CN115851725B

CN115851725B - SiRNA for inhibiting expression of helicase V gene and application thereof

Info

Publication number: CN115851725B
Application number: CN202211334013.3A
Authority: CN
Inventors: 黄茂; 尹浩帆; 黄志坚
Original assignee: Zhuhai Peoples Hospital
Current assignee: Zhuhai Peoples Hospital
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2024-06-11
Anticipated expiration: 2042-10-28
Also published as: CN115851725A

Abstract

The invention belongs to the technical field of biological medicines, and discloses siRNA for inhibiting the expression of a helicase V gene and application thereof. The siRNA comprises siRNA-2 and/or siRNA-3; the nucleotide sequences of the sense strand and the antisense strand in the siRNA-2 are shown as SEQ ID NO. 3-4; the nucleotide sequences of the sense strand and the antisense strand in the siRNA-3 are shown in SEQ ID NO. 5-6. The siRNA has the performance of interfering and inhibiting the expression of the helicase V gene, can inhibit the proliferation of neurogenic tumor cells such as neuroblastoma and the like, and can promote the apoptosis of the neurogenic tumor cells. The siRNA can be applied to preparing an uncoiling enzyme V gene expression inhibitor, a medicament for promoting tumor cell apoptosis and a medicament for inhibiting tumor cell clone formation.

Description

SiRNA for inhibiting expression of helicase V gene and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to siRNA for inhibiting expression of a helicase V gene and application thereof.

Background

Neuroblastoma (NB), one of the neurogenic tumors, originates in the nerve spine of the sympathetic nervous system, is the most common malignant extracranial solid tumor in children, accounting for 10% of the incidence of malignant tumors in children, 15% of the deaths of tumors in children. According to INRG (International Neuroblastoma Risk Group) classification systems, NB can be divided into high-risk, medium-risk, low-risk, and extremely low-risk groups; wherein the very low risk group, such as part of the M-S (INRG stages)/IV-S stage (INSS stages) patients, regress themselves without treatment of the tumor. For the high-risk group, even though high-intensity comprehensive treatment including chemotherapy, surgery, radiotherapy, autologous stem cell transplantation and immunotherapy is given, the 2-year survival rate of Neuroblastoma (NB) in the high-risk group is only 19%. It follows that Neuroblastoma (NB) is a very significant category of diseases with clinical and biological heterogeneity, and clinical oncologists have been devoted to their research on control, hope to find accurate and effective therapeutic targets for NB. NB is classified into ganglioneuroma, ganglioneuroblastoma intermixed, ganglioneuroblastoma nodular and neuroblast oma by histological classification.

Nucleic acid interference (RNA interference, RNAi) is a sequence-specific RNA degradation process that theoretically provides a relatively easy and straightforward way for knockdown or silencing of any gene. In naturally occurring RNA interference, double-stranded RNA is cleaved by the action of ribonuclease III/helicase-Dicer into small interfering RNA (siRNA) molecules, a double-stranded RNA (dsRNA) of 19-23 nucleotides (nt) with two nucleotide overhangs at the 3' end. These siRNAs are integrated into a multicomponent ribonuclease known as an RNA-induced silencing complex (RISC), and one strand of the siRNA remains attached to the RISC, which strand directs the complex to a cognate RNA complementary to the leading single strand siRNA (ss-siRNA) sequence in the RISC. Such siRNA directed endonucleases can digest homologous RNA, thereby inactivating it. Studies have revealed that chemically synthesized 21-25 nucleotide sirnas exhibit very pronounced RNAi effects in mammalian cells, and that the thermodynamic stability of siRNA hybridization (at the ends or in the middle) plays an important role in determining the function of the molecule, and that nucleic acid interference techniques have broad application prospects.

Therefore, the RNAi technology is adopted to interfere the expression of the cancer related genes, so that the tumor cells are inhibited, and the RNAi technology is a tumor treatment strategy with great potential.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides an siRNA for inhibiting the expression of a helicase V gene and application thereof, wherein the siRNA has the effects of interfering and inhibiting the expression of the helicase V gene, and can inhibit the increase of at least neurogenic tumor cells such as neuroblastoma and promote the apoptosis of neurogenic tumor cells such as neuroblastoma.

In earlier studies, it has been found that the helicase V (FUBP 1) gene is highly expressed in Neuroblastoma (NB) tumor tissue and cell lines, and that inhibition of FUBP1 can inhibit proliferation of neuroblastoma cells, promote apoptosis, and have a high correlation.

Therefore, the invention screens the siRNA targeting the helicase V gene, and selects the siRNA with RNAi activity from a plurality of candidate siRNA sequences. The two optimal small interfering fragments siRNA-2 and siRNA-3 are finally obtained through further screening and optimization by testing in a mammalian cell culture to determine whether the candidate siRNA sequence has expected interference effect.

