| 1. Expression and significance of cyclooxygenase-2 in cervical carcinoma of premenopausal patientsObjective To investigate the relationship between the expression of cyclooxygenase-2 (COX-2) in cervical carcinoma and the menstrual cycle and its clinical significance. Methods Cervical carcinoma specimens were obtained from 47 premenopausal patients. The phase of menstrual cycle was determined by case history and HE staining with uterine endometrium. COX-2 and proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical SABC staining. The expression of COX-2 was obtained with the use of a computerized image analysis system, and the label index (LI) of PCNA was counted under microscope. Results The expression of COX-2 was mainly in carcinoma area. There was no difference of the positive rate between proliferating phase and secretory phase, but the expression of COX-2 was significantly higher during proliferative phase than secretory phase. The PCNA LI was positive correlated with the expression of COX-2. The expression of COX-2 was enhanced in carcinoma from the patients with lymph node metastasis and local infiltration in muscle layer. There was no significant correlation between the expression of COX-2 and the FIGO stage and differentiated degree. Conclusions The expression of COX-2 in cervical carcinoma was regulated possibly by the intermittent change of female hormone, and may play an important role in the regulation of the growth and metastasis of cervical carcinoma.2. The influence of estrogen and progesterone on the expression of cyclooxygenase-2 in Hela cellsObjective To investigate the regulatory effect of 17-βestradiol (E2) and medroxyprogesterone acetate (MPA) on the expression of COX-2 in Hela cells. Methods Hela cells were incubated with alcohol, E2, E2+MPA, MPA for 24 hours respectively. Western blot and reverse transcriptase– polymerase chain reaction (RT– PCR) was used to assay the effects of E2 and MPA on the expression of COX-2 protein and mRNA. The expression of COX-2 and PCNA protein in Hela cells was detected by immunocytochemistry SABC staining. The expression of PCNA was obtained with the use of a computerized image analysis system, Results COX-2 was expressed in normal cultural Hela cells. Incubated with E2 could significantly enhanced the expression of COX-2 protein and mRNA. On the contrary, E2+MPA and MPA alone could decrease the expression of COX-2. The OD value of immunostaining of PCNA, E2 group was higher than the control, E2+MPA and MPA group's OD value was decreased significantly than the control and E2. Conclusions Sexual hormone can regulate the expression of COX-2 in Hela cells. E2 can elevate, but MPA and E2+MPA can down-regulate its expression, and maybe therefore influence the proliferation of Hela cells.3. Construction of a siRNA eukaryotic expression vector for COX-2 and examine its effect in Hela cellsObjective To construct a eukaryotic expression vector expressing siRNA for COX-2 in eukaryotic cells. And then investigate its effects on the expression of COX-2 in Hela cells. Methods The full length of COX-2 gene sequence was analyzed with software of Ambion Corporation and the principle for design siRNA. Computer aided designing two COX-2 specific siRNA sequences. The synthesized siRNA sequences, included a loop, were cloned into Pgenesil-1 vector and called Pgenesil-C1 and Pgenesil-C2. P-Control, a same long random sequence inserted into the same position, as the negative control. After gene sequence was confirmed by cleavage of restriction enzymes and DNA sequence analyzed, P-Control, Pgenesil-C1 and Pgenesil-C2 was transiently transfected into Hela cells with LipofectamineTM2000, respectively. 48h after transfecting, RT-PCR and Western blot was used to assay the inhibition effect on the expression of COX-2. Results Cleavage of restriction enzymes and DNA sequence analyzed demonstrated that the siRNA sequences were cloned into the Pgenesil-1 vector successfully. After transfected with Pgenesil-C1 and Pgenesil-C2 for 48h, the expression of COX-2 mRNA and protein in Hela cells were inhibited significantly. The effect of Pgenesil-C1 (87%) was more obviously than Pgenesil-C2 (53%). Conclusions The eukaryotic expression vectors expressing siRNA for COX-2 were constructed successfully. Pgenesil-C1 can inhibit the expression of COX-2 significantly.4. Effects of the COX-2 expression down-regulated by RNA interference on cell cycle of cervical carcinoma Hela cellsObjective To investigate the change of the cell cycle of Hela cells after down-regulated the expression of COX-2 by RNA interference. Methods A eukaryotic expression plasmid of siRNA targeting on COX-2, Pgenesil-C1 was transfected into Hela cells with LipofectamineTM2000 and P-Control was transfected as a control. 48h and 96h after transfection respectively, MTT method was used to assay Hela cells proliferation level. The cell cycle of Hela cells was determined by flow cytometry. The changes of the expression of cyclinD1 and cyclin E protein were detected with Western blot. Results After transfected with Pgenesil-C1, the cell proliferation rate was decreased obviously. Flow cytometry assay demonstrated that after transfected with Pgenesil-C1 for 48h and 96h, The percentage of Hela cells in G0/G1 phase was increased, in S phase and G2/M phase was decreased at the same time. The expression of cyclinD1 and cyclin E protein was decreased significantly. The apoptosis ratio was 3.25% after transfected for 96h, increased obviously than the control. Conclusions Down-regulated the expression of COX-2 by RNA interference through a eukaryotic expression plasmid, Pgenesil-C1 can decrease cell proliferation rate. The cell cycle was arrested at G0/G1 phase maybe by inhibited the expression of cyclinD1 and cyclin E protein.5. Influences of the COX-2 expression down-regulated on apoptosis of cervical carcinoma cells induced by cisplatinObjective To investigate the changes of the expression of apoptosis-related genes after the expression of COX-2 was down-regulated by RNA interference in Hela cells. And then explore the influence of COX-2 on the sensitivity to cisplatin and its mechanism. Methods A eukaryotic expression plasmid of siRNA targeting on COX-2, Pgenesil-C1 was constructed. Than Pgenesil-C1 was transfected into Hela cells with LipofectamineTM2000 and P-Control was transfected as a control. Transfected cells were selected with G418. After treated with cisplatin of different concentration, the viability of Hela cells was examined by MTT assay. The apoptosis ratio was determined by flow cytometry. RT-PCR was used to assay the changes of the expression of Survivin,Bax and Bcl-2 mRNA. Results After transfected with Pgenesil-C1, MTT assay demonstrated that the viability of Hela cells was decreased along with the concentration of CDDP increasing. The Pgenesil-C1 group decreased more obviously than the control. Flow cytometry assay proved that the apoptosis ratio of the Pgenesil-C1 group was raised up significantly compared with the control. The expression of Survivin and Bcl-2 mRNA was decreased obviously compared with the control and the P-Control group. There was no significantly change of the expression of Bax mRNA. Conclusions After the expression of COX-2 was suppressed by RNA interference with a eukaryotic expression plasmid, Pgenesil-C1, the sensitivity of Hela cells to cisplatin was increased. This change maybe related to the down-regulated expression of Survivin and Bcl-2, instead of Bax. |
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