Gene silencing of Col1α1 by RNAi in rat myocardium fibroblasts
-
Guang-Mou Zhang
,
Zi-Wei He
Abstract
Objective
The collagen type I alpha 1 (Col1α1) not only acts as a scaffold for the cell matrix, but also is involved in myocardial fibrosis. The purpose of this study is to screen an efficient shRNA specific to rat Col1α1 gene.
Methods
The shRNA expression plasmids targeting Col1α1 were constructed and their knockdown efficiency was examined by qPCR and Western blot.
Results
Three shRNA expression plasmids targeting Col1α1 were successfully constructed, as suggested by analysis of restriction enzyme digestion and DNA sequencing. The Col1α1 shRNA-expressing plasmids were then transfected into rat cardiac fibroblasts with high efficiency. The qPCR and Western blotting results demonstrated that Col1α1 expression at both mRNA and protein level was significantly inhibited (p < 0.05) in shRNA-transfected cells compared to scrambled shRNA-transfected cells.
Conclusion
A Col1α1-specific shRNA with high efficiency of gene silencing is successfully obtained. The construction of Col1α1 shRNA-expressing plasmid lays the foundation for the further studies on the role of Col1α1 gene in myocardial fibrosis.
Öz
Amaç
Kollajen tip I alfa 1 (Col1α1) sadece hücre matrisi için bir iskele görevi görmez, aynı zamanda miyokard fibrozisinde rol oynar. Bu çalışmanın amacı, sıçan Col1α1 genine özgü etkili bir shRNA’yı taramaktır.
Yöntem
Col1α1’i hedefleyen shRNA ekspresyon plazmidleri üretildi ve nakavt verimleri qPCR ve Western blot ile incelendi.
Bulgular
Restriksiyon enzimi kesimi ve DNA dizilimi analizinin gösterdiği gibi Col1α1’i hedef alan üç shRNA ekspresyon plazmidi başarıyla yapıldı. Col1α1 shRNA ifade eden plazmidler daha sonra, yüksek verimde sıçan kardiyak fibroblastlarına transfekte edildi. qPCR ve Western blot sonuçları, hem mRNA hem de protein seviyesinde Col1α1 ekspresyonunun, shRNA ile transfekte edilmiş hücrelerde, karıştırılmış shRNA ile transfekte edilmiş hücrelere kıyasla, önemli ölçüde inhibe edildiğini (p < 0.05) göstermiştir.
Sonuç
Col1α1’e özgü gen susturma etkinliği yüksek bir shRNA başarıyla elde edildi. Col1α1 shRNA eksprese eden plazmidin üretimi, Col1α1 geninin miyokardiyal fibrozisteki rolü üzerine yapılacak sonraki araştırmaların temelini oluşturmaktadır.
Introduction
Collagen type I is a trimeric structure formed by two collagen type I alpha 1 (Col1α1) and one collagen type I alpha 2 (Col1α2). It is characterized by intense and strong resistance to traction, and 85% extracellular collagen in cardiac muscle cells is collagen type I. It acts as not only as a scaffold for the cell matrix but also a transmitter of myocardial contraction force, playing an important role in maintaining the compression and integrity of ventricular wall structures [1], [2], [3]. As the main matrix-producing cells, cardiac fibroblasts play a pivotal role in maintaining the integrity of the matrix network and mediating the proliferation of cardiomyocytes through fibronectin/β1-integrin [4], [5].
Mature collagen fibers are highly stable, with a half-life of 80–120 days. Homeostatic control of myocardial extracellular matrix is ongoing in the process of synthesis and degradation of matrix proteins [6], [7]. Break of a tight balance between synthesis and degradation of collagen metabolism will directly lead to abnormalities in cardiac structure and function. Myocardial fibrosis interferes with the contractile coupling of the myocardium during systole and diastole, which directly affects the systolic and diastolic functions of the myocardium [8], [9], [10]. Disruption of the collagen network in myocardial fibrosis results in abnormal myocardial contractile function, and collagen fibers and their receptors play a significant role in maintaining the homeostasis of the heart [11], [12], [13]. However, the significance of collagen type I in myocardial fibrosis has not fully understood.
