SiRNA of targeted RNR, pharmaceutical composition containing siRNA and application of siRNA in preparation of antitumor drugs

Abstract

The invention particularly relates to siRNA (Ribonucleic Acid) of targeted RNR (Ribonucleic Acid), a pharmaceutical composition containing the siRNA and application of the siRNA in preparation of anti-tumor drugs. The siRNA provided by the invention comprises at least one of si-RRM2 # 1 and si-RRM2 # 2, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3 to SEQ ID NO.6. The siRNA provided by the invention can effectively inhibit RRM2 expression, so that proliferation of esophageal cancer cells is remarkably inhibited, and cell apoptosis is induced. Meanwhile, by combining the compound with chemotherapy drugs, the sensitivity of esophageal cancer cells to the chemotherapy drugs can be further improved, then the treatment effect of the chemotherapy drugs on the esophageal cancer cells is enhanced, and the problem that the chemotherapy drugs are prone to drug resistance, and consequently esophageal cancer treatment fails is solved. The invention provides a brand new esophageal cancer treatment strategy combining siRNA and chemotherapeutic drugs, and the siRNA and chemotherapeutic drugs can be further applied to treatment of other specific RNR target tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to siRNA targeting RNR, a pharmaceutical composition containing the siRNA, and its application in the preparation of anti-tumor drugs. Background Art

[0002] Esophageal cancer is one of the cancers with high incidence and high fatality rate globally. It is highly invasive and has a very low survival rate. In China, the number of new cases and the number of fatal cases of esophageal cancer rank first in the world, and 85% of the patients have squamous cell carcinoma of the esophagus as the pathological type. Currently, the main treatment methods for esophageal cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and multimodal therapy, etc. However, in China, 60% - 70% of esophageal cancer patients are already in the advanced stage at the time of initial diagnosis and are difficult to undergo surgery and radiotherapy. Therefore, chemotherapy has become the core treatment method. Currently, the classic first-line chemotherapy regimen is 5-fluorouracil (5-FU) combined with cisplatin (FP regimen), but this regimen has relatively large toxic and side effects. In contrast, oxaliplatin combined with fluorouracil (FOLFOX regimen) has gradually gained favor due to its lower toxicity. In addition, the combination of taxane drugs (such as paclitaxel and albumin-bound paclitaxel) and platinum drugs also shows good efficacy. For patients who fail first-line treatment, second-line drugs such as taxane drugs, docetaxel, and S-1 have gradually become popular. At the same time, the "platinum rechallenge" strategy is still effective in some patients. However, during the chemotherapy process of esophageal cancer cells, due to various mechanisms such as gene mutations of tumor cells, overexpression of drug efflux pumps, changes in apoptosis pathways, and enhanced DNA repair ability, tumor cells are prone to develop drug resistance, resulting in the five-year survival rate of advanced esophageal cancer patients being still lower than 20%. Therefore, how to further improve the efficacy of chemotherapy drugs and reduce their toxic and side effects is an urgent problem to be solved in current clinical treatment.

[0003] Ribonucleotide reductase (RNR) is an important enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs) and plays a crucial role in regulating DNA synthesis. This enzyme is composed of two key subunits: the large subunit RRM1 and the small subunit RRM2. Research shows that the high expression of RRM2 not only promotes the proliferation and metastasis of tumor cells but also enhances the resistance of tumor cells to chemotherapy drugs. In view of this, RRM2 has become a potential target that has attracted much attention in the field of tumor treatment in recent years. By targeting and intervening in it through various ways, it is expected to effectively inhibit tumor growth and spread and significantly improve the effect of anti-tumor treatment.

[0004] As a tool capable of specifically silencing gene expression, small interfering RNA (siRNA) has shown great potential in anti-cancer therapy. siRNA binds to the target mRNA, induces its degradation, and then specifically inhibits the expression of specific genes. By targeting genes directly related to the survival, proliferation, invasion, metastasis, and drug resistance of tumor cells, siRNA can further enhance the anti-tumor effect of chemotherapeutic drugs, providing new strategies for tumor treatment.