The invention provides an siRNA for inhibiting the expression of a helicase V gene, which comprises siRNA-2 and/or siRNA-3;

The nucleotide sequences of the sense strand and the antisense strand in the siRNA-2 are shown as SEQ ID NO. 3-4;

the nucleotide sequences of the sense strand and the antisense strand in the siRNA-3 are shown in SEQ ID NO. 5-6.

The nucleotide sequence of siRNA-2 is:

Sense strand: 5'-UCAACCAGAUGCUAAGAAA-3' (SEQ ID NO: 3);

Antisense strand: 5'-AGUUGGUCUACGAUUCUUU-3' (SEQ ID NO: 4).

The nucleotide sequence of siRNA-3 is:

Sense strand: 5'-CAACUACAACUCAAACUAA-3' (SEQ ID NO: 5);

Antisense strand: 5'-GUUGAUGUUGAGUUUGAUU-3' (SEQ ID NO: 6).

As can be seen from the above sequences, the interfering fragments siRNA-2 and siRNA-3 are small double-stranded RNAs (dsRNAs) of 19 nucleotides (nt). In certain particular aspects, the siRNA may lack or possess chemical modification.

Further, at least one of the siRNA-2 or siRNA-3 has a chemical modification. The chemical modification method can increase the stability of siRNA, raise the resistance to hydrolysis of ribozyme, reduce immune stimulating reaction, prolong the action time of siRNA in interfering with gene expression down regulation, and make the action efficient and specific.

The chemical modification may be located on the guide and/or passenger strand, on one or more nucleotides at the 3 'or 5' end and/or on one or more nucleotides constituting the internal backbone.

Still further, when the chemical modification occurs on at least one of the ribose, base, or phosphate of a nucleotide, the chemical modification includes, but is not limited to: substitution of at least one of the 2' -OH groups of ribose with at least one of the 2' -O-methyl RNA (2 ' OMe), 2' -O ' -methoxyethyl (2 ' MOE), 2' -fluoro (2F), or 2' -fluoro-beta-arabinonucleotide (FANA) groups, conversion of the 2' -oxyalkyl group to an aminoethyl-, guanidinoethyl-, cyanoethyl-, or allyl group, substitution of the phosphodiester groups with phosphorothioates, alkylation or thiolation of one or more nucleotides of the siRNA, substitution of ribonucleotides with deoxyribonucleotides, or substitution of nucleotides with Locked Nucleic Acids (LNA).

The test results of the invention show that double-stranded siRNA such as siRNA-2, siRNA-3 and the like can have the effects of interfering and inhibiting the expression of the helicase V gene, so that the expression quantity of the helicase V gene is reduced. Each double-stranded siRNA can only knock down the helicase V gene, but unexpectedly, the interference fragment compound formed by the siRNA-2 and the siRNA-3 can achieve the almost complete knocking down state of the helicase V gene, which shows that the interference effect generated by the compound formed by the siRNA-2 and the siRNA-3 is more ideal compared with that of single double-stranded siRNAs such as the siRNA-2, the siRNA-3 and the like, and has more laboratory and clinical application values.

Based on this, the present invention provides an interfering agent that inhibits the expression of the helicase V gene, comprising the siRNA.

Further, the siRNA is siRNA-2 and siRNA-3. The unique advantages of siRNA can lead the siRNA to adopt double-chain combination of a plurality of siRNAs to target disease induction genes, and the interference agent greatly improves the capability of knocking out the helicase V gene by forming the siRNA-2 and the siRNA-3 into a compound, and improves the efficacy of the interference agent.

The invention also provides application of the siRNA in preparation of an inhibitor of the expression of the helicase V gene. In the prior report, no expression inhibitor of the helicase V gene has been disclosed, so that the siRNA can be used for preparing the expression inhibitor of the helicase V gene, and thus, the siRNA has important application in biological and medical related research and treatment.

The invention also provides application of the siRNA in preparing anti-tumor drugs.

Early experiments demonstrated that the helicase V gene (FUBP) is another important oncogene independent of N-Myc and that upregulation of the expression of the helicase V gene in cancer patients is significantly associated with poor clinical survival. Helicase V also regulates gene expression in various tumor tissues and cell lines including liver cancer, squamous cell carcinoma, renal cell carcinoma, breast cancer, prostate cancer, bladder cancer, and non-small lung cancer, and promotes proliferation, cell cycle, invasion, and metastasis of the above-mentioned tumor cells.

