miR-21 promotes keratinocyte migration and re-epithelialization during wound healing

MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-β1. Similar to the effect of TGF-β1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-β1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-β-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-β1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.     

miR-155靶向SOCS1对骨肉瘤Saos2细胞的增殖、侵袭和迁移能力的影响

目的:探讨microRNA-155(miR-155)对骨肉瘤Saos2细胞增殖、侵袭和迁移的影响以及其作用机制。方法:利用实时荧光定量(qRT-PCR)实验检测miR-155在正常成骨细胞与骨肉瘤Saos2细胞中的表达水平,以及miR-155-mimic、miR-155-inhibitor的转染效率。采用CCK-8实验检测细胞的增殖能力,Transwell实验和划痕实验分别检测Saos2细胞的侵袭和迁移能力,Western blot检测细胞内的STAT3磷酸化水平以及SOCS1表达水平,双荧光素酶报告基因实验进行靶基因验证。结果:miR-155在骨肉瘤Saos2细胞中表达明显高于正常成骨细胞(P0.001)。在分别转染miR-155-mimic和miR-155-inhibitor后,Saos2细胞内miR-155表达水平明显上调和下降(P0.001)。过表达miR-155可促进Saos2细胞增殖、侵袭和迁移,降低SOCS1的蛋白水平,上调STAT3的磷酸化水平,差异均具有统计学意义。相反,降低miR-155水平可抑制Saos2细胞的增殖、侵袭和迁移能力,差异均具有统计学意义。结论:骨肉瘤Saos2细胞中高表达的miR-155可以通过抑制SOCS1表达来激活STAT3信号通路进而促进细胞的增殖、侵袭和迁移,因此,靶向抑制miR-155表达可以作为潜在治疗骨肉瘤的途径。     

A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR₁), and by PAR₁ inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR₁-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.     

Downregulation of CD9 in Keratinocyte Contributes to Cell Migration via Upregulation of Matrix Metalloproteinase-9

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.     

Switch from αvβ5 to αvβ6 integrin is required for CD9-regulated keratinocyte migration and MMP-9 activation

Our previous research found that tetraspanin CD9 is downregulated in migrating epidermis during wound healing, and CD9 downregulation contributes to keratinocyte migration via matrix metalloproteinase-9 (MMP-9) activation. However, little is known about the mechanisms involved in CD9-regulated keratinocyte migration and MMP-9 activation. In this study, we revealed that the expressions of integrin subunits β5 and β6 were regulated by CD9. Furthermore, CD9 silencing triggered the switch from αvβ5 to αvβ6 integrin in HaCaT keratinocytes and CD9 overexpression reversed the switch. Importantly, integrin αvβ6 functional blocking antibody 10D5 significantly inhibited CD9 silencing-induced keratinocyte migration and MMP-9 activation, suggesting that the switch from αvβ5 to αvβ6 integrin plays a key role in CD9-regulated cell migration and MMP-9 activation in keratinocytes.     

MiR-519d-3p Suppresses Invasion and Migration of Trophoblast Cells via Targeting MMP-2

Our study was approved by the Medical Ethics Committee of Tang Du Hospital, Fourth Military Medical University and complied strictly with national ethical guidelines. Preeclampsia (PE) is a specific clinical disorder characterized by gestational hypertension and proteinuria and is a leading cause of maternal and perinatal mortality worldwide. The miR-519d-3p is upregulated in the maternal plasma of patients with PE which indicates a possible association between this microRNA and the pathogenesis of PE. No studies to date have addressed the effect of miR-519d-3p on the invasion and migration of trophoblast cells. In our study, we found that miR-519d-3p expression was elevated in placental samples from patients with PE. In vitro, overexpression of miR-519d-3p significantly inhibited trophoblast cell migration and invasion, whereas transfection of a miR-519d-3p inhibitor enhanced trophoblast cell migration and invasion. Luciferase assays confirmed that matrix metalloproteinase-2 (MMP-2) is a direct target of miR-519d-3p. Quantitative real-time PCR and western blot assays showed that overexpression of miR-519d-3p downregulated MMP-2 mRNA and protein expression. Knockdown of MMP-2 using a siRNA attenuated the increased trophoblast migration and invasion promoted by the miR-519d-3p inhibitor. In placentas from patients with PE or normal pregnancies, a negative correlation between the expression of MMP-2 and miR-519d-3p was observed using the Pearson correlation and linear regression analysis. Our present findings suggest that upregulation of miR-519d-3p may contribute to the development of PE by inhibiting trophoblast cell migration and invasion via targeting MMP-2; miR-519d-3p may represent a potential predictive and therapeutic target for PE.     