[0005] Currently, there is no research report on the specific small interfering RNA (siRNA) therapy targeting the RRM2 target. At the same time, there is also a lack of exploration on the combined use of siRNA targeting RRM2 and chemotherapeutic drugs (such as 5-fluorouracil) in anti-tumor treatment, especially in the treatment of esophageal cancer. Therefore, it is of great significance to research and develop a specific siRNA therapy targeting RRM2 and combine it with chemotherapeutic drugs for combined use, in order to play a synergistic role in the treatment of tumors such as esophageal cancer, improve the treatment effect, and reduce chemotherapy drug resistance. This research not only has the potential to provide new strategies and methods for tumor treatment, but also may bring better treatment options and survival prognosis for patients. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention aims to provide siRNAs targeting RNR, pharmaceutical compositions containing the siRNAs, and their applications in the preparation of anti-tumor drugs. The present invention designed si-RRM2#1 and si-RRM2#2 targeting the small subunit RRM2 of RNR, both of which can effectively inhibit the expression of RRM2, and then significantly inhibit the proliferation of esophageal cancer cells and induce apoptosis. At the same time, the present invention combines si-RRM2#1 or si-RRM2#2 with chemotherapeutic drugs, and through experiments, it is proved that it can further reduce the half-maximal inhibitory concentration of esophageal cancer cells to chemotherapeutic drugs, and exacerbate the apoptosis and cell cycle arrest of esophageal cancer cells induced by chemotherapeutic drugs, thereby enhancing the therapeutic effect of chemotherapeutic drugs on esophageal cancer cells, and solving the problem that the failure of esophageal cancer treatment is easily caused by chemotherapy drug resistance.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide an siRNA targeting RNR, the siRNA specifically targets the RNR target, and includes at least one of si-RRM2#1 and si-RRM2#2;

[0009] The sense strand of the si-RRM2#1 is as shown in SEQ ID NO.3, and the antisense strand is as shown in SEQ ID NO.4;

[0010] The sense strand of the si-RRM2#2 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO.6.

[0011] Preferably, the siRNA specifically targets the RNR target and is used to specifically silence the expression of the RNR target gene.

[0012] Preferably, the siRNA targets the RRM2 of the RNR small subunit.

[0013] Preferably, the siRNA is obtained by chemical synthesis, in vitro transcription or in vivo expression system.

[0014] The second object of the present invention is to provide a pharmaceutical composition, comprising:

[0015] a. at least one siRNA targeting RNR as described in any one of claims 1 to 4; and

[0016] b. at least one chemotherapeutic drug;

[0017] The chemotherapeutic drug is selected from 5-fluorouracil, cis-dichlorodiammineplatinum (II), etoposide and docetaxel.

[0018] Preferably, the chemotherapeutic drug is 5-fluorouracil.

[0019] Preferably, the composition further comprises at least one pharmaceutically acceptable carrier, diluent or excipient.

[0020] Preferably, the siRNA targeting RNR and the chemotherapeutic drug in the pharmaceutical composition are used in combination, and the administration forms include direct co-blending administration, liposome encapsulation administration, nanoparticle encapsulation administration, aptamer-mediated targeted administration, antibody-mediated targeted administration and administration through a hydrogel composite delivery system.

[0021] Another object of the present invention is to provide the application of the above-mentioned siRNA targeting RNR or pharmaceutical composition in the preparation of anti-tumor drugs, and the tumors include esophageal cancer, lung cancer, pancreatic cancer, gastric cancer, breast cancer, colorectal cancer and thyroid cancer.

[0022] Another object of the present invention is to provide the application of the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs, and the tumors include esophageal cancer, lung cancer, pancreatic cancer, gastric cancer, breast cancer, colorectal cancer and thyroid cancer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention designs a brand-new siRNA targeting the small subunit RRM2 of RNR, including si-RRM2#1 and si-RRM2#2, both of which can effectively inhibit the expression of RRM2, thereby significantly inhibiting the proliferation of esophageal cancer cells and inducing apoptosis.

