showed that the fluorescence signal of naked FAM-siBraf ( Figure 5 siBraf in it) was significantly weakened after 6 hours of incubation, indicating that siBraf began to degrade, and no signal could be detected after 24 hours. In contrast, in the TDN-siBraf group, the FAM-siBraf dissociated from TDN still showed an obvious fluorescence signal after 24 hours ( Figure 5 ). This indicates that TDN-siBraf can protect siBraf from rapid degradation.

[0077] Experimental Example 2. Uptake by thyroid cancer cells

[0078] Detect the entry of TDN-siBraf into thyroid cancer cells. Immunofluorescence was used to detect the entry of TDN-siBraf into thyroid cancer cells. siBraf and S1 were labeled with FAM (green fluorescence) and Cy5 (red fluorescence), respectively. Cy5-TDN ( Figure 6 in which is TDN) was synthesized according to the method described in Comparative Example 1, and Cy5-TDN-FAM-siBraf ( Figure 6 in which is TDN-siBraf) was synthesized according to the method described in Example 1. Con was the cell control group without drug administration.

[0079] OCUT-2C and KTC-1 cells were respectively inoculated into 10 mm confocal culture dishes and cultured for 12 hours, followed by 24-hour serum-free starvation treatment. After removing the original medium, the cells were placed in a medium containing 1% FBS, and Cy5-TDN (100 nM), FAM-siBraf (100 nM) or Cy5-TDN-FAM-siBraf (100 nM) was added and incubated for 24 hours. Subsequently, the two cell lines were fixed with 4% paraformaldehyde for 30 minutes. After washing with PBS, the cells were sealed with an anti-fluorescence quenching mounting medium containing DAPI (Biosharp). Finally, confocal microscopy was used for observation and image acquisition.

[0080] It was found that significant co-localization of TDN-Cy5 and FAM-siBraf was observed in both cell lines in the TDN-siBraf group. In addition, the Cy5 fluorescence intensity of TDN and the TDN-siBraf group was comparable. At the same time, compared with the weaker signal detected in the siBraf group, the fluorescence intensity of FAM-siBraf in the cells treated with TDN-siBraf was significantly enhanced ( Figure 6 ). It shows that TDN-siBraf can be better taken up by thyroid cancer cells compared with siBraf.

[0081] Experimental Example 3. In vivo biodistribution and local enrichment of TDN-siBraf

[0082] Establishment of subcutaneous tumor model: Nude mice were anesthetized by inhaling isoflurane with an anesthesia machine. 5×105 OCUT-2C cells were suspended in 100 μL PBS and injected subcutaneously into the right abdomen of each mouse. All mice were raised under specific pathogen-free (SPF) conditions. The body weight and tumor volume of the mice were measured every 2 days. The tumor volume was calculated according to the following formula: Tumor volume = 1 / 2 × length × (shortest width)2. When the tumor volume reached 40-60 mm 3 , all the mice were randomly divided into four groups.

[0083] Each group was injected with 50 μL of normal saline (Con), FAM-siBraf (250 nM, siBraf), Cy5-TDN (250 nM, TDN), or Cy5-TDN-FAM-siBraf (250 nM, TDN-siBraf) around the tumor. The biodistribution and local enrichment of simple FAM-siBraf and Cy5-TDN-FAM-siBraf in a mouse model bearing OCUT-2C tumors were evaluated using an IVIS kinetic system, and the observation time lasted for 48 hours.

[0084] The FAM fluorescence in the siBraf group and the TDN-siBraf group initially concentrated in the tumor tissue. However, as the observation time extended, the FAM signal in the siBraf group rapidly spread throughout the body and mainly accumulated in the facial and abdominal regions. Compared with siBraf, the fluorescence intensity in the TDN-siBraf group was significantly stronger in the tumor area, indicating its higher local enrichment ability at the tumor site ( Figure 7 ).

[0085] Experimental Example 4. In vitro regulation of the proliferation, migration, and apoptosis of thyroid cancer cells by TDN-siBraf

[0086] OCUT-2C and KTC-1 cells were seeded in 6-well plates and cultured for 12 hours. After removing the original medium, Cy5-TDN (100 nM, Figure 8 TDN therein), FAM-siBraf (100 nM, Figure 8 siBraf therein), or Cy5-TDN-FAM-siBraf (100 nM, Figure 8 TDN-siBraf therein) was added and incubated for 24 hours. Subsequently, the proteins were collected, the concentration was determined by BCA, then WB was performed, and finally, band analysis was carried out using ImageJ. Con was the cell control group without drug administration.

