Mir-331-3p as biomarker for predicting malaria transmission and application of mir-331-3p

Abstract

The invention relates to mir-331-3p as a biomarker for predicting malaria transmission and application of the mir-331-3p, and belongs to the technical field of biology. The invention aims to provide a more accurate biomarker, and solves the technical defect that the existing index cannot effectively evaluate the malaria spreading risk. The invention provides a host-derived miR-331-3p (miR-331-3p) used as a malaria transmission prediction marker. The detection kit containing the marker is constructed by detecting the expression level of miR-331-3p in host serum and combining the characteristic that the miR-331-3p regulates and controls the development of plasmodium in mosquito bodies in a cross-species manner. Compared with traditional gametophyte detection, the method has the advantages that the sensitivity is improved, and the technical bottleneck that the gametophyte rate and the transmission capacity are unhooked is broken through; 2) non-invasive detection is realized, and only trace serum samples are needed; and 3) establishing a host-medium bidirectional regulation and control model, and providing a new target spot for malaria transmission blocking.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to mir-331-3p as a biomarker for predicting malaria transmission and application thereof. Background Art

[0002] Malaria is transmitted through the bites and blood-sucking of Anopheles mosquitoes. Blocking and inhibiting the development of gametocytes that enter the mosquito's body can effectively control or even block mosquito-borne transmission. Therefore, the traditional view is that gametocytes are the core factor affecting the transmission of malaria. The high or low gametocyte rate in malaria patients is positively correlated with the transmission ability of malaria. Gametocytes are also often used as the main indicator for predicting transmission.

[0003] Studies on the transmission of three strains of murine malaria (Pb ANKA, Py BY265, and Py 17XNL) have consistently found that parasite infection in Anopheles mosquitoes is not necessarily positively correlated with the prevalence of gametocytes in the host's blood. Specifically, the gametocyte prevalence in mice infected on days 4 to 6 was nearly three times higher than in mice infected on days 1 to 3, yet malaria transmission capacity was significantly reduced. Gametocytes enter the mosquito's system along with blood components, suggesting that host blood components, in addition to gametocytes, may also play a role in malaria transmission. Summary of the Invention

[0004] In light of this, the present invention, through a series of screening studies, discovered that Plasmodium infection can induce elevated expression of host miR-331-3p. Furthermore, miR-331-3p, upon entering the mosquito body through blood feeding, can play a cross-species role in promoting Plasmodium infection in mosquitoes. The present invention aims to provide a biomarker for predicting malaria transmission, and a second objective is to investigate the application of miR-331-3p in predicting malaria transmission.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a mir-331-3p as a biomarker for predicting malaria transmission, wherein the biomarker is mir-331-3p, and the nucleic acid sequence thereof is shown in SEQ ID NO: 13;

[0007] Furthermore, the use of said biomarkers in predicting the spread of malaria;

[0008] Furthermore, the use of the biomarker in detecting the spread of malaria;

[0009] The present invention further provides a kit for predicting the spread of malaria, comprising a probe for detecting the expression level of mir-331-3p, primers, enzyme and buffer;

[0010] Preferably, the forward primer sequence is shown in SEQ ID NO: 19;

[0011] Furthermore, the detection method of the kit includes the following steps:

[0012] S1: Obtain biological samples from the host;

[0013] S2: detecting the expression level of mir-331-3p in the biological sample, wherein the sequence of mir-331-3p is shown in SEQ ID NO: 13;

[0014] S3: Compare the test results with the threshold to predict the risk of malaria transmission;

[0015] Preferably, the biological sample is any one of serum, whole blood, and plasma.

[0016] The beneficial effects of the present invention are:

[0017] As a biomarker for predicting malaria-infected mosquitoes, miR-331-3p has the following advantages:

[0018] 1. Breakthrough of traditional detection technology bottleneck

[0019] Compared to traditional methods that rely on gametocyte prevalence, the use of host-derived miR-331-3p as a biomarker overcomes the technical challenge of decoupling gametocyte prevalence from transmissibility. This significantly improves sensitivity, enabling the detection of early or low-level infections.