[0025] Secondly, the present invention also provides a pharmaceutical composition for the combined use of si-RRM2#1 or si-RRM2#2 with chemotherapeutic drugs. Experimental results show that the combined use of si-RRM2#1 or si-RRM2#2 with chemotherapeutic drugs can further improve the sensitivity of esophageal cancer cells to chemotherapeutic drugs, thereby enhancing the therapeutic effect of chemotherapeutic drugs on esophageal cancer cells and solving the problem of treatment failure of esophageal cancer caused by drug resistance of chemotherapeutic drugs.

[0026] The present invention provides a brand-new treatment strategy for esophageal cancer by combining siRNA with chemotherapeutic drugs, and it can further be applied to the treatment of other tumors specifically targeting the RNR target, especially the RRM2 target. Brief Description of the Drawings

[0027] Figure 1 It is a diagram showing the expression of RRM2 in esophageal cancer;

[0028] Figure 2 It is a diagram showing the silencing efficiency of si-RRM2;

[0029] Figure 3 It is a diagram showing the trend of CCK-8 cell viability and cell morphology;

[0030] Figure 4 It is a diagram showing the results of the colony formation assay;

[0031] Figure 5 It is a diagram showing the effect of si-RRM2 on enhancing the inhibition of the growth and proliferation of esophageal cancer cell line KYSE30 by chemotherapeutic drugs;

[0032] Figure 6 It is a diagram showing the effect of si-RRM2 on enhancing the inhibition of the growth and proliferation of esophageal cancer cell line KYSE410 by chemotherapeutic drugs;

[0033] Figure 7 It is a diagram showing the results of the EdU cell proliferation assay;

[0034] Figure 8 It is a diagram showing the effect of si-RRM2 on enhancing the inhibition of colony formation of esophageal cancer cells by 5-FU;

[0035] Figure 9 It is a diagram showing the results of the apoptosis assay;

[0036] Figure 10 It is a diagram showing the changes in the cell cycle, apoptosis, and cyclin expression of esophageal cancer cells. Detailed Description of the Invention

[0037] The following is a further detailed description of the above content of the present invention in the form of specific embodiments by way of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples only.

[0038] 1. Reagents and raw materials

[0039] The normal esophageal epithelial cell line HEEC was purchased from BeiNa Biotechnology, with the catalog number BNCC359279;

[0040] The esophageal cancer cell lines KYSE30 and KYSE410 were purchased from Wuhan Ponsure Biotechnology Co., Ltd., with the catalog numbers CL-0577 and CL-0586 respectively;

[0041] The siRNA fragments were synthesized by Suzhou GenePharma Co., Ltd.;

[0042] The transfection reagent Lipofectamine RNAiMAX was purchased from Thermo Fisher, catalog number 13778150;

[0043] The cell culture RNA extraction kit was purchased from Chengdu Foji Biotechnology Co., Ltd., catalog number RE-03111;

[0044] The TaKaRa reverse transcription kit was purchased from Takara Bio Inc. (Dalian), catalog number RR037A;

[0045] The 5-fluorouracil (5-FU) was purchased from Tianjin KingYork Pharmaceutical Co., Ltd., specification 10mL: 0.25g * 5 vials;

[0046] The cisplatin (cis-dichlorodiammineplatinum (II), CDDP) was purchased from Jiangsu Hansoh Pharmaceutical Group Co., Ltd., specification 6ml: 30mg * 5 bottles;

[0047] The etoposide was purchased from Qilu Pharmaceutical (Hainan) Co., Ltd., specification 5mL: 0.1g * 5 vials;

[0048] The docetaxel was purchased from Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd., specification 1mL: 20mg * 5 vials;

[0049] The CCK-8 kit was purchased from Beyotime Biotechnology, catalog number C0038;

[0050] The EdU-594 cell proliferation detection kit was purchased from Beyotime Biotechnology, catalog number C0071S;

[0051] The Annexin V-FITC apoptosis detection kit was purchased from Beyotime Biotechnology, product number C1062L;

[0052] The cell cycle detection kit was purchased from Beyotime Biotechnology, product number C1052.

