RNA interference; Hypertension; Angiotensin Ⅱ receptor; carrier
Gene silencing and screening of rat AT 1 receptor targeted by high-efficiency shRNAs
Objective: To study the regulatory effect of short hairpin RNA(shrna) on the expression of rat angiotensin Ⅱ1(at1) receptor gene in mammalian cells, and to explore the correlation between the characteristics of high-efficiency shrna and its function. Methods: Four shRNAs expression vectors targeting rat angiotensin Ⅱ receptor were constructed. ShRNAs target four different regions of the same gene. The constructed pGenesil 1shRNA plasmid and scrambled plasmid were used to transfect ra t C6 glioma cells. Cultured cells were collected at different stages for RTPCR and protein blot analysis. Results: After 24 hours, AT 1 mRNA levels in all treatment groups did not decrease significantly. After 48 hours, the AT 1 mRNA level in lead treatment group decreased to (55.7 7.6)% of that in control group, and reached the lowest point (43.7 8.2)% after 72 hours. Other groups had no obvious inhibitory effect on the expression of AT 1 receptor mRNA (P>0.05). AT 1 mRNA and protein levels were similar in the end. At 24h and 48h, the AT 1 protein was (46.9 4.2)% and (37.0 3.7)% compared with the control group, respectively, and the maximum decrease was observed after 72h incubation [28.14.0 compared with the control group]. Conclusion: Apart from some general rules, we believe that the ring structure of mRNA in the targeted region of siRNA and the better accessibility of siRNA targeting mRNA do have a strong influence on silencing.
Keywords RNAi hypertension; Angiotensin Ⅱ receptor; vector
Objective: To study the effectiveness of shRNA in regulating the expression of rat angiotensin ⅱ 1 receptor gene in mammalian cells, and to explore the relationship between the characteristics of effective shRNA and its function. Methods: Four shRNA expression vectors targeting at different regions of rat AT 1 receptor gene were designed and constructed, transfected into rat glioma C6 cells, and the transfected cells were collected at different times. RTPCR and Western blot were performed. Results: After 24 hours of transfection, the mRNA level of AT 1 did not decrease significantly in all treatment groups. Compared with the control group, 48h after transfection, the mRNA level of angiotensin Ⅱ receptor in Pb group decreased to (55.7 7.6)%. After 72 h, it reached the lowest point (43.7 8.2)%. The expression of AT 1 receptor mRNA in other groups was not significantly inhibited (P > 0.05). The results of inhibiting angiotensin ⅱ receptor mRNA gene and its protein in rats are similar. Compared with the control group, the angiotensin Ⅱ receptor protein in lead exposed group decreased to (46.9 4.2)% and (37) at 24 h and 48 h, respectively. 72 h after transfection, the maximum reduction was (28.14.0)% 4.0)%. Conclusion: In addition to the general principle, the circular structure of target mRNA and whether shRNA can reach the target site play an important role in the success of gene interference experiment.
RNA interference; Hypertension; Angiotensin Ⅱ receptor; carrier
Gene interference can specifically degrade the RNAi of the target gene, thus reducing or silencing the expression of the target gene in tissues or cells [1]. Several single-stranded nucleotides encoding rat AT 1R mRNA short hairpin RNA(shRNA) were selected and synthesized, annealed to form a double-stranded structure, and cloned into a plasmid vector. After constructing four expression vectors encoding AT 1R shRNA, C6 cells were transfected. According to the functional expression of shRNA plasmids with different nucleotide sequences in mammalian cells, the differences of their targeting effects on AT 1R in silent rats were studied.
1 materials and methods
1. 1 plasmid was purchased from Wuhan Jingsai Biotechnology Co., Ltd., and restriction endonucleases BamH, Hind, Sal and Pst were purchased from Dalian Bao Biotechnology Co., Ltd.; T4 DNA ligase and buffer were purchased from New England Biolabs. C6 cells of rat glioma cell line were purchased from China Type Collection. Cell culture medium DMEM was purchased from Hyclone Company. Transfection reagent Metafectamine was purchased from Biotex, Germany. All DNA synthesis and primer synthesis services are provided by Shanghai Boya Biotechnology Co., Ltd.