Meanwhile, the helicase V gene (FUBP 1) also serves as a transcription factor for regulating HIF1 alpha expression, and can proliferate and inhibit apoptosis by enhancing glycolysis and ATP generation in tumor cells. Based on the siRNA has the function of well inhibiting the expression of the helicase V gene, the siRNA can generate an anti-tumor effect and can be applied to the preparation of anti-tumor drugs.

Further, the tumor is a neurogenic tumor.

Further, the neurogenic tumor is a neuroblastoma.

The invention verifies the anti-tumor effect of the siRNA in human tumor cells (human neuroblastoma cells), and the result shows that the siRNA can obviously inhibit the proliferation of the human neuroblastoma cells and promote the apoptosis of the tumor cells.

In clinical use, the siRNA of the present invention may be dissolved in sterile water without RNase, gently mixed, then mixed with small interfering RNA transfection reagent RNAi-Mate, incubated for 30 minutes at room temperature to form a complex, and then administered to organisms.

The invention also provides application of the siRNA in preparing a medicament for promoting tumor cell apoptosis. The invention is verified by experiments, and the result shows that the apoptosis number of tumor cells is obviously increased after the siRNA is added.

The invention also provides application of the siRNA in medicines for inhibiting tumor cell clone formation. The invention is verified by a cell plate cloning experiment, and the result shows that after the siRNA is added, the proliferation of tumor cells is obviously inhibited.

The invention also provides an anti-tumor pharmaceutical composition, which comprises the siRNA and pharmaceutically acceptable auxiliary materials.

Further, the pharmaceutically acceptable auxiliary materials comprise at least one of solvent, filler, lubricant, disintegrating agent, buffering agent, cosolvent, antioxidant, antibacterial agent, emulsifying agent, adhesive or suspending agent.

Still further, the buffer is a buffer solution at an acidic pH. The buffer solution of acidic pH is in particular a citrate or histidine buffer with or without the addition of an inorganic or organic salt selected from salts of which the cations are polyamines, in particular from spermine, spermidine or putrescine, or from salts of which the cations are metal cations, in particular from salts of zinc, cobalt, copper, manganese, calcium, magnesium or iron, in particular salts of manganese, zinc, magnesium alone or in combination of two or three.

The invention also provides a recombinant vector containing the siRNA. After the recombinant vector transfects tumor cells, the high expression of the helicase V gene in the tumor cells can be inhibited, so that the growth of the tumor cells is inhibited.

The construction method of the recombinant vector specifically comprises the following steps: the siRNA sequence for specifically inhibiting the expression of the helicase V gene is designed into a hairpin siRNA insert fragment which can be inserted into a vector, and then the hairpin siRNA insert fragment is connected with a pGPU6/GFP/Neo plasmid vector to obtain an interference small molecule fragment for resisting tumor biological behaviors.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention designs a double-chain siRNA for interfering the expression of an helicase V gene, which comprises siRNA-2 and/or siRNA-3, wherein both the siRNA-2 and the siRNA-3 have certain interference efficiency, the interference effect of a compound formed by the siRNA-2 and the siRNA-3 is more ideal, the expression level of the helicase V gene in cells can be effectively reduced, and a foundation is provided for researching the function and signal transduction path of the helicase V gene;

(2) The research of the invention shows that the RNA interference silent uncoiling enzyme V can obviously inhibit tumor formation, promote apoptosis, inhibit proliferation, inhibit cell clone formation, inhibit cell migration and/or cell invasion, and can be used for preparing products for preventing and/or treating tumors;

(3) The siRNA provided by the invention can specifically and efficiently inhibit mRNA and protein expression of the helicase V gene, reduce cell proliferation, increase apoptosis and reduce cell migration and invasion capacity. The method is applied to research of tumor pathogenesis and tumor treatment, and has important significance.

Drawings

FIG. 1 shows the mRNA expression level of FUBP in SK-N-BE2 cells treated with different interference fragments in example 4;

FIG. 2 shows the mRNA expression level in SK-N-BE2 cells treated with control fragment NC, or a complex composed of siRNA-2 and siRNA-3, in example 4;

FIG. 3 shows the protein expression levels of FUBP in SK-N-BE2 cells treated with different interference fragments in example 4;

FIG. 4 shows the protein expression level of FUBP1 in SK-N-BE2 cells treated with control fragment NC, or a complex of siRNA-2 and siRNA-3, as in example 4;

FIG. 5 shows the results of a plate cloning experiment of SK-N-BE2 cells treated with control fragment NC, or a complex composed of siRNA-2 and siRNA-3, in example 4;

FIG. 6 shows apoptosis results of SK-N-BE2 cells treated with control fragment NC or a complex of siRNA-2 and siRNA-3 of example 4 by flow cytometry.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following embodiments, and any modifications, substitutions, and combinations made without departing from the spirit and principles of the present invention are included in the scope of the present invention.

