Application of reagent for knocking down or knocking out p21 and / or CCNG2 gene in preparation of anti-aging product

Abstract

The invention provides application of a reagent for knocking down or knocking out p21 and / or CCNG2 genes in preparation of an anti-aging product, and belongs to the technical field of biological medicines. According to the knockdown or knockout p21 and / or CCNG2 gene provided by the invention, the cell cycle arrest condition caused by UVB irradiation is effectively relieved, the effect of relieving or alleviating the cell aging state is achieved, and the treatment effect on light aging is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of reagents of CCNG2 gene in the preparation of anti-aging products. Background Art

[0002] Aging is the process of gradual functional and structural decline of organisms over time. It is a complex natural phenomenon involving multiple changes at the cellular level, including telomere shortening, DNA damage accumulation, mitochondrial dysfunction, etc. From a physiological point of view, the body's metabolism slows down and organ function declines; at the molecular level, gene expression changes and protein homeostasis is unbalanced. These changes lead to various manifestations of aging.

[0003] Skin aging is a type of aging, which is mainly caused by two factors: endogenous and exogenous. Endogenous aging is determined by genetic and physiological processes, which makes the skin naturally thinner and less elastic; exogenous aging is mainly affected by environmental factors, among which ultraviolet radiation is the most important factor, which is photoaging. Common symptoms of photoaging include dry and rough skin, obvious wrinkles, large wrinkle areas, deep grooves, lack of sebum, etc. Its typical clinical manifestations are wrinkles, sagging skin, capillary dilation, uneven skin pigmentation, etc. Its mechanism is mainly that ultraviolet radiation causes DNA damage in skin cells, activates signaling pathways, leads to overexpression of matrix metalloproteinases (MMPs), and decomposes collagen and elastic fibers.

[0004] Photoaging models are important tools for studying the mechanisms of skin aging and preventive measures. In order to study photoaging, researchers have established a variety of models. Common in vitro cell models include human skin fibroblasts or keratinocytes, which simulate the photoaging process through ultraviolet irradiation to observe changes in cell morphology, function, and gene expression. In vitro cell models can be used to deeply study the mechanism of photoaging, thereby providing a theoretical basis for the development of anti-photoaging drugs, skin care products, etc., and are also important for understanding changes in the skin during the aging process.

[0005] The prominent feature of cell senescence is that the DNA replication process is blocked when cells face a series of endogenous and exogenous stresses. One of the basic characteristics of senescent cells is irreversible cell cycle arrest and the expression of cell cycle inhibitory proteins in large quantities. Among them, p21, as the main marker of senescent cells, is encoded by the CDKN1A gene. The aging process is closely related to cyclin-dependent kinases (CDKs). p21 is a CDK inhibitor that can specifically bind to CDK2, CDK4, and CDK6, and maintain the stagnant state of the cell cycle by inhibiting the phosphorylation modification of cyclins by these kinases. This mechanism is an important response of cells to internal and external stimuli such as DNA damage and oxidative stress. During the process of cell senescence, the expression of p21 usually increases significantly. Highly expressed p21 causes cells to stagnate in the G1 phase, effectively preventing cells from entering the S phase for DNA replication. This cell cycle arrest is a typical feature of senescent cells, which means that cells lose their ability to grow and proliferate after experiencing physiological or pathological stress. In addition, p21 is not only involved in maintaining cell cycle arrest, but also plays a key role in establishing the senescence-associated secretory phenotype (SASP) through protein Rb-dependent transcription (involving specific SMAD and STAT transcription factors). The initiation of this transcriptional program by p21 is the primary response to cellular stress that occurs simultaneously with cell cycle arrest, and the resulting immediate early secretory protein group is called the p21-activated secretory phenotype (PASP), which contains hundreds of factors, among which the chemokine CXCL14 can recruit macrophages to monitor stressed cells with elevated p21 expression.

[0006] CCNG2 is an atypical member of the Cyclin family and one of the key factors in cell cycle regulation. The cell cycle is a complex and highly regulated precision process designed to ensure that cells complete replication and division at the right time. Unlike classic Cyclin proteins (such as CyclinA, B, D, E, etc.), CCNG2 does not form an activation complex with cell cycle-dependent kinases (CDKs), but inhibits cell proliferation by negatively regulating the cell cycle. The core hallmark of cell senescence is the irreversible arrest of the cell cycle, and CCNG2 plays an important role in this process. It inhibits CDK activity by upregulating the expression of p21 and p27, thereby blocking cells from entering the S phase. The expression of CCNG2 is regulated by a variety of internal and external signals. Under normal physiological conditions, its expression level is strictly controlled to maintain precise regulation of the cell cycle. However, when cells are stimulated by external growth factors, stress damage or endogenous damage, the expression level of CCNG2 changes, which directly affects the cell proliferation ability by regulating the cell cycle process in the G2 / M phase. During the aging process, the cell cycle is susceptible to various internal and external factors, which manifests as cell cycle arrest or decreased proliferation capacity. CCNG2 may participate in the regulation of this process by affecting the function of cell cycle checkpoints, promoting or inhibiting cell proliferation during the aging process, thereby affecting the development of aging. In addition, CCNG2 is also involved in the regulation of intracellular transcription factors. By binding to transcription factors, it regulates gene expression including cell cycle proteins and cell cycle checkpoint factors, and plays a wide range of roles in the cell cycle regulatory network. When cells respond to internal and external stresses such as DNA damage and oxidative stress, CCNG2 expression usually increases, participates in stress response regulation, helps cells maintain internal environmental stability, and protects cell survival and function. It may play an important protective role by regulating stress responses during the aging process.

