Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion / 生理学报

The purpose of this study was to investigate the effects of ubiquitin C-terminal hydrolase-L1 (UCHL1) on non-small cell lung cancer cell line A549. UCHL1 gene knockout A549 cell line was constructed by CRISPR-CAS9 gene editing technique. The mRNA and protein levels of UCHL1 were examined by RT-PCR and Western blot, respectively. Cell proliferation and cycles were analyzed by CCK-8 method and flow cytometry, respectively. The sensitivity of A549 cells to cisplatin was detected by CCK-8 method. Migration ability of A549 cells was detected by scratch assay and Transwell test, and p-Erk expression level was assessed by Western blot. The results showed that UCHL1 gene knockout A549 cells were successfully constructed by CRISPR-CAS9 gene editing technique. After UCHL1 gene knockout, there was no significant change in cell proliferation and cell cycle ratios in A549 cells. UCHL1 gene knockout A549 cells exhibited decreased sensitivity to cisplatin and migration activity, as well as increased p-Erk expression level. These results suggest that the loss of UCHL1 gene function may reduce the sensitivity and migration ability of A549 cells, and this effect may be related to the activation of Erk1/2 signaling pathway.

Uchl1 and its associated proteins were involved in spermatocyte apoptosis in mouse experimental cryptorchidism / 生理学报

Uchl1 was found to be involved in spermatocyte apoptosis. The aim of the present study was to test whether Uchl1 and its associated proteins Jab1 and p27(kip1) were involved in spermatogenic damages in response to heat-stress in cryptorchidism. Hematoxylin and eosin (HE) staining and DNA end labeling (TUNEL) were used to observe morphological and apoptotic characteristics of spermatogenic cells; Immunohistochemical analysis was used to detect changes of Uchl1 and its associated proteins Jab1 and p27(kip1) in response to heat-stress from cryptorchidism leading to spermatocyte losses; And protein affinity analysis (pull-down) and immunofluorescence co-localization were used to verify the relevance among the three proteins in spermatocytes. The results showed that, Jab1 and p27(kip1), in parallel to Uchl1, increased in spermatocytes of apoptotic appearances in response to heat-stress, but not in multinucleated giant cells; Jab1 bound to Uchl1 in testis protein extracts, and co-localized with Uchl1 and p27(kip1) specifically in spermatocytes with apoptotic appearances. These results suggest that the accumulation of Uchl1 protein is involved in the heat-stress-induced spermatocyte apoptosis through a new pathway related with Jab1 and p27(kip1), but not the formation of multinucleated giant cells.

Gene expression profiles of peri-implantation endometrium of high responders during controlled ovarian hyperstimulation / 上海第二医科大学学报

Objective To investigate the gene expression profiles of peri-implantation endometrium of high responders during controlled ovarian hyperstimulation. Methods High responders with cancelled embryo-transfer during controlled ovarian hyperstimulation (high responder group, n=4) and healthy fertile volunteers (control group, n=3) were performed endometrial biopsies during peri-implantation. Histologic changes of endometrium were observed by HE staining, genes of differential expression were screened with microarrays Affymetrix U133A 2.0 and identified by Real-time PCR. The biological process analysis was performed by online biological information analysis tool PANTHER. Results The ehdometrium was in mid-secretory phase in control group, while development delay was found in some glandular organs in endometrium of high responder group. Three hundred and sixty-four genes of differential expression were screened, among which 233 were up-regulated genes and 131 were down-regulated genes. OPN, PLA2G2, DPPIV, IGFBP5 and SSAT were identified as endometrial function-related genes, whose Real-time PCR findings were positively correlated to gene signal values detected by microarray(r=0.44, P<0.01). PANTHER analysis indicated that genes of differential expression participated in the biological processes of cytokine signal transduction and immunological regulation. Conclusion Ovarian high response affects the gene expression profiles of peri-implantation endometrium, which may be one of the causes of sub-optimal endometrial receptivity.

Ubiquitin carboxyl-terminal hydrolase L1 contributes to the oocyte selective elimination in prepubertal mouse ovaries / 生理学报

Apoptosis of abnormal oocytes is essential for defective oocyte elimination during prepubertal ovary development, and the ubiquitin system regulates the cell apoptosis via the degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the ubiquitin system, and the UCH-L1-dependent apoptosis is important for spermatogenesis. In the present study, the change in the number of follicles and the expression of UCH-L1 in oocytes were determined in prepubertal mouse ovaries by immunohistochemical techniques. A significant decrease in the follicular pool was found in prepubertal mouse ovaries during the period of day 21 to day 28 after birth, and accordingly, the UCH-L1 protein expression was increased, to some degree in association with Jun activation domain-binding protein 1 (Jab1) and cyclin-dependent kinase inhibitor p27(Kipl). The increased UCH-L1 protein, together with the corresponding changes of Jab1 was detected in morphologically abnormal oocytes of prepubertal ovaries. Through the immunofluorescent colocalization, UCH-L1 was shown concentrating in abnormal oocytes, and a parallel change in Jab1 was also seen. The affinity analysis confirmed the interaction between UCH-L1 and Jab1 in ovaries. These results suggest that UCH-L1 plays an important role, possibly in association with Jab1 and p27(Kipl), in selective elimination of abnormal oocytes during mouse prepubertal development.