1.2 method
The shRNA of 1.2. 1 target gene AT 1R mRNA in GenBank was constructed, and the gene sequence of rat AT 1R (serial number NM_030985) was selected, and the sequence with the length of 2 1 nucleotide ending in TT was selected by online design software siRNA targeting instrument. The GC content shall not exceed 60% at most. The four nucleotide sequences of the target gene with four or more consecutive A or T bases were eliminated and verified by BLAST, and the forward and reverse sequence single-stranded DNA encoding the shRNA sequence was chemically synthesized as follows: A pair of nonspecific sequences were simultaneously designed for the nucleotide sequence of the target gene as a positive control. The synthesized shRNA sequence was annealed, connected with the linearized pGenesil 1 plasmid, and transformed into a positive clone, which was identified by Pstⅰⅰ and Salⅰⅰ digestion and sequencing. The different target gene sequence plasmids mentioned above are called pGenesilA(Pa), pGenesilB(Pb), pGenesilC(Pc) and pGenesilD(Pd) respectively.
1.2.2 Cell culture and transfection culture plates were inoculated with the same number of cells at a density of 2× 108/L, and the cells were transfected at a ratio of 1∶6 according to the transfection scheme of the reagent provider.
The primer sequence of1.2.3 rtpcraft1gene is: 5' tgttcctccttcttcattttct3', 5'ctttgcttggttact.
The primer sequences of CCT TCA 3' and internal reference GAPDH are 5' ttcaagcacagtcaag3ag3' and 5'ttagtggggtct, respectively.
CGC TCC 3’。 The 25 μL reaction volume includes 5 μL cDNA, and each primer is 0.25 μL,10.5 μ l MgCl2, 0.25 μL Taq DNA polymerase, 0.5 μL dNTP mixture, 5 μL 10× buffer. The reaction conditions are: annealing at 94℃ for 3 min, then annealing at 94℃ for 65438. After 30 cycles at 55℃ 15 s and 72℃ 15 s, the product was subjected to agarose gel electrophoresis.
1.2.4 Western blot analysis showed that C6 cells were harvested at different times after transfection with pGensilAT 1shRNA plasmid and control plasmid. After washing with PBS solution, the cells were dissolved by loading buffer solution. After boiling the solution for 65438 00 minutes, centrifuge the supernatant and discard the precipitate. Determination of protein concentration. Cell lysates were separated by electrophoresis. After 50 g/L skim milk was sealed, anti-AT 1 antibody (1:1000) and anti-β actin antibody (1:1500) were added, and incubated at room temperature1.5 h.
Statistical processing: According to the analysis results of RTPCR and Western Blot, the gray value was measured by image processing software ImageTool 3.0, and the relative value was compared with the internal reference. Statistical software SPSS 1 1.5 was used to analyze the variance between groups. The significance level was p < 0.05.
Two results
2. 1 different shRNA expression plasmid targets and secondary structures We selected some sequences of rat angiotensin Ⅱ receptor mRNA sequences, namely 556~575 bp, 469~488 bp, 595~6 14 bp and 663~682 bp, as gene silencing targets (Figure 1
The sense chain of AT 1shRNA is the same as the target gene sequence of rat AT 1 gene, except that there are two continuous thymidine missing at the 3' end, and the antisense strand is complementary to the target sequence. The RNA produced by transcription is expected to fold into short hairpin siRNA. Theoretically, the transcribed shRNA should be able to cleave the target gene mRNA, but the shuffled control plasmid should not be used for at 65438.
2.2 AT 1 mRNA level and protein expression were transfected with four groups of positive plasmids for 24 h, and the AT 1 mRNA level in each group did not decrease significantly. After 48 h, compared with the control group, the expression level of AT 1 mRNA in the transfected Pb plasmid group decreased to (55.7 7.6)%. The expression level of AT 1 receptor mRNA in C6 glioma cells transfected with recombinant siRNA was not significantly inhibited (p > 0.05). The results showed that Pb plasmid significantly inhibited the expression of AT 1 receptor mRNA, while the other three groups of plasmids had no obvious effect on inhibiting AT 1 receptor mRNA (Figures 3 and 4). Western Blot analysis of Pb plasmid with effective inhibitory effect on AT 1 receptor mRNA. At 24 h and 48 h, the expression of AT 1 protein was (46.9 4.2)% and (37.0 3.7)% respectively. Compared with the control group, the maximum expression level decreased 72 h after transfection (only (28.65438 0.4)% of the control group). The results showed that the protein level of AT 1 was decreased in the cells transfected with Pb plasmid, but the expression of AT 1 gene could not be decreased by transfection with shuffled shRNA plasmid and control group (Figure 5). All the results clearly show that transfection of Pb plasmid can inhibit the expression of AT 1 gene.
