Application of siRNA (small interfering ribonucleic acid) targeting IFI44 (interferon 44) in preparation of medicine for inhibiting malignant progression of bladder cancer

Abstract

The invention relates to the technical field of biological medicines, and provides application of siRNA (small interfering Ribonucleic Acid) targeting IFI44 in preparation of medicines for inhibiting malignant progression of bladder cancer. The siRNA is selected from siRNA (Ribonucleic Acid) 1 or siRNA 2; the siRNA1 comprises a positive-sense strand of which the nucleotide sequence is as shown in SEQ ID NO.1 and an antisense strand of which the nucleotide sequence is as shown in SEQ ID NO.2; the siRNA2 comprises a positive-sense strand of which the nucleotide sequence is as shown in SEQ ID NO.3 and an antisense strand of which the nucleotide sequence is as shown in SEQ ID NO.4. The siRNA targeting IFI44 is applied to preparation of drugs for inhibiting the malignant progression of bladder cancer, and proliferation and migration of bladder cancer cells are inhibited and apoptosis of the bladder cancer cells is promoted by reducing the expression level of IFI44, so that the malignant progression of the bladder cancer is inhibited.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of siRNA targeting IFI44 in preparation of a drug for inhibiting malignant progression of bladder cancer. BACKGROUND

[0002] Bladder cancer is a malignant tumor originating from the mucosal epithelial tissue of the bladder, has a high incidence in the urinary system malignant tumors, is prone to occur in the middle-aged and old people, and the incidence of male is significantly higher than that of female. The incidence is related to various factors, long-term smoking is the most important risk factor, in addition, long-term contact with industrial chemicals, chronic cystitis, chronic irritation such as bladder stones, and genetic factors can also induce bladder cancer.

[0003] At present, although some progress has been made in the treatment of bladder cancer, such as surgical treatment, chemotherapy, etc., there are still obvious defects. First, for non-muscular invasive bladder cancer, transurethral resection of bladder tumor is the main operation, however, the tumor recurrence rate after operation is high, and some patients may also have tumor progression; for muscle invasive bladder cancer, although radical cystectomy can more thoroughly remove the tumor, the surgical trauma is large, and the patients after operation have to face the lifestyle changes brought by urinary diversion surgery, and may have various complications such as infection and anastomotic fistula; secondly, although the commonly used drugs for chemotherapy such as gemcitabine and cisplatin can inhibit tumor growth to some extent and prolong the survival period of patients, the chemotherapy drugs will also cause damage to normal cells while killing tumor cells, resulting in adverse reactions of patients. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides application of siRNA targeting IFI44 in preparation of a drug for inhibiting malignant progression of bladder cancer, which aims to solve the problems mentioned in the background.

[0005] In a first aspect, the application provides application of siRNA targeting IFI44 in preparation of a drug for inhibiting malignant progression of bladder cancer, wherein the siRNA is selected from siRNA1 or siRNA2. The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 2. The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 4.

[0006] Further, the siRNA targeting IFI44 inhibits the malignant progression of bladder cancer by reducing the expression level of IFI44, thereby inhibiting the proliferation and migration of bladder cancer cells and promoting the apoptosis of bladder cancer cells.

[0007] Further, the bladder cancer cells are T24 cells or J82 cells.

[0008] Further, the drug further comprises a pharmaceutically acceptable carrier.

[0009] Further, the dosage form of the drug is a nanoparticle.

[0010] In a second aspect, the application provides a use of a nanomaterial ZIF-8 encapsulating siRNA targeting IFI44 in the preparation of a drug for inhibiting the malignant progression of bladder cancer, wherein the siRNA is selected from siRNA1 or siRNA2. The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 2. The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 4.

[0011] Further, the nanomaterial ZIF-8 encapsulating siRNA targeting IFI44 further comprises an RGD targeting peptide.