The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.

Example 1: design and Synthesis of siRNA

The invention screens and designs siRNA targeting helicase V gene to obtain three blunt-end siRNA double-stranded sequences shown in table 1, which are named siRNA-1 (si 1), siRNA-2 (si 2) and siRNA-3 (si 3) respectively, and the siRNA sequences are synthesized by Shangzhou Ruibo biotechnology Co.

TABLE 1siRNA double-stranded sequences

Among the above nucleotide sequences, the sense strand and antisense strand sequences in siRNA-1 (si 1) are represented by SEQ ID NO:1-2, the sense strand and antisense strand sequences in siRNA-2 (si 2) are represented by SEQ ID NO:3-4, and the sense strand and antisense strand sequences in siRNA-3 (si 3) are represented by SEQ ID NO:5-6, respectively.

The chemical synthesis method of siRNA is that 5 nucleotide monomers (A, U, C, G and dT) are prepared into 0.2mol/L solution by acetonitrile, 5 nucleotide monomers, acetonitrile, dichloroacetic acid and tetrazole are respectively installed at the corresponding positions of a synthesizer, wherein the nucleotide monomers, acetonitrile and tetrazole should be added with proper molecular sieves, and the specific synthesis steps are as follows:

(1) Treating the nucleotide or oligonucleotide bound to the solid support with dichloroacetic acid to remove the Dimethoxytrityl (DMT) protecting group on the 5 'hydroxyl functional group, exposing the reactive 5' end;

(2) Coupling reaction: is catalyzed by tetrazole to form 3 '. Fwdarw.5' phosphate chain;

(3) Blocking reaction: unreacted 5' hydroxyl groups are completed with acetic anhydride under the catalysis of N2 methylimidazole.

And synthesizing the required siRNA by adopting an RNA synthesizer according to a designed program.

Example 2: chemical modification of siRNA

For the siRNA synthesized in the embodiment 1, the chemical modification method can be adopted to increase the stability of the siRNA, improve the resistance of the siRNA to hydrolysis of ribozyme in vivo, reduce the immune stimulation reaction, prolong the action time of siRNA to interfere with the down regulation of gene expression, and ensure that the action has high efficiency and specificity.

The site of chemical modification may be located on the guide and/or passenger strand, on one or more nucleotides at the 3 'or 5' end and/or on one or more nucleotides constituting the internal backbone.

When a chemical modification occurs on at least one of the ribose, base, or phosphate of a nucleotide, the chemical modification includes, but is not limited to: substitution of at least one of the 2' -OH groups of ribose with at least one of the 2' -O-methyl RNA (2 ' OMe), 2' -O ' -methoxyethyl (2 ' MOE), 2' -fluoro (2F), or 2' -fluoro-beta-arabinonucleotide (FANA) groups, conversion of the 2' -oxyalkyl group to an aminoethyl-, guanidinoethyl-, cyanoethyl-, or allyl group, substitution of the phosphodiester groups with phosphorothioates, alkylation or thiolation of one or more nucleotides of the siRNA, substitution of ribonucleotides with deoxyribonucleotides, or substitution of nucleotides with Locked Nucleic Acids (LNA).

Example 3: siRNA transfected cells

Since it is currently not very well possible to predict which one of the candidate siRNA sequences targeting the mRNA sequence of a disease gene is the siRNA with the most RNAi activity, it is necessary to test in mammalian cell culture to determine if the expected interference effect occurs, and the typical detection method is:

Mammalian cells were passaged into 6-well plates and interference experiments were performed until the cells grew to a density of about 50%. 10. Mu.L of transfection reagent Hiperfect (Siemens) was added to 100. Mu.L of OPTI-MEN per well, 5. Mu.L of siRNA synthesized in example 1 (final concentration 100 nM) was added to 100. Mu.L of OPTI-MEN, and after 5min of standing, the two were mixed and then mixed and left for 15min. The resulting mixture was uniformly dropped into a 6-well plate culture dish (total culture solution: 2 mL), and the culture was continued for 48 hours, to extract total RNA, followed by verification of target mRNA and protein levels by fluorescent quantitative PCR and western blot (western blot), respectively.

