siRNAs, compositions, and applications for inhibiting MCM7

Abstract

This invention discloses an siRNA for inhibiting MCM7, a composition thereof, and its application. The siRNA designed and validated in this invention can efficiently inhibit MCM7 gene expression, thereby inhibiting DNA synthesis, cell proliferation, and cell clone generation in cancer cells, achieving the purpose of preventing and treating tumors. This invention provides a new target and candidate drug for cancer prevention or treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to siRNA for inhibiting MCM7, compositions and uses thereof. BACKGROUND

[0002] MCM complex is composed of MCM2-MCM7 subunits, has helicase activity in cells, opens DNA double strands before DNA replication, and participates in DNA replication initiation (Bik Tye, Annual Review of Biochemistry, 1999). Moreover, MCM complex also plays an important role in regulating cell proliferation, DNA damage repair and cell cycle.

[0003] Small interfering RNA (siRNA) is a double-stranded RNA with 20-25 nucleotides, which is first discovered in the post-transcriptional gene silencing phenomenon of plants. It is reported that artificially synthesized siRNA can silence the expression of specific genes in mammalian cells (Thomas Tuschl et al., Nature, 2001; Thomas Tuschl et al., Science, 2001; Thomas Tuschl et al., Cell, 2002). Since siRNA can target and interfere at the gene level without relying on the crystal structure of target protein, scientists have studied a series of methods for inhibiting the expression of target genes by using siRNA for RNA interference (RNAi), so as to carry out gene function research and disease treatment.

[0004] Since different sites on the target gene have different sequences, different secondary structures and different thermodynamic properties, the possibility and degree of interference of different sites by siRNA will be very different. In addition, the activity of the same siRNA in different types of cells may also be different. Therefore, for any target gene in any cell, the design, testing and obtaining of high-activity siRNA is a process of creative invention.

[0005] At present, there is no report about siRNA for inhibiting MCM7 gene of cancer cells such as liver cancer, gastric cancer and prostate cancer. SUMMARY

[0006] The primary purpose of the present application is to provide siRNA for inhibiting MCM7.

[0007] Another purpose of the present application is to provide the use of the above-mentioned siRNA.

[0008] Still another object of the present application is to provide a method for preventing or treating tumors / cancer by using siRNA to specifically target MCM7 gene.

[0009] The inventors designed and tested many RNA interference fragments against MCM7 gene, but most of the siRNAs had low interference efficiency and could not effectively proceed to later tumor treatment research. The inventors creatively invented some efficient siRNA sequences for interfering MCM7 gene through exploration research. This is crucial for the application of RNA interference, that is, the effects of siRNAs at different sites of target genes are quite different, which may be related to the secondary structure and thermodynamic properties of siRNA, base distribution and other factors.

[0010] The inventors further found that by changing one or more bases of the sense strand of siRNA, the sense strand and the antisense strand form an incomplete complementary pairing, thus changing the thermodynamic properties of the whole double-stranded RNA and improving the efficiency of the antisense strand entering the RNA interference complex protein, thereby improving the efficiency of siRNA in inhibiting the target MCM7 gene.

[0011] To achieve the above object, the technical solution adopted by the present application is:

[0012] The inventors designed siRNA targeting MCM7 protein. The siRNA inhibits the expression of human MCM7 gene and thus inhibits the synthesis of MCM7 protein, thereby disrupting the formation of the entire MCM complex (MCM2-MCM7), and thus inhibiting DNA replication and cell proliferation to achieve the purpose of preventing or treating tumors / cancer.

[0013] By inhibiting the expression of any one subunit of the MCM complex through siRNA, the formation of the entire complex can be inhibited, and thus cell proliferation can be inhibited to produce an anti-tumor effect.