[0012] Further, the preparation method of the nanomaterial ZIF-8 encapsulating siRNA targeting IFI44 comprises the following steps: Step S1: the siRNA, Zn 2+ and 2-methylimidazole are prepared by a co-precipitation method to obtain ZIF-8@IFI44 siRNA; Step S2: polyethylene glycol is added, stirred at 4℃ overnight, centrifuged several times, and then dispersed in 10mM MES again, and then EDC and NHS are added, activated at 37℃ water bath for 25min to obtain ZIF-8@IFI44 siRNA@PEG; Step S3: RGD targeting peptide is added, stirred at 4℃ for 24h, then centrifuged and washed, and then dispersed in ultrapure water to obtain ZIF-8@IFI44 siRNA@PEG-RGD.

[0013] The application has the following technical effects: (1) The siRNA targeting IFI44 is applied in the preparation of a drug for inhibiting the malignant progression of bladder cancer, which reduces the expression level of IFI44 to inhibit the proliferation and migration of bladder cancer cells, promotes the apoptosis of bladder cancer cells, and inhibits the malignant progression of bladder cancer, thereby providing an efficient and low-toxicity treatment scheme for bladder cancer.

[0014] (2) The nanometer material ZIF-8 wrapped with siRNA targeting IFI44 (ZIF-8@IFI44 siRNA@PEG-RGD) has strong targeting property and low toxicity and side effects, the PEG modification prolongs the blood circulation time and reduces immune clearance, the RGD peptide precisely combines with the integrin on the surface of bladder cancer cells, thereby improving the enrichment efficiency and delivery efficiency, and providing the possibility for personalized treatment, and having high clinical conversion value. BRIEF DESCRIPTION OF DRAWINGS

[0015] The exemplary embodiments of the present application can be more completely understood in reference to the following drawings: Figure 1 is a preparation process flow chart of the nanometer material ZIF-8 wrapped with siRNA targeting IFI44 according to an embodiment of the present application.

[0016] Figure 2 is a morphological analysis result chart of ZIF-8@IFI44 siRNA1@PEG-RGD by field emission transmission electron microscopy according to embodiment 1 of the present application, wherein: Figure 2 A in is a transmission electron microscope image, and the transmission electron microscope shows that ZIF-8@IFI44 siRNA@PEG-RGD is a regular rhombohedral dodecahedron; Figure 2 B in is a dynamic light scattering particle size distribution chart; Figure 2 C in is a Zeta potential distribution chart.

[0017] Figure 3 is a chart for detecting the release of IFI44 siRNA2 in ZIF-8@IFI44 siRNA2@PEG-RGD by dialysis according to embodiment 2 of the present application.

[0018] Figure 4 is a chart for analyzing the mRNA and protein levels of IFI44 in T24 cells and J82 cells according to embodiment 3 of the present application, wherein: Figure 4 A in is the mRNA level of IFI44, and * indicates p<0.05 and *** indicates p<0.001; Figure 4 B in is the protein level of IFI44, and GAPDH is used as the internal reference protein.

[0019] Figure 5 is a chart for analyzing the mRNA and protein levels of IFI44 in T24 cells and J82 cells according to embodiment 4 of the present application, wherein: Figure 5 A in is the mRNA level of IFI44; Figure 5 B in FIG. 7 is the protein level of IFI44, with GAPDH as the internal reference protein.

[0020] Figure 6 FIG. 8 is the CCK8 experimental analysis result and the Transwell migration experiment analysis result of Example 5 of the present application, wherein: Figure 6 A in FIG. 8 is a CCK8 experimental analysis result graph, ns indicates no significant difference, and *** indicates p<0.001. Figure 6 B in FIG. 8 is a cell number statistical result graph of the Transwell migration experiment, ns indicates no significant difference, and *** indicates p<0.001. Figure 6 C in FIG. 8 is a cell staining microscope image of the Transwell migration experiment, scale bar: 200 μm.

[0021] Figure 7 FIG. 9 is a graph of the analysis result of the apoptosis experiment by flow cytometry analysis of Example 6 of the present application, wherein: Figure 7 A in FIG. 9 is a flow cytometry scatter plot. Figure 7 B in FIG. 9 is a statistical graph of the apoptosis rate, ns indicates no significant difference, and *** indicates p<0.001.

