The Value Of Mesothelin In Diagnosis, Prognositic And Gene Therapy Of Epithelial Ovarian Cancer And It's Mechanisms In Cell Cycle And Anoikis

Ovarian cancer is the most common female malignancy and is the leading cause of death from gynecological malignancies. The majority of patients are diagnosed with advanced epithelial ovarian cancer. Only 30% patients with advanced-stage ovarian cancer survive 5 years after initial diagnosis. But the survival rate of patients diagnosed in early stage can reach up to 90%. New tumor marker and new therapy are very in need.Mesothelin(MSLN) is a differentiation antigen that is present on normal mesothelial cells of the pleura, peritoneum, and percardium but overexpressed in several human cancers, including mesotheliomas, pancreatic adenocarcinomas, and ovarian cancers. MSLN has two isotypes, one can released into blood circulation, and is called soluble MSLN related peptides/proteins(SMRP). The molecular weight of SMRP is significantly smaller than CA125, which is commonly used as tumor marker for ovarian cancer diagnosis, so SMRP is supposed to has higher sensivity than CA125 and can be a new marker for ovarian cancer.It is suggested that MSLN may enhance cell proliferation and adhesion ability, however not much is known about it's function in ovarian cancer. Because it's high expression in cancers(include ovarian cancer) and limited expression on normal tissues, MSLN is a promising factor responsible for tumorigenesis and metastasis in abdominal cavity of ovarian cancer. Thus, MSLN is also a promising target for ovarian cancer immunotherapy. To understand the biological significance of MSLN in ovarian cancer, will provide ovarian cancer potent evidence for diagnosis, controling metastasis in abdominal cavity and new therapy.Cell anchorage-dependent growth and anchorage-independent growth ability are both important in tumorigenesis. Normal cells grow only when they adhere to other cells and extracellular matrix(ECM), that is called anchorage-dependent growth. Some cells can still grow and proliferate even after missing the signal transmission with other cells and ECM, this is named anchorage-independent growth. Increased anchorage-independent growth ability is a characteristic of malignant cells and plays important role in tumorigenesis and metastasis.Cell cycle is the key point in modulate cell proliferation and loss of it is considerable reason for tumorigenisis. Cyclin and cyclin-dependent kinase(CDK) regulat cell cycle when they synthesised into dimer. Cell cycle will be initiated by the dimer and enter into different stage. Cyclin D1 is significant element among cyclin family and promote cell cycle positively. Cyclin D1 is over-expressed in many malignent neoplasm and considered responsible for tumorigenesis.Anoikis is a special type apoptosis, refers to cells go to death after separated from theire original living enviroment. Most malignant tumor cells have powerful ability to resist anoikis, and can maintain viable after droping from tumor tissues and then plant to other positions. Anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim have adverse affect in apoptosis.This research first studied the diagnositic and prognositic value of SMRP in ovarian cancer and evaluate SMRP's diagnostic effect. In vitro, MSLN biological function and detailed mechanisms were approached as follows:anchorage-dependent growth, anchorage-independent growth ability, cell cycle and anoikis were tested. Then cyclin D1, Bcl-2 and Bim were detected. In vivo, MSLN-scilencing chornic lentivirus was injected to understand role of MSLN in gene therapy. Xenograft tissue cell cycle and apoptosis were mesearued to reveal detailed machanism of MSLN.PartⅠDiagnostic and Prognostic Value of Serum MSLN/SMRP in Patients with Epithelial Ovarian CancerObjective:In order to investigate the role that serum SMRP played in the diagnoswas and prognostic in epithelial ovarian cancer, serum SMRP concentration