[0014] In one aspect of the present application, siRNA is provided, which can inhibit the expression of MCM7 gene and is composed of a sense strand and an antisense strand, wherein the siRNA is selected from:

[0015] siRNA-1: sense strand: 5'-GGACUUAAUUUGUGAGAAU-3';

[0016] antisense strand: 5'-AUUCUCACAAAUUGAGUCC-3';

[0017] or

[0018] siRNA-2: sense strand: 5'-GGAAGUGGUAAAUAAAGAU-3';

[0019] Sense strand: 5'-AUCUUUAUUUACCACUUCC-3' Antisense strand: 5'-GGGAAAGUGAAUUAAAGTT-3'

[0020] or has 80% or more, or better, 90% or more homology with the sequence of the sense strand or antisense strand of the siRNA-1 or siRNA-2, or the base thereof can be modified into a derivative of nucleic acid, and has the same function.

[0021] Further, two deoxyribonucleotides dT or dN in single-stranded overhanging structure are added to the 3' end of the sense strand and antisense strand of the siRNA.

[0022] Further, the siRNA prevents or treats tumor / cancer by inhibiting the expression of the MCM7 gene.

[0023] Further, the tumor / cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, large intestine cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, esophageal cancer or brain tumor.

[0024] Further, the tumor can slow down or stop growing, shrink or disappear due to the inhibition of the expression of the MCM7 gene.

[0025] Further, the MCM7 gene is selected from human MCM7 gene.

[0026] Further, the sequence of the siRNA can be locally modified at some sites, as long as it does not affect its binding and inhibition of the target.

[0027] Further, the base of the siRNA sequence and its locally modified sequence can be modified into a derivative of nucleic acid, as long as it does not affect its binding and inhibition of the target.

[0028] In another aspect of the present application, the use of siRNA in the preparation of a drug or composition for preventing or treating tumor / cancer is provided.

[0029] Further, the tumor / cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, large intestine cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, esophageal cancer or brain tumor.

[0030] Further, the tumor / cancer can slow down or stop growing, shrink or disappear due to the inhibition of the expression of the MCM7 gene.

[0031] Further, the concentration of siRNA is 5-150 nM, preferably 10-100 nM, more preferably 15-60 nM, and most preferably 20-40 nM.

[0032] It can also have the following additional technical features:

[0033] The siRNA can inhibit the expression of MCM7 gene, and is composed of a sense strand and an antisense strand,

[0034] The siRNA is selected from the group consisting of:

[0035] siRNA-1: sense strand: 5'-GGACUUAAUUUGUGAGAAU-3';

[0036] antisense strand: 5'-AUUCUCACAAAUUGAGUCC-3';

[0037] or

[0038] siRNA-2: sense strand: 5'-GGAAGUGGUAAAUAAAGAU-3';

[0039] antisense strand: 5'-AUCUUUAUUUACCACUUCC-3';

[0040] or has 80% or more, or better, 90% or more homology with the sequence of the sense strand or the antisense strand of the siRNA-1 or siRNA-2, or the base thereof can be modified into a derivative of nucleic acid and has the same function.

[0041] Further, two deoxyribonucleotides dT or dN in single-stranded overhanging structure are added to the 3' end of the sense strand and the antisense strand of the siRNA.

[0042] Further, the MCM7 gene is selected from the human MCM7 gene.

[0043] Further, the siRNA can be locally modified at some sites as long as the binding and inhibition to the target are not affected.

[0044] Further, the base of the siRNA sequence and the locally modified sequence thereof can be modified into a derivative of nucleic acid as long as the binding and inhibition to the target are not affected.

[0045] In another aspect of the present application, a medicine or composition for preventing or treating tumor / cancer is provided, and the medicine or composition comprises:

[0046] the siRNA described above;

[0047] or an expression system capable of expressing the siRNA described above.

[0048] Further, the tumor / cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colon cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, esophageal cancer or brain tumor.

[0049] The concentration of the further siRNA is 5-150 nM, preferably 10-100 nM, more preferably 15-60 nM, and most preferably 20-40 nM.

[0050] Further, the tumor can slow down or stop growing, shrink or disappear due to the inhibition of the expression of the MCM7 gene.

[0051] Further, the drug or composition described above further comprises:

[0052] a pharmaceutically acceptable carrier;

[0053] other active ingredients for preventing or treating tumors.

