Dissertation > Medicine, health > Oncology > Department of Otolaryngology tumor > Pharyngeal tumors

The Changes Expression and the Role of STAT3,MMP-2, Cyclind1in Egcg Antisense Nasopharyngeal Carcinoma CNE2Cell Line

Author WangWenHua
Tutor HeXiaoSong
School Guilin Medical College,
Course Surgery
Keywords EGCG STAT3 MMP-2 CyclinD1 Nasopharyngeal Carcinoma
CLC R739.63
Type Master's thesis
Year 2013
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Objective:Nasopharyngeal carcinoma is one of the most common malignant tumors in Guangdong, Guangxi and Fujian Of the South China’s area, the main treatment is radiotherapy plus chemotherapy and other comprehensive treatment currently, but yet the5year survival rate is only about50%, and the therapeutic effect and survival time of incurably ill patients is worse. This study was designed to investigate the effects of epigallocatechin gallate (Epigallocatechin-3-gallate, EGCG) on the growth of human nasopharyngeal carcinoma cell line CNE2in vitro, and through the analysis of the changes of the expression of STAT3, MMP-2, CyclinD1, to explore the mechanism of EGCG against nasopharyngeal carcinoma, for the development and search for new drugs to improve the treatment of nasopharyngeal carcinoma.Methods:1.Nasopharyngeal carcinoma cell line CNE2were cultured in vitro and treated with various concentrations of EGCG (0,40,80,160mg/L) for24h,48h and72h, respectively, the morphological changes of cells were observed by inverted microscope;2. After, MTT assay was used to determine the inhibitory rate of CNE2cell proliferation.3. Then the CNE2cells of every group treated with different concentrations of EGCG (0、40、80、160mg/L) for48h, apoptosis and apoptosis rate by Hoechst33258staining.4. The change of the CNE2cell invasion were observed by Transwell cell invasion assay after EGCG treatment at different concentration.5. The change of the CNE2cell cycle were analyze by Flow Cytometry after EGCG treatment at different concentration and different time.6. The expression of STAT3、CyclinD1、MMP-2were detected by Reverse Transcription Polymerase Chain Response (RT-PCR)Results:1. Observed under an inverted microscope, we found that the CNE2cell division phase decrease, some cells round, and with the EGCG concentration increased and prolonged duration of action, more and more cells floating and necrosis.2. The results of MTT showed that, with the increasing of the concentration of EGCG and the prolongation of action time, inhibitory effect of EGCG on the proliferation of nasopharyngeal carcinoma cell line CNE2continues to increase, and in a dose-and time-dependent manner (p<0.05).3. The results of Hochest33258staining showed that, EGCG after treatment of CNE2cells appeared typical morphologic changes of apoptosis such as nuclear fragmentation, nuclear condensation. Compared with the control group, with the increase of EGCG concentration, the number of apoptotic cells increased (P<0.05).4. Flow cytometry showed:after24h and48h of the CNE2cells were treatment by different concentrations of EGCG (0,40,80,160mg/L) respectively, the percentage of G1/G0phase cells arrest in respectively:(46.67±0.38)%、 (55.20±0.33)%、(62.33±0.28)%、(68.65±0.40)%and (49.12±0.28)%、(57.73±0.45)%、(64.36±0.26)%、(69.62±0.33)%. Compared with the control group, the percentage of G1/GO cells of the experimental group increased significantly (P<0.05); the results of comparison between any two group in the experimental groups were significantly different (P<0.05), and in a concentration and time dependent manner.5. The results of Transwell cell invasion assay showed that: with EGCG concentration increased, compared with the control group, the invasion of CNE2cells in a concentration-dependent weakened (p<0.05).6. The results of RT-PCR test showed that after24h and48h of the EGCG intervention, with the increase in concentration and acting time of EGCG, the relative expression of STAT3, MMP-2,and CyclinD1mRNA was gradually reducing and was down-regulated in a dose-dependent manner and time-dependent manner(P<0.05).Conclusions:1. The expression of STAT3mRNA was down-regulated in concentration-dependent and time-dependent by EGCG, which may serve as one of the mechanisms of the biological effects of EGCG anti nasopharyngeal carcinoma.2. The invasion of the CNE2cell was inhibited by EGCG, which may be related to the expression of MMP-2mRNA was reduction in dose-dependent and time-dependent manner.3. The proliferation of the CNE2cell was inhibited and apoptosis was induced by EGCG, which may be related to the expression of CyclinDl mRNA was reduction in dose-dependent and time-dependent manner.

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