BACKGROUND:The degradation of extracellular matrix, which is mediated by matrix metal oproteinases, plays a crucial role in the corneal neovascularization. Tissue factor pathway inhibitor 2, a new type serine proteinase inhibitor, can effectively inhibit the activity of matrix metal oproteinases.
OBJECTIVE:To elucidate the effect of tissue factor pathway inhibitor 2 on the expressions of matrix metal oproteinases in keratocytes in vitro.
METHODS:Rabbit keratocytes were primarily cultured and subcultured in vitro. Plasmid vector pBos-Cite-neo/TFPI-2 was transfected into keratocytes with Lipofectamine 2000. The positive cells were selected using G418.
RESULTS AND CONCLUSION:The results of reverse transcription-polymerase chain reaction, western blot analysis and gelatinase zymography analysis showed that, expression of mRNA and protein of tissue factor pathway inhibitor 2 was upregulated in the transfected keratocytes (P<0.05), while activity of matrix metal oproteinase 1 and 2 was significantly decreased (P<0.05). Experimental findings indicate that that tissue factor pathway inhibitor 2 strongly inhibits the activity of matrix metal oproteinase 1 and 2 in keratocytes.

events were associated with promestriene use. Conclusion The premestriene capsule was safe and effective in the treatment of postmenopausal atrophic vaginitis.

OBJECTIVETo investigate the effect of microRNA-140 (miR-140) on the migration and invasion of colorectal cancer (CRC) cells and the possible mechanism.

METHODSmiR-140 mimics, miR-140 specific inhibitor or small interfering RNA (siRNA) against Smad3 were transfected into human CRC cell line RKO cells respectively, using Oligofectamine or Lipofectamine2000. Quantitative real-time PCR (real-time PCR) was used to measure the expression levels of miR-140 and Smad3 mRNA. Smad3 protein was analyzed by Western blot. The in vitro cell migrating and invasive abilities were determined by wound-healing and Transwell chamber assay after up-regulating or down-regulating miR-140 or knocking down Smad3.

RESULTSThe Western blot assays showed that the Smad3 protein level was significantly reduced after up-regulating miR-140 (0.04 ± 0.01), compared with that of (0.47 ± 0.02, P < 0.05) in the control group and that of (0.52 ± 0.06) in the negative control group (P < 0.05 for both). The results of real-time PCR indicated that no significant difference was found in the levels of Smad3 mRNA between miR-140 transfection and NC groups (1.11 ± 0.13 vs. 1.00 ± 0.06, P > 0.05). The wound-healing assay showed that the migrating ability was dramatically attenuated by miR-140 compared with that in the control and NC groups, whereas no significance was found when compared with that of the Smad3 siRNA transfected cells. The number of cells migrating through Transwell chamber without matrigel in the miR-140 group was (76.2 ± 4.4), remarkably lowered than that in the control (267.1 ± 4.9) and NC (336.1 ± 5.7) groups (P < 0.05 for both), but no significant difference between the miR-140 (76.2 ± 4.4) and Smad3 siRNA (83.5 ± 7.3) groups. Transwell chamber with matrigel assay showed that number of cells penetrating through the membrane was (109.5 ± 7.4) in the miR-140 group, significantly lower than that in the control (403.1 ± 5.1) and NC (392.6 ± 8.4) groups (P < 0.05 for both), while Smad3 siRNA transfection had a similar effect (138.8 ± 3.6)(P > 0.05). Down-regulation of miR-140 increased the level of smad3 protein expression, and partially reversed the inhibition of the cell migration and invasion mediated by miR-140. Co-transfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the abilities of cell migration and invasion.

CONCLUSIONSmiR-140 regulates the Smad3 expression at the post-transcriptional level. miR-140 suppresses the migrating and invasive abilities of CRC cells, possibly through down-regulation of Smad3. The findings of this study suggest that miR-140 may have a unique potential as a possible biomarker candidate for diagnosis and therapy of tumor metastasis.

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; MicroRNAs ; Neoplasm Invasiveness ; RNA, Messenger ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Smad3 Protein ; genetics ; metabolism ; Transfection ; Up-Regulation