Application of NDUFS6 target spot in resisting neuroblastoma

Abstract

The invention discloses application of an NDUFS6 target spot in resisting neuroblastoma, and relates to the technical field of biological medicines. The invention discloses an application of an NDUFS6 inhibitor in preparation of antitumor drugs, especially neuroblastoma. The invention discloses the regulation and control effect of NDUFS6 on tumor-related phenotypes of NB cells for the first time, realizes the inhibition effect on neuroblastoma by inhibiting the gene level and protein level of NDUFS6, and provides new target selection and medicine for treatment of neuroblastoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of the NDUFS6 target in the treatment of neuroblastoma. Background Technology

[0002] Neuroblastoma (NB) is the most common extracranial solid malignant tumor in children. Originating from sympathetic adrenal progenitor cells, it primarily occurs in the adrenal medulla and exhibits high heterogeneity. High-risk NB (HRNB) patients, after induction chemotherapy, surgery, high-dose chemotherapy combined with autologous stem cell salvage therapy and anti-GD2 immunotherapy, have a 5-year event-free survival rate of only 51%, and a 4-year overall survival rate of only 20% for relapsed patients. Furthermore, patient response to treatment varies greatly, and this heterogeneity severely limits the development of universal therapies. More effective drugs are urgently needed for HRNB.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide the application of NDUFS6 as a target in the treatment of neuroblastoma. This invention reveals for the first time the regulatory role of NDUFS6 on the tumor-associated phenotype of NB cells, and achieves an inhibitory effect on neuroblastoma by inhibiting both the NDUFS6 gene and protein levels, thus providing a new target and drug for the treatment of neuroblastoma.

[0005] The extremely complex heterogeneity of neuroblastoma is a major obstacle to the treatment of refractory and recurrent tumors. In the future, tumor treatment may develop towards personalized treatment. Analyzing the heterogeneity and pathogenesis of neuroblastoma and developing effective drugs for a large number of patients as quickly as possible are currently hot research topics.

[0006] In a first aspect, the present invention provides the use of an inhibitor of NDUFS6 in the preparation of an antitumor drug, wherein the tumor is neuroblastoma.

[0007] This invention, for the first time, inhibits the proliferation, invasion, and migration of neuroblastoma at the gene level by knocking down the expression of the NDUFS6 gene in neuroblastoma cells. At the protein level, this invention utilizes computer technology for virtual drug screening, using the NDUFS6 protein molecule as a target, and screens for compounds that strongly bind to the NDUFS6 protein through two-dimensional and three-dimensional molecular docking technology; and verifies the inhibitory effect of these compounds on neuroblastoma.

[0008] The results of the embodiments of this invention fully demonstrate that NDUFS6 can serve as a potential therapeutic target for neuroblastoma, and inhibitors that inhibit this target can be used to formulate drugs against neuroblastoma. This invention provides a new therapeutic target and treatment regimen for the treatment of neuroblastoma.

[0009] Optionally, in some embodiments of the present invention, the inhibitor may inhibit, weaken, interfere with, or silence the expression of NDUFS6, which may be inhibiting, weakening, interfering with, or silencing the expression of NDUFS6 at the gene level, such as at the RNA level, or inhibiting, weakening, interfering with, or silencing the expression of NDUFS6 at the protein level.

[0010] Optionally, in some embodiments of the present invention, the inhibitor may bind to the NDUFS6 protein to inhibit or attenuate its activity.

[0011] Optionally, in some embodiments of the present invention, the inhibitor is selected from 1,4-bD-Xylopentaose, Guanosine-5'-triphosphate (disodium salt), Forsythoside I, Xylotetraose, Nicotiflorin, Isomaltotetraose, Fodipir, Tubuloside A, NADPH (tetrasodium salt), Deferoxamine (mesylate), GDP-α-D-mannose (disodium), and Solasonine.

[0012] Optionally, in some embodiments of the present invention, the inhibitor may be a protein degrading agent, such as a protein degradation-targeting chimera (PROTAC).

[0013] Optionally, in some embodiments of the present invention, the expression of the NDUFS6 gene may be suppressed, weakened, interfered with, or silenced.

[0014] Optionally, in some embodiments of the present invention, the inhibitor is selected from siRNA and shRNA.

[0015] The core molecules of different RNA technologies are easily obtained by those skilled in the art through conventional techniques based on the nucleic acid sequence of the NDUFS6 gene.

[0016] NDUFS6 gene ID: 4726 (https: / / www.ncbi.nlm.nih.gov / datasets / gene / 4726 / ) NDUFS6 protein ID: CAG33161 (https: / / www.ncbi.nlm.nih.gov / protein / CAG33161.1)

[0017] Optionally, in some embodiments of the present invention, the above-mentioned NDUFS6 gene and its protein are human NDUFS6 gene and its protein.

[0018] On the other hand, the present invention provides a drug for inhibiting neuroblastoma, comprising an inhibitor that targets NDUFS6 to inhibit the expression or activity of NDUFS6.

