CN114958855B - siRNA and SIRT6 low expression cell line for promoting endothelial cell apoptosis - Google Patents
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Abstract
The invention discloses an siRNA and SIRT6 low expression cell line for promoting endothelial cell apoptosis. The invention utilizes the RNA interference (RNAi) technology to obtain SIRT6 knockdown siRNA and shRNA, the sequence of the siRNA is shown in SEQ ID NO 1-2, the sequence of the shRNA is shown in SEQ ID NO 5-6, and the SIRT6 knockdown can obviously promote the oxidative stress induced endothelial cell apoptosis. In addition, the invention constructs a micro-vascular endothelial cell model with low SIRT6 expression, which can be used for researching the effect of SIRT6 in the generation and development of the inter-endothelial apoptosis, and is helpful for revealing the mechanism of the generation and development of the endothelial apoptosis and providing a useful clue for treating cardiovascular diseases caused by the endothelial apoptosis. The invention has important application value.
Description
Technical Field
The invention relates to an siRNA and SIRT6 low expression cell line for promoting endothelial cell apoptosis, belonging to the technical fields of medical molecular biology and genetic engineering.
Background
In recent years, despite the rapid development of treatments for cardiovascular diseases, they remain a high mortality disease, and new treatments and interventions are therefore needed to treat cardiovascular diseases and improve prognosis. Vascular endothelial cells are the innermost cells of the blood vessel, and are capable of regulating the exchange of body fluids, nutrients and metabolites, and are critical for maintaining vascular health. In recent years, there is growing evidence that endothelial dysfunction is a major contributor to diseases such as atherosclerosis, myocardial infarction, hypertension, diabetes, etc. Therefore, maintaining endothelial cell function, protecting vascular endothelial barrier is an important means for preventing and treating cardiovascular related diseases.
Endothelial apoptosis is a programmed death that maintains cellular homeostasis in response to external stimuli and to accomplish replacement of old and new cells. The physiological function of endothelial cells involves a balance between pro-apoptotic and anti-apoptotic signals, and disturbance of this balance is one of the important contributors to endothelial dysfunction, manifested by increased vascular permeability, inflammatory cell infiltration, and endothelial-dependent vasodilation disorders. Thus, maintaining a balance between endothelial cell apoptosis and anti-apoptosis is a potential target for the prevention and treatment of a variety of cardiovascular diseases. Then, there is currently no clinically effective intervention target for endothelial apoptosis. Elucidating regulatory molecules and signaling pathways involved in endothelial apoptosis of cardiovascular diseases is of great importance for the prevention and treatment of cardiovascular diseases.
Sirtuin6 (SIRT 6), one of the important members of the Sirtuin protein family, has NAD + A variety of catalytic activities for dependent deacetylation, mono-ADP ribosylation and de-long chain fatty acylation. SIRT6 has been shown to maintain genome stability, glucose/lipid metabolic homeostasis, and to inhibit inflammation and oxidative stress as a longevity protein, playing a beneficial role in age-related disorders. Recent studies have shown that SIRT6 is involved in the development of a variety of cardiovascular diseases such as atherosclerosis, heart failure, cardiac hypertrophy, and myocardial ischemia reperfusion injury. Importantly, SIRT6 can inhibit apoptosis, inflammation and aging of endothelial cells, maintaining endothelial cells and vascular function. However, the specific mechanism by which SIRT6 inhibits endothelial apoptosis and related organ damage is not yet known. Furthermore, finding new targets for SIRT6 to regulate endothelial apoptosis and exploring related molecular signaling pathways remains a key challenge.
Small interfering RNAs (Small interfering RNAs, sirnas) can induce degradation by recognizing complementary specific mrnas, thereby inducing specific gene silencing and inhibiting expression of target genes. siRNA can specifically reduce or shut down expression of a specific gene, and thus has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. The plasmid, slow virus and adenovirus of the short hairpin RNA (shRNA) gene are transfected or infected into cells, so that siRNA is generated in the cells, the effective delivery of target cells is achieved, and the expression of the target genes is continuously inhibited. Compared with other vectors, the viral shRNA vector can directly and efficiently infect cells to carry out gene silencing research, has stable transfection effect, and can obtain RNA interference effect with long-term stability.
