SiRNA for knocking down ZIC2 gene expression and application thereof

Abstract

The invention belongs to the technical field of disease treatment medicines, and particularly relates to siRNA for knocking down ZIC2 gene expression and application of the siRNA. The sequence of the siRNA is shown as SEQ ID NO: 1-2 or SEQ ID NO: 3-4, and the siRNA can effectively knock down the expression of a ZIC2 gene, so that the proliferation capacity of liver cancer cells is remarkably reduced, cell apoptosis is promoted, tumor cell invasion is reduced, and the aim of treating cancers is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease treatment drugs, and specifically relates to a siRNA for knocking down ZIC2 gene expression and an application thereof. Background Art

[0002] The ZIC2 gene is a gene that is closely related to various physiological and pathological processes such as nervous system development and cancer. ZIC2 belongs to the Zinc Finger transcription factor family and plays an important role in various types of tumors, including affecting the growth and metastasis of tumor cells by regulating cell cycle, migration, proliferation and other mechanisms. The overexpression of ZIC2 is closely related to the occurrence and progression of malignant tumors such as liver cancer and lung cancer. Therefore, ZIC2 has become a potential target in tumor treatment.

[0003] Intervention of the ZIC2 gene has become one of the important strategies to inhibit tumor cell growth. In recent years, RNA interference (RNAi), as an important gene silencing technology, has been widely used in gene function research and disease treatment. RNA interference is a natural process that inhibits the expression of specific genes through double-stranded RNA (dsRNA), which can achieve efficient and specific gene silencing at the post-transcriptional level. RNAi-mediated gene silencing is mediated by small interfering RNA (siRNA). siRNA molecules activate the action of RNA-induced silencing complex (RISC) by complementary binding to the target gene mRNA, thereby leading to the degradation of the target mRNA.

[0004] However, the ZIC2 gene has a high homology, and designing siRNA sequences with strong specificity and stability and achieving tumor cell killing remains a technical challenge. Summary of the invention

[0005] The present invention develops a siRNA for knocking down the expression of the ZIC2 gene. The siRNA can efficiently and specifically knock down the expression of the ZIC2 gene, thereby achieving the killing of tumor cells and further playing a role in cancer treatment.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a siRNA for knocking down ZIC2 gene expression, selected from siRNA1 or siRNA2, the sequence of siRNA1 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of siRNA2 is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0008] Another aspect of the present invention provides a drug for treating cancer, comprising the siRNA of the present invention.

[0009] Preferably, the above-mentioned medicine further comprises a pharmaceutically acceptable carrier.

[0010] In another aspect, the present invention provides a use of the siRNA of the present invention in the preparation of a drug for treating cancer, wherein the cancer is a cancer that overexpresses the ZIC2 gene.

[0011] Preferably, the application includes: application of the siRNA of the present invention in the preparation of a drug for inhibiting tumor cell proliferation.

[0012] More preferably, the application includes: use of the siRNA of the present invention in the preparation of a drug for delaying the G1 phase process and thereby inhibiting the proliferation of HepG2 cells.

[0013] Preferably, the application includes: application of the siRNA of the present invention in the preparation of a drug for promoting apoptosis of tumor cells.

[0014] Preferably, the application includes: application of the siRNA of the present invention in the preparation of a drug for inhibiting tumor cell invasion.

[0015] More preferably, the above cancer is liver cancer or lung cancer.