Extraction of Total RNA

The cell lines were collected into a 1.5mL RNA-free centrifuge tube, 1mL Trizol was added, and the mixture was thoroughly mixed and allowed to stand at room temperature for 5min. 0.2mL of chloroform (chloroform) was added thereto, and the mixture was vigorously shaken for 15s and allowed to stand at room temperature for 2 minutes. Centrifuge at 4℃12000 r/min.times.15 min, aspirate the supernatant (note not to aspirate the lower layer) into another 1.5mL centrifuge tube. Isopropanol in the same amount as the supernatant was added, mixed gently and allowed to stand at room temperature for 10min. Centrifuge at 4℃for 12000r/min X10 min, discard supernatant. 1mL of pre-chilled 75% ethanol (with DEPC water) was added, the pellet was gently washed, centrifuged at 4℃and 7500r/min X5 min, and the supernatant discarded. Air-dried, and dissolved in 20. Mu.L of DEPC water (dissolution promotion at 65 ℃ C. For 10-15 min). The concentration (unit: μg/μL) and purity of the extracted RNA were determined by an ultraviolet spectrophotometer, and when OD260/OD280 was between 1.8 and 2.0, it was shown that the purity of the extracted RNA was good.

Reverse transcription of total RNA to cDNA

After extracting total RNA from the above cells, 2. Mu.L of the above extracted total RNA was aspirated, and the RNA concentration was measured (Nanodrop spectrophotometer), followed by reverse transcription (reagent) according to the reaction system in Table 2RT REAGENT KIT) to obtain cDNA.

TABLE 2 reverse transcription reaction system

Reagent(s)	Dosage of
		Total RNA	500ng
5×PrimeScript Buffer	2μL
		H₂O	Make up to a total volume of 10. Mu.L

Wherein the reverse transcription conditions are: the water bath was set at 37℃for 15min and 85℃for 5s, and the product was stored at-80 ℃.

Real-time fluorescent quantitative PCR

And (3) using the cDNA obtained by the reverse transcription as a template, automatically designing PCR primers, and amplifying the helicase V gene, wherein the amplified product fragment product is 70-150bp, and the primer sequences of the amplified helicase V gene and the internal reference gene beta-actin are shown in Table 3.

TABLE 3 fluorescent quantitative PCR primer sequences

Gene	Upstream primer sequences	Downstream primer sequences
			FUBP1	5’-GCGTCTAAAGGTTTCCGCGA-3’	5’-ACCAGCAATGCCATAGAGGTG-3’
β-actin	5’-GCACTCTTCCAGCTTCCTT-3’	5’-GTTGGCGTACAGGTCTTTGC-3’

Among the above primer sequences, the upstream primer and the downstream primer sequences of the amplified helicase V gene (FUBP) are represented as SEQ ID NO. 7-8, respectively, and the upstream primer and the downstream primer sequences of the amplified reference gene beta-actin are represented as SEQ ID NO. 9-10, respectively.

After the completion of the primer design, real-time quantitative PCR detection was performed according to the reaction system shown in Table 4.

TABLE 4 fluorescent quantitative PCR reaction System

The reaction conditions for setting the fluorescent quantitative PCR are as follows: denaturation (95 ℃ C. 30s,1 cycle); annealing/extension (95 ℃ C. 5s,60 ℃ C. 30s,40 cycles). In the analysis result, a control sample was used as a calibration sample (Calibrator), and the remaining samples were compared according to the ratio of the calibration sample.

Western blot (western blot) detection

Preparing a Western Blot related solution:

① 2 x sample lysis buffer: 10% SDS 4mL, 2mL of glycerol, 2.5 mol/L Tris-HCl (pH 6.8) 2.5mL and 0.5mL of deionized water were mixed to give a total volume of 9mL lysis buffer, which was stored at room temperature for further use.

② 10 X protein denaturation buffer: 1mL of 0.4% bromophenol blue and 1mL of beta-mercaptoethanol are mixed evenly to obtain 2mL of denaturation buffer solution, and the mixture is preserved at room temperature for standby.

③ SDS electrophoresis buffer: 3.02g of Tris, 1.0g of SDS and 18.8g of glycine are taken, 900mL of double distilled water is added and stirred until complete dissolution is achieved, then the volume is fixed to 1L, and the mixture is stored at room temperature.

④ Electrotransport buffer (ph=8.3): glycine 14.41g and Tris 3.03g were dissolved in 700mL double distilled water, stirred until complete dissolution, then methanol 200mL was added, the volume was fixed to 1L, and stored at 4 ℃.

⑤ TBST buffer: 2.42g Tris and 8g NaC are added into 900mL double distilled water, stirred until the mixture is completely dissolved, the pH value is adjusted to 7.6, the volume is fixed to 1L, and the mixture is stored at room temperature.

⑥ Sealing liquid: 7-10% of experimental skim milk powder was dissolved in TBST buffer.

