Hyaluronic acid as a medium for human sperm migration tests

Hyaluronic acid was used as a substitute for human cervical mucus as a medium for sperm migration since its viscosity, molecular weight and structure are similar to constituent glycoproteins of human cervical mucus. Parameters of sperm motility were comparable in human cervical mucus and hyaluronate as measured by a computerized sperm motion analysis system (Hamilton-Thorn 2030 and Str?mberg-Mika System). Sperm migration rates were also similar in human cervical mucus, bovine mucus and hyaluronic acid and the reproducibility was best in hyaluronic acid. Sperm survival for 24 h was maintained in human cervical mucus and hyaluronic acid but not in bovine mucus. The use of hyaluronic acid in sperm migration tests is recommended.     

Collective cell migration of the nephric duct requires FGF signaling

Background : During the course of development, the vertebrate nephric duct (ND) extends and migrates from the place of its initial formation, adjacent to the anterior somites, until it inserts into the bladder or cloaca in the posterior region of the embryo. The molecular mechanisms that guide ND migration are poorly understood. Results : A novel Gata3‐enhancer‐Gfp‐based chick embryo live imaging system was developed that permits documentation of ND migration at the individual cell level for the first time. FGF Receptors and FGF response genes are expressed in the ND, and FGF ligands are expressed in surrounding tissues. FGF receptor inhibition blocked nephric duct migration. Individual inhibitors of the Erk, p38, or Jnk pathways did not affect duct migration, but inhibition of all three pathways together did inhibit migration of the duct. A localized source of FGF8 placed adjacent to the nephric duct did not affect the duct migration path. Conclusions : FGF signaling acts as a “motor” that is required for duct migration, but other signals are needed to determine the directionality of the duct migration pathway. Developmental Dynamics 244:157–167, 2015. © 2014 Wiley Periodicals, Inc.     

HOXA10 controls proliferation,migration and invasion in oral squamous cell carcinoma

Although HOX genes are best known for acting in the regulation of important events during embryogenesis, including proliferation, differentiation and migration, alterations in their expression patterns have been frequently described in cancers. In previous studies we analyzed the expression profile of the members of the HOX family of homeobox genes in oral samples of normal mucosa and squamous cell carcinoma (OSCC) and identified differently expressed genes such as HOXA10. The present study aimed to validate the increased expression of HOXA10 in OSCCs, and to investigate the effects arising from its knockdown in OSCC cells. The levels of HOXA10 mRNA were determined in human OSCC samples and cell lines by quantitative PCR, and HOXA10-mediated effects on proliferation, apoptosis, adhesion, epithelial-mesenchymal transition (EMT), migration and invasion were studied in HSC-3 tongue carcinoma cells by using retrovirus-mediated RNA interference. Higher expression of HOXA10 mRNA was observed in OSCC cell lines and in tumor tissues compared to normal controls. HOXA10 knockdown significantly reduced the proliferation of the tumor cells which was accompanied by increased levels of p21. HOXA10 silencing also significantly induced the expression of EMT markers and enhanced the adhesion, migration and invasion of HSC-3 cells. No effects on cell death were observed after HOXA10 knockdown. The results of the current study confirm the overexpression of HOXA10 in OSCCs, and further demonstrate that its expression is functionally associated with several important biological processes related to oral tumorigenesis, such as proliferation, migration and invasion.     

Decreased expression of miR-378 correlates with tumor invasiveness and poor prognosis of patients with glioma

microRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in a variety of biological process. It has been reported that dysregulation of miRNA is always associated with cancer progression and development, and miR-378 aberrant expression has been found in some types of cancers. However, the association of miR-378 and glioma has not been evaluated. In this work, we measured the expression of miR-378 in glioma tissues and non-neoplastic brain tissues was measured using real-time PCR, and found that miRNA-378 expression level was significantly lower in glioma tissues compared with non-neoplastic brain tissues. Patients with lower miR-378 expression level had significantly poorer overall survival. Multivariate Cox regression analysis showed that miR-378 expression was an independent prognostic factor for 5-year overall survival. Over-expression of miR-378 inhibits glioma cell migration and invasion. In conclusion, our results indicated that miR-378 may serve as a tumor suppressor and play an important role in inhibiting tumor migration and invasion. Our work implicates the potential effect of miR-378 on the prognosis of glioma.     