[0053] 2. Instruments and equipment

[0054] The NanoDrop micro UV-Vis spectrophotometer was purchased from Thermo Fisher Scientific, product number 840-317400;

[0055] The microplate reader was purchased from Tecan Switzerland, model Sunrise;

[0056] The ZOE fluorescence cell imager was purchased from Bio-Rad Laboratories, Inc., USA;

[0057] The BD flow cytometer was purchased from BD, USA, model CANTO PLUS;

[0058] The real-time fluorescence quantitative PCR instrument was purchased from Thermo Fisher Scientific, model QuantStudio Dx;

[0059] The iBright intelligent imaging system was purchased from Thermo Fisher Scientific, model CL1000.

[0060] Example 1 Expression of RRM2 gene in normal cells and esophageal cancer cells

[0061] As Figure 1 (A) shows, the analysis results of the transcriptome database of GSE9982 esophageal cancer cell line (website: https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE9982) by the inventors of the present invention show that the expression level of RRM2 in all esophageal cancer cell lines is significantly higher than that of human normal esophageal epithelial cells.

[0062] To further verify this result, the present invention uses qPCR experiments to detect the expression of RRM2 in esophageal cancer cells, specifically including the following steps:

[0063] Cell treatment: Inoculate esophageal cancer cells (KYSE30, KYSE-410) into a 6-well plate at a density of 4×10 5 cells / well, culture in RPMI-1640 medium containing 10% fetal bovine serum, and place at 37°C, 5% CO 2In the incubator, when the cells grow to the logarithmic growth phase, wash them twice with PBS.

[0064] RNA extraction and detection: Lyse the cells and extract total RNA according to the instructions of the RNA extraction kit from Foji Biology. Detect the purity and concentration of RNA by NanoDrop to ensure that the OD260 / 280 value is between 1.8 and 2.0.

[0065] Reverse transcription and qPCR: Use the TaKaRa reverse transcription kit and operate according to the instructions to reverse transcribe 1 μg of RNA into cDNA and construct a qPCR reaction system. Detect by qPCR and record the Ct values of RRM2 and GAPDH.

[0066] Data analysis: Calculate the relative expression level of RRM2 (2^-△△Ct) using the △△Ct method. GAPDH was selected as the internal reference to calculate the relative expression level of the gene.

[0067] The experimental results are as Figure 1 (B) shown. It was found that the expression level of RRM2 in esophageal cancer cell lines KYSE30 and KYSE410 was significantly higher than that in the normal esophageal epithelial cell line HEEC. From Figure 1 The results can be seen that RRM2 was significantly up-regulated in Esophageal Squamous Cell Carcinoma (ESCC) cells, suggesting that it may play a key role in the occurrence and development of esophageal cancer.

[0068] Example 2 Verification of the efficiency of silencing RRM2 by qPCR

[0069] (1) Construction of siRNA targeting RRM2

[0070] According to the mRNA sequence of the RRM2 gene (refer to the NCBI database, https: / / www.ncbi.nlm.nih.gov / gene / 6241, Gene ID: 6241), a highly conserved region suitable for interference was selected as the target, and specific primers were designed using Primer-BLAST for qPCR detection of the mRNA expression level of RRM2. The sense strand of the RRM2 primer is shown as SEQ ID NO.1, and the antisense strand is shown as SEQ ID NO.2. According to the siRNA design principle, 2 candidate siRNA sequences were synthesized by Suzhou GenePharma Co., Ltd. for experimental verification. The sense strand of si-RRM2#1 is shown as SEQ ID NO.3, and the antisense strand is shown as SEQ ID NO.4; the sense strand of si-RRM2#2 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO.6; among them, the capital letters C, G, T, A, and N represent the base composition of nucleotides; the letter N represents U. si-NC was selected as the normalization control (i.e., SEQ ID NO.7 and SEQ ID NO.8).