Western Blot specific procedure:

(1) Taking a 6-hole plate inoculated with cells, washing the cells with phosphate buffer solution (PBS solution) or normal saline for 3 times when the cells are in a logarithmic growth phase, adding 100 mu L of cell lysate (containing 1% of PMSF and phosphatase inhibitor) into each hole, collecting the lysate into an EP tube of 0.5mL by using a cell scraper, and boiling for 30min at 100 ℃;

(2) Protein quantification of the collected lysates was performed using BCA kit (keyl corporation);

(3) Detecting the concentration of extracted protein by an ultraviolet spectrophotometer, and according to the protein volume: adding protein loading buffer solution (10×; beta-mercaptoethanol, 0.4% bromophenol blue) into SDS-PAGE protein loading buffer solution with volume=4:1, boiling at 100deg.C for 10min, packaging, and storing at-80deg.C in refrigerator;

(4) Polyacrylamide gel (SDS-PAGE gel): SDS-PAGE gels of corresponding concentrations were prepared at different molecular weight sizes of the target proteins. The glass plate for glue preparation is cleaned, dried, aligned, placed into a clamp for clamping, vertically clamped on a frame, and prepared for glue filling (clamping hard to prevent glue leakage). Firstly, adding separation gel, firstly, quickly and slowly, preventing bubbles from generating, generally adding deionized water when the separation gel is added to a position 1.5cm away from the upper end, sealing, standing for 40min, then discarding the deionized water, adding concentrated gel to the top end, inserting a comb, and preparing for loading. Adding 50 mug of sample into each hole, loading about 15-20 mug of sample into each hole, concentrating for 30min by 80V electrophoresis, regulating voltage to 120V after bromophenol blue enters the separating gel, stopping electrophoresis when bromophenol blue approaches the bottom of the separating gel,

(5) Transferring: placing PVDF film with proper size into methanol for activation for 10min, covering PVDF film on gel after electrophoresis, covering filter paper and sponge on non-contact surface of PVDF film and gel, placing into electrotransfer tank for electrotransfer, and transferring constant current for 300mA×180min. Transferring the protein on the gel to a PVDF film soaked in methanol (keeping low temperature), and properly increasing or reducing the electrotransformation time according to the molecular weight;

(6) After electrotransformation, taking out the PVDF film, putting the PVDF film into a sealing solution (TBST buffer solution containing 7-10% of skimmed milk) prepared at present, sealing for 1h at room temperature, and taking the front of the film upwards;

(7) Incubating primary antibody: after the sealing is finished, taking out, and putting the membrane into a container containing a proper amount of 1 XTBE for washing the membrane for 5min multiplied by 3 times; preparing a corresponding primary antibody diluted by 1 XTBST containing 5% of skimmed milk powder; placing the washed membrane on a flat plate, enabling the front surface of the membrane to be upward, and uniformly dripping diluted antibody on the membrane; incubate for about 2h at room temperature and then place at 4℃overnight.

(8) Incubating the secondary antibody: the PVDF membrane was removed and washed 3 times with 1 XTBE for 10min each with shaking. Preparing 1 XTBST diluted corresponding secondary antibody containing 5% skimmed milk powder; uniformly dripping the diluted secondary antibody on the membrane, and incubating for 1h at room temperature or 4 ℃ for 4h;

(9) After the TBST film is washed for 3 times, mixing A, B liquid of ECL luminous liquid according to the specification of a chemiluminescent kit (prepared before use); sucking the washed film with filter paper at a corner; placing the front surface of the film upwards on a plastic film, and dripping A, B mixed solution on the film; and photographing and storing by the gel imaging system, and analyzing results.

Example 4: tumor cell validation assay

Experimental cell material: SK-N-BE2 cells (human neuroblastoma cells, cultured in DMEM medium and 10% fetal bovine serum) were cultured at 37℃and 5% carbon dioxide concentration.

Real-time fluorescent quantitative PCR detection

The above-mentioned human neuroblastoma cells SK-N-BE2 were passaged into 6-well plates, cultured to 60% density, siRNA (siRNA-1, siRNA-2 and siRNA-3) interfering with the expression of the helicase V gene (FUBP 1) and control fragment NC (sense strand: 5'-UUCUCCGAACGUGUGUCACGUTT-3' (SEQ ID NO: 11); antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 12)) were added to SK-N-BE2 cells as in the method of siRNA transfected cells in example 3, and mRNA was extracted after further culture for 48 hours, and the mRNA expression level of the helicase V gene in SK-N-BE2 cells was detected by a real-time fluorescent quantitative PCR detection method.

As shown in FIG. 1, compared with the control fragments NC and siRNA-1 (si 1), the single interference fragment siRNA-2 (si 2) and siRNA-3 (si 3) have the effects of interference and knockout, so that the mRNA expression amount of the helicase V gene is reduced, namely the effective fragment. As can be seen from FIG. 2, when two effective fragments siRNA-2 (si 2) and siRNA-3 (si 3) (i.e., si2+3) are added simultaneously, the complex formed by the two fragments has better interference capability, so that the expression of the mRNA of the helicase V gene can be knocked out almost completely.

