CN117737068A

CN117737068A - Small nucleic acid molecules for the treatment of cutaneous melanoma

Info

Publication number: CN117737068A
Application number: CN202410103654.0A
Authority: CN
Inventors: 沈聪聪; 陈晓栋; 顾黎雄; 王新; 崔晓美
Original assignee: Affiliated Hospital of Nantong University
Current assignee: Affiliated Hospital of Nantong University
Priority date: 2024-01-25
Filing date: 2024-01-25
Publication date: 2024-03-22

Abstract

The invention discloses a small interfering nucleic acid molecule, which consists of a sense strand and an antisense strand of the following sequences: the sense strand is GAUUUGUGGUUUUGUAN and the antisense strand is UAGAACAGUACCACAACAAUCNN; or the sense strand is CGAGUGACUGAGACCUANN, and the antisense strand is UAGGUCUCUCAGUGGUCACUCGNN, wherein N is dT; n represents the number of N, and N is 0 or 2. The small interfering nucleic acid molecule can be used for preparing medicines for treating skin melanoma.

Description

Small nucleic acid molecules for the treatment of cutaneous melanoma

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a small interfering nucleic acid molecule and application thereof in preparing a medicine for treating skin melanoma.

Background

Cutaneous melanoma is a highly malignant tumor originating from the basal layer melanocytes of the epidermis, with a high mortality rate leading among various types of cutaneous malignant tumors. Studies have shown that the incidence and mortality of cutaneous melanoma has increased year by year in recent years worldwide, with lower deadly age compared to other solid tumors. The pathogenesis of cutaneous melanoma is complex, involving a variety of cell signaling pathways, the specific molecular mechanisms of which are to be studied further. Early surgical excision is the most effective means of treating cutaneous melanoma, but because of its high invasiveness, many patients already have tumor metastasis at the time of visit, presenting great difficulty in treatment. In recent years, although new therapeutic approaches such as targeted therapy and immunotherapy have been increasingly applied to the treatment of metastatic skin melanoma, there are still many limitations that the treatment of advanced skin melanoma remains very difficult to a great extent, and the survival rate of patients for 5 years is low. Therefore, further research on pathogenesis of skin melanoma and exploration of new therapeutic targets are important for improving curative effect of advanced skin melanoma and prolonging life of patients.

Ubiquitin-proteasome pathway (UPP) is an important protein degradation regulating system in cells that can affect or regulate a variety of cellular activities by polyubiquitination of substrate proteins and degradation by proteasomes, including: gene transcription, cell cycle regulation, immune response, cellular receptor function, tumor growth, inflammatory processes, and the like. The UPP pathway is also a dynamic protein bidirectional modification regulation system, in vivo, ubiquitin is modified by a ubiquitin ligase system (E1-E2-E3), and deubiquitinase (deubiquitinating enzymes, DUB) family is responsible for hydrolyzing an ester bond, a peptide bond or an isopeptide bond at the carboxyl terminal of ubiquitin to specifically hydrolyze ubiquitin molecules from ubiquitin-linked protein or precursor protein, so as to play a role in deubiquitination, and reverse regulation on protein degradation, thereby influencing the function of the protein. DUBs are a large family of proteases, the human genome encoding approximately 100 deubiquitinating enzymes, and are largely divided into five families, the ubiquitin carboxy-terminal hydrolase (ubiquitin carboxy terminal hydrolases, UCH) family, the ubiquitin-specific protease (Ubiquitin Specific Peptidase, UBP) family, the Otubaim (OTU) family, the Josephin domain protein family, and the JAMM family, respectively. The UBP family is the most member and structurally diverse class of currently known deubiquitinating enzymes. UBP not only reverses ubiquitination of proteins, but also has many different functions such as protein trafficking, apoptosis, chromatin remodeling, DNA damage repair, and cell cycle regulation, and is particularly involved in cancer-related cell signaling. Since components of the ubiquitin mechanism are involved in different types of cancers, targeted UBP family proteins can be used for anti-tumor therapy.

Ubiquitin-specific peptidase 41 (Ubiquitin Specific Peptidase, UBP 41) is one of the important members of the UBP family, but its function in tissues and cells is relatively less studied. Recent studies show that UBP41 is also abnormally high in human tumors, and its high expression promotes proliferation, migration and induction of apoptosis of cancer cells, and is associated with poor prognosis of some malignant tumors, suggesting that targeting UBP41 may become a tumor treatment strategy, but UBP41 has not been reported in cutaneous melanoma.

Disclosure of Invention

In order to obtain RNAi drugs capable of effectively treating skin melanoma, the inventor tries to design and screen a series of small interfering nucleic acid molecules (siRNA) by taking UBP41 genes as targets, and experiments show that some siRNA molecules can specifically inhibit UBP41 expression, and two groups of small interfering nucleic acid molecules can not only effectively inhibit UBP41 genes, but also achieve the effect of treating skin melanoma. Based on the findings, the technical scheme of the invention is as follows.

In a first aspect the invention provides a small interfering nucleic acid molecule for use in the treatment of cutaneous melanoma, consisting of a sense strand and an antisense strand of the sequence:

sense strand: 5 '-GAUUUGUGGUUACUUUUCUANN-3',

antisense strand: 5 '-UAGAACAGUAACACAAAUCNn-3'; or alternatively

Sense strand: 5'-CGAGUGACACUGAGACCUANN-3',

antisense strand: 5 '-UAGGUCUCUCAGUGUCUCCGNn-3'.

Wherein N is deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and N is 0 or 2.

In one embodiment, the n is 0, i.e., the small interfering nucleic acid molecule consists of a sense strand and an antisense strand of the sequence:

sense strand: 5'-GAUUGUGGUUACUGUUCUA-3' (SEQ ID NO: 1),

antisense strand: 5'-UAGAACAGUAACCACAAUC-3' (SEQ ID NO: 2); or alternatively

Sense strand: 5'-CGAGUGACACUGAGACCUA-3' (SEQ ID NO: 3),

antisense strand: 5'-UAGGUCUCAGUGUCACUCG-3' (SEQ ID NO: 4).

In another embodiment, the N is dT and N is 2, i.e., the small interfering nucleic acid molecule consists of a sense strand and an antisense strand of the sequence:

sense strand: 5 '-GAUUUGUGGUUACUUUUCUANN-3' (SEQ ID NO: 5),

can also be expressed as 5 '-GAUUUGUGGUUUUUUCUADTdT-3' (SEQ ID NO: 5),

antisense strand: 5 '-UAGAACAGUAACACAAAUCNN-3' (SEQ ID NO: 6),

can also be represented as 5 '-UAGAACAGUAACACAAAUCdTTT-3' (SEQ ID NO: 6).

Sense strand: 5 '-CGAGUGAACCUGAGACCUANN-3' (SEQ ID NO: 7),

can also be represented as 5 '-CGAGUGACACUGACCUAdTTT-3' (SEQ ID NO: 7),

antisense strand: 5 '-UAGGUCUCUCAGUGUCUCCGNN-3' (SEQ ID NO: 8),

can also be expressed as 5 '-UAGGUCUCUCAGUGGdTTT-3' (SEQ ID NO: 8).

In a second aspect, the invention provides the use of a small interfering nucleic acid molecule as described above in the manufacture of a medicament for inhibiting UBP41 expression.

Preferably, the above-mentioned drugs are used for inhibiting proliferation, metastasis, invasion and promotion of apoptosis of skin melanoma cells.

In a third aspect, the present invention provides a medicament for treating cutaneous melanoma, comprising the small interfering nucleic acid molecule as described above as a pharmaceutically active ingredient.

Alternatively, the medicament is a pharmaceutical composition comprising a pharmaceutically acceptable carrier in addition to a therapeutically effective amount of the small interfering nucleic acid molecule as described above as active ingredient.

In one embodiment, the medicament is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule described above, one or more additional pharmaceutical ingredients for inhibiting UBP41 expression.

In another embodiment, the medicament is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule, one or more additional pharmaceutical ingredients for treating cutaneous melanoma.

The above medicine can be in injection form, and is suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.

In vitro experiments prove that the small interfering nucleic acid molecules SEQ ID NO. 1-2 and SEQ ID NO. 3-4 screened by the invention can specifically and downwardly regulate UBP41 gene expression, and can inhibit proliferation, transfer, invasion or promote technical effects of apoptosis of skin melanoma cells, which suggests that the small interfering nucleic acid molecules can be used as siRNA molecules for treating skin melanoma.

Drawings

Fig. 1 shows the expression of UBP41 according to the present invention in skin melanoma tissue and paracancerous tissue, wherein P <0.05.

FIG. 2 shows the expression of UBP41 in skin melanoma cell lines A375 and A875 according to the present invention, in comparison with HaCaT, a normal human skin cell, wherein P <0.05, represents the comparison of UBP41 expression in A375 cells with HaCaT cells; #P <0.05 represents the expression of UBP41 in A875 cells compared to HaCaT cells.

Fig. 3 is a graph showing inhibition of UBP41 mRNA expression in a375 cells by siRNA molecules directed to UBP41 in part, wherein P <0.05, compared to the normal group.

Fig. 4 is a graph showing inhibition of UBP41 mRNA expression in a875 cells by siRNA molecules directed to UBP41 in part, wherein P <0.05, compared to the normal group.