[0071] Sequence information of the RRM2 specific primer:

[0072] Sense strand: 5’-AGTTCCTCACGGAGGCCTT-3’ (SEQ ID NO.1);

[0073] Antisense strand: 5’-CTCTGATACTCGCCTACTCGC-3’ (SEQ ID NO.2);

[0074] Sequence information of si-RRM2#1:

[0075] Sense strand: 5’-NCAAGAAACGAGGACNGANTT-3’ (SEQ ID NO.3);

[0076] Antisense strand: 5’-ANCAGNCCNCGNNNCNNGAGC-3’ (SEQ ID NO.4);

[0077] Sequence information of si-RRM2#2:

[0078] Sense strand: 5’-GAGGAGAGACNAAGAGAAATT-3’ (SEQ ID NO.5);

[0079] Antisense strand: 5’-NNNCNCNNACNCNCNCCNCTT-3’ (SEQ ID NO.6);

[0080] Sequence information of si-NC:

[0081] Sense strand: 5’-NCAAGAAACGAGGACNGANTT-3’ (SEQ ID NO.7);

[0082] Antisense strand: 5’-ANCAGNCCNCGNNNCNNGAGC-3’ (SEQ ID NO.8).

[0083] (2) Verification of the interference efficiency of siRRM2

[0084] Inoculation and culture of cells: Inoculate esophageal cancer cell lines KYSE30 and KYSE410 cells in 6-well plates, adjust the cell density to 4×10 5 cells / well, culture in RPMI-1640 medium containing 10% fetal bovine serum, and place in an incubator at 37°C and 5% CO 2 so that the confluence reaches 70% - 80% after 24 hours.

[0085] siRNA transfection: Prepare the siRNA transfection mixture according to the instructions of the transfection reagent Lipofectamine RNAiMAX. The specific operation is as follows:

[0086] Dilute and mix the RRM2-specific siRNA (experimental group si-RRM2#1, si-RRM2#2) or negative control siRNA (si-NC group) and the transfection reagent respectively. After standing for 5 - 10 minutes, add the mixture to the cells in serum-free medium and mix gently. After incubating for 4 - 6 hours, change to complete medium containing 10% FBS and continue to culture for 48 hours.

[0087] RNA extraction and detection: Use an RNA extraction kit and operate according to the instructions to extract the total RNA in the cells. Use NanoDrop to detect the concentration and purity of RNA (A260 / A280 value is 1.8 - 2.0).

[0088] Reverse transcription and qPCR: Use the TaKaRa reverse transcription kit and operate according to the instructions to reverse transcribe 1μg RNA into cDNA and construct a qPCR reaction system. Through qPCR detection, record the Ct values of RRM2 and GAPDH.

[0089] Data analysis: Calculate the relative expression level of RRM2 (2^-△△Ct) using the △△Ct method, and GAPDH is selected as the internal reference to calculate the relative expression level of the gene.

[0090] The experimental results are as Figure 2As shown in the figure, the results showed that after transfection of two siRNAs targeting RRM2 (si-RRM2#1 and si-RRM2#2) into cells, both siRNAs exhibited high silencing efficiency, significantly reducing the mRNA expression levels of RRM2 in KYSE30 and KYSE410 cells, and the silencing efficiencies reached 82.2% and 89.8% respectively. It was shown that si-RRM2#1 and si-RRM2#2 could effectively and precisely interfere with the expression of RRM2.

[0091] Example 3 Silencing RRM2 Inhibits the Growth and Proliferation of Esophageal Cancer Cells

[0092] Cell seeding and transfection: KYSE30 and KYSE410 cells were seeded in 96-well plates at a density of 7000 cells / well, and the cells in the 96-well plates were transfected according to the method in Example 2 to construct a control group (si-NC) and experimental groups (si-RRM2#1, si-RRM2#2).

[0093] CCK-8 assay for cell viability: After transfection, the 96-well plates were placed in an incubator at 37°C and 5% CO 2 for 48 hours. Then, 10 μL of CCK-8 solution was added to each well and incubation continued for 1 - 1.5 hours. Subsequently, the OD value (wavelength 450 nm) was read on an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Sunrise), and the absorbance of each well was measured to calculate the cell viability. According to the formula of cell viability (%) = (OD 实验组 -OD 空白 ) / (OD 对照组 -OD 空白 )×100%, the cell viability was calculated. Among them, OD 实验组 was the OD value of the experimental group cells, OD 对照组 was the OD value of the control group cells, and OD 空白 was the OD value of the blank well without cells.

