siRNA molecules for the treatment of ovarian cancer

Abstract

The present invention discloses a siRNA molecule for treating ovarian cancer, which is a siRNA molecule for inhibiting the NEK6 gene, and is composed of a sense strand and an antisense strand of the following sequence: the sense strand is 5'-CAGAUGAUCAAGUACUUUANn-3', and the antisense strand is 5'-UAAAGUACUUGAUCAUCUGNn-3'; or the sense strand is 5'-CGGAGAGGACAGUAUGGAANn-3', and the antisense strand is 5'-UUCCAUACUGUCCUCUCCGNn-3'. Wherein, N in the sense strand and the antisense strand is the same or different, and each is independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and n is 0, 1 or 2. The siRNA molecule of the present invention can be used to prepare a drug for treating ovarian cancer.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a siRNA molecule for inhibiting NEK6 gene and use of the siRNA molecule in preparing a drug for treating ovarian cancer. Background Art

[0002] Ovarian cancer is one of the common malignant tumors of female reproductive organs, and its incidence rate ranks third after cervical cancer and uterine body cancer (Gu et al., Int J Gynecol Cancer, 2017, 27: 77-84; Kaldawy et al., Gynecol Oncol, 2016, 143: 433-438.). Among ovarian cancers, epithelial cancer is the most common, but the mortality rate of ovarian epithelial cancer ranks first among all types of gynecological tumors, posing a serious threat to women's lives. Due to the complexity of ovarian embryonic development, tissue anatomy and endocrine function, early symptoms are atypical, and it is quite difficult to distinguish the tissue type and benign and malignant nature of ovarian tumors before surgery. Ovarian epithelial cancer is very easy to metastasize and invade. Only 30% of patients find that the tumor is confined to the ovary during surgery, and early diagnosis is a major problem (Weidle et al., Cancer Genomics Proteomics, 2016, 13: 407-423; Sulaiman et al., Front Pharmacol, 2016, 7: 271). Ovarian malignancies, especially epithelial cancers, are difficult to detect early, and the cause is unknown. Except for hereditary ovarian cancer, there is no primary prevention measure. Early diagnosis and early treatment are currently advocated to strive for early detection of lesions. Therefore, discovering biological markers for early diagnosis of ovarian cancer and finding new molecular therapeutic targets are the primary tasks in conquering ovarian cancer, and are also the hot spots in the current research on the molecular pathological mechanisms of ovarian cancer.

[0003] NIMA-related kinase (never in mitosis gene A-related expressed kinase, NEK) consists of 11 members, which are located in different cell regions and participate in cell biological processes such as cell cycle, cell cycle restriction points, ciliary biology, aging, telomere maintenance and chemical resistance (Kristensen et al., Nat Rev Genet, 2019, 20 (11): 675-691). NEK kinase is expressed at a low level in the G1 phase of the cell cycle, increased in the S phase and G2 phase, and decreased in expression after the cells enter mitosis. Compared with other phases, NEK activity peaks in the S phase and G2 phase, and assists centrosome separation in the G2 / M phase. Therefore, overexpression of NEK leads to chromosomal instability and aneuploidy in cancer cells. In addition, overexpression of NEK activates several oncogenic pathways, allowing normal cells to be transformed into cancer cells by acquiring proliferation, invasion and drug resistance (Li et al., Cell Death Dis, 2020, 11 (5): 399). Since the NEK kinase family is one of the less studied kinase families, the precise molecular mechanism of tumorigenesis is still unclear (Liang et al., Onco Targets Ther, 2020, 13: 6279-6288). Recent studies have shown that the NEK family is abnormally expressed in a variety of tumors, such as breast cancer, lung cancer, prostate cancer, pancreatic cancer, gastric cancer, colorectal cancer, and hepatocellular carcinoma. However, in addition to the more in-depth research in breast cancer (Yan et al., Front Oncol, 2021, 11: 1-13), the molecular mechanism of the NEK family in promoting the growth of other malignant tumors has not been reported. NIMA-associated kinase 6 (NEK6) is a recently discovered serine / threonine protein kinase that can regulate the cell cycle and has an important regulatory effect on a variety of signaling pathway kinases and transcription factors. It is one of the important members of the NEK family (Cao et al., Med (US), 2018, 97: 1-5). The researchers found that the expression of NEK6 in 133 breast cancer specimens was higher than that in adjacent tumor tissues, and the high expression of NEK6 was related to the histological grade, tumor size and TNM stage of breast cancer, indicating that NEK6 expression is an independent prognostic predictor for breast cancer, suggesting that it may become a potential therapeutic target for breast cancer (Sabir et al., PLoS ONE, 2015, 10: 1-19). In addition, the researchers also found that NEK6 is highly expressed in prostate cancer, and NEK6 overexpression stimulates the cytoskeleton, differentiation and immune signaling pathways (Zhang et al., Oncol Rep, 2018, 39: 1023-1033). NEK6 is also highly expressed in hepatocellular carcinoma, colon cancer and gastric cancer. Studies have shown that NEK6 can be used as one of the targeted molecules for tumor inhibition. Summary of the invention

[0004] In clinical medical research, the inventors unexpectedly discovered that the NEK6 gene is highly expressed in ovarian cancer patients, and the expression level of the NEK6 gene is more significant compared with patients with other female reproductive organ malignancies such as cervical cancer and uterine body cancer, suggesting that NEK6 can not only be used as one of the biomarkers for diagnosing ovarian cancer, but also has the potential to be a target for ovarian cancer treatment.

