CN111676249A - Construction and application of RNA interference expression vector of targeting AQP4 gene - Google Patents
Construction and application of RNA interference expression vector of targeting AQP4 gene Download PDFInfo
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Abstract
The invention relates to the technical field of biomedicine, in particular to construction and application of an RNA interference expression vector of a targeted AQP4 gene. The invention provides an RNA interference expression vector of a targeted rat AQP4 gene, which comprises a DNA fragment which is designed according to a rat AQP4 gene sequence and can generate siRNA for interfering AQP4 protein expression; the invention also provides a method for constructing the vector, which comprises the following steps: a sense strand and an antisense strand of the siRNA are designed according to mRNA of AQP4, the middle is connected by a ring structure formed by 10 deoxynucleotides, an RNA poly III polymerase transcription termination site is connected behind the sense strand and the antisense strand, meanwhile, BamHI and XhoI enzyme cutting points are respectively designed at two ends of a template, the designed siRNA is synthesized into a double-stranded DNA sequence, and the double-stranded DNA sequence is connected with a carrier to obtain the RNA interference expression carrier of the targeted AQP4 gene. The vector constructed by the invention can be applied to preparation of a preparation for treating rat brain injury and research of the effect of AQP4 in body disease occurrence.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to construction and application of an RNA interference expression vector of a targeted AQP4 gene.
Background
Aquaporins (AQPS) are a group of proteins related to water permeability, wherein AQP1 is discovered firstly, and then 13 aquaporins including AQP0-AQP12 are discovered in succession, wherein AQP1, AQP3, AQP4, AQP5, AQP8 and AQP9 are mainly present in the brain tissue of mammals, particularly the presence and expression of AQP4 are most important, and are involved in the development of cerebral edema and various nervous system diseases. A large number of researches show that the expression increase of AQP4 is the main reason of cerebral edema caused by craniocerebral trauma, and the severity of the cerebral edema can be effectively evaluated by detecting the level of AQP4 in the cerebrospinal fluid after the craniocerebral trauma.
Numerous studies have shown that AQP4 expression plays a positive role in cytotoxic brain edema and a reverse role in vasogenic brain edema. In cytotoxic cerebral edema, the blood brain barrier is not destroyed, and water molecules enter the brain tissue through AQP4, so when the expression of AQP4 is increased, more water enters the brain tissue, and the caused cerebral edema is more serious. Papadopoulos et al[1]It is proposed that upregulation of AQP4 expression reduces the degree of cerebral edema in vasogenic cerebral edema. This is probably due to disruption of the blood brain barrier, penetration of serum proteins and isotonic fluids into the intercellular spaces in order to adapt to changes in hydrostatic pressure, resulting in swelling of the intercellular spaces. Therefore, the formation of the angiogenetic cerebral edema does not participate in AQP4, but the discharge of water molecules in the development process of the angiogenetic cerebral edema requires AQP4 as a mediation. AQP4 therefore appears to have an inhibitory effect on brain edema during this process.
Karmacharya et al[2]The research shows that the low-intensity ultrasonic stimulation applied to a rat brain edema model can reduce the local aggregation of AQP4, and compared with a control group, the water content of brain tissues and the table of AQP4 can be obviously reducedSo as to achieve the purpose. The other aspects are as follows: the treatment methods of sub-low temperature treatment, bone flap removing and pressure reducing, dexamethasone and the like can relieve cerebral edema, improve prognosis and improve survival rate by inhibiting the expression of AQP 4. It is therefore speculated that AQP4 is a therapeutic target to alleviate brain edema due to a variety of causes.
Currently, the research on AQP4 has been not limited to its transport effect on water, but has been directed to more research. Ishiyama et al[3]The detection result shows that the Meniere disease can specifically change the expression of AQP4 in supporting cells and mitochondrial proteins, so that the detection of the change of the expression level of AQP4 has extremely important significance for the diagnosis of the Meniere disease. Yasui[4]The AQP4 is found to play a key role in the process of the lymphatic system disorder, and various nerve function degeneration diseases and mental diseases caused by the lymphatic system pathway dysfunction can be better treated by developing an anti-AQP 4 medicament in the future. In addition, AQP4 is involved in pathological processes of diseases such as inflammatory brain diseases, neuromyelitis optica, Alzheimer's disease, etc., and is not described in detail herein. Although much effort has been made in basic research on AQP4, clinical transformation rate is still low, and few studies on the diagnosis and treatment of clinical diseases using AQP4 as a target have been reported, and in future studies, AQP4 can be brought closer to the clinic to develop a therapeutic agent and a diagnostic method for AQP 4.
The small RNA interference (RNAi) technology is a new method for suppressing gene expression developed in recent years, and is a post-transcriptional gene silencing mechanism (PTGS) triggered by double-stranded RNA (dsRNA). The high specificity of RNAi action may specifically inhibit pathogenic mutant alleles without affecting normal allele functions, so RNAi has wide application foreground in gene therapy. The RNAi mechanism of action is that after dsRNA is introduced into cells, under the action of Dicer enzyme (RNase III specific nucleotidase), the dsRNA is degraded into 21-23bp siRNA with 2 protruding basic groups at the 3' end, and is combined with intracellular exonuclease, unwinding enzyme, Argonaute protein and the like to form a ribonucleotide protein complex-RISC (RNA-inducing complementary complex) with a plurality of subunits, thereby efficiently and progressively mediating the degradation of endogenous homologous mRNA of the cells. In mammalian cells, introduction of long double-stranded RNA leads to extensive inhibition of gene expression, direct application of short double-stranded siRNA mediates RNAi with high efficiency, and avoids nonspecific gene inhibition effects. The short double-chain siRNA is easy to synthesize by a chemical method, simple and convenient to operate, high in transfection efficiency, small in toxic and side effects on cells or tissues, convenient to prepare in large scale and convenient to apply clinically, and has irreplaceable advantages in the aspect of drug development.
[1]Papadopoulos M C，Manley G T，Krishna S.Aquaporin4facilitatesreabsorption of excess fluid in vasogenic brain edema[J].FASEB J，2004，18(11)：1291-1293.
[2]Karmacharya M B，Kim K H，Kim S Y，et al.Low intensity ultrasoundinhibits brain edema formation in rats：potential action aqp4 membranelocalization[J].Neuropathology and applied neurobiology，2015，41(4)：e80-94.
[3]Ishiyama G，Lopez I A，Sepahdari A R，et al.Meniere’s disease：histopathology cytochemistry and imaging[J].Annals of the New York Academy ofSciences，2015，1343(1)：49-57.
[4]Yasui M.Roles of Aquaporins in brain disorders[J].Brain and Nerve，2015， 67(6)：733-738.
Disclosure of Invention
The technical problem to be solved by the invention is to construct an RNA interference expression vector targeting a rat AQP4 gene, and apply the RNA interference expression vector to research on diseases of which pathogenesis is related to AQP 4.
The specific scheme of the invention is as follows:
an RNA interference expression vector targeting a rat AQP4 gene, wherein the sequence of the RNA interference expression vector comprises a DNA fragment which is designed according to the sequence of the rat AQP4 gene and can generate siRNA for interfering the expression of AQP4 protein.
The construction method of the RNA interference expression vector targeting the rat AQP4 gene comprises the following steps:
design of AQP4mRNA (AQP 4-siRNA) sequence interfered by small molecule
The AQP4mRNA sequence (Gene ID: 612628) of rat was obtained through Gene Bank, several alternative AQP 4-siRNAs were designed according to the Gene design principle, and the optimal sequence was selected through preliminary experiments. Designing 2 complementary DNA template single strands according to each AQP4-siRNA sequence, wherein the DNA template single strands comprise a sense strand and an antisense strand, the middles of the DNA template single strands are connected by a ring structure formed by 10 deoxynucleotides, the rear part of the DNA template single strands is connected with an RNA poly III polymerase transcription termination site, meanwhile, the two ends of the template are respectively designed with BamHI enzyme cutting points and XhoI enzyme cutting points, Green Fluorescent Protein (GFP) is used as a positive control, and Scramble II is used as a negative control;
② constructing a plasmid vector for expressing AQP4-siRNA
Synthesizing 2 single-stranded DNA templates (including sense strand and antisense strand), taking equivalent DNA template synthetic fragments, annealing to generate double-stranded DNA, carrying out 4% agarose nucleic acid electrophoresis, purifying electrophoresis products, connecting the obtained double-stranded DNA with carrier fragments, synthesizing a plasmid carrier containing AQP4-siRNA, and comparing transfection effects of different carriers.
Furthermore, in the construction method of the RNA interference expression vector targeting the rat AQP4 gene, the double-stranded DNA linked vector in the step (II) is one of lentivirus, liposome or retrovirus.
The RNA interference expression vector of the target rat AQP4 gene is applied to preparing a preparation for treating rat brain injury.
The RNA interference expression vector targeting the rat AQP4 gene is applied to research on the effect of AQP4 in the occurrence of body diseases.
Has the advantages that:
the AQP4 gene is a target point for treating the currently researched hot disease, and the expression of a target gene can be more efficiently, specifically and conveniently inhibited by applying siRNA mediated RNAi technology than any previous means. The invention provides the siRNA sequence capable of efficiently inhibiting the expression of the AQP4 gene, and acts on brain injury cells, thereby laying an important foundation for the clinical treatment of the AQP4-siRNA applied to the pathological processes of diseases such as brain inflammatory diseases, neuromyelitis optica, Alzheimer disease and the like.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows: synthesis and vector construction of AQP4-siRNA
Selection of interference sites for AQP4 and design of oligodeoxynucleotides
Searching a complete mRNA sequence of rat AQP4 in an NCBI database, taking the complete mRNA sequence as a template, simultaneously designing and synthesizing a DNA fragment of siRNA aiming at the rat AQP4 sequence by using online siRNA tat finder software (http:// genomic. jp/sidedirect /) according to the requirement of the siRNA on the sequence by starting expression of an interference vector promoter by referring to an siRNA design principle. The sequence obtained by analysis of the sequence of AQP4mRNA1 (Gene ID: 612628) was imported on-line, the target Gene sequence with a GC content between 40% and 55% was selected as a potential preference and BLAST search was performed in the GenBank expressed sequence tag (EsT) database to compare the selected sequence with the corresponding genomic database and exclude sequences homologous to other coding sequences to determine it as a specific sequence.
In order to improve the efficiency of correctly annealing sequences into double-stranded DNA molecules, DNA containing siRNA sequences capable of generating interference target gene expression is designed into oligonucleotide sequences of a sense strand and an antisense strand according to the following principle, the oligonucleotide sequences are connected by a ring structure formed by 10 deoxynucleotides in the middle, an RNA poly III polymerase transcription termination site is connected behind the oligonucleotide sequences, meanwhile, BamHI and XhoI enzyme cutting sites are respectively designed at two ends of a template, Green Fluorescent Protein (GFP) is used as a positive control, and Scramble II is used as a negative control.
The above sequence is synthesized into single-stranded oligonucleotide by chemical synthesis method.
Annealing of DNA fragments to become double stranded
An equal amount of 100mmol/L of the DNA synthesis fragment was taken, incubated at 95 ℃ for 5min in annealing buffer (100mmol/L NaCl), slowly cooled to room temperature, and then purified using a DNA purification kit.
3. Construction of expression vectors
The obtained double-stranded DNA is connected with a carrier fragment to synthesize a plasmid carrier (lentivirus, liposome and retrovirus) containing AQP4-siRNA, and the transfection effects of different carriers are compared. It was transformed into DH 5. alpha. competent cells, and recombinant clone screening and vector DNA sequencing were performed to confirm the correctness of the inserted single stranded DNA sequence.
4. Efficient AQP4-siRNA sequence screening
Transfecting the constructed plasmid vector expressing the AQP4-siRNA into a glial cell, extracting plasmids by using a QIAGENPmid kit (Cat.No:12123) kit, selecting cells with good growth state for inoculation, transfecting when the cell density is 80%, selecting 1 best AQP4-siRNA sequence by fluorescent quantitative PCR when the cell density reaches 90%, and extracting and purifying the plasmids for later use.