[0022] Figure 8 FIG. 10 is a graph of the analysis result of the malignant progression pathway of the bladder cancer cells of Example 7 of the present application, wherein: Figure 8 A in FIG. 10 is a KEGG enrichment analysis result graph. Figure 8 B in FIG. 10 is the change in the total and phosphorylation of the key genes PI3K and AKT in the PI3K-AKT pathway after knocking down IFI44 in T24 and J82 cells. Figure 8 C in FIG. 10 is the change in the total and phosphorylation of the key genes PI3K and AKT in the PI3K-AKT pathway after co-incubation of T24 cells with phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA2, ZIF8@IFI44 siRNA2@PEG-RGD. Figure 8 D in FIG. 10 is the change in the total and phosphorylation of the key genes PI3K and AKT in the PI3K-AKT pathway after co-incubation of J82 cells with phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA2, ZIF-8@IFI44 siRNA2@PEG-RGD.

[0023] Figure 9 is the result of tumor volume analysis of mice in Example 8 of the present application, ns represents no significant difference, *** represents p<0.001, wherein: Figure 9 A in is the result of tumor volume record of mice in each group; Figure 9 B in is the tumor volume of mice after dissection in each group; Figure 9 C in is the tumor weight of mice after dissection in each group.

[0024] Figure 10 is a general schematic diagram of the tumor of mice after dissection in each group in Example 8 of the present application.

[0025] Figure 11 is the result of immunohistochemical analysis in Example 9 of the present application, scale: 20 μm.

[0026] Figure 12 is the result of weight record of mice in Example 8 of the present application and the result of detection of ALT, AST, CREA and UREA in Example 10, wherein: Figure 12 A in is the result of weight record of mice; Figure 12 B in is the result of detection of ALT; Figure 12 C in is the result of detection of AST; Figure 12 D in is the result of detection of CREA; Figure 12 E in is the result of detection of UREA.

[0027] Figure 13 is a result of HE staining in Example 11 of the present application, scale: 20 μm. DETAILED DESCRIPTION

[0028] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs; the terms used herein are only for the purpose of describing specific examples, and are not intended to limit the present application.

[0030] The application provides application of the siRNA targeting IFI44 in preparation of a drug for inhibiting malignant progression of bladder cancer, and the siRNA is selected from siRNA1 or siRNA2. The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 2. The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 4.

[0031] In some embodiments, the siRNA targeting IFI44 inhibits malignant progression of bladder cancer by reducing the expression level of IFI44, inhibiting proliferation and migration of bladder cancer cells, and promoting apoptosis of bladder cancer cells.

[0032] In some embodiments, the bladder cancer cells are T24 cells or J82 cells.

[0033] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0034] In some embodiments, the dosage form of the drug is a nanoparticle.

[0035] In some embodiments, the application provides application of the nanomaterial ZIF-8 encapsulating the siRNA targeting IFI44 in preparation of a drug for inhibiting malignant progression of bladder cancer, and the siRNA is selected from siRNA1 or siRNA2. The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 2. The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO. 4.

[0036] In some embodiments, the nanomaterial ZIF-8 encapsulating the siRNA targeting IFI44 further comprises an RGD targeting peptide.

[0037] In some embodiments, the preparation method of the nanomaterial ZIF-8 encapsulating the siRNA targeting IFI44 specifically comprises the following steps. Step S1: siRNA, Zn 2+ and 2-methylimidazole are prepared by a co-precipitation method to obtain ZIF-8@IFI44 siRNA. Step S2: Add polyethylene glycol, stir overnight at 4°C, centrifuge several times, re-disperse in 10 mM MES, add EDC and NHS, activate in a 37°C water bath for 25 min, obtain ZIF-8@IFI44 siRNA@PEG; Step S3: Add RGD targeting peptide, centrifuge and wash after stirring at 4°C for 24 h, re-disperse in ultrapure water, obtain ZIF-8@IFI44 siRNA@PEG-RGD.

[0038] Materials and methods: (1) Cell culture: T24 cells and J82 cells were maintained under standard laboratory conditions and cultured in RPMI 1640 medium (GIBC, USA). The cells were incubated with culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C in a humidified environment containing 5% carbon dioxide.