[0005] Since RNA interference technology has become a new gene therapy method and siRNA has been approved by the FDA, we have tried to design and screen hundreds of siRNA molecules with NEK6 gene as the target, and found that some of them can specifically inhibit NEK6 expression, and two groups of siRNA molecules can effectively inhibit NEK6 gene. Therefore, the first object of the present invention is to provide a siRNA molecule for inhibiting NEK6 gene, which is composed of the sense strand and antisense strand of the following sequence:

[0006] Sense strand: 5'-CAGAUGAUCAAGUACUUUANn-3',

[0007] Antisense strand: 5'-UAAAGUACUUGAUCAUCUGNn-3', or

[0008] Sense strand: 5'-CGGAGAGGACAGUAUGGAANn-3',

[0009] Antisense strand: 5′-UUCCAUACUGUCCUCUCCGNn-3′.

[0010] Wherein, N in the sense strand and the antisense strand is the same or different, and each is independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and n is 0, 1 or 2.

[0011] In one embodiment, the n is 0, that is,

[0012] Sense strand: 5'-CAGAUGAUCAAGUACUUUA-3' (SEQ ID NO: 1),

[0013] Antisense strand: 5'-UAAAGUACUUGAUCAUCUG-3' (SEQ ID NO: 2), or

[0014] Sense strand: 5'-CGGAGAGGACAGUAUGGAA-3' (SEQ ID NO: 3),

[0015] Antisense strand: 5'-UUCCAUACUGUCCUCUCCG-3' (SEQ ID NO: 4).

[0016] The siRNA molecule is the backbone sequence of the group of siRNA molecules.

[0017] In another preferred embodiment, N is dT, n is 2, that is,

[0018] Sense strand: 5'-CAGAUGAUCAAGUACUUUAdTdT-3' (SEQ ID NO: 5),

[0019] Antisense strand: 5'-UAAAGUACUUGAUCAUCUGdTdT-3' (SEQ ID NO: 6), or

[0020] Sense strand: 5'-CGGAGAGGACAGUAUGGAAdTdT-3' (SEQ ID NO: 7),

[0021] Antisense strand: 5'-UUCCAUACUGUCCUCUCCGdTdT-3' (SEQ ID NO: 8).

[0022] The second object of the present invention is to provide use of any of the above siRNA molecules or a mixture of the two in preparing a drug for inhibiting NEK6 expression.

[0023] Optionally, the above-mentioned drug is an anti-tumor drug.

[0024] Preferably, the drug is a drug for treating ovarian cancer.

[0025] Among them, siRNA molecules can be used as effective ingredients to inhibit the growth and metastasis of ovarian cancer cells, or promote the apoptosis of ovarian cancer cells.

[0026] The third aspect of the present invention provides a drug for treating ovarian cancer, which comprises the above-mentioned siRNA molecule as a drug active ingredient.

[0027] Optionally, the above-mentioned medicine is a pharmaceutical composition, which, in addition to comprising a therapeutically effective amount of the above-mentioned siRNA molecule as an active ingredient, further comprises a pharmaceutically acceptable carrier.

[0028] In one embodiment, the above-mentioned drug is a pharmaceutical composition, which, in addition to a therapeutically effective amount of the above-mentioned siRNA molecule, further comprises one or more other pharmaceutical ingredients for inhibiting the expression of NEK6.

[0029] In another embodiment, the above-mentioned drug is a pharmaceutical composition, which, in addition to a therapeutically effective amount of the above-mentioned siRNA molecule, further comprises one or more other pharmaceutical ingredients for treating ovarian cancer.

[0030] In one embodiment, the above-mentioned drug is an injection form or a gel. The injection form is suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.

[0031] In vitro experiments have shown that the siRNA molecules provided by the present invention can specifically downregulate the expression of the NEK6 gene, thereby achieving the technical effects of inhibiting the growth and metastasis of ovarian cancer cells and promoting the apoptosis of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a bar graph of the siRNA molecules (si-NEK6-1, si-NEK6-2 and si-NEK6-3) of the present invention inhibiting the mRNA expression level of the NEK6 target gene in SK-OV-3 and A2780 ovarian cancer cells. Compared with the negative control group si-NC, the two siRNA molecules (si-NEK6-1, si-NEK6-2) significantly inhibited the mRNA expression level of the NEK6 gene, where *P<0.05, compared with SK-OV-3 cells treated with si-NC, #P<0.05, compared with A2780 cells treated with si-NC.