[0087] OCUT-2C and KTC-1 cells were seeded in 10-mm confocal culture dishes and cultured for 12 hours. After removing the original medium, Cy5-TDN (100 nM, Figure 8 TDN therein), FAM-siBraf (100 nM, Figure 8 siBraf therein), or Cy5-TDN-FAM-siBraf (100 nM, Figure 8 TDN-siBraf therein) was added and incubated for 24 hours. Subsequently, the two cell lines were fixed with 4% paraformaldehyde for 30 minutes. After washing with PBS, immunofluorescence staining was performed using a Braf antibody, and the cells were mounted with an anti-fluorescence quenching mounting medium containing DAPI (Biosharp). Finally, observation and imaging were carried out using a confocal microscope. Con was the cell control group without drug administration.

[0088] Western blot results showed that in both cell lines, naked siBraf produced a slight knockdown effect on Braf gene expression compared with the control group, while the Braf expression level in the TDN-siBraf group was significantly reduced ( Figure 8 A and 8C). Similarly, immunofluorescence results ( Figure 8 These results indicate that TDN-siBraf can effectively inhibit the expression of Braf gene, and has a synergistic effect compared with the use of TDN and siBraf alone.

[0089] Cell migration was assessed using Chemotaxicell chambers. OCUT-2C and KTC-1 cells were plated at 1 × 10 5 The density of cells was inoculated in the upper chamber of the chamber, and TDN-siBraf (100nM, without fluorescence), siBraf (100nM, without fluorescence and without sticky ends) and TDN (100nM, without fluorescence and without sticky ends) were added to the lower chamber and incubated for 24 hours. No drug was added as a control (Con). Subsequently, the cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with Giemsa staining solution. Images were taken with an inverted microscope, and the migrated cells were counted and analyzed using ImageJ software. The experimental results showed that in the Transwell experiment, after 24 hours of incubation, the number of migrated tumor cells in the siBraf group was reduced compared with the control group and the TDN group, while the number of migrated cells in the TDN-siBraf group was significantly reduced in both cell lines, and it had a stronger inhibitory effect than the siBraf group, with a significant difference ( Figure 9 A and 9B). This indicates that TDN-siBraf has a synergistic inhibitory effect on the migration of thyroid cancer cells.

[0090] In the cell proliferation experiment, the two cell lines were seeded in a 96-well plate at a density of 2×104 cells per well and incubated overnight. The next day, the cells were treated with culture medium containing TDN, siBraf or TDN-siBraf (all at a concentration of 100nM, without fluorescence, and siBraf and TDN without sticky ends), and the medium was changed and the drug was added every day for 4 days. No drug was added as a control (Con). Cell Counting Kit-8 (Dojindo) was used to analyze cell proliferation every day according to the instructions of the kit. The results of the CCK8 proliferation experiment showed that the use of TDN or siBraf alone had little effect on the proliferation of the two cell lines, and after 48 hours of incubation, the TDN-siBraf group showed a significantly stronger inhibitory effect on cell proliferation ( Figure 9 C and 9D).

[0091] Similarly, apoptosis was evaluated using an apoptosis detection kit (UE Landy). The cells were fed with fresh medium every other day. On the 4th day, the cells were prepared for sample collection, washed with cold PBS, and resuspended in 1X binding buffer at a concentration of 1×106 cells / mL. AnnexinV-FITC and propidium iodide (PI) were added, and the cells were incubated for 15 minutes at room temperature in the dark. After staining, the cells were analyzed by flow cytometry (Cytek) within 1 hour. The results of the flow cytometry apoptosis assay were also consistent with the above findings. In OCUT-2C cells, the proportion of late apoptotic cells in the TDN-siBraf treatment group increased slightly. In KTC-1 cells, the proportion of apoptotic cells in the TDN and siBraf groups was on average less than 5%( Figure 9 E).

[0092] The above experiments showed that compared with TDN and siBraf alone, the TDN-siBraf of the present invention could significantly inhibit the migration and proliferation of thyroid cancer cells, better promote the apoptosis of thyroid cancer cells, and had a synergistic effect.

