WO2020124525A1

WO2020124525A1 - Use of microrna-7 in preparation of anti-rotavirus medicament

Info

Publication number: WO2020124525A1
Application number: PCT/CN2018/122565
Authority: WO
Inventors: 周艳; 李鸿钧; 陈林林; 解裕萍; 胡晓青; 吴晋元; 尹娜; 孙茂盛
Original assignee: 中国医学科学院医学生物学研究所
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2020-06-25

Abstract

The present invention relates to the field of biomedicine, and in particular, to use of MicroRNA-7 in preparation of an anti-rotavirus medicament. It has been found for the first time that rotavirus NSP5 is a target of miR-7, and the combination of miR-7 and NSP5 can inhibit the replication of rotavirus. The introduction of over-expressed miR-7 mimics in the body of a suckling mouse can inhibit the replication of rotavirus and diarrhea caused by rotavirus infection, thereby being applied to the preparation of an anti-rotavirus medicament.

Description

Application of MicroRNA-7 in the preparation of anti-rotavirus drugs

Technical field

The application of the present invention relates to the field of biomedicine, especially the application of MicroRNA-7 in the preparation of anti-rotavirus drugs.

Background technique

Rotavirus (RV) is one of the main pathogens that cause diarrhea in infants and young children under five years old. The number of gastroenteritis cases in children caused by RV infection in the world still reaches about 125 million each year, resulting in the death of about 400,000 children. There is no effective targeted treatment drug. With the deep exploration in the field of RNA, the role of non-coding RNA in the life process is gradually revealed. Among them, miRNAs and siRNAs can play an important role in many biological processes because they can regulate the post-transcriptional expression of genes. The miRNA can bind to the complementary sequence of the target mRNA, thereby triggering the degradation or translation inhibition of the target mRNA molecule. It has found a new way to inhibit the expression of disease-causing genes, and has great potential for the development of small RNA drugs.

MicroRNAs (miRNAs) are a class of non-coding small RNAs of about 22 bases in length. They control the expression of more than one-third of genes in eukaryotes at the post-transcription level by inhibiting translation and causing mRNA degradation. Almost all biological processes such as cell differentiation and development, proliferation, apoptosis and metabolism. In the process of virus infection, cells can suppress the infection and proliferation of foreign invading viruses by modulating the host cell's microRNA to target viral genes or their own signaling pathway genes. MicroRNA-7 (miR-7) is a small RNA that was discovered in 2001 and has evolved conservatively. In human cells, the primary transcription precursor of miR-7 (pri-miR-7) is encoded by chromosomes 9, 15 and 19, transported and finally processed into the same mature 23-base miR -7 sequence. The regulatory role of miR-7 in the development of human brain, pancreas, thymus and other tissues and organs, the growth, migration and escape of various tumors, and the pathogenesis of diabetes has been systematically and deeply studied. The mechanism of action in virus infection and replication is less studied. In 2014, Zhang et al. found that siRNAs synthesized in vitro specifically targeting poliovirus (PV) 5'-UTR can not only effectively inhibit the replication of PV and virus titers, but also significantly promote miR-7 in host cells. The expression is up-regulated. Recent studies have also found that miR-7 can affect the replication of white spot syndrome virus (WSSV) in crabs by targeting and suppressing the expression of immune-related factor Myd88. However, it is unclear whether miR-7 regulates rotavirus replication and its mechanism of action, and whether it can be used as a small RNA molecule drug to treat rotavirus infection has not been reported.

The interaction between the virus and the host cell is a regulation strategy of the virus and the host cell formed during the long-term evolution. During the process of virus infection, the cell can invade the virus by regulating the microRNA of the host cell to target the virus gene or its own signaling pathway gene. The infection and proliferation are suppressed. There are relatively few reports on the antiviral effect of host microRNAs after rotavirus infection, and the role is not yet clear. Studies on the use of small RNA as an anti-rotavirus infection drug have not been reported.

Summary of the invention

The purpose of the present invention is to provide the application of MicroRNA-7 in the preparation of anti-rotavirus drugs.

The present invention finds that miR-7 has a significant regulation target gene NSP5 (SED NO.3), miR-7 targets 17bp-36bp of the NSP5 gene of the rotavirus ZTR-68 strain, and miR-7-NSP5 functions The locus can be used as a new and effective anti-rotavirus therapeutic target.

Application of miR-7 small RNA mimics in the preparation of anti-rotavirus drugs.

Wherein, the miR-7 small RNA mimic is mml-miR-7 sense single-stranded oligonucleotide.

The sense single-stranded oligonucleotide of miR-7 can be artificially synthesized or directly commercially available. The sense single-stranded oligonucleotide of miR-7 can also be modified by PNA, LNA or 2-Ome.

In the present invention, the registration number of miR-7 is (MI0007581, http://www.miRbase.org). The precursor of miR-7 is miR-7-1, and the accession number is (MIMAT0006159, http://www.miRbase.org).

In the present invention, the isolated wild-type rotavirus ZTR-68 strain was infected with sensitive cells MA104 cells for deep sequencing analysis, and the microRNAs expression profile during rotavirus infection was analyzed, and it was found that miR-7 was infected. After the virus was increased by nearly 400 times. Through qRT-PCR verification, we found that miR-7 was indeed upregulated nearly 20-fold after rotavirus ZTR-68 infection. After different types of rotavirus strains infected MA104 cells, miR-7 expression was up-regulated to varying degrees. ZTR-68 strain rotavirus and its expression level gradually increased with the time of viral infection and the amount of MOI infected.

Through transfection of miR-7 mimic, the expression of miR-7 was up-regulated in MA104 cells. After 16 hours of infection with rotavirus, compared with the control group, the expression of infected rotavirus protein decreased, suggesting that miR-7 Virus proliferation plays a negative regulatory role and may have antiviral effects. We predicted the target genes of miR-7, and the results showed that miR-7 could bind to the NSP5 gene of rotavirus. The function of NSP5 protein is to interact with NSP2 to promote the formation of virus pool and virus replication during rotavirus proliferation. Through a series of experiments, we discovered for the first time that NSP5 is indeed the target gene of miR-7. MiR-7 can combine with the NSP5 gene to affect the expression of NSP5 protein, affect the formation of the virus pool during virus replication, and further inhibit virus replication. . Through the establishment and evaluation of the suckling mouse model, miR-7 can indeed significantly affect the replication of the virus and suppress the diarrhea caused by the viral infection after being introduced into the intestinal tract of suckling rats.

Rotavirus belongs to the genus Reovirus, and is an encapsulated icosahedral virus. The genome is discontinuous and consists of 11 segments of double-stranded RNA, encoding 6 structural proteins (VP1-4, 6, 7) and 6 non-structural proteins (NSP1-6). Rotavirus non-structural protein NSP5 is encoded by the 11th gene in the viral RNA genome and is a highly phosphorylated dimeric protein rich in threonine (25% of total amino acids) and serine residues. The molecular weight after translation modification conditions is 26-35Kd. During virus replication, by interacting with NSP2, under Ca2+ regulated by NSP4, NSP5 rich in serine-threonine shifts between hypophosphorylated hyperphosphorylated isomers during the replication cycle and participates in the virus pool The success of. The virus pool is a place for rotaviruses to prevent the innate immune monitoring of genome replication, which is very important for virus replication. Studies have shown that blocking the production of NSP5 by siRNA can inhibit viral replication, the production of structural and non-structural proteins, the synthesis of viral genome dsRNA and progeny viruses. Therefore, NSP5 is very important in rotavirus replication. The microRNA that inhibits rotavirus replication with NSP5 as the target has not been discovered and reported. The present invention first discovered that rotavirus NSP5 is the target of miR-7, and the combination of miR-7 and NSP5 can inhibit rotavirus replication. Over-expression mimics of miR-7 introduced into suckling mice can inhibit rotavirus replication and diarrhea caused by rotavirus infection.

RNA interference technology has been widely used in various antiviral studies, such as HIV-1, HCV and influenza viruses. miR-7 has the potential to become an anti-rotavirus replication small RNA drug, and the miR-7-NSP5 action site may be a new and effective anti-rotavirus therapeutic target.

BRIEF DESCRIPTION

Figure 1: Time-sensitive expression detection of miR-7: After ZTR-68 infected MA104 cells at different time periods (0, 12h, 24h, 36h and 48h), RT-PCR was used to detect the expression level of miR-7 (*p<0.01)

Figure 2: RT-PCR detection of miR-7 expression level after infection of MA104 cells with different MOI (0.1, 0.5, 1 and 2) ZTR-68 rotavirus

Figure 3: RT-PCR detection of miR-7 expression level after infection of MA104 cells infected with different G-type rotavirus with 0.5MOI

Figure 4: RT-PCR detection of miR-7 expression level after 36 hours of ZTR-68 infection in HT-29 cells and Caco-2 cells

Figure 5: qRT-PCR detection of rotavirus NSP3 expression after miR-7 up-regulation and down-regulation (*P<0.01)

Figure 6 Immunofluorescence to detect the effect of miR-7 up-regulation on rotavirus replication (A) Rotavirus-specific immunofluorescence to detect the effect of miR-7 up-regulation on viral protein expression (red is RV, blue is DAPI ); (B) Statistics of positive cells in rotavirus immunofluorescence test results after up-regulation and down-regulation of miR-7 molecules (average of 10 random fields)

Figure 7 Plaque detection of rotavirus titer of progeny after miR-7 up-down regulation

Figure 8: Analysis of the relationship between miR-7 and rotavirus NSP5 gene target (A) miRanda software predicts the binding of miR-7 to rotavirus NSP5 gene; (B) Dual luciferase reporter system detects miR-7 and NSP5 Construction of mutant plasmids; (C) Dual luciferase reporter system to detect the binding of miR-7 to NSP5 (**P<0.05)

Figure 9 Westernblot detection of NSP5 protein expression in different time periods (12h, 24h, 36h, 48h) after transfection of miR-7 mimics and inhibitors

Figure 10 Westernblot detection of NSP5 eukaryotic expression plasmid and miR-7 mimic co-transfected 293 cells to detect the expression of NSP5 protein

Figure 11 Transmission electron microscope observation of the formation of intracellular virus pool after miR-7 was up-regulated

Fig. 12 Diarrhea scores of suckling rats after miR-7 up and down

Figure 13 HE staining to observe the small intestinal villi lesions in neonatal rats after miR-7 up-down regulation

detailed description

Example 1 Time-sensitive expression detection of miR-7 during rotavirus replication

We infected wild-type rotavirus ZTR-68 isolated from the research group with sensitive RNA MA104 cells and then performed small RNA deep sequencing analysis to study the expression profile of microRNAs during rotavirus infection. We found that 40 microRNAs were found in rotavirus The expression level changes after infection. After comparison with multiple databases (miRBase database, Rfam database, GeneBank database, etc.) and RT-PCR verification, it was found that the expression level of miR-7 was significantly increased in the 40 differentially expressed microRNAs. The total RNA of the cells was extracted at 12, 24, 36, and 48h after MA104 cells were infected with ZTR-68 (MOI = 0.1). It was found by qRT-PCR that the expression level of miR-7 was up-regulated with the prolongation of viral infection time. The up-regulation factor continued to increase until it reached its peak after 36 hours of rotavirus infection (Figure 1).

Example 2 Detection of miR-7 expression levels in MA104 cells infected with different G type and same MOI and same G type and different MOI rotavirus

We used the G1 type ZTR-68 rotavirus to infect MA104 cells with different MOI (0.1, 0.5, 1 and 2), and extracted total cellular RNA and qRT-PCR to detect the expression of miR-7 24h after infection. The results showed that as the MOI of rotavirus infection increased, the up-regulation of miR-7 showed a rising trend (Figure 2). Choose G1, G2, G4, and G9 genotypes of rotavirus to infect MA104 cells with MOI of 0.5. Infection of MA104 cells with different genotypes of rotavirus can cause upregulation of miR-7 expression, and G1 rotavirus The most significant (Figure 3).

Example 3 Detection of miR-7 expression level after rotavirus infection of different host cells

Caco-2 and HT-29 cells were infected with ZTR-68 virus (MOI=0.1), and the total RNA of cells was extracted 36h after infection, and the expression of miR-7 was detected by qRT-PCR. The results also indicate that rotavirus infection can cause the expression of miR-7 in host cells to be significantly up-regulated (Figure 4).

Example 4 The role of miR-7 in rotavirus replication

We synthesized miR-7 inhibitors and mimics, which were transfected into host cells (MA104 cells) respectively, and were infected with rotavirus 24 hours after transfection. After verification by qRT-PCR experiments, compared with the control MA104 cells, the expression level of miR-7 was significantly up-regulated and down-regulated. Viral RNA was extracted from cells after different times of RV infection (12h, 24h, 36h and 48h), and the rotavirus genome NSP3 was detected by probe method to determine the rotavirus copy number. NSP3 results showed that the reduction of miR-7 group at the same time point increased the copy number of NSP3 faster than that of miR-7 group, and the upregulation of miR-7 group inhibited rotavirus proliferation (Figure 5). After 16 hours of RV infection, the expression of rotavirus protein was detected by immunofluorescence. The results showed that compared with the control, the protein synthesis of the virus was reduced after miR-7 was up-regulated, and the protein synthesis of the virus was increased after miR-7 was down-regulated (Figure 6). The virus supernatant was collected 72h after infection with RV virus, and blindly transmitted for three generations in MA104 cells with up-regulated miR-7 and down-regulated miR-7 to collect the virus. The titration of the virus harvested after miR-7 was up-regulated and down-regulated was detected by the plaque method. The results showed that compared with the control cell progeny virus, after up-regulation of miR-7, the proliferation of RV progeny virus decreased compared with the control, miR-7 was down After -7, the titer of the RV progeny virus increased (Figure 7).

Example 5 miR-7 targeting rotavirus NSP5 gene regulates virus proliferation

After determining the regulatory effect of miR-7 on rotavirus proliferation, we used miRanda software to analyze the relationship between miR-7 and rotavirus 11 genes. The prediction results show that miR-7 targets 17bp-36bp of the NSP5 gene of the rotavirus ZTR-68 strain (Figure 8A). The dual luciferase reporter system was used to detect the regulatory effect between miR-7 and the target gene NSP5: we cloned the target sequence (NC) and mutant sequence (NSP5Mut) of the target gene NSP5 into the luciferase expression plasmid, and miR -7 molecules of mimics and control microRNA sequences were co-transfected into HEK293 cells, and the relative change of luciferase activity was detected by luciferase detection kit to identify whether miR-7 had a regulatory effect on the target gene. The results showed that miR-7 It can significantly regulate the expression of luciferase with the target gene NSP5. After the binding site was mutated, the regulatory relationship disappeared, indicating that miR-7 regulates the expression of NSP5 through this binding site (Figure 8B, 8C).

After transfection of miR-7 mimics and inhibitors in MA104 cells for 24h, they were infected with rotavirus and extracted total cell proteins at 12-48h respectively and tested whether the expression of NSP5 protein was regulated by miR-7 in different time periods. The results showed that miR-7 significantly inhibited the expression of rotavirus NSP5 protein 24 hours after virus infection (Figure 9), further proving that NSP5 is the target gene of miR-7.

In order to further verify that miR-7 can regulate the expression of NSP5 gene, we also constructed a pEGFP-N2-NSP5 eukaryotic expression vector. When co-transforming MA104 cells with miR-7 mimics and inhibitor for 48h, we can find that miR-7 -7 can significantly inhibit the expression of rotavirus NSP5 protein (Figure 10).

Since NSP5 is the main regulatory protein that forms the rotavirus virus pool, we performed transmission electron microscopy observation on the cells after the rotavirus infected MA104 cells for 12 hours. The results showed that the virus formed a large number of virus pool-like structures in the cells transfected with NC, and there were a large number of virus particles in the virus pool. Compared with the NC group, after up-regulating miR-7, the number of intracellular virus pools was reduced, and the structure of the virus pool was incomplete. The number of intracellular virus down-regulating miR-7 was higher, and the number and morphology of virus pools were not significantly different from NC (Figure 11) ).

Example 6 The role of miR-7 in rotavirus infection model

In addition to in vitro studies, in order to further clarify the antiviral effect of miR-7 on rotavirus and the role in the prevention and treatment of diarrhea caused by rotavirus infection, we established a model of diarrhea in suckling mice. At an infectious dose of 200ID50, rotavirus was intragastrically infected to suckling rats born at 5 days. After 24 hours of infection, suckling rats began to develop diarrhea. After confirming that the model is established successfully, start the experiment. In a preferred embodiment of the present invention, the miR-7 overexpression mim-7 mimir-7 and miR-7 expression inhibitor miR-7 are purchased from Shanghai Gemma Gene Company, miR-7 mimir-7 and expression inhibitor miR-7 antagomir was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

We synthesized miR-7 agonist miR-7, a miRNA agonist for animal experiments with special chemical modification, miR-7 antagomir, a miRNA antagonist for animal experiment with special chemical modification, and miRNAnegativecontrol with special chemical modification. , Administered by gavage. 24 hours after dosing, 200 ID50 of rotavirus was intragastrically infected, and normal feeding was observed. According to the scoring rules of BOSHUIZEN JA and others for diarrhea in suckling rats, the diarrhea in suckling rats is scored (0 to 4) according to the color, hardness, quantity, etc. of the feces, the score of no fecal discharge is 0, and the score of brown shaped stool Points, brown soft stool score 2 points, yellow soft stool score 3 points, yellow watery stool score 4 points, perianal fecal pollution score 4 points, greater than 2 points are considered to have diarrhea. According to the degree of diarrhea of the suckling rats, the diarrhea score and diarrhea rate of each group of suckling rats were counted. The NC group and the miR-7 antagomir group had significant diarrhea after 24 hours of challenge, with a score of 4 points, and the miR-7 antagomir group had a higher degree of diarrhea than the NC group. There was no significant diarrhea in the miR-7 agomir group after challenge. After 48 hours of challenge, the suckling rats were euthanized, and their intestinal tissues were taken for HE staining to detect pathological changes. Taking the suckling mice in the uninfected virus group as a control, it was found that after the rotavirus infection in the NC group, the intestinal villi showed obvious swelling and damage, and a large number of vacuolated cells were visible on the top of the intestinal villi. The small intestinal villi in the antagomir group that down-regulated miR-7 also showed obvious swelling and breakage, and a large number of vacuolated cells were visible on the top of the small intestine villi, which was more severe than the NC group. In the miR-7's agomir group, the small intestinal villi showed slight swelling and inflammatory cell infiltration, but no damage was found (Figure 13). Combined with diarrhea scores, miR-7 can significantly inhibit rotavirus replication and reduce or suppress diarrhea caused by rotavirus infection in suckling mice.

Claims

The application of MicroRNA-7 in the preparation of anti-rotavirus drugs is characterized in that miR-7 has a significant regulatory target gene NSP5 (SED NO.2), and miR-7 is targeted to rotavirus ZTR-68 strain The 17bp-36bp of the NSP5 gene and the miR-7-NSP5 action site can be used as new and effective anti-rotavirus therapeutic targets for the preparation of anti-rotavirus drugs.
The application of MicroRNA-7 according to claim 1 in the preparation of anti-rotavirus drugs, characterized in that the miR-7 small RNA mimics are used in the preparation of anti-rotavirus drugs.
The application of MicroRNA-7 according to claim 2 in the preparation of anti-rotavirus drugs, characterized in that the miR-7 small RNA mimic is mml-miR-7 sense single-stranded oligonucleotide.
The application of MicroRNA-7 according to claim 2 in the preparation of anti-rotavirus drugs, characterized in that the sense single-stranded oligonucleotide of miR-7 can be artificially synthesized or directly commercially available, miR-7 The sense single-stranded oligonucleotide can also be modified by PNA, LNA or 2-Ome.