MicroRNA-200a inhibits epithelial-mesenchymal transition in human hepatocellular carcinoma cell line

Objective: Our study investigated the role of microRNA (miR)-200a and its molecular targets in hepatocellular carcinoma (HCC) cells. Methods: An inhibitor of miR-200a was transiently transfected into the hepatocellular carcinoma cell line, MHCC-97L. The effect of this transfection on mRNA levels of epithelial-mesenchymal transition (EMT)-related genes was measured by fluorescence-based quantitative real-time polymerase chain reaction (qRT-PCR). Further, protein levels of EMT-related genes, cell proliferation and apoptosis-related markers were assessed by Western blot analysis in these transfected cells. MTT and wound-healing assay were used to evaluate the proliferation and migration of MHCC-97L cells in presence and in absence of miR-200a inhibitor. Results: Compared with miR-NC control group, qRT-PCR results in anti-miR-200a group revealed a significant reduction in the mRNA levels of E-cadherin, with a concomitant increasing in vimentin mRNA level (all P < 0.05). Western blot results showed higher E-cadherin and Caspase-3 protein expressions in anti-miR-200a group compared to miR-NC group (P < 0.05). In addition, vimentin and Ki-67 protein expression was found sharply decreased in anti-miR-200a group compared to miR-NC group (P < 0.05). Consistent with this, wound-healing and MTT assay showed that migration and proliferation capacity of MHCC-97L cells in anti-miR-200a group is significantly increased compared with miR-NC group (both P < 0.05). Conclusion: Our study reveals an important role of miR-200a in inhibiting EMT, proliferation and migration in HCC cells, suggesting the possibility of miR-200a-based therapeutics in HCC.     

Ifi204基因表达对大鼠血管外膜成纤维细胞凋亡和迁移的影响

目的探讨ifi204基因过表达与沉默对大鼠血管外膜成纤维细胞(VAF)凋亡和迁移的影响。方法原代培养大鼠VAF,利用携带ifi204基因的慢病毒颗粒或空载体慢病毒颗粒感染VAF,分别作为实验组(ifi204 lv组)和阴性对照组(blank lv组)。用ifi204基因小干扰RNA瞬时干预VAF使其沉默(ifi204 siRNA组)、空白小干扰RNA作为阴性对照组(control siRNA组),未经处理的VAF作为空白对照组(negative组)。流式细胞仪检测细胞凋亡,细胞划痕法和Transwell法测定细胞迁移情况,用real-time PCR和Western blot分别检测mRNA和蛋白表达。结果与blank lv组、control siRNA组、ifi204 siRNA组和negative组相比,ifi204 lv组的P204、P53 mRNA和蛋白表达及细胞凋亡率明显上调(P0.05),细胞迁移速度降低(P0.05)。转染ifi204 siRNA可抑制P204、P53的mRNA和蛋白表达(P0.05),提高细胞活力,抑制细胞凋亡(P0.05),加快细胞迁移速度(P0.05)。结论 ifi204基因表达对大鼠VAF细胞凋亡和迁移的影响可能与激活P53表达有关。     

miR-580对乳腺癌细胞系中Twist1的调节作用

目的：探讨miR-580在MCF-10A细胞系中对Twist1的调节。方法：本实验利用生物信息学方法预测Twist1的靶miRNA是miR-580。首先,采用qPCR法检测在MCF-10A系列细胞系中Twist1及miR-580的表达。然后,在MCF-10A细胞系中分别转染miR-580类似物和miR-580抑制物后,利用RT-PCR、Western blot、t检验分析Twist1的表达及细胞迁移能力的变化。最后利用荧光素酶实验验证miR-580通过结合在Twist1的3'UTR调节其表达。结果：1)在MCF-10A细胞系中Twist1与miR-580的表达呈负相关;2)在MCF-10A细胞系中转染miR-580类似物后,Twist1的表达下调;在MCF-10A细胞系中转染miR-580抑制物后Twist1的表达上调;3)在MCF-10A细胞系中引入miR-580类似物后细胞迁移能力降低;4)miR-580直接结合在Twist1的3'UTR。结论：miRNA-580在MCF-10A细胞系中通过结合在Twist1的3'UTR负向调节Twist1的表达从而克制细胞的迁移。     

1α,25-Dihydroxyvitamin D3 inhibits the invasive potential of human breast cancer cellsin vitro

Using the Boyden chamber invasion assay, the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on the invasiveness of the highly invasive, oestrogen receptor-negative human breast cancer cell line MDA-MB-231 was examined. The MDA-MB-231 cells were shown to contain high-affinity receptors for 1,25(OH)₂D₃ with a K_d of 1.5 × 10^–11 M. When the cells were treated with 1,25(OH)₂D₃ for 4 days before the assay was performed, a dose-dependent inhibition of their invasive potential was demonstrated. Fifty per cent inhibition of invasion was obtained with a concentration of 13 pM of 1,25(OH)₂D₃. However, when the cells were treated for only 6 h during the assay, no inhibitory effect was seen. The process of migration was also affected by treatment with 1,25(OH)₂D₃ for 4 days, although the inhibition was not of the same magnitude as seen for the invasion. Fifty per cent inhibition of migration occurred at a concentration of 3.2 nM of 1,25(OH)₂D₃ (250 times higher than in the invasion assay). Inhibition of invasion and migration was not due to the known anti-proliferative effect of 1,25(OH)₂D₃, as no growth reduction could be demonstrated with treatment up to 5 days. Based on the present investigation it can therefore be concluded that 1,25(OH)₂D₃ is able to inhibit tumour cell invasiveness by a mechanism which is not exclusively based on its anti-proliferative and anti-migrative effects.