[0093] Experimental Example 5. In vitro regulation of mitochondrial function and apoptosis of thyroid cancer cells by TDN-siBraf

[0094] Two thyroid cancer cell lines, KTC-1 and OCUT-2C, were cultured separately and treated with medium containing TDN, siBraf, or TDN-siBraf (all at a concentration of 100 nM, without fluorescence and without sticky ends for siBraf and TDN) for 4 days, with the medium changed and the drugs added every other day. First, the cationic dye JC-1 was used to evaluate mitochondrial dysfunction, and the change in mitochondrial membrane potential (MMP) was measured by confocal microscopy. Theoretically, high MMP can cause JC-1 to aggregate in the mitochondrial matrix to form red aggregates, while low MMP shows green monomers. The results showed that after treatment with siBraf, the ratio of aggregates / monomers decreased slightly. Notably, after treatment with TDN-siBraf, the ratio of JC-1 aggregates / monomers in both cell lines decreased significantly, and compared with the control group, the TDN group, and the siBraf group, the MMP decreased significantly, with statistical differences( Figure 10 ). This indicates that compared with TDN and siBraf alone, the TDN-siBraf of the present invention can significantly inhibit the mitochondrial activity of thyroid cancer cells.

[0095] In addition, transmission electron microscopy (TEM) was used to further evaluate the change in mitochondrial morphology. The results showed that compared with the other three groups, the number of mitochondria in the two cell lines treated with TDN-siBraf showed a downward trend( Figure 11). At the same time, significant changes in mitochondrial morphology were observed. In the TDN-siBraf group, mitochondria were significantly swollen, mitochondrial cristae collapsed or disappeared to varying degrees, and the internal structure of mitochondria was unclear.

[0096] To further verify that TDN-siBraf-mediated thyroid cancer cell toxicity is associated with the induction of DNA damage and apoptosis, γH2AX was selected as a key biomarker of cell damage. The expression of γH2AX was detected by immunofluorescence staining. The results showed that TDN-siBraf treatment significantly increased the expression of γH2AX in both cell lines compared with the other three groups ( Figure 12 ), indicating that TDN-siBraf has a significant effect in promoting DNA damage in thyroid cancer cells.

[0097] The above experimental results show that compared with the use of TDN and siBraf alone, TDN-siBraf has a significant damaging effect on the mitochondria and DNA of thyroid cancer cells, and plays a synergistic role.

[0098] Test Example 6: In vivo experiment

[0099] 1. Main experimental materials: Animals: BALB / c female mice (18-20g). Reagents: TUNEL kit; Immunofluorescence antibodies: Braf, Ki67, MMP9.

[0100] 2. Experimental setup and model construction:

[0101] 2.1 Experimental groups (4 groups*3 mice)

[0102] A: Control group: normal saline

[0103] B: TDN group: TDN (250nM)

[0104] C: siBraf group: siBraf (250 nM)

[0105] D: TDN+siBraf group: TDN-siBraf (250nM)

[0106] TDN, siBraf and TDN-siBraf are not fluorescent, and siBraf and TDN do not have sticky ends.

[0107] Subcutaneous tumor model establishment: Mice were anesthetized by inhalation of isoflurane using an anesthesia machine. 5×10 5OCUT-2C cells were suspended in 100 μL of PBS and injected subcutaneously into the right flank of each mouse. All mice were housed under specific pathogen-free (SPF) conditions. The body weight and tumor volume of the mice were measured every 2 days, and the tumor volume was calculated according to the following formula: tumor volume = 1 / 2 × length × (shortest width)2. When the tumor volume reached 40 - 60 mm 3 , all the mice were randomly divided into four groups. Each group was injected with 50 μL of normal saline, TDN (250 nM), siBraf (250 nM), or TDN-siBraf (250 nM) around the tumor every 2 days.

[0108] 2.2 Specimen collection and tissue staining

[0109] On the 20th day after tumor inoculation, the mice were euthanized, and the tumor tissues were collected for weighing and photographing. After routine fixation, the tissues were sectioned for further analysis. The tumor sections were stained with hematoxylin-eosin (H&E), immunohistochemistry (Braf, Ki-67, MMP9), and TUNEL staining to evaluate the anti-tumor effect. Under an inverted microscope, the number of positive stainings in the same part of each group of mice in 3 fields of view was counted for statistical analysis.

[0110] 2.3 Safety assessment

[0111] On the day of the end of the experiment, mouse plasma and serum samples were collected, and white blood cell counts (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cell indices (RBC, HCT, HGB), liver function markers (ALT, AST), and kidney function markers (UREA, Cr) were measured and analyzed. In addition, the main organs, including the liver, kidney, spleen, and heart, were removed and stained with hematoxylin-eosin (H&E) to evaluate the biosafety of TDN-siBraf.

[0112] 3. Experimental results

[0113] 3.1 Anti-tumor effect of TDN-siBraf in vivo

[0114] The results showed that there was no significant difference in the body weight of the four groups of mice during the whole treatment period ( Figure 13 A), indicating that the TDN-siBraf system had low toxicity. The tumor growth curve showed ( Figure 13 B) that from the 14th day, TDN-siBraf showed a significant inhibitory effect on tumor progression, and the tumor size was significantly smaller than that of other groups. On the 20th day, all the mice were euthanized for further analysis. The in vitro tumor images and tumor weight statistics showed ( Figure 14) In the TDN-siBraf group, tumor growth was significantly inhibited, and the size and mass of the tumors were smaller than those in other groups, with significant differences. This indicates that TDN-siBraf exerted a synergistic anti-tumor effect compared with the use of TDN and siBraf alone.

[0115] 3.2 Safety assessment

[0116] H&E staining was performed on the main organs to evaluate biological safety. The results showed ( Figure 15 ) that there was less tissue degeneration in the liver, kidney, and heart in all four groups, and there was no significant difference in the density of immune cells in the spleen among the groups. In addition, there was no significant difference in body weight change among the groups. Routine blood tests and liver and kidney function tests were performed on the mice on the day of euthanasia. The results of hematological analysis showed that the TDN-siBraf system had no adverse effects on the main indicators ( Figure 16 A). The detection of white blood cell count and classification showed that there was no significant difference among the groups ( Figure 16 B). The experimental results indicate that the TDN-siBraf prepared by the present invention has good in vivo safety.

[0117] 3.3 Tissue staining and TUNEL staining were used to detect apoptosis of thyroid cancer cells in each group

[0118] The results are shown in the figure. HE staining showed ( Figure 15 the first column) that the stromal density of the tumor tissue in the TDN-siBraf group was lower and the necrotic foci were more dispersed, indicating a more ideal therapeutic effect. To evaluate the knockdown efficiency of Braf expression in thyroid cancer tissues, immunohistochemistry (IHC) analysis was performed. The results were as Figure 17 shown, and the results showed that the Braf expression level in the TDN-siBraf group was significantly decreased. IHC staining also showed that the staining intensities of Ki-67 and MMP9 in the TDN-siBraf group were significantly decreased, indicating a decrease in tumor proliferation and invasion ability. At the same time, the results of TUNEL staining showed that the apoptosis level in the TDN-siBraf group was significantly increased, while almost no obvious apoptotic signals were seen in the TDN group and the siBraf group. The experimental results show that the TDN-siBraf group can significantly promote apoptosis of thyroid cancer cells and play a synergistic effect compared with the use of TDN and siBraf alone.

[0119] Experimental example 7: TDN-siBraf exerts an anti-tumor effect by inhibiting MEK-ERK

[0120] Partial tissues from subcutaneous tumors of nude mice treated with TDN-siBraf and siBraf were used for transcriptome sequencing. The tissues were lysed with Trizol reagent, and the cell lysates were stored at -80 °C for sequencing. RNA sequencing was performed using the Illumina HiSeq X10 platform (Illumina). Gene expression values were transformed into log10[TPM (transcripts per million reads) + 1], and gene expression was quantified by the FPKM method. Significance analysis was performed based on p-value and false discovery rate (FDR) analysis. Differentially expressed genes were defined as fold change > 2 or < 0.5, and FDR < 0.05. Bioinformatics analysis was performed using the OECloud tool provided by https: / / cloud.oebiotech.com. Samples were extracted from mouse tumor tissues and subjected to transcriptome analysis. Subsequently, RNA sequencing was used to compare the gene expression profiles between the TDN-siBraf group and the siBraf group. The results of KEGG analysis showed that the most significantly upregulated pathways could be divided into four main components ( Figure 18 A), among which the pathway related to necroptosis was significantly upregulated. Among the significantly downregulated pathways, the MAPK signaling pathway decreased significantly after treatment with TDN-siBraf ( Figure 18 B), suggesting that TDN-siBraf could inhibit MEK-ERK signal transduction. Further GO and KEGG pathway analysis results showed that the expression level of SLC7A11 decreased significantly ( Figure 18 C and 18D). As a downstream target of the MAPK pathway, the reduction of SLC7A11 further supported the inhibitory effect of TDN-siBraf on the MEK-ERK signaling cascade.

[0121] Two thyroid cancer cell lines, KTC-1 and OCUT-2C, were cultured separately and treated with medium containing TDN, siBraf, or TDN-siBraf (concentration of 100 nM, without fluorescence and without sticky ends for siBraf and TDN) for 2 days. Immunofluorescence staining results showed that compared with the other three groups, pretreatment with TDN-siBraf could significantly reduce the expression levels of p-MEK and p-ERK in both cell lines ( Figure 19 ). In addition, the experiment also found that after treatment with TDN-siBraf, p-ERK was mainly localized in the cytoplasm, while in other groups, p-ERK was mainly located in the nucleus ( Figure 19 D, indicated by the arrow). Meanwhile, Western blot results showed that compared with the control group, the TDN group, and the siBraf group, pretreatment with TDN-siBraf could significantly reduce the expression of p-MEK and p-ERK in both cell lines ( Figure 20)。In contrast, among the four groups of treatments for the two cell lines, there were no significant differences in the expression levels of total MEK and ERK.

[0122] In summary, the TDN-siBraf complex of the present invention can be enriched at the tumor site, effectively taken up by cancer cells, inhibit Braf expression, inhibit the proliferation and invasion of cancer cells, promote apoptosis, inhibit the activation of the MEK-ERK signaling pathway, increase the levels of mitochondrial dysfunction and DNA damage, and exhibit good synergistic anti-tumor effects. At the same time, the TDN-siBraf complex of the present invention has good biosafety and biocompatibility. Therefore, the TDN-siBraf complex of the present invention is suitable for the preparation of thyroid cancer-related drugs and has good industrial application prospects.

Claims

1. A tetrahedral framework nucleic acid complex for Braf gene silencing, characterized in that: It is a tetrahedral framework nucleic acid carrying siRNA; the forward sequence of the siRNA is shown as SEQ ID NO.8, and the reverse sequence is shown as SEQ ID NO.

7.

2. The tetrahedral framework nucleic acid complex according to claim 1, characterized in that: The four single-stranded DNA sequences of the tetrahedral framework nucleic acid are shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

3. The tetrahedral framework nucleic acid complex according to claim 1 or 2, characterized in that: The molar ratio of the tetrahedral framework nucleic acid to the siRNA is 1:

1.

4. A method for preparing the tetrahedral framework nucleic acid complex according to any one of claims 1 to 3, characterized in that: It includes the following steps: (1) four single-stranded DNA sequences of a tetrahedral framework nucleic acid are added to a TM buffer, maintained at 90 to 95°C for 10 to 30 minutes, and then rapidly cooled to 0 to 4°C and maintained for more than 20 minutes to obtain a tetrahedral framework nucleic acid with sticky ends; (2) The tetrahedral framework nucleic acid with sticky ends is mixed with siRNA and then incubated to obtain the product.

5. The method according to claim 4, characterized in that: In step (1), the final concentration of the four single-stranded DNA sequences is 1 μM; And / or, in step (1), the pH value of the TM buffer is 8.

0.

6. The method according to claim 4, characterized in that: In step (2), the molar ratio of the tetrahedral framework nucleic acid with sticky ends to the siRNA is 1:1; And / or, in step (2), the incubation is at 25-37° C. for 20-30 min.

7. Use of the tetrahedral framework nucleic acid complex according to any one of claims 1 to 3 in the preparation of a drug for Braf gene silencing.

8. Use of the tetrahedral framework nucleic acid complex according to any one of claims 1 to 3 in the preparation of a drug for preventing and / or treating a disease associated with Braf gene silencing; Preferably, the disease associated with Braf gene silencing is cancer.

9. The use according to claim 8, characterized in that: The disease associated with Braf gene silencing is thyroid cancer; Preferably, the thyroid cancer is papillary thyroid carcinoma or anaplastic thyroid carcinoma.

10. A drug for preventing and / or treating thyroid cancer, characterized in that: The invention is prepared by taking the tetrahedral framework nucleic acid complex described in any one of claims 1 to 3 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

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