Example two: rat AQP4-siRNA interference carrier interfering AQP4 expression
The best rat AQP4-siRNA interference vector explored in the example I is used for transfecting glial cells, RNA and protein of the cells are harvested, and the change of mRNA level and protein level expression quantity of AQP4 is detected, so that whether the AQP4-siRNA interference vector can silence AQP4 genes in the cells is proved.
Example three: application of rat AQP4-siRNA interference vector in animal model
1. Establishing animal model of brain trauma
Using PinPointTMA craniocerebral trauma impactor (Hatteras, USA) establishes a moderate cerebral trauma model according to an improved Feeney method (moderate cerebral trauma parameters: impact speed is 2.5m/s, impact depth is 4.0mm, impact time is 0.85s, impact head diameter is 4mm, and cortical medullary injury is generated during trauma but no obvious intracerebral injury exists). Rats were first anesthetized intraperitoneally with 10% chloral hydrate (0.4ml/100 g). Then disinfecting the rat head with iodophor or iodine skin disinfectant, preparing skin, cutting the scalp along the median line to about 2.0cm, and applying dental bench-type electric drill (307-2B, Kazaki precision tool technology, Inc. of Shanghai Ling) to treatDrilling a skull with a drill bit with the diameter of 1.5mm at a position 2.5mm behind bregma and 2.5mm to the right of a sagittal line to ensure the integrity of dura mater, drilling a bone window with the diameter of about 5mm by using a mosquito vascular clamp, fixing a rat in a prone position, fixing the head in a stereotaxic instrument (ST-7Setagaya-Ku, Tokyo, Japan), performing brain trauma according to parameters of a moderate brain trauma model, closing the bone window by bone wax after trauma, suturing the scalp, and performing heat preservation treatment.
Except for the treatment mode of opening the skull window without impacting in the control group, the other operations are the same as those in the trauma group; the placebo treatment group uses a micro-syringe (Ningbo city Zhenhai glass instrument factory) with the specification of 25 mu L to suck 20 mu L of liposome solution without RNAi-AQP4 interference fragment to inject into the cerebral ventricles at a constant speed within 20min, then pulls out after leaving a needle for 5min, then seals the bone window with bone wax, sutures the scalp after hemostasis, sterilizes the operative area, marks the rat and then keeps the temperature with a hot water bag; the operation of the treatment group with the interfering agent was identical to that of the placebo treatment group except that the injected drug was a liposome-encapsulated RNAi-AQP4 interfering agent.
2. Multi-modality MRI examination
6 rats in each group were anesthetized with isoflurane (Aerorane, Baite International Ltd.), placed in a Veprott V-1 animal anesthesia machine (Vetequip, USA) for 7.0T multi-mode MRI scan, a BRUKER 7.0T small animal MRI scanner (BRUKER, GERMANY) was used, the rats were placed in a dedicated phased array experimental animal coil in the supine position, the scan was centered on apparent crossing, the scan parameters Localizer: TR 100ms, TE 3ms, layer thickness 1mm, interval 0mm, visual field 40mm × 40mm, matrix 256 × 6. T multi-mode MRI scan2TR 4000ms, TE 35ms, layer thickness 1mm, spacing 0mm, field of view 35mm × 35mm, matrix 256 ×, SWI TR 700ms, TE 12ms, layer thickness 1mm, spacing 0mm, field of view 35mm × 35mm, matrix 384 ×, DWI TR 3000ms, TE22ms, layer thickness 1mm, spacing 0mm, field of view 35mm × 35mm, matrix 128 × 128, respectively selected with b value of 0s/mm2、1000s/mm2And (6) imaging. And transmitting the obtained image to a post-processing workstation after scanning is finished, measuring the rADC value of the damage area of the maximum layer of the high signal, and according to a formula: ADC (mm)2In (S1/S0)/(b0-b1), S1 is the signal intensity of b 1000, S0 is the signal intensity of b0, and In is the natural logarithm.