[0020] 2. Non-invasive testing and clinical practicality

[0021] The test requires only a trace amount of serum or plasma sample, avoiding the risks of traditional invasive testing and overcoming the risks associated with traumatic testing. It is particularly suitable for children and critically ill patients. The test process is simple and rapid, utilizing proven qPCR technology, eliminating the need for complex antibody development and shortening the timeline for clinical application. In clinical serum and plasma samples, microRNAs are stably present in a form that is resistant to RNases and is not easily degraded.

[0022] 3. Innovation of cross-species regulatory mechanisms

[0023] After entering the mosquito's bloodstream, host miRNA (miR-331-3p) regulates the expression of mosquito immune genes (such as spaetzle) across the host's phylum, establishing a bidirectional "host-vector" interaction model and providing a new target for blocking transmission. This mechanism breaks through the traditional strategy of unilaterally intervening with either the malaria parasite or the vector, achieving dual regulation.

[0024] 4. Technical compatibility and scalability

[0025] The detection kit can integrate multiple miRNA markers to form a multi-indicator joint detection system, further improving prediction accuracy. The regulation method is not only applicable to Plasmodium yoelii but can also be expanded to Plasmodium falciparum, showing broad application potential.

[0026] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0028] Figure 1 The figure is the heat map of miR enrichment with significant differential expression;

[0029] Figure 2 is the effect of microRNA inhibitors on the number of oocysts;

[0030] Figure 3 Effects of DEPC and microRNA analogs on the number of oocysts

[0031] Figure 4 The effect of microRNA analogs on the number of Plasmodium falciparum oocysts.

[0032] Figure 5 This is a heat map showing significant enrichment of host miR-affected mosquito gene changes;

[0033] Figure 6 Identify host miRs that regulate mosquito target genes for RNAi;

[0034] Figure 7 Identify host miRNA-regulated mosquito target genes for dual-luciferase activity. DETAILED DESCRIPTION

[0035] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0036] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0037] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0038] Example 1

[0039] 1. Experimental Materials

[0040] (1) Test mosquito vectors, malarial parasites and mice

[0041] 1. Anopheles stephensi: This is the Hor strain that has been bred and raised in our laboratory for a long time.

[0042] 2. Plasmodium yoelii: BY265 strain, blood-transmitted using Kunming strain mice once a week and mosquito-transmitted once every 5 weeks.

[0043] 3. Mice: Kunming strain mice were provided by the Experimental Animal Center of our university, weighing 16-20 g.

[0044] (2) Main reagents

[0045] TRIzol (Ambion, Invitrogen)

[0046] miRcute enhanced miRNA cDNA first-strand synthesis kit and miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0047] Mmu-mir-331-3P inhibitor: Shanghai Gene Synthesis

[0048] (3) Main instruments

[0049] Quantitative PCR instrument (BIO-RAD CFX96, Beijing SaiBio Technology Co., Ltd. )

[0050] 2. Experimental Methods

[0051] (1) Administration of Mmu-mir-331-3P analogs

[0052] 1. Drug preparation: Before opening the tube, centrifuge at 3000g for 1 minute. Slowly open the tube cap and add 150ul DEPC water to 1OD of Mmu-mir-331-3P analog. Vortex to dissolve and prepare a 20um solution.

[0053] 2. Administration: Take 200ul of the 20um Mmu-mir-331-3P analog prepared according to the above method, add 200ul of PBS, mix well, and inject through the tail vein of the mouse.

[0054] (II) Observation of the effect of Mmu-mir-331-3P analogs on promoting mosquito infection with Plasmodium

[0055] 1. Preparation of mice infected with Plasmodium: Inoculate healthy mice intraperitoneally as usual. Three days later, select mice with parasitemia of more than 10% as blood donors after blood tests.

[0056] 2. Preparation of blood donor mice: Blood was collected from 6 mice infected with Plasmodium, mixed and collected, and transferred to 5 new mice via the tail vein. 2 hours later, the Mmu-mir-331-3P analog or control prepared according to the above-mentioned administration method was injected via the tail vein to prepare for blood donation.

[0057] 3. Mosquito-borne disease grouping and drug administration

[0058] Infection group + control group: The blood donor mice prepared as above provided blood to Anopheles stephensi for about 2 hours, and the blood-satiated female mosquitoes were selected for routine breeding.

[0059] Infection group + mir-331-3P analog group: infection and medication were the same as before.

[0060] The above groups of mosquitoes were all raised in a mosquito breeding room at 24-25°C and a relative humidity of 70%-80%.

[0061] 4. Identification of the role of Mmu-mir-331-3P in promoting mosquito infection with Plasmodium

[0062] The stomachs of the two groups of mosquitoes infected on the 7th day were dissected to observe the morphology and size of the oocysts and calculate the infection rate. Figure 6 shown.

[0063] Example 2 Quantitative PCR detection of changes in mir-331-3p expression in mice infected with Plasmodium

[0064] 1. Whole Blood RNA Extraction

[0065] (1) Take about 200ul of mouse whole blood, add 1ml of Trizol, and quickly and thoroughly lyse it. Centrifuge at 12000g, 4℃ for 10min, and remove residual tissue. Take the supernatant and transfer it to a new EP tube. (Add Tiangen External Reference CR100-01, dilute the current stock solution 1000 times, and add 1ul to each tube. Mix thoroughly after adding the external reference.)

[0066] (2) Place at room temperature for 5 minutes to allow the sample to fully lyse.

[0067] (3) Add 0.2 ml of chloroform (5:1), shake vigorously for 15 seconds, and let it stand at room temperature for 10 minutes.

[0068] (4) Centrifuge at 12,000 g for 15 min at 4°C. Carefully aspirate the colorless supernatant and transfer it to a new tube. (When removing the supernatant, ensure that the amount obtained in each tube is consistent and avoid chloroform.)

[0069] (5) Add 0.5 ml of isopropanol (2:1) and let it stand at room temperature for 5 to 10 minutes.

[0070] (6) Centrifuge at 12,000 g for 10 min at 4°C, remove the supernatant, and add 1 ml of 75% ethanol (prepared with DEPC water) to the precipitate.

[0071] (7) Wash the precipitate by centrifugation at 7500 g for 5 min at 4°C, remove the supernatant, and air-dry.

[0072] (8) Dissolve the total RNA in an appropriate amount of DEPC water (15 μl) and determine the purity and concentration of the total RNA.

[0073] (9) Store at -80℃ until ready for use.

[0074] 2. miRNA cDNA First Strand Synthesis

[0075] Reverse transcription was performed using the Tiangen miRcute Enhanced miRNA cDNA First-Strand Synthesis Kit

[0076]

[0077] 3. miRNA fluorescence quantitative detection

[0078] The miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green) was used to detect the expression changes of mir-331-3p in mice infected with Plasmodium.

[0079]

[0080] Forward Primer sequence: CCCTGGGCCTATCCTAGAA (SEQ ID NO: 19)

[0081] The reverse primer sequence was provided by Tiangen miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green, FP411).

[0082] Example 3 Changes in microRNA expression in hosts infected with Plasmodium

[0083] Since the contrast between the gametocyte rate of mice infected with Py BY265 on day 3 and day 5 is most obvious compared with that of infected mosquitoes, this example selected mice infected with Plasmodium on day 3 and day 5 for whole blood small RNA sequencing. The results showed that there were 18 microRNAs significantly upregulated by more than 1.5 times in mice on day 3, such as Figure 1 The specific sequence is as follows:

[0084] mmu-miR-674-5p: sequence as shown in SEQ ID NO: 1,

[0085] mmu-let-7d-5p: sequence as shown in SEQ ID NO: 2,

[0086] mmu-miR-486b-5p: sequence as shown in SEQ ID NO: 3,

[0087] mmu-miR-486a-5p: sequence as shown in SEQ ID NO: 4,

[0088] mmu-miR-505-5p: sequence as shown in SEQ ID NO: 5,

[0089] mmu-miR-196b-5p: sequence as shown in SEQ ID NO: 6,

[0090] mmu-miR-450b-3p: sequence as shown in SEQ ID NO: 7,

[0091] mmu-miR-8114: sequence as shown in SEQ ID NO: 8,

[0092] mmu-miR-18a-3p: sequence as shown in SEQ ID NO: 9,

[0093] mmu-miR-1306-3p: sequence as shown in SEQ ID NO: 10,

[0094] mmu-miR-25-5p: sequence as shown in SEQ ID NO: 11,

[0095] mmu-miR-379-5p: sequence as shown in SEQ ID NO: 12,

[0096] mmu-miR-331-3p: sequence as shown in SEQ ID NO: 13,

[0097] mmu-miR-6915-5p: sequence as shown in SEQ ID NO: 14,

[0098] mmu-miR-3057-5p: sequence as shown in SEQ ID NO: 15,

[0099] mmu-miR-1938: sequence as shown in SEQ ID NO: 16,

[0100] 1_387: sequence as shown in SEQ ID NO: 17,

[0101] 17_10002: The sequence is shown in SEQ ID NO:18.

[0102] Example 4 Functional screening of the 18 MiRNAs enriched in Example 3

[0103] By administering mir inhibitors, we conducted a preliminary functional screening of 18 miRNAs and found that the administration of mir-331-3p inhibitors can reduce the number of malarial parasite oocysts in mosquitoes and inhibit malarial parasite infection of mosquitoes, while the administration of analogs leads to an increase in the number of oocysts. Further using an in vitro blood feeding platform, we observed that mir-331-3p analogs can promote the development of human falciparum malarial parasites, leading to an increase in the number of oocysts. The mir-331-3p of malarial parasite-infected hosts has the effect of promoting malarial parasite infection of mosquitoes. The results suggest that miR-331-3p can be used to design new indicators for predicting malaria transmission. Figure 2 、 3 , as shown in 4.

[0104] Example 5 Screening and identification of host mir-331-3P-regulated mosquito target genes

[0105] Transcriptome sequencing was used to screen for changes in the expression of mosquito genes that may be targeted and regulated by mir. The results showed that the mosquito was significantly regulated within 24 hours of infection with Plasmodium, which is the critical time for the zygote to cross the mosquito gastric epithelial cells. Combined with the predicted target genes that the mir sequence seed region may bind to the Anopheles stephensi genome, important target-regulated mosquito genes were enriched, especially immune-related genes for follow-up research. Using RNAi to identify, it was found that knocking out the spaetzle molecule in the mosquito toll signaling pathway can increase the number of oocysts, thereby reversing the phenomenon that mir inhibitor administration prevents oocyst development, indicating that this molecule is a cross-kingdom regulated target gene of mir-331-3p, such as Figure 5 and6 shown.

[0106] Example 6 Dual luciferase verification of the regulatory effect of microRNA on target genes

[0107] The dual luciferase assay was further used to verify the interaction between mir and mosquito spaetzle. The mir analog, negative control, plasmids with constructed spazle sequence, or mutant sequence were introduced into 293 cells respectively. The results showed that in mice; (1) the fluorescence ratio of the mir analog + spaetzle group was significantly reduced compared with the negative control + spaetzle group; (2) the fluorescence ratio of the mir analog + spaetzle mutant group was not significantly changed compared with the negative control + spaetzle mutant group. This indicates that mouse mir-331-3p and the mosquito target gene spaetzle have a direct interaction, and the seed region at the 3' end of mir-331-3p is complementary to the CDS region of the mosquito target gene spaetzle. The results are as follows: Figure 7 shown.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. mir-331-3p as a biomarker for predicting malaria transmission is characterized by: The biomarker is mir-331-3p, and its nucleic acid sequence is shown in SEQ ID NO:

13.

2. Use of the biomarker according to claim 1 in predicting the spread of malaria.

3. Use of the biomarker according to claim 1 in detecting malaria transmission.

4. A kit for predicting the spread of malaria, characterized in that The invention comprises a probe for detecting the expression level of mir-331-3p according to claim 1, primers, enzyme and buffer.

5. The kit according to claim 4, wherein: The forward primer sequence is shown in SEQ ID NO:

19.

6. The detection method of the kit according to claim 5, characterized in that: S1: Obtain biological samples from the host; S2: detecting the expression level of mir-331-3p in the biological sample, wherein the sequence of mir-331-3p is shown in SEQ ID NO: 13; S3: Compare the test results with the threshold to predict the risk of malaria transmission.

7. The detection method according to claim 6, wherein: The biological sample is any one of serum, whole blood, and plasma.

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