Disclosure of Invention
The invention aims to realize SIRT6 low expression by using an siRNA, thereby promoting endothelial cells to undergo apoptosis under the condition of oxidative stress and constructing a stable micro-vascular endothelial cell line with SIRT6 low expression.
In order to achieve the above object, the present invention provides a substance capable of inhibiting SIRT6 expression, comprising:
siRNA comprising a sense strand shown in SEQ ID NO. 1 and an antisense strand shown in SEQ ID NO. 2;
or taking the siRNA as a target point, and synthesizing shRNA by a shRNA expression system, wherein the shRNA comprises the sequence shown in SEQ ID NO:5 and the sense strand shown in SEQ ID NO: 6.
The invention also provides an expression vector, which comprises a sequence shown as SEQ ID NO:1-2 or SEQ ID NO: 5-6.
Preferably, the expression vector is selected from viral, fungal or bacterial expression vectors.
The invention also provides a cell containing the expression vector, and the cell is a human microvascular endothelial cell HMEC-1.
The invention also provides a preparation method of the recombinant lentivirus expression vector, which comprises the following steps:
step 1: setting SEQ ID NO:5 and the single stranded DNA molecule shown in SEQ ID NO:6 annealing the single-stranded DNA molecule to form a DNA double-stranded fragment with cohesive ends;
step 2: restriction enzyme 1 and restriction enzyme 2 carry out enzyme digestion on a lentiviral vector pHBLV-U6-MCS-PGK-PURO, and the DNA double-stranded fragment obtained in the step 1 is connected into the vector through T4ligase to obtain the recombinant lentiviral vector.
The invention also provides a construction method of the SIRT6 low-expression microvascular endothelial cell line, which comprises the following steps:
step 1: the recombinant lentiviral vector prepared by the preparation method is combined with a virus packaging auxiliary plasmid pSPAX2 and pMD2G to form a three-plasmid lentivirus system for virus packaging, and 293T cells are transfected to obtain virus liquid;
step 2: and (3) infecting the microvascular endothelial cell line by using the virus liquid obtained in the step (1), and screening to obtain the microvascular endothelial cell line with SIRT6 low expression.
The invention also provides the use of an agent capable of inhibiting SIRT6 expression as described above in any one of the following A1) to A6):
a1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
a2 A) of: preparing a product for constructing a microvascular endothelial cell model with SIRT6 low expression;
a3 A) of: preparing a product for constructing an animal model of SIRT6 low expression;
a4 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
a5 A) of: constructing a micro-vascular endothelial cell model with SIRT6 low expression;
a6 A) of: an animal model of SIRT6 low expression is constructed.
The invention also provides the application of the recombinant lentivirus expression vector prepared by the preparation method in any one of the following B1) and B6):
b1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
b2 A) of: preparing a product for constructing a microvascular endothelial cell model with SIRT6 low expression;
b3 A) of: preparing a product for constructing an animal model of SIRT6 low expression;
b4 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
b5 A) of: constructing a micro-vascular endothelial cell model with SIRT6 low expression;
b6 A) of: an animal model of SIRT6 low expression is constructed.
The invention also provides the use of the above-described cells in any one of the following C1) C4):
c1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
c2 A) of: preparing a product for constructing an animal model of SIRT6 low expression;
c3 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
c4 A) of: an animal model of SIRT6 low expression is constructed.
The invention also provides application of the SIRT6 low-expression microvascular endothelial cell line constructed by the construction method in any one of the following D1) D4):
d1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
d2 A) of: preparing a product for constructing an animal model of SIRT6 low expression;
d3 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
d4 A) of: an animal model of SIRT6 low expression is constructed.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses RNAi technology to knock down SIRT6 gene, which can promote the endothelial cell apoptosis of HMEC-1 under the condition of oxidative stress; the invention utilizes a micro-vascular endothelial cell model with SIRT6 low expression constructed by a recombinant lentiviral vector, can be used for researching the effect of SIRT6 in the occurrence and development of the inter-endothelial apoptosis, is helpful for revealing the mechanism of the occurrence and development of the endothelial apoptosis, and provides a useful clue for treating cardiovascular diseases caused by the endothelial apoptosis; therefore, the invention has important application value.