[0039] (2) Synthesis of IFI44 knockdown plasmid: The knockdown primer siRNA for IFI44 was synthesized by General Biosystems, and modified with CY5 fluorescence at the 5' end, the nucleotide sequence: IFI44 siRNA1, sense strand: 5'-AGGAUAACCUAGACGACAUAATT-3' (SEQ ID NO. 1), antisense strand: 5'-UUAUGUCGUCUAGGUUAUCCUTT-3' (SEQ ID NO. 2); IFI44 siRNA2, sense strand: 5'-ACCGAGCGGUAUAGGAUAUAUTT-3' (SEQ ID NO. 3), antisense strand: 5'-AUAUAUCCUAUACCGCUCGGUTT-3' (SEQ ID NO. 4).

[0040] (3) RT-PCR (reverse transcription polymerase chain reaction) and qRT-PCR (quantitative reverse transcription polymerase chain reaction): Total RNA was obtained from cultured cells using the standard Trizol protocol; cDNA was then synthesized using the kit "TransGen Biotech Transcript®II All-in-One First Strand cDNA" for qPCR; RT-qPCR was then performed using ChamQ Universal SYBR qPCR Master Mix; 2 -ΔΔCT Method was used for data analysis, and the results were shown as relative expression levels, with β-Actin as an internal control.

[0041] (4) Western Blot: Cells were collected and 2x protein loading buffer was added to extract protein, which was boiled at 100°C for 10 min; the gel was run at 150V for 1.5h, and then transferred to NC membrane at 350mA for 90 min; the membrane was blocked with 5% skim milk for 2h, and then incubated with various primary antibodies overnight; the membrane was washed with 1x TBST for 3 times, each for 10 min; the secondary antibody was incubated for 2h, and then exposed to an imaging instrument.

[0042] (5) Transmission electron microscopy and determination of particle size and potential: Field emission transmission electron microscopy (transmission electron microscopy) (JEM-F200, Japan) was used to scan the morphology of each group of ZIF-8 materials to see if they were clear spherical boundaries, and then a particle size and potential measuring instrument (Zetasizer Nano ZS90, UK) was used to measure the particle size and potential of each group of ZIF-8 materials.

[0043] (6) Stability test: ZIF-8@IFI44 siRNA@PEG-RGD was incubated with 10% fetal bovine serum or phosphate buffer (0.01M, pH=7.4) at 37°C for different times, and then the nanoparticle size was monitored at different intervals using dynamic light scattering (DLS) technology.

[0044] (7) Cell uptake experiment: Bladder cancer cells were seeded into a confocal culture dish, and after cell adhesion, the cells were treated with ZIF-8 and ZIF-8@IFI44 siRNA@PEG-RGD for 4 hours; then, the cells were incubated with Lyso Tracker Green (Beyotime, China) for 1 hour; Hoechst 33342 was used for nuclear staining for 10 minutes; finally, a confocal laser scanning microscope (Germany Zeiss) was used to collect images.

[0045] (8) Cell proliferation and migration assay: Colony formation assay and CCK-8 assay were used to evaluate cell proliferation ability; Transwell migration assay was used to evaluate cell migration ability. In the colony formation assay, bladder cancer cells were seeded at 1x10 3Cells were placed in 6-well plates at a concentration of 1000 cells and grown for 7 to 14 days. The process included fixing cells with 4% paraformaldehyde for 30 minutes and staining with 0.1% crystal violet for 15 minutes. In the CCK-8 assay, 2000 cells were placed in 96-well plates and observed at 0, 24, 48, 72, and 96 hours. At each time point, CCK-8 reagent (MedChemExpress, China) was added and incubated for 2 hours, after which the absorbance at 450 nm was measured using a microplate reader. In the Transwell migration assay, 600 µL of culture medium containing 20% ​​serum was added to the lower chamber, and 3 × 10⁶ cells were added to each well. 4 Bladder cancer cells were added to the upper chamber of 200 µL serum-free medium and cultured for 48 hours. After treatment with 4% paraformaldehyde, cells that migrated to the lower side of the chamber were stained with crystal violet and counted by photograph.

[0046] (9) Apoptosis experiment: Apoptosis was assessed using the Annexin V-FITC / PI kit. 50 × 10⁻⁶ 4 Cells from different groups were placed in 6-well plates and incubated for one day. Cells were collected, resuspended in buffer, stained with FITC for 10 minutes, stained with PI for 5 minutes, and finally detected by flow cytometry.