FIG. 5 shows the inhibition of mRNA expression of siUBP41 in A375 cells by SiUBP41-1 (SEQ ID NO: 1-2) and siUBP41-2 (SEQ ID NO: 3-4) screened according to the present invention, wherein P <0.05, compared to the normal group. Wherein, normal group means not receiving siRNA molecule treatment, siNC is negative control.

FIG. 6 shows the inhibition of mRNA expression of the siUBP41 in A875 cells by the siRNA molecules siUBP41-1 and siUBP41-2 screened according to the invention, wherein P <0.05, compared to the normal group.

FIG. 7 shows inhibition of protein expression of UBP41 by siUBP41-1 and siUBP41-2 selected according to the invention in A375 and A875 cells, wherein P <0.05, compared to the normal group.

FIG. 8 is a graph showing the inhibition of proliferation of A375 cells by siUBP41-1 and siUBP41-2 screened according to the present invention over time, wherein P <0.05, compared to the normal group.

FIG. 9 is a graph of inhibition of proliferation of A875 cells over time for siUBP41-1 and siUBP41-2 selected according to the invention, wherein P <0.05, compared to the normal group.

FIG. 10 is a statistical bar graph of the inhibition of A375 and A875 cell migration by siUBP41-1 and siUBP41-2 screened according to the invention, wherein P <0.05, compared to the normal group.

FIG. 11 is a statistical bar graph of the inhibition of A375 and A875 cell invasion by siUBP41-1 and siUBP41-2 screened according to the invention, wherein P <0.05, compared to the normal group.

FIG. 12 is a statistical bar graph of the promotion of apoptosis of A375 and A875 by SiUBP41-1 and SiUBP41-2 screened according to the invention, wherein P <0.05, compared to the normal group.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The inventor discovers that a small interfering nucleic acid molecule is a double-chain siRNA molecule (SEQ ID NO:1-2 and SEQ ID NO: 3-4) consisting of a sense strand and an antisense strand, which are named siUBP41-1 and siUBP41-2 in examples, can specifically target UBP41 genes, block mRNA transcription of the UBP41 genes and prevent translation of the genes, thereby fundamentally inhibiting the expression of the UBP41, achieving the purpose of inhibiting skin melanoma, and can be used for improving the treatment effect of the skin melanoma.

As used herein, the terms "small interfering nucleic acid", "siRNA sequence", "siRNA molecule", "double stranded siRNA", or "RNAi molecule" are interchangeable, and refer to the same meaning and scope. Wherein the siRNA is a double-stranded structure formed by annealing a sense strand and an antisense strand.

In this context, sometimes UBP41 protein is used in combination with its coding gene (DNA) name for ease of description, and it will be understood by those skilled in the art that they represent different substances in different descriptive contexts. For example, the UBP41 gene refers to a genetic DNA encoding UBP41 protein.

Similarly, for ease of description, RNA such as siRNA is sometimes mixed with the names of its encoding genes, and those skilled in the art will appreciate that they represent different substances in different descriptive contexts. Those skilled in the art will readily understand their meaning depending on the context and context.

For example, sense and antisense RNA SEQ ID NOs 1 to 8 in the above RNA molecule may be expressed as a DNA form:

SEQ ID NO:1：GATTGTGGTTACTGTTCTA，

SEQ ID NO:2：TAGAACAGTAACCACAATC，

SEQ ID NO:3：CGAGTGACACTGAGACCTA，

SEQ ID NO:4：TAGGTCTCAGTGTCACTCG。

SEQ ID NO. 5: GATTGTGGTTACTGTTCTANN (where n=dt),

may also be expressed as gattgtgtgttadtdt,

SEQ ID NO. 6: TAGAACAGTAACCACAATCNN (where n=dt),

also denoted as TAGACACAGAACAATCdTTT,

SEQ ID NO. 7: CGAGTGACACTGAGACCTANN (where n=dt),

also expressed as CGAGTGACACACCTGAGACCTADGT,

SEQ ID NO. 8: TAGGTCTCAGTGTCACTCGNN (where n=dt),

also denoted as TAGGTCTAGGTCTAGTCTACTCGdTTT.

The preparation of the above siRNA molecules may be accomplished by a variety of methods, such as: chemical synthesis methods, in vitro transcription, enzymatic cleavage of long-chain dsRNA, expression of RNA by vectors, synthesis of RNA expression elements by PCR, etc., the advent of these methods provides researchers with alternative space and better gene silencing efficiency.

The siRNA molecule can be used as a pharmaceutical active ingredient for treating skin melanoma, and can be prepared into RNAi drugs.

Optionally, the RNAi agent may contain, in addition to a therapeutically effective amount of the RNAi molecule siUBP41-1 or siUBP41-2, other inhibitors of UBP41 expression as auxiliary active ingredients, in order to enhance the cutaneous melanoma treatment effect of the siRNA molecule.