3 processing and analysis of images
Selecting a region of interest (ROI) layer corresponding to an edema zone around a wound core region for pathological observation, wherein the ROI is as small as possible (the ROI is uniformly selected to be 0.6mm at this time)2) (ii) a Each ROI measurement needs to be performed by at least 2 experienced physicians, 2 physicians are not aware of the design of the experiment and the expected results in advance, and then the results are analyzed to select ROIs while avoiding bleeding regions as much as possible (SWI images show low signal) to avoid affecting the accuracy of the ADC.
The calculation formula is as follows:
rs-T2WI(T2WI relative high signal area) ═ × 100% (high signal area in the wound area/total area of ipsilateral cerebral hemisphere);
rs-SWI (SWI relatively high signal area) ═ 100% of (low signal area in wound area/total area of ipsilateral cerebral hemisphere);
rs-DWI (DWI relatively high signal area) ═ 100% of (high signal area in wound area/total area of ipsilateral cerebral hemisphere);
r-ADC (relative ADC value) × (ADC value of wound side ROI/ADC value of contralateral mirror region ROI) × 100%.
(ADC (mm2/S) ═ In (S1/S0)/(b0-b1), S1 is the signal intensity of b ═ 1000, S0 is the signal intensity of b ═ 0, In is the natural logarithm)
4. Pathological morphology observation (optical lens)
After 7.0T multi-mode MRI scanning of each group of rats is finished, 2 rats are randomly selected from the rats, 1% sodium pentobarbital (0.4ml/100g) is subjected to intraperitoneal injection and excessive anesthesia, then the rats are fixed on an animal experiment table in a supine position, the chest is opened from the lower part of the sternal xiphoid process after skin preparation, the heart is exposed, important thoracic cavity and abdominal great vessels are intentionally avoided in the chest opening process, a scalp vein needle is connected with a 50ml syringe to be inserted into the left ventricle after the heart is fully dissociated, the left ventricle is required to be connected with a 50ml syringe, the left ventricle is required to run along the left ventricular outflow tract, the right ventricle is required to be prevented from being inserted, the right ventricle is required to be cut, dark red blood flows out, about 120ml of physiological saline is injected into the left ventricle to flow out clear liquid from the right ventricle, 150ml of 4% paraformaldehyde solution is injected at constant speed until the lung of the rat is white, the liver is pink, the four limbs and, coronal section was dissected with the largest wound center seen with naked eyes as the center. Routine paraffin embedding and sectioning, routine HE staining, optical microscopy of wound center and surrounding tissues and photography. After HE staining, the cytoplasm was red and the nucleus blue.
5. Statistical analysis of data
rs-DWI、r-ADC、rs-T2All the data of the measured data, such as the expression of WI, rs-SWI and AQP4, are as follows
Statistical analysis was performed using SPSS 21.0 software, and data were analyzed by oneway-ANOVA for inter-group comparisons and LSD for intra-group comparisons, with statistical significance of P < 0.05.
6. Results
6.1 MM-MRI Performance
No abnormality in the imaging performance of the control group, T2The WI and DWI have no brain edema signal, the SWI have no abnormal low signal, the iconography performance of the wound group has no statistical significance (P is less than 0.05) compared with each index of the placebo treatment group, and compared with the wound group: rs-DWI and rs-T2WI increased gradually over time, with 6h increase being most pronounced (P < 0.05) and reaching a maximum at 12 h. The r-ADC rose at 1h, reached the highest at 6h, then began to decline, 12h to the lowest (P < 0.05). The placebo-treated group and the trauma group varied in accordance with the changes of rs-DWI, r-ADC and rs-T2Differences in WI and rs-SWI are not statistically significant (P)>0.05). Comparing the interferon treatment group with the trauma group, rs-DWI and rs-T2WI decreased significantly (P < 0.05) at 6h and 12 h. r-ADC rose back at 6h and 12h (P < 0.05); no statistical significance was observed at each time point of rs-SWI (P)>0.05), see tables 1-4.
Table 1: experiment of each group of rs-T at different time points2WI expression (%) (N ═ 6)
Table 2: various time points were tested for rs-DWI expression (%) (N ═ 6)
Table 3: various time points experiments were performed on each group of r-ADC expression (%) (N ═ 6)
Table 4: various time points were tested for rs-SWI expression (%) (N ═ 6)
6.2 pathological manifestations
Control group: no obvious cellular edema is seen, and no abnormal morphology of glial cells and vascular endothelial cells is seen.