[0007] Studies have shown that CCNG2 and p21 are closely linked in the process of cell aging. CCNG2 can upregulate the expression of p21 through p53-dependent or -independent mechanisms. Under conditions of DNA damage or oxidative stress, CCNG2 can activate the p53 signaling pathway and indirectly promote the transcription of the p21 gene. The synergistic effect of CCNG2 and p53 can enhance the transcription level of p21, effectively block CDK activity, and induce cell cycle arrest. Other studies have shown that in some cells, CCNG2 may also directly promote p21 expression through non-p53-dependent pathways, such as activating TGF-β or PI3K / Akt pathways. p21 is not only a downstream effector molecule of CCNG2, but may also provide feedback regulation on the function of CCNG2. Studies have found that increased expression of p21 can stabilize the protein level of CCNG2 and further enhance the cell cycle inhibitory effect. This positive feedback effect is crucial in the process of cell stress response and aging, and helps maintain the homeostasis of cells under stressful environments. CCNG2 blocks CDK activity by upregulating p21, causing cells to permanently stagnate in the G1 or G2 phase, thereby inducing cell senescence. In addition, CCNG2 and p21 jointly participate in the formation of the senescence-associated secretory phenotype (SASP), which affects the functional state of surrounding cells.

[0008] RNA interference (RNAi) is a highly conserved sequence-specific gene expression regulation mechanism mediated by double-stranded RNA (dsRNA), which plays an important role in the antiviral defense of organisms and the maintenance of genome stability. The core process of this mechanism is that if the endogenous or exogenous double-stranded RNA has a homologous complementary sequence with the target gene's transcription product mRNA, it can specifically mediate the degradation of the mRNA in the cell, thereby effectively blocking the expression of a specific gene. This process belongs to sequence-specific post-transcriptional gene silencing (PTGS). RNAi technology has achieved remarkable results in the study of gene function in a variety of organisms. In actual operation, researchers design and construct small interfering RNA (siRNA) for the target gene. With the help of RNAi technology, the expression of the gene can be specifically inhibited. By systematically observing and analyzing the phenotypic changes of the organism caused by the inhibition of gene expression, the functional characteristics of the gene can be deeply analyzed. Compared with traditional gene knockout technology, RNAi technology has unique advantages. Gene knockout technology usually takes a long time to achieve permanent closure of a gene expression, while RNAi technology shows high efficiency and flexibility. In a relatively short period of time, such as within a week or even just two days, RNAi technology can achieve controllable silencing of up to 10 gene expressions. This feature makes RNAi technology extremely valuable in disease model construction and subsequent mechanism research, and provides strong technical support for in-depth exploration of the pathogenesis of diseases. At present, RNAi technology has been widely used in the research field of the pathogenesis of various diseases, greatly promoting people's understanding and recognition of the molecular mechanisms of diseases. In addition, other methods that can achieve knockdown or knockout, such as: shRNA, CRISPRi technology, antisense RNA technology, inhibitors, TALENs technology, ZFN technology, CRISPR / Cas9 and other technologies can also achieve similar effects.

[0009] The present invention takes photoaging as the main model for skin aging research. By utilizing the principle of RNAi technology to design and synthesize siRNA sequences corresponding to the target genes P21 and CCNG2, the silencing effect of each siRNA on the target gene is verified in a cell model of photoaging. At the same time, the effects on photoaging are studied by detecting cell proliferation, cell senescence, cell cycle, ROS generation, and the expression of extracellular matrix-related genes and proteins. Summary of the invention

[0010] The object of the present invention is to provide siRNA sequences, compounds or structures for regulating the expression of P21 and CCNG2, which are used for treating aging and aging-related diseases.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides an application of a reagent for knocking down or knocking out p21 and / or CCNG2 genes in the preparation of anti-aging products.

[0013] Preferably, the reagent is selected from one or more of siRNA, shRNA, CRISPRi technology, antisense RNA technology, TALENs technology, ZFN technology, and CRISPR / Cas9 for knocking down or knocking out p21 and / or CCNG2 genes; preferably, siRNA is used to knock down p21 and / or CCNG2 genes.

[0014] Preferably, the aging includes cell aging, organ aging and individual aging.

[0015] Preferably, the nucleotide sequence of the siRNA targeting knockdown of p21 is shown as SEQ ID NO: 5-24, preferably, the nucleotide sequence is SEQ ID NO: 23-24; the nucleotide sequence of the siRNA targeting knockdown of CCNG2 is shown as SEQ ID NO: 25-44, preferably, the nucleotide sequence is SEQ ID NO: 29-30.

[0016] Preferably, the anti-aging product is used to improve skin photoaging; preferably, the skin photoaging refers to the appearance of wrinkles, sagging, pigmentation and dryness on the skin; preferably, the skin photoaging is photoaging of dermal fibroblasts; the mode of causing the photoaging of dermal fibroblasts is ultraviolet irradiation, and the ultraviolet irradiation is UVB light source irradiation.

[0017] The present invention also provides a siRNA for knocking down p21 and CCNG2 genes, the nucleotide sequence of the siRNA for knocking down p21 gene is shown as SEQ ID NOs: 5 to 24, preferably, the nucleotide sequence of the siRNA for knocking down p21 gene is shown as SEQ ID NOs: 23 to 24; the nucleotide sequence of the siRNA for knocking down CCNG2 gene is shown as SEQ ID NOs: 25 to 44, preferably, the nucleotide sequence of the siRNA for knocking down CCNG2 gene is shown as SEQ ID NOs: 29 to 30.

