[Effects of Cellular Density on the Induction of Suspension Globe of Ovarian Cancer Stem Cells].

OBJECTIVE: To determine the effect of cellular density on the separation and identification of cancer stem cells from human ovarian clear cell carcinoma cell line ES-2 and adenocarcinoma cell line A2780. METHODS: ES-2 and A2780 cells were cultured with human recombinant epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and bovine serum albumin and insulin in serum free medium. The cancer stem cells were obtained through serial passages. Changes in cell morphology,expressions of surface marker CD133 and CD44,and soft AGAR clone forming in the stem cells were examined under different cell density,either in serum-supplemented medium (SSM group) or in serum free medium (SFM group). RESULTS: Under the density of 2×104 mL-1,ES-2 cells survived in SFM,but did not form stem cells. When the density increased to 5×104 mL-1 or 1×105 mL-1,ES-2 cells survived in SFM,proliferated and formed stem cells. Compared with adherent cells,the suspension globe of stem cells expressed high levels of CD133 and CD44 ( P<0.05),with proliferation and clone forming ability after serial passages. The stem cell balls under the density of 5×104 mL-1 had stronger ability of tumor formation. A2780 cells formed suspension globe under the density of 1×104 mL-1 and 3×104 mL-1,but larger and more transparent balls were observed under the density of 3×104 mL-1 density. No suspension globe was formed under the density of 5×104 mL-1. More CD133+/CD44+cells were detected by flow cytometry under the density of 3×104 mL-1,compared with that under the density of 1×104 mL-1 ( P<0.05). Tumor stem cells grew faster under the density of 3×104 mL-1. CONCLUSION: The optimal density for identifying stem cells from human ovarian cancer is 5×104 mL-1 for ES-2 and 3×104 mL-1 for A2780,respectively.

Has­miR­125a and 125b are induced by treatment with cisplatin in nasopharyngeal carcinoma and inhibit apoptosis in a p53­dependent manner by targeting p53 mRNA.

MicroRNA (miRNA) is a class of non­coding RNA, which targets mRNAs of interest and suppresses its expression by degradation or translational inhibition. miRNA (miR)­125a and miR­125b were previously demonstrated to translationally and transcriptionally inhibit the expression of p53. The observed downregulation of the protein level of p53 in cisplatin­treated patients with nasopharyngeal carcinoma (NPC) indicates the association between cisplatin resistance, miR­125a and miR­125b. In the present study, through the detection of the expression levels of miR­125a and miR­125b, a significant upregulation of these miRs was demonstrated in cisplatin­treated patients with NPC. As a consequence, the protein expression level of p53 decreased notably. To confirm the induction of miR­125a and miR­125b by treatment with cisplatin, a cisplatin­resistant TW03 cell model (TW03/DDP) was constructed. As expected, in the TW03/DDP cells, the expression levels of miR­125a and miR­125b were upregulated, and this caused downregulation of p53. Ectopic expression of these miRNAs in the TW03 cell model sensitized TW03 to cisplatin by decreasing the protein expression levels of p53, whereas ectopic expression in the antisense oligos of these microRNAs demonstrated the opposite effect. In addition, the present demonstrated that the cisplatin­induced expression of miR­125a and miR­125b inhibited cisplatin­induced apoptosis in the TW03 cells by decreasing the protein expression levels of p53. Taken together, the present study revealed for the first time, to the best of our knowledge, that induction of the expression of miR­125a and miR­125b by treatment with cisplatin resulted in resistance to the cisplatin drug in the NPC cells.

Comparative proteomic analysis suggests that mitochondria are involved in autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD), characterized by ectatic collecting duct, is an infantile form of PKD occurring in 1 in 20 000 births. Despite having been studied for many years, little is known about the underlying mechanisms. In the current study, we employed, for the first time, a MS-based comparative proteomics approach to investigate the differently expressed proteins between kidney tissue samples of four ARPKD and five control individuals. Thirty two differently expressed proteins were identified and six of the identified protein encoding genes performed on an independent group (three ARPKD subjects, four control subjects) were verified by semi-quantitative RT-PCR, and part of them were further validated by Western blot and immunohistochemistry. Moreover, similar alteration tendency was detected after downregulation of PKHD1 by small interfering RNA in HEK293T cell. Interestingly, most of the identified proteins are associated with mitochondria. This implies that mitochondria may be implicated in ARPKD. Furthermore, the String software was utilized to investigate the biological association network, which is based on known and predicted protein interactions. In conclusion, our findings depicted a global understanding of ARPKD progression and provided a promising resource of targeting protein, and shed some light further investigation of ARPKD.