Drawings
FIG. 1 is a graph showing the interference effect of RT-qPCR identification SIRT6 siRNA on SIRT6 gene;
FIG. 2 is a graph showing the effect of RTqPCR in identifying SIRT6shRNA interference on SIRT6 gene;
FIG. 3 shows the inhibitory effect of Western blot identification SIRT6shRNA on SIRT6 protein;
FIG. 4 is a graph showing the identification of gene expression of SIRT6shRNA on HMEC-1 apoptosis markers under oxidative stress by RT-qPCR;
FIG. 5 shows protein expression of a biomarker for identifying the endothelial apoptosis of SIRT6 shRNA-interfered HMEC-1 in the presence of oxidative stress by Western blot;
in the figures above, P <0.001 shows that there was a significant difference between the two groups after statistical analysis.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Example 1: design and synthesis of SIRT6 gene mRNA targeting siRNA (SIRT 6 siRNA for short)
By looking up the sequence of human SIRT6 in NCBI GenBank database (www.ncbi.nlm.nih.gov/gene), siRNA targeting SIRT6 gene was designed as follows: (1) Designing an interfering target region on the cDNA sequence of SIRT6 to be located 50-100nt downstream of the initiation codon (AUG) while avoiding intron sequence design; (2) Starting from the transcription initiation codon (AUG) of human SIRT6, AA (N) was found 19 ) TT or NA (N) 21 ) Or NAR (N) 17 ) YNN, wherein N represents any nucleotide, R represents purine (A or G), Y represents pyrimidine (C or U); (3) Design was performed avoiding the 5'UTR and the 3' UTR, i.e., the non-coding regions of transcripts; (4) The region within 50-100bp of the start codon and the stop codon is avoided for design; (5) Design avoiding extension of 4 or more identical bases; (6) Avoiding G or C base content<30% or>60% of the area is designed; (7) avoiding single nucleotide polymorphism sites; (8) Homology alignment was performed on the gene library data using NCBI BLAST (www.ncbi.nlm.nih.gov/BLAST /) to avoid off-target effects.
According to the above principle, the sense strand and the antisense strand of SIRT6 siRNA and control siRNA (siRNA NC) were designed, respectively, and the above double-stranded siRNA was synthesized by Shanghai qing biosciences limited.
The purities of SIRT6 siRNA and siRNA NC were both greater than 99%,2 OD/tube. The method can be directly used for cell transfection by using water without nuclease for dissolution. The nucleotide sequences of the sense and antisense strands of SIRT6 siRNA and siRNA NC are shown in table 1.
TABLE 1 nucleotide sequences of SIRT6 siRNA and siRNA NC
Example 2: detection of SIRT6 siRNA effect on SIRT6 interference in human microvascular endothelial cell HMEC-1
1. Transfection
(1) HMEC-1 was inoculated into 6-well plates and cultured in MCDB131 medium containing 10% fetal bovine serum, and transfection was started when the cell density was about 70% at about 80%.
(2) By means of Lipofectamine TM 3000 transfection reagents (Invitrogen, USA) 2 siRNAs (SIRT 6 siRNA and siRNA NC) were transfected into HMEC-1 cells, respectively, as follows: (1) 125 μl of Opti-MEM was taken TM Medium and 3.75. Mu.l Lipofectamine TM 3000 The Reagent is in an EP pipe, and is blown and evenly mixed; (2) mu.l of Opti-MEM was taken TM Blowing Medium and 5 mug siRNA into another EP tube, and mixing uniformly; (3) the two-tube systems of (1) and (2) were mixed and after incubation for 10-15min at room temperature, the transfection system was added to each well for transfection.
(3) The cells were placed at 37℃in 5% CO 2 Is cultured in an incubator and is continuously transfected for 48-72 hours.
2. cDNA acquisition
Total RNA of the above-described HMEC-1 transfected with SIRT6 siRNA and siRNA NC was extracted with Trizol (Japanese Takara Co., ltd.) respectively, and subjected to reverse transcription to obtain cDNA. The specific operation steps of reverse transcription are as follows:
(1) Digestion of DNA: mu.g of HEMC-1 total RNA transfected with siRNA, 2. Mu.l of 5X gDNA Eraser Buffer and 1. Mu.l of gDNA Eraser were added to the PCR tube and made up to 10. Mu.l with nuclease-free water. The reaction system was then thoroughly mixed and centrifuged and incubated at 42℃for 2min in a metal bath to digest the DNA.
(2) Preparing a reverse transcription reaction system: mu. l PrimeScriptRT Buffer 2, 4. Mu.l RT primer Mix, 1. Mu.l RNase Free dH were added to each tube after digestion of DNA in step (1) 2 O and 1 mu l PrimeScriptRT Enzyme Mix I, and blow-mixing and centrifuging.
(3) Placing the reaction system in a PCR instrument, performing reverse transcription reaction according to the conditions of 37 ℃,15min,85 ℃ and 5s to obtain the cDNA of the HMEC-1 after siRNA transfection, and preserving at 4 ℃.
3. qPCR identification of SIRT6 siRNA interference effect on SIRT6 gene
Preparation of qRT PCR reaction System (10. Mu.l): 0.3. Mu.l of upstream primer, 0.3. Mu.l of downstream primer, 5. Mu. l TB Green Premix Ex TaqII (Tli RNaseH Plus), 4. Mu.l of RNase free dH 2 O and 0.4. Mu.l of the cDNA template composition obtained in step 2. And (3) placing the prepared reaction system on a fluorescent quantitative PCR instrument for qPCR amplification reaction. The beta actin gene is used as an internal reference gene, and a 2delta delta CT method is adopted to detect the relative expression quantity of mRNA of SIRT6 gene in the siRNA transfected human microvascular endothelial cell HMEC-1.
The upstream primer for amplifying SIRT6 gene is as follows: 5'GCCTGGTCATCGTCAACCTG 3' (SEQ ID NO: 9) and the downstream primer 5'TCATGACCTCGTCAACGTAGC 3' (SEQ ID NO: 10).
The upstream primer for amplifying the beta-actin gene was 5'GTTGTCGACGACGAGCG 3' (SEQ ID NO: 11) and the downstream primer was 5'GCACAGAGCCTCGCCTT 3' (SEQ ID NO: 12).
4. Experimental results
The detection results are shown in FIG. 1. The results show that compared with siRNA NC, SIRT6 mRNA expression level is obviously reduced after SIRT6 siRNA transfects human microvascular endothelial cell HMEC-1.
Example 3: construction of SIRT6 gene targeting lentiviral vector and identification of SIRT6 gene interference effect
1. Construction of SIRT6shRNA and shRNA NC recombinant lentiviral vector
(1) Determining interference target point, designing and synthesizing primer
In view of the significant effect of SIRT6 siRNA on SIRT6 expression in example 2, a short hairpin RNA (SIRT 6 shRNA) was designed based on the SIRT6 siRNA sequence, with the upstream primers: 5'GATCCGCTACGTTGACGAGGTCATGACTCGAGTCATGACCTCGTCAACGTAGCTTTTTTG 3' (SEQ ID NO: 5), the downstream primer is: 5'AATTCAAAAAAGCTACGTTGACGAGGTCATGACTCGAGTCATGACCTCGTCAACGTAGCG 3' (SEQ ID NO: 6).
Short hairpin RNAs (shRNA NCs) were designed based on sirnc sequences, with the upstream primers: 5'GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC 3' (SEQ ID NO: 7), the downstream primer is: 5'AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG 3' (SEQ ID NO: 8).
(2) Primer annealing to form a double-stranded fragment with cohesive ends
The two sets of primers were diluted to 100. Mu.M, respectively, and then a reaction system (20. Mu.l) was prepared: 1 μl upstream primer, 1 μl downstream primer, 2 μl 10 x oligo Buffer, 16 μl ddH 2 O. The two pairs of primers synthesized in (1) were annealed to form a double-stranded fragment with cohesive ends in an annealing procedure of 95℃for 10min, 75℃for 10min, 55℃for 10min, 35℃for 10min, and 15℃for 10 min.
(3) Recombinant lentiviral vector construction
(1) And (3) carrier enzyme cutting: the following reagents were sequentially added to prepare a reaction system (40. Mu.l reaction system): 1 μl vector DNA (1 μg/μl), 4 μl 10 Xbuffer, 32 μl ddH 2 O, 1.5. Mu.l of restriction enzyme 1, 1.5. Mu.l of restriction enzyme 2. Gently blowing and mixing, placing in a water bath kettle at 37 ℃ to react for 1-2h, and carrying out enzyme digestion on the carrier pHBLV-U6-MCS-PGK-PURO. After completion, agarose gel electrophoresis was performed to collect the target fragment.
(2) The interference fragment is linked to the vector: a ligation reaction system (20. Mu.l) was configured: the double-stranded fragment obtained in step (2) (4. Mu.l), the digested vector obtained in step (1) (50 ng or more), T4ligase buffer (2. Mu.l), T4ligase (1. Mu.l), and the like were digested with ddH 2 O is fixed to volume of 20 mul, and at 16 ℃ overnight, double-stranded DNA target fragments corresponding to SIRT6shRNA and shRNA NC are connected to the pHBLV-U6-MCS-PGK-PURO carrier after enzyme digestion, thus obtaining the SIRT6shRNA and shRNA NC recombinant lentiviral carrier.
2. Preparation of viral concentrate
(1) 293T cells (American ATCC cell bank) were seeded in 6-well plates at 37℃with 5% CO 2 In the incubator (C), the cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum and 1% diabody (streptomycin and penicillin) and transfected after the cell density reached 70-80%.
(2) Preparing a cotransfection system: 10 mu G of pSPAX2, 5 mu G of pMD2G, 10 mu G of recombinant lentiviral vector SIRT6shRNA or shRNA NC obtained by the above steps, 75 mu l of Lipofiter TM Transfection reagent, after mixing, incubated at room temperature for 15min and slowly added dropwise to 293T cells of step (1) at 37℃with 5% CO 2 Is cultured in a cell culture incubator containing 10% fetal bovine serum FBS, and fresh complete medium is replaced 16h after transfection.
(3) Collecting viruses: two virus supernatants were collected 48h and 72h after transfection (fresh complete medium was replaced after 48h collection) in 50mL centrifuge tubes, centrifuged at 4 ℃,2000g for 10min, and the pellet (cell debris) was removed; and placing the virus stock supernatant into an overspeed centrifuge tube, centrifuging for 120min at 4 ℃ and 82700g to obtain virus precipitate, re-suspending the virus precipitate by using a complete culture medium, and then subpackaging the re-suspension into sterilized virus tubes to obtain SIRT6shRNA 1 and shRNANC virus concentrated solution, and storing in a refrigerator at-80 ℃.
3. Cell infection
Human microvascular endothelial cells HMEC-1 (American ATCC cell bank) were infected with SIRT6shRNA and shRNANC virus concentrates prepared in step 2 above. The method comprises the following specific steps of;
(1) HMEC-1 cells were seeded in 6-well plates at 37℃with 5% CO 2 Is cultured in MCDB131 microvascular endothelial cell culture medium containing 10% fetal bovine serum and 1% double antibody (streptomycin and penicillin), and is transfected after cell density reaches 70-80%.
(2) Adding 30 μl of SIRT6shRNA and shRNA NC virus concentrate into the culture medium in the step (1); after 12h, fresh complete medium was changed; positive selection with puromycin was performed 48h after 48h post transfection followed by amplification culture.
4. RT qPCR identification of interference effect of SIRT6shRNA on SIRT6 gene
(1) Total RNA of human microvascular endothelial cells (HMEC-1) infected with SIRT6shRNA and shRNA NC virus concentrate, respectively, was extracted with Trizol (Japanese Takara Co.) and subjected to reverse transcription to obtain cellular cDNA. The specific steps of reverse transcription are as follows:
(1) digestion of DNA: mu.g of total RNA of HEMC-1 after virus infection, 2. Mu.l of 5X gDNA Eraser Buffer and 1. Mu.l of gDNA Eraser were added to the PCR tube and made up to 10. Mu.l with nuclease-free water. The reaction system was then thoroughly mixed and centrifuged and incubated at 42℃for 2min in a metal bath to digest the DNA.
(2) Preparing a reverse transcription reaction system: mu. l PrimeScriptRT Buffer 2, 4. Mu.l RT primer Mix, 1. Mu.l RNase Free dH were added to each tube after digestion of DNA in step (1) 2 O and 1 mu l PrimeScriptRT Enzyme Mix I, and blow-mixing and centrifuging.
(3) The reaction system is placed in a PCR instrument, reverse transcription reaction is carried out according to the conditions of 37 ℃,15min,85 ℃ and 5s, thus obtaining the cDNA of HMEC-1 after virus infection, and the cDNA is preserved at 4 ℃.
(2) qPCR identification of SIRT6shRNA interference effect on SIRT6 gene
Preparation of qRT PCR reaction System (10. Mu.l): 0.3. Mu.l of upstream primer, 0.3. Mu.l of downstream primer, 5. Mu. l TB Green Premix Ex TaqII (Tli RNaseH Plus), 4. Mu.l of RNase free dH 2 O and 0.4. Mu.l of the cDNA template composition obtained in step (1). And (3) placing the prepared reaction system on a fluorescent quantitative PCR instrument for qPCR amplification reaction. The beta actin gene is used as an internal reference gene, and the relative mRNA expression quantity of SIRT6 gene in the HMEC-1 infected by SIRT6shRNA virus is detected by adopting a 2delta delta CT method.
The upstream primer for amplifying SIRT6 gene is as follows: 5'GCCTGGTCATCGTCAACCTG 3' (SEQ ID NO: 9), the downstream primer is: 5'TCATGACCTCGTCAACGTAGC 3' (SEQ ID NO: 10).
The upstream primer for amplifying the beta actin gene is as follows: 5'GTTGTCGACGACGAGCG 3' (SEQ ID NO: 11), the downstream primer is: 5'GCACAGAGCCTCGCCTT 3' (SEQ ID NO: 12).
(3) Experimental results
The detection results are shown in FIG. 2. The result shows that compared with the HMEC-1 after the shRNA NC virus infection, the expression quantity of SIRT6 mRNA in cells after the SIRT6shRNA virus infection is obviously reduced, and the interference effect of the SIRT6shRNA is obvious.
5. Western blot identification of SIRT6shRNA inhibition effect on SIRT6 protein
(1) Western blot specific procedure
Extracting HMEC-1 cell total protein infected by SIRT6shRNA and shRNANC virus concentrate respectively by neutral lysate (RIPA: PMSF=100:1), denaturing at 99 ℃ for 10min, and performing SDS-PAGE electrophoresis at constant pressure of 120V and 60 min; constant current 300mA,60min to transfer protein to PVDF film; sealing the PVDF film for 40min at room temperature by using a rapid sealing liquid; adding a primary anti-dilution liquid through 1000: incubating the blocked PVDF membranes at 4 ℃ with SIRT6 rabbit primary antibody (abcam, USA) and beta actin rabbit primary antibody (abcam, USA) after 1 dilution; recovering primary antibody, washing the membrane with 1 XTBE solution 3 times each for 15min; adding secondary antibody diluent into the mixture, and passing the mixture through 5000:1 diluted rabbit secondary antibody (company Cell Signaling Technology in U.S.) was incubated for 1h at room temperature; washing the membrane with 1 XTBST solution on a shaker for 3 times each for 15min; a chemiluminescent developer is prepared and developed by a Bio-Rad chemiluminescent imaging system.
(2) Experimental results
The detection results are shown in FIG. 3. The results show that the protein content of SIRT6 in HMEC-1 after the SIRT6shRNA virus concentrate is infected is obviously reduced.
Example 4: effect of SIRT6 knockdown on oxidative stress-induced apoptosis of human microvascular endothelial cell HMEC-1
1. Human microvascular endothelial cell HMEC-1 oxidative stress model
The SIRT6 knockdown human microvascular endothelial stable cell line constructed in example 2 and the control cell line were treated at 5% CO 2 ，95％N 2 After culturing in serum-free medium for 12 hours, fresh medium containing 10% fetal bovine serum is replaced, and H with concentration of 400 μm is added 2 O 2 Culturing for 6 hours.
2. RTqPCR identification of biomarker changes in SIRT6 shRNA-interfered HMEC-1 endothelial apoptosis upon oxidative stress
(1) Total RNA of human microvascular endothelial cells (HMEC-1) infected with SIRT6shRNA and shRNA NC virus concentrate, respectively, was extracted with Trizol (Japanese Takara Co.) and subjected to reverse transcription to obtain cellular cDNA. The specific steps of reverse transcription are as follows:
(1) digestion of DNA: mu.g of total RNA of HEMC-1 after virus infection, 2. Mu.l of 5X gDNA Eraser Buffer and 1. Mu.l of gDNA Eraser were added to the PCR tube and made up to 10. Mu.l with nuclease-free water. The reaction system was then thoroughly mixed and centrifuged and incubated at 42℃for 2min in a metal bath to digest the DNA.
