Cell Wound Scratch Assay Research Articles

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.

Inhibition of miR-205 Impairs the Wound-Healing Process in Human Corneal Epithelial Cells by Targeting KIR4.1 (KCNJ10)

The aim of the study was to test the hypotheses that injury stimulates the expression of miR-205, which in turn inhibits KCNJ10 channels by targeting its 3' UTR, thereby facilitating the wound-healing process in human corneal epithelial cells (HCECs). A stem-loop qRT-PCR was used to examine the miR-205 expression. BrdU cell proliferation assay and wound scratch assay were applied to measure the effect of miR-205 mimic or antagomer in HCECs. The patch-clamp technique, dual luciferase reporter assay, and Western blot analysis were employed to test whether miR-205 regulates KCNJ10, one of the target genes of miR-205. Both of the primary human and mouse corneal epithelial cells (pH/MCECs) were employed to further confirm the observations obtained in HCECs. The scratch injury in pH/MCECs increased the expression of miR-205 and decreased the expression of KCNJ10 within 24 hours. The notion that miR-205 may target KCNJ10 was supported by dual luciferase reporter assay showing an inhibition effect of miR-205 on 3' UTR of KCNJ10. Application of miR-205 antagomer significantly delayed the regrowth in wounded HCECs. However, inhibition of KCNJ10 partially abolished the effect from miR-205 antagomer and restored the healing process. Moreover, overexpression miR-205 antagomer enhanced the protein expression of KCNJ10 but not KCNJ16. In addition, patch-clamp demonstrated that inhibition of endogenous miR-205 expression increased Ba²⁺-sensitive inwardly rectifying K⁺ channels. In addition, an electrophysiological study of pHCECs showed the presence of KCNJ10-like 20 pS K⁺ channels and scratch injury significantly decreased the Ba²⁺-sensitive inwardly rectifying K⁺ currents. miR-205 stimulates wound healing by inhibiting its target gene KCNJ10.

LL‐37/CpG oligonucleotide complex formation and its effects on prostate cancer progression

LL‐37 is an antimicrobial DNA‐binding peptide that has been implicated in anti‐self DNA and anti‐tumor immune responses. The interaction between LL‐37 and DNA is not well‐characterized. We have used electrophoretic mobility shift assays (EMSA) and western blotting to investigate the interaction between LL‐37 and unmethylated CpG sequences known to activate Toll‐like receptor 9 (TLR9) pathways. TLR9 activation by CpG promotes weak anti‐tumor TH1 immune responses that are enhanced by LL‐37. In addition, we have tested the effects of LL‐37/CpG complexes in a prostate cancer progression model. By EMSA, we observe that LL‐37 is aggregated (has low gel mobility) in the absence of DNA, but that LL‐37/DNA association occurs with increased LL‐37 mobility, suggesting that CpG binding disrupts LL‐37 aggregation. Increases in LL‐37 concentration lead to reduced mobility, indicative of further complexation. We previously found that CpG/LL‐37 increases prostate cancer cell growth (PC‐3 cell line) and invasion through a matrigel matrix (LNCaP cell line). Currently, we are testing the efficacy of CpG/LL‐37 treatments on migration and anchorage‐independent growth in a prostate cancer progression model (PC‐3 cell line) using a cell scratch wound assay and soft agar assay. This work was supported by NIH EARDA grant 5G11HD037064‐08 and a Virginia Foundation of Independent Colleges Summer Undergraduate Research Fellowship.

Toll-like receptor 4 signaling in dysfunction of cardiac microvascular endothelial cells under hypoxia/reoxygenation

This study was designed to detect the role of Toll-like receptor 4 (TLR4) signaling in the dysfunction of cardiac microvascular endothelial cells (CMECs) after hypoxia/reoxygenation (H/R). The cell viability of CMECs was measured by MTT assay. The migration of CMECs was detected by cell scratch wound assay. The expressions of TLR4, nuclear factor-kappa B (NF-κB) and eNOS were analyzed by Western blot. Secretions of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by NO detection kit and ELISA. Lipopolysaccharide (LPS) incubation increased the expressions of TLR4, NF-κB, IL-6 and TNF-α in CMECs (P < 0.05 vs. control). The CMECs after H/R injury had impaired cell viability (P < 0.01 vs. control) and migration ability (P < 0.05 vs. control). Moreover, the expressions of TLR4, NF-κB, IL-6 and TNF-α were elevated after H/R in CMECs (P < 0.01 vs. control), while NO and the eNOS expression were significantly decreased. In contrast, administration of the TLR4-neutralizing antibody MTS510 prior to H/R injury down-regulated the expressions of IL-6 and TNF-α and attenuated the dysfunction of CMECs. TLR4 and its signaling components can be activated by LPS and H/R in CMECs. Blocking the TLR4 signal pathway before H/R injury attenuates CMEC dysfunction.