CN106176795B - Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts - Google Patents
Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts Download PDFInfo
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Abstract
The invention discloses a kind of application of prawn miR-35 in prawn is antiviral and inhibition human tumour shifts.Prawn miR-35 is the distinctive miRNA of prawn, its base sequence is 5 '-AACUGUGAGAAGUUCCGGU-3 ', shrimp disease prevention and treatment is directed to White Spot Syndrome Virus (WSSV), and the tumour cell for inhibiting human tumour transfer to be directed to is breast cancer cell MDA-MB-231.
Description
Technical field
The present invention relates to the application of a miRNA, more particularly, to a kind of prawn miR-35 prawn is antiviral and suppression
Application in human tumour transfer processed, this, which is more advantageous to, understands the mechanism that miRNA regulates and controls across species.
Background technique
MicroRNA (miRNA) is small non-coding RNA, is about made of 18-25 nucleotide, and many miRNA are in difference
It is all highly conserved in species.MiRNA is the important factor of controlling gene expression, horizontal tune after their participation genetic transcriptions
Control.They hold non-coding region (3 ' UTR) to bind directly by seed sequence and target gene 3 ', to inhibit the translation of target gene
Or mediate the degradation of target gene.
Prawn has very big commercial value as important one of aquatic products.However, due to prawn white spot syndrome disease
Disease of prawn caused by virus infections such as malicious (WSSV) becomes the thorny problem of prawn culturing.It is right after virus infection prawn
Significant change occurs for the express spectra of miRNA in shrimp body, illustrates that miRNA may participate in the antiviral immunity reaction of prawn.Therefore, may be used
Host and virus miRNA as target spot, to be drawn by the infection and duplication that influence viral in prawn body to mitigate virus infection
The disease of prawn risen, while improving the immune defense ability of prawn.
In the past studies have shown that the miR-168a of rice is able to suppress mammal LDL 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 and silver
Flower coding miR-2911, can wide application in influenza virus (including H1N1, H5N1 and H7N9)；The miRNA of external source can be with
Fetus is passed to by placenta, influences the gene expression of fetus.This illustrates that Mirnas of plant is able to enter in animal body simultaneously " transboundary "
The physiological activity of animal is influenced, biological function is played.Early-stage study the result shows that, the CHI3L1 albumen of macrophages secrete can
To promote the migration of tumour cell.It therefore, can be using the CHI3L1 albumen in macrophage as the target spot of oncotherapy.
Summary of the invention
It is existing in the prior art above-mentioned it is an object of the invention to overcome in order to solve the problems, such as background technique
Deficiency, and screen and acquire a miRNA (miRNA-35), this miRNA can inhibit WSSV virus to the infection of prawn and
It can inhibit the migration of tumour cell by acting on 3 ' UTR of CHI3L1.
Prawn miR-35 of the invention is the distinctive miRNA of prawn, and obtaining its base sequence by sequencing 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).
The tumour cell that the application that the present invention inhibits human tumour to shift is directed to is breast cancer cell MDA-MB-231.
The target gene of the prawn miR-35 inhibiting effect is the M2 after the induction differentiation of human monocyte cell line THP-1 cell
CHI3L1 gene in type macrophage.
The beneficial effects of the present invention are:
Prawn miRNA-35 of the present invention can not only inhibit virus in the intracorporal infection of prawn and duplication, play antiviral work
With, moreover it is possible to it interacts with 3 ' UTR of CHI3L1mRNA, the expression of CHI3L1 gene in low macrophage is struck, to influence tumour
The transfer of cell.
Detailed description of the invention
Fig. 1 is influence of the miR-35 to WSSV copy number in prawn body.
Fig. 2 is influence of the miR-35 to the prawn death rate.
Fig. 3 is influence of the miR-35 to CHI3L1 gene expression amount in M2 type macrophage.
Fig. 4 is influence of the miR-35 to CHI3L1 expressing quantity in M2 type macrophage.
Fig. 5 is the influence that miR-35 migrates breast cancer cell MDA-MB-231.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
The embodiment of the present invention is as follows:
Embodiment first prepares experiment sample:
Prawn miR-35, negative control miRNA and control miRNA are specifically to utilize In vitro
Transcription T7Kit (Takara), which is transcribed in vitro, to be obtained.
Preparation is used 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 '.
Preparing the base sequence for inhibiting control miRNA, the control miRNA of human tumour transfer embodiment is
SEQ ID NO.3:5 '-UUCUCCGAACGUGUCACGUUU-3 '.
Embodiment 1
1) preparation experiment group and control group:
Experimental group by prawn miR-35 and the WSSV virus liquid for being diluted in phosphate buffer solution (PBS) while being injected into
In prawn body, prawn miR-35 injects the dosage of 30 μ g miRNA, contains 10 in WSSV virus liquid4A WSSV virion；
First control group by negative control miRNA and is diluted in the WSSV virus liquid of phosphate buffer solution (PBS) simultaneously
It is injected into prawn body, negative control miRNA injects the dosage of 30 μ g miRNA, contains 10 in WSSV virus liquid4A WSSV disease
Virion；
The WSSV virus liquid for being diluted in phosphate buffer solution (PBS) is individually injected into prawn body by the second control group,
Contain 10 in WSSV virus liquid4A WSSV virion；
Phosphate buffer solution (PBS) is individually injected into prawn body by third control group, and WSSV is free of in PBS solution
Virion.
2) experiment of both then carrying out in the following way:
A) in experimental group, the first control group, the second control group and third control group, injection prawn for 24 hours, 36h and 48h
Afterwards, the cheek silk of each group prawn is collected, the genome in cheek silk is extracted, viral copies therein are detected by real-time PCR
Number.
Viral copy number result is as shown in Figure 1, visible in figure inject prawn for prawn miR-35 and WSSV virus liquid simultaneously
Later, the intracorporal viral copy number of prawn more only injects WSSV virus liquid group and while injecting negative control miRNA and WSSV disease
Venom group is decreased significantly, and illustrates that prawn miR-35 can effectively inhibit the infection and duplication of virus.
B) in experimental group, the first control group, the second control group and third control group, injection prawn 12h, for 24 hours, 36h,
After 48h and 72h, the death rate of each group prawn is counted.
The mortality results of prawn are as shown in Fig. 2, visible in figure inject prawn miR-35 and WSSV virus liquid pair simultaneously
After shrimp, the death rate of prawn more only injects WSSV virus liquid group and while injecting negative control miRNA and WSSV virus liquid group
It is decreased significantly, illustrates that prawn miR-35 can effectively improve the survival rate of prawn.
Embodiment 2
1) M2 type macrophage is obtained, M2 type macrophage is differentiated by the induction of THP-1 monocyte, specific method
Are as follows: THP-1 cell 6h is first handled 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 control group:
Experimental group: prawn miR-35 is transfected into M2 type macrophage using lipo RNAiMAX transfection reagent (Invitrogen)
In cell, after transfecting 48h, cell and its culture supernatant are collected.
First control group: control miRNA is transfected into using lipo RNAiMAX transfection reagent (Invitrogen)
In M2 type macrophage, after transfecting 48h, cell and its culture supernatant are collected.
Second control group: M2 type macrophage is without any processing, after 48h, collects cell and its culture supernatant.
3) experiment of both then carrying out in the following way:
A) for experimental group, the first control group and the second control group, the RNA in the M2 type macrophage of collection is extracted,
And the expression of CHI3L1 gene is detected, and as a result as shown in figure 3, in figure after visible transfection prawn miR-35, M2 type macrophage
Significant downward has occurred in the expression quantity of intracellular CHI3L1 gene mRNA levels, illustrates that prawn miR-35 can effectively inhibit
The expression of CHI3L1 gene.
B Western blot test) is carried out to the M2 type macrophage of collection, detects the variation of CHI3L1 protein level.
As a result as shown in figure 4, in figure after visible transfection prawn miR-35, the expression quantity of CHI3L1 protein level in M2 type macrophage
Significant downward has occurred, further illustrates that prawn miR-35 can effectively inhibit the expression of CHI3L1 gene.
C) the M2 type macrophage culture supernatant of collection is trained in transwell altogether with breast cancer cell MDA-MB-231
It supports, wherein transwell upper chamber spreads the MDA-MB-231 cell of serum free medium resuspension, and room is M2 type under transwell
Macrophage culture supernatant.Co-culture 12h after, detection migrate to the MDA-MB-231 cell of transwell lower membrane, and with show
Micro mirror counts.
The breast cancer cell MDA-MB-231 migration results situation obtained is counted as shown in figure 5, visible when M2 type is huge in figure
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 inhibit the migration of breast cancer cell MDA-MB-231.
As seen from the above-described embodiment, prawn miRNA-35 of the invention can inhibit viral in the intracorporal infection of prawn and multiple
System plays antivirus action, moreover it is possible to interact with 3 ' UTR of CHI3L1mRNA, strike the table of CHI3L1 gene in low macrophage
It reaches.
The present invention relates to RNA sequence it is as follows:
SEQ ID NO.1: prawn miR-35
Source: artificial synthesized
Sequence: 5 '-AACUGUGAGAAGUUCCGGU-3 '；
SEQ ID NO.2: negative control miRNA
Source: artificial synthesized
Sequence: 5 '-UAAGCGUAGUAUUGCAGCG-3 '；
SEQ ID NO.3:control miRNA
Source: artificial synthesized
Sequence: 5 '-UUCUCCGAACGUGUCACGUUU-3 '.
Claims (1)
1. a kind of prawn miR-35 is in prawn disease and inhibits the application in human tumour transfer pharmacy, base sequence is
SEQ ID NO.1:5 '-AACUGUGAGAAGUUCCGGU-3 ', the shrimp disease refer to White Spot Syndrome Virus
(WSSV)；The human tumour is breast cancer cell MDA-MB-231.
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| 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 |
| CN109609504B (en) * | 2019-01-10 | 2022-03-22 | 汕头大学 | Application of miR-276 simulant in preparation of broad-spectrum white spot syndrome virus resistant medicine |
| 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 |
| CN116262919A (en) * | 2021-12-14 | 2023-06-16 | 成都凌泰氪生物技术有限公司 | Nucleic acid molecule based on miRNA-2911 |
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| 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 |
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|---|---|---|---|---|
| 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 |
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