[0054] Further, the pharmaceutically acceptable carrier and / or excipient includes, but is not limited to, buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological saline, excipients, fillers, coagulants and harmonizers, surfactants, diffusing agents, antifoaming agents.

[0055] Further, the other active ingredients for preventing or treating tumors include: chemotherapeutic agents, radiotherapeutic agents or antibody drugs.

[0056] Further, the form of the drug or composition is suitable for: direct naked RNA injection, direct injection of RNA wrapped in liposomes, direct injection of RNA wrapped in proteins or polypeptides, gold-coated RNA gene gun bombardment, bacterial plasmid expression of RNA or viral expression of RNA.

[0057] Further, the siRNA drug or composition can be in any form of solid, liquid, gel, semi-liquid, aerosol.

[0058] Further, the MCM7 gene is selected from the human MCM7 gene.

[0059] The use of siRNA can effectively inhibit the expression of the MCM7 gene and the synthesis of the protein, achieving the purpose of treating tumors / cancer.

[0060] The beneficial effects of the present application are:

[0061] The present application changes the 6th base of the 5' end of the sense strand of the siRNA-1 sequence, so that the sense strand and the antisense strand form an incomplete complementary pairing, thus changing the thermal dynamic properties of the entire double-stranded RNA, improving the efficiency of the antisense strand entering the RNA interference complex protein, and thereby improving the efficiency of siRNA-1 in inhibiting the target MCM7 gene.

[0062] Compared with conventional gene knockout technology, the siRNA provided by the application has the advantages of simple operation, short test period, high inhibition effect of more than 90% on MCM7 at mRNA and protein levels, high inhibition efficiency, good specificity, and the like, and can effectively inhibit DNA replication, proliferation and clonal formation ability of cancer cells, has important significance for developing new anticancer gene drugs and improving cancer treatment effect, and has significant clinical application prospect and economic value. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 Silencing effect of siRNA-1 specific to MCM7 on MCM7 mRNA level of HepG2 and Hep3B liver cancer cells.

[0064] Figure 2 Figures A to C of the drawing are respectively silencing effects of siRNA-1 specific to MCM7 on MCM7 protein level of HepG2 and Hep3B liver cancer cells and siRNA-2 on MCM7 protein level of HepG2 liver cancer cells, wherein β-actin is an internal reference protein.

[0065] Figure 3 EdU positive cell fluorescence microscope graph of siRNA-1 inhibiting DNA replication of HepG2 liver cancer cells, wherein Figure 3 Figures A, C and E are respectively EdU positive cell fluorescence microscope graph of negative control group cells NC transfected HepG2 liver cancer cells, Hochst stained cell nucleus DNA graph, EdU positive cell fluorescence microscope graph and Hochst stained cell nucleus DNA graph overlap graph of the transfected cells; wherein Figure 3 Figures B, D and F are respectively EdU positive cell fluorescence microscope graph of siRNA-1 transfected HepG2 liver cancer cells, Hochst stained cell nucleus DNA graph, EdU positive cell fluorescence microscope graph and Hochst stained cell nucleus DNA graph overlap graph of the transfected cells.

[0066] Figure 4 Figure is a proportion statistical graph of EdU incorporation positive HepG2 cells.

[0067] Figure 5 Figures A to E are respectively growth curves of siRNA-1 inhibiting proliferation of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and MCF7 breast cancer cells.

[0068] Figure 6Figures A to E are graphs showing the number of cancer cell clones after transfection of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and MCF7 breast cancer cells with siRNA-1 or negative control NC, respectively.

[0069] Figure 7 Figures A to E are graphs showing the proportion of the total area of cancer cell clones to the total area of the wells after transfection of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and MCF7 breast cancer cells with siRNA-1 or negative control NC, respectively. DETAILED DESCRIPTION

[0070] The technical solutions in the present application will be described below in connection with the examples, but the present application is not limited thereto.

[0071] Example 1 siRNA design

[0072] According to the basic principles of siRNA target sequences, siRNA sequences (1 21 -nucleotide) expressed by the MCM7 gene transcript (NM_001278595.1) were designed and synthesized, i.e. the sense strand and the antisense strand of siRNA, and the base sequences thereof are as follows:

[0073] siRNA-1 sense strand: 5'-GGACUUAAUUUGUGAGAAUdTdT-3'; (SEQ ID NO. 1)

[0074] siRNA-1 antisense strand: 5'-AUUCUCACAAAUUGAGUCCdTdT-3' (SEQ ID NO. 2).

[0075] or

[0076] siRNA-2 sense strand: 5'-GGAAGUGGUAAAUAAAGAUdTdT-3' (SEQ ID NO. 3);

[0077] siRNA-2 antisense strand: 5'-AUCUUUAUUUACCACUUCCdTdT-3' (SEQ ID NO. 4).

[0078] The base sequence of the negative control RNA (NC) is as follows:

[0079] Sense strand: 5'-CUCUUAGCCAAUAUUCGCUdTdT-3' (SEQ ID NO. 5);

[0080] Antisense: 5'- AGCGAAUAUUGGCUAAGAGdTdT-3' (SEQ ID NO. 6).

[0081] Two deoxyribonucleotides (dT or dN) in single-stranded overhang structure are added to the 3' end of the siRNA sequence and the sense and antisense strands of the control RNA in the present application to enhance the stability of the siRNA in vivo and in vitro and prevent degradation by nucleases.

[0082] The siRNA sequence in the present application can be partially modified at some sites as long as it does not affect the binding and inhibition of the target.

[0083] The siRNA sequence and its partially modified sequence in the present application can have the bases modified into derivatives of nucleic acids as long as it does not affect the binding and inhibition of the target.

[0084] Example 2 Transfection of siRNA in cells

[0085] Lipofectamine RNAiMax was used as the transfection reagent, and the procedure was in accordance with the operating procedures of Thermo Fisher Scientific. The cell lines used were HepG2 and Hep3B liver cancer cell lines, SGC-7907 gastric cancer cell line, PC3 prostate cancer cell line and MCF7 breast cancer cell line.

[0086] The experimental methods in the present application not specified in the specific conditions are usually in accordance with the conventional conditions such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or those suggested by the manufacturer.

[0087] The transfection procedure was as follows: the above different cell lines were inoculated in 12-well plates and cultured at 37°C, 5% CO2 overnight to allow the cells to grow to a density of 40-50%. The siRNA and negative control RNA (NC) prepared in the present application were transfected into different cell lines according to the operating procedures of Lipofectamine RNAiMax (Thermo Fisher Scientific). The cells were collected after transfection, and qRT-PCR and western blotting were used to further detect the interference effect of the siRNA.

[0088] Example 3 Detection of siRNA inhibition of MCM7 mRNA expression

[0089] Methods: Cells were collected after transfection, total RNA was extracted, reverse transcribed, and real-time quantitative PCR was performed to detect the expression level of MCM7 mRNA in cancer cells after siRNA treatment.

[0090] The siRNA and negative control RNA (NC) prepared in this invention were transfected into the HepG2 liver cancer cell line, and the same method was used to transfect the Hep3B liver cancer cell line. 24 hours after transfection, cells were collected, and an appropriate amount of cells were reseeded into 6-well plates. Total RNA was extracted from the cells 72 hours later. Reverse transcription was performed, and MCM7 mRNA was detected by real-time quantitative PCR.

[0091] 1. Total RNA extraction

[0092] (1) Collect tumor cells into centrifuge tubes, centrifuge at 800 rpm for 3 min, discard the supernatant, wash once with PBS and transfer to EP tubes;

[0093] (2) Centrifuge again at 800 rpm for 3 min, discard the supernatant, add 0.5 ml TRIzol, repeatedly pipette to dissolve the tumor cells, and let stand at room temperature for 5-10 min;

[0094] (3) Add 0.2 ml chloroform / ml TRIzol, shake vigorously to mix for 15 seconds, and let stand at room temperature for 10 minutes;

[0095] (4) Centrifuge at 12000 rpm and 4℃ for 15 min;

[0096] (5) After centrifugation, the liquid is divided into three layers, from bottom to top: phenol / chloroform layer, intermediate protein layer, and upper colorless aqueous phase. RNA is stored in the upper aqueous phase.

[0097] (6) Transfer the upper aqueous phase to a new EP tube, taking care to avoid aspirating the intermediate protein;

[0098] (7) Add 0.5 ml / ml of pre-cooled isopropanol TRIzol, mix by inversion, and let stand at room temperature for 10 min;

[0099] (8) Centrifuge at 12000 rpm and 4℃ for 10 min;

[0100] (9) Discard the supernatant and wash the RNA precipitate with 75% ethanol (750 μl anhydrous ethanol and 250 μl freshly prepared DEPC water). Centrifuge at 12,000 rpm and 4°C for 5 min.

[0101] (10) Discard the supernatant and dry it in a clean bench for about 3 minutes until the RNA becomes translucent.

[0102] (11) Add 15-20 μl of 1‰ DEPC water to dissolve the RNA precipitate, measure the concentration and OD value with a UV spectrophotometer, store at -70℃ or use directly for reverse transcription reaction.

[0103] 2. Reverse transcription into cDNA

[0104] Pre-deformed at high temperature for 5 minutes, then rapidly frozen on ice. Reverse transcription reaction system:

[0105]

[0106] Reaction conditions: 37℃ for 15 min, 50℃ for 5 min, 95℃ for 5 min, and hold at 4℃. The synthesized cDNA can be used immediately for downstream experiments or stored at -20℃.

[0107] 3. Real-time Quantitative PCR

[0108] After diluting the reverse-transcribed cDNA sample at an appropriate ratio, the following PCR reaction system was prepared using THUNDERBRID SYBR qPCR Mix:

[0109]

[0110] in:

[0111] The upstream primer sequence is 5'-GTGAAGGATCCTGCGACACA-3' (SEQ ID NO.7);

[0112] The downstream primer sequence is 5'-ACACGCGTTCTTTTGTTCCG-3' (SEQ ID NO.8);

[0113] The upstream primer sequence for the internal control is 5'-ACACGCGTTCTTTTGTTCCG-3' (SEQ ID NO.9);

[0114] The downstream primer sequence for the internal control is 5'-GGACTCCATGCCCAGGAAGGAA-3' (SEQ ID NO.10).

[0115] result: Figure 1 A and B indicate that, compared to the control group NC, transfection of cancer cells with siRNA-1 effectively inhibited the expression of MCM7 mRNA in HepG2 and Hep3B liver cancer cells, with a silencing effect of over 90%.

[0116] Example 4: Detection of siRNA inhibition of MCM7 protein expression

[0117] Methods: The siRNA and negative control RNA (NC) prepared in this invention were transfected into the hepatocellular carcinoma cell line HepG2, and the same method was used to transfect the hepatocellular carcinoma cell line Hep3B. After 24 hours of transfection, the cells were collected, and an appropriate amount of cells were re-seeded into 12-well plates. After 72 hours, the cells were collected for Western blotting experiments.

[0118] 1. Remove the culture medium, add an appropriate amount of 2×laemmli buffer, gently shake the 12-well plate to lyse the cells, collect them into PE tubes, rub the DNA against the wells of the PE tube rack to break up the DNA, and boil at 95℃ for 2 minutes.

[0119] 2. The boiled and denatured samples were subjected to polyacrylamide gel electrophoresis and Western blotting, and then the PVDF membrane was blocked with 5% skim milk at room temperature for 0.5 hours.

[0120] 3. Select a suitable primary antibody (mouse anti-human MCM7 monoclonal antibody, Santa Cruz Biotechnology), dilute it appropriately, and incubate it with a PVDF membrane overnight at 4°C.