[0018] The present invention also provides a siRNA reagent for knocking down p21 and / or CCNG2 genes, wherein the siRNA has one or more of 2'-O-methylation modification, 2'-methoxyethoxy modification and cholesterol modification, and further optionally, the siRNA terminal group is selected from the group consisting of cholesterol, bile acid, fatty acid, polyethylene glycol, antibody, polymer, and N-acetylgalactosamine.

[0019] The present invention also provides a pharmaceutical composition, comprising the double-stranded siRNA or the modified double-stranded siRNA reagent, and a pharmaceutically acceptable carrier.

[0020] The present invention also provides an anti-aging product, comprising the above double-stranded siRNA or the above modified double-stranded siRNA reagent.

[0021] Preferably, the administration method of the anti-aging product is selected from one of intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, epidermal smear, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0022] The reagent (siRNA) for knocking down or knocking out p21 and CCNG2 genes provided by the present invention has a good inhibitory effect on the two targets, and to a certain extent, effectively alleviates the cell cycle arrest caused by UVB irradiation, and has the effect of alleviating or reducing the cell aging state. Knocking down p21 or CCNG2 alone can partially increase the proliferation rate of photoaged fiber cells and partially reduce aging indicators. At the same time, knocking down can further increase the proliferation rate of fiber cells and further reduce aging indicators, which is manifested as an increase in the proportion of photoaged fiber cells in the S phase, reducing ROS, increasing CoL-1 gene expression, and reducing MMP1 and IL-1 gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of p21 & CCNG2 on the proliferation of photoaged HDF cells in Example 2 (Note: compared with the control group, Pa < 0.05; compared with the model group, Pb < 0.05; compared with the model group, Pc < 0.05);

[0024] Figure 2 The effect of p21 & CCNG2 on photoaged HDF cell senescence in Example 2;

[0025] Figure 3 This is the statistical analysis of the senescence of photoaged HDF cells in Example 2;

[0026] Figure 4 Statistical analysis of the cell (S) cycle of photoaged HDF cells in Example 2 (Note: Pa < 0.05 compared with the control group; Pb < 0.05 compared with the model group);

[0027] Figure 5 Statistical analysis of the cell (G0 / G1) cycle of photoaged HDF cells in Example 2 (Note: Pa < 0.05 compared with the control group; Pb < 0.05 compared with the model group);

[0028] Figure 6Analysis of ROS production in photoaged HDF cells in Example 2 (Note: P**<0.01 compared with the control group; P**<0.01 compared with the model group);

[0029] Figure 7 The effect of p21 and CCNG2 on the expression of extracellular matrix-related mRNA (COL-1) in photoaged HDF cells in Example 2 (Note: Pa < 0.05 compared with the control group; Pb < 0.05 compared with the model group);

[0030] Figure 8 The effect of p21 and CCNG2 on the expression of extracellular matrix-related mRNA (MMP-1) in photoaged HDF cells in Example 2 (Note: Pa < 0.05 compared with the control group; Pb < 0.05 compared with the model group);

[0031] Fig. 9 The effect of p21 and CCNG2 on the expression of extracellular matrix-related mRNA (IL-1) in photoaged HDF cells in Example 2 (Note: Pa < 0.05 compared with the control group; Pb < 0.05 compared with the model group);

[0032] Fig.10 Schematic diagram of the binding mode of the modified siRNA in Example 4;

[0033] Fig.11 The following are the back skin conditions of each group of mice after the experiment in Example 5. Specific embodiments

[0034] In order to facilitate the understanding of the present application, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0037] As used in this application, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members or features.

[0038] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0039] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items. "Any and all combinations" include any combination of two related listed items, any more related listed items, or all related listed items. For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B".

[0040] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0041] Unless otherwise specified, the term "nucleic acid" as used in this application refers to deoxyribonucleotides, ribonucleotides or modified nucleotides and polymers thereof in single-stranded or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring and non-naturally occurring, identical to the reference nucleic acid sequence. They have similar binding properties and they are metabolized in a similar manner as reference nucleotides.

[0042] Unless otherwise specified, the term "nucleotide" in this application includes those with natural bases (standard) and modified bases well known in the art. Nucleotides can be unmodified or modified in the sugar, phosphate and / or base moieties (also interchangeably referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc.). Eckstein et al., International PCT Publication No. WO 92 / 07065; Usman et al., International PCT Publication No. WO 93 / 15187;). Some non-limiting examples of base modifications that can be introduced into nucleic acid molecules include hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., nucleoside thymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine and pseudouridine), propyne, etc. (Burgin et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). In this regard, "modified bases" refer to nucleotide bases other than adenine, guanine, cytosine and uracil, and their equivalents.

[0043] "RNA" refers to a molecule containing at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the αβ-D-furanose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and the addition, deletion, substitution and / or alteration of one or more nucleotides. The nucleotides in the RNA molecules of the present invention may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs of naturally occurring RNA.

[0044] Unless otherwise specified, the term "modified nucleotide" in this application refers to a nucleotide having one or more modifications to a nucleoside, a nucleobase, a pentose ring or a phosphate group. For example, modified nucleotides include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate and cytidine monophosphate, as well as deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxyribonucleotides containing deoxycytidine monophosphate. Modifications include modifications that occur in nature by modification by enzymes that modify nucleotides (e.g., methyltransferases). Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-natural modifications in nucleotides include 2' modifications such as 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2 including those containing -O-2'-bridges, 2'-LNA and 2'-O-(N-methylcarbamate) or base analogs.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1 siRNA synthesis and screening

[0047] The target human p21 mRNA is disclosed in NCBI Reference Sequence: NM_000389.5 (CDKN1A) and is 2117 base pairs in length, SEQ ID NO: 1:

[0048]

[0049] The target human CCNG2 mRNA is disclosed in NCBI Reference Sequence: NM_004354.3 and is 5471 base pairs in length, SEQ ID NO: 2:

[0050]

[0051] A series of siRNAs were designed and synthesized for the two targets p21 and CCNG2.