Western blot (western blot) detection

The above-mentioned human neuroblastoma cells SK-N-BE2 were passaged into 6-well plates, cultured to a density of 60%, siRNA (siRNA-1, siRNA-2 and siRNA-3) interfering with the expression of the helicase V gene (FUBP) and control fragment NC (sense strand: 5'-UUCUCCGAACGUGUGUCACGUTT-3' (SEQ ID NO: 11); antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 12)) were added to SK-N-BE2 cells by the method of siRNA-transfected cells in example 3, and protein was recovered after further culturing for 48 hours, and the protein expression level of the helicase V gene in SK-N-BE2 cells was detected by the western blotting (western blot) method.

As shown in FIG. 3, compared with the control fragments NC and siRNA-1 (si 1), the single interference fragment siRNA-2 (si 2) and siRNA-3 (si 3) have the effects of interference and knockout, so that the expression quantity of the helicase V gene is reduced, namely the effective fragment. As can be seen from FIG. 4, when two effective fragments siRNA-2 (si 2) and siRNA-3 (si 3) (i.e., si2+3) are added simultaneously, the complex formed by the two fragments has better interference capability, so that the expression of the helicase V gene can be knocked out almost completely.

Cell plate cloning experiments

Cell clone formation rate, i.e., cell seeding survival, refers to the number of cells that adhere to and form clones after seeding the cells. Cells after adherence do not necessarily each proliferate and form clones, but cells forming clones must be cells that are adherent and proliferation-competent, so that the rate of colony formation can truly reflect cell population dependence and proliferation capacity.

The specific experimental steps of the cell plate cloning experiment are as follows:

(1) Each group of cells in the logarithmic growth phase was taken, digested with 0.25% trypsin and blown into single cells, and the cells were suspended in DMEM medium of 10% fetal bovine serum for use.

(2) The cell suspension is diluted by multiple gradient, and each group of cells is respectively inoculated into a dish containing 2mL of pre-temperature culture solution with 37 ℃ according to the gradient density of 50 cells per dish, and the cells are gently rotated to be uniformly dispersed. Culturing in a cell culture incubator with 37 deg.C, 5% CO ₂ and saturated humidity for 2-3 weeks.

(3) It is often observed that the culture is terminated when macroscopic clones appear in the culture dish. The supernatant was discarded and carefully rinsed 2 times with PBS. Cells were fixed by adding 5mL of 4% paraformaldehyde for 15 min. Then removing the fixing solution, adding a proper amount of crystal violet for dyeing for 10-30min, then slowly washing off the dyeing solution by using running water, and drying in air.

(4) The plate was inverted and a clear film with a grid was superimposed and clones were counted directly with the naked eye or the number of clones greater than 10 cells were counted under a microscope (low power microscope).

Cell plate cloning experiments were performed using SK-N-BE2 cells treated with control fragment NC, or a complex composed of siRNA-2 (si 2) and siRNA-3 (si 3), as the material. The experimental results are shown in FIG. 5, and the cloning efficiency of SK-N-BE2 cells treated with the complex of siRNA-2 (si 2) and siRNA-3 (si 3) is significantly lower than that of SK-N-BE2 cells treated with the control fragment NC, indicating that proliferation of tumor cells SK-N-BE2 is significantly inhibited after the addition of the effective interfering fragment (complex of siRNA-2 and siRNA-3).

Annexin V-FITC/PI double-dye flow cytometry for measuring apoptosis

Annexin v is a Ca ²⁺ -dependent phospholipid binding protein with a molecular weight of 35 to 36KDa. During apoptosis, phosphatidylserine (PS) inside the cell membrane is turned outside the membrane, and Phosphatidylserine (PS) in the cell membrane is turned outside from inside to outside of the lipid membrane as a marker of early apoptosis, while annexin v has a specific high affinity with Phosphatidylserine (PS). Thus, early apoptosis of cells can be detected using a flow cytometer with FITC (fluorescein isothiocyanate) -labeled annexin v as a fluorescent probe. Propidium iodide (Propidium Iodide, PI) is a nucleic acid dye that is impermeable to the intact cell membrane. When the cell is in a state of late apoptosis or has died, the cell membrane integrity is destroyed and the cell membrane permeability is enhanced. PI is able to permeate the cell membrane and stain the nucleus red. Thus, late apoptotic cells or dead cells can be detected by PI staining. Cells at different stages of apoptosis can be distinguished by double labelling cells with annexin v and PI.