[0094] Microscopic observation: The white light channel in the ZOE fluorescence cell imager was used to observe the cell morphology, and records and photographs were taken.

[0095] Colony formation assay: KYSE30 and KYSE410 cells were seeded in 6-well plates at a density of 1000 cells / well and transfected as described above. After transfection, the 6-well plates were placed in an incubator at 37°C and 5% CO 2 for culture. The experiment was terminated when the cell colonies grew to a size visible to the naked eye and the experimental results could be clearly observed. The cells were washed several times with PBS, fixed with 4% paraformaldehyde for 30 minutes, the paraformaldehyde was aspirated, the cells were washed 3 times with PBS, stained with 0.1% crystal violet for 30 minutes. The crystal violet was removed, the cells were washed with running water until the background was clean, dried, photographed, and the number of colonies was counted.

[0096] The experimental results are as Figure 3 and Figure 4 shown. It can be seen that transfection with siRRM2 significantly inhibited the growth of KYSE30 and KYSE410 cells in a time-dependent manner. Microscopic observation of cell morphology revealed that compared with the spindle or rhomboid morphology of cells in the si-NC group, the number of cells in the si-RRM2#1 and si-RRM2#2 groups was significantly reduced, the morphology changed from rhomboid to round, and shrinkage was presented. The results of the cell colony formation assay showed that compared with the si-NC group, the number of colonies and the colony formation area in the si-RRM#1 and si-RRM2#2 groups were significantly reduced. It was indicated that silencing RRM2 significantly interfered with the growth and proliferation of esophageal cancer cells.

[0097] Example 4 Silencing RRM2 Enhances the Inhibitory Effect of Chemotherapeutic Drugs on the Growth of Esophageal Cancer Cells

[0098] Cell seeding and transfection: KYSE30 and KYSE410 cells were seeded in 96-well plates at a density of 7000 cells / well and transfected with si-RRM2#1 and si-RRM2#2 as described above. After transfection, the 96-well plates were placed in an incubator at 37 °C and 5% CO 2 .

[0099] Drug treatment and combination: In the KYSE30 cell transfection group, different concentrations of 5-FU (5, 10, 20, 40 μM), different concentrations of cisplatin (1.25, 2.50, 5, 10, 20 μM), different concentrations of etoposide (5, 10, 20, 40, 80 μM), and different concentrations of docetaxel (0.9375, 1.875, 3.75, 7.5, 15 nM) were combined for 48 hours; in the KYSE410 cell transfection group, different concentrations of 5-FU (5, 10, 20, 40 μM), different concentrations of cisplatin (2.50, 5, 10, 20, 40 μM), different concentrations of etoposide (5, 10, 20, 40, 80, 160 μM), and different concentrations of docetaxel (6.25, 12.5, 25, 50, 100 nM) were combined for 48 hours.

[0100] CCK-8 assay for cell viability: After culturing for 48 hours, the liquid in the well plates was removed. The pre-warmed blank medium and CCK8 reagent were mixed into a detection solution at a ratio of 9:1, and then 100 μL of the detection solution was added to each well of the 96-well plates. The reaction was carried out for 1 - 1.5 hours, and the OD value (wavelength 450 nm) was read on an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance value of each well was read, and the cell viability was calculated. The calculation method of cell viability was the same as that in Example 3.

[0101] Control group setting: A blank control group was set up in a 96-well plate, that is, only blank medium was added, without transfection and drug treatment, to correct the baseline of OD values; a negative control group was set up in the transfection group, that is, non-specific siRNA (si-NC) was transfected to evaluate the effect of transfection itself on cell viability.

