SiRNA for schistosoma japonicum hsc20 gene expression and application thereof

Abstract

This invention relates to the fields of molecular biology and biomedicine, and discloses siRNA for the expression of the *Schistosoma japonicum* Hsc20 gene and its applications. The siRNA contains a nucleotide sequence that hybridizes to a target sequence of the *Schistosoma japonicum* Hsc20 gene, wherein the target sequence is selected from the sequences shown in SEQ ID NO. 1-SEQ ID NO. 4. The siRNA specifically inhibiting the expression of the *Schistosoma japonicum* Hsc20 gene provided by this invention can be used to interfere with the transcription and expression of the *Schistosoma japonicum* Hsc20 gene and the growth and development of *Schistosoma japonicum*. In vitro experiments have confirmed that the siRNA provided by this invention can efficiently silence the *SjHsc20* gene; and in vivo RNA interference experiments in mice show that this siRNA can induce 50.62% (…) P The insect reduction rate was <0.05% and 44.29% ( P A liver oocyte reduction rate of <0.01% is suitable for preparing drugs to treat schistosomiasis.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and biomedicine, and in particular to a siRNA expressing the Hsc20 gene of Schistosoma japonicum and its applications. Background Technology

[0002] Schistosomiasis japonicus is a serious zoonotic parasitic disease. Besides humans, Schistosoma japonicum can infect more than 40 species of mammals, including cattle, sheep, and pigs, causing emaciation, anemia, and reduced working capacity in infected animals. In economically important animals, this manifests as decreased milk production in dairy cows, infertility or abortion in cows, and in severe cases, death. It is one of the major diseases hindering the development of cattle and sheep farming. Epidemiological surveys show that buffalo, cattle, and sheep are the main sources of infection for schistosomiasis japonicus. The prevalence of animal schistosomiasis japonicus seriously threatens public health. Effectively blocking the transmission of schistosomiasis japonicus in animals is crucial for its eradication.

[0003] Heat shock cogmate protein 20 (Hsc20) is a J-type chaperone protein, also known as heat shock cognate B (HscB), which binds to and regulates the ATPase and peptide-binding activity of Hsp70. Hsp70 has biological functions in assisting in the folding of nascent proteins and correcting misfolded proteins, and it possesses strong immunogenicity; heterologous HSP70 can elicit a strong Th1 immune response in the host. In *E. coli*, Hsc20, along with Hsc66 (an Hsp70-like molecular chaperone), participates in the biosynthesis of iron-sulfur proteins. Iron-sulfur proteins are essential for many key cellular biological processes, playing important roles in various cellular processes such as DNA replication and repair, oxidative respiration, and photosynthesis. Furthermore, studies have found that Hsc20 deficiency leads to impaired Fe-S cluster biosynthesis, defects in erythrocyte hemoglobinization, and impaired cell development, broadly interfering with hematopoiesis in vivo.

[0004] RNA interference (RNAi) is an evolutionarily conserved molecular regulatory mechanism characterized by the introduction of exogenous double-stranded RNA (dsRNA) to achieve sequence-specific silencing of target genes in vivo or in vitro. This process primarily occurs at the post-transcriptional level, involving the synergistic effects of multiple conserved protein factors. Epigenetic regulation of gene expression can be achieved through mRNA degradation or translational repression pathways. The core effector molecule in this process is siRNA, which is produced by the cleavage of initial dsRNA by Dicer enzymes (RNase III family endonucleases), forming a small double-stranded molecule (21-23 nucleotides in size). After unwinding, the antisense strand of the siRNA integrates into the RNA-induced silencing complex (RISC), recognizing the open reading frame or untranslated region of the target mRNA through strict base complementarity pairing, and activating the endonuclease activity of the Argonaute (AGO) protein, ultimately leading to the specific degradation of the target gene mRNA. This mechanism requires siRNA to have high sequence specificity, and its silencing efficiency is also closely related to the physicochemical properties of siRNA, such as GC content, thermodynamic stability and terminal modification. Current technology lacks siRNA that can significantly inhibit the transcription of the Hsc20 gene of Schistosoma japonicum.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide siRNA expressing the Hsc20 gene of Schistosoma japonicum and its application. This siRNA can significantly inhibit the transcription of the Hsc20 gene of Schistosoma japonicum and can be used to prepare drugs for treating schistosomiasis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A siRNA that specifically inhibits the expression of the Schistosoma japonicum Hsc20 gene, wherein it contains a nucleotide sequence that hybridizes with a target sequence of the Schistosoma japonicum Hsc20 gene, said target sequence being selected from the sequences shown in SEQ ID NO.1-SEQ ID NO.4.

[0009] The siRNA that specifically inhibits the expression of the Schistosoma japonicum Hsc20 gene, wherein the siRNA comprises a first strand and a second strand, the first strand and the second strand being complementary to form an RNA dimer, and the RNA sequence of the first strand being consistent with the target sequence of the Schistosoma japonicum Hsc20 gene, wherein the first strand and the second strand of the siRNA are selected from one or any combination of two or more of the following:

[0010] The nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6;

[0011] The nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8;

[0012] The nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10;

[0013] The nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.12.

[0014] The siRNA that specifically inhibits the expression of the Hsc20 gene of Schistosoma japonicum, wherein the first and second strands of the siRNA are the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8.

[0015] A drug for treating schistosomiasis, wherein the active ingredient of the drug is: siRNA that specifically inhibits the expression of the Schistosoma japonicum Hsc20 gene by RNA interference, the siRNA containing a nucleotide sequence that hybridizes with a target sequence of the Schistosoma japonicum Hsc20 gene, the target sequence being selected from the sequences shown in SEQ ID NO.1-SEQ ID NO.4.

[0016] The drug for treating schistosomiasis, wherein the first and second strands of the siRNA are selected from one or any combination of two or more of the following:

[0017] The nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6;

[0018] The nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8;

[0019] The nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10;

[0020] The nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.12.

[0021] The use of an siRNA that specifically inhibits the expression of the Hsc20 gene of Schistosoma japonicum as described in this invention in the preparation of drugs for the treatment or prevention of schistosomiasis.

[0022] Beneficial effects: The siRNA specifically inhibiting the expression of the Hsc20 gene in Schistosoma japonicum provided by this invention can be used to interfere with the transcription and expression of the Hsc20 gene in Schistosoma japonicum and the growth and development of Schistosoma japonicum; in vitro experiments have confirmed that the siRNA provided by this invention can efficiently silence the SjHsc20 gene; and in vivo RNA interference experiments in mice show that this siRNA can induce mice to obtain 50.62% ( P The insect reduction rate was <0.05% and 44.29% ( P A liver oocyte reduction rate of <0.01% is suitable for preparing drugs to treat schistosomiasis. Attached Figure Description

[0023] Figure 1 This is a comparison of the effects of real-time quantitative PCR analysis on the in vitro RNA interference of insects at different time points, as presented in this invention. Detailed Implementation

[0024] This invention provides siRNA expressing the Hsc20 gene of Schistosoma japonicum and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples provide further detailed description of the invention. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the scope of protection of the invention.

[0025] In the following examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Mr. Green and J. Sambrook, 3rd ed., translated by He Fuchu, Chen Wei and Yang Xiaoming, Beijing: Science Press, 2017).

[0026] Example 1: In vitro RNA interference

[0027] 1.1 Design of siRNA molecules

[0028] Four target sequences (SEQ ID NO.1-SEQ ID NO.4, see Table 1 below) and four pairs of siRNA molecules (SEQ ID NO.5-SEQ ID NO.12, see Table 1 below) that specifically hybridize with the target sequences were designed based on the Hsc20 gene sequence of Schistosoma japonicum (GeneBank Sjp_0008430). Irrelevant siRNA molecule was used as an irrelevant control. The siRNA was designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0029] Table 1. siRNA sequence and corresponding target gene sequence of Schistosoma japonicum Hsc20 gene.

[0030]

[0031] 1.2 Parasite collection and culture and in vitro RNA interference

[0032] Fifteen 6-8 week old male BALB / c mice were infected with 40 cercariae per mouse via abdominal patch method using Schistosoma japonicum cercariae. Five mice were sacrificed at each time point (days 21 and 28) after infection, and the parasites were collected via portal vein perfusion. The mice were washed three times with RPMI 1640 medium containing penicillin and streptomycin (1000 units each); then transferred to disposable culture dishes and washed once with RPMI 1640 complete medium (containing 10% fetal bovine serum and 1000 units of penicillin and streptomycin), and cultured in a CO2 incubator (37°C, 5% CO2).

