| Objective:To investigate the relationship between expression level of MAPK and the risk factors for bladder cancer and clinical prognosis, to evaluate the safety and effectiveness of laparoscopic radical resection of the bladder, and to investigate the biological MAPK gene on proliferation and apoptosis of bladder cancer cells.Methods:Part I:The high risk factors of bladder cancer and the level of MAPK expression in peripheral blood of Han population(1) From January 2012 to March 2016,42 cases who were above the age of 70 year old and 89 patients under the age of 70 were collected in department of Urology our hospital.On the other hand, the age,number,and sex matched healthy control were collected at the same time.(2) The smoking history, drinking history, toxic activity, and statistical analysis were recorded and statistically analyzed by using Logistic and single factor to sum up the possible carcinogenic factors;(3) The expression levels of MAPK in peripheral blood of patients and healthy controls were detected by enzyme-linked immunosorbent assay (ELISA).Part II:Short term and long term outcome of laparoscopic radical resection of bladder cancer in elderly patients and nonelderly patients with bladder cancer were collected and analysed.(1) The patients with bladder cancer were divided into 70 groups according to their age and two groups under the age of 70 years. Two groups of patients were treated with laparoscopic radical resection of bladder;(2) The safety of the treatment, the success rate of the operation, the recurrence rate and the survival rate after surgery were evaluated;(3) Immunohistochemistry staining was performed to detect the expression of MAPK in the tissues adjacent to the cancer tissues and adjacent tissues, and the positive area percentage was statistically analyzed;Part III:Study on the mechanism of MAPK in bladder cancer(1) Construction of MAPKsiRNA and it was infected into the bladder cancer cell SIU87;(2) Detection of apoptosis and cell cycle of bladder cancer cell by flow cytometry;(3) Western blot was used to detect the level of Bax/Bcl-2 expression in cells;(4) The invasion ability of bladder cancer cell was detected by Transwell assay;ResultsPart I:(1) All of the clinical data was recorded of the 131 bladder cancer patients including sex, age, weight, BMI and other clinical indexes were stastically analyzed, which showed that compared with 131 healthy controls, the BMI in patients with bladder cancer were significantly higher, the difference was statistically significant (P<0.05). In terms of age, weight and other aspects, no significant statistical difference was found (P>0.05).(2) Clinical history analysis includes the following basic history:gender, family history, history of radiation exposure, smoking history, drinking history, toxic substance exposure, high fat diet. We found that in patients with high fat diet, exposure to toxic substances, and a family history of bladder cancer patients were significantly more than the control group, the difference was statistically significant (P<0.05).(3) The MAPK level in peripheral blood of patients and healthy control was compared. The results showed that, compared with the control group, in the patients with bladder cancer, the MAPK expression level in peripheral blood increased significantly, the difference was statistically significant (P<0.05).(4) A further study was performed in both groups. The patients was divided into two groups, in one group,the patients were older than 70 years old and the other group was younger than 70 years. The peripheral MAPK expression level in two groups showed no significant statistical significance (P>0.05).(5) The patients were detected by ultrasound, in patients older than 70years old, the average size was (6.9 ±2.1) cm3, while in patients less than 70 years old,the average tumor size was (6.3 ± 1.9) cm3, the difference was no statistically significant difference (P>0.05).(6) The age and sex is as a proportion of the conditions,a balanced test was performed in the group of patients and healthy control.The results showed that age(P = 0.241>0.05), gender (P= 0.894>0.05) in the two groups were no statistically significant, which indicated that the base line data was comparable in the two groups.(7) The significant test of the correlation between case group and healthy control group was performed.The education level and occupation belong to the grade classification index, and the rank sum test is adopted. From table 1-11, 1-10,we speculated that smoking (P<0.001) is a statistical significance risk factors.(8) The homogeneity test of variance was used on analysed the high fat diet time, blood glucose and other data statistically. The data was input in SPSS 20 statistical software to draw the results (see table 1-11),we found that the high fat diet time has statistical significance, and the blood sugar is not statistically significant (P>0.05).(9) Level of Education was evaluated by two classification and rank sum test, see table 1-12,which shows that:Z=1.839<1.96, P=0.048<0.05.A significant difference was found which indicated that education is one of the risk factors for bladder cancer.(10) Rank sum test was further performed, the results show that P<0.001, which shows that the difference between the two groups is significant, occupation is one of the factors associated with bladder cancer.