[0047] (10) Pharmacokinetic assay and biodistribution of ZIF-8@IFI44 siRNA@PEG-RGD in mice: Healthy female mice were randomly assigned to three groups (n=3), each receiving a tail vein injection of either IFI44 siRNA or ZIF-8@IFI44siRNA@PEG-RGD at a dose of 1 nmol of siRNA. Blood samples were collected at predetermined intervals using heparinized tubes via the orbital vein. The fluorescence intensity of CY5 in the blood was calculated using an ELISA reader to assess pharmacokinetics. In the biodistribution study, healthy female BALB / c nude mice with subcutaneous tumors were randomly assigned to two groups (n=3), each receiving an intravenous injection of either IFI44 siRNA or ZIF-8@IFI44 siRNA@PEG-RGD. Twenty-four hours later, the fluorescence intensity of CY5 in major organs and tumors was measured using a small animal CT / in vivo imaging system (Milabs BV).

[0048] (11) Antitumor efficacy of ZIF-8@IFI44 siRNA@PEG-RGD: In the mouse xenograft tumor experiment, the nude mice received subcutaneous injection of about 5 million T24 cells; then, on the 9th day, the xenograft tumor mice were divided into five groups (n=5) by random allocation; on the 10th day, the mice were injected with phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA or ZIF-8@IFI44 siRNA@PEG through the tail vein, 1 nmol of siRNA was injected every three days, for a total of six cycles; on the 25th day, the mice were sacrificed, and their main organs and tumors were harvested and preserved with 4% paraformaldehyde for IHC and HE staining.

[0049] (12) Immunohistochemistry: The paraffin-embedded sections were deparaffinated and rehydrated, and then subjected to antigen retrieval with EDTA (pH=9.0); then, they were immersed in a 3% hydrogen peroxide solution for 15 minutes; after blocking with 5% milk for 2 hours, the sections were incubated with the primary antibody at 4°C overnight, and the next day, the sections were incubated with the secondary antibody for 1 hour, then stained with DAB and counterstained with hematoxylin. The degree of staining was evaluated using a scoring system, and the proportion of positive cells was evaluated using the following scale: 0 (0%), 1 (1-24% positive cells), 2 (25-49% positive cells), 3 (50-74% positive cells), and 4 (≥75% positive cells). The staining intensity was 0 for negative, 1 for weak, 2 for moderate, and 3 for strong. The immunoreactivity score was obtained by multiplying the degree score by the intensity score.

[0050] Example 1: Preparation of nanomaterial ZIF-8 (zeolitic imidazolate framework-8) encapsulating IFI44-targeted siRNA, the preparation process is as shown in Figure 1 , specifically: (1) IFI44 siRNA1, Zn 2+ and 2-methylimidazole to obtain ZIF-8@IFI44 siRNA1 by co-precipitation; (2) Add polyethylene glycol, stir at 4°C overnight, centrifuge several times, re-disperse in 10 mM MES, then add EDC and NHS, activate in a 37°C water bath for 25 min, to obtain ZIF-8@IFI44 siRNA1@PEG; (3) Add RGD-targeted peptide, centrifuge and wash after stirring at 4°C for 24 h, re-disperse in ultrapure water to obtain ZIF-8@IFI44 siRNA1@PEG-RGD.

[0051] The results of morphological analysis of ZIF-8@IFI44 siRNA1@PEG-RGD by field emission transmission electron microscopy (TEM) are as shown in Figure 2A, the results show that ZIF-8@IFI44 siRNA1@PEG-RGD is rhombus; the particle size and potential detection results of ZIF-8@IFI44 siRNA1@PEG-RGD by the particle size and potential detector are shown in Figure 2 B and Figure 2 C, the results show that the average diameter of ZIF-8@IFI44 siRNA1@PEG-RGD is about 165.3 nm, and the average potential is-19.2 mV; this indicates the successful construction of ZIF-8@IFI44 siRNA1@PEG-RGD.