[0052] The groups are as follows:

[0053] Table 1 siRNA sequences designed for p21 and CCNG2 targets

[0054]

[0055]

[0056] Human dermal fibroblasts (HDF), as the main component of the dermis of the skin, play a role in generating extracellular matrix including collagen, which is beneficial to maintaining the health of the skin. HDF is also the main target of skin photoaging.

[0057] Human dermal fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibodies (penicillin and streptomycin) in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), the transfection reagent was used for transfection. Specifically: the cell concentration was adjusted to 1×10 6 / ml, add 1ml of cell solution to each well of a 6-well plate and RNAiMAXReagent transfection reagent. After standing at room temperature for 5 minutes, add 5μL 10μM siRNA and incubate at 37℃, 5% CO2 for 48h. In addition to the experimental group, the following control groups are also set up for each cell plating: positive control group (siRNA targeting known efficient knockdown genes), negative control group (irrelevant siRNA), Mock transfection reagent control group, NC untreated control group (no siRNA). Both the experimental group and the control group have 3 replicates. See the table below for grouping details:

[0058] Table 2 Experimental groups

[0059]

[0060]

[0061] Real-time quantitative PCR analysis of target mRNA levels

[0062] 1. Lyse the cells 48 hours after transfection and extract total cellular RNA using the Trizol method.

[0063] 2. Reverse transcription (Takara kit, cat. no. 638313).

[0064] 3. Fluorescence quantitative qPCR.

[0065] The β-Actin gene was used as the internal reference gene, and the real-time PCR kit SYBR Premix (2×) was used to perform the real-time fluorescence quantitative PCR reaction. The PCR reaction was performed using the Rotor-Gene-Q fluorescence quantitative PCR instrument of Qiagen Company, USA. The primers used were:

[0066] Table 3 Primers

[0067]

[0068] After the PCR reaction, the Ct error of 9 replicates of a sample (3 transfection replicates and 3 qPCR replicates for each sample) was within ±0.5. The average expression level of the target gene of the preferred siRNA relative to the NC group (the relative expression level of mRNA in the NC untreated control group is 1) is shown in the following table:

[0069] Table 4 The average expression levels of target genes in P21 siRNA group relative to NC group

[0070]

[0071]

[0072] Table 5 Average expression levels of target genes in CCNG2 siRNA group relative to NC group

[0073] Group CCNG2 mRNA expression level Standard error NC untreated control group 1.00 0.11 Mock Transfection Reagent Set 1.03 0.09 Negative control group 0.99 0.07 Positive control group 1.03 0.02 CCNG2siRNA1 group 0.84 0.11 CCNG2siRNA2 group 0.81 0.20 CCNG2siRNA3 group 0.41 0.12 CCNG2siRNA4 group 0.69 0.21 CCNG2siRNA5 group 0.67 0.09 CCNG2siRNA6 group 0.75 0.17 CCNG2 siRNA7 group 0.79 0.16 CCNG2 siRNA 8 group 0.63 0.05 CCNG2siRNA9 group 0.55 0.11 CCNG2 siRNA10 group 0.70 0.18

[0074] The results showed that the negative control and positive control groups of the two experiments were normal, and the results of the transfection reagent group showed that the transfection reagent did not affect the expression of the two targets; for p21, p21siRNA1 and p21 siRNA 10 had the best target inhibition effect, and for CCNG2, CCNG2 siRNA3 had the best target inhibition effect.

[0075] Example 2

[0076] Skin aging is a natural physiological process that manifests itself in the form of wrinkles, sagging skin, pigmentation, and dry skin. Skin aging is actually a very complex biological process that is closely related to many different factors, such as genetic genes, external environment, and personal lifestyle.

[0077] At present, there are a series of strategies to alleviate skin aging, such as sun protection, skin moisturizing, antioxidant maintenance, physical exercise, healthy diet, regular work and rest, skin care measures (including cosmetic care and professional care at beauty salons), and medical cosmetic methods (such as laser therapy, microneedle stimulation technology, filler injections, etc.). However, although each of these strategies can play a certain role, in general, they often only relieve symptoms on the surface and it is difficult to fundamentally solve the problem of skin aging.

[0078] Construction of in vitro skin photoaging cell model

[0079] When constructing the model, HDF cells with vigorous cell proliferation activity are selected. When the cell confluence reaches 45-55%, they can be used for UVB lamp irradiation. Disinfect the UVB lamp with alcohol one day in advance and place it on the clean bench overnight. During the experiment, first remove the culture medium in the culture dish, add a thin layer of PBS solution, turn off the alcohol lamp, increase the wind speed of the clean bench to ensure sterility, remove the culture dish cover, and place the UVB lamp parallel to the culture medium at a height of 10 cm. Use an ultraviolet light intensity meter to measure the irradiation intensity to ensure that the irradiation dose is 90mJ / cm 2 After irradiation, the PBS in the culture dish was discarded, and H-DMEM complete medium was added again, and the subsequent culture conditions remained unchanged.

[0080] Experimental Grouping

[0081] The experiment was divided into three groups, namely the control group, the model group and the experimental group. The model group and the experimental group were irradiated with UVB under the same conditions, and the irradiation conditions were as described above; for the experimental group, p21siRNA 10 and CCNG2siRNA3 were transfected 24 hours before UVB irradiation for pretreatment, while the model group was added with an equal amount of PBS solution; for the control group, except for not giving UVB irradiation, the other conditions were the same as the model group.