LncRNA CRNDE promotes cell proliferation,migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis

Ovarian cancer seriously threatens the health of women. LncRNA CRNDE is known to be upregulated in ovarian cancer. However, the mechanism by which CRNDE regulates the progress of ovarian cancer is largely unknown. MTT assay was applied to measure the cell viability. Colony formation assay was used to measure the cell proliferation. Cell migration was tested by wound healing, and Transwell assay was performed to detect cell invasion. In addition, the expression of miR-423-5p, CRNDE and FSCN1 were detected by RT-qPCR and western blotting, respectively. Meanwhile, dual-luciferase reporter assay and RIP assay were performed to explore the correlation between miR-423-5p and CRNDE (or FSCN1). CRNDE and FSCN1 were upregulated in ovarian cancer cells (SKOV3, CAOV-3, IGROV1, A2780 and C13K), while miR-423-5p was downregulated. Moreover, silencing of FSCN1/CRNDE significantly decreased proliferation, migration and invasion of ovarian cancer cells (SKOV3 and CI3K) via suppressing MMP-2 and MMP-9. In addition, CRNDE could sponge miR-423-5p, and FSCN1 was confirmed to be the direct target of miR-423-5p. Furthermore, CRNDE knockdown-induced inhibition of FSCN1 was notably reversed by miR-423-5p downregulation. Knockdown of CRNDE inhibited cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis. Thus, CRNDE may serve a new target for ovarian cancer.

     

miR‐221 promotes keratinocyte proliferation and migration by targeting SOCS7 and is regulated by YB‐1

Proliferation and migration of keratinocytes are vital processes for the successful epithelization specifically after wounding. MiR‐221 has been identified to play a potential role in promoting wound regeneration by inducing blood vessel formation. However, little is known about the role of miR‐221 in the keratinocyte proliferation and migration during wound healing. An in vivo mice wound‐healing model was generated; the expression levels of miR‐221 were assessed by qRT‐PCR and fluorescence in situ hybridization. Initially, we found that miR‐221 was upregulated in the proliferative phase of wound healing. Further, in an in vivo wound‐healing mice model, targeted delivery of miR‐221 mimics accelerated wound healing. Contrastingly, inhibition of miR‐221 delayed healing. Additionally, we observed that overexpression of miR‐221 promoted cell proliferation and migration, while inhibition of miR‐221 had the opposite effects. Moreover, we identified SOCS7 as a direct target of miR‐221 in keratinocytes and overexpression of SOCS7 reversed the effects of miR‐221 in HaCaT keratinocytes. Finally, we identified that YB‐1 regulates the expression of miR‐221 in HaCaT keratinocytes. Overall, our experiments suggest that miR‐221 is regulated by YB‐1 in HaCaT keratinocytes and acts on SOCS7, thereby playing an important role in HaCaT keratinocyte proliferation and migration during wound healing.     

miR-155对肝癌细胞增殖的影响

目的:在肝癌细胞系中过表达miR-155,研究其对肝癌细胞增殖的影响。方法:将pc DNA3.0-miR-155表达载体瞬时转染Huh7.5.1及Hcclm3肝癌细胞系,通过实时定量PCR技术对miR-155在转录水平的表达进行检测,采用CCK8法及克隆形成实验检测miR-155过表达后对Huh7.5.1及Hcclm3肝癌细胞系增殖的影响。结果:转染细胞后72 h,经实时定量PCR检测,Huh7.5.1及Hcclm3肝癌细胞中成熟miR-155的表达分别上调约431及16倍(P0.01),说明其能有效高表达;CCK8法及克隆形成实验结果显示,miR-155能够明显促进肝癌细胞增殖(P0.01)。结论:pc DNA3.0-miR-155转染Huh7.5.1及Hcclm3肝癌细胞系后能高效表达成熟miR-155,同时证明过表达miR-155能使肝癌细胞的增殖受到非常明显的促进。     

Hypoxia induces epidermal keratinocyte matrix metalloproteinase-9 secretion via the protein kinase C pathway

Hypoxia promotes keratinocyte migration on wound bed connective tissues and is a profound biological signal that transforms a basal keratinocyte, destined to differentiate, into a motile cell that is essential for re-epithelialization. In this study, we examined the effect of hypoxia on keratinocyte-derived collagenases associated with keratinocyte migration. Cells plated on various connective tissue matrices under normoxic and hypoxic conditions, demonstrated a two-fold increase in the 92 kDa, type IV collagenase (MMP-9) when examined by quantitative zymography and ELISA. Western blotting and ELISA demonstrated a two-fold increase in tissue inhibitor of metalloproteinase (TIMP-1), an enzyme that binds to MMP-9 and inhibits its activity. The hypoxia-induced increase in cell motility could be inhibited by a neutralizing antibody to MMP-9. Northern blotting demonstrated that MMP-9 and TIMP-1 mRNA increased 2.5- to 4-fold, 2-12 h after the cells were made hypoxic. The hypoxia-induced changes in MMP-9 and TIMP-1 were inhibited by staurosporine and bisindolylmaleimide, inhibitors of protein kinase C (PKC), but not by inhibitors of tyrosine phosphorylation and the mitogen-activated protein kinase pathway. Inhibition of PKC also inhibited hypoxia-induced keratinocyte migration on type I collagen. These data provide evidence that hypoxia-induced keratinocyte migration is mediated by increased cellular secretion of MMP-9 via the PKC pathway.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.     