was determined in samples from three groups with ovarian cancer, benign ovarian tumor and healthy female volunteers. The difference of serum SMRP levels has been compared among the three groups, and various subtypes of ovarian cancer, across stages and grades were considered. Serum SMRP levels were also measured before and after surgical cytoreduction in patients with EOC. Serum CA125 level was evaluated at equal pace in candidates mentioned above.Methods:The SMRP level of 56 ovarian cancer patients,38 ovarian benign tumor patients and 34 healthy volunteers was was determined by a commercial ELISA assay, the MESOMARK kit, supplied by Fujirebio Diagnostics. Serum CA125 concentration was routinely assayed by chemiluminescence. The statistical analysis of the data was done using SSPS version 13.0 for windows. Measurement data comparison, numeration data comparison, correlation analysis and diagnostic ability analysis were applied. A p value less than 0.05 resulting from a two-sided test was considered as statistically significant.Results:1. The median SMRP values measured in patients with ovarian cancer were significantly higher than in patients with either benign ovarian tumor(Wilcoxon z=6.973, P<0.001) or healthy volunteers (z=7.153, P<0.001)after rank transformation. However, the SMRP levels between patients with benign ovarian tumor and healthy volunteers were not statistically significantly different (Wilcoxon z=1.895, P=0.058>0.05).2. There was no significant difference between the histologic subtypes of ovarian cancer(Kruskal-Wallwas H Test,χ2=5.736, P=0.057>0.05).3.The SMRP values measured in stageⅡ-ⅣEOC patients significantly higher than in stageⅠEOC patients, (Wilcoxon z=3.184, P=0.001<0.05).4.There was significant difference between the differentiation grades of ovarian cancer,(Kruskal-Wallwas H Test,χ2=26.221, P=0.000<0.05).The higher differentiation grade correspond the lower level of serum SMRP. 5.There was no significant difference between the histologic subtypes of benign ovarian tumor. (Kruskal-Wallwas H Testχ2=6.948, P=0.225>0.05).6.Serum SMRP levels significantly decreased after surgical cytoreduction than preoperative in patients with EOC(z=5.129, P=0.000<0.05).7.Diagnostic ability of serum SMRP in distinguishing ovarian cancer from other samples. Receiver operating characteristic (ROC) curve analysis showed the diagnostic ability was promising. The area under the ROC curve(AUC) was 0.938, standard error was 0.026 and 95% confidence interval (CI) was 0.888-0.988.8.The assessment of the ELISA assay which detect serum SMRP as a biomarker for ovarian diseases. Optimal cutoff values which define the best diagnostic efficiency were calculated according to Youden-index (YI= sensitivity+specificity-1), we made cut-off=1.6432nM. Consequently we get the criterion that means patients with SMRP serum concentrations more than 1.6432nM were judged positive/anomalism, less than or equal to 1.6432nM were judged negative/normal, with sensitivity at 0.7941, specificity at 1.9.To assess the clinical potential of SMRP and CA125 in differentiation EOC from benign ovarian tumor (using pathologic diagnosis as the Golden standard), the receiver operating characterwastic (ROC) curve were made for the two tumor markers. The area under the receiver operating characteristic (ROC) curve (AUC) for serum SMRP was 0.925 with a cut-off value of 1.6432nM for differentiating EOC patients from benign ovarian tumor patients, whereas the AUC for CA125 was 0.941 with a cut-off value of 35.0u/ml. Both SMRP and CA125 showed proximalis distinguish ability. The diagnostic test evaluating index was comparison of SMRP and CA125. The sensitivity (Se) was proximalis. SMRP has higher specificity(Sp), omission diagnose rate(β), positive predictive value(PV+), correct ratio (π), Youden-index (YI), lower mistake diagnose rate(α) and negative predictive value(PV-) than CA125.10.Correlations between serum SMRP and CA125 values were