[0016] The beneficial effects of the present invention include: the siRNA provided by the present invention can effectively knock down the expression of the ZIC2 gene, resulting in a significant decrease in the proliferation ability of liver cancer cells, and promoting cell apoptosis and reducing tumor cell invasion, thereby achieving the purpose of treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The secondary structure diagram of siRNA; A: ZIC2siRNA1; B: ZIC2siRNA2;

[0018] Figure 2 Representative images of HepG2 cells transfected with ZIC2 siRNA under bright field and fluorescence microscopy;

[0019] Figure 3 The results of fluorescence real-time quantitative PCR measurement of the inhibitory effect of ZIC2 siRNA on ZIC2 mRNA expression; 1: control group (no siRNA transfection); 2: negative control group (transfection of non-target siRNA); 3: ZIC2 siRNA1 group; 4: ZIC2 siRNA2 group;

[0020] Figure 4AThe results of Western blot detection of the inhibitory effect of ZIC2 siRNA on ZIC2 protein expression; 1: negative control (transfection of non-target siRNA); 2: ZIC2 siRNA1; 3: ZIC2 siRNA2;

[0021] Figure 4B This is a bar graph of the inhibitory effect of ZIC2 siRNA on ZIC2 protein expression detected by Western blot; 1: negative control (transfection of non-target siRNA); 2: ZIC2 siRNA1; 3: ZIC2 siRNA2;

[0022] Figure 5 The results of the CCK8 method for detecting the inhibition of cancer cell proliferation by ZIC2 siRNA; 1: control group (no siRNA transfection); 2: negative control (transfection of non-target siRNA); 3: ZIC2 siRNA1; 4: ZIC2 siRNA2;

[0023] Fig. 6A This is the result of AnnexinV-PI double staining flow cytometry to detect apoptosis in the control group (not transfected with siRNA);

[0024] Figure 6B This is the result of AnnexinV-PI double staining flow cytometry detection of apoptosis in the negative control group (transfected with non-target siRNA);

[0025] Figure 6C This is the result of AnnexinV-PI double staining flow cytometry to detect ZIC2siRNA1 apoptosis;

[0026] Fig.6D This is the result of AnnexinV-PI double staining flow cytometry to detect ZIC2siRNA2 apoptosis;

[0027] Figure 7 This is the result of fluorescence real-time quantitative PCR to measure the inhibitory effect of ZIC2 siRNA on MMP-9 mRNA expression;

[0028] Fig. 8A This is the cell cycle diagram of the control group (not transfected with siRNA);

[0029] Figure 8B This is the cell cycle diagram of the negative control group (transfected with non-target siRNA);

[0030] Figure 8C This is the cell cycle diagram of ZIC2siRNA1;

[0031] Fig.8D This is the cell cycle diagram of ZIC2siRNA2. DETAILED DESCRIPTION

[0032] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0033] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0034] The embodiment of the present invention provides a siRNA for knocking down the expression of the ZIC2 gene, which is selected from siRNA1 or siRNA2, the sequence of siRNA1 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of siRNA2 is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0035] It should be noted that the siRNA provided by the present invention can effectively knock down the expression of the ZIC2 gene, resulting in a significant decrease in the proliferation ability of liver cancer cells and promoting cell apoptosis; in addition, the siRNA in the present invention can reduce the invasion of tumor cells, thereby achieving the purpose of treating cancer.

[0036] The embodiment of the present invention further provides a drug for treating cancer according to the present invention, including the siRNA according to the present invention.

[0037] It should be noted that, based on the inhibitory effect of the siRNA of the present invention on tumor cells, the siRNA of the present invention can be prepared into a drug for treating cancer.

[0038] In some specific examples, the above-mentioned drug further includes a pharmaceutically acceptable carrier.

[0039] It should be noted that the drug may also include other pharmaceutically acceptable carriers, such as buffer solutions and the like.

[0040] The embodiment of the present invention also provides a use of the siRNA of the present invention in preparing a drug for treating cancer, wherein the cancer is a cancer that overexpresses the ZIC2 gene.

[0041] In some specific examples, the application includes: the application of the siRNA of the present invention in the preparation of a drug for inhibiting the proliferation of tumor cells. Specifically, the siRNA of the present invention can achieve the purpose of treating cancer by inhibiting the proliferation of tumor cells.

[0042] In some specific examples, the application includes: the use of the siRNA of the present invention in the preparation of a drug for delaying the G1 phase process to inhibit the proliferation of HepG2 cells. Specifically, the siRNA of the present invention can delay the G1 phase process to inhibit the proliferation of HepG2 cells and thus achieve the purpose of treating cancer.