3 discussion
Angiotensin Ⅱ is the main effector molecule of renin-angiotensin system, which has the functions of regulating blood pressure, water-sodium balance and nerve function. Studies have confirmed that angiotensin ⅱ is involved in the pathophysiological process of arteriosclerosis, myocardial infarction, vascular and myocardial remodeling and congestive heart failure. Angiotensin Ⅱ induces cardiomyocytes by directly activating AT 1R and indirectly stimulating the release of some growth factors and cytokines. Vascular cell hypertrophy and proliferation [2]. At the same time, it can also stimulate AT2, activate the biological effect opposite to AT 1, lead to vasodilation, increase bradykinin production, and inhibit fibrosis and vascular remodeling [3]. In this study, a short hairpin RNA expression plasmid of AT 1 was constructed, and the expression of AT 1 mRNA in mammalian cells was inhibited by gene silencing technology, hoping to block AT65438+.
The experimental results showed that the mRNA expression of AT 1 gene decreased in the early stage, and the corresponding protein also decreased with the inhibition of mRNA level. The results showed that shRNA sequence driven by U6 promoter could effectively and specifically inhibit the expression of AT 1 gene in transfected C6 cells. Therefore, the expression of shRNA can inhibit the expression of AT 1 gene related to the pathophysiology of hypertension in cells.
At present, there is no * * * opinion or standard to select siRNA sequence [5]. In order to optimize the efficiency of siRNA-induced gene silencing, many research groups have studied the length, secondary structure and sequence specificity of siRNA double strands. It is generally believed that [6], the siRNA sequence should be selected after 100 bases downstream of the start codon of the target gene. The sequence of siRNA should avoid the 5' or 3' non-coding region of the start codon. The sequence of sense chain should be AA(N 19)TT, where n represents any nucleotide, that is, the selected siRNA should have two 3' overhangs of uridine. The length of the selected siRNA should be 2 1 nucleotide. There are many factors that determine the success of RNA interference research. Including the position of the target site in the tertiary structure of mRNA, transfection efficiency and specificity of double-stranded shRNA [7-8]. When comparing the four plasmids designed in this experiment, we noticed that the targeted cleavage sites of Pc and Pd are located at the core of the secondary structure of the target gene mRNA, especially Pd. This may be one of the reasons why the short hairpin RNA produced by plasmid transfection can not enter the target mRNA cleavage site after combining with RISC (RNA-induced gene silencing complex), which leads to the failure of the experiment. However, the targeted cleavage sites of Pa and Pb are located on the secondary structure surface of the target gene mRNA, but the plasmid Pa is also ineffective. The possible reasons are as follows: ① the low GC content of plasmid Pa will lead to the decrease of mRNA recognition efficiency of the target gene; ② The efficiency of hybridization with RISC decreased. In addition, we also found that the local secondary structure of the target gene cut by Pb plasmid is looser than that cut by Pa plasmid, that is, the hydrogen bond at the target site of the former is relatively weaker than that of the latter. Among these factors, we think the most important factor is whether siRNA can enter the target site and whether the structure of target site mRNA is loose enough to allow targeted cleavage of siRNA and RISC complex.
In short, when designing short hairpin RNA, do not choose non-coding region except that the target sequence is in the open reading frame; The GC content should be about 50%; Avoid at least 100 bases after the start codon; In addition to searching for 5 ′ aa (n19) sequence and carrying out some general rules such as BLAST [9], we believe that whether siRNA can enter the target cleavage site and whether the secondary structure of the target site is loose is the most important factor for the rational design of effective shRNA and the success of the experiment.
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