(2) Preparing a reverse transcription reaction system: mu. l PrimeScriptRT Buffer 2, 4. Mu.l RT primer Mix, 1. Mu.l RNase Free dH were added to each tube after digestion of DNA in step (1) 2 O and 1 mu l PrimeScriptRT Enzyme Mix I, and blow-mixing and centrifuging.
(3) The reaction system is placed in a PCR instrument, reverse transcription reaction is carried out according to the conditions of 37 ℃,15min,85 ℃ and 5s, thus obtaining the cDNA of HMEC-1 after virus infection, and the cDNA is preserved at 4 ℃.
(2) qPCR to identify the Effect of SIRT6shRNA on HMEC-1 apoptosis marker expression under oxidative stress
(1) Preparation of qRT PCR reaction System (10. Mu.l): 0.3. Mu.l of upstream primer, 0.3. Mu.l of downstream primer, 5. Mu. l TB Green Premix Ex TaqII (Tli RNaseH Plus), 4. Mu.l of RNase free dH 2 O and 0.4. Mu.l of the cDNA template composition obtained in step (1).
(2) And (3) placing the prepared reaction system on a fluorescent quantitative PCR instrument for qPCR amplification reaction. The beta actin gene is used as an internal reference gene, and the relative mRNA expression quantity of SIRT6 gene in the HMEC-1 infected by SIRT6shRNA virus is detected by adopting a 2delta delta CT method.
The upstream primer for amplifying Bax gene is: 5'CCCGAGAGGTCTTTTTCCGAG 3' (SEQ ID NO: 13), the downstream primer is: 5'CCAGCCCATGATGGTTCTGAT 3' (SEQ ID NO: 14);
the upstream primer for amplifying the Caspase-3 gene is as follows: 5'
CATGGAAGCGAATCAATGGACT 3' (SEQ ID NO: 15), the downstream primer is: 5'
CTGTACCAGACCGAGATGTCA 3’(SEQ ID NO：16)；
The upstream primer for amplifying the beta actin gene is as follows: 5'GTTGTCGACGACGAGCG 3' (SEQ ID NO: 11), the downstream primer is: 5'GCACAGAGCCTCGCCTT 3' (SEQ ID NO: 12).
(3) Experimental results
The expression of biomarkers of endothelial apoptosis is shown in figure 4 (SIRT 6 is SIRT6 knockdown human microvascular endothelial stable cell line, shSIRT6 NC is control cell line). The results show that, through H 2 O 2 After oxidative stress treatment, compared with the HMEC-1 infected by the ShSIRT6 NC and SIRT6shRNA virus concentrated solution, the mRNA expression of apoptosis biomarkers Bax and Caspase-3 is obviously increased, namely the SIRT6shRNA can obviously promote the apoptosis of endothelial cells induced by oxidative stress.
3. Western blot identification of biomarker changes of endothelial apoptosis of SIRT6 shRNA-interfered HMEC-1 in oxidative stress
(1) Western blot specific procedure
Extracting HMEC-1 cell total protein infected by SIRT6shRNA and shRNANC virus concentrate respectively by neutral lysate (RIPA: PMSF=100:1), denaturing at 99 ℃ for 10min, and performing SDS-PAGE electrophoresis at constant pressure of 120V and 60 min; constant current 300mA,60min to transfer protein to PVDF film; sealing the PVDF film for 40min at room temperature by using a rapid sealing liquid; adding a primary anti-dilution liquid through 1000: after 1 dilution, bax rabbit primary antibody (Abcam Co., USA), caspase-3 rabbit primary antibody (Abcam Co., USA) and beta-actin rabbit primary antibody (Abcam Co., USA) were incubated overnight at 4deg.C on the blocked PVDF membrane; recovering primary antibody, washing the membrane with 1 XTBE solution 3 times each for 15min; adding secondary antibody diluent into the mixture, and passing the mixture through 5000:1 diluted rabbit secondary antibody (company Cell Signaling Technology in U.S.) was incubated for 1h at room temperature; washing the membrane with 1 XTBST solution on a shaker for 3 times each for 15min; a chemiluminescent developer is prepared and developed by a Bio-Rad chemiluminescent imaging system.
