CN116536315A

CN116536315A - SiRNA of targeted PSME3 gene and application thereof

Info

Publication number: CN116536315A
Application number: CN202310521476.9A
Authority: CN
Inventors: 胡超; 陈雪; 胡杰; 叶美希; 王熙瑞; 胡玲
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2023-05-10
Filing date: 2023-05-10
Publication date: 2023-08-04

Abstract

The invention belongs to the technical field of gene targeting drugs, and particularly relates to a PSME3 gene targeting siRNA and application thereof. The siRNA is selected from siRNA1 or siRNA2, wherein the sense strand and the antisense strand of siRNA1 are respectively shown in SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the sense strand and the antisense strand of siRNA2 are shown in SEQ ID NO:3 and SEQ ID NO: 4. The siRNA can target a PSME3 gene of a human or a PSME3 gene of a mouse, so that the mRNA level of the PSME3 gene in cancer cells can be effectively reduced, the protein expression level of the PSME3 gene in the cancer cells can be effectively reduced, and the purposes of effectively inhibiting proliferation of the cancer cells and retarding the growth cycle of the cancer cells can be achieved.

Description

SiRNA of targeted PSME3 gene and application thereof

Technical Field

The invention belongs to the technical field of gene targeting drugs, and particularly relates to a PSME3 gene targeting siRNA and application thereof.

Background

PSME3, also known as REGγ,11 Sγ, PA28 γ, PSME3 or Ki (GeneID: 10197), is one of the 11S proteasome activator family members that can degrade a substrate protein by binding to the α subunit of the 20S core particle, feeding the substrate protein into the 20S β subunit protease catalytic site. The PSME3-20S protein degradation system may modulate physiological responses by degrading some proteins with important biological functions, thereby eliciting diseases, particularly cancers.

In recent years, it has been found that PSME3 can regulate cell cycle and apoptosis by degrading cyclin-dependent kinase inhibitors such as p21, p16, p19 and p14, and the absence of these cyclin inhibitors can induce normal cells to be transformed into cancer cells, thereby causing cancer to occur. Meanwhile, PSME3 can promote the ubiquitination degradation of p53 through combining with p53 (p 53 is a tumor suppressor, which can accelerate apoptosis and inhibit tumorigenesis) and MDM2, thereby promoting tumor development; in addition, studies on the expression level of PSME3 in tumor tissues of human and mouse have found that PSME3 is expressed at a higher level in lung cancer, colon cancer, thyroid cancer and liver cancer, and these findings confirm the important link between PSME3 and cancer, making PSME3 a potential cancer label. In addition, PSME3 can also be used for promoting the generation of skin cancer by degrading GSK3 beta (GSK 3 beta is a serine/threonine kinase, has the functions of regulating and controlling a plurality of signal proteins and transcription factors besides regulating and controlling glycogen synthesis, regulating and controlling cell differentiation, proliferation, survival and apoptosis, and has a certain inhibition effect on the generation of cancer), up-regulating the expression level of beta-catenin, and promoting the expression of target genes CyclinD1 and c-Myc downstream of Wnt/beta-catenin signal channels. These results indicate that PSME3 plays a very important regulatory role in cellular activity and human disease, and is a potential target for diagnosis and treatment of cancer or other diseases.

Therefore, it is necessary to develop a way to increase the practical application of PSME3 target based on PSME3 gene target.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide an siRNA targeting PSME3 gene, which can target PSME3 gene of human or PSME3 gene of mouse, thereby effectively inhibiting expression of PSME3 gene, and thus inhibiting proliferation of cancer cells and blocking growth cycle of cancer cells.

In order to achieve the above purpose, the present invention may adopt the following technical scheme:

in one aspect, the present invention provides an siRNA targeting PSME3 gene, selected from siRNA1 or siRNA2, wherein the sense strand and the antisense strand of siRNA1 are as shown in SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the sense strand and the antisense strand of siRNA2 are shown in SEQ ID NO:3 and SEQ ID NO: 4.

In another aspect, the present invention provides a nucleic acid molecule targeting a PSME3 gene, which is obtained by chemical modification of the siRNA targeting the PSME3 gene.

In another aspect, the present invention provides a recombinant expression vector comprising the above siRNA targeting PSME3 gene or the above nucleic acid molecule targeting PSME3 gene.