[0082] siRNA transfection experiments

[0083] 5×10 4 The cells were seeded in 12-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibodies (penicillin and streptomycin) at 37°C in a 5% CO2 incubator for 12 h. When the cells were in the logarithmic growth phase and in good condition (70% confluence), 5 μL of Lipofectamine 2000 (Invitrogen, Cat#11668019) was added to each well. RNAiMAX Reagent transfection reagent was allowed to stand at room temperature for 5 min, and then p21 siRNA 10 (0.1 μmol) or CCNG2 siRNA 3 (0.1 μmol) or p21 siRNA 10 (0.1 μmol) and CCNG2 siRNA 3 (0.1 μmol) were transfected as needed according to different experimental groups. Fresh complete culture medium was replaced 6 h after transfection, and cultured for 18 h at 37°C and 5% CO2.

[0084] Detection of HDF cell proliferation activity

[0085] HDF cells were collected at 5×10 3 The density of cells was spread in a 96-well plate. According to the experimental group, the experimental group cells were transfected with p21siRNA10, CCNG2siRNA3, and p21siRNA10 and CCNG2siRNA3 at the same time 24 hours before illumination (refer to the above for the transfection experimental steps), and the rest of the treatments remained the same. On the day of illumination, the 96-well plate was taken out, the culture medium was removed, and the cover of the 96-well plate was removed after adding an appropriate amount of PBS solution. The model group and the experimental group were placed under the UVB lamp correspondingly, and the irradiation operation was performed according to the established standard conditions. After the irradiation process was completed, the PBS solution in the well was quickly discarded, 100μl of H-DMEM complete culture medium was added to each well, and the 96-well plate was steadily returned to the incubator to maintain the culture environment for 48h. After 48h, 10μl of WST-8 staining solution was added to each well, and the 96-well plate was returned to the incubator again and left to stand for about 2h. The absorbance OD value at a wavelength of 450nm was accurately detected using an enzyme marker. It is particularly important to note that a blank control well is specially set up, which contains only cell culture medium and WST-8 staining solution, and does not contain any cells, in order to ensure the accuracy and reliability of the experimental data.

[0086] The results are as follows Figure 1 As shown, the cell proliferation activity of the model group cells irradiated with UVB was significantly decreased compared with the control group; while the cell proliferation activity of the three groups of HDF cells transfected with p21siRNA, CCNG2siRNA and two siRNAs at the same time showed an upward trend to varying degrees. All three groups were significantly different from the model group, and the cell activity of the group transfected with p21siRNA10 and CCNG2siRNA3 at the same time showed the greatest upward trend.

[0087] HDF cell senescence detection

[0088] The experimental results of cell proliferation activity indicate that HDF cells have undergone cell senescence after UVB irradiation, and the cell replication rate has decreased. In order to further confirm that the cells have entered the state of senescence, we used the gold standard of cell senescence detection, β-galactosidase staining, to further verify and further clarify whether knocking down p21 and CCNG2 can alleviate or reduce the state of cell senescence.

[0089] HDF cells were cultured at a rate of 5 × 10 4 The cells were plated at a density of 100 μg / well in a 6-well plate and divided into experimental groups. The experimental group cells were transfected with p21 siRNA 10 and CCNG2 siRNA 3 24 h before illumination (refer to the above for the transfection experimental steps). The rest of the treatments remained the same. Cell samples were prepared 72 h after illumination treatment. The steps were as follows:

[0090] 1) After discarding the cell culture medium in the culture medium, wash with an appropriate amount of PBS solution, add 1 ml of fixative solution, and fix at room temperature for 15 minutes;

[0091] 2) Aspirate and discard the cell fixative solution, wash with an appropriate amount of PBS solution, and repeat the washing three times;

[0092] 3) Aspirate and discard the PBS solution, and add 1 ml of the prepared β-galactosidase staining working solution to each well.

[0093] Table 6 Configuration of β-galactosidase staining working solution

[0094] Reagent name volume β-Galactosidase staining solution A 100μl β-Galactosidase staining solution B 100μl β-Galactosidase staining working solution C 9.3ml X-Gal Solution 500μl

[0095] 4) Seal the 6-well plate with plastic wrap and incubate it in a non-CO2 incubator at 37°C overnight;

[0096] 5) Counting under an optical microscope was used to perform statistics and analysis of the data.

[0097] The results are as follows Figure 2 , 3 As shown: Under the microscope, very few cells in the control group were stained blue, while most of the cells in the model group were stained blue, indicating that the cells had undergone aging changes. In the group transfected with p21 siRNA 10 and CCNG2 siRNA3, the blue staining of cells was greatly improved compared with the model group, and most of the cells in the group transfected with p21 siRNA 10 and CCNG2 siRNA3 were not stained blue. This group had the best effect in reducing cell aging, so this group was used as the experimental group for other experiments. After statistical analysis of the blue staining of cells ( Figure 3 ), we found that the difference between the model group and the control group was significant, and the difference between the experimental group and the model group was also significant.

[0098] Cell cycle detection

[0099] UVB radiation can cause a significant decline in cell proliferation activity, which is closely related to the state of the cells. After UVB irradiation, the proliferation activity of cells in the model group decreased sharply, and we then tested the cell cycle.

