MicroRNA-215-3p suppresses the growth and metastasis of cervical cancer cell via targeting SOX9.

OBJECTIVE: The aim of the current study was to investigate the potential roles of miR-215-3p in the progression of cervical cancer. PATIENTS AND METHODS: The levels of miR-215-3p in both cervical cancer tissues and cell lines were detected using quantitative Real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, migration and invasion assays were applied to investigate the role of miR-215-3p on the growth and aggressiveness of cervical carcinoma SiHa cell. The expression of SRY-Box 9 (SOX9) was assessed by Western blotting assay. The Xenograft model and lung metastasis model were applied to reveal the impact of miR-215-3p on the growth and distant metastasis of cervical carcinoma cell in vivo. Moreover, miR-215-3p and a SOX9 siRNA were co-transfected into the SiHa cell to investigate the underlying mechanism of miR-215-3p-SOX9 on cervical cancer tumorigenesis. RESULTS: We used genome-wide gene expression analysis using clinical cervical cancer samples to identify that miR-215-3p was down-regulated in cervical cancer. We then collected 31 pairs of cervical cancer and the corresponding non-cancerous tissues to determine miR-215-3p level and indicated that miR-215-3p was significantly down-expressed in cervical cancer. Furthermore, the functional analysis suggested that over-expression of miR-215-3p suppressed the aggressiveness of SiHa cell, whereas down-regulation led to the opposite results. We identified SOX9 as a direct target of miR-215-3p, and its level was negatively related to the level of miR-215-3p in cervical carcinoma tissue. Up-regulation of SOX9 reversed the suppressive impact of miR-215-3p on cervical carcinoma cell, and down-regulation of SOX9 reversed the promote effects of miR-215-3p CONCLUSIONS: These findings showed the important role of the miR-215-3p/SOX9 axis in the progression of cervical carcinoma.

EGFR tyrosine kinase inhibitor AG1478 inhibits cell proliferation and arrests cell cycle in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC), which occurs with a high incidence in southern China and southeast Asia, is of epithelial origin with overexpression of EGF receptor. To study the effect of inhibition of EGFR signaling on nasopharyngeal carcinoma cell proliferation and cell cycle distribution, EGFR tyrosine kinase inhibitor AG1478 was employed to treat Nasopharyngeal Carcinoma CNE2 cells. The results showed that AG1478 inhibited proliferation of CNE2 cells. Immunoblot showed that AG1478 inhibited EGFR phosphorylation in CNE2 cells without reduced expression of EGFR protein. The activation of Akt and MAPK which are downstream molecules of EGFR signaling pathway, were also inhibited by AG1478. AG1478 induced cell cycle arrest in G1 phase, and the levels of protein p27 were significantly up-regulated. We concluded that inhibition of the EGFR signaling induced cell cycle arrest in G1 phase in CNE2 cells and p27 up-regulation was involved in this process. The EGFR kinase specific inhibitor is of potential to be developed into drugs for NPC treatment.

[Mechanism of apoptosis induced by squamocin in leukemia cells].

AIM: To investigate the mechanism of apoptosis of HL60 cells induced by the annonaceous acetogenin, squamocin. METHODS: Induction of apoptosis was determined through Hoechst33258 dye staining and DNA agarose gel electrophoresis. Expression of the proteins was detected using Western blot analysis. Caspase-3 activity was detected using caspase-3 kit. RESULTS: Treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation, DNA fragmentation, cleavage of the death substrate poly(ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. Stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. CONCLUSION: These results suggest that apoptosis of HL-60 cells induced by squamocin require caspase-3 activation, and could be related to SAPK activation.

Apoptosis induced by ceramide in hepatocellular carcinoma Bel7402 cells.

AIM: To study the biological function of ceramide signaling in Bel7402 cells. METHODS: Inhibition of cell growth was assayed using MTT method. Morphologic assessment of apoptosis was performed with fluorescence microscope. DNA fragmentation was detected by electrophoresis and flow cytometry. The levels of protein p53, Bcl-2, and Bax were measured with Western blot. RESULTS: Bel7402 cells treated with C2-ceramide underwent cell proliferation inhibition. IC50 value was 14.28 mumol.L-1. After treatment of Bel7402 with ceramide, the morphologic changes including reduction in volume, nuclear chromatin condensation, fluorescence strength were observed. SubG1 peaks were detected on flow cytometry (FCM). Agarose gel electrophoresis of DNA from cells treated with ceramide revealed "ladder" pattern. The Western blot assay from cell extracts showed that the levels of protein p53 were decreased after ceramide treatment. The levels of protein Bcl-2 were decreased also. But the levels of Bax protein showed no difference between untreated cells and treated cells. CONCLUSION: Ceramide induces apoptosis in Bel7402 cells, related to Bcl-2 down-regulation.