LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.     

Circ-8073 regulates CEP55 by sponging miR-449a to promote caprine endometrial epithelial cells proliferation via the PI3K/AKT/mTOR pathway

Circular RNAs (circRNAs) are a large class of endogenous non-coding RNAs that function as regulators in various cells and tissues. Here, the function and mechanism of circRNA8073 (Circ-8073) on endometrial epithelial cells (EECs) and the development of endometrial receptivity were investigated in dairy goats. Circ-8073 could bind to and inhibit miR-449a activity. Circ-8073 binding to the target site of miR-449a had a negative feedback relationship. Centrosomal protein55 (CEP55) was a direct target gene of miR-449a, and Circ-8073 could increase the expression levels of CEP55 by sponging miR-449a in EECs in vitro. Circ-8073/miR-449a/CEP55 could promote EECs proliferation via the PI3K/AKT/mTOR pathway. In addition, CEP55 could regulate the expression levels of vascular endothelial growth factor (VEGF) and forkhead box M1 (FOXM1) in EECs, which contributed to the development of endometrial receptivity. These findings showed that Circ-8073 regulated CEP55 by sponging miR-449a to promote EEC proliferation via the PI3K/AKT/mTOR pathway, suggesting that it could function as a regulator in the development of endometrial receptivity in dairy goats.     

Cloning of leukemia inhibitory factor (LIF) and its expression in the uterus during embryonic diapause and implantation in the mink (Mustela vison)

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice. Whether LIF plays a role in termination of embryonic diapause and initiation of implantation in carnivores, especially in species with obligate delayed implantation such as the mink, is not known. The objectives of this study were to clone the LIF coding sequence in the mink and determine its mRNA abundance in the uterus through embryonic diapause, implantation, and early postimplantation. We show that the mink LIF cDNA contains 609 nt encoding a deduced protein of 203 amino acids. The homologies are 80.6, 90, 88.2, 87.6, and 86.8% in coding sequence and 79.2, 90.1, 91, 90.1 and 85.4% in amino acid sequence with mouse, human, pig, cow, and sheep respectively. Glycosylation sites and disulfide bonds present in other species are generally conserved in the mink LIF sequence. Quantitation by polymerase chain reaction amplification indicates that LIF mRNA is expressed in mink uterus just prior to implantation and during the first two days after implantation, but not during diapause or later after implantation pregnancy. The abundance of LIF mRNA was significantly higher in the uterus at the embryo expansion stage (P < 0.05) than at days 1–2 of postimplantation. By immunohistochemical localization it was shown that LIF is expressed in the uterine epithelial glands at time of embryonic expansion and in early postimplantation. The coincidence of LIF expression with implantation in this species suggests that LIF is involved in the implantation process, and may be a maternal signal which terminates obligate embryonic diapause. Mol. Reprod. Dev. 51:13–21, 1998. © 1998 Wiley-Liss, Inc.     

A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli

Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His₆-tag and Trx-His₆-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His₆-hLIF fusion protein was not as soluble as the Trx-His₆-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (>95% pure) His₆-hLIF and Trx-His₆-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His₆-hLIF and Trx-His₆-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.