[0052] Example 2: (1) IFI44 siRNA2, Zn 2+ and 2-methylimidazole to obtain ZIF-8@IFI44 siRNA2 by co-precipitation method; (2) Add polyethylene glycol, stir overnight at 4℃, centrifuge several times, and re-disperse in 10 mM MES, then add EDC and NHS, activate in 37℃ water bath for 25 min, to obtain ZIF-8@IFI44 siRNA2@PEG; (3) Add RGD targeting peptide, centrifuge and wash after stirring at 4℃ for 24 h, re-disperse in ultrapure water to obtain ZIF-8@IFI44 siRNA2@PEG-RGD.

[0053] Dialysis method is used to detect the release of IFI44 siRNA2 in ZIF-8@IFI44 siRNA2@PEG-RGD (10 mM phosphate buffer with PH=6, 37℃, 200 rpm shaking table), as shown in Figure 3 The results show that under acidic conditions, the cumulative release of IFI44 siRNA2 reaches 83.20% within 48 h.

[0054] Example 3: Different concentrations (10, 20, 40, 60 nM) of ZIF-8@IFI44 siRNA1@PEG-RGD were co-incubated with T24 cells and J82 cells for 48 h, and then the mRNA and protein expression levels of IFI44 were analyzed.

[0055] The results of mRNA and protein level analysis of IFI44 in T24 cells and J82 cells are shown in Figure 4The results show that, due to the effective endosome escape ability of ZIF-8@IFI44 siRNA1@PEG-RGD, the mRNA and protein expression levels of IFI44 in T24 cells and J82 cells can be significantly reduced with the increase of the concentration of ZIF-8@IFI44 siRNA1@PEG-RGD, and nearly complete inhibition is achieved at the concentration of 40 nM of ZIF-8@IFI44 siRNA1@PEG-RGD.

[0056] Example 4: 40 nM of ZIF-8@IFI44 siRNA2@PEG-RGD, and multiple groups of controls (phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA2) were respectively co-incubated with T24 cells and J82 cells for 48 hours, followed by analysis of the mRNA and protein expression levels of IFI44.

[0057] The results of the analysis of the mRNA and protein levels of IFI44 in T24 cells and J82 cells are shown in Figure 5 The results show that, compared with the phosphate buffer, ZIF-8, and ZIF-8@siNC, the mRNA and protein expression levels of IFI44 of ZIF-8@IFI44 siRNA2 are reduced, and the mRNA and protein expression levels of IFI44 of ZIF-8@IFI44 siRNA2@PEG-RGD are reduced most significantly. This indicates that the IFI44 siRNA2 wrapped by ZIF-8 has the function of knocking down IFI44; and the addition of RGD targeting peptide can enhance the uptake efficiency of bladder cancer cells to the nanomaterial, and the knocking down efficiency is more significant.

[0058] Example 5: After 40 nM of ZIF-8@IFI44 siRNA2@PEG-RGD and multiple groups of controls (phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA2) were respectively co-incubated with T24 cells and J82 cells for 48 hours, the proliferation and migration abilities of bladder cancer cells were evaluated by CCK8 experiment and Transwell migration experiment.

[0059] The results of the CCK8 experiment analysis are shown in A of Figure 6 The results show that ZIF-8@IFI44 siRNA2 can inhibit the proliferation ability of bladder cancer cells, and ZIF-8@IFI44 siRNA2@PEG-RGD has the most significant effect on inhibiting the proliferation of bladder cancer cells.

[0060] The results of the Transwell migration experiment analysis are shown inFigure 6 The results show that ZIF-8@IFI44 siRNA2 can inhibit the migration ability of bladder cancer cells, and ZIF-8@IFI44 siRNA2@PEG-RGD has the most significant effect on inhibiting the migration of bladder cancer cells.

[0061] Example 6: After 40 nM of ZIF-8@IFI44 siRNA2@PEG-RGD and multiple groups of controls (phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA2) were co-incubated with T24 cells and J82 cells for 48 hours, apoptosis experiments were performed.

[0062] The analysis results of the apoptosis experiment by flow cytometry analysis are shown in Figure 7 The results show that ZIF-8@IFI44 siRNA2 can increase the apoptosis of bladder cancer cells, and ZIF-8@IFI44 siRNA2@PEG-RGD further increases the apoptosis rate of bladder cancer cells.