The term "(therapeutic effect enhancement" or "improvement" is used herein to mean a statistically significant amount of improvement. In some embodiments, "enhancing" or "increasing" generally refers to an increase of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, an increase 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.

In another embodiment, the medicament comprises, in addition to a therapeutically effective amount of the RNAi molecules, one or more UBP41 gene inhibitors and/or pharmaceutical ingredients for treating cutaneous melanoma. Such pharmaceutical compositions may have the effect of treating cutaneous melanoma.

It should be understood that the term "or" as used herein sometimes means "and/or," and the term "or" sometimes also means "and/or. The term "and/or" as used in phrases herein such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The term "effective amount" as used herein refers to the amount of treatment required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a drug to provide the desired effect. Thus, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a particular effect when administered to a typical subject. In various contexts, an effective amount as used herein also includes an amount sufficient to delay the progression of a disease state, alter the course of a disease state (e.g., without limitation, slow the progression of a disease state), or reverse a disease state. It should be appreciated that there are many ways known in the art to determine an effective amount for a given application. For example, pharmacological methods for dose determination may be used in a therapeutic setting. In the context of therapeutic or prophylactic applications, the amount of composition administered to a subject will depend on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, weight and tolerance to drugs. It also depends on the extent, severity and type of the disease. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. For example, a therapeutically effective amount of an RNAi molecule can be determined by clinical examination. Suitable effective dosage amounts also take into account the therapeutic factors such as the dosage form, constitution, weight, age, condition course, site of administration, etc. of the individual to be administered.

The dosage range of the agent depends on potency, including amounts sufficient to produce the desired effect, e.g., slowing the progression of cutaneous melanoma, and 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 one skilled in the art. In the event of any complications, the dosage may also be adjusted by the individual physician.

The dosage form of the medicament of the present invention may be in various forms as long as it is suitable for administration of the corresponding disease and maintains the activity of the RNA molecule appropriately. For example, for injectable delivery systems, the dosage form may be a lyophilized powder.

Optionally, any pharmaceutically acceptable carrier and adjuvant may be included in the above pharmaceutical dosage forms, provided that it is suitable for the respective delivery system and properly maintains the activity of the RNAi molecule.

The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers are well known in the art and include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, including for example, aqueous solutions (such as water or physiological buffered saline) or other solvents or vehicles (such as glycols, glycerol, oils (such as olive oil) or injectable organic esters). Excipients may be selected, for example, to achieve delayed release of the agent or to selectively target one or more cells, tissues or organs. For the purposes of the present invention, the pharmaceutical compositions may be in the form of dosage units, such as powder for injection, solutions, injections and the like.

The preparation of pharmacological compositions comprising an active ingredient dissolved or dispersed therein is well known in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions, however, solid forms suitable for solution or suspension in a liquid prior to use may also be prepared. The formulation may also be emulsified or in the form of a liposome composition. The active ingredient may be admixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance or maintain the effectiveness of the active ingredient. The therapeutic compositions 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 the polypeptide) formed with inorganic acids, for example hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid vehicles are sterile aqueous solutions that do not contain any substance other than the active ingredient and water, or include a buffer such as sodium phosphate at physiological pH, physiological saline, or both, such as phosphate buffered saline. Still further, the aqueous carrier may comprise more than one buffer salt, as well as salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. In addition to water, the liquid composition may also comprise a liquid phase. Examples of such other liquid phases are glycerol, vegetable oils (e.g. cottonseed oil) and water-oil emulsions. The amount of active agent used in the present invention that is effective to treat a particular disease or condition, such as DCM, will depend on the nature of the disease or condition and can be determined by standard clinical techniques.

As used herein, the terms "treat," "treatment," or "ameliorating," when used in reference to a disease, disorder, or medical condition, such as cutaneous melanoma, refer to the therapeutic treatment of the condition, wherein the purpose is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of the symptoms or condition. The term "treating" includes reducing or alleviating at least one adverse reaction or symptom of the disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the disorder is reduced or stopped. That is, "treatment" includes not only improvement of symptoms or markers, but also stopping or at least slowing the progression or worsening of symptoms that would be expected without treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of deficiency, stabilization (i.e., not worsening) or decreased state of UBP41 gene expression levels, delay or slowing of myocardial apoptosis or pyrosis, and increased longevity as compared to that expected in the absence of treatment.

A therapeutically effective amount is an amount of an agent sufficient to produce a statistically significant, measurable change in inhibition of UBP41 gene expression, promotion of cardiomyocyte growth, and the like. Such effective amounts can be measured in clinical trials and animal studies.