The control fragment NC, or a complex pair composed of siRNA-2 (si 2) and siRNA-3 (si 3), is adopted for SK-N-BE2

Treating the cells, and after the cell treatment is finished, digesting and collecting the cells by using pancreatin (without EDTA); washing the cells three times with PBS, and then centrifuging at 2000rpm for 5min to collect 10 to 50 tens of thousands of cells; mu. LAnnexinV-FITC and 5 mu L Propidium Iodide were added to 500. Mu.l Binding Buffer at a ratio of 1:100, and the cells were resuspended in the prepared dye solution; the reaction was carried out at room temperature for 15min in the absence of light, and the detection was carried out by an up-flow cytometer within 1 hour. Statistical treatment statistical results are expressed as x+/-s, are analyzed by SPSS statistical software, the data are compared pairwise by adopting paired data t test, and the comparison among multiple groups is performed by ANOVA.

Flow cytometry analysis: setting of the cross gate and adjustment of fluorescence compensation were performed with a previously prepared normal culture group, an annexin V-FITC single-stain group, a PI single-stain group, a positive annexin V-FITC/PI double-stain group (25. Mu.M colchicine treatment). Meaning represented by each quadrant of the cross gate: upper left quadrant-necrotic cells (annexin v-FITC staining negative, PI staining positive); upper right quadrant-late apoptotic cells (annexin v-FITC staining positive, PI staining positive); lower left quadrant-normal living cells (Annexin V-FITC staining negative, PI staining negative); lower right quadrant-early apoptotic cells (annexin v-FITC staining positive, PI staining negative).

As shown in FIG. 6, compared with the SK-N-BE2 cells treated by the control fragment NC, the number of the SK-N-BE2 cells treated by the complex composed of siRNA-2 (si 2) and siRNA-3 (si 3) in the late apoptosis stage is obviously increased, and the number of the SK-N-BE2 cells is increased from 1.48% to 5.22%, which shows that after the effective interference fragment (the complex composed of siRNA-2 and siRNA-3) is added, the apoptosis process of the tumor cells SK-N-BE2 is obviously accelerated. The result shows that the expression of the helicase V gene (FUBP 1) is reduced, so that the apoptosis of tumor cells can be promoted, and the result is consistent with the Western Blot detection result.

Example 5: construction of siRNA recombinant vector

The embodiment provides an siRNA recombinant vector, the construction method of which comprises the following steps: the siRNA sequences (siRNA-2 and siRNA-3 in example 1) that specifically inhibited the expression of the helicase V gene were designed as hairpin siRNA inserts that could be inserted into the vector and then ligated with pGPU6/GFP/Neo plasmid vector to give interfering small molecule fragments for antitumor biological behavior. After the recombinant vector transfects tumor cells, the high expression of the helicase V gene in the tumor cells can be inhibited, so that the growth of the tumor cells is inhibited.

Example 6: antitumor pharmaceutical composition

This example provides an anti-tumor pharmaceutical composition comprising siRNA-2 and siRNA-3 of example 1, and further comprising a buffer solution comprising citrate. The antitumor pharmaceutical composition can be used for inhibiting proliferation of at least neurogenic tumor cells such as neuroblastoma, and promoting apoptosis of tumor cells.

Claims

1. The application of siRNA for inhibiting the expression of helicase V gene in preparing a medicament for promoting tumor cell apoptosis is characterized in that the siRNA consists of siRNA-2 and siRNA-3;

the sequence of the sense strand in the siRNA-2 is shown as SEQ ID NO. 3, and the antisense strand is a sequence reversely complementary with the SEQ ID NO. 3;

the sequence of the sense strand in the siRNA-3 is shown as SEQ ID NO. 5, and the antisense strand is a sequence reversely complementary to the SEQ ID NO. 5;

The tumor is neuroblastoma.

2. The use according to claim 1, wherein at least one of said siRNA-2 or siRNA-3 has a chemical modification.

3. An agent for inhibiting the expression of a helicase v gene comprising the siRNA of claim 1 or 2.

4. Use of the siRNA of claim 1 or 2 for the preparation of an inhibitor of helicase v gene expression.

5. Use of the siRNA of claim 1 or 2 for the preparation of an anti-tumor medicament; the tumor is neuroblastoma.

6. Use of the siRNA of claim 1 or 2 for the preparation of a medicament for inhibiting the formation of tumor cell clones; the tumor is neuroblastoma.

7. An anti-tumor pharmaceutical composition comprising the siRNA of claim 1 or 2, further comprising a pharmaceutically acceptable adjuvant; the tumor is neuroblastoma.

8. A recombinant vector comprising the siRNA of claim 1 or 2.