[0063] The results of Example 5 and Example 6 show that ZIF-8@IFI44 siRNA2 can inhibit the malignant progression of bladder cancer cells in vitro, and the inhibitory effect is more significant after the addition of RGD ligand.

[0064] Example 7: In order to explore why knocking down IFI44 can inhibit the malignant progression of bladder cancer cells, IFI44 siRNA2 was used to knock down IFI44, and controls were set up for RNA sequencing analysis. KEGG enrichment analysis is shown in Figure 8 The results show that knocking down IFI44 mainly affects the PI3K-AKT signaling pathway.

[0065] Then, IFI44 siRNA1 and IFI44 siRNA2 were used to knock down IFI44, and controls were set up to verify the Western Blot (protein blotting method) on T24 cells and J82 cells with knocked down IFI44, as shown in Figure 8 The results show that the phosphorylation of PI3K and AKT, the key genes in the PI3K-AKT pathway, is reduced, which indicates that IFI44 (knockdown) affects the malignant progression of bladder cancer by affecting the PI3K-AKT signaling pathway.

[0066] After co-incubating 40 nM ZIF-8@IFI44 siRNA2@PEG-RGD with multiple control groups (phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA2) with T24 cells and J82 cells for 48 hours, Western blotting experiments were performed.

[0067] Western blotting results are as follows: Figure 8 As shown in C and D, the results show that ZIF-8@IFI44 siRNA2 can inhibit the phosphorylation of PI3K / AKT, while ZIF-8@IFI44 siRNA2@PEG-RGD further inhibits its phosphorylation. This further demonstrates on nanomaterials that IFI44 affects the malignant progression of bladder cancer by influencing the PI3K-AKT signaling pathway.

[0068] Example 8: To evaluate the antitumor efficacy of ZIF-8@IFI44 siRNA1@PEG-RGD, a xenograft bladder cancer model was established by injecting T24 cells into the axillary region of mice. The mice were divided into five groups. Starting on day 10, each group was injected with phosphate-buffered saline, ZIF-8@siNC, ZIF-8@IFI44 siRNA1, and ZIF-8@IFI44 siRNA1@PEG-RGD at 1 nmol via tail vein injection every three days. Each group received six injections. The volume of the tumor and the weight of the mice were measured and recorded.

[0069] The tumor volume records for each group of mice are as follows: Figure 9 As shown in Figure A, the results indicate that ZIF-8@IFI44 siRNA1 can inhibit the growth of bladder cancer tumors, and ZIF-8@IFI44 siRNA1@PEG-RGD has the strongest inhibitory effect on the growth of bladder cancer tumors.

[0070] Mouse weight recording results are as follows Figure 12 As shown in A, the results indicate that the body weight of mice in each group was not affected, demonstrating safety.

[0071] Once the largest tumor in the mice exceeded 1.5 cm in diameter, all mice were euthanized for tissue sampling. Figure 10 As shown; the volume and weight of the tumors in each group of mice after dissection were measured, as shown. Figure 9The results are shown in FIGS. 8A and 8B, and the results show that the bladder cancer tumor volume and weight of the mice injected with ZIF-8@IFI44 siRNA1 and ZIF-8@IFI44 siRNA1@PEG-RGD are reduced, which indicates that ZIF-8@IFI44 siRNA1 has an anti-tumor property, and the anti-tumor property of ZIF-8@IFI44 siRNA1@PEG-RGD is the best.

[0072] Example 9: The tumor bodies of the mice in each group of Example 8 after dissection were embedded for immunohistochemical analysis.

[0073] The results of the immunohistochemical analysis are shown in FIGS. 9A and 9B, and the results show that the protein level of IFI44 of the mice injected with ZIF-8@IFI44 siRNA1 and ZIF-8@IFI44 siRNA1@PEG-RGD is significantly reduced compared with the mice injected with phosphate buffer, ZIF-8, ZIF-8@siNC, which indicates that the knockdown of IFI44 in vivo is successful. And through Ki67 detection, the mice injected with ZIF-8@IFI44 siRNA1 and ZIF-8@IFI44 siRNA1@PEG-RGD both show a decrease in the proliferation index Ki67, that is, an anti-tumor effect. Figure 11 The results of the immunohistochemical analysis prove that ZIF-8@IFI44 siRNA1 has an anti-tumor effect, and the anti-tumor effect of ZIF-8@IFI44 siRNA1@PEG-RGD is the best.