The efficacy of a given treatment for (e.g., inhibiting UBP41 gene expression and/or treating skin melanoma) may be determined by a clinician of skill. However, a treatment is considered "effective treatment" if, for example, any or all signs or symptoms of inhibiting UBP41 gene expression and/or treating cutaneous melanoma are altered in a beneficial manner, or other clinically acceptable symptoms are ameliorated or even reduced, e.g., by at least 10% after treatment with an agent as described herein. Efficacy can also be measured by failure of an individual to worsen, assessed by the need for hospitalization or medical intervention (i.e., cessation of progression of disease). Methods of measuring these indicators are known to those skilled in the art and/or described herein.

An effective amount for treating a disease refers to an amount that when administered to a mammal in need thereof is sufficient to result in the term as defined herein being effective for treating the disease. The efficacy of an agent can be determined by assessing, for example, physical indicators of inhibition of UBP41 gene expression and/or treatment of cutaneous melanoma.

The agents may be administered intravenously by subcutaneous injection, intramuscular injection, or by gradual infusion over time. The agents given for a given route, e.g., useful in the methods and compositions described herein, may be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and if desired, may be delivered by peristaltic pump, intravenous port means, or by other means known to those of skill in the art. Preferably, the patient to be treated for cutaneous melanoma is administered intravenously or intramuscularly. Topical administration directly to sites of high UBP41 gene expression, such as skin tissue, is also specifically contemplated.

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

The composition is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount and time of administration will depend on the subject to be treated, the ability of the subject's body to utilize the active ingredient, and the degree of therapeutic effect desired.

The precise amount of active ingredient, such as RNAi, to be administered depends on the judgment of the physician 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 one or more hour intervals by subsequent injections or other administrations. Alternatively, it is contemplated that continuous intravenous infusion is sufficient to maintain the concentration in the blood within a range designated for in vivo treatment.

The present invention has verified the inhibition of UBP41 gene and inhibition of proliferation, metastasis, invasion of skin melanoma cells, or promotion of apoptosis of skin melanoma cells by individual siRNA molecules such as siUBP41-1 and siUBP41-2 at the cellular level.

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below. It will be appreciated by those skilled in the art that the following examples are provided for illustration of the invention and are not intended to be limiting thereof.

Examples

The siRNA and PCR primers herein were synthesized by the bomek biotechnology company.

In the examples of the present invention, if no specific explanation is given for the experimental operating temperature, this temperature is usually referred to as room temperature (10-30 ℃).

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

Statistical analysis: numerical variables in this study were all expressed as mean ± standard error, two-tailed Student's t for comparison between the two groups and ANOVA for comparison between the three groups. Statistical differences were considered when P <0.05.

Example 1

Sirna design:

based on the coding region of UBP41 gene sequence (genbank number AF 079564) in NCBI database, specific siRNA sequences were designed and showed 100% homology with UBP41 gene by sequence homology analysis.

Sirna sequence:

the siRNA sequences obtained according to the design are shown in Table 1.

Sirna structure:

all siRNA molecules are double-stranded and consist of a sense strand and an antisense strand with reverse complements of the sequences, and a protruding overhang structure dC, dG, dA, dT, dCdC, dGdG, dAdA or dTdT of 1-2 bases can be further contained at the 3 '-end of the sense strand and the 5' -end of the antisense strand.

Table 1: designed siRNA sequence list

Molecular numbering	Sense strand (5 '-3')	Antisense strand (5 '-3')
			siUBP41-1	GAUUGUGGUUACUGUUCUA	UAGAACAGUAACCACAAUC
siUBP41-2	CGAGUGACACUGAGACCUA	UAGGUCUCAGUGUCACUCG
			siUBP41-3	GAGUGACACUGAGACCUAA	UUAGGUCUCAGUGUCACUC
siUBP41-4	GGAUGUGCUUGAUGGAGAU	AUCUCCAUCAAGCACAUCC
			siUBP41-5	GCAUGAGGCUCUUCACCAA	UUGGUGAAGAGCCUCAUGC
siUBP41-6	CACUGAGACCUAAGUCCAA	UUGGACUUAGGUCUCAGUG
			siUBP41-7	GGCCGACAGAUGUGGAGAA	UUCUCCACAUCUGUCGGCC
siUBP41-8	GGAACACGUGCUUCAUGAA	UUCAUGAAGCACGUGUUCC
			siUBP41-9	GCCGACAGAUGUGGAGAAA	UUUCUCCACAUCUGUCGGC
siUBP41-10	UGUGCUUGAUGGAGAUGAA	UUCAUCUCCAUCAAGCACA
			siUBP41-11	CAAAGAUCUUGGUGCUCCA	UGGAGCACCAAGAUCUUUG
siUBP41-12	AGACCUGGACUUAAGAGAA	UUCUCUUAAGUCCAGGUCU
			siUBP41-13	CCAGGAUUCGAACCAGCAA	UUGCUGGUUCGAAUCCUGG
siUBP41-14	CGUGCUUCAUGAACUCAAU	AUUGAGUUCAUGAAGCACG
			siUBP41-15	CGUGUACAGAUUGUGGUUA	UAACCACAAUCUGUACACG
siUBP41-16	CGAGGUGAACCGAGUGACA	UGUCACUCGGUUCACCUCG
			siUBP41-17	GGCUGGUCUUCGAAACCUU	AAGGUUUCGAAGACCAGCC
siUBP41-18	AGACCCAGAUCCAGAGAUA	UAUCUCUGGAUCUGGGUCU