In yet another aspect, the present invention provides a composition for inhibiting expression of a PSME3 gene, comprising the above-described siRNA targeting the PSME3 gene or the above-described nucleic acid molecule targeting the PSME3 gene or the above-described recombinant expression vector.

In yet another aspect, the present invention provides an agent for inhibiting expression of a PSME3 gene, comprising the above-described siRNA targeting a PSME3 gene or the above-described nucleic acid molecule targeting a PSME3 gene or the above-described recombinant expression vector or the above-described composition for inhibiting expression of a PSME3 gene; and a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides an application of the above siRNA targeting PSME3 gene or the above nucleic acid molecule targeting PSME3 gene or the above recombinant expression vector or the above composition for inhibiting PSME3 gene expression in preparing a PSME3 gene expression inhibitor.

In a further aspect, the present invention provides the use of an siRNA targeting a PSME3 gene as described above or a nucleic acid molecule targeting a PSME3 gene as described above or a recombinant expression vector as described above or a composition for inhibiting expression of a PSME3 gene as described above in the preparation of a medicament for treating a disease caused by abnormal expression of a PSME3 gene.

The beneficial effects of the invention at least comprise: the siRNA provided by the invention can target a PSME3 gene of a human body and a PSME3 gene of a mouse, so that the mRNA level of the PSME3 gene in cancer cells can be effectively reduced, the protein expression level of the PSME3 gene in the cancer cells can be effectively reduced, and the purposes of effectively inhibiting proliferation of the cancer cells and retarding the growth cycle of the cancer cells are achieved.

Drawings

FIG. 1 shows the proliferation of liver cancer cell lines Hepa1-6 of PSME3-496 transfected mice;

FIG. 2 is a flow cytometry detection of liver cancer cell line Hepa1-6 cell cycle status of PSME3-496 transfected mice;

FIG. 3 is a histogram of liver cancer cell lines Hepa1-6 from PSME3-496 transfected mice;

FIG. 4 shows the mRNA expression levels of PSME3 after transfection of the liver cancer cell lines Hepa1-6 of mice with PSME3-223 and PSME3-496, respectively;

FIG. 5 shows the mRNA expression levels of PSME3 after transfection of the human hepatoma cell lines HepG2 with PSME3-223 and PSME3-496, respectively;

FIG. 6 shows protein expression levels of PSME3 after transfection of SiRNAs into liver cancer cell lines Hepa1-6 of mice with PSME3-223 and PSME3-496, respectively.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of features, numbers, operations, elements, materials, or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, elements, materials or combinations thereof may be present or may be added. As used herein, "/" may be interpreted as "and" or "as appropriate.

The embodiment of the invention provides a PSME3 gene targeted siRNA, which is selected from siRNA1 or siRNA2, wherein the sense strand and the antisense strand of the siRNA1 are respectively shown as SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the sense strand and the antisense strand of siRNA2 are shown in SEQ ID NO:3 and SEQ ID NO: 4. The siRNA1 (hereinafter also referred to as PSME 3-223) or siRNA2 (hereinafter also referred to as PSME 3-496) of the invention can target the PSME3 gene of human and mice at the same time, and can effectively reduce the mRNA level of the PSME3 gene in cancer cells, thereby effectively reducing the protein expression level of the PSME3 gene in cancer cells, further effectively inhibiting proliferation of cancer cells and retarding the growth cycle of cancer cells.

In addition, siRNA is a small interfering RNA, sometimes referred to as short interfering RNA or silencing RNA, a class of double stranded RNA molecules, 20-25 base pairs in length, similar to miRNA, and operates within the RNA interference (RNAi) pathway. It interferes with post-transcriptional degraded mRNA of a specific gene expressing a nucleotide sequence complementary to it, thereby preventing translation.

Another embodiment of the present invention provides a nucleic acid molecule targeting PSME3 gene, which is obtained by chemical modification of the siRNA targeting PSME3 gene described above. It should be noted that, because the stability of general siRNA is poor, it is easily degraded in vivo by nuclease and is not easily absorbed by tissues, so that its application in vivo is limited. The siRNA can be chemically modified to increase the stability of the siRNA, and can effectively inhibit the expression of a target gene. In addition, chemical modification is a means known in the art, such as phosphate backbone modification, ribose modification, base modification, etc., and may be selected by those skilled in the art according to the specific circumstances.

Another embodiment of the present invention provides a recombinant expression vector comprising the above siRNA targeting PSME3 gene or the above nucleic acid molecule targeting PSME3 gene. The siRNA or nucleic acid molecules described above may be expressed in combination with an expression vector, such as a lentiviral expression vector, as known in the art. In addition, in addition to the expression of the siRNA or the nucleic acid molecules and the expression vector forming the recombinant expression vector, other nucleic acid sequences capable of targeted inhibition of the PSME3 gene can be added into the expression vector to form the recombinant expression vector for coaction.

In yet another embodiment, the present invention provides a composition for inhibiting expression of a PSME3 gene, comprising the above-described siRNA targeting the PSME3 gene or the above-described nucleic acid molecule targeting the PSME3 gene or the above-described recombinant expression vector. The above-described siRNA or nucleic acid molecules targeting the PSME3 gene or the above-described recombinant expression vectors may be combined with other PSME3 gene-targeting active agents to form a composition that co-inhibits PSME3 gene expression. It should be noted that other active substances targeting PSME3 gene may be other nucleic acid molecules or chemical molecules.

In yet another embodiment, the present invention provides an agent for inhibiting expression of PSME3 gene, comprising the above siRNA targeting PSME3 gene or the above nucleic acid molecule targeting PSME3 gene or the above recombinant expression vector or the above composition for inhibiting expression of PSME3 gene; and a pharmaceutically acceptable carrier. Specifically, the above-described siRNA, nucleic acid molecules, recombinant expression vectors or compositions may be formulated with other pharmaceutically acceptable carriers, such as buffers, as known in the art, for use in inhibiting PSME3 gene expression.

In still another embodiment, the present invention provides an application of the above siRNA targeting PSME3 gene or the above nucleic acid molecule or recombinant expression vector targeting PSME3 gene or the composition for inhibiting PSME3 gene expression in preparing a PSME3 gene expression inhibitor. Specifically, based on the fact that the siRNA targeting the PSME3 gene can inhibit the expression of the PSME3 gene, the nucleic acid molecules modified by the siRNA and the recombinant expression vectors and the compositions formed by the nucleic acid molecules can also inhibit the expression of the PSME3 gene, and based on the fact that the siRNA, the nucleic acid molecules modified by the siRNA and the recombinant expression vectors and the compositions formed by the nucleic acid molecules can be applied to preparation of the PSME3 gene expression inhibitor.

In still another embodiment, the present invention provides an application of the above siRNA targeting PSME3 gene or the above nucleic acid molecule targeting PSME3 gene or the above recombinant expression vector or the above composition for inhibiting PSME3 gene expression in preparing a medicament for treating a disease caused by abnormal expression of PSME3 gene. As described above, the PSME3 gene may degrade a substrate protein by binding to the alpha subunit of the 20S core particle, feeding the substrate protein into the 20S beta subunit protease catalytic site. That is, the PSME3-20S protein degradation system may regulate physiological responses by degrading some proteins with important biological functions, thereby causing diseases. Therefore, the PSME3 gene can be prepared into a recombinant expression vector or composition for treating diseases caused by abnormal expression of the PSME3 gene by using the siRNA and the nucleic acid molecules modified by the siRNA.

It should be noted that the term "abnormal expression of PSME3 gene" as used above refers to an expression level exceeding a healthy level, and the healthy level of PSME3 expression varies slightly from individual to individual.

In some embodiments, the disease caused by abnormal expression of PSME3 gene is cancer. In addition to cancer, other diseases caused by abnormal expression of the PSME3 gene may be used, such as systemic lupus erythematosus (PSME 3 gene is originally found in serum of systemic lupus erythematosus patients), myocarditis, dilated cardiomyopathy, and the like.

It should be noted that, in addition to being a potential target of cancer, PSME3 has been found by the prior studies to promote replication of coxsackievirus to enhance its infection efficacy and promote myocarditis and dilated cardiomyopathy by promoting degradation of p 53; the CK1 delta-MDM 2-p53 pathway can also be inhibited by degrading casein kinase 1 delta (CK 1 delta), thereby promoting aging; it is also possible to reduce lipid metabolism in the liver by inhibiting autophagy by degrading silencing information regulator 1 (SriT 1); the activity of FoxO1 can be regulated and controlled through degrading Protein Kinase A (PKA), and the angiogenesis induced by VCAM1 is promoted.

In addition, PSME3 can regulate the activity of nuclear factor kB (NF kB) signal path by degrading nuclear factor kB inhibitor epsilon (IkB epsilon), thereby promoting the occurrence of inflammatory-related colon cancer and experimental enteritis, etc. In addition to protein degradation through proteasome activation, PSME3 can regulate vital activity independent of proteasome activation. For example, REGγ can act on promyelocytic leukemia Protein (PML) to accumulate in large amounts in nucleosomes, thereby regulating the number of PML nucleosomes; PSME3 has different intracellular distributions in different mitotic phases, and has a certain effect on maintaining the stability of centromeres and chromosomes of cells; in response to DNA damage, PSME3 can aggregate at the site of DNA damage as a targeting protein for ATM proteins, mediating proteasome accumulation, thereby degrading damage-related proteins.

That is, the siRNA of the present invention can treat diseases related thereto, such as the above-mentioned diseases caused by PSME3 gene, by targeted inhibition of PSME3 gene.

In some embodiments, the cancer includes, but is not limited to, lung cancer, colon cancer, thyroid cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, endometrial cancer, cervical cancer, brain cancer, and lymphatic cancer.

For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.

In the following examples, experimental data were analyzed by GraphpadPrism 5.0 software and expressed as Mean ± standard deviation (Mean ± SD). Data comparison between the two groups used independent sample t-test, with P <0.05 indicating statistical differences.

In the following examples, high sugar DMEM complete medium was used purchased from Gibco,11995065; both Hepa1-6 and HepG2 cells used were purchased from Shanghai national institute of sciences cell Bank.

1. Design and synthesis of PSME3 siRNAs

EXAMPLE 1 design and Synthesis of PSME3 siRNAs

And designing the siRNA sequence of the PSME3 gene according to the siRNA design principle. Two sets of double stranded siRNAs were designed based on PSME3 mRNA sequences of human (NM-176863.3, NM-005789.4) and mouse (NM-011192.4) as shown in Table 1. The two groups of siRNAs aiming at PSME3 designed by the invention can target PSME3 genes of human and mice at the same time; the Shanghai Ji Ma pharmaceutical technology Co.Ltd was commissioned for synthesis.

TABLE 1siRNA sequences

2. PSME3 siRNAs validation

In the following examples, cell resuscitations, passaging and culturing were performed according to the following steps:

(1) Taking out the frozen Hepa1-6 and HepG2 cells from the liquid nitrogen, melting in a water bath at 37 ℃, transferring the liquid into a centrifuge tube in an ultra-clean bench by using a liquid transfer gun, centrifuging at 1000rpm for 3 min;

(2) The supernatant was discarded, 1ml of high-sugar DMEM complete medium (containing 10% fetal calf serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin) was added for resuspension, transferred to a flask, 4ml of high-sugar DMEM complete medium was supplemented, and placed in a cell incubator at 37℃with 5% CO ₂ Culturing;

(3) The next day, fresh high-sugar DMEM complete culture medium is replaced, and an inverted optical microscope is used for observing the cell morphology and growth condition;

(4) To the bottom of the culture flask with full cells, the culture medium is sucked and removed, and 3ml of PBS is added for washing once;

(5) Removing PBS, adding 1ml 0.25% pancreatin, digesting for 5min at 37deg.C, adding 1ml (equal volume) high sugar DMEM, transferring to centrifuge tube, centrifuging at 1000rpm for 3 min;

(6) Sucking and removing the supernatant, adding 1ml of high-sugar DMEM complete medium to re-suspend the cells, taking 200-500 μl and adding into a culture flask, and supplementing 5ml of high-sugar DMEM complete medium;

(7) Placing the cells into a cell culture box for culturing for 2-3 days until the cell state is stable and good, and using the cells for subsequent experiments.

In the following examples, cell transfection was performed according to the following procedure:

(1) 1 day before cell transfection, cells were trypsinized and plated (cell culture plates of different sizes, e.g., 6-, 12-, 24-, 48-, and 96-well cell culture plates);

(2) The next day, cells were rinsed once with PBS before transfection, and then allowed to rest by addition of appropriate amounts of Opti-MEM (Life Technologies company) medium as shown in Table 2;

(3) Using Lipo3000 (Life Technologies company) cell transfection reagent, transfection solutions were prepared according to table 2;

TABLE 2 transfection nucleic acid doses and required transfection reagent doses for different culture wells

(4) After the liquid of the pipe A and the liquid of the pipe B are respectively prepared, lightly and uniformly mixing the two pipes, standing for 15min, uniformly adding the two pipes of mixed liquid into corresponding holes, and continuously culturing;

(5) 4h-6h after transfection, gently sucking the transfection solution, washing with PBS preheated at 37 ℃ for 2 times, changing into a complete culture medium preheated at 37 ℃ and continuing to culture; the subsequent experiments of example 2, example 3, example 4 and example 5 were performed within 24h-72h after transfection.

In the following examples, the following double strands were used as unrelated controls, positive control, sense strand:

UUCUCCGAACGUGUCACGUTT, antisense strand: ACGUGACACGUUCGGAGAATT.

Example 2CCK-8 detection of cell proliferation

(1) Day before transfection, 2X 10 ⁵ Hepa1-6 cells were seeded in 6-well plates and transfected with PSME3-496, irrelevant control Negative Control (NC) and transfection reagent only (Mock), respectively;

(2) 24 hours after transfection, the cells were digested with 0.25% pancreatin and were treated as per 2X 10 ³ Inoculating the individual cells into a 96-well plate, and detecting the proliferation of the individual cells in each group of 4 complex wells for 1 Day (Day, D), 2D, 3D and 4D;

(3) At each indicated time, 10 μ l Cell Counting Kit-8 reagent (CCK-8, dojindo, japan) was added to each well and the cells were incubated at 37℃for 1 hour, after which the absorbance at 450nm was measured using a Microplate reader (Bio-Tek, USA) and 630nm wavelength was used as reference wavelength.

As shown in FIG. 1, the number of liver cancer cells in the PSME3-496 group is moderately lower than that in the NC group and the Mock group, and the increase of the liver cancer cells in the PSME3-496 group is obviously slowed down after 3D days, which indicates that after the liver cancer cell line Hepa1-6 cells of the mice are transfected by the PSME3-496, the proliferation of the liver cancer cells can be obviously inhibited, and the number of the liver cancer cells can be reduced.

Example 3 flow cytometry detection of cell cycle

(1) Day before transfection, 1X 10 ⁵ The Hepa1-6 cells were seeded in 12-well plates and transfected with PSME3-496, independent control Negative Control (NC) and transfection reagent only (Mock), respectively, 3 replicates per group;

(2) After 48h, the cells were digested with 0.25% pancreatin, centrifuged at 2000rpm for 3min, and the supernatant was discarded;

(3) Washing the cells with pre-chilled PBS once, centrifuging at 2000rpm for 3min, and discarding the supernatant;

(4) Re-suspending the cells with 200 μl of pre-chilled PBS, then adding 600 μl of pre-chilled absolute ethanol, gently mixing upside down, and overnight at 4deg.C;

(5) Taking out the cell suspension, centrifuging at 3000rpm for 3min, and discarding the supernatant;

(6) The cells were resuspended in 1ml pre-chilled PBS, centrifuged at 2000rpm for 3min and the supernatant discarded;

(7) The cells were resuspended in 200. Mu.l of pre-chilled PBS per tube, then RNase was added at a final concentration of 50. Mu.g/ml, gently mixed, at 4℃for 30min;

(8) Adding propidium iodide (PI, shanghai Biyun Biotechnology Co., ltd.) solution with final concentration of 50 μg/ml into each tube, mixing gently, and keeping away from light at 4deg.C for 30min;

(9) At the end of incubation, the cell suspension was filtered through a 200 mesh screen and analyzed on-machine.

Flow cytometry results are shown in FIG. 2, histogram statistics are shown in FIG. 3, and the percentage of tumor cells in G0 and G1 (G0/G1) phases increases after PSME3-496 treatment of the Hepa1-6 cells relative to NC and Mock control groups, and the proportion of cells in S phase and G2/M phase decreases, indicating that cell cycle progression is blocked, resulting in cell cycle arrest.

The cell cycle refers to a process from the start of the first division to the next division of cells that divide continuously. The cell cycle is divided into G1, S, G and M phases in time sequence, and a small part of cells stay in the G0 phase and are not split any more; in fig. 2, G1 (early stage of DNA synthesis): a gap period between the completion of mitosis and the completion of DNA replication; s (DNA synthesis phase): DNA replication and chromosome doubling; g2 (late stage of DNA synthesis): a period of time before DNA replication is complete until mitosis begins; m (mitosis): cell division begins to end; stage G0: after the end of the M phase, the cells temporarily exit the cell cycle and become terminated, no longer dividing cells.

Example 4 relative expression level test of Gene

(1) Different transfected cells were obtained by transfecting Hepa1-6 cells and HepG2 cells using PSME3-496, PSME3-223, irrelevant control Negative Control (NC) and transfection reagent only (Mock), respectively, according to the method of example 3 above;

(2) The Trizol method extracts the total RNA of the transfected cells, and comprises the following specific steps:

(a) If the Trizol reagent is 300. Mu.l, 60. Mu.l chloroform (1/5 Trizol reagent amount) is added, vortex shaking is carried out for 2-3 min, and the mixture is kept stand on ice for 5min;

(b) Centrifuge at 12000 Xg for 15min at 4℃and carefully aspirate the upper colorless clear liquid, transfer to new RNase Free EP tube;

(c) Adding equal volume isopropanol, mixing, standing at-20deg.C for 30min, and precipitating.

(d) Centrifuging at 12000 Xg for 30min at 4deg.C, and removing supernatant;

(e) Adding 500 μl of precooled 75% ethanol for cleaning, and centrifuging at 7500×g for 5min;

(f) Absorbing and discarding the supernatant, centrifuging briefly, absorbing and discarding water drops remained on the tube wall, drying at room temperature for 4min, and adding a proper amount of RNase Free water according to the size of RNA precipitation for dissolution; using a NanoDrop2000 instrument to detect the concentration of RNA, and preserving the RNA in a refrigerator at the temperature of minus 80 ℃ for subsequent experiments;

(3) Reverse transcription of RNA:

mRNA was reverse transcribed into cDNA using a PrimeScript RT Master Mix (Takara) kit, as follows:

(a) Preparing a reverse transcription reaction solution (see table 3) on ice, and reversely transcribing mRNA into cDNA;

TABLE 3 reverse transcription reaction solution

(b) Gently mixing, and performing reverse transcription on a PCR apparatus according to the following procedure (Table 4);

TABLE 4 reverse transcription procedure

37℃	15min
		85℃	5sec
4℃	∞

(c) 70 μl dH was added to the reverse transcription product ₂ O dilution for subsequent experiments;

(4) Detecting the expression of the genes mice Psme3 (mma-Psme 3) and human PSME3 (hsa-PSME 3) by using a real-time quantitative PCR (qRT-PCR) technology; wherein, the murine Gapdh (mmu-Gapdh) and the human GAPDH (hsa-GAPDH) are used as the internal references, and the primers are shown in Table 5;

TABLE 5 primer list

The method comprises the following specific steps:

(a) The following qRT-PCR reaction system was formulated on ice as shown in Table 6;

TABLE 6qRT-PCR reaction System

(b) qRT-PCR reactions were performed according to the following procedure (Table 7);

TABLE 7qRT-PCR reaction procedure

(c) Finally according to 2 ^-ΔΔCt The method calculates the relative expression level of the gene.

After two groups of siRNAs PSME3-223 and PSME3-496 are transfected into liver cancer cell lines Hepa1-6 cells of mice, the expression of the gene of the Psme3 (mma-Psme 3) of the mice is shown in figure 4, and compared with the NC group and the Mock group, the expression level of mRNA of the PSME3 can be obviously reduced by two groups of PSME3-223 and PSME3-496, wherein the PSME3-223 group is superior to the PSME3-496 group.

After two groups of siRNAs PSME3-223 and PSME3-496 are transfected into human hepatoma cell line HepG2 cells, the expression of human PSME3 (hsa-PSME 3) gene is shown in FIG. 5, and the two groups of PSME3-223 and PSME3-496 can obviously reduce the expression level of PSME3 mRNA compared with the NC group and the Mock group, wherein the PSME3-223 group is superior to the PSME3-496 group.

EXAMPLE 5Western blot detection of target protein expression

(1) Cell transfection: different transfected cells were obtained by transfecting Hepa1-6 cells using PSME3-496, PSME3-223, independent control Negative Control (NC) and transfection reagent only (Mock), respectively, as described in example 3 above.

(2) Sample preparation: the transfected cells of each group of example 3 were collected, a certain amount of cell lysate (protease inhibitor and phosphatase inhibitor were added just before use) was added, and the lysed cells were repeatedly blown on ice with a pipette; centrifuging at 12000 Xg for 5min (4 ℃), and sucking supernatant to obtain total protein; protein concentration was measured by BCA (bi yun biotechnology limited) protein quantification and protein samples from the same experiment were diluted to the same concentration; after adding 5 Xloading buffer, denaturing in boiling water bath for 5min, and preserving the prepared sample at-80deg.C.

(2) Westernblot detection: preparing 12% SDS-PAGE gel, taking 20 mug total protein for electrophoretic separation, and transferring the protein to a nitrocellulose membrane by a semi-dry method; after blocking with TBST containing 5% bsa for 2h, the corresponding primary antibodies were added: PSME3 (Wuhan Sanying Co., 1:500 dilution) and GAPDH (Shanghai Biyun Biotechnology Co., ltd., 1:1000 dilution), were incubated overnight at 4 ℃; the next day the membranes were washed 3 times with TBST for 10min each, then HRP-labeled secondary antibody (Promega Co., U.S.A.) was added for 1h at room temperature, the membranes were washed 3 times with TBST, and finally the ECL chemiluminescence developed on a chemiluminescent instrument.

As shown in FIG. 6, after transfection of liver cancer cell lines Hepa1-6 cells of mice with two groups of siRNAs, PSME3-223 and PSME3-496, the NC group and Mock group were significantly brighter than those of the PSME3-223 and PSME3-496 groups, indicating that the two groups of siRNAs, PSME3-223 and PSME3-496, significantly inhibited protein expression of PSME 3.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

1. An siRNA targeting PSME3 gene, selected from the group consisting of siRNA1 and siRNA2, wherein the sense strand and the antisense strand of siRNA1 are set forth in SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the sense strand and the antisense strand of siRNA2 are shown in SEQ ID NO:3 and SEQ ID NO: 4.

2. A PSME3 gene-targeting nucleic acid molecule, which is chemically modified by the PSME3 gene-targeting siRNA of claim 1.

3. A recombinant expression vector comprising the siRNA targeting the PSME3 gene of claim 1 or the nucleic acid molecule targeting the PSME3 gene of claim 2.

4. A composition for inhibiting expression of a PSME3 gene, comprising the siRNA targeting a PSME3 gene of claim 1 or the nucleic acid molecule targeting a PSME3 gene of claim 2 or the recombinant expression vector of claim 3.

5. An agent for inhibiting expression of a PSME3 gene, comprising the siRNA targeting a PSME3 gene of claim 1 or the nucleic acid molecule targeting a PSME3 gene of claim 2 or the recombinant expression vector of claim 3 or the composition for inhibiting expression of a PSME3 gene of claim 4; and a pharmaceutically acceptable carrier.

6. Use of the siRNA targeting a PSME3 gene of claim 1 or the nucleic acid molecule targeting a PSME3 gene of claim 2 or the recombinant expression vector of claim 3 or the composition for inhibiting PSME3 gene expression of claim 4 in the preparation of a PSME3 gene expression inhibitor.

7. Use of the siRNA targeting the PSME3 gene of claim 1 or the nucleic acid molecule targeting the PSME3 gene of claim 2 or the recombinant expression vector of claim 3 or the composition for inhibiting expression of the PSME3 gene of claim 4 in the manufacture of a medicament for treating a disease caused by abnormal expression of the PSME3 gene.

8. The use according to claim 7, wherein the disease caused by abnormal expression of PSME3 gene is cancer.

9. The use according to claim 8, wherein the cancer comprises lung cancer, colon cancer, thyroid cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, endometrial cancer, cervical cancer, brain cancer and lymphatic cancer.