[0033] Figure 2 The siRNA (si-NEK6-1, si-NEK6-2) of the present invention inhibits the protein expression level of the NEK6 target gene in SK-OV-3 and A2780 ovarian cancer cells. Compared with the untreated normal cell control (Control, Con) and the negative control group si-NC, siRNA (si-NEK6-1, si-NEK6-2) significantly inhibited the expression level of NEK6 protein, where *P<0.05, compared with SK-OV-3 cells treated with si-NC, #P<0.05, compared with A2780 cells treated with si-NC.

[0034] Figure 3 The results of the siRNA (si-NEK6-1, si-NEK6-2) of the present invention inhibiting the proliferation of SK-OV-3 and A2780 ovarian cancer cells. Compared with the untreated normal cell control (Con) and the negative control group si-NC, siRNA (si-NEK6-1, si-NEK6-2) significantly inhibited the growth of cells at 48h and 72h, where *P<0.05, compared with SK-OV-3 cells treated with si-NC, #P<0.05, compared with A2780 cells treated with si-NC.

[0035] Figure 4 The figure is the result of the siRNA (si-NEK6-1, si-NEK6-2) of the present invention inhibiting the migration of SK-OV-3 and A2780 ovarian cancer cells. Compared with the untreated normal cell control (Con) and the negative control group si-NC, siRNA (si-NEK6-1, si-NEK6-2) significantly inhibited the invasion of cells, where *P<0.05, compared with SK-OV-3 cells treated with si-NC, #P<0.05, compared with A2780 cells treated with si-NC.

[0036] Figure 5 The figure is the result of the siRNA (si-NEK6-1, si-NEK6-2) of the present invention inhibiting the invasion of SK-OV-3 and A2780 ovarian cancer cells. Compared with the untreated normal cell control (Con) and the negative control group si-NC, siRNA (si-NEK6-1, si-NEK6-2) significantly inhibited the invasion of cells, where *P<0.05, compared with SK-OV-3 cells treated with si-NC, #P<0.05, compared with A2780 cells treated with si-NC.

[0037] Figure 6 The siRNA (si-NEK6-1, si-NEK6-2) of the present invention promotes apoptosis of SK-OV-3 and A2780 ovarian cancer cells. Compared with the untreated normal cell control (Con) and the negative control group si-NC, siRNA (si-NEK6-1, si-NEK6-2) significantly promoted cell apoptosis, where *P<0.05, compared with SK-OV-3 cells treated with si-NC, #P<0.05, compared with A2780 cells treated with si-NC. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0039] The present inventors have discovered an siRNA molecule, a double-stranded siRNA molecule consisting of a sense chain SEQ ID NO: 5 and an antisense chain SEQ ID NO: 6, or a double-stranded siRNA molecule consisting of a sense chain SEQ ID NO: 7 and an antisense chain SEQ ID NO: 8, respectively named si-NEK6-1 and si-NEK6-2 in the examples, which can specifically target the NEK6 gene, block the mRNA transcription of the NEK6 gene, and prevent the translation of the gene, thereby fundamentally inhibiting the expression of NEK6, and ultimately achieving the purpose of inhibiting tumors.

[0040] In this article, the terms "siRNA", "siRNA sequence", "siRNA molecule", "double-stranded siRNA", or "double-stranded siRNA molecule" are interchangeable and have the same meaning and scope. Among them, siRNA is a double-stranded structure formed by annealing of the sense strand and the antisense strand.

[0041] In this article, sometimes for the sake of simplicity of description, the names of NEK6 protein and its encoding gene (DNA) are mixed. Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context and context. For example, for NEK6, when used to describe the function or category of NEK kinase, it refers to the protein; when described as a gene, it refers to the gene encoding the NEK kinase.

[0042] Similarly, for the sake of simplicity of description, RNA, such as siRNA, is sometimes used interchangeably with the names of its encoding genes, and those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context and background.

[0043] For example, sometimes for the purpose of convenience of description or making sequence lists, the sense and antisense strands of the RNA molecules SEQ ID NOs: 1-8 may be expressed as deoxyribonucleic acid DNA:

[0044] SEQ ID NO:1:CAGATGATCAAGTACTTTA,

[0045] SEQ ID NO:2: TAAAGTACTTGATCATCTG,

[0046] SEQ ID NO:3: CGGAGAGGACAGTATGGAA,

[0047] SEQ ID NO:4: TTCCATACTGTCCTCTCCG.

