The Expression And The Functional Analysis Of Peptidy-prolyl Cis/trans Isomerase (Pin1) In Cervical Cancer And Cervical Intraepithelial Neoplasia

Chapter 1: The Expression of Peptidy-prolyl Cis/trans Isomerase Pin1 in Cervical Cancer and Cervical Intraepithelial NeoplasiaObjective: To study the expression of pin1 in the cervical cancer andcervical intraepithelial neoplasia.Methods: The Pin1 mRNA and protein expression were examined in cervical cancer and cervical intraepithelial neoplasia samples with RT-PCR and immunocytochemical analysis.Results: 1. The mean rate of Pin1/actin mRNA was 1.92 ± 0.48(n=10), 1. 03 + 0. 40(n=10), 0. 42±0. 26(n=10), 0. 39 + 0. 16(n=10), 0. 33 ± 0. 19(n=10), respectively, in cervical cancer, CIN III, CIN II, CIN I and negative group. The mean rate of Pin1/actin in cervical cancer was significantly higher than that in other groups (P<0. 01) ; the mean rate of Pin1/actin in CIN III was also significantly higher than that in CIN II, CIN I and negative control group (P <0. 05).2. The intensity of Pin1 immunopositive cells was 175. 50 ± 15. 44(n=41) ;154. 39 ± 18. 56(n=60) ;138. 89 ±11.46(n=30) ; 125. 17 ± 15. 24 (n=10) , respectively, in cervical cancer , CIN II+CIN III, CIN I and negative group. There was a significant difference of Pin1 protein expression among these groups (P<0. 001). Conclusions: The Pin1 was overexpressed in cervical cancer and cervical intraepithelial neoplasia, and the expression lever was correlated with pathological progress.Chapter2: A Study of the Expression and the Relation BetweenPin1 and Cyclin D1 in Cervical CancerObjective: To study the expression of Pin1, Cyclin D1 and their relation in cervical cancer tissue.Methods: The Pin1, Cyclin D1 mRNA and protein expression were examined in cervical cancer samples with and RT-PCR immunocytochemical analysis.Results: 1. The intensity of Pin1 immunopositive cells (165. 97±14. 42, n=41) in cervical cancer was significantly higher than that in negative group(137. 09 ±13. 96, n=10) (P <0. 01). The intensity of it in stage II (170. 96 ± 13. 60, n=11) was significantly higher than that in stage I (163. 05 ± 13. 81, n=30) ( P<0. 05) ; The intensity of Pin1 immunopositive cells in cervical cancer kith lymph node metastasis (173. 59 ± 12. 47, n=7) was also significantly higher than that in patients with no lympn node metastasis (164. 40± 14. 67, n=34 ) ; There was no significant difference of Pin 1 protein expression among histological types and grades.2. There was no significant difference between the cervical cancer group and the negative group in the intensity of Cyclin D1 immunopositive cells. The intensity of Cyclin D1 immunopositive cells in cervical glandular cancer (131.28 ± 12.47, n=11) was significantly higher than that in cervical squmous cancer (125. 04 ± 12. 93, n=30) ( P<0. 05). 3. There were 10 cases with Pin1 mRNA positive and 5 cases with Cyclin D1 positive among 15 cervical cancer samples. There were with 3 Cyclin D1 positive among Pin1 positive patients, 2 Cyclin D1 positive among Pin1 negative patients. There was no significant correlation between the expression of Pin1 and the Cyclin D1 mRNA.Conclusions: Pin1 was overexpressed in cervical cancer; there was no significant correlation between the expression of Pin1 and Cyclin D1 in cervical cancer tissue.cheapter 3: A Study of the Expression and Relation of Pin1, p16INK4a, Ki-67 in Cervical Intraepithelial NeoplasiaObjective: To study and compare the expression of Pin1, p16INK4a, Ki-67 in cervical Intraepithelial neoplasiaMethods: Immunocytochemical analysis of Pin1, p16INK4a, Ki-67 expression was performed in 30 CIN IIIsamples, 30 CIN II,30 CIN I and 10 negative samples. The status of HPV was examined by polymerase chain reaction analysis of the extracted DNA.Results: 1. The intensity of Pin1 immunopositive cells was 154. 39 ± 18. 56, 138.89 ± 11.46, 125.17 ± 15.24, respectively, in CIN III + CIN II , CIN I and negative group. There was a significant difference of Pin1 expression among these groups(P<0. 05). 2. The percentage of p16INK4a immunopositive cells in CINs was significantly higher than that in negative group (P=0. 018). Similarly, there was also a significant difference of p16INK4a immunopositive cells among CINs (P=0.025). The percentage of Ki-67 immunopositive cells in CINs was significantly higher than that in negative control group (P=0. 