The siRNA molecules A, U, C, G in table 1 above may contain various chemical modifications that may improve the stability and activity of the siRNA.

Example 2

1. Cell culture:

human skin melanoma cells A375, A875 and normal skin cells HaCaT were cultured in DMEM medium (ThermoFisher Scientific) containing 10% fetal bovine serum (Lonsera), and 1% penicillin-streptomycin (ThermoFisher Scientific) was added thereto at 37℃with 5% CO ₂ Is cultured in an incubator of (a).

siRNA synthesis:

the siRNAs in Table 1 (including siRNA molecules containing 1-2 base overhang structures) were synthesized by annealing the sense strand and the corresponding antisense strand, respectively, to siRNA duplex, and were configured to have a concentration of 20. Mu.M prior to transfection.

In addition, sirnas that are different from the human UBP41 gene were designed and synthesized as siNC of negative control, which has a sense strand (UUCUCCGAACGUGUCACGUdTdT) and an antisense strand (acgugacguucggagaadtdt). The sense strand and the corresponding antisense strand were annealed to siNC double strands, respectively, and were configured to have a concentration of 20 μm prior to transfection.

siRNA transfection:

the cells obtained in step 1 were plated onto a cell culture plate and incubated in DMEM medium containing 10% fetal bovine serum at 37, °C 5% CO ₂ Culturing overnight in an incubator. Transfection of reagents with cells3000 (ThermoFisher Scientific company), siRNA and SiNC transfection were separately transfected into cells according to their instructions.

Example 3

The siRNA with high inhibition efficiency is screened by a real-time quantitative PCR method.

1. Cell culture and transfection:

skin melanoma cells A375, A875 were cultured as in example 2, and cells A375, A875 in the logarithmic phase were inoculated into 96-well plates, and after 24 hours of cell growth and up to about 75% confluence, siRNA molecules (including siRNA molecules containing 1-2 base overhang structures) in Table 1 were transfected into cells, respectively, and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a).

Rna extraction and RT-qPCR reaction:

after 48 hours of siRNA transfection of cells, the cells were collected and total RNA of the cells was extracted using an RNA extraction reagent Trizol (ThermoFisher Scientific Co.) according to the instructions.

The gene specific primer is used to detect the mRNA expression level of UBP41 gene in the sample, and the housekeeping gene GAPDH is used as an internal reference. Quantitative PCR reactions were performed using SYBR Green RT-qPCR kit (ThermoFisher Scientific company) according to the manufacturer's instructions.

The primer sequences used were as follows:

UBP41：

upstream: 5'-GTTCCTTCGCTTTCTTCT-3';

downstream: 5'-TCCTACTGTCTTCCCGTT-3'.

GAPDH：

Upstream: 5'-GGAGCGAGATCCCTCCAAAAT-3';

downstream: 5'-GGCTGTTGTCATACTTCTCATGG-3'.

After the reaction is finished, 2 is used for exporting data ^-ΔΔCt The experimental result is analyzed by a method, and the method for calculating the inhibition rate of the siRNA is as follows: (1-2 ^-ΔΔCt Value) x 100%.

As shown in fig. 1, UBP41 is significantly highly expressed in skin melanoma tissue compared to paracancerous tissue.

As shown in fig. 2, UBP41 is significantly highly expressed in melanoma cell lines a375 and a875 compared to normal human skin cells HaCaT.

As shown in fig. 3, the designed siRNA targeting UBP41 can inhibit mRNA expression of UBP41 in a375 cells to some extent, especially the inhibition effect of siUBP41-1 and siUBP41-2 is most remarkable, compared to normal group and negative control siNC.

As shown in fig. 4, the designed siRNA targeting UBP41 can inhibit mRNA expression of UBP41 in a875 cells to some extent, especially the inhibition effect of siUBP41-1 and siUBP41-2 is most remarkable, compared to normal group and negative control siNC.

As shown in fig. 5, siUBP41-1 and siUBP41-2 significantly inhibited mRNA expression of UBP41 in a375 cells compared to normal and negative controls siNC. Wherein, normal group means not receiving siRNA molecule treatment, siNC is negative control, the following.

As shown in fig. 6, siUBP41-1 and siUBP41-2 significantly inhibited mRNA expression of UBP41 in a875 cells compared to normal and negative controls siNC.

The results showed that siUBP41-1 and siUBP41-2 have an effect of highly inhibiting UBP41 in skin melanoma cells. It is suggested that siUBP41-1 and siUBP41-2 which inhibit UBP41 expression with high efficiency can be used as potential medicines for treating skin melanoma.

Example 4

The condition that the siRNA molecules inhibit the expression level of UBP41 protein in skin melanoma cells is detected by a Western blot (Western blot) method.

Skin melanoma cells A375, A875 were cultured as in example 2, and cells A375, A875 in logarithmic growth phase were inoculated into 6-well plates, and after 24 hours of cell growth and up to about 75% confluence, siRNA molecule siUBP41 selected in example 3 was transfected into cells, and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a).

After 48h of cell transfection, the cells were lysed with RIPA protein lysate (Beyotime Corp.) and quantified with BCA, about 30. Mu.g of the protein was separated by SDS-polyacrylamide gel electrophoresis, and then the protein was transferred to PVDF membrane (Merck Millipore Corp.) and blocked with 5% skim milk at room temperature for 2h, and then each of them was treated with primary antibodies: murine anti-human UBP41 antibody (proteontech, 1:1,000 dilution); GAPDH antibody (Proteintech, 1:20,000 dilution) served as an internal control. After TBST wash, horseradish peroxidase-conjugated secondary antibody was used for incubation, and TBST was used for 5min×3 times. Western blot detection was performed using BeyoECL Plus (Beyotime Corp.) and histogram analysis was performed using Image J software to scan protein band gray values.

As shown in fig. 7, siUBP41-1 and siUBP41-2 significantly inhibited UBP41 protein expression in cells compared to negative control siNC treated a375, a875 cells.

Example 5

The effect of siRNA on proliferation of skin melanoma cells after inhibition of UBP41 expression was examined by CCK-8.

Skin melanoma cells A375, A875 were cultured as in example 2, and cells A375, A875 in logarithmic growth phase were inoculated into 96-well plates, and after 24 hours of cell growth and up to about 75% confluence, siRNA molecule siUBP41 selected in example 3 was transfected into cells and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a).

The cells of each experimental group after 24h, 48h, 72h and 96h transfection were taken and subjected to cell proliferation assay, which comprises the following steps: mu.L of CCK-8 (Beyotime Co.) was added to each well, and the incubator incubated for 4 hours, and OD was measured at a wavelength of 450nm using an enzyme-labeled instrument, to prepare a growth curve.

As shown in fig. 8 and 9, siUBP41-1 and siUBP41-2 significantly inhibited proliferation of cells compared to negative control siNC treated a375, a875 cells. Both the cell proliferation rates of a375 and a875 decrease more and more over time.

Example 6

The effect of siRNA on skin melanoma cell migration after inhibition of UBP41 expression was examined using a cell streak assay.

Skin melanoma cells A375, A875 were cultured as in example 2, and cells A375, A875 in the logarithmic phase were inoculated into 3.5cm dishes with a cell density of 5X 10 ⁵ After 24h of cell growth, cells/well were streaked onto the cell monolayer with a 200. Mu.L pipette tip to a cell wound, washed with PBS, and the siRNA molecules siUBP41-1 and siUBP41-2 selected in example 3 were transfected into cells and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a). At 0h or 48h, cells were observed under a microscope and photographed.

As shown in fig. 10, siUBP41-1 and siUBP41-2 significantly inhibited migration of a375 and a875 cells compared to negative control siNC treated a375, a875 cells.

Example 7

The effect of siRNA on skin melanoma cell invasion following inhibition of UBP41 expression was examined using a Transwell assay.

Skin melanoma cells A375, A875 were cultured as in example 2, and the cells A375, A875 having logarithmic growth phase were collected and centrifuged at 1,000rpm for 5min, followed by 3X 10 ⁵ The concentration of cells/mL was resuspended in serum-free DMEM. Matrigel (BD company) was preheated at 37 ℃ and its concentration was adjusted to 1mg/mL with serum free DMEM. After 300. Mu.L of pre-chilled DMEM containing 10% FBS was added to the 24-well plate, pre-chilled transwells were placed in the 24-well plate, and 100. Mu.L of matrigel (1 mg/mL) was added to the upper chamber of each Transwell. After incubation for 4-5 h at 37℃the 200. Mu.L of resuspended cells were added to the upper chamber of each Transwell and incubated at 37℃with 5% CO ₂ The culture was continued for 24 hours. The siRNA molecules siUBP41-1 and siUBP41-2 selected in example 3 were transfected into cells and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a). After 48h of transfection, the cells were washed with PBS, fixed in 70% pre-chilled ethanol for 1h, and then stained with 0.5% crystal violet at room temperature for 20min. After washing with PBS, the non-invasive cells on the side of the upper chamber were rubbed with a clean cotton swab, and the affected cells were observed and photographed under a microscope.

As shown in fig. 11, siUBP41-1 and siUBP41-2 significantly inhibited invasion of a375 and a875 cells compared to negative control siNC treated a375, a875 cells.

Example 8

The effect of siRNA on skin melanoma cell apoptosis after inhibition of UBP41 expression was examined using Annexin V-FITC/PI and flow cytometry.

Skin melanoma cells A375, A875 were cultured as in example 2, and A375, A875 cells having a logarithmic growth phase were collected and inoculated into 6-well plates at a cell density of 5X 10 ⁵ Individual cells/well, then 5% co at 37 °c ₂ Culturing in an incubator for 24 hours. The sample obtained in example 3 was subjected to the screeningSiUBP41-1 and SiUBP41-2 were transfected into cells and placed at 37℃with 5% CO ₂ Is cultured in an incubator of (a). 48h after transfection of the cells, the cells were washed with PBS, resuspended in 100. Mu.L of PBS, and 5. Mu.L of PI and Annexin V-FITC were added to each well. After incubation at 37℃for 48h in the absence of light, cells were washed with PBS, resuspended in 100. Mu.L of PBS and finally assayed by flow cytometry (BD Biosciences).

As shown in fig. 12, siUBP41-1 and siUBP41-2 significantly promoted apoptosis of a375 and a875 cells compared to negative control siNC treated a375, a875 cells.

As can be seen from the above experiments, siRNA molecules siUBP41-1 and siUBP41-2 targeting UBP41 exhibit activity of highly inhibiting the expression of UBP 41; they can inhibit proliferation, migration, invasion of skin melanoma cells and promote skin melanoma cells, and thus have potential for application in the treatment of skin melanoma.

Claims

1. A small interfering nucleic acid molecule for use in treating cutaneous melanoma, comprising a sense strand and an antisense strand of the sequence:

sense strand: 5 '-GAUUUGUGGUUACUUUUCUANN-3',

antisense strand: 5 '-UAGAACAGUAACACAAAUCNn-3'; or alternatively

Sense strand: 5'-CGAGUGACACUGAGACCUANN-3',

antisense strand: 5 '-UAGGUCUCUCAGUGUCUCCGNn-3',

2. The small interfering nucleic acid molecule of claim 1, wherein n is 0, i.e.

Sense strand: 5'-GAUUGUGGUUACUGUUCUA-3' (SEQ ID NO: 1),

antisense strand: 5'-UAGAACAGUAACCACAAUC-3' (SEQ ID NO: 2); or alternatively

Sense strand: 5'-CGAGUGACACUGAGACCUA-3' (SEQ ID NO: 3),

antisense strand: 5'-UAGGUCUCAGUGUCACUCG-3' (SEQ ID NO: 4).

3. The small interfering nucleic acid molecule of claim 1, wherein N is dT and N is 2, the sense strand: 5 '-GAUUUGUGGUUACUUUUCUANN-3' (SEQ ID NO: 5),

antisense strand: 5 '-UAGAACAGUAACACAACAAUCNN-3' (SEQ ID NO: 6); or alternatively

Sense strand: 5 '-CGAGUGAACCUGAGACCUANN-3' (SEQ ID NO: 7),

antisense strand: 5 '-UAGGUCUCUCAGUGUCCUCGNN-3' (SEQ ID NO: 8).

4. Use of a small interfering nucleic acid molecule according to any of claims 1-3 for the manufacture of a medicament for inhibiting UBP41 gene expression.

5. The use according to claim 4, wherein the medicament is for inhibiting proliferation and metastasis of skin melanoma cells or promoting apoptosis of skin melanoma cells.

6. A medicament for treating cutaneous melanoma, characterized by comprising as a pharmaceutically active ingredient a small interfering nucleic acid molecule according to any one of claims 1 to 3.

7. The medicament according to claim 6, characterized in that it is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule according to any of claims 1 to 3 as active ingredient, a pharmaceutically acceptable carrier.

8. The agent of claim 6, which is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule of any one of claims 1-3, one or more additional pharmaceutical ingredients that inhibit UBP41 expression.

9. The medicament of claim 6, which is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of the small interfering nucleic acid molecule of any one of claims 1-3, one or more additional pharmaceutical ingredients for the treatment of cutaneous melanoma.

10. The medicament according to any of claims 6 to 9, wherein the medicament is in an injectable form, suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.