[0121] 4. The next day, wash with TBST 3 times, 10 minutes each time;

[0122] 5. Select an HRP-labeled anti-mouse IgG secondary antibody corresponding to the host species of the primary antibody, dilute it appropriately, and incubate it with a PVDF membrane at room temperature for 1 hour.

[0123] 6. Wash with TBST 3 times, 10 minutes each time;

[0124] 7. ECL solution was used for imaging to detect the expression of MCM7 protein in tumor cells.

[0125] result: Figure 2 A, B, and C show that, compared to the control group NC, siRNA-1 transfection of cancer cells effectively inhibited the expression of MCM7 protein in HepG2 and Hep3B cells; siRNA-2 transfection of cancer cells effectively inhibited the expression of MCM7 protein in HepG2 cells; the silencing effect of both siRNA-1 and siRNA-2 was over 90%.

[0126] Example 5: siRNA inhibits DNA replication in cancer cells

[0127] Methods: The siRNA and negative control RNA (NC) prepared in this invention were transfected into the HepG2 liver cancer cell line, and the same method was used to transfect the Hep3B liver cancer cell line. Twenty-four hours after transfection, cells were collected, and an appropriate amount of cells were reseeded into 96-well plates. Twelve hours later, mimosine reagent was added to the cells and incubated for 24 hours to synchronize the cells at the G1 and S phase boundary.

[0128] Cells were washed three times with fresh culture medium, each time with a three-minute interval, to release them from mimosine inhibition. Cells were cultured in fresh culture medium for 3.5 hours, followed by 0.5 hours of culture with 50 mmol / L EdU (5-Ethynyl-2'-deoxyuridine, a thymidine analog). Cells were fixed and stained, and the proportion of cells showing EdU incorporation was observed and counted under a fluorescence microscope.

[0129] result: Figure 3 and Figure 4 The results showed that after siRNA-1 transfection of cancer cells, the proportion of EdU-incorporated positive cells was significantly reduced compared with the negative control NC, indicating that siRNA-1 significantly inhibited DNA replication in cancer cells.

[0130] The fresh culture medium used was Gibco RPMI 1640.

[0131] Example 6: siRNA inhibits cancer cell proliferation

[0132] Methods: The siRNA and negative control RNA (NC) prepared in this invention were transfected into different cancer cell lines, respectively. Cells were collected 24 hours after transfection. An appropriate amount of cells was divided into five equal parts and re-seeded in 12-well plates. Cells were counted continuously for five days, with one well selected for cell counting each day. Cell growth curves after transfection were plotted.

[0133] Result: As Figure 5 The results showed that siRNA-1 could effectively inhibit the proliferation of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells, and MCF7 breast cancer cells.

[0134] Similarly, siRNA-2 can also effectively inhibit the proliferation of the aforementioned cancer cells.

[0135] Example 7: siRNA inhibits cancer cell clone generation

[0136] Methods: The siRNA and negative control RNA (NC) prepared in this invention were transfected into the different cancer cell lines described above, respectively. Cells were collected 24 hours after transfection. Cells were seeded into 6-well plates at a cell density of 0.4 × 10⁻⁶ cells / well. 3 Cells / well. After 14 days of culture, fix with methanol and stain with crystal violet.

[0137] Result: As Figure 6 and Figure 7 show, Figure 6As shown in A to E, after siRNA-1 transfection of cancer cells, the number of cancer cell clones was significantly reduced compared with the negative control NC. Figure 7 As shown in A to E, after siRNA-1 transfected cancer cells, the proportion of the total clonal area to the total well area decreased compared with the negative control NC. This indicates that siRNA can effectively inhibit the clonal generation ability of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells, and MCF7 breast cancer cells.

[0138] Example 8: Application of MCM7siRNA

[0139] The present invention relates to the application of MCM7siRNA in the preparation of drugs for the prevention or treatment of tumors / cancers, wherein the cancer is selected from liver cancer, stomach cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal carcinoma, ovarian cancer, skin cancer, esophageal cancer, or brain tumor.