[0048] SEQ ID NO:5: CAGATGATCAAGTACTTTANN (wherein N=dT),

[0049] It can also be expressed as CAGATGATCAAGTACTTTAdTdT,

[0050] SEQ ID NO:6: TAAAGTACTTGATCATCTGNN (wherein N=dT),

[0051] It can also be expressed as TAAAGTACTTGATCATCTGdTdT,

[0052] SEQ ID NO:7: CGGAGAGGACAGTATGGAANN (wherein N=dT),

[0053] It can also be expressed as CGGAGAGGACAGTATGGAAdTdT,

[0054] SEQ ID NO:8: TTCCATACTGTCCTCTCCGNN (wherein N=dT),

[0055] It can also be expressed as TTCCATACTGTCCTCTCCGdTdT.

[0056] There are many methods for preparing siRNA molecules, such as chemical synthesis, in vitro transcription, enzyme cleavage of long-chain dsRNA, vector expression of RNA, PCR synthesis of RNA expression elements, etc. The emergence of these methods provides researchers with options to better obtain gene silencing efficiency.

[0057] The above siRNA molecules, such as si-NEK6-1 and si-NEK6-2, can be used as active pharmaceutical ingredients in the treatment of ovarian cancer, and can be made into RNAi drugs.

[0058] Optionally, in addition to a therapeutically effective amount of the RNAi molecule siNEK6-1 or siNEK6-2, the RNAi drug may also contain other NEK6 expression inhibitors as auxiliary active ingredients to enhance the therapeutic effect of the siRNA molecule on ovarian cancer.

[0059] The term "(therapeutic effect) enhancement" or "improvement" is used herein to indicate a statistically significant amount of improvement. In some embodiments, "enhancement" or "increase" or "improvement" generally refers to an improvement of at least 10% compared to a reference level (e.g., there is no given treatment or medicament), and can include, for example, an improvement of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more.

[0060] In another embodiment, the above-mentioned medicine contains, in addition to the above-mentioned RNAi molecule in a therapeutically effective amount, one or more NEK6 gene inhibitors and / or drug components for treating ovarian cancer. Such a pharmaceutical composition may have the effect of treating ovarian cancer.

[0061] As used herein, the term "effective amount" refers to the therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a drug that provides the desired effect. Therefore, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a specific effect when applied to a typical subject. In various contexts, the effective amount as used herein also includes an amount sufficient to delay the development of a disease condition, change the course of a disease condition (for example, but not limited to, slowing the progression of a disease condition), or reverse a disease condition. It should be understood that there are many ways known in the art to determine the effective amount for a given application. For example, pharmacological methods for dose determination can be used in a therapeutic context. In the context of therapeutic or preventive applications, the amount of the composition applied to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as overall health, age, sex, weight, and tolerance to the drug. It also depends on the extent, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. For example, the therapeutically effective amount of an RNAi molecule can be determined by clinical investigation. The appropriate effective dosage also takes into account therapeutic factors such as the dosage form, the constitution of the individual being administered, weight, age, course of the condition, and site of administration.

[0062] The dosage range of the agent depends on the efficacy, including an amount sufficient to produce the desired effect, such as slowing the development of ovarian cancer or even causing reversal of the condition. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition and sex of the patient and can be determined by a person skilled in the art. The dosage can also be adjusted by an individual physician in the event of any complications.

[0063] The dosage form of the siRNA drug of the present invention can be in various forms, as long as it is suitable for the administration of the corresponding disease and properly maintains the activity of the RNA molecule. For example, for an injection administration system, the dosage form can be a lyophilized powder. In addition to the injection dosage form, the drug can also be a gel.

[0064] Optionally, the above-mentioned pharmaceutical dosage form may contain any pharmaceutically acceptable carrier and adjuvant, as long as they are suitable for the corresponding administration system and properly maintain the activity of the RNAi molecule.

[0065] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers are well known in the art, and these carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and harmless to the patient, including, for example, aqueous solutions (such as water or physiologically buffered saline) or other solvents or vehicles (such as glycols, glycerol, oils (such as olive oil) or injectable organic esters). Excipients can be selected, for example, to achieve delayed release of the medicament or to selectively target one or more cells, tissues or organs. For the purposes of the present invention, the pharmaceutical composition can be in dosage unit form, such as powder injections, solutions, injections, etc.

[0066] The preparation of pharmacological compositions comprising active ingredients dissolved or dispersed therein is well known in the art and does not need to be limited based on the formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions, however, solid forms of solutions or suspensions suitable for being placed in liquids before use can also be prepared. The preparation can also be emulsified or present in the form of liposome compositions. The active ingredient can be mixed with a pharmaceutically acceptable and compatible excipient for the active ingredient, and in an amount suitable for the treatment methods described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, etc. and combinations thereof. In addition, if necessary, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., which enhance or maintain the effectiveness of the active ingredient. The therapeutic composition as described herein may include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of polypeptides), which are formed by inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed by free carboxyl groups can also be derived from inorganic bases, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any substance except the active ingredient and water, or contain buffers such as sodium phosphate, saline or both at physiological pH, such as phosphate buffered saline. Further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. In addition to water, the liquid composition can also contain a liquid phase. Examples of such other liquid phases are glycerol, vegetable oils (e.g., cottonseed oil) and water-oil emulsions. The amount of the active agent such as siRNA used in the present invention to effectively treat a specific disease or condition ovarian cancer will depend on the nature of the disease or condition, and can be determined by standard clinical techniques.