(2) Experimental results
The expression of biomarkers of endothelial apoptosis is shown in figure 5 (SIRT 6 is SIRT6 knockdown human microvascular endothelial stable cell line, shrut 6 NC is control cell line). The results show that, through H 2 O 2 After the treatment of the oxidative stress, the reaction mixture,compared with the ShSIRT6 NC, protein expression of apoptosis biomarkers Bax and Caspase-3 in the HMEC-1 after being infected by the SIRT6shRNA virus concentrate is obviously increased, namely the SIRT6shRNA can obviously promote apoptosis of endothelial cells induced by oxidative stress.
The SIRT6 knockdown human microvascular endothelial stable cell line constructed by the invention is a SIRT6 low-expression cell line, and lays a foundation for deeply researching the molecular mechanism of endothelial cell apoptosis.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.
Sequence listing
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Claims (6)
1. Use of a substance capable of inhibiting SIRT6 expression, characterized in that the substance capable of inhibiting SIRT6 expression is:
siRNA, the siRNA is a sense strand shown in SEQ ID NO. 1 and an antisense strand shown in SEQ ID NO. 2;
or taking the siRNA as a target point, and synthesizing shRNA by a shRNA expression system, wherein the shRNA is SEQ ID NO:5 and the sense strand shown in SEQ ID NO:6, an antisense strand shown in FIG. 6;
the application is selected from any one of the following A1) -A6):
a1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
a2 A) of: preparing a product for constructing a microvascular endothelial cell model with SIRT6 low expression;
a3 A) of: preparing a product for constructing an animal model of SIRT6 low expression on microvascular endothelial cells;
a4 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
a5 A) of: constructing a micro-vascular endothelial cell model with SIRT6 low expression;
a6 A) of: an animal model of SIRT6 low expression in microvascular endothelial cells is constructed.
2. Use of an expression vector comprising the amino acid sequence set forth in SEQ ID NO:1-2 or SEQ ID NO: 5-6;
the application is selected from any one of the following B1) -B6):
b1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
b2 A) of: preparing a product for constructing a microvascular endothelial cell model with SIRT6 low expression;
b3 A) of: preparing a product for constructing an animal model of SIRT6 low expression on microvascular endothelial cells;
b4 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
b5 A) of: constructing a micro-vascular endothelial cell model with SIRT6 low expression;
b6 A) of: an animal model of SIRT6 low expression in microvascular endothelial cells is constructed.
3. The use according to claim 2, wherein the expression vector is selected from viral, fungal or bacterial expression vectors.
4. The use according to claim 2, wherein the method of preparing the expression vector comprises the steps of:
step 1: setting SEQ ID NO:5 and the single stranded DNA molecule shown in SEQ ID NO:6 annealing the single-stranded DNA molecule to form a DNA double-stranded fragment with cohesive ends;
step 2: restriction enzyme 1 and restriction enzyme 2 carry out enzyme digestion on a lentiviral vector pHBLV-U6-MCS-PGK-PURO, and the DNA double-stranded fragment obtained in the step 1 is connected into the vector through T4ligase to obtain the recombinant lentiviral vector.
5. Use of a microvascular endothelial cell line with low SIRT6 expression, said cell line comprising an expression vector comprising the sequence set forth in SEQ ID NO:1-2 or SEQ ID NO: 5-6;
the application is selected from any one of the following C1) -C4):
c1 A) of: preparing a product for promoting the apoptosis of microvascular endothelial cells under oxidative stress conditions;
c2 A) of: preparing a product for constructing an animal model of SIRT6 low expression on microvascular endothelial cells;
c3 A) of: promoting the apoptosis of microvascular endothelial cells under the condition of oxidative stress in vitro;
c4 A) of: an animal model of SIRT6 low expression in microvascular endothelial cells is constructed.
6. The use of claim 5, wherein the method of constructing a microvascular endothelial cell line with low SIRT6 expression comprises the steps of:
step 1: the recombinant lentiviral vector prepared by the preparation method of claim 4 is combined with a virus packaging auxiliary plasmid pSPAX2 and pMD2G to form a three-plasmid lentivirus system for virus packaging, and then 293T cells are transfected to obtain virus liquid;
step 2: and (3) infecting the microvascular endothelial cell line by using the virus liquid obtained in the step (1), and screening to obtain the microvascular endothelial cell line with SIRT6 low expression.
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