[0100] According to 5×10 4 HDF cells were plated in a 6-well plate at a density of 1 / well. The experimental group cells were transfected with p21 siRNA 10 and CCNG2 siRNA 3 24 hours before illumination (refer to the above for the transfection experimental steps). The model group cells and the experimental group cells were exposed to UVB light. The rest of the treatments were the same for each group. Cell samples were prepared 48 hours after illumination treatment. The steps are as follows:

[0101] 1) Use trypsin to digest the HDF cells treated in the previous step. After digestion and centrifugation, discard the supernatant, add 1 ml of pre-cooled PBS to resuspend the cells, and centrifuge at 1000g for 5 minutes;

[0102] 2) Add 1 ml of pre-cooled 70% ethanol solution to the precipitate after centrifugation in the above step, gently blow and mix evenly, and place at 4°C to fix overnight; then centrifuge and discard the ethanol solution and add 1 ml of pre-cooled PBS solution to resuspend the cells, and centrifuge at 1000g for 5 minutes for later use;

[0103] 3) Preparation of propidium iodide staining working solution

[0104] Table 7 Configuration of propidium iodide staining working solution

[0105] Reagents volume Staining Buffer 6ml Propidium iodide staining solution (20×) 300μl RNase A (50×) 120μl Final volume 6.42ml

[0106] 4) Add 0.5 ml of propidium iodide staining working solution to the spare cell sample, slowly resuspend the cell pellet, and incubate in a 37°C water bath in the dark for 30 minutes. After the incubation, use a flow cytometer to detect the cell cycle.

[0107] 5) The flow cytometer detection conditions are: red fluorescence is detected at an excitation wavelength of 488 nm, and the results are analyzed using software.

[0108] By analyzing the cell cycle, we found that some of the cell cycle processes of cells irradiated by UVB were blocked. Fig.10 , 11 The results in the above table were statistically analyzed for the proportion of cells in the S phase after 48 hours of illumination ( Figure 4 ), the proportion of S phase cells in the model group increased significantly compared with the control group, and the difference was significant. Similarly, we counted the G0 / G1 phase cells and found that ( Figure 5), compared with the control group, the proportion of cells in the G0 / G1 phase in the model group increased, and some cells entered the cell cycle arrest and stagnated in the S phase. Further, we observed the proportion of cells in the S phase and G0 / G1 phase in the groups pre-treated by p21 siRNA 10 and CCNG2 siRNA3 transfection. The results showed that compared with the model group, the proportion of cells in the S phase in the experimental group was significantly reduced, while the proportion of cells in the G0 / G1 phase was significantly increased. This indicates that the pre-treatment operation of p21 siRNA 10 and CCNG2 siRNA 3 transfection effectively alleviated the cell cycle arrest caused by UVB irradiation to a certain extent.

[0109] Detection of ROS generation

[0110] In order to further explore the possible mechanism by which p21 and CCNG2 alleviate skin photoaging in HDF cells, given that UVB-induced ROS is the initiator of skin photoaging, we used flow cytometry to detect the ROS content of each group of HDF cells after UVB irradiation.

[0111] According to 5×10 4 HDF cells were plated in 6-well plates at a density of 1:1000. The experimental group cells were transfected with p21 siRNA10 and CCNG2 siRNA3 24 h before illumination (refer to the above for the transfection experimental steps). The model group cells and the experimental group cells were exposed to UVB, and the rest of the treatments were the same for each group. On the day of illumination, the H2DCF-DA probe was diluted to a final concentration of 10 μmol / ml in a serum-free medium at a ratio of 1:1000. 1 ml of the diluted H2DCF-DA probe (MedChemExpress, HY-D0940) was added to the 6-well plate, and then the 6-well plate was placed in a cell culture incubator at 37 ° C for about 1 h. After the incubation, the liquid in the 6-well plate was aspirated and discarded, and the serum-free cell culture medium was used to wash it three times to fully remove the H2DCF-DA probe that was not loaded into the cells. After sufficient washing, the serum-free cell culture medium was aspirated and discarded, and 1 ml of PBS solution was added to each well. As described above, the model group and the experimental group were exposed to UVB, and the conditions were the same as before and kept consistent. After irradiation, the cells were fully digested to make a single cell suspension and collected in a flow cytometer. The ROS production was detected by flow cytometry 30 minutes after irradiation. The difference in fluorescence intensity after stimulation was detected using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0112] The results are as follows Figure 6As shown: After UVB irradiation, the ROS content of HDF cells in the model group increased sharply; while the ROS content of the experimental group transfected with p21siRNA10 and CCNG2siRNA3 pre-treated cells decreased significantly. Quantitative analysis found that compared with the control group, the increase in ROS content of HDF cells in the model group was significantly different; compared with the model group, the change in ROS content of HDF cells in the experimental group was also significantly different.

[0113] qRT-PCR detection

[0114] Skin photoaging has many characteristics, among which the reduction of extracellular matrix content is a more prominent feature. Human dermal fibroblasts (HDF cells) are distributed in the dermis of skin tissue and play an important role in the regulation of extracellular matrix. In order to further clarify the effect of UVB on HDF cells and the repair mechanism of transfection of p21siRNA10 and CCNG2siRNA3 pretreatment (transfection experimental steps refer to the above), we detected the changes of three factors including MMP-1, COL-1, and IL-1 from a genetic perspective by qRT-PCR detection.

[0115] 1. Extraction of RNA from HDF cells: 5×10 4 HDF cells were plated in 6-well plates at a density of 10 / well and divided into experimental groups. For the experimental group, cells were transfected with p21 siRNA 10 and CCNG2 siRNA 3 24 hours before illumination. The rest of the treatments were the same as in the previous experiment. The treatments were performed according to the following steps:

[0116] 1) Aspirate the culture medium in the 6-well plate on ice, wash twice with PBS solution, add 500 μl Trizol to each well, blow and aspirate to remove the cells, and then transfer the suspension to a 1.5 ml RNase-free centrifuge tube;

[0117] 2) Add 100 μl of chloroform to each tube and shake thoroughly for 15 seconds, then let stand at room temperature for 3 minutes;

[0118] 3) Set the low-temperature centrifuge in advance and cool it to 4°C, centrifuge at 12000rpm for 15min, and carefully transfer the supernatant to a new 1.5ml RNase-free centrifuge tube;

[0119] 4) Add 250 μl of isopropanol to the supernatant to precipitate RNA, mix thoroughly and place at room temperature for about 10 minutes, then centrifuge again at 12,000 rpm for 10 minutes;

[0120] 5) After discarding the supernatant, add 1 ml of 80% ethanol solution to the precipitate and wash the RNA by turning it upside down;

[0121] 6) Centrifuge again at 4°C, 7500 rpm for 5 min;

[0122] 7) Discard the supernatant, place at room temperature for 3-5 minutes to evaporate the ethanol, and add 20 μl DEPC water to dissolve the RNA;

[0123] 8) NanoDrop2000 was used to detect the concentration and quality of the extracted RNA samples (when NanoDrop2000 was used to measure the concentration, the RNA 260 / 280 = 1.9-2.0, 260 / 230>2 had good purity, indicating that there were few residual proteins and phenolic substances).

[0124] 2. RNA reverse transcription:

[0125] First, remove genomic DNA. The reaction system is as follows:

[0126] Table 8 Reaction system for removing genomic DNA

[0127] Reagents volume 5×gDNA Eraser Buffer 2μl gDNA Eraser 1μl Total RNA 1 μg <![CDATA[RNase Free dH2O]]> Make up to a total volume of 10ul Total 10ul

[0128] After the reaction system is mixed, it is placed in a PCR instrument at 42°C for 2 minutes and then temporarily stored at 4°C.

[0129] Table 9 Reverse transcription system

[0130] Reagents volume The above reaction solution 10μl <![CDATA[5×PrimeScript TM Buffer]]> 4μl <![CDATA[PrimeScript TM RT Enzyme MixI]]> 1μl RT Primer Mix 1μl <![CDATA[RNase Free dH2O]]> 4μl

[0131]

[0132] The solution of the system was mixed and placed in a PCR instrument. The reaction was carried out at 37°C for 15 minutes and at 85°C for 5 seconds. The reverse transcribed cDNA sample was then stored at -20°C for a long term.

[0133] 3. qRT-PCR detection: The reaction system is as follows:

[0134] Table 10 Reaction system for qRT-PCR detection

[0135] Reagents volume 2xSYBR Green qPCR mix 10μl Forwardprimer 1μl Reverseprimer 1μl cDNA (diluted 20 times) 5μl <![CDATA[RNaseFreedH2O]]> 3μl Total 20ul

[0136] The primer sequences are shown in Table 11 below. The qRT-PCR reaction program is 95° C., 10 min, 95° C., 15 s, 60° C., 60 s, and the cycle is continued for 40 times.

[0137] Table 11 Primer sequence information for qRT-PCR

[0138]

[0139] The results are as follows Figures 7 to 9As shown in the figure: compared with the control group, the expression level of COL-1mRNA in the model group decreased; compared with the model group, the expression level of COL-1mRNA in the experimental group increased, and the differences between the model group and the control group and between the experimental group and the model group were significant. The expression level of MMP-1mRNA was just the opposite of COL-1mRNA: compared with the control group, the expression level of MMP-1mRNA in the model group increased; compared with the model group, the expression level of MMP-1mRNA in the experimental group showed a downward trend; the differences between the model group and the control group and between the experimental group and the model group were significant. At the same time, we also found that pretreatment with transfection of p21siRNA10 and CCNG2siRNA3 can alleviate the inflammatory response caused by photoaging of HDF cells. The expression level of IL-1mRNA in HDF cells was detected: compared with the control group, the expression level of IL-1mRNA in the model group increased; compared with the model group, the expression level of IL-1mRNA in the experimental group showed a downward trend, and the differences between the model group and the control group and between the experimental group and the model group were significant.

[0140] Example 4 siRNA mimic synthesis

[0141] Preparation process:

[0142] Monomers and reagents were purchased from Haijing Gaochuang or other qualified companies. The modified p21 siRNA 10 and CCNG2 siRNA 3 are shown in Table 12 below, where s represents 2'-O-methyl modification, m represents 2'-methoxyethoxy modification, and chol represents cholesterol modification. Fig.10 shown.

[0143] Table 12 siRNA sequences with modifications

[0144]

[0145]

[0146] Preparation process:

[0147] Step 1a. Synthesis using a 12-channel synthesizer (HJ-12): including deprotection, coupling, oxo / thio modification, capping, etc. Specifically:

[0148] Prepare DCA toluene solution as a deprotecting agent, prepare 0.25M, ETT (0.25M, 5-ethylthiotetrazolyl N / A acetonitrile) as a coupling agent, prepare 0.05M iodine pyridine / water solution as an oxidant, prepare hydrogenated xanthin as a thioagent, and CH3CN / imidazole solution containing 20% ​​acetyl chloride as a capping agent, and place them in the designated reagent position corresponding to the HJ-12 synthesizer. Set the synthesis program to enter the specified oligonucleotide base sequence, start cyclic oligonucleotide synthesis, each step coupling time is 6 minutes, and the galactose ligand corresponds to the L and S monomers coupling time of 15 minutes. After automatic circulation, the oligonucleotide solid phase synthesis is completed.

[0149] Step 1b. Ammonolysis and desiliconization.

[0150] The aminolysis was carried out with 160 mL / mmol AMA, 60°C, 3 h. The desiliconization was carried out with 20 mL / mmol DMSO, 280 mL / mmol TEA, 93.3 mL / mmol TEA*3HF (≤15°C), 40°C, 4 h. After the desiliconization reaction was completed, the system was diluted with purified water, the pH of the system was adjusted to about 7.0 with acetic acid aqueous solution, and a centrifuge was used for centrifugal sedimentation to collect the solid.

[0151] Step 1c. Purification.

[0152] Agilent HPLC purification system, 45°C hot ethanol dissolved the sample, mobile phase A: 200mM TEAA in H2O, mobile phase B: prepared acetonitrile, elution gradient: 20-80% 90min, linear flow rate: 160-200cm / h (10mL / min), purification column using C8 (using C8 for cholesterol) or C18 column (using C18 without cholesterol), column temperature: 40°C.

[0153] Step 1d. Desalting and freeze-drying.

[0154] Purified water was used for ultrafiltration desalination, and the equipment was 3KDa TFF for membrane ultrafiltration desalination.

[0155] Finally, double-stranded small nucleic acids were prepared by annealing and freeze-drying.

[0156] Example 5 Treatment of skin photoaging

[0157] Construction of skin photoaging model: 8-week-old male C57bL / n mice were used, 6 mice in each group, as shown in Table 13 below, and balbc mice were irradiated with UVB (310nm, 20W) at a distance of 30cm, and irradiated for 20min at a fixed time every day for 4 weeks.

[0158] Treatment plan: In the treatment group (Model+p21 siRNA&CCNG2 siRNA), 100μg of modified p21siRNA 10 and 100μg of modified CCNG2 siRNA3 dissolved in 100μl of normal saline were injected at multiple points on the back of each group. The drugs were administered once a week for a total of 4 times; the Model group was given an equal amount of normal saline every week.

[0159] After 4 weeks, the Model group mice had a large number of thick wrinkles on their backs, and their skin became thickened, keratinized, and dry. The treatment group (Model+p21 siRNA&CCNG2 siRNA) had significantly fewer skin wrinkles, less skin thickening, less keratinization, and less dry and cracked skin than the Model group. Fig.11 shown.

[0160] Visual scoring of photoaging mice was performed according to the following criteria:

[0161] 0 points: Smooth without any wrinkles; fine lines run through the entire body

[0162] 1 point: fine lines

[0163] 2 points: A few shallow wrinkles; all fine lines disappear

[0164] 3 points: Shallow wrinkles across the skin of the back

[0165] 4 points: Deep, rough wrinkles with sagging

[0166] 5 points: Increased deep wrinkles

[0167] 6 points: Severe wrinkles on the surface; lesions develop.

[0168] The results are shown in Table 13. The score of the Model group increased significantly compared with that of the Control group, and the skin aged seriously. The visual score of the treatment group decreased significantly compared with that of the Model group.

[0169] Table 13 Wrinkle Scores

[0170]

[0171]

[0172] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. Use of a reagent for knocking down or knocking out p21 and / or CCNG2 genes in the preparation of anti-aging products.

2. The use according to claim 1, characterized in that: The reagent is selected from one or more of siRNA, shRNA, CRISPRi technology, antisense RNA technology, TALENs technology, ZFN technology, and CRISPR / Cas9 for knocking down or knocking out p21 and / or CCNG2 genes; preferably, siRNA is used to knock down p21 and / or CCNG2 genes.

3. The method according to claim 1, characterized in that The aging includes cell aging, organ aging and individual aging.

4. The use according to claim 2, characterized in that: The nucleotide sequence of siRNA targeting knockdown of p21 is shown as SEQ ID NO: 5-24, preferably, the nucleotide sequence is SEQ ID NO: 23-24; the nucleotide sequence of siRNA targeting knockdown of CCNG2 is shown as SEQ ID NO: 25-44, preferably, the nucleotide sequence is SEQ ID NO: 29-30.

5. The use according to claim 1, characterized in that: The anti-aging product is used to improve skin photoaging; preferably, the skin photoaging refers to the appearance of wrinkles, sagging, pigmentation and dryness on the skin; preferably, the skin photoaging is photoaging of dermal fibroblasts; the mode of causing the photoaging of dermal fibroblasts is ultraviolet irradiation, and the ultraviolet irradiation is UVB light source irradiation.

6. A siRNA for knocking down p21 and CCNG2 genes, characterized in that: The nucleotide sequence of the siRNA for knocking down the p21 gene is shown as SEQ ID NOs: 5 to 24, preferably, the nucleotide sequence of the siRNA for knocking down the p21 gene is shown as SEQ ID NOs: 23 to 24; the nucleotide sequence of the siRNA for knocking down the CCNG2 gene is shown as SEQ ID NOs: 25 to 44, preferably, the nucleotide sequence of the siRNA for knocking down the CCNG2 gene is shown as SEQ ID NOs: 29 to 30.

7. The siRNA reagent for knocking down p21 and / or CCNG2 gene according to claim 6, characterized in that: The siRNA has one or more of 2'-O-methylation modification, 2'-methoxyethoxy modification and cholesterol modification. Further optionally, the siRNA terminal group is selected from the group consisting of cholesterol, bile acid, fatty acid, polyethylene glycol, antibody, polymer, and N-acetylgalactosamine.

8. A pharmaceutical composition, characterized in that The invention comprises the double-stranded siRNA according to claim 6 or the modified double-stranded siRNA agent according to claim 7, and a pharmaceutically acceptable carrier.

9. An anti-aging product, characterized in that: The double-stranded siRNA reagent comprises the double-stranded siRNA of claim 6 or the modified double-stranded siRNA reagent of claim 7.

10. The anti-aging product according to claim 9, characterized in that The administration method of the anti-aging product is selected from one of intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, epidermal smear, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

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