[0033] The siRNA was introduced into cultured parasites using an immersion method. First, the dried siRNA powder was removed from a -20°C freezer and centrifuged in a low-temperature high-speed centrifuge to allow the powder to settle at the bottom of the centrifuge tube. It was then carefully removed and diluted on an ice box. Following the manufacturer's instructions, 125 μL of DEPC water was added to 1 OD of siRNA to prepare a 40 μM solution. Next, preheated culture medium was placed in 12-well plates. 1 mL of RPMI 1640 complete medium and 1 OD of siRNA were added to each well. Three pairs of parasites were added to each well, and the plate was gently shaken to mix the siRNA thoroughly with the medium. A blank control and an irrelevant RNAi control were also included.

[0034] 1.3 SjHsc20-specific siRNA screening and interference effects

[0035] RNA was extracted from the parasites in each interference group and the interference control group, reversed to cDNA, and then detected by qRT-PCR. PSMD4 of *Schistosoma japonicum* was used as an internal reference gene, and the expression level of the Sj-Hsc20 gene in the irrelevant RNA control group was used as a reference. Two... -ΔΔCt The transcriptional level of the SjHsc20 gene in worms from different interference groups was analyzed.

[0036] Sj-PSMD4

[0037] Upstream primer: 5'-CCTCACCAACAATTTCCACATCT-3';

[0038] Downstream primer: 5'-GATCACTTATAGCCTTGCGAACAT-3'.

[0039] SjHsc20

[0040] Upstream primer: 5'-AAAAGCTCGCGTTGGATGCTT-3';

[0041] Downstream primer: 5'-GAAGACTTGAAGTCCGAAATCGCG-3'.

[0042] The results are as follows Figure 1 As shown, it contains two sub-figures, corresponding to the 21-day-old male and female synocassis of Schistosoma japonicum (Schistosoma japonicum). Figure 1 (as shown in A) and 28-day-old Schistosoma japonicum synocassis ( Figure 1 (As shown in B); the horizontal axis represents different treatment groups, including four siRNAs targeting the Sj-Hsc20 gene (siRNA-33, siRNA-205, siRNA-474, siRNA-718), an irrelevant control siRNA (siRNA NC), and a blank control (Blank); the vertical axis represents the relative expression level, reflecting the transcriptional level of the Sj-Hsc20 gene, with lower values ​​indicating a more significant gene silencing effect.

[0043] from Figure 1 As shown in Figure A, in the blank control group, the relative expression level of the Sj-Hsc20 gene was approximately 1.0, serving as a baseline reference reflecting the basal transcriptional level of this gene in 21-day-old worms under normal culture conditions. In the irrelevant control group (siRNA NC), the relative gene expression level was similar to that of the blank control group, ranging from approximately 0.95 to 1.0, with no statistically significant difference. P >0.05 indicates that the irrelevant siRNA did not specifically interfere with the transcription of the Sj-Hsc20 gene, ruling out non-specific silencing effects. In the siRNA-718 group, the relative gene expression level was approximately 0.55-0.6, which was significantly lower than that of the control group, reaching a statistically significant level. P <0.05 indicates that the siRNA can effectively inhibit the transcription of the Sj-Hsc20 gene in 21-day-old worms, with a silencing efficiency of approximately 40%-45%. The siRNA-205 group showed the most significant decrease in relative gene expression (approximately 0.10-0.15) among all siRNA treatment groups, with a statistically significant difference. P <0.0001), with a silencing efficiency as high as 90%-95%, significantly superior to other siRNA molecules, exhibiting the strongest early silencing activity. However, in the siRNA-33 and siRNA-474 groups, the relative gene expression levels showed no change compared to the blank control and siRNA NC group, indicating that these two siRNA groups had extremely weak silencing effects on the Sj-Hsc20 gene in 21-day-old worms.

[0044] from Figure 1 As can be seen from B, in the siRNA-718 group, the relative gene expression level was approximately 0.03, which was significantly lower than that in the control group, and the difference was statistically highly significant. P<0.0001), the silencing efficiency increased to 97%, and the silencing effect was significantly enhanced compared to the 21-day-old worms. In the siRNA-33 group: the relative gene expression level was approximately 0.02, showing the largest decrease compared to the 21-day-old worms, and the difference was statistically significant. P <0.001 indicates that the siRNA's silencing effect on mature worms (28 days) is significantly better than that on juvenile worms (21 days). In the siRNA-205 group, the relative gene expression level was only 0.01-0.02, which was more than 98% lower than that of the control group, and the statistical difference was extremely significant. P With a silencing efficiency of up to 98.37% (<0.0001), it exhibits extremely strong and sustained silencing ability, making it the most stable and significant silencing molecule among all tested siRNAs. In the siRNA-474 group, the relative gene expression level was approximately 0.6-0.7, showing a greater decrease compared to 21-day-old worms, and the silencing efficiency increased to 30%-40%, indicating that this siRNA has a better silencing effect on mature worms (28 days) than on young worms (21 days).

[0045] comprehensive Figure 1 From A and B, we can draw the following conclusions:

[0046] Target specificity: All siRNAs targeting the Sj-Hsc20 gene provided in this invention can inhibit gene transcription to varying degrees, while the unrelated control siRNAs have no significant effect, proving that the silencing effect is sequence specific, which is attributed to the complementary base pairing between the siRNA and the target gene, consistent with the molecular mechanism of RNAi.

[0047] Time-dependent effects: The silencing effect of most siRNAs increased with prolonged worm culture time, especially siRNA-205, siRNA-33, and siRNA-718, whose silencing efficiency was significantly higher in 28-day-old worms than in 21-day-old worms. This may be because as worms mature, increased cellular metabolic activity leads to improved siRNA uptake efficiency, or increased expression of RNAi pathway-related proteins (such as Dicer and AGO), thereby enhancing the silencing effect.

[0048] Molecular differences: The silencing efficiency of different siRNAs varied significantly. siRNA-205 exhibited the best silencing activity, effectively inhibiting Sj-Hsc20 gene transcription in 21-day and 28-day-old worms, with a stable and sustained silencing effect. siRNA-718 was the next best, showing strong silencing ability in mature worms. siRNA-33 and siRNA-474 had relatively weaker silencing efficiencies. This difference may be related to the sequence characteristics of the siRNA, such as GC content, thermodynamic stability, and the location of the target sequence in the gene. These factors affect the binding efficiency of siRNA to target mRNA and the assembly activity of the RISC complex.

[0049] Application potential: The high silencing efficiency of siRNA-205 provides a key basis for subsequent in vivo experiments and drug development. It can effectively silence target genes at different developmental stages of the parasite, laying the molecular basis for interfering with the growth and development of schistosomiasis and reducing infectivity.

[0050] Example 2: In vivo RNA interference

[0051] 1. Methods and Steps

[0052] Fifteen 6-week-old BALB / c mice were randomly divided into three groups: siRNA-205 interference group, irrelevant siRNA control group, and PBS blank control group, with five mice in each group. Each mouse was challenged with 40±2 Schistosoma japonicum cercariae via abdominal skin patch method. Starting 12 days post-infection, the three groups of mice were injected intravenously with siRNA-205 (1 OD / 125 μL / mouse), irrelevant siRNA (1 OD / 125 μL / mouse), or PBS (125 μL / mouse), respectively. Injections were given every 4 days for a total of 7 times. At 42 days post-infection, the mice were euthanized, and the parasites were collected by portal vein perfusion to calculate the parasite load. At the same time, the livers of the mice were harvested for hepatic egg count.

[0053] Insect reduction rate = (1 - average insect load in the interference group / average insect load in the control group) × 100%;

[0054] Liver ovulation reduction rate = (1 - EPG in the interference group / EPG in the control group) × 100%;

[0055] 2. Results: The results of the in vivo interference experiment showed that the injection of siRNA numbered 205 into the tail vein of mice induced partial reduction of worms and eggs, as shown in Table 2.

[0056] Table 2. Changes in parasite load and liver egg count in mice induced by SjHsc20 gene silencing.

[0057]

[0058] As shown in Table 2, only siRNA-205 targeting the SjHsc20 gene can produce a significant and stable insect-suppressing and egg-suppressing effect. Irrelevant siRNAs have no such effect, proving that this effect is a specific result of SjHsc20 gene silencing, rather than a non-specific effect of exogenous RNA.

[0059] The data in Table 2 also show that silencing the SjHsc20 gene can simultaneously inhibit the survival and reproduction of schistosomes. These two processes are key links in the transmission and pathogenesis of schistosomiasis (adult survival is the basis for persistent infection, and eggs are the core of pathogenicity). Therefore, the SjHsc20 gene is an effective target for the treatment of schistosomiasis, providing a core target basis for subsequent drug development.

[0060] The in vitro experiments in Example 1 have confirmed that siRNA-205 can efficiently silence the SjHsc20 gene, and the data in Table 2 further verify that it can still exert significant activity in live animal models (insect reduction rate >50%, egg reduction rate >40%, both of which are statistically significant), solving the key problem of whether in vitro activity can be converted into in vivo effect, and providing direct in vivo experimental evidence for siRNA-205 as an active ingredient in anti-schistosomiasis drugs.

[0061] In summary, this invention is the first to identify the Hsc20 gene as a target gene for the prevention and control of schistosomiasis japonicus. Existing research on schistosomiasis primarily focuses on traditional targets such as proteases and surface antigens. However, this invention, through prediction and verification of the Hsc20 gene function, discovers its involvement in the synthesis of iron-sulfur proteins, which are essential proteins for key life processes in schistosomiasis, including DNA replication and oxidative respiration. Silencing the Hsc20 gene can lead to metabolic disorders and inhibited growth and reproduction in schistosomiasis. This selection of a target gene breaks through traditional research approaches.

[0062] Furthermore, through in vitro and in vivo experiments, this invention provides the first-ever confirmation that the Hsc20 gene is a key gene for the growth, development, and reproduction of Schistosoma japonicum. Example 1 shows that silencing this gene significantly inhibits gene transcription in the parasite; Example 2 demonstrates that it can induce a parasite reduction rate of up to 50.62% and an egg reduction rate of 44.29% in mice, proving the significant application value of this target gene. There are no prior art reports on the function of the Hsc20 gene in Schistosoma japonicum; this invention fills a gap in this field.

[0063] This application screened the optimal siRNA molecules (siRNA-205, SEQ ID NO.7 and SEQ ID NO.8) through in vitro experiments. These molecules effectively silenced target genes in 21-day and 28-day schistosomes, achieving a silencing efficiency of up to 98.37%, and also demonstrated significant effects in in vivo experiments. This screening method based on schistosomes at different developmental stages takes into account the life cycle characteristics of Schistosoma, ensuring the sustained effectiveness of siRNA in vivo, which is superior to existing screening strategies that target only a single developmental stage.

[0064] This invention applies RNAi technology to the silencing of the Hsc20 gene in Schistosoma japonicum, constructing a complete technical system encompassing target gene selection, siRNA design, in vitro screening, and in vivo validation. Furthermore, this invention utilizes siRNA as the active ingredient in drugs for treating schistosomiasis, providing a novel anti-schistosomiasis drug. siRNA drugs offer advantages such as high sequence specificity, minimal side effects, and low likelihood of inducing drug resistance, enriching the technical means of schistosomiasis treatment and providing a new approach for novel drug development.

[0065] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An siRNA that specifically inhibits the expression of Hsc20 gene of Schistosoma japonicum, characterized in that, The siRNA comprises a first strand and a second strand, which are complementary to form an RNA duplex, and the RNA sequence of the first strand is consistent with the target sequence of the Hsc20 gene of Schistosoma japonicum; the first strand and the second strand of the siRNA are the nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO.

8.

2. A medicament for treating schistosomiasis, characterized by, The active ingredient of the drug is the siRNA for specifically inhibiting the expression of the Hsc20 gene of Schistosoma japonicum according to claim 1.

3. Use of the siRNA for specifically inhibiting the expression of the Hsc20 gene of Schistosoma japonicum according to claim 1 in the preparation of a drug for treating or preventing schistosomiasis.

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