[0043] In some specific examples, the application includes: the application of the siRNA of the present invention in the preparation of a drug for promoting apoptosis of tumor cells. Specifically, the siRNA of the present invention can achieve the purpose of treating cancer by promoting apoptosis of tumor cells.

[0044] In some specific examples, the application includes: the application of the siRNA of the present invention in the preparation of a drug for inhibiting tumor cell invasion. Specifically, the siRNA of the present invention can achieve the purpose of treating cancer by inhibiting tumor cell invasion.

[0045] In some specific examples, the cancer is liver cancer or lung cancer.

[0046] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0047] 1. siRNA sequence design

[0048] According to the bioinformatics method, the human Zic2 mRNA sequence (NM_007129.5) was obtained from genebank. According to the siRNA sequence design principle, siRNA was designed using siRNA online design software such as siDirect (http: / / sidirect2.rnai.jp / ) and siRNA Wizard v3.1 (http: / / www.invivogen.com / sirnawizard / siRNA.php). The BLAST software provided by NCBI GenBank (written by the U.S. National Library of Medicine and the National Institutes of Health) was used to perform homology analysis on the selected target sequence, and the RNAfold Web Server software was used to predict the secondary structure of the mRNA target site of the selected target sequence. After excluding the regions with too complex secondary structures, two siRNA sequences, ZIC2 siRNA1 and ZIC2 siRNA2, were preliminarily screened out, and Shanghai Jima Pharmaceutical Technology Co., Ltd. was commissioned to synthesize the above siRNA sequences. The secondary structures of the two siRNA sequences are as follows: Figure 1 The specific sequence is shown in Table 1 below.

[0049] Table 1 2 siRNA sequences

[0050]

[0051] In addition, the minimum free energy (MFE) of ZIC2siRNA1 and ZIC2siRNA2 were -1.80 kcal / mol and 0 kcal / mol, respectively, the frequencies of MFE structures were 73.35% and 99.01%, and the free energies of thermodynamic clusters were -1.99 kcal / mol and -0.01 kcal / mol, respectively, indicating that ZIC2siRNA has a high quality, a stable secondary structure, and a high probability of base pairing, indicating that the siRNA can play an effective interference role in cells.

[0052] 2. ZIC2 siRNA transfection into HepG2 cells

[0053] HepG2 cells in logarithmic growth phase were cultured at 1×10 6 The cells were cultured in a 6-well plate at a density of 100 cells / well, and 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to culture until the confluence reached about 60%-80%. RNAiMAX transfection kit instructions for siRNA transfection, the steps are as follows:

[0054] (1) Discard the supernatant and dilute 9 μL of each with serum-free Opti-MEM medium RNAiMAX transfection reagent and 3 μL of 10 μM siRNA were added to 150 μL of diluted siRNA. Mix with RNAiMAX transfection reagent (volume ratio 1:1), and incubate the transfection mixture at 25°C for 5 minutes;

[0055] (2) Add the transfection mixture to the cells in step (1); set up a negative control group transfected with non-target siRNA (5'-TTCTCCGAACGTGTCACGT-3', SEQ ID NO: 5); set up 3 replicate wells in each of the siRNA group and the negative control group, and place the cells at 37°C, 5% CO 2 incubator with appropriate conditions for 48 h;

[0056] (3) Observe under a microscope and judge the transfection efficiency by fluorescence. The transfection efficiency of ZIC2siRNA1 and ZIC2siRNA2 can reach more than 80% and 70%, respectively. Figure 2 shown.