[0102] The experimental results are as Figure 5 and Figure 6 shown. After constructing transient interference models of KEYSE30 and KYSE410, 4 commonly used clinical chemotherapy drugs, 5-FU, cisplatin, etoposide, and docetaxel, were used to treat the cells. From Figure 5 and Figure 6 the CCK8 experimental results, it can be seen that compared with other first-line anti-cancer drugs for esophageal cancer, 5-FU has the strongest effect on inhibiting cell growth in RRM2 gene knockdown cells, and the combined effect is the best. This result indicates that targeted silencing of RRM2 can enhance the therapeutic effect of 5-FU on esophageal cancer cells, has potential clinical application value, and provides a new combination treatment strategy.

[0103] Example 5 Silencing of RRM2 enhances the inhibitory effect of 5-FU on the proliferation of esophageal cancer cells

[0104] Cell plating and transfection: KYSE30 and KYSE410 were plated in a 96-well plate at a density of 7000 / well. After the cells adhered, KYSE30 and KYSE410 cells were transfected with si-RRM2#2 according to the above transfection method.

[0105] Drug treatment and grouping: The transfected cells were divided into four groups, a blank control group (added with blank medium RPMI-1640), an si-RRM2#2 group, a 5-FU single-use group (10 μM), and a combined group (si-RRM2#2 + 5-FU), and the drug treatment was carried out for 24 hours.

[0106] EdU cell proliferation assay: The EdU-594 cell proliferation assay kit was used to detect cell proliferation according to the instructions. Prepare a 2-fold concentrated EdU working solution, add 100 μL to each well, mix with the culture medium, and incubate at 37 °C for 2 hours. Remove the above liquid, add 4% paraformaldehyde to fix the cells for 15 minutes, and wash the cells 3 times. Add an appropriate immunostaining washing solution to permeabilize the cells. After 10 - 15 minutes, wash the cells 1 - 2 times with PBS containing 3% BSA. Add 50 μL of reaction solution to each well for staining, wash the cells 3 times, add nuclear staining solution, incubate at room temperature in the dark for 10 minutes, observe the fluorescence staining of the cells using Cytation, and take pictures and record in different channels. Hoechst 33342 is blue fluorescence, with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm (blue light). The maximum excitation wavelength of Azide 594 is 590 nm, and the maximum emission wavelength is 615 nm (red light). Quantitative analysis was performed using imageJ, and statistical graphs were plotted using GraphPad Prism 8.

[0107] Colony formation assay: KYSE30 and KYSE410 cells were plated in 6-well plates at a density of 1000 cells / well. After transfection for 6 hours, the cells were treated with si-RRM2#2, 5-FU (10 μM), or combined treatment for 14 days, and then colony formation was detected.

[0108] The experimental results are as Figure 7 and Figure 8 shown. The cells treated with si-RRM2#2 showed significant proliferation inhibition after 5-FU treatment, and the proportion of EdU-positive cells decreased significantly. Further colony formation assay results showed that compared with the group treated with 5-FU alone, the number of colonies in the group treated with the combination of si-RRM2#2 and 5-FU decreased significantly, and the area of colony formation also decreased significantly. These results indicate that silencing RRM2 can not only inhibit the proliferation of esophageal cancer cells but also enhance the inhibitory effect of 5-FU on cell proliferation.

[0109] Example 6 Silencing RRM2 enhances 5-FU-induced apoptosis and cell cycle arrest

[0110] Cell plating and transfection: The KYSE30 and KYSE410 cells to be detected were plated in 6-well plates, and the KYSE30 and KYSE410 cells were transfected with si-RRM2#2 according to the above transfection method.

[0111] Drug treatment and cell collection: After transfection for 6 hours, 10, 40 μM of 5-FU solution was added to the corresponding wells for treatment, and the 6-well plates were continued to be placed at 37 °C, 5% CO 2Cultivate in an incubator for 48 hours. After 48 hours, collect the floating cells in the supernatant. Meanwhile, wash the adherent cells once with PBS, add an appropriate amount of trypsin without EDTA for digestion to detach the cells from the wall. Incubate in an incubator until the adherent cells can be detached by gentle pipetting, then aspirate the trypsin cell digestion solution. Transfer the detached cells together with the cells collected from the previous supernatant into a centrifuge tube, centrifuge, discard the supernatant, and collect the cells.