Matrix metalloproteinase activity is necessary for thymosin beta 4 promotion of epithelial cell migration

Studies from our laboratory provide substantial evidence that thymosin beta 4, (Tbeta(4)), an actin-sequestering protein, promotes corneal wound healing through its ability to stimulate epithelial cell migration. Matrix metalloproteinases (MMPs), which are expressed in a wide variety of tissues including the cornea, also play a key role in epithelial cell migration and wound healing. In this study we investigated the role of MMPs in Tbeta(4)-stimulated corneal epithelial cell migration. In Boyden chamber assays, XG076, an inhibitor of the conversion of pro- to active MMPs, had no effect on epithelial cell migration stimulated by exogenous activated MMP-1. However, in in vitro migration assays where the activation of pro-MMPs was blocked, XG076 significantly inhibited cell migration and wound healing in the presence or absence of Tbeta(4). GM6001, a broad-spectrum inhibitor of active MMPs and selective MMP inhibitors, also suppressed Tbeta(4)-stimulated cell migration. Tbeta(4) upregulated MMP-1 gene and protein expression in primary human corneal epithelial cells and in transformed human corneal epithelial cells following scrape wounding. From these results we conclude that MMP catalytic activity is necessary for Tbeta(4) promotion of epithelial cell migration. These novel findings are the first to demonstrate a functional link between the two.     

miR-96-5p regulates wound healing by targeting BNIP3/FAK pathway

Cutaneous wound healing is a highly orchestrated basic biological process and one of the key processes in restoring skin integrity. The role of microRNAs (miRNAs) during this process has raised numerous attention and is poorly explored. The aim of this study is to investigate the potential function of BCL2 interacting protein (BNIP3) and its target miRNA, miR-96-5p, in cutaneous wound healing. The results demonstrated that BNIP3 was significantly increased and miR-96-5p was obviously decreased during wound healing. Overexpression of BNIP3 significantly increased, while inhibition of BNIP3 decreased cell proliferation and migration of human primary keratinocytes. miR-96-5p was predicted to be a target miRNA for BNIP3 and luciferase reporter assay confirmed that miR-96-5p directly targeted the 3′-untranslated region of BNIP3. Moreover, miR-96-5p overexpression significantly decreased, while miR-96-5p inhibition dramatically increased BNIP3 protein expression and focal adhesion kinase (FAK) pathway activation. Furthermore, miR-96-5p inhibited cell proliferation and migration of human primary keratinocytes. Overall, our findings suggest that miR-96-5p might be critical in the regulation of wound healing by mediating BNIP3 and FAK pathway.     

MicroRNA-21 promotes the proliferation and inhibits apoptosis in Eca109 via activating ERK1/2/MAPK pathway

The aim of this study was to investigate how miR-21 promotes proliferation and inhibits apoptosis in esophageal squamous cell carcinoma (ESCC). MTT, wound healing assay and cell cycle showed that proliferation and migration of ESCC cell line Eca109 cells were increased in miR-21 mimics group, and decreased in anti-miR-21 Oligonucleotide (AMO) group after transfection into Eca109 cells with miR-21 mimics, AMO and scramble sequence, respectively. Cell apoptosis assay indicated that cell apoptosis can be obviously inhibited by overexpression of miR-21 and promoted by downregulation of miR-21. Meanwhile, western-blot results showed that p-ERK1/2 expression was elevated in miR-21 mimics group, whereas decreased in AMO group. Furthermore, the ERK1/2, a key component of MAPK signaling pathway, was knocked down, and overexpressed successfully using shRNA-ERK1/2 and overexpressing plasmids containing full length cDNA of ERK1/2, respectively. It was observed that shRNA-ERK1/2 can significantly decreased the level of miR-21 expression, while overexpression of ERK1/2 can up-regulate expression of miR-21. As further confirmation, Eca109 cells were treated with gradient concentration of U0126, a kind of MEK inhibitor, and expression of miR-21 was subsequently examined. It was found that U0126 can significantly decreased endogenous expression of miR-21. In parallel, U0126 decreased cell proliferation, migration and increased the apoptosis in Eca109 cells, with the expression of miR-21 being reduced significantly in U0126 group as compared with control groups. Our findings indicated that miR-21 promoted the proliferation, migration and inhibited apoptosis of Eca109 cells through activating ERK1/2/MAPK pathway, and that targeting miR-21 could be a promising therapeutic strategy in ESCC.