The placebo treatment group and the wound group have similar performances, edema and fuzzy gaps can be seen at the periphery of vascular endothelium 1h after the wound, and mild edema can be seen in part of glial cells, and the main pathological condition is angiogenetic edema accompanied by a small amount of intracellular edema. TP can see obvious swelling of cell volume after 6h of trauma, and the obvious swelling is mainly manifested by mixed edema which mainly takes intracellular edema as a main part; the 12hTP cell swelling degree and the angiogenetic edema are obviously increased compared with the former, the pathological condition is mixed edema, but the intracellular edema and the angiogenetic edema degree are further aggravated than 1h and 6h, and the edema degree reaches the peak at 12 h.
Treatment group with interferents: TP angiogenetic edema and a small amount of intracellular edema are not obviously relieved at 1 h; the intracellular edema is obviously relieved within 6 hours, and the angiogenetic edema can be relieved to a certain extent; the degree of intracellular edema and angiogenetic edema are obviously relieved within 12 hours.
The experiment in example one shows that the edema changes of the placebo-treated group and the trauma group are basically consistent, and the differences of the rs-DWI, the r-ADC, the rs-T2WI and the rs-SWI are not statistically significant (P > 0.05). In the interferon treatment group, compared with the trauma group, rs-SWI at each time point has no statistical significance (P >0.05), which means that the interferon has no treatment effect on the core injury area. Data changes of rs-DWI and rs-T2WI in 1h have no statistical significance, which shows that AQP4-RNAi has poor treatment effect on early-stage traumatic angiogenetic edema, is obviously reduced in 6h and 12h, has statistical significance (P is less than 0.05) in difference, and shows that the gene interference agent has certain treatment effect on intracellular edema and late-stage angiogenetic edema. The AQP4-siRNA encapsulated by liposome is applied to a rat brain trauma model, and has important significance for researching the effect of AQP4 in brain diseases.
Claims (5)
1. An RNA interference expression vector targeting rat AQP4 gene, characterized in that: the sequence of the RNA interference expression vector comprises a DNA fragment which is designed according to the sequence of rat AQP4 gene and can generate siRNA for interfering the expression of AQP4 protein.
2. The method for constructing an RNA interference expression vector targeting rat AQP4 gene according to claim 1, characterized in that: the method comprises the following steps:
design of small molecule interference AQP4-siRNA sequence
The AQP4mRNA sequence of rats was obtained by Gene Bank, Gene ID: 612628, alternative AQP4-siRNA is designed according to the gene design principle, and the optimal sequence is selected through preliminary experiments. Designing 2 complementary DNA template single strands according to each AQP4-siRNA sequence, wherein the DNA template single strands comprise a sense strand and an antisense strand, the middles of the DNA template single strands are connected by a ring structure formed by 10 deoxynucleotides, the rear part of the DNA template single strands is connected with an RNA poly III polymerase transcription termination site, meanwhile, the two ends of the template are respectively designed with BamHI enzyme cutting points and XhoI enzyme cutting points, Green Fluorescent Protein (GFP) is used as a positive control, and Scramble II is used as a negative control;
② constructing a plasmid vector for expressing AQP4-siRNA
Synthesizing 2 single-stranded DNA templates, taking equivalent DNA template synthesis fragments, annealing to generate double-stranded DNA, carrying out 4% agarose nucleic acid electrophoresis, purifying electrophoresis products, connecting the obtained double-stranded DNA with carrier fragments, synthesizing a plasmid carrier containing AQP4-siRNA, and comparing transfection effects of different carriers.
3. The method for constructing RNA interference expression vector of claim 2, which targets the rat AQP4 gene, characterized in that: the vector for connecting the double-stranded DNA in the second step is one of lentivirus, liposome or retrovirus.
4. The RNA interference expression vector of claim 1 targeting rat AQP4 gene is used for preparing a preparation for treating rat brain injury.
5. The RNA interference expression vector of claim 1, which targets the rat AQP4 gene, is used for researching the role of AQP4 in the occurrence of body diseases.
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