[0067] As used herein, the terms "treat", "therapy", "treatment" or "improvement" when used to refer to a disease, disorder or medical condition such as ovarian cancer, refer to the therapeutic treatment of a disorder, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a symptom or disorder. The term "treatment" includes reducing or alleviating at least one adverse reaction or symptom of a disorder. If one or more symptoms or clinical markers are reduced, the treatment is generally "effective". Alternatively, if the progression of the disorder is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also includes stopping or at least slowing down the progression or deterioration of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, relief of one or more symptoms, reduction in the degree of defect, stabilization (i.e., no deterioration) or a decreased state of NEK6 gene expression levels, delay or slowing of cardiomyocyte apoptosis or pyroptosis, and increased lifespan compared to the lifespan expected in the absence of treatment.

[0068] The therapeutically effective amount is an amount of the agent sufficient to produce a statistically significant, measurable change in inhibiting NEK6 gene expression, promoting normal cell growth, etc. Such an effective amount can be measured in clinical trials and animal studies.

[0069] The efficacy of a given treatment for (e.g., inhibition of NEK6 gene expression and / or ovarian cancer) can be determined by a clinician with technical skills. However, if, for example, any or all signs or symptoms of inhibition of NEK6 gene expression and / or treatment of ovarian cancer are altered in a beneficial manner, or other clinically acceptable symptoms are improved or even alleviated, for example, at least 10% after treatment with an agent described herein, the treatment is considered to be "effective treatment," as the term is used herein. Efficacy can also be measured by the failure of an individual to deteriorate, as assessed by the need for hospitalization or medical intervention (i.e., cessation of progression of the disease). Methods for measuring these indicators are known to those skilled in the art and / or described herein.

[0070] An effective amount for treating a disease is an amount sufficient to result in effective treatment of the disease as that term is defined herein when administered to a mammal in need thereof. The efficacy of an agent can be determined by assessing, for example, physical indicators of inhibiting NEK6 gene expression and / or treating ovarian cancer.

[0071] The above-mentioned agents can be administered by subcutaneous injection, intramuscular injection, intravenous injection or intravenous infusion gradually over time. Given an appropriate formulation for a given route, for example, the agents that can be used in the methods and compositions described herein can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavitary, and if necessary, can be delivered by means of a peristaltic pump, a venous port, or by other means known to those skilled in the art. It is preferred to administer intravenously or intramuscularly to the ovarian cancer patient to be treated. It is also particularly contemplated that the NEK6 gene is directly administered to a site of high expression, such as ovarian tissue.

[0072] For example, therapeutic compositions comprising at least one pharmaceutical agent may be conventionally administered in unit doses. When used in relation to therapeutic compositions, the term "unit dose" refers to physically discrete units suitable as unit dosage forms for administration to a subject, each unit containing a predetermined quantity of active material, in association with the required physiologically acceptable diluent (i.e., carrier or vehicle), calculated to produce the desired therapeutic effect.

[0073] The compositions are administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount and timing of administration will depend upon the subject to be treated, the subject's body's ability to utilize the active ingredients, and the extent of the therapeutic effect desired.

[0074] The exact amount of active ingredient, such as RNAi, that needs to be administered depends on the physician's judgment and varies from individual to individual. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable dosing regimens are also variable, but are represented by an initial administration, followed by repeated administration at intervals of one or more hours by subsequent injections or other administrations. Alternatively, it is contemplated that continuous intravenous infusion is sufficient to maintain the concentration in the blood within the range specified for in vivo treatment.

[0075] The present invention has verified at the cellular level the inhibitory effect of individual siRNA molecules such as siNEK6-1 and siNEK6-2 on the NEK6 gene and the inhibitory effect on the proliferation, metastasis and invasion of ovarian cancer cells, or the promoting effect on the apoptosis of ovarian cancer cells.

[0076] To make the present invention more clearly understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0077] Example

[0078] The siRNA and PCR primers in this article were synthesized by Biomimetic Biotechnology Co., Ltd.

[0079] In the embodiments of the present invention, if there is no specific description on the experimental operation temperature, the temperature generally refers to room temperature (10-30° C.).

[0080] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0081] Statistical analysis: In this study, all numerical variables were expressed as mean ± standard error. The two-group comparison was performed using the two-tailed Student's t test, and the three-group comparison was performed using the ANOVA test. When P < 0.05, it was considered statistically significant.

[0082] Example 1: siRNA synthesis and cell transfection

[0083] 1. Cell Culture

[0084] Human ovarian cancer cell lines SK-OV-3 and A2780 (collected by the Affiliated Hospital of Nantong University) were cultured in DMEM medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum at 37°C and 5% CO. 2 Culture in an incubator.

[0085] 2. Design and synthesize siRNA molecules

[0086] Design and synthesize siRNA sequences targeting NEK6 gene as follows:

[0087] Sense strand: 5'-CAGAUGAUCAAGUACUUUAdTdT-3' (SEQ ID NO: 5),

[0088] Antisense strand: 5'-UAAAGUACUUGAUCAUCUGdTdT-3' (SEQ ID NO: 6),

[0089] The siRNA molecule was named si-NEK6-1.

[0090] Sense strand: 5'-CGGAGAGGACAGUAUGGAAdTdT-3' (SEQ ID NO: 7),

[0091] Antisense strand: 5'-UUCCAUACUGUCCUCUCCGdTdT-3' (SEQ ID NO: 8),

[0092] The siRNA molecule was named si-NEK6-2.

[0093] Sense strand: 5'-CUCCGAGAAGUUACGAGAAdTdT-3',

[0094] Antisense strand: 5'-UUCUCGUAACUUCUCGGAGdTdT-3',

[0095] The siRNA molecule was named si-NEK6-3.

[0096] The siRNA has a sense strand and an antisense strand. The sense strand and the corresponding antisense strand are annealed to form siRNA double strands, and the concentration is configured to be 20 μM before transfection.

[0097] In addition, siRNA with different origin from human gene was designed and synthesized as negative control (si-NC), and its sequence is:

[0098] Sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3',

[0099] Antisense strand: 5′-ACGUGACACGUUCGGAGAAdTdT-3′.

[0100] The si-NC has a sense strand and an antisense strand. The sense strand and the corresponding antisense strand are annealed to form a si-NC double strand, and the concentration is set to 20 μM before transfection.

[0101] 3. Transfect cells with siRNA

[0102] The cells obtained in step 1 were divided into 5×10 4 The cells were plated in 24-well cell culture plates and incubated in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 Culture in an incubator overnight. 2000 (Thermo Fisher Scientific, USA). According to the instructions, siRNA and si-NC were transfected into SK-OV-3 and A2780 cells, respectively. Normally cultured cells without any treatment were set as untreated groups.

[0103] Example 2: Real-time quantitative PCR detection of mRNA expression levels

[0104] The cells were transfected with siRNA according to the method of Example 1. After 48 hours of cell transfection, the cells were collected and Total cellular RNA was extracted using ELISA kit (Thermo Fisher Scientific, USA) according to its instructions.

[0105] Gene-specific primers were used to detect the mRNA expression level of the NEK6 gene in the samples, and the housekeeping gene GAPDH was amplified as an internal reference. The NEK6 gene and the internal reference gene GAPDH were amplified for each sample, and each reaction was performed in triplicate. According to the manufacturer's instructions, the first-strand cDNA was synthesized using a reverse transcription kit (Thermo Fisher Scientific, USA), and then the quantitative PCR reaction was performed using the DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific, USA).

[0106] Real-time quantitative PCR primers for detecting NEK6 gene:

[0107] The upstream primer is: 5'-TCTTGAAGCAACTGAACCA-3',

[0108] The downstream primer is: 5'-CCAACTCCAGCACAATGT-3'.

[0109] Real-time quantitative PCR primers for detecting the housekeeping gene GAPDH:

[0110] The upstream primer is: 5'-GGAGCGAGATCCCTCCAAAAT-3',

[0111] The downstream primer is: 5'-GGCTGTTGTCATACTTCTCATGG-3'.

[0112] After the reaction is completed, export the data using 2 -ΔΔCt Analyze the experimental results and draw graphs, such as Figure 1 As shown, compared with the negative control si-NC, the three siRNA molecules si-NEK6-1, si-NEK6-2 and si-NEK6-3 all inhibited the mRNA expression level of the NEK6 gene in ovarian cancer cells SK-OV-3 and A2780, among which si-NEK6-1 and si-NEK6-2 had particularly significant inhibitory effects. For example, in SK-OV-3 cells, si-NEK6-1 and si-NEK6-2 inhibited the mRNA level of NEK6 by 86% and 89%, respectively, and in A2780 cells, si-NEK6-1 and si-NEK6-2 inhibited the mRNA level of NEK6 by 84% and 80%, respectively, which significantly inhibited its mRNA expression level, and the differences were statistically significant (P<0.05). The following examples focus on si-NEK6-1 and si-NEK6-2.

[0113] Example 3: Western blot detection of protein expression levels

[0114] According to the method of Example 1, cells were cultured at 1.5×10 6 1000 cells / well were plated in a 6-well plate, and cells were transfected after 24 hours of growth to a confluence of about 75%. 48 hours after cell transfection, cells were lysed with pre-cooled cell protein lysis buffer (Promega, USA), placed at 4°C, centrifuged at 10,000rpm for 20 minutes, and the supernatant was diluted for polyacrylamide gel electrophoresis, and the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Merck, USA), and blocked with 5% skim milk at room temperature for 2 hours, and then rabbit anti-human NEK6 monoclonal antibody (Proteintech, 1:500 dilution) and mouse anti-human GAPDH monoclonal antibody (Proteintech, 1:50,00 dilution) were used as internal references. After washing with TBST, incubated with horseradish peroxidase-conjugated secondary antibody (NEK6 with goat anti-rabbit IgG-HRP, 1:5,000 dilution), and washed with TBST 3 times (5min / time). Detected with BeyoECL Plus kit (Beyotime).

[0115] like Figure 2 As shown in the figure, compared with the negative control si-NC, the two siRNAs significantly inhibited the expression level of NEK6 protein in ovarian cancer cells A2780 and SK-OV-3. For example, in SK-OV-3 cells, si-NEK6-1 and si-NEK6-2 inhibited the protein level of NEK6 by 52% and 58%, respectively, and in A2780 cells, si-NEK6-1 and si-NEK6-2 inhibited the protein level of NEK6 by 56% and 54%, respectively, which significantly inhibited its protein expression level, and the differences were statistically significant (P<0.05).

[0116] Example 4: Cell proliferation detection

[0117] Cell culture and siRNA transfection were performed according to the method of Example 1. Before transfection, when the cell confluence reached about 80%, the cells in the logarithmic growth phase were plated in a 96-well cell culture plate, and repeated for 3 wells as untransfected samples (i.e., samples transfected at 0 h). The cells of each experimental group after transfection for 24 h, 48 h, and 72 h were respectively taken for MTT cell proliferation assay, and the assay method was as follows: 100 μL of DMEM and 10 μL of MTT (Beyotime) were added to each well, incubated in an incubator for 4 h, and the OD value was measured at a wavelength of 570 nm using an enzyme marker (Themo Fisher Scientific, USA) to prepare a growth curve.

[0118] like Figure 3As shown, compared with the untreated normal cell control (Con) and the negative control si-NC, si-NEK6-1 and si-NEK6-2 inhibited cell proliferation in A2780 and SK-OV-3 cells, especially at 48h and 72h.

[0119] Example 5: Cell migration assay

[0120] According to the method of Example 1, ovarian cancer cells SK-OV-3 and A2780 were respectively plated into 24-well cell culture plates and cultured for 24 hours before transfection. After 48 hours of cell transfection, the cells were cultured in DMEM and resuspended to adjust the concentration to 5×10 5 cells / mL. The inner chamber of the Transwell plate (Sigma-Aldrich, USA) was pre-incubated with DMEM medium for 1 hour, and the upper and lower chambers were separated by an 8μm polycarbonate membrane. 200μL of cell resuspension and 600μL of DMEM medium containing 10% FBS or conditioned medium (the supernatant of cells treated for 48 hours above) were added to each upper chamber. After 24 hours, the cells remaining in the upper chamber were removed with a cotton swab, and the cells that migrated to the lower chamber were fixed with 4% pre-cooled paraformaldehyde for 30 minutes. Finally, the cells were stained with 0.5% crystal violet for 10 minutes. The cells were counted in a 200-fold magnification field of view, and 5 fields of view were counted under each condition. The number of migrated cells was calculated and a bar graph was made.

[0121] like Figure 4 As shown, compared with the untreated normal cell control (Con) and the negative control si-NC, si-NEK6-1 and si-NEK6-2 significantly inhibited the migration of A2780 and SK-OV-3 cells, and the difference was statistically significant (P<0.05).

[0122] Example 6: Cell invasion assay

[0123] According to the method of Example 1, ovarian cancer cells SK-OV-3 and A2780 were respectively plated into 24-well cell culture plates and cultured for 24 hours before transfection. After 48 hours of cell transfection, the cells were cultured in DMEM and resuspended to adjust the concentration to 5×10 6cells / mL. The inner chamber of the Transwell plate (Sigma-Aldrich, USA) was pre-incubated with DMEM medium for 1 hour, and the upper and lower chambers were separated by an 8μm polycarbonate membrane coated with 100μL of Matirgel (0.5mg / mL, BD, USA). 300μL of cell resuspension and 600μL of DMEM medium containing 10% PBS or conditioned medium (the supernatant of cells treated for 48 hours above) were added to each upper chamber. After 24 hours, the cells remaining in the upper chamber were removed with a cotton swab, and the cells that migrated to the lower chamber were fixed with 4% pre-cooled paraformaldehyde for 30 minutes. Finally, the cells were stained with 0.5% crystal violet for 10 minutes. The cells were counted in a 200-fold magnification field of view, and 5 fields of view were counted under each condition. The number of invasive cells was calculated and a bar graph was made.