calculated using Spearman's rank test, and there wasn't significant correlations between the two markers (p=0.334>0.05).11.Combine serum SMRP and CA125 may outperform SMRP or CA 125 alone, we obtained the sensitivity at 97.9%, and specificity at 81.6% in distinguishing EOC from benign ovarian tumor.Conclusion:1.Serum SMRP level evaluated by ELISA kit was significantly elevated in patients with EOC than in patients with either benign ovarian masses or in healthy volunteers.2.The diagnostic test evaluating showed that serum SMRP has a promising sensitivity and specificity. When compared with CA125, SMRP has higher specificity and the sensitivity was proximalis. These results have identified serum SMRP as a potential biomarker for EOC, which could be useful for early diagnosis.3.The rapid decrease in SMRP levels after surgery in patients with EOC suggests that serum SMRP may be a useful test to monitor treatment response in epithelial ovarian cancer.PartⅡInfluence and Mechanism of Mesothelin on Ovarian Cancer Cell Proliferation and Cell CycleObjective:To evalute the effect of MSLN on SKOV3 cell proliferation, including influence on cell anchorage-dependent, anchorage-independent growth ability and cell cycle. Cyclin D1 was tested to find out the mechanism of MSLN in enhence proliferation.Methods:1.Negative control vector lentivirus(LV-MSLN-neg) and RNAi lentivirus aim at MSLN(LV-MSLN-RNAi) were used to transfect SKOV3 cells(SKOV3-MSLN-neg, SKOV3-MSLN-RNAi). The transfect efficiency was investigated by immunofluorescence microscope and calculated. SKOV3 cells were conducted as blank contrlo, SKOV3-MSLN-neg as negative group and SKOV3-MSLN-RNAi as test group.2.Total protein of the three kinds cells(SKOV3-MSLN-neg, SKOV3-MSLN-RNAi,SKOV3) were extracted and quantitated by Western Blot to evaluate MSLN gene scilencing effect.3. Cell counting test was to measure the proliferation of the cells, and plate clone formation test and soft agar clone formation test were used to view the anchorage-dependent and anchorage-independent growth ability.4. Cell cycle was detected by flow cytometry. SKOV3-MSLN-neg, SKOV3-MSLN-RNAi and SKOV3 cells were serum-starved for 24 hours, then cultured in 10% fetal bovein serum containing medium for 24 hours. Cells were collected and tested by flow cytometry.5. Total protein of the three kinds cells(SKOV3-MSLN-neg, SKOV3-MSLN-RNAi,SKOV3) commonly cultured to subconfluence were extracted and cyclin D1 expression was quantitated by Western Blot.In order to investigate the dynamic change of cyclin D1, SKOV3-MSLN-neg, SKOV3-MSLN-RNAi,SKOV3 cells were serum-starved for 24 hours, then given 2% fetal bovein serum contaning medium, and cultured for 0,2,4,6 hours, cyclin D1 expression at each time was measured in the end by Western Blot.Results:1.The transfect efficiency was about 80-90% and was up to standard.2.MSLN expression were significantly decreased after LV-MSLN-RNAi transfection, and the inteference efficiency reachs 90%.3.Cell proliferation tested by cell counting shows:cell numbers in SKOV3-MSLN-neg, SKOV3-MSLN-RNAi and SKOV3 were (9.89±2.0)×105, (18.9±2.24)×105, (19.81±2.5)×105 respectively. Cells grow slowly in SKOV3-MSLN-RNAi compare to other two groups(F=29.76, P<0.05). There wasn't significant difference between SKOV3-MSLN-neg and SKOV3(P=0.531).4.Clone formation efficiency in test group was(15.85±2.5)%, in negative contol was (28.3±2.7)% and blank control was (29.13±1.98)%. The plate clone formation efficiency in test group was fewer than in other two groups(F=47.62, P<0.05). Clone formation efficiency in blank control and negative control has no difference(P=0.612). 