In the si-RNA-p15 groups the MTT recovered to 834% or 784%, respectively, with TGF-1 or TGF-2 treatment (Figure 5B, Table 3). DISCUSSION In this study we found that TGF-1 or TGF-2 treatments of primary cultured human limbal epithelial cells stimulated production of the CDKIs p57 and p15 and inhibited proliferation of these cells. and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-1 or TGF-2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-1 or TGF-2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot Pseudouridine and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus. Conclusions These findings demonstrate that TGF-1 and/or TGF-2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process. The transforming growth factor (TGF)- family comprises a large number of structurally related growth factors, each capable of regulating an array of cellular processes including proliferation, lineage determination, differentiation, motility, and cell death [1]. TGF- is one of a few known negative regulators of epithelial cell growth, yet the mechanisms by which it affects cell cycle arrest in epithelial cells are poorly understood [2]. Previous reports have shown that TGF- induces cell migration after wounding and inhibits proliferation of corneal epithelial cells either in vivo or in vitro [3C5]. It has been demonstrated that TGF- antagonizes the ability of epidermal growth Rabbit polyclonal to ADCK4 factor to stimulate corneal epithelial proliferation [5,6]. TGF- comprises three closely related isoforms in mammals known as TGF-1, TGF-2, and TGF-3 which have been detected in corneal epithelium and stroma [7]. Among them, TGF-1 and TGF-2 are the predominant forms Pseudouridine in the ocular surface and they play important roles as negative modulators of corneal cell proliferation [8]. In addition, they induce G1-phase arrest in limbal basal cells through an autocrine or a paracrine mechanism [9]. Hayashida-Hibino et al. [10] have demonstrated that TGF-1 control the differentiation and proliferation of corneal epithelial cells through downregulation of various targets, including plasminogen activator inhibitor type 2, transferrin, integrin 3, and cyclin D1. However, the effects of TGF-1 and TGF-2 on cell cycle regulation in primary cultured human limbal epithelial cells have not been fully clarified. Cell division consists of two consecutive processes; mitosis (M) and interphase (including G1, S, and G2 phases). M, G1, S, and G2 form the cell cycle. In the process of cell cycle regulation, cyclin-dependent kinase (CDK) inhibitors (CDKI) play an important role. Two major classes of CDKI have been identified: Cip/Kip and INK4. The Cip/Kip family contains the more general CDKI consisting of p21, p27, and p57, which specifically inactivate G1 cyclinE/CDK2 and cyclinD/CDK4/6. Member of the INK4 family, including p15, p16, p18, and p19, possess 4 ankyrin repeats and inhibit the G1 cyclinD/CDK4/6 complexes [11]. The expression of p15, p21, and p27 increases in response to TGF- in many cell types [12]. Increased levels of these CDKIs result in a major inhibition of CDK activities associated with the early G1 phase progression, thus locking the cell cycle prior to the G1 restriction point [11,13]. It has been reported that p15, p16, p21, p27, and p57 are expressed by mammary epithelial cells [14C18]. However, CDKI expression in the human limbal epithelial cells has not been completely investigated. RNA interference (RNAi) is a Pseudouridine phenomenon where double-stranded RNA Pseudouridine (dsRNA) induces Pseudouridine the sequence-dependent degradation of a cognate mRNA in cells [19]. It has been found that mRNA produced by the RNAi-targeted gene is absent from the cytoplasm and reduced in the nucleus. So RNAi exerts its effect during or following RNA processing, but before protein translation [20]. Several techniques have been developed to improve the effects of RNAi including short interfering RNAs (siRNAs), hairpinRNAs (hpRNAs), tiny non-coding RNAs (tncRNAs), and small modulatory RNA (smRNA) [21], among which siRNA is the most popular method. SiRNA uses dsRNAs of 21C22 nucleotides in length and silences genes.