[0124] like Figure 5 As shown, compared with the untreated normal cell control (Con) and the negative control si-NC, si-NEK6-1 and si-NEK6-2 significantly inhibited the invasion of SK-OV-3 and A2780 cells, and the difference was statistically significant (P<0.05).

[0125] Example 7: Cell apoptosis detection

[0126] Cell culture and siRNA transfection were performed according to the method of Example 1.

[0127] The cell apoptosis was detected and analyzed using Annexin V-FITC apoptosis assay kit (Beyotime). SK-OV-3 and A2780 ovarian cancer cells were transfected for 48 h, digested with 0.25% trypsin and washed twice with PBS buffer. 5 Each cell was resuspended in 500 μL of binding buffer and stained with 5 μL of Annexin V-FITC according to the operating instructions, and then 5 μL of propidium iodide (PI) solution was added and incubated at room temperature in the dark for 15 min. Cells in different experimental groups were analyzed for cell apoptosis using flow cytometry (BD Company, USA), and the cell apoptosis rate was calculated and a bar graph was made.

[0128] like Figure 6 As shown, compared with the untreated normal cell control (Con) and the negative control si-NC, si-NEK6-1 and si-NEK6-2 significantly promoted the apoptosis of SK-OV-3 and A2780 cells, and the difference was statistically significant (P<0.05).

[0129] It can be seen from the above experiments that siRNA targeting NEK6 (si-NEK6-1 and si-NEK6-2) showed high efficiency in inhibiting NEK6 expression activity; siRNA molecules (si-NEK6-1 and si-NEK6-2) can inhibit the proliferation, migration and invasion of SK-OV-3 and A2780 cells, and significantly promote the apoptosis of SK-OV-3 and A2780 ovarian cancer cells, and therefore have the potential to be used in anti-tumor, especially ovarian cancer treatment.

Claims

1. A siRNA molecule for inhibiting the NEK6 gene, consisting of a sense strand and an antisense strand of the following sequence: Sense strand: 5'-CAGAUGAUCAAGUACUUUANn-3', Antisense strand: 5'-UAAAGUACUUGAUCAUCUGNn-3', or Sense strand: 5'-CGGAGAGGACAGUAUGGAANn-3', Antisense strand: 5'-UUCCAUACUGUCCUCUCCGNn-3', in, N in the sense strand and the antisense strand is the same or different and is independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, which is 0, 1 or 2.

2. The siRNA molecule according to claim 1, characterized in that The n is 0, that is, Sense strand: 5'-CAGAUGAUCAAGUACUUUA-3' (SEQ ID NO: 1), Antisense strand: 5'-UAAAGUACUUGAUCAUCUG-3' (SEQ ID NO: 2), or Sense strand: 5'-CGGAGAGGACAGUAUGGAA-3' (SEQ ID NO: 3), Antisense strand: 5'-UUCCAUACUGUCCUCUCCG-3' (SEQ ID NO: 4).

3. The siRNA molecule according to claim 1, characterized in that The N is dT, n is 2, that is, Sense strand: 5'-CAGAUGAUCAAGUACUUUAdTdT-3' (SEQ ID NO: 5), Antisense strand: 5'-UAAAGUACUUGAUCAUCUGdTdT-3' (SEQ ID NO: 6), or Sense strand: 5'-CGGAGAGGACAGUAUGGAAdTdT-3' (SEQ ID NO: 7), Antisense strand: 5'-UUCCAUACUGUCCUCUCCGdTdT-3' (SEQ ID NO: 8). 4 . Use of any one of the siRNA molecules according to claim 1 in the preparation of a drug for inhibiting NEK6 expression, wherein the drug is a drug for treating ovarian cancer.

5. A drug for treating ovarian cancer, characterized in that: The method comprises the siRNA molecule according to any one of claims 1 to 3 as a pharmaceutical active ingredient.

6. The drug according to claim 5, characterized in that The pharmaceutical composition comprises, in addition to a therapeutically effective amount of the siRNA molecule according to any one of claims 1 to 3 as an active ingredient, a pharmaceutically acceptable carrier.

7. The drug according to claim 5, characterized in that The pharmaceutical composition comprises, in addition to a therapeutically effective amount of the siRNA molecule according to any one of claims 1 to 3, one or more other pharmaceutical ingredients for inhibiting the expression of the NEK6 gene.

8. The drug according to claim 5, characterized in that The pharmaceutical composition comprises, in addition to a therapeutically effective amount of the siRNA molecule according to any one of claims 1 to 3, one or more other pharmaceutical ingredients for treating ovarian cancer.

9. The drug according to claim 5, characterized in that The drug is in the form of an injection or a gel.

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