In this study, we aimed to screen an efficient shRNA specific to rat collagen type I alpha 1 (Col1α1) gene. Three interference target sequences were designed and synthesized, and shRNA mammalian expression plasmids for Col1α1 interference were constructed and identified. A stable transfection system was established by transfection of rat cardiac fibroblasts, providing a basis for further study on the role of Col1α1.
Materials and methods
Reagents and tools
Sac I (Thermo Fisher Scientific Inc., Waltham, MA, USA); BSA (New England Biolabs, NEB, USA); DL2000 (TianGen Biotech Co., Ltd., Beijing, China); DNA ligase (TransGen Biotech, Beijing, China); plasmid maxi preparation kit (ComWin Biotech Co., Ltd., Beijing, China); plasmid mini preparation kit and DNA recovery kit (Generay, Inc., Shanghai, China); Lipo2000, fetal bovine serum (FBS) and trypsin (Invitrogen Inc., Carlsbad, CA, USA); Opti-MEM (Gibco BRL, Grand Island, NY, USA); RNA extraction reagent Trizol (Aidlab Biotechnologies Co., Ltd., Beijing, China); reverse transcription and quantitative polymerase chain reaction (qPCR) reagents (Vazyme Biotech Co., Ltd., Nanjing, China); DNTP and DNA Marker (TianGen Biotech Co., Ltd., Beijing, China); Protein lysate (Beyotime Biotechnology Co., Ltd., Shanghai, China); SDS (Sinopharm Group Co., Ltd., Shanghai, China); acrylamide (Amersco Inc., Solon, OH, USA); PVDF membrane (Millipore, Billerica, MA, USA); rabbit polyclonal antibody Col1α (Abcam Inc., Cambridge, MA, USA); mouse antibody actin (Boshide Biotech Co., Ltd., Wuhan, China). Synthesis of primers was performed by Tsingke Biological Technology Co., Ltd. (Beijing, China).
Design of targeted short hairpin RNA (shRNA)
The siRNA sequence was designed by retrieving Col1α mRNA (NM_053304.1) sequence from Pubmed. According to the principle for designing siRNA, three targeted shRNA interfering Col1α were designed. Meanwhile, a scrambled siRNA (Scr) sequence with no homology to any gene was designed. The algorithm for designing siRNA which were applied in the current study is from online tool (https://rnaidesigner.thermofisher.com/rnaiexpress/) [14]. Using this tool, three of target sequences and related siRNA candidates were obtained. The selected Col1α sequences were as follows: Scr, TCTGAAGGCGAGAGTGATA; Col1α1-1, TGAAGGGACACAGAGGTTT; Col1α1-2, GGTGATACTGGTGTGAAAG; Col1α1-3, TGGTGCTACTGGTTTCCCT (Table 1).
Oligo DNA sequence targeting rat Col1α1.
| Name of primer | Sequence |
|---|---|
| Scr-A | 5′-TTTGTCTGAAGGCGAGAGTGATATTCAAGACG TATCACTCTCGCCTTCAGATTTTTTG-3′ |
| Scr-B | 5′-AGCTCAAAAAATCTGAAGGCGAGAGTGATACGTCTTGAATATCACTCTCGCCTTCAGA-3′ |
| Col1α1-1-1.2-A | 5′-TTTGTGAAGGGACACAGAGGTTTTTCAAGACGAAACCTCTGTGTCCCTTCATTTTTTG-3′ |
| Col1α1-1-1.2-B | 5′-AGCTCAAAAAATGAAGGGACACAGAGGTTTCGTCTTGAAAAACCTCTGTGTCCCTTCA-3′ |
| Col1α1-2-1.2-A | 5′-TTTGGGTGATACTGGTGTGAAAGTTCAAGACGCTTTCACACCAGTATCACCTTTTTTG-3′ |
| Col1α1-2-1.2-B | 5′-AGCTCAAAAAAGGTGATACTGGTGTGAAAGCGTCTTGAACTTTCACACCAGTATCACC-3′ |
| Col1α1-3-1.2-A | 5′-TTTGTGGTGCTACTGGTTTCCCTTTCAAGACGAGGGAAACCAGTAGCACCATTTTTTG-3′ |
| Col1α1-3-1.2-B | 5′-AGCTCAAAAAATGGTGCTACTGGTTTCCCTCGTCTTGAAAGGGAAACCAGTAGCACCA-3′ |
Construction of Col1α recombinant plasmid vector
Three DNA sequences were annealed and connected. Plasmid PG1.2 was digested by BSAI and then recovered. The linearized plasmid PG1.2 was connected with the diluted DNA annealing fragments with T4 DNA ligase. Converted with competence DH5α, positive clones were determined by digestion with enzyme Sac I (#ER1131, Thermo Fisher Scientific Inc., Waltham, MA, USA) and DNA sequencing. For enzyme digestion, 1 unit of Sac I was used to digest 1 μg of plasmid DNA in a 30 μL reaction for 60 min, and the digested DNA products were run on a 1.5% agarose gel electrophoresis, followed by exposure to UV light.
ShRNA transfection
Rat cardiac fibroblasts in the logarithmic growth period were inoculated into a 6-well plate (2×105/well) at 37°C in 5% CO2 and saturated humidity overnight. Two hours before transfection, cells were cultured in serum-free opti-MEM medium. The experimental groups were divided into shRNA-1 group, shRNA-2 group, shRNA-3 group and transfected empty liposome control group. With 100 μL opti-MEM dissolved in 6 μL lipo2000 and 4 μg RNA-interfering plasmid, the liquid was gently mixed and placed at room temperature for 5 min, then the diluent of lipo2000 and DNA were mixed and place at room temperature for 20 min. Subsequently, 200 μL mixture was added to each well separately, and cells were cultured in a full medium with serum after 6 h [15].
Observation of transfection efficiency
After transfection of rat cardiac fibroblasts for 24 h, the transfection effect was observed under fluorescent microscope and photographed.
qPCR
After transfection of rat cardiac fibroblasts for 48 h, 1 mL Trizol was added to each well and lysed for 10 min. The lysate was collected in EP tubes without enzyme, then 200 μL chloroform was added and reversely mixed for 5 min. After centrifugation at 12,000 rpm for 10 min, the supernatant was discarded. Then 75% ethanol was added, the supernatant was discarded after centrifugation at 12,000 rpm for 10 min. The RNA was dried for 5–10 min, and 20 μL enzyme-free water was added to dissolve and precipitate. The concentration and purity were detected using ultraviolet spectrophotometer. According to the instruction of reverse transcription kit, reverse transcription of RNA into cDNA was carried out. Q-PCR was performed after diluting cDNA 10 times. Using the method of SYBR, β-actin was taken as an internal reference, the primer sequences were shown in Table 2. Reaction conditions: 2 min at 50°C, 10 min at 95°C, 30 s at 95°C, 30 s at 60°C, 40 cycles. A melting curve was set up and analyzed by relative quantitative 2−ΔΔCt [16].
Primer sequence for qPCR detection of rat Col1α1.
| Name of primer | Sequence | Product size | |
|---|---|---|---|
| β-Actin | Forward | 5′-CACGATGGAGGGGCCGGACTCATC-3′ | 240 bp |
| Reverse | 5′-TAAAGACCTCTATGCCAACACAGT-3′ | ||
| Rat Col1α1 | Forward | 5′-ATCCTGCCGATGTCGCTATCC-3′ | 220 bp |
| Reverse | 5′-CTCGTGCAGCCATCCACAAGC-3′ | ||
BCA protein quantitative method
After transfection of rat cardiac fibroblasts for 72 h, the total protein in each group was extracted using RIPA protein lysate plus PMSF. The concentration of protein in each group was determined by BCA protein quantitative detection method. Briefly, cultured rat cardiac fibroblasts were washed twice with cold PBS (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 140 mM NaCl, pH 7.4) and then proteins were extracted with RIPA (Vazyme Biotech, Nanjing, China) and centrifuged at 15,000×g for 10 min at 4°C [17]. The protein concentration was measured using a Pierce™ BCA Protein Assay Kit (#23225, Thermo Fisher Scientific Inc., Waltham, MA, USA) the manufacturer’s instructions. Then the protein was denatured by being boiled with addition of 5× SDS-PAGE loading buffer was added and boiled to make it denature, then cooled to room temperature and kept −20°C.
Western blotting
The Western blotting analysis was performed as described [17]. Briefly, equal amounts of protein (40 μg) were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then the gel was taken for transferring to a PVDF membrane (Millipore), followed by blocking with 5% skim milk for 2 h and incubated with primary antibody (dilution ratio: β-actin, 1:200; Col1α1, 1:5000) at 4°C overnight. The PVDF membrane was washed with PBST (PBS with 0.1% Tween-20, pH 7.4) five to six times and cultured with HRP-labeled secondary antibody (dilution ratio: 1:50,000) for 2 h. After that, ECL working liquid was added for visualization and exposure, and the gray value of gel was analyzed by BandScan.
Statistical analysis
All data were expressed as mean±standard deviation, and the experiments were repeated three times. GaphPad Prism6 was adopted for analysis. The comparison between groups was performed by one-way analysis of variance. p<0.05 was considered statistically significant.
Results
Identification of shRNA-encoding plasmids
After digestion with single restriction enzyme Sac I, a DNA fragment of 600 bp containing shRNA sequence could be observed in recombinant plasmids pG1.2-Col1α1-1, -2, and -3 (Figure 1), suggesting that the shRNAs of Co11α1 have been cloned into the shRNA expression vector pG1.2. In line with this, DNA sequencing analysis confirmed that the insertion sequence was consistent with the Col1α1 mRNA sequence (data not shown).
Restriction digestion of recombinant plasmid shRNA by Sac I.
A DNA fragment of 600 bp containing shRNA sequence could be observed in recombinant plasmids pG1.2-Col1α1-1, -2, and -3.
Transfection efficiency of shRNA
The shRNA expression plasmids pG1.2-Col1α1-1, -2, -3, and the scrambled shRNA control pG1.2-Scr were transfected into rat cardiac fibroblasts. The green fluorescent protein (GFP) could be observed for all four plasmid-transfected fibroblasts, and the fluorescence intensity in Col1α1 shRNA-transfected cells were comparable with scrambled shRNA-transfected cells, suggesting efficiency in both cells (Figure 2).
Expression of GFP in each group under the ordinary optical microscope and fluorescence microscope separately.
(A) pG1.2-Scr; (B) pG1.2-Col1α1-1; (C) pG1.2-Col1α1-2; (D) pG1.2-Col1α1-3.
RNAi inhibits Col1α1 mRNA expression in cultured rat cardiac fibroblasts
Two days post transfection, the gene silencing effects of shRNA on Col1α1 were examined. As shown in Figure 3, compared with the scrambled shRNA-transfected cardiac fibroblasts, cells transfected with Col1α1-1 and -2 shRNA showed a significant decrease in mRNA levels of Col1α1. In contrast, Col1α1-3 did not significantly downregulate the expression Col1α1 mRNA.
qPCR detection for mRNA expression of Col1α1 in rat myocardium fibroblasts transfected with shRNA expression plasmids. (1) pG1.2-Scr; (2) pG1.2-Col1α1-1; (3) pG1.2-Col1α1-2; (4) pG1.2-Col1α1-3.
*p<0.05 and **p<0.01 versus scrambled shRNA (pG1.2-Scr) group.
RNAi inhibits Col1α1 protein expression in cultured rat cardiac fibroblasts
To further confirm the effects of shRNAs on Col1α1 mRNA expression in cultured rat cardia fibroblasts, we examined the expression of Col1α1 at protein level by Western blotting. As shown in Figure 4A and B, the introduction of Col1α1-1 and -2 shRNAs significantly reduced the expression of Col1α1 protein, when compared to control cells. Again, Col1α1-3 shRNA did not affect the protein expression of Col1α1. Taken together, our data suggest that the Col1α-specific shRNA expression plasmids are successfully constructed, and two of shRNAs could efficiently silence the expression of target gene Col1α1.
Western blotting analysis for protein expression of Col1α1 in rat myocardium fibroblasts transfected with shRNA expression plasmids.
(A) Col1α1 expression at protein levels was examined with Western blot. (B) The levels of Col1α1 protein were quantified by densitometry and normalized to β-actin. (1) pG1.2-Scr; (2) pG1.2-Col1α1-1; (3) pG1.2-Col1α1-2; (4) pG1.2-Col1α1-3. *p<0.05 and ***p<0.001 versus scrambled shRNA (pG1.2-Scr) group.
Discussion
Collagen type I is the most important component of extracellular matrix proteins. In fibroblasts and myofibroblasts, two types of collagen polypeptides are encoded by Col1α1 and Col1α2 genes separately [18], [19]. In the process of myocardial fibrosis, the expression of Collagen type I is significantly increased. In the model of myocardial fibrosis induced by hypertension and myocardial infarction, Collagen type I fibers are dense and elongated. Myocardial infarction leads to the death of many myocardial cells, which triggers inflammatory responses and further changing the degradation of normal intercellular matrix and the production of various cell matrix components [20].
Dai et al. [21], [22] amplified the promoter of Col1α1 from rat genome to construct eukaryotic expression vector of enhanced green fluorescent protein (EGFP) driven by the promoter of Col1α1. After transfection, rat osteosarcoma cell line ROS17/2.8 with stable transfection of CollA1-EGFP was selected to investigate the specific mechanism for the changes of Col1α1 promoter in osteoblasts under microgravity. Yu et al. [23] studied the effect of Col1α1-targeted interference on invasion and metastasis of human breast cancer by transfecting human breast cancer cell line MDA-MB-231 with Col1α1-targeted shRNA eukaryotic expression vector. Qi et al. [24] established stably transfected Chinese hamster ovary cell line CHO with eukaryotic expression vector of Col1α1 aminophenol 1000-1189, and found that Col1α1 interacted with the surface of Rat-1 fibroblasts, which laid the foundation for the study of Col1α1 and receptor complexes on the surface of fibroblasts.
Col1α1-encoded Collagen type I deposition is an important sign of myocardial fibrosis. In order to explore the role of Col1α1 in the occurrence and development of myocardial fibrosis in future, the present study screened out the most effective interference sites to obtain eukaryotic expression vector for Col1α1 interference with higher specificity and efficiency. More importantly, rat fibroblast cell line was stably transfected with eukaryotic expression vector of Col1α1, which providing a basis for the study on the role of Col1α1 in the pathophysiological process of myocardial fibrosis induced by TGF-β.
Acknowledgements
The authors would like to greatly thank Dr. Yang Hai-Jie from School of Life Science and Technology, Xinxiang Medical University for helping us prepare the manuscript.
Funding: The present study was supported by China National Students’ training program for innovation and Entrepreneurship (grant no. 201610472041).
Availability of data and materials: All data generated or analyzed during this study are included in this published article.
Authors’ contributions: ZH and YL were responsible for the analysis and interpretation of data of the manuscript; JL was responsible for statistical analysis; GZ was responsible for design and drafting of the manuscript.
Consent for publication: Written informed consent was obtained.
Conflict of interest: The authors declare that they have no competing interests.
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©2019 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
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Artikel in diesem Heft
- Frontmatter
- Review Article
- Mitochondrial dysfunction and energy deprivation in the mechanism of neurodegeneration
- Research Articles
- Cancer diagnosis via fiber optic reflectance spectroscopy system: a meta-analysis study
- Development of molecularly imprinted Acrylamide-Acrylamido phenylboronic acid copolymer microbeads for selective glycosaminoglycan separation in children urine
- Assessment of LXRα agonist activity and selective antiproliferative efficacy: a study on different parts of Digitalis species
- Computational assessment of SKA1 as a potential cancer biomarker
- In vitro apoptotic effect of Zinc(II) complex with N-donor heterocyclic ligand on breast cancer cells
- A single-tube multiplex qPCR assay for mitochondrial DNA (mtDNA) copy number assessment
- A case–control study on effects of the ATM, RAD51 and TP73 genetic variants on colorectal cancer risk
- Effects of α-lactalbumin and sulindac on primary and metastatic human colon cancer cell lines
- The role of interleukin-9 and interleukin-17 in myocarditis with different etiologies
- Gene silencing of Col1α1 by RNAi in rat myocardium fibroblasts
- A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays
- Role of SNPs of CPTIA and CROT genes in the carnitine-shuttle in coronary artery disease: a case-control study
- Interleukin-6 signaling pathway involved in major depressive disorder: selective serotonin reuptake inhibitor regulates IL-6 pathway
- Simultaneous comparison of L-NAME and melatonin effects on RAW 264.7 cell line’s iNOS production and activity
- Data-mining approach for screening of rare genetic elements associated with predisposition of prostate cancer in South-Asian populations