[0019] Optionally, in some embodiments of the present invention, the inhibitor may bind to the NDUFS6 protein to inhibit or attenuate its activity.

[0020] Optionally, in some embodiments of the present invention, the inhibitor is selected from 1,4-bD-Xylopentaose, Guanosine-5'-triphosphate (disodium salt), Forsythoside I, Xylotetraose, Nicotiflorin, Isomaltotetraose, Fodipir, Tubuloside A, NADPH (tetrasodium salt), Deferoxamine (mesylate), GDP-α-D-mannose (disodium), and Solasonine.

[0021] Optionally, in some embodiments of the present invention, the inhibitor may inhibit, weaken, interfere with, or silence the expression of the NDUFS6 gene.

[0022] Optionally, in some embodiments of the present invention, the drug is selected from siRNA and shRNA.

[0023] Optionally, in some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0024] On the other hand, the present invention provides a method for treating neuroblastoma, comprising: administering the drug as described above to the subject requiring treatment.

[0025] Optionally, in some embodiments of the present invention, the subject is selected from humans or non-human primates such as monkeys and orangutans. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Statistical analysis of qPCR results for NDUFS6 overexpression and knockdown stable transgenic lines; A. qPCR results of NDUFS6 overexpression and knockdown stable transgenic lines constructed from SK-N-BE(2) cell line. The difference in NDUFS6 mRNA level between the OE group and the OENC group was extremely significant (P=0.0015), significantly higher; the difference in NDUFS6 mRNA level between the KD group and the KDNC group was extremely significant (P<0.0001), significantly lower; B. qPCR results of NDUFS6 overexpression and knockdown stable transgenic lines constructed from SH-SY5Y cell line. The difference in relative NDUFS6 mRNA expression level between the OE group and the OENC group was extremely significant (P<0.0001), significantly higher; the difference in relative NDUFS6 mRNA expression level between the KD group and the KDNC group was extremely significant (P=0.0067), significantly lower.

[0028] Figure 2 : WB bands and statistical results of NDUFS6 overexpression and knockdown stable transgenes; The relative expression level of NDUFS6-FLAG protein in the OE group of SK-N-BE(2) was significantly higher than that in the OENC group (P=0.0493); The relative expression level of NDUFS6 protein in the KD group of SK-N-BE(2) was not significantly different from that in the KDNC group (P=0.7482); The relative expression level of NDUFS6-FLAG protein in the OE group of SK-N-BE(2) was significantly higher than that in the OENC group (P=0.0030); The relative expression level of NDUFS6-FLAG protein in the KD group of SK-N-BE(2) was significantly higher than that in the KDNC group.

[0029] Figure 3 CCK8 cell proliferation curve; A and B are SK-N-BE(2) cell proliferation curves; C and D are SH-SY5Y cell proliferation curves. The absorbance values ​​of the two cell types at 450 nm were detected for 4 consecutive days. KD represents knockdown, KDNC represents the corresponding empty vector control; OE represents overexpression, OENC represents the corresponding empty vector control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0030] Figure 4: Bar chart showing clone formation samples and clone count statistics; A is a bar chart showing the overexpression and knockdown stable transgenes of SK-N-BE(2) cells. The number of clones in the OE group was higher than that in the OENC group, with a highly significant difference (P = 0.0003), and the number of clones in the KD group was higher than that in the KDNC group, with a highly significant difference (P = 0.0027); B is a bar chart showing the overexpression and knockdown stable transgenes of SH-SY5Y cells. The number of clones in the OE group was higher than that in the OENC group, with a significant difference (P = 0.0365), and the number of clones in the KD group was higher than that in the KDNC group, with a highly significant difference (P = 0.0003). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0031] Figure 5 : Transwell invasion and migration experiment results and statistical charts; A shows the invasion status and number of SK-N-BE(2) cells in the NDUFS6OE and OENC groups after 24 h; B shows the migration status and number of SK-N-BE(2) cells in the NDUFS6KD and KDNC groups after 48 h; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

[0032] Figure 6 : Transwell invasion and migration assay results and statistical graphs; C shows the invasion status and cell number statistics of SH-SY5Y cells in the NDUFS6 OE and OENC groups after 48 h; D shows the migration status and cell number statistics of SH-SY5Y cells in the NDUFS6 KD and KDNC groups after 48 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

[0033] Figure 7 The results of the scratch assay are shown in the following figures: A shows the scratch pattern and relative migration area of ​​SK-N-BE(2) cells in the NDUFS6 OE and OENC groups at 0 h and 36 h (P<0.0001); B shows the scratch pattern and relative migration area of ​​SK-N-BE(2) cells in the NDUFS6 KD and KDNC groups at 0 h and 36 h (P<0.0001); *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0034] Figure 8The results of the scratch assay are shown in the graphs. C represents the scratch pattern and relative migration area of ​​SH-SY5Y cells in the NDUFS6 OE and OENC groups at 0h and 36h (P<0.0001); D represents the scratch pattern and relative migration area of ​​SH-SY5Y cells in the NDUFS6 KD and KDNC groups at 0h and 24h (P<0.0001). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0035] Figure 9 : A simulation diagram of the molecular docking of the drug Xylotetraose (Docking score: -9.266); the left side is the 2D interaction diagram, and the right side is the 3D interaction diagram. In the 3D diagram, the C backbone of the Human NDUFS6 protein is shown in green, N atoms in blue, O atoms in bright red, and H atoms in white. HY-N6840 is shown as a gray-white columnar assembly structure. Hydrogen bond lengths are shown as red dashed lines; the longer the bond length, the weaker the hydrogen bond interaction. HY-N6840 can form 6 hydrogen bonds with TYR46, GLN44, GLN60, and GLU62 of the Human NDUFS6 protein; the specific distances are shown in the 3D interaction diagram.

[0036] Figure 10 Line graph of relative inhibition rate as a function of drug concentration in SK-N-BE(2) drug sensitivity test; A Figure 1 ,4-bD-Xylopentaose GI50 (μM) is 1.245; Guanosine-5'-triphosphate (disodium salt) GI50 (μM) is 0.8258; B chart Forsythoside I GI50 (μM) <0.0137; Xylotetraose GI50 (μM) <0.0137; Nicotiflorin GI50 (μM) <0.0137; Isomaltotetraose GI50 (μM) <0.0137.

[0037] Figure 11Line graphs showing the relative inhibition rate of SH-SY5Y drug susceptibility test as a function of drug concentration: Figure A: Fmoc-Ala-Glu-Asn-Lys-NH2 GI50 (μM) is 3.879; Deferoxamine (mesylate) GI50 (μM) is 3.44; Solasonine GI50 (μM) is 12.44; Figure B: Fodipir GI50 (μM) is 105.8; Guanosine-5'-triphosphate (disodium salt) GI50 (μM) is 61; Tubuloside A GI50 (μM) is 64.8. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Example 1

[0041] Construction of stable transgenes with NDUFS6 overexpression and knockdown.

[0042] To verify the function of NDUFS6 in NB cells, this example used two NB cell lines, SK-N-BE(2) and SH-SY5Y, and constructed stable transgenic lines of SK-N-BE(2) with NDUFS6 overexpression, SK-N-BE(2) with NDUFS6 knockdown, SH-SY5Y with NDUFS6 overexpression, and SH-SY5Y with NDUFS6 knockdown. qPCR results showed that all four stable transgenic lines were successfully constructed. Figure 1 )

[0043] The target sequence of the siRNA used for NDUFS6 knockdown is: CACATGCGGTTACTGTGGGCT.

[0044] Western blot analysis was used to detect the expression levels of NDUFS6 protein in NDUFS6 overexpression or knockdown SK-N-BE(2) and SH-SY5Y cells to verify the construction of stable transgenic cells. The results showed that exogenous NDUFS6 protein overexpression was detected in both the overexpression and knockdown groups, but there was no significant difference between the knockdown and knockdown control groups.

[0045] Example 2

[0046] The regulatory effect of NDUFS6 on the proliferation of SH-SY5Y and SK-N-BE(2) cells.

[0047] The proliferation ability of stable transgenic cells with NDUFS6 overexpression and knockdown was detected using the Cell Counting Kit 8 (CCK8) assay to verify the regulatory effect of NDUFS6 on cell proliferation. CCK8 results showed that increased NDUFS6 expression in SK-N-BE(2) cells led to a relative increase in cell proliferation rate, suggesting that NDUFS6 overexpression promotes cell proliferation; decreased NDUFS6 expression led to a relative decrease in cell proliferation rate, suggesting that NDUFS6 knockdown inhibits cell proliferation. Figure 3 ).

[0048] Example 3

[0049] The colony formation assay was used to detect the ability of stable cell lines overexpressing and knockdown of NDUFS6 to proliferate from single or very few cells into visible cell clusters, thereby verifying the effect of NDUFS6 on cell proliferation. According to the statistical analysis of the colony formation assay, after NDUFS6 expression was increased in SK-N-BE(2) cells, the number of cell clusters formed by colonies was relatively high within a certain period of time (P = 0.0003), which suggests that the cell proliferation capacity was enhanced, so overexpression has a promoting effect on cell proliferation; after NDUFS6 expression was decreased, the number of cell clusters formed by colonies was relatively low within a certain period of time (P = 0.0027), which suggests that the cell proliferation capacity was weakened, so overexpression has an inhibitory effect on cell proliferation. Increased NDUFS6 expression in SH-SY5Y cells resulted in a relatively high number of cell clumps formed within a certain timeframe (P = 0.0365), suggesting enhanced cell proliferation; therefore, overexpression promotes cell proliferation. Conversely, decreased NDUFS6 expression resulted in a relatively low number of cell clumps formed within a certain timeframe (P = 0.0003), indicating weakened cell proliferation; therefore, decreased expression inhibits cell colony formation. Figure 4 ).

[0050] Example 4

[0051] The regulatory role of NDUFS6 in the invasion and migration of SH-SY5Y and SK-N-BE(2) cells.

[0052] The above cell experiments have preliminarily demonstrated the promoting effect of NDUFS6 on NB cell proliferation. In order to further explore the regulatory effect of increased NDUFS6 expression on other tumor-related phenotypes of NB cells, this example then verified the effect of NDUFS6 on the invasion and migration ability of NB cell lines SK-N-BE(2) and SH-SY5Y cells. Transwell invasion and migration assays were performed on stable cell lines with NDUFS6 overexpression and knockdown. The results showed that in the invasion assay, overexpression of NDUFS6 promoted the invasion of SK-N-BE(2) cells and SH-SY5Y cells (P < 0.0001; P < 0.0001), while knockdown of NDUFS6 weakened the invasion of tumor SK-N-BE(2) cells and SH-SY5Y cells (P < 0.0001; P = 0.0051). In the migration assay, overexpression of NDUFS6 promoted the migration of SK-N-BE(2) cells and SH-SY5Y cells (P < 0.0001; P < 0.0001), while knockdown of NDUFS6 weakened the migration of SH-SY5Y cells (P < 0.0001), but did not significantly weaken the migration of SK-N-BE(2) cells. Figures 5-6 ).

[0053] Example 5

[0054] The scratch healing test can be used as a supplementary detection method to more comprehensively assess cell migration ability. A scratch of uniform width is created on the bottom of a dish covered with cells to observe cell migration towards the center. The scratch healing test results suggest that overexpression of NDUFS6 significantly promotes the migration ability of SK-N-BE(2) cells and SH-SY5Y cells, while knockdown of NDUFS6 significantly weakens the migration ability of SK-N-BE(2) cells and SH-SY5Y cells. Figures 7-8 ).

[0055] Example 6

[0056] Virtual drug screening results

[0057] Two hundred drug molecules from the MCE library were selected based on calculations showing they could dock with the NDUFS6 protein molecule; all had docking scores greater than 4.25. The docking score is a numerical value reflecting the intermolecular binding ability in virtual drug screening; a value greater than 4.25 indicates binding ability, greater than 5 indicates strong binding ability, and greater than 7 indicates extremely strong binding ability. The docking scores were sorted in ascending order, and the top 30 drugs were selected. The lowest docking score was 6.753, and the highest was 9.266. The table below shows the 30 drugs with strong or extremely strong binding ability. Figure 9This is a molecular docking simulation diagram of the drug Xylotetraose.

[0058] Table 1. Information on the top 30 drugs by virtual drug screening scores.

[0059]

[0060] Example 7

[0061] Cellular drug sensitivity test results

[0062] (a) Results of SK-N-BE(2) in the MYCN-positive NB cell line

[0063] In the SK-N-BE(2) cell line, the top 30 drugs with the highest docking fractions were selected for validation. The relative inhibition rates of compounds 1,4-bD-Xylopentaose, Guanosine-5'-triphosphate (disodium salt), Forsythoside I, Xylotetraose, Nicotiflorin, and Isomaltotetraose reached over 50% at 48 h with increasing concentration. Among them, 1,4-bD-Xylopentaose and Guanosine-5'-triphosphate (disodium salt) showed a relative inhibition rate close to 70% at 48 h and exhibited a steep increasing trend with increasing concentration. The relative inhibition rates of Forsythoside I, Xylotetraose, Nicotiflorin, and Isomaltotetraose fluctuated between 50-60% at 48 h, showing little change with concentration. 1,4-bD-Xylopentaose and Guanosine-5'-triphosphate (disodium salt) showed more significant inhibitory effects on MYCN-amplified tumor cells. Figure 10 ).

[0064] (ii) Results in the MYCN-negative NB cell line SH-SY5Y

[0065] In the SH-SY5Y cell line, the relative inhibition rates of compounds Fodipir, Guanosine-5'-triphosphate (disodium salt), Tubuloside A, Fmoc-Ala-Glu-Asn-Lys-NH2, NADPH (tetrasodium salt), Deferoxamine (mesylate), GDP-α-D-mannose (disodium), and Solasonine reached over 50% with increasing drug concentration after 48 hours. Among them, the relative inhibition rates of Fmoc-Ala-Glu-Asn-Lys-NH2, Deferoxamine (mesylate), and Solasonine were close to 100% after 72 hours. The relative inhibition rates of Fodipir, Guanosine-5'-triphosphate (disodium salt), and Tubuloside A still showed a steep increasing trend with increasing drug concentration after 72 hours.

[0066] In summary, this invention, for the first time, inhibits the proliferation, invasion, and migration of neuroblastoma at the gene level by knocking down the expression of the NDUFS6 gene in neuroblastoma cells. At the protein level, this invention utilizes computer technology for virtual drug screening, using the NDUFS6 protein molecule as a target, and screens for compounds that strongly bind to the NDUFS6 protein through two-dimensional and three-dimensional molecular docking techniques; and verifies the inhibitory effect of these compounds on neuroblastoma.

[0067] The results of the embodiments of this invention fully demonstrate that NDUFS6 can serve as a potential therapeutic target for neuroblastoma, and inhibitors that inhibit this target can be used to formulate drugs against neuroblastoma. This invention provides a new therapeutic target and treatment regimen for the treatment of neuroblastoma.

[0068] Experimental methods involved in Examples 1-7:

[0069] (I) Construction of stable cell lines

[0070] 1. Spread wild-type SH-SY5Y and SK-N-BE(2) into a six-well plate and change the medium when the growth reaches 40%-50%.

[0071] 2. Add approximately 50 μL of virus solution (MOI 20), 0.5 μL of polybreane transfection reagent, and 1 ml of culture medium to each well.

[0072] 3. Change the medium after 16 hours. 48 hours after the medium change, add 2 ml / well of puromycin-containing medium (50 ml + 10 μl puromycin). Continue culturing in this medium for 4-5 days to kill cells that failed to transfect, until all wild-type cells in the blank control group have died. Continue culturing for 1 day, then switch to regular medium. Passaging is not affected during this process.

[0073] (II) Extraction of Cell Proteins

[0074] 1. Prepare RIPA protein lysis buffer on ice. The ratio of RIPA to protease inhibitor and phosphorylase inhibitor is 100:1. 150 μL is needed for each well of a six-well plate.

[0075] 2. Culture the cells in a six-well plate. When the cells reach 100% confluence, remove them, discard the culture medium, and wash twice with 1 ml PBS at 4°C.

[0076] 3. Add 150 μL of the prepared RIPA protein lysis buffer to each well of a six-well plate, distribute evenly, and incubate on ice for 5 minutes.

[0077] 4. With the tip of a 200ul pipette inverted, scrape the bottom of a six-well plate back and forth in different directions on the inverted, pre-chilled metal frame of the EP tube. This will cause the lysed cell fluid to pool on one side, and then transfer it to a 1.5ul EP tube using a 200ul pipette.

[0078] 5. Incubate on ice for 30 minutes to further lyse the protein.

[0079] 6. Centrifuge at 4℃, 16.5*10^3 g, for 20 min.

[0080] 7. Use a 200ul pipette tip to aspirate the flocculent precipitate and retain the supernatant.

[0081] 8. Take 3 μL of the retained liquid for subsequent BCA concentration determination.

[0082] 9. Measure the liquid volume using a 200ul syringe and add 5 × SDS as directed.

[0083] 10. Heat the remaining liquid protein at 95°C for 10 minutes.

[0084] (III) Cell RNA Extraction

[0085] 1. Culture the cells in a six-well plate. When the cells reach 100% confluence, remove them, discard the culture medium, and wash twice with 1 ml of ice-cold PBS.

[0086] 2. Perform the following procedures on ice to minimize RNA lysis. Add 1 ml of Trizol reagent, lyse cells for 2 min, and transfer the lysis buffer to an EP tube.

[0087] 3. Add 200 μL of chloroform, gently invert and mix 50 times, then let stand for 5 minutes.

[0088] 4. Centrifuge at 4℃, 12*10^3 g, for 15 min.

[0089] 5. Transfer the supernatant into a new EP tube (be careful not to aspirate the middle membrane), add an equal volume of isopropanol, invert and mix several times, and let stand on ice for 20 minutes.

[0090] 6. Centrifuge at 4℃, 12*10^3 g, for 15 min, and remove the supernatant.

[0091] 7. Add 1ml of 75% alcohol. A white RNA precipitate will be visible at the bottom of the tube. Gently invert the tube to wash the RNA while avoiding breaking it.

[0092] 8. Centrifuge at 4℃, 12*10^3 g, for 5 min to remove the supernatant.

[0093] 9. Add 1ml of 100% alcohol and gently invert.

[0094] 10. Centrifuge at 4℃, 12*10^3 g, for 5 min to remove the supernatant.

[0095] 11. Air dry at room temperature for 15 minutes, then dissolve the RNA in 20 μL of DEPC water on ice for 10 minutes. Store at -80°C.

[0096] (iv) RNA reverse transcription

[0097] 1. RNA concentration determination: Thaw the sample RNA on ice, gently tap the bottom of the EP tube to mix the RNA, take 1 μL of each sample and drop it onto the cleaned spot of the Nanodrop2000 instrument, measure the concentration, and dilute the RNA with an appropriate amount of DEPC water to a concentration of 500-1000 μg / μL for later use.

[0098] 2. Thaw the template RNA on ice. After thawing the 5× FastKing-RT SuperMix and RNase-Free ddH,O at room temperature, quickly place them on ice and vortex to mix.

[0099] 3. Prepare the reverse transcription reaction system on ice as shown in the table below.

[0100] Table 2. Components of the Reverse Transcription System

[0101]

[0102] 4. Perform the reverse transcription reaction as shown in the table below.

[0103] Table 3 Reverse Transcription Procedure

[0104]

[0105] 5. Add 180 μL of DEPC water to dilute the cDNA solution, and store it in a freezer at -20°C.

[0106] (v) Detection of target gene RNA level using RT-qPCR

[0107] 1. Select the target gene and use the Primer Bank primer library. Human primer pairs were identified, and primers were synthesized by a biotechnology company (see table). GAPDH was used as an internal control for RT-qPCR.

[0108] Table 4 Human Gene Primer Information Table

[0109]

[0110] 2. Prepare the reaction system using the Universal Blue qPCR SYBR Green Master Mix kit (11184ES08), as shown in Table 5.

[0111] Table 5 RT-qPCR Primer List

[0112]

[0113] 3. Use the Applied Biosystems QuantStudio 3 Real-Time PCR instrument to perform real-time PCR detection. The procedure is shown in Table 6.

[0114] Table 6 RT-qPCR reaction procedure

[0115]

[0116] 4. Observe the melting curve, and after confirming that there are no abnormal peaks, export the data and use the 2−ΔΔCT method in Excel software to calculate the relative expression level of the target gene.

[0117] (vi) Protein concentration determination

[0118] 1. Prepare BCA reagents: 200 μL of reagent A per sample, and 4 μL of reagent B per sample, with an A:B ratio of 50:1. Prepare the standard at the specified concentration of 0.5 mg / mL according to the table below, and add it directly to the 96-well plate.

[0119] Table 7 Standard Product Configuration

[0120]

[0121] 2. Thaw the protein sample to be used for concentration measurement on ice, dilute it 10 times with PBS, mix well, and add 10 μL of diluted protein solution to the corresponding well of a 96-well plate.

[0122] 3. Add 200 μL of the prepared BCA reagent to each well. Do not let the pipette tip touch the protein sample. If it does, replace the pipette tip.

[0123] 4. In the heating chamber, at 37 degrees Celsius, for 30 minutes.

[0124] 5. Measure the absorbance (note that you should not touch the bottom of the 96-well plate with your hand).

[0125] 6. Plot a standard curve based on the measured concentration, derive the formula for absorbance versus protein concentration, calculate the protein concentration, and restore it to the concentration before dilution.

[0126] 7. Calculate the protein loading amount of 25ug per sample, set the total loading volume, and calculate the 1X loading supplement volume per sample.

[0127] (vii) Western Blot Experiment

[0128] 1. Prepare the separating gel first: 4 ml separating gel buffer + 4 ml separating gel solution + 80 μL modified ammonium persulfate solution.

[0129] 2. Flattening the adhesive: Add 1ml of isopropyl alcohol, flatten the adhesive, and after 15 minutes, pour off the isopropyl alcohol. Clean the interface 3 times with ddH2O, pour out the water, and use lens paper to absorb the residual water in the tank.

[0130] 3. Prepare the stacking gel: 1 ml stacking gel solution + 1 ml colored stacking gel buffer + 20 μl modified ammonium persulfate solution (coagulant).

[0131] 4. Add a second layer of adhesive: Prepare double the volume of the second layer of adhesive, adding it until it overflows.

[0132] 5. Insert the comb: Select a comb of the appropriate thickness according to the size of the board, wash it clean, let it dry, align it with the top layer of glue, and let it sit for 15 minutes.

[0133] 6. While waiting, the protein sample can be thawed on ice. After it returns to a liquid state, reheat the protein at 95°C for 5 minutes. After cooling, centrifuge to remove the water droplets from the top of the tube to the bottom. Prepare the protein sample and 1X loading according to the previously calculated concentration, mix well and centrifuge, and place on ice for later use.

[0134] 7. Prepare the electrophoresis buffer: add 900 ml of ddH2O to 100 ml of 10X glycine electrophoresis buffer and mix well.

[0135] 8. Install the electrophoresis rack: Wet the glass plate and the slot with electrophoresis solution. Align the front and back plates horizontally with the notch in the clips, press them firmly, transfer them to the electrophoresis tank, add electrophoresis solution, and observe for leakage. If there is no leakage, the comb can be pulled out. Note that the pulling out process should be done slowly and vertically to avoid deformation of the glue hole.

[0136] 9. Sample loading: Add the calculated volume of protein. During the loading process, be careful to avoid generating air bubbles. Any residual protein at the bottom of the EP tube should be rinsed with a small amount of electrophoresis buffer and added to the same well. The loading process should be quick to prevent the loaded sample from spreading excessively to both sides of the gel well, which could lead to connected bands in the final result.

[0137] 10. Electrophoresis: First, adjust to 25V for 10 minutes to flatten the protein sample, then switch to 80V for about 25 minutes to run the stacking gel; then adjust to 120V for about 35 minutes to run the separating gel to 1 / 2.

[0138] 11. Transfer preparation: Prepare ethanol-based ice-free transfer solution, cut PVDF membrane (size: 5.5×8.5cm), activate with methanol for 1 minute before use, shake on a shaker, then immerse in the transfer solution, remove and pry open the glass plate, cut off excess gel, transfer to the black filter paper of the transfer clamp, align the gel position, cover with the PVDF membrane, remove air bubbles, clamp the transfer clamp, and install the transfer tank in sequence.

[0139] 12. Transfer: Use constant current mode, 300mA for 45min, ice bath.

[0140] 13. Washing the membrane: After the time is up, disassemble the membrane transfer frame, observe the membrane transfer, transfer the membrane to the washing tank, and wash it once with TBST for 5 minutes.

[0141] 14. Sealing: Seal with 5% skim milk powder for 1.5 hours.

[0142] 15. Film cutting: Cut the film according to the molecular weight.

[0143] 16. Washing the membrane: Wash the membrane with TBST for 10 minutes each time, for a total of 3 washes.

[0144] 17. Apply 4 ml of primary antibody per tank and incubate overnight at 4°C on a shaker.

[0145] 18. Recover the primary antibody.

[0146] 19. Washing the membrane: Wash the membrane with TBST for 10 minutes each time, at a rapid speed of 230 rpm / min, for a total of 3 washes.

[0147] 20. Apply secondary antibody: 6 ml / tank, incubate on a shaker at room temperature for 1 hour.

[0148] 21. Recover the secondary antibody.

[0149] 22. Washing the membrane: TBST washing, 10 min / time, fast 230 rpm / min, for a total of 3 washes.

[0150] 23. Prepare the exposure solution fresh at a 1:1 ratio.

[0151] 24. Use BIO-RAD ChemiDoc, expose the image using Image Lab software, and save the image after taking the picture.

[0152] 25. Use ImageJ software to analyze the gray values. Divide the gray value of the target protein by the gray value of the inner reference band in the corresponding lane, and use this as the relative expression level of the target protein in that group.

[0153] (viii) Detection of CCK8 proliferation activity

[0154] 1. Five cell proliferation detection time points were set: 0h, 24h, 48h, 72h and 96h.

[0155] 2. Set up 5-6 accessory wells for each experimental group or control group. When the cells grow to a sufficient number, wash, digest with trypsin, count, add 2200 cells to each well of the 96-well plate of SH-SY5Y, and add 1700 cells and 100μl of cell culture medium to each well of SK-N-BE(2).

[0156] 3. After the cells have adhered, remove the 96-well plate from the incubator at the set time point, discard the culture medium, and add 110 μl of premixed CCK8 reagent per well (100 μl of culture medium mixed with 10 μl of CCK8 reagent) under light-protected conditions.

[0157] 4. After incubating in an incubator for 2 hours, the OD value was measured using an ELISA reader as a control time point for proliferation detection.

[0158] 5. Detect OD values ​​at 24h, 48h, 72h and 96h respectively. Use Excel software to calculate the relative OD values ​​at 24h, 48h, 72h and 96h by comparing the OD values ​​at the time points, and plot the results.

[0159] (ix) Cloning experiments

[0160] 1. After digestion and centrifugation, resuspend the cells in 2 ml of culture medium per T25, and count them using a counting chamber and an automated cell counter to determine the cell suspension concentration.

[0161] 2. Based on the measured concentration, calculate the number of cells to be added to each well of the 6-well plate. Add 1200 cells to each well of SK-N-BE(2) and 1700 cells to each well of SH-SY5Y. Place the cells to be seeded in an EP tube containing 1000 μL of culture medium and then transfer it to the corresponding well of the 6-well plate. Gently pipette to mix and ensure that the cells are evenly distributed at the bottom of the well. Culture at 37°C and 5% CO2 for 14 days for SK-N-BE(2) and 19 days for SH-SY5Y. Minimize movement during the first 7 days. Change the medium on the 7th day and add or change the medium 1-2 days later.

[0162] 3. Once the clones reach a considerable size, fix and stain them. Remove the six-well plate, wash once with ice-cold PBS, add 1 ml of 4% paraformaldehyde per well, fix for 20 min, wash once with PBS, add 1 ml of 1% crystal violet per well, stain for 15 min, wash away excess dye with ddH2O, and dry in a 37℃ oven for 1 h.

[0163] 4. Take photos with a regular camera.

[0164] (x) Transwell migration experiment

[0165] 1. Starvation treatment of cells: SH-SY5Y cells were cultured in medium containing 3% FBS, and SK-N-BE (2) cells were cultured in serum-free medium for 24 hours.

[0166] 2. Pre-hydration of the upper chamber basement membrane: Add 100 μL of culture medium to the center of the upper chamber, observe to confirm that there is no leakage in the upper chamber, and aspirate the liquid from the upper chamber after standing for more than 3 hours.

[0167] 3. After digesting SH-SY5Y and SK-N-BE(2) with 0.25% trypsin, the number of cells was counted. (The result was 1.5 × 10⁻⁶). 5 SH-SY5Y cells, 1×10 5 SK-N-BE(2) cells were mixed in 200 μL of culture medium. SH-SY5Y cells were mixed in medium containing 3% FBS and SK-N-BE(2) cells were mixed in serum-free medium. 200 μL of cell suspension was added vertically and slowly in the center of the upper chamber to avoid generating air bubbles.

[0168] 4. Use a 24-well plate in the lower chamber and add 600uL of culture medium. Use SH-SY5Y with 20% FBS medium and SK-N-BE(2) with 10% serum medium. Incubate at 37°C for 48h.

[0169] After 3.48 hours, wash twice with PBS, fix with 4% paraformaldehyde for 20 minutes, and wash once with PBS.

[0170] After staining with 5.0.1% crystal violet at room temperature for 15 min, thoroughly wash with PBS and gently wipe away the upper layer of cells with a slightly moistened small cotton swab.

[0171] 6. Take photos under a microscope and count the images.

[0172] (xi) Transwell invasion experiment

[0173] The general steps are the same as those of the Transwell transfer experiment, with the following differences:

[0174] 1. When hydrating the basement membrane, add ice-cold medium premixed with Matrigel at a ratio of 1:15 (Matrigel: medium) on ice. After standing for more than 3 hours, carefully aspirate the liquid in the upper chamber with the tip of a 200ul pipette. Do not aspirate completely to avoid damaging the gel layer that has solidified on the membrane.

[0175] 2. SH-SY5Y culture for 48h, SK-N-BE(2) culture for 24h.

[0176] (xii) Scratch test

[0177] 1. Prepare a six-well plate: On the back of the bottom of a new six-well plate, use the thick end of a marker to draw three lines horizontally and evenly spaced along a sterile ruler. The lines at the top and bottom ends should be far from the edge to avoid excessive light refraction when taking photos.

[0178] 2. Digest, centrifuge, and resuspend the cells. Seed the cells in a six-well plate and use the "cross" shaking method to distribute the cells evenly.

[0179] 3. Once the cells have grown to full size (the cells are packed together and there are almost no gaps at the bottom of the plate), use a 200µl yellow pipette tip to draw a line along the vertical mark in the center of the six-well plate, passing it across the bottom of the well. Apply even pressure from left to right during the drawing process to ensure a smooth line.

[0180] 4. Aspirate the culture medium and floating cell clumps from the wells, wash once with PBS, and add fresh culture medium.

[0181] 5. Microscopic photography: Take photos using a 10 x 20x microscope at 0 h, 24 h, 48 h, and 72 h (for some cells, this may need to be increased by 12 h, 36 h, and 60 h).

[0182] (xiii) Statistical Analysis

[0183] 1. Statistical Analysis Methods: This section analyzes the differences between groups based on RT-qPCR results of SH-SY5Y and SK-N-BE(2) NDUFS6 overexpression and knockdown, WB gray values, colony formation results, Transwell results, and scratch assay results. The Shapiro-Wilk test was used to determine whether the data conformed to a normal distribution, and the F-test was used to determine the homogeneity of variance. After testing, it was found that all data could not simultaneously conform to a normal distribution. Data with homogeneous variances were subjected to unpaired t-tests, while data with unequal variances were subjected to Welch-corrected unpaired t-tests. The CCK8 experimental results were analyzed using two-way ANOVA.

[0184] 2. Significance judgment: P < 0.05 is considered to be statistically significant.

[0185] 3. Visualization of differential analysis: The test results are visualized using Graphpad Prism software (V 10.1.2). Line charts are drawn to display the CCK8 statistical results, and bar charts are drawn to display the statistical results of RT-qPCR, WB, colony formation, Transwell, and scratch assay.

[0186] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of NDUFS6 inhibitors in the preparation of antitumor drugs, characterized in that, The tumor is a neuroblastoma.

2. The application according to claim 1, characterized in that, The inhibitor can bind to the NDUFS6 protein to inhibit or attenuate its activity.

3. The application according to claim 2, characterized in that, The inhibitors are selected from 1,4-bD-Xylopentaose, Guanosine-5'-triphosphate (disodium salt), Forsythoside I, Xylotetraose, Nicotiflorin, Isomaltotetraose, Fodipir, Tubuloside A, NADPH (tetrasodium salt), Deferoxamine (mesylate), GDP-α-D-mannose (disodium), and Solasonine.

4. The application according to claim 1, characterized in that, It can inhibit, weaken, interfere with, or silence the expression of the NDUFS6 gene.

5. The application according to claim 4, characterized in that, The inhibitors are selected from siRNA and shRNA.

6. A drug for inhibiting neuroblastoma, characterized in that, It includes inhibitors that target NDUFS6 to inhibit the expression or activity of NDUFS6.

7. The drug according to claim 6, characterized in that, The inhibitor can bind to the NDUFS6 protein to inhibit or attenuate its activity.

8. The drug according to claim 6 or 7, characterized in that, The inhibitors are selected from 1,4-bD-Xylopentaose, Guanosine-5'-triphosphate (disodium salt), Forsythoside I, Xylotetraose, Nicotiflorin, Isomaltotetraose, Fodipir, Tubuloside A, NADPH (tetrasodium salt), Deferoxamine (mesylate), GDP-α-D-mannose (disodium), and Solasonine.

9. The drug according to claim 6 or 7, characterized in that, The inhibitor can suppress, weaken, interfere with, or silence the expression of the NDUFS6 gene.

10. The medicament according to claim 8, characterized in that, The drug is selected from siRNA and shRNA.

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