028). The rate of HPV-DNA positive in CIN samples was significantly higher than it in negative group (P=0. 011). There was no significant difference of Ki-67 and HPV-DNA expression among CINs.3. The intensity of Pin1 immunopositive cells in pl6INK4a immunopositive samples was significantly higher than that in pl6INK4a immunonegative samples (P<0. 001) ; The intensity of Pin1 immunopositive cells in Ki-67 immunopositive samples was significantly higher than that in Ki-67 immunonegative samples (P<0. 001) ; There was no significant difference of Pin1 expression between HPV-DNA positive and HPV-DNA negative samples.Conclution: The expression of Pin1 correlateed with p16INK4a and Ki-67 expression in cervical Intraepithelial neoplasia.Part Two : The Expression and Function Analysis of Pin1 in the Liquid-Based Cervical CytologyObjectives: To study the potential of Pin1 as a biomaker for cervicalIntraepithelial neoplasia. Methods: Immunocytochemical analysis of Pin1 expression was performed in 67 HSIL cases, 79 LSIL cases, 57 ASC-US cases, 35 ASC-H cases, 33 AGC cases. All the archived slides were first decoloried and then immunostained with primary anti-Pin1 antibody. The immunoreactive was assessed as the number of immunopositive cells/1,000 cells.Results: 1. The median and the inter-quartil range of Pin1 immunopositive cells/1, 000 cells were 3. 1, 4.7; 1.4, 1.7; 1.0, 0.95; 1.4, 1.0; 1.6, 1.3, respectively, in HSIL, LSIL, ASC-US, ASC-H and AGC slides. Comparison of the number of Pin1 immunopositive cells/1, 000 cells exhibited a significantly higher median in HSIL than that in others cytological groups (P<0. 01).2. The median and the inter-quartil range of Pin1immunopositive cells/1,000 cells were 1.35, 1.2; 1.2, 1. 78; 4. 2, 2. 3; 8. 6, 3. 9; 8. 5, 3. 9, respectively, in negtive group, CIN I, CIN II, CIN III and SCC group. The median counts in CIN III and SCC were significantly higher than that in other groups, P<0. 01.3. Recetive-operating characteristic curves yielded a test accuracy of 0. 765, 0. 934, 0. 978 for CIN I, CIN II, CIN III respectively. Thresholds for 95% sensitivity were at 0. 59, 1.65, 3. 98 immunopositive cells/1, 000 cells for CIN I, CIN II, CIN III respectively.Conclusions: Pin1 immunocytochemistry could be used as an adjuvant biomarker to liquid-based cervical cytology because it has a good diagnostic accuracy to discriminate the advanced pathologic stages from CIN I. Part Three: The Effect of Juglone, Pin1 Inhibitor, Prohibiting the Cell Proliferation of Cervical Cancer Lines in vitroObjective: To study the effect of Juglone in the inhibition of the proliferation and cycle distribution of cervical cancer cell lines in vitro .Methods: The human cervical cancer cells HeLa, SiHa C33a, Caski were employed in the studies. After treatment with Juglone and CTX, the inhibitory effect on the proliferation of cancer cells was determined by MTT assay, and the cell cycle distribution was detected by flow cytometry.Results: 1. Juglone and CTX both inhibited the proliferation of the tumor cells in a dose-dependent manner. The inhibitory effect of Juglone was stronger than that of CTX (P <0. 01).2. Juglone and CTX both could induce apoptosis in HeLa SiHa C33a and Caski cell lines. The Juglone had a stronger effect than CTX in inhibiting cancer cell proliferation.3. Juglone changed the cell cycle distribution of Hela , with the rate of G2/M is more higher than G1 after treatment.Conclution: Juglone can inhibit the mitosis and proliferation of the cervical cancer cells in vitvo. Summary1. The Pin1 was overexpressed in cervical cancer and cervical Intraepithelial neoplasia, and the expression lever was correlated with pathological progress.2. Pin1 was overexpressed in cervical cancer. There was no significant correlation between the expression of Pin1 and Cyclin D1 in cervical cancer tissue.3. The expression of Pin1 correlates with p16INK4a and Ki-67 expression in cervical Intraepithelial neoplasia.4. Pin1 immunocytochemistry could be used as an adjuvant biomarker to liquid-based cervical cytology because it has a good diagnostic accuracy to discriminate the advanced pathologic stages from CIN I.5. Juglone can inhibit the division and proliferation of the cervicalcancer cells in vitvo.