[0112] Cell apoptosis and cycle detection: Process according to the Annexin V-FITC double staining cell apoptosis detection kit or the cycle detection reagent instruction manual. Use a BD flow cytometer to detect cell apoptosis and cell cycle.

[0113] Protein extraction and Western Blotting detection: Collect cells according to the above method, extract total cell protein, measure the protein concentration and perform denaturation treatment. After subjecting the protein sample to SDS electrophoresis, transfer the protein to a PVDF membrane, block it with 5% skim milk powder at room temperature for 1 hour, incubate with the primary antibody corresponding to the detected protein, and incubate overnight at 4°C. After 24 hours, wash the membrane 3 times with TBST, incubate with the secondary antibody at room temperature for 1 hour, and wash 3 times with TBST.

[0114] Developing and result analysis: Use the ECL method for developing, and detect the expression levels of apoptosis-related protein Cleaved-PARP and cell cycle protein CyclinD1 in esophageal cancer cells after treatment with si-RRM2#2 alone, 5-FU alone, or in combination.

[0115] From Figure 9 and Figure 10 it can be seen that both silencing RRM2 and 5-FU treatment can activate the expression of Cleaved-PARP, but in the combined group, the expression level of Cleaved-PARP is further increased. Compared with the control group, the expression levels of CyclinD1 in the 5-FU group and the si-RRM2#2 group are decreased, and in the combined group, the expression of CyclinD1 protein is further decreased, causing the cells to be arrested at the G1 / G0 phase. These results indicate that silencing RRM2 in esophageal cancer cells can promote cell apoptosis, arrest the cells at the G1 / G0 phase, and thus improve the sensitivity of esophageal cancer cells to 5-FU.

[0116] The above embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A siRNA targeting RNR, characterized in that The siRNA specifically targets the RNR target, including at least one of si-RRM2#1 and si-RRM2#2; The sense strand of the si-RRM2#1 is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; The positive strand of the si-RRM2#2 is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.

6.

2. The siRNA targeting RNR according to claim 1, characterized in that The siRNA is specific to the RNR target site and is used to specifically silence the expression of the RNR target site gene.

3. The siRNA targeting RNR according to claim 1, characterized in that The siRNA targets the RNR small subunit RRM2.

4. The siRNA targeting RNR according to claim 1, characterized in that The siRNA is obtained by chemical synthesis, in vitro transcription or in vivo expression system.

5. A pharmaceutical composition, characterized in that Include: a. at least one siRNA targeting RNR as described in any one of claims 1 to 4; and b. At least one chemotherapy drug; The chemotherapy drug is selected from 5-fluorouracil, cis-diamminedichloroplatinum (II), etoposide and docetaxel.

6. The pharmaceutical composition according to claim 5, characterized in that The chemotherapy drug is 5-fluorouracil.

7. The pharmaceutical composition according to claim 5, characterized in that The composition further comprises at least one pharmaceutically acceptable carrier, diluent or excipient.

8. The pharmaceutical composition according to claim 5, characterized in that The siRNA targeting RNR and the chemotherapeutic drug in the pharmaceutical composition are used in combination, and the administration forms thereof include direct blending administration, liposome encapsulation administration, nanoparticle encapsulation administration, aptamer-mediated targeted administration, antibody-mediated targeted administration and administration via a hydrogel composite administration system.

9. Use of the siRNA targeting RNR according to any one of claims 1 to 4 in the preparation of anti-tumor drugs, characterized in that: The tumors include esophageal cancer, lung cancer, pancreatic cancer, gastric cancer, breast cancer, colorectal cancer and thyroid cancer.

10. Use of the pharmaceutical composition according to any one of claims 5 to 8 in the preparation of anti-tumor drugs, characterized in that: The tumors include esophageal cancer, lung cancer, pancreatic cancer, gastric cancer, breast cancer, colorectal cancer and thyroid cancer.

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