5.Cell anchorage-independent growth ability of test group(8.38±0.82)% was decreased than blank control (18.38±0.50)% and negative control(19.13±0.67)%(F=78.98, P<0.05). No difference was found between blank control and negative control(P=0.441).6.Cell cycle measured by flow cytometry:G1 stage (88.53±1.41)%, S stage (7.35±0.57)% in test group, G1 stage (66.31±1.65)%, S stage (20.88±2.64) % in negative control, and G1 stage (66.69±2.00), S stage (20.78±2.67)% in blank control group. S stage percentage in test group was smaller than in blank and negative control (F=154.282, P<0.05). S stage percentage in blank and negative control has no difference(P=0.913).7.Cyclin D1 expression was down-regulated in test group, compare to other two groups(Chi-Square 17.784,P=0.000<0.05). The OD value in test group, blank contol and negative control was respectively 0.042±0.006,0.917±0.226 and 0.884±0.252. There wasn't difference between blank control and negative control (Z=-0.929, P=0.387)When changed to 2% fetal bovien serum, the OD values in test group at 0,2, 4,6 hours points were:0.006±0.001,0.187±0.006,0.387±0.018, and 0.639±0.037. In blank control, they were 0.191±0.119, 0.528±0.027,0.941±0.018,0.036±0.006, and in negative control were 0.188±0.116,0.545±0.032,0.926±0.015,0.036±0.007. At 0,2,4 hours point cyclin D1 expression in test group were all down-regulated compare to other two groups. After changed to 2% fetal bovien serum for 6 hours, cyclin D1 decreased in blank control and negative control, on the contrary, increased in test group(P<0.05).Conclusion:1.MSLN can enhance cell anchorage-dependent and anchorage-independent growth ability. When MSLN was silenced by RNAi, anchorage-dependent and anchorage-independent growth ability both decreased.2. MSLN modulates cell cycle in SKOV3 cells through promoting cells into S stage.3. Cyclin D1 total amount increasing or synthesis ahead of schedule may play important role in cell cycle chang promoted by MSLN.PartⅢThe Influence and Mechanism of MSLN on Ovarian Cancer Cell AnoikisObjective:To observe the affect of MSLN on ovarian cancer cell line SKOV3 anoikis. Analyse the biological funcion of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim in the processe of MSLN-participate ovarian cancer cell line anoikis.Methods:1.Anoikis model development and test:poly-hema was a kind of anionic polymerization, when spreaded to cell culture dish, cells will be detached from adhesion.2 ml,10g/1 concentration of poly-hema was uesed twice. The dish need to be washed by PBS before cells planted to it. SKOV3-MSLN-neg, SKOV3-MSLN-RNAi and SKOV3 cells were planted to the dish,3×105 /cm. After 72 hours culture, cells were collected and anoikis was tested by flow cytometry.3.Cell total protein was isolated after cultured in poly-hema spread dish, and Bcl-2 and Bim will be quantitated by Western Blot.Results:1. Cell anoikis tested by flow cytometry:after suspension cultured for 72 hours, the apoptosis percentage in test group, blank control and negative control were (32.197±1.77)%, (14.93±0.94)% and (15.49±0.91)%. Apoptosis percentage in test group was higher than other two groups (F=80.07, P=0.000<0.05), but there wasn't difference between blank control and negative control (P=0.739<0.05)2. Bcl-2 and Bim expression in anoikis:The OD values of Bcl-2 in test group, blank control group and negative group were 0.712±0.116,0.93±0.025,0.82±0.093, there wasn't difference in these three groups (Chi-Squwere 2.293, P=0.318)The OD values of Bim in test group, blank control group and negative group were 0.982±0.012,0.724±0.025,0.739±0.025. Bim expression in test group was up-regulated(Chi-Squwere 17.646, P=0.00). Bim expression in blank control and negative control has no significant difference (P=0.826)Conclusion:1. MSLN can enhance the ability of resistant to anoikis in ovarian cancer cell line SKOV3, and MSLN-scilencing can weaken the ability of resistant to anoikis accordingly.2. Bim, a pro-apoptotic protein, particpates the ability of resistant to anoikis. Increased expression of Bim was responsible for the depressed ability of resistant to anoikis.Part IV Gene Therapy Value and Mechanism of MSLN in Ovarian Cancer Xenografts in Nude MiceObjective:to investigate the effect of MSLN-targeted gene therapy in vivo. Cell cycle and apoptosis of xenografts tissue were measured to revealed the biological mechanism of MSLN in gene therapy. In order to study the safty of chronic lentivirus in gene therapy, toxicity test was performed.Methods:1. Development of ovarian cancer xenograft in nude mice:Femal BALB/c nude mice,4-6weeks old and 13.5-16.5g weigh, live in clearning environment. SKOV3 cells, 1×107 number each mouse, were injected into the peritoneal cavity of nude mice.2. MSLN-scilencing chronic lentivirus therapy:Nude mice were separated into 3 groups randomly, and there were 5 mice in each group. Lentivirus were injected every other day after cells injection. PBS injection was used as blank control, and empty vector lentivirus as negative control. Fourteen days later, xenograft formation will be investigated.3. MSLN expression effect by lentivirus injecion in xenografts:total protein of xenograft tIssue was isolated and Western Blot was used to quantitated the MSLN expression.4. Cell cycle and apoptosis of xenograft tissue tested by flow cytometry: xenograft tissue was abtained according to the requirment when mice were killed. Tissues, washed with sodium chloride twice, immobilizated by cold alcohol, were sent to the machine. 5.Toxicity test:Nude mice were separated into 3 groups randomly, and there were 5 mice in each group. LV-MSLN-RNAi and LV-MSLN-neg were injected, and PBS was injected as control. Three weeks later, change of body weight was tested and then mice were killed. Investigate changes in nude mice organs(heart, liver, spleen, kidney) by nake eye and HE chromoscopy.Results:1.The body weight of the mice in blank control group and negative group increased quickly than in test group after cell injection for 7 days. Tumor formation rate in three groups has no difference (P=0.222>0.05).The average tumor weight (F=43.2, P<0.05), tumor number(F=31.13, P<0.05) and number of the tumor positions (F=44.98, P<0.05) were all suppressed in test group compared to other two groups.2.MSLN interference efficiency in LV-MSLN-RNAi and LV-MSLN-neg was 75.6% and 8.9%, compared with control group treated by PBS. The expression of MSLN treated by LV-MLSN-shRNA chronic lentivirs was significantly lower than the negative control group and the blank control group (P<0.05). The negative control group and the blank control group have no siginificant difference in the interference efficiency (P>0.05).3. Cell cycle of xenografts tissue:G1 stage (80.78±0.64)%, S stage (11.20±0.71)% in test group, Gl stage(58.74±1.50)%, S stage(31.89±2.16) % in negative control, and G1 stage (60.13±2.32)%, S stage (33.19±2.01) % in blank control group. S stage percentage in test group was smaller than in blank and negative control (F=154.282,P<0.05). S stage percentage in in blank and negative control has no difference(P=0.913).4. Apoptosis of xenografts tissue:the apoptosis percentage in test group, blank control group, and negative control group was (29.49±1.7)%, (17.41±1.52), (21.50±1.19)%. The apoptosis percentage was obviously higher in test group than in other two groups (F=11.972,P=0.000<0.05), as to blank control and negative control, no difference was observed (P=0.165)5.Toxicity test:changes of body weight in LV-MSLN-RNAi injection, LV-MSLN-neg injection and PBS injection was (1.44±0.34), (1.64±0.29) and (1.43±0.43) (F=10.35, P>0.05). No significant changes happened in heart, liver, spleen, kidney tissue by nake eye and HE chromoscopy observation.Conclusion:1. The metastasis and implation of xenografts in abdominal cavity were effected by MSLN scilencing chronic lentivirus injection. Chronic lentivirus injection as gene therapy was effective for ovarian cancer in animal test.2. MSLN scilencing chronic lentivirus injection can suppress cell proliferation and induced apoptosis in xenografts.3. Toxicin was not found in experimental animals treated by chronic lentivirus, so chronic lentivirs therapy was a safe and reliable method.