[0140] In summary, the siRNA of this invention effectively inhibits the expression of the MCM7 gene, thereby reducing the synthesis of the MCM7 protein. The inhibition effect of siRNA is as high as 90% or more, with extremely high inhibition efficiency and good specificity. At the same time, it can effectively inhibit the DNA replication, proliferation and clone formation ability of cancer cells. It is of great significance for the development of new anti-cancer gene drugs and the improvement of cancer treatment effects, and has significant clinical application prospects and economic value.

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. SEQUENCE LISTING <110> Enzhi (Guangzhou) Pharmaceutical Technology Co., Ltd. Enkang Pharmaceutical Technology (Guangzhou) Co., Ltd. Foshan Interpharm Technology Co., Ltd. and Guangzhou Intergene Technology Co., Ltd. <120> siRNAs that inhibit MCM7, their compositions and applications <130> <160> 10 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial primers <400> 1 ggacuuaauu ugugagaaut t 21 <210> 2 <211> twenty one <212> DNA <213> Artificial primers <400> 2 auucucacaa auugagucct t 21 <210> 3 <211> twenty one <212> DNA <213> Artificial primers <400> 3 ggaaguggua aauaaagaut t 21 <210> 4 <211> twenty one <212> DNA <213> Artificial primers <400> 4 aucuuuauuu accacuucct t 21 <210> 5 <211> twenty one <212> DNA <213> Artificial primers <400> 5 cucuuagcca auauucgcut t 21 <210> 6 <211> twenty one <212> DNA <213> Artificial primers <400> 6 agcgaauauu ggcuaagagt t 21 <210> 7 <211> 20 <212> DNA <213> Artificial primers <400> 7 gtgaaggatc ctgcgacaca 20 <210> 8 <211> 20 <212> DNA <213> Artificial primers <400> 8 acacgcgttc ttttgttccg 20 <210> 9 <211> 20 <212> DNA <213> Artificial primers <400> 9 acacgcgttc ttttgttccg 20 <210> 10 <211> twenty two <212> DNA <213> Artificial primers <400> 10 ggactccatg cccaggaagg aa 22

Claims

1. A siRNA, characterized in that, The siRNA can inhibit the expression of MCM7 gene, and is composed of a sense strand and an antisense strand, The siRNA is selected from the group consisting of: siRNA-1: sense strand: 5'-GGACUUAAUUUGUGAGAAU-3'; antisense strand: 5'-AUUCUCACAAAUUGAGUCC-3'; or siRNA-2: sense strand: 5'-GGAAGUGGUAAAUAAAGAU-3'; antisense strand: 5'-AUCUUUAUUUACCACUUCC-3'.

2. The siRNA of claim 1, wherein Two deoxyribonucleotides dT or dN in single-stranded overhang structure are added to the 3' end of the sense strand and the antisense strand of the siRNA.

3. Use of the siRNA according to claim 1 or 2 in the preparation of a medicament for preventing or treating cancer, wherein the cancer is selected from liver cancer, gastric cancer, prostate cancer or breast cancer.

4. A medicament for preventing or treating cancer, characterized by, The medicament comprises: the siRNA according to claim 1 or 2; or an expression system capable of expressing the siRNA according to claim 1 or 2.

5. The medicament according to claim 4, characterized in that, The cancer is selected from liver cancer, gastric cancer, prostate cancer or breast cancer.

6. The medicament according to claim 4, characterized in that, Further comprising: a pharmaceutically acceptable carrier; other active ingredients for preventing or treating tumors.

7. The medicament according to claim 6, characterized in that, The pharmaceutically acceptable carrier comprises buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological saline, excipients, fillers, coagulants and harmonizing agents, surfactants, diffusing agents, antifoaming agents.

8. The medicament according to claim 6, characterized in that, The other active ingredients for preventing or treating tumors comprise chemotherapeutic agents, radiotherapeutic agents or antibody drugs.

9. The medicament according to any one of claims 4 to 8, characterized in that, The medicament is selected from any of solid, liquid, gel, semi-liquid, and aerosol forms.

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