(11) After a significant test, in this group of patients in the final confirmation of the 5 risk factors for the occurrence of bladder cancer. In order to further explore the correlation factors and correlation, the correction of confounding factors was performed. It was included in a variety of factors analysis, and ultimately constructed the non conditional Logistic regression model.Bladder cancer occurred as the independent variable, the dependent factor was the dependent variable, the "back"method was used for screening,the probability of the selection criteria was 0.05, excluding the standard 0.05. The results showed that smoking, occupational nature, exposure time of toxic substances were statistically significant (P<0.05).(12) Among the patients with bladder cancer and effects factors on the expression level of MAPK, a COX multi factor regression analysis was performed.The results showed that in the patients with bladder cancer, the serum levels of MAPK and BMI, TNM staging, T staging, lymph node metastasis, the difference was statistically significant (P<0.05).Part ?:(1)The baseline and preoperative characteristics of patient are summarized in Table1. Patients in the elderly group had a mean age of 73 years (range 70-76 years) and those in the nonelderly group had a mean age of 58 years (range 37-69 years; P= 0.000). Elderly patients was significantly more score ? of ASA (50% versus 20.2%, P= 0.001) and comorbidity rate (73.8% versus 21.3%, P= 0.000), particularly for hypertension (23.8% versus 9.0%, P=0.021) and type 2 diabetes mellitus (19.0% versus 5.6%, P=0.037), than that of the nonelderly patients. There were significantly fewer elderly patients than nonelderly patients with an ASA score of ? (45.2% versus 78.7%, P= 0.000).(2) The pathological data of the two groups are summarized in Table 2. There was no significant difference in the variation of pathological data between the twogroups.(3) The intraoperative data, postoperative course, and 90-day postoperative complications are summarized in Table 3. There were no significant differences between both groups regarding operative time, operative blood loss, rate of blood transfusion, time to resumption of oral intake, length of postoperative stay, and 90-day postoperative complications. There were no incidences of 90-day postoperative death in any patients. There were lesser patients who underwent adjuvant chemotherapy in the elderly group than in the nonelderly group, although this difference was not significant (P= 0.055).(4) The median follow-up period was similar between the two groups. Assessment of long-term outcomes is shown in Figures 1 and 2.There were no significant differences between the elderly and nonelderly groups in terms of 5-year OS rate (P = 0.274) and 5-year DFS rate (P= 0.110). Recurrent tumors developed in 33.3% of patients in the elderly group and in 21.3% of patients in the nonelderly group. There were no significant differences in the sites of recurrence and time to first recurrence between the two groups.(5) Multivariate analysis revealed that depth of tumor invasion and lymph node metastasis were associated with OS and it also revealed that depth of tumor invasion, tumor grade, and lymph node metastasis were associated with DFS.(6) Immunohistochemical staining showed that the expression level of MAPK in bladder cancer tissue was significantly higher than that in adjacent normal tissues., the difference was statistically significant (P<0.05).Part?:(1) We use Western blot method to detect MAPK protein expression level in bladder cancer cell. In BIU87 cells, it can be observed a higher level expression of MAPK; therefore, we use the BIU87 cells for further analysis.(2) In order to explore the function of MAPK in BIU87 cells, we constructed the shRNA, and finally found that the MAPK expression in transfected cells was down regulated. According to the need, we constructed three shRNA sequences of human MAPK gene and a non-specific antagonistic shRNA sequence as the negative control.We finally cloned it into the slow virus vector. After recombinant and package into HEK-293T cells,BIU87 cells were subsequently infected with RNAi or negative control virus in MAPK.Western blotting and real-time fluorescence quantitative PCR was furthered used to confirm the results of RNAi silencing MAPK as shown in figure 2-3A. MAPK-Ri-3 was identified as the most effective type of infection, and we selected them for follow-up experiments.(3) In order to further study the effect of MAPK down-regulation on the proliferation of bladder cancer cells, we used CCK-8 method to analyze the proliferation of BIU87 cells infected with MAPK-Ri-3. As shown in figure 2-4A, we found that in MAPK-Ri-3 infected cells, cells with wild type (WT) and shRNA antagonist (NEG) virus infected cells, cell adhesion decreased, so its growth was significantly impaired. These data indicate that the increase of MAPK expression can promote the proliferation of bladder cancer cells, and inhibit the proliferation of bladder cancer cells after MAPK.(4)Then, the potential inhibition of MAPK gene silencing on cell cycle progression was studied. As shown in figure 2-4B, and WT (46.2 ± 2.8%) compared with the negative control group (44.9 ± 1.7%), the expression of MAPK can inhibit cell proliferation, and the cell staged on Gl phase increased significantly (64.9 ± 2.9%) and the number of cells in S phase and G2-M phase was decreased. These data suggest that MAPK may cause cell cycle arrest in the G1 phase, which may be associated with MAPK-Ri-3 cell proliferation.(5) In order to further study the mechanism of MAPK gene on cell apoptosis, we use V/PI Annexin double staining method (Figure 2-5A) to detect the apoptosis rate. In MAPK-Ri-3 cells, the apoptosis rate increased to 49.6 ± 1.5%, compared with WT (4.3 ± 0.5%) and negative control group (5 ± 1%). The difference was statistically significant (P<0.05). These data suggest that MAPK may play an anti-apoptotic role in bladder cancer cells.(6)In order to investigate the effect of MAