CN106176795A - The prawn miR 35 application in prawn antiviral and suppression people's neoplasm metastasis - Google Patents

The prawn miR 35 application in prawn antiviral and suppression people's neoplasm metastasis Download PDF

Info

Publication number
CN106176795A
CN106176795A CN201610565800.7A CN201610565800A CN106176795A CN 106176795 A CN106176795 A CN 106176795A CN 201610565800 A CN201610565800 A CN 201610565800A CN 106176795 A CN106176795 A CN 106176795A
Authority
CN
China
Prior art keywords
prawn
mir
application
wssv
mirna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610565800.7A
Other languages
Chinese (zh)
Other versions
CN106176795B (en
Inventor
章晓波
陈玉磊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN201610565800.7A priority Critical patent/CN106176795B/en
Publication of CN106176795A publication Critical patent/CN106176795A/en
Application granted granted Critical
Publication of CN106176795B publication Critical patent/CN106176795B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Abstract

The invention discloses a kind of prawn miR 35 application in prawn antiviral and suppression people's neoplasm metastasis.Prawn miR 35 is the distinctive miRNA of prawn, its base sequence is 5 ' AACUGUGAGAAGUUCCGGU 3 ', shrimp disease preventing and treating is directed to white spot syndrome virus (WSSV) (WSSV), suppression people's neoplasm metastasis for tumor cell be breast cancer cell MDA MB 231.

Description

Prawn miR-35 application in prawn antiviral and suppression people's neoplasm metastasis
Technical field
The present invention relates to the application of a miRNA, especially related to a kind of prawn miR-35 and at prawn antiviral and pressed down Application in people's neoplasm metastasis processed, this is more beneficial for understanding the mechanism that miRNA regulates and controls across species.
Background technology
MicroRNA (miRNA) is little non-coding RNA, is about made up of 18-25 nucleotide, and a lot of miRNA are in difference It species is all high conservative.MiRNA is the important factor that controlling gene is expressed, the tune of level after they participation genetic transcriptions Control.They by Seed Sequences and target gene 3 ' end non-coding region (3 ' UTR) directly in conjunction with, thus suppress the translation of target gene Or the degraded of mediation target gene.
Prawn, as one of important aquatic products, has the biggest commercial value.But, owing to prawn white spot syndrome is sick The disease of prawn that the viruses such as poison (WSSV) infect and cause becomes the thorny problem of prawn culturing.After virus infects prawn, right The express spectra generation significant change of miRNA in shrimp body, illustrates that miRNA may participate in the antiviral immunity reaction of prawn.Therefore, may be used Using by host and virus miRNA as target spot, by affect the infection of virus in prawn body and replicating and alleviate virus infection and draw The disease of prawn risen, improves the immune defence ability of prawn simultaneously.
Research in the past shows, the miR-168a of Oryza sativa L. can suppress mammal low density lipoprotein receptor adaptin base Because of the expression of (LDLRAP1), the decline of this gene expression can promote the rising of liver low density cholesterol level；Gold silver The miR-2911 of flower coding, it is possible to wide application is in influenza virus (including H1N1, H5N1 and H7N9)；The miRNA of external source is permissible Pass to fetus by Placenta Hominis, affect the gene expression of fetus.This explanation Mirnas of plant can enter in animal body also " transboundary " Affect the physiologically active of animal, play biological function.Early-stage Study result shows, the CHI3L1 albumen of macrophages secrete can To promote the migration of tumor cell.Therefore, it can the CHI3L1 albumen in macrophage as the target spot of oncotherapy.
Summary of the invention
In order to solve problem present in background technology, it is an object of the invention to overcome present in prior art above-mentioned Deficiency, and screen and acquire a miRNA (miRNA-35), this miRNA can suppress the infection to prawn of the WSSV virus, again The migration of tumor cell can be suppressed by acting on CHI3L1 3 ' UTR.
The prawn miR-35 of the present invention is the distinctive miRNA of prawn, and obtaining its base sequence by order-checking is SEQ ID NO.1:5 '-AACUGUGAGAAGUUCCGGU-3 '.
The application of prawn disease of the present invention is directed to white spot syndrome virus (WSSV) (WSSV).
The present invention suppress people's neoplasm metastasis application for tumor cell be breast cancer cell MDA-MB-231.
The target gene of described prawn miR-35 inhibitory action is the M2 after the differentiation of human monocyte cell line's THP-1 cell induction CHI3L1 gene in type macrophage.
The invention has the beneficial effects as follows:
Prawn miRNA-35 of the present invention is possible not only to the infection in prawn body of the suppression virus and duplication, plays antiviral and makees With, moreover it is possible to interact with CHI3L1mRNA 3 ' UTR, strike the expression of CHI3L1 gene in low macrophage, thus affect tumor The transfer of cell.
Accompanying drawing explanation
Fig. 1 is that miR-35 is on the impact of WSSV copy number in prawn body.
Fig. 2 is the miR-35 impact on prawn mortality rate.
Fig. 3 is that miR-35 is on the impact of CHI3L1 gene expression amount in M2 type macrophage.
Fig. 4 is that miR-35 is on the impact of CHI3L1 expressing quantity in M2 type macrophage.
Fig. 5 is the impact that breast cancer cell MDA-MB-231 is migrated by miR-35.
Detailed description of the invention
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Embodiments of the invention are as follows:
Embodiment first prepares experiment sample:
Prawn miR-35, negative control miRNA and control miRNA are the most all to utilize In vitro Transcription T7Kit (Takara) in vitro transcription obtains.
Preparation is for the negative control miRNA of prawn disease embodiment, and the base sequence of negative control miRNA is SEQ ID NO.2:5 '-UAAGCGUAGUAUUGCAGCG-3 '.
Preparation for suppressing the base sequence of control miRNA, the control miRNA of people's neoplasm metastasis embodiment is SEQ ID NO.3:5 '-UUCUCCGAACGUGUCACGUUU-3 '.
Embodiment 1
1) preparation experiment group and matched group:
Experimental group, is injected simultaneously into prawn miR-35 with the WSSV virus liquid being diluted in phosphate buffered solution (PBS) In prawn body, prawn miR-35 injects the dosage of 30 μ g miRNA, containing 10 in WSSV virus liquid4Individual WSSV virion；
First matched group, by negative control miRNA be diluted in the WSSV virus liquid of phosphate buffered solution (PBS) simultaneously Being expelled in prawn body, negative control miRNA injects the dosage of 30 μ g miRNA, containing 10 in WSSV virus liquid4Individual WSSV is sick Virion；
Second matched group, is individually expelled to the WSSV virus liquid being diluted in phosphate buffered solution (PBS) in prawn body, Containing 10 in WSSV virus liquid4Individual WSSV virion；
3rd matched group, is individually expelled to phosphate buffered solution (PBS) in prawn body, without WSSV in PBS solution Virion.
2) then according in the following manner carry out of both experiment:
A) in experimental group, the first matched group, the second matched group and the 3rd matched group, at injection prawn 24h, 36h and 48h After, collect the cheek silk of each group of prawn, extract the genome in cheek silk, detect viral copies therein by real-time PCR Number.
Viral copy number result is as it is shown in figure 1, be injected simultaneously into prawn by prawn miR-35 with WSSV virus liquid seen from figure Afterwards, the viral copy number in prawn body is more only injected WSSV virus liquid group and injects negative control miRNA Yu WSSV disease simultaneously Venom group is all decreased significantly, and illustrates that prawn miR-35 can effectively suppress infection and the duplication of virus.
B) in experimental group, the first matched group, the second matched group and the 3rd matched group, injection prawn 12h, 24h, 36h, After 48h and 72h, count the mortality rate of each group of prawn.
The mortality results of prawn as in figure 2 it is shown, in figure visible prawn miR-35 and WSSV virus liquid is injected simultaneously into right After shrimp, the mortality rate of prawn is more only injected WSSV virus liquid group and injects negative control miRNA and WSSV virus liquid group simultaneously All it is decreased significantly, illustrates that prawn miR-35 can be effectively improved the survival rate of prawn.
Embodiment 2
1) obtaining M2 type macrophage, M2 type macrophage is differentiated by the induction of THP-1 mononuclear cell, concrete grammar For: first process THP-1 cell 6h with 320nM propylene glycol methyl ether acetate (PMA), then with 20ng/ml interleukin-4 (IL-4) THP-1 cell 18h is continued with interleukin-13 (IL-13).
2) preparation experiment group and matched group:
Experimental group: M2 type is huge to be bitten to utilize lipo RNAiMAX transfection reagent (Invitrogen) to be transfected into by prawn miR-35 In cell, after transfection 48h, collect cell and culture supernatant thereof.
First matched group: utilize lipo RNAiMAX transfection reagent (Invitrogen) to be transfected into by control miRNA In M2 type macrophage, after transfection 48h, collect cell and culture supernatant thereof.
Second matched group: M2 type macrophage is left intact, after 48h, collects cell and culture supernatant thereof.
3) then according in the following manner carry out of both experiment:
A) for experimental group, the first matched group and the second matched group, the RNA in the M2 type macrophage of collection is all extracted, And detecting the expression of CHI3L1 gene, result is as it is shown on figure 3, in figure after visible transfection prawn miR-35, M2 type is huge to be bitten The expression of intracellular CHI3L1 gene mRNA levels there occurs notable downward, illustrates that prawn miR-35 can effectively suppress The expression of CHI3L1 gene.
B) the M2 type macrophage collected is carried out Western blot test, the change of detection CHI3L1 protein level. Result as shown in Figure 4, in figure after visible transfection prawn miR-35, the expression of CHI3L1 protein level in M2 type macrophage There occurs notable downward, further illustrate prawn miR-35 and can effectively suppress the expression of CHI3L1 gene.
C) by the M2 type macrophage culture supernatant collected and breast cancer cell MDA-MB-231 training altogether in transwell Supporting, wherein on transwell, the MDA-MB-231 cell that serum-free medium is resuspended is spread in room, and under transwell, room is M2 type Macrophage culture supernatant.After co-culturing 12h, detection migrates to the MDA-MB-231 cell of transwell lower membrane, and with aobvious Micro mirror counts.
Breast cancer cell MDA-MB-231 migration results situation that counting obtains as it is shown in figure 5, in figure visible when M2 type huge After transfecting prawn miR-35 in phagocyte, the transfer ability of the breast cancer cell MDA-MB-231 co-cultured therewith is remarkably decreased, Illustrate that prawn miR-35 can effectively suppress the migration of breast cancer cell MDA-MB-231.
As seen from the above-described embodiment, the prawn miRNA-35 of the present invention can suppress virus infection in prawn body and answer System, plays antivirus action, moreover it is possible to interact with CHI3L1mRNA 3 ' UTR, strikes the table of CHI3L1 gene in low macrophage Reach.
The RNA sequence that the present invention relates to is as follows:
SEQ ID NO.1: prawn miR-35
Source: synthetic
Sequence: 5 '-AACUGUGAGAAGUUCCGGU-3 '；
SEQ ID NO.2: negative control miRNA
Source: synthetic
Sequence: 5 '-UAAGCGUAGUAUUGCAGCG-3 '；
SEQ ID NO.3:control miRNA
Source: synthetic
Sequence: 5 '-UUCUCCGAACGUGUCACGUUU-3 '.

Claims (5)

1. prawn miR-35 application in prawn disease and suppression people's neoplasm metastasis.
Prawn miR-35 the most according to claim 1 is the distinctive miRNA of prawn, and its base sequence is SEQ ID NO.1: 5’-AACUGUGAGAAGUUCCGGU-3’。
The application of prawn disease the most according to claim 1 is directed to white spot syndrome virus (WSSV) (WSSV).
The most according to claim 1 suppression people's neoplasm metastasis application for tumor cell be breast cancer cell MDA- MB-231。
Application in suppression people's neoplasm metastasis the most according to claim 1, it is characterised in that prawn miR-35 inhibitory action Target gene be human monocyte cell line's THP-1 cell induction differentiation after M2 type macrophage in CHI3L1 gene.
CN201610565800.7A 2016-07-18 2016-07-18 Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts Active CN106176795B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610565800.7A CN106176795B (en) 2016-07-18 2016-07-18 Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610565800.7A CN106176795B (en) 2016-07-18 2016-07-18 Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts

Publications (2)

Publication Number Publication Date
CN106176795A true CN106176795A (en) 2016-12-07
CN106176795B CN106176795B (en) 2018-12-14

Family

ID=57493178

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610565800.7A Active CN106176795B (en) 2016-07-18 2016-07-18 Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts

Country Status (1)

Country Link
CN (1) CN106176795B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106615619A (en) * 2016-12-19 2017-05-10 浙江大学 Feed containing miRNA-34 (micro Ribonucleic Acid-34) as well as production method and anti-virus and anti-cancer effects thereof
CN109609504A (en) * 2019-01-10 2019-04-12 汕头大学 A kind of miR-276 analogies are preparing the application in wide spectrum white spot syndrome virus resisting drug
CN109907162A (en) * 2019-03-29 2019-06-21 盐城工学院 A kind of anti-Leucoplakia feed addictive based on miR-35 and its application in aquaculture
CN109943562A (en) * 2019-03-06 2019-06-28 汕头大学 A kind of miR-2 antisense nucleic acid is preparing the application in wide spectrum white spot syndrome virus resisting preparation
WO2023109814A1 (en) * 2021-12-14 2023-06-22 成都凌泰氪生物技术有限公司 Use of mirna-2911 molecule as nucleic acid stabilizer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102190718A (en) * 2010-12-03 2011-09-21 武汉大学 Recombined litopenaeus setiferus protein SF-P9, preparation method and application thereof
CN103966230A (en) * 2014-05-20 2014-08-06 中国水产科学研究院南海水产研究所 Sequence of penaeus monodon cell cycle protein E gene as well as preparation method and application thereof
CN104004764A (en) * 2014-05-20 2014-08-27 中国水产科学研究院南海水产研究所 Sugpo prawn cyclin H gene sequence as well as preparation method and application of sugpo prawn cyclin H gene sequence

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102190718A (en) * 2010-12-03 2011-09-21 武汉大学 Recombined litopenaeus setiferus protein SF-P9, preparation method and application thereof
CN103966230A (en) * 2014-05-20 2014-08-06 中国水产科学研究院南海水产研究所 Sequence of penaeus monodon cell cycle protein E gene as well as preparation method and application thereof
CN104004764A (en) * 2014-05-20 2014-08-27 中国水产科学研究院南海水产研究所 Sugpo prawn cyclin H gene sequence as well as preparation method and application of sugpo prawn cyclin H gene sequence
CN104099339A (en) * 2014-05-20 2014-10-15 中国水产科学研究院南海水产研究所 Penaeus monodon cell cycle protein E gene sequence and preparation method and application thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106615619A (en) * 2016-12-19 2017-05-10 浙江大学 Feed containing miRNA-34 (micro Ribonucleic Acid-34) as well as production method and anti-virus and anti-cancer effects thereof
CN109609504A (en) * 2019-01-10 2019-04-12 汕头大学 A kind of miR-276 analogies are preparing the application in wide spectrum white spot syndrome virus resisting drug
CN109943562A (en) * 2019-03-06 2019-06-28 汕头大学 A kind of miR-2 antisense nucleic acid is preparing the application in wide spectrum white spot syndrome virus resisting preparation
CN109943562B (en) * 2019-03-06 2023-03-10 汕头大学 Application of miR-2 antisense nucleic acid in preparation of broad-spectrum White Spot Syndrome Virus (WSSV) resistant preparation
CN109907162A (en) * 2019-03-29 2019-06-21 盐城工学院 A kind of anti-Leucoplakia feed addictive based on miR-35 and its application in aquaculture
WO2023109814A1 (en) * 2021-12-14 2023-06-22 成都凌泰氪生物技术有限公司 Use of mirna-2911 molecule as nucleic acid stabilizer

Also Published As

Publication number Publication date
CN106176795B (en) 2018-12-14

Similar Documents

Publication Publication Date Title
CN106176795A (en) The prawn miR 35 application in prawn antiviral and suppression people's neoplasm metastasis
Pascutti et al. Impact of viral infections on hematopoiesis: from beneficial to detrimental effects on bone marrow output
Aivaliotis et al. How do cytokines trigger genomic instability?
Avdic et al. Viral interleukin-10 expressed by human cytomegalovirus during the latent phase of infection modulates latently infected myeloid cell differentiation
He et al. MicroRNAs activate natural killer cells through Toll-like receptor signaling
Huang et al. Efficient human cytomegalovirus reactivation is maturation dependent in the Langerhans dendritic cell lineage and can be studied using a CD14+ experimental latency model
Duan et al. Nasopharyngeal carcinoma progression is mediated by EBER-triggered inflammation via the RIG-I pathway
Ojha et al. Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections
Kim et al. Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish cells by AmiRNA
Chen et al. Defective antiviral responses of induced pluripotent stem cells to baculoviral vector transduction
Zhang et al. EBV‑BART‑6‑3p and cellular microRNA‑197 compromise the immune defense of host cells in EBV‑positive Burkitt lymphoma
Zheng et al. Immunosuppressive Tumor Microenvironment and Immunotherapy of Epstein–Barr Virus-Associated Malignancies
Amsden et al. Antiviral activities of interleukin-27: a partner for interferons?
Xu et al. Development of a primary culture system for haematopoietic tissue cells from Cherax quadricarinatus and an exploration of transfection methods
Zhang et al. Mouse circulating extracellular vesicles contain virus‐derived siRNAs active in antiviral immunity
CN109593723B (en) Mesenchymal stem cell for inhibiting immune reaction and preparation method and application thereof
CN102406653B (en) Anti-virus effect, implementation method and purpose of miRNA(ribose nucleic acid)
Grewal et al. The role of microRNAs in the pathogenesis of HIV-related lymphomas
Gentili et al. Natural killer cells in SARS-CoV-2-vaccinated subjects with increased effector cytotoxic cd56dim cells and memory-like CD57+ NKG2C+ CD56dim cells
CN103898050A (en) Recombinant mesenchymal stem cell capable of efficiently secreting nitric oxide as well as preparation method and application of recombinant mesenchymal stem cell
Chang et al. Pathogenesis of virus-associated human cancers: Epstein–Barr virus and hepatitis B virus as two examples
CN113234676B (en) Method for promoting duck T cell proliferation and application thereof
CN101948544B (en) FAT10 gene siRNA recombination analogue virus as well as preparation method and application thereof
Wei et al. Synergistic inhibition of avian leukosis virus subgroup J replication by miRNA-embedded siRNA interference of double-target
CN101979595B (en) Dual expression vector for immunostimulatory RNA and HBV target gene silence RNA, construction and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant