T Zhang1, W Li2, X Qiu1, B Liu1, G Li1, C Feng1, J Liao1, K Lin1. 1. Department of Urology, Second Guangdong Provincial People's Hospital, Guangzhou 510317, China. 2. Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Abstract
OBJECTIVE: To construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED. OBJECTIVE: Models of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively. OBJECTIVE: The primarily cultured CCSMCs from diabetic rats with ED showed a high α-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED. OBJECTIVE: We successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.
OBJECTIVE: To construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED. OBJECTIVE: Models of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively. OBJECTIVE: The primarily cultured CCSMCs from diabetic rats with ED showed a high α-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED. OBJECTIVE: We successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.
Authors: Hong Yue; Maria Febbraio; Philip A Klenotic; David J Kennedy; Yueheng Wu; Shaoxian Chen; Amira F Gohara; Oliver Li; Adam Belcher; Bin Kuang; Thomas M McIntyre; Roy L Silverstein; Wei Li Journal: Arterioscler Thromb Vasc Biol Date: 2019-02 Impact factor: 8.311
Authors: Y Kouidrat; D Pizzol; T Cosco; T Thompson; M Carnaghi; A Bertoldo; M Solmi; B Stubbs; N Veronese Journal: Diabet Med Date: 2017-07-18 Impact factor: 4.359