[0057] 3. Detection of ZIC2 mRNA expression after siRNA interference

[0058] (1) HepG2 cells in the logarithmic growth phase were cultured at 1×10 6 The cells were cultured in a 6-well plate at a density of 100 cells / well, and 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to culture until the confluence reached about 60%-80%. A control group (untransfected with siRNA), a negative control group (transfected with non-target siRNA), a ZIC2siRNA1 group, and a ZIC2siRNA2 group were set up respectively; Perform siRNA transfection according to the instructions of RNAiMAX transfection kit (refer to step 2 above for details);

[0059] (2) The transfected HepG2 cells were collected, 1 mL of Trizol was added, homogenized, and placed at room temperature for 5 min. 0.2 mL of chloroform was added, mixed, and placed at room temperature for 5 min. The mixture was centrifuged at 12,000 rpm at 4°C for 15 min. The colorless upper aqueous phase was aspirated into a new centrifuge tube, 0.5 mL of isopropanol was added, mixed, and placed at 4°C for 10 min. The mixture was centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was discarded. 1 mL of 75% ethanol was added, mixed, and centrifuged at 7,500 g at 4°C for 5 min. The supernatant was discarded. After the precipitate was slightly dry, 20 μL of RNase-free H 2 O, RNA purity was 498 ng / μL using the spectrophotometer (Nanodrop 8000) from ThermoScientific at OD260 / 280, and stored at -80°C for future use;

[0060] (3) Total RNA was extracted to remove genomic DNA. The reaction system was as shown in Table 2. The reaction system (reaction solution) was mixed and incubated in a 42° C. water bath for 2 min. After rewarming, the reaction solution was rapidly cooled to 4° C. to obtain the reaction solution.

[0061] Table 2 Reaction system for removing genomic DNA

[0062] 5XgDNA Eraser Buffer 2.0μL gDNA Eraser 1.0μL Total RNA 1.0μL <![CDATA[RNase-Free dH 2 The]]> 6.0μL Total volume 10μL

[0063] (4) The total RNA was reverse transcribed into cDNA under the following reaction conditions: 37°C, 15 min; 85°C, 5 s; 4°C, rapid cooling. The cDNA synthesis system is shown in Table 3.

[0064] Table 3 cDNA synthesis system

[0065] The reaction solution in step (3) 10μL PrimeScript RT Enzyme Mix I 1.0μL RT Primer Mix 1.0μL 5XPrimeScript Buffer 2 4.0μL <![CDATA[RNase-Free dH 2 The]]> 4.0μL Total volume 20μL

[0066] (5) Using the reverse transcribed cDNA in (4) as a template, the interference efficiency of siRNA was determined by real-time fluorescence quantitative PCR; the ZIC2 primers were: Forward: 5'-TCCCTTCCGGAGTCTTTGAA-3', Reverse: 5'-TTCAAAGACTCCGGAAGGGA-3'; the reaction conditions were: 95°C 5 min → (95°C, 30 s; 60°C, 30 s; 72°C, 10 s) 40 cycles → 72°C 10 min; wherein the real-time fluorescence quantitative PCR reaction system is shown in Table 4 below.

[0067] Table 4 Real-time fluorescence quantitative PCR reaction system

[0068] SYBR Premix Ex TaqII 12.5μL PCR Forward Primer 1.0μL PCR Reverse Primer 1.0μL cDNA solution 2.0μL <![CDATA[dH 2 The]]> 8.5μL Total volume 25μL

[0069] Test results such as Figure 3 As shown, the results showed that the inhibition rates of ZIC2 siRNA1 and ZIC2 siRNA2 on ZIC2 mRNA were 80% and 70%, respectively. Figure 3 The inhibition rate of the negative control was 0, indicating that ZIC2siRNA1 and ZIC2siRNA2 can effectively inhibit the expression of ZIC2 gene at the transcriptional level.

[0070] 4. Detection of ZIC2 protein expression after siRNA interference

[0071] (1) HepG2 cells in the logarithmic growth phase were cultured at 1×10 6The cells were cultured in a 6-well plate at a density of 100 cells / well, and 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to culture until the confluence reached about 60%-80%. A control group (untransfected with siRNA), a negative control group (transfected with non-target siRNA), a ZIC2siRNA1 group, and a ZIC2siRNA2 group were set up. Perform siRNA transfection according to the instructions of RNAiMAX transfection kit (refer to step 2 above for details);

[0072] (2) Cell lysis: 48 hours after transfection, cells were collected and centrifuged at 1000 rpm for 5 min. The collected cells were placed on ice. 100 μL of lysis buffer (10 μL PMSF + 990 μL PIRA, mixed) was added to each well of cells. The cells were fully lysed by pipetting several times and transferred to a 1.5 ml centrifuge tube and placed on ice.

[0073] (3) Protein concentration determination: After sufficient lysis, centrifuge at 12,000 rpm for 5 min, take the supernatant, and determine the protein content according to the instructions of the BCA protein concentration determination kit of Biyuntian Company. Add 5× protein loading buffer at a volume ratio of 1:4, denature at 99°C for 10 min, and quickly cool in an ice bath;

[0074] (4) Preparation of concentrated gel: Fix the cleaned glass plate on the gel-making mold, and align the bottom of the glass plate to prevent gel leakage; prepare 10 mL of 10% separation gel according to the mass of the target protein according to the kit instructions (BCA protein concentration determination kit), mix well and immediately pour into the double-layer glass plate, add 1 mL of anhydrous ethanol to press the gel, and solidify at room temperature;

[0075] (5) Preparation of separation gel: After the separation gel solidifies, pour out the anhydrous ethanol and absorb the residual ethanol with filter paper; prepare 3 mL of 5% concentrated gel according to the instructions, mix well and immediately pour it onto the top of the separation gel. Once it is full, immediately insert a Teflon comb and let it stand at room temperature for 30 min to solidify;

[0076] (6) Sample loading: Add enough SDS-PAGE electrophoresis buffer, pull out the comb steadily, and add the sample into the well, using the pre-stained protein marker as a reference;

[0077] (7) Electrophoresis: Set the voltage to 80 V. When the marker enters the separation gel, adjust the voltage to 120 V. The bromophenol blue runs to the bottom of the gel and the electrophoresis is terminated.

[0078] (8) Transfer: Separate the glass plates, take out the gel, cut the gel according to the size range of the target protein molecular weight and the internal reference protein molecular weight (β-actin), measure the length and width of the gel, cut out the PVDG membrane of the same size as the gel, activate it with methanol for 10 seconds, soak it in the transfer buffer for 5 minutes before use; place the following between the plastic racks in the order from negative to positive: foam, qualitative filter paper, gel, PVDF membrane, qualitative filter paper, foam. Gently press and roll to remove bubbles, put the clip into the electrophoresis tank, add enough transfer solution, and conduct constant current at 250mA and 4°C for about 1 hour;

[0079] (9) Blocking: After the transfer, transfer the membrane to TBST (9.0 g NaCL + 3.0 g Trisbase + 1 mL Tween-20 + 1 mL concentrated hydrochloric acid + ddH2O to 1000 mL, mix well) containing 5% skim milk powder and block on a shaker at room temperature for about 1 h;

[0080] (10) Incubation with primary antibody: refer to the instructions of the primary antibodies (ZIC2 antibody and β-actin antibody) of abcam, dilute the primary antibody with primary antibody diluent according to the appropriate ratio; add the diluted primary antibody and incubate at 4°C overnight; after blocking, wash the membrane three times with TBST, each time for 10 min;

[0081] (11) Incubation with secondary antibody: dilute the secondary antibody from Abcam with TBST at a ratio of 1:5000, incubate on a shaker at room temperature for 1 h, and wash the membrane three times with TBST for 10 min each time after the incubation.

[0082] (12) Development: Evenly drop ECL developer on the membrane and develop on the ChemiDoc MP developer.

[0083] Test results such as Figure 4A and Figure 4B As shown, the results showed that the expression of ZIC2 protein in the ZIC2siRNA1 group and the ZIC2siRNA2 group was significantly lower than that in the negative control group, indicating that ZIC2siRNA1 and ZIC2siRNA2 effectively inhibited the expression of the ZIC2siRNA2 gene at the protein level, and further indicating that ZIC2siRNA can inhibit the expression of the ZIC2siRNA gene at the protein level.

[0084] 5. Proliferation examination of HepG2 cells after siRNA interference

[0085] HepG2 cells in logarithmic growth phase were cultured at 4×10 4The cells were cultured in a 96-well plate at a density of 100 μL / well, and 100 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to culture until the confluence reached about 60%-80%. A control group (untransfected with siRNA), a negative control group (transfected with non-target siRNA), a ZIC2siRNA1 group, and a ZIC2siRNA2 group were set up. RNAiMAX transfection kit instructions for siRNA transfection, the steps are as follows:

[0086] (1) According to RNAiMAX transfection reagent instructions, dilute 1.5 μL with 25 μL serum-free Opti-MEM medium RNAiMAX transfection reagent and 0.5 μL of 10 μM siRNA were added to 25 μL of diluted siRNA. Mix with RNAiMAX transfection reagent (volume ratio 1:1), incubate at room temperature for 5 minutes to obtain the transfection mixture.

[0087] (2) Add the transfection mixture to the cells, with 3 replicate wells in each of the siRNA group and the negative control group (non-target siRNA cells). Place the cells at 37°C and 5% CO 2 After 48 h in an incubator with the same conditions, the culture medium was replaced with DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. A control group (cells not transfected with siRNA) and a blank control group (without cells, only culture medium) were set up. 10 μL of CCK8 solution was added to each well and incubated for another 1 h.

[0088] (3) Place the plate in the plate reader of Thermo Fisher Scientific's microplate reader (Multiskan FC), read the absorbance (OD value) at a wavelength of 450 nm and record it; the percentage of live cells is calculated as the ratio of the absorbance of (treated group - blank group) to (control group - blank group).

[0089] Test results such as Figure 5 As shown, the results showed that both ZIC2 siRNAs had an inhibitory effect on the proliferation of HepG2 cells, and the proliferation inhibition rates were 64.09%±0.91% and 57.78%±0.27%, respectively, indicating that ZIC2 siRNA can inhibit the growth of liver cancer cells.

[0090] 6. AnnexinV-PI double staining flow cytometry detection of siRNA interference on apoptosis of HepG2 cells

[0091] (1) HepG2 cells in the logarithmic growth phase were cultured in a 6-well plate at a density of 1×106 cells / well, and 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added, and cultured to about 60%-80% confluency. A control group (untransfected with siRNA), a negative control group (transfected with non-target siRNA), a ZIC2siRNA1 group, and a ZIC2siRNA2 group were set up respectively. Perform siRNA transfection according to the instructions of RNAiMAX transfection kit (refer to step 2 above for details);

[0092] (2) 48 h after transfection, cells were collected and washed with PBS, centrifuged at 1000 rpm for 5 min, washed twice, and resuspended in 200 μL 1× binding buffer to adjust the cell density to 1×10 6 Pieces / mL;

[0093] (3) According to the grouping, 5 μL Annexin V-FITC dye and 5 μL PI were added and gently mixed. The cells were incubated at room temperature in the dark for 15 min, and 300 μL PBS was added to resuspend the cells. The stained cells were detected using a Beckman Coulter flow cytometer (CytoFLEX); Annexin V / PI cells were living cells, and Annexin V+ / PI cells were early apoptotic cells.

[0094] Test results such as Figures 6A to 6D As shown in the results, the proportion of dead cells (early apoptosis + late apoptosis + necrotic cells) in the ZIC2siRNA1 and ZIC2siRNA2 experimental groups were 44.53% and 31.61%, respectively, while the proportion of dead cells in the control group and the negative control group were 22.93% and 24.35%, respectively, indicating that the apoptosis proportion of HepG2 cells increased significantly after ZIC2siRNA treatment.