[0074] Example 10:

[0075] To study whether ZIF-8@IFI44 siRNA1@PEG-RGD treatment of bladder cancer in mice causes damage to the liver and kidney of the mice, in the above xenotransplant tumor bladder cancer model, after treatment with phosphate buffer, ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA, ZIF-8@IFI44 siRNA@PEG-RGD, the mice were sacrificed on the 28th day, and at the same time, the blood was taken from the eye orbit, and the ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREA (creatinine), and UREA (urea) in the blood were detected to exclude the existence of hepatotoxicity and nephrotoxicity. Used to prove the safety of the drug. The detection results of ALT, AST, CREA, and UREA are shown in FIGS. 10A and 10B.

[0076] Figure 12 ​The results show that ZIF-8@IFI44 siRNA1@PEG-RGD does not affect the liver and kidney indexes of mice after intravenous injection, which indicates the safety of ZIF-8@IFI44 siRNA1@PEG-RGD.

[0077] Example 11: To further study whether ZIF-8@IFI44 siRNA1@PEG-RGD treatment of bladder cancer in mice causes damage to the heart, liver, spleen, lung and kidney of mice, HE staining (hematoxylin-eosin staining) experiments were performed to paraffin-embed the heart, liver, spleen, lung and kidney of mice after treatment, followed by HE staining.

[0078] The results of HE staining verify the damage of internal organs, as shown in Figure 13 The results show that there is no difference in the heart, liver, spleen, lung and kidney of mice after injection of ZIF-8, ZIF-8@siNC, ZIF-8@IFI44 siRNA1 and ZIF-8@IFI44 siRNA1@PEG-RGD compared with injection of phosphate buffer, so the organs of mice are not damaged after tail vein injection of ZIF-8@IFI44 siRNA1@PEG-RGD, which confirms the safety of ZIF-8@IFI44 siRNA1@PEG-RGD.

[0079] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of an siRNA targeting IFI44 in the manufacture of a medicament for inhibiting the malignant progression of bladder cancer, characterized in that: The siRNA is selected from siRNA1 or siRNA2. The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.

2. The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.

4.

2. Use according to claim 1, characterized in that: The siRNA targeting IFI44 inhibits the malignant progression of bladder cancer by reducing the expression level of IFI44, thereby inhibiting the proliferation and migration of bladder cancer cells and promoting the apoptosis of bladder cancer cells.

3. Use according to claim 2, wherein: The bladder cancer cells are T24 cells or J82 cells.

4. Use according to claim 3, wherein: The drug further comprises a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The dosage form of the drug is a nanoparticle.

6. The use of a nanomaterial ZIF-8 encapsulating an siRNA targeting IFI44 in the preparation of a drug for inhibiting the malignant progression of bladder cancer, characterized in that: The siRNA is selected from siRNA1 or siRNA2. The siRNA1 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 1 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.

2. The siRNA2 comprises a sense strand with a nucleotide sequence as shown in SEQ ID NO. 3 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO.

4.

7. Use according to claim 6, wherein: The nanomaterial ZIF-8 encapsulating the siRNA targeting IFI44 further comprises an RGD targeting peptide.

8. Use according to claim 7, wherein: The preparation method of the nanomaterial ZIF-8 encapsulating the siRNA targeting IFI44 comprises the following steps: Step S1: The siRNA, Zn 2+ ZIF-8@IFI44 siRNA was prepared by co-precipitation method with 2-methylimidazole Step S2: add polyethylene glycol, stir at 4℃ overnight, centrifuge several times, re-disperse in 10mM MES, then add EDC and NHS, activate in a 37℃ water bath for 25min, and obtain ZIF-8@IFI44 siRNA@PEG; Step S3: add RGD targeting peptide, centrifuge and wash after stirring at 4℃ for 24h, re-disperse in ultrapure water, and obtain ZIF-8@IFI44 siRNA@PEG-RGD.

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