[0095] 7. Detection of the expression of MMP-9 mRNA by siRNA interference in HepG2 cells

[0096] According to the method for detecting the expression of ZIC2 mRNA after siRNA interference in step 3 above, the expression of MMP-9 mRNA, an invasion molecule of HepG2 cells, was detected by siRNA interference; wherein the primers for MMP-9 are:

[0097] ForwardPrimer5′-GAAGATGCTGCTGTTCAGCG-3′;

[0098] ReversePrimer5′-ACTTGGTCCACCTGGTTCAA-3′.

[0099] Test results such as Figure 7 As shown, the results showed that compared with the control group and the negative control group, the RNA expression of MMP-9 in the ZIC2siRNA1 group and the ZIC2siRNA2 group was significantly inhibited, indicating that ZIC2siRNA1 and ZIC2siRNA2 had a significant inhibitory effect on the invasion of HepG2 cells.

[0100] 8. siRNA interference in cell cycle detection of HepG2 cells

[0101] (1) HepG2 cells in the logarithmic growth phase were cultured at 1×10 6 The cells were cultured in a 6-well plate at a density of 100 cells / well, and 2 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to culture until the confluence reached about 60%-80%. A control group (untransfected with siRNA), a negative control group (transfected with non-target siRNA), a ZIC2siRNA1 group, and a ZIC2siRNA2 group were set up. Perform siRNA transfection according to the instructions of RNAiMAX transfection kit (refer to step 2 above for details);

[0102] (2) 48 h after transfection with ZIC2 siRNA, HepG2 cells were collected; washed twice with pre-cooled PBS, centrifuged at 1000×g for 5 min, and the supernatant was discarded. The cells were resuspended in 300 μL 1× PBS, mixed, and fixed with 70% ethanol at 4°C for 20 min, centrifuged at 1500 rpm for 5 min, washed 2-3 times with PBS, resuspended in 300 μL PBS, added with 20 μg / mL RNaseA, and incubated at 37°C for 30 min; 50 μg / mL PI was added for staining for 30 min, and directly analyzed using Beckman Coulter flow cytometer (CytoFLEX).

[0103] Test results such as Figures 8A to 8D As shown in the figure, the proportion of cells in G1, S and G2 / M phases in the negative control group was not significantly different from that in the control group, while in the ZIC2siRNA1 and ZIC2siRNA2 treatment groups, the proportion of cells in G1 phase increased significantly, and the proportion of cells in S phase decreased to 28.02% and 25.76%, respectively. These results indicate that ZIC2siRNA delays the progression of G1 phase and thus effectively inhibits the proliferation of HepG2 cells.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An siRNA for knocking down ZIC2 gene expression, characterized in that: Selected from siRNA1 or siRNA2, the sequence of siRNA1 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of siRNA2 is shown in SEQ ID NO:3 and SEQ ID NO:

4.

2. A drug for treating cancer, characterized in that: Comprising the siRNA according to claim 1.

3. The drug according to claim 2, characterized in that The medicament also includes a pharmaceutically acceptable carrier.

4. Use of the siRNA according to claim 1 in the preparation of a drug for treating cancer, wherein the cancer is a cancer that overexpresses the ZIC2 gene.

5. The use according to claim 4, characterized in that: Applications include: Use of the siRNA described in claim 1 in the preparation of a drug for inhibiting tumor cell proliferation.

6. The use according to claim 5, characterized in that: Applications include: application of the siRNA described in claim 1 in the preparation of a drug for delaying the G1 phase process and thereby inhibiting the proliferation of HepG2 cells.

7. The use according to claim 4, characterized in that: Applications include: Use of the siRNA described in claim 1 in the preparation of a drug for promoting apoptosis of tumor cells.

8. The use according to claim 4, characterized in that: Applications include: Use of the siRNA described in claim 1 in the preparation of a drug for inhibiting tumor cell invasion.

9. The use according to any one of claims 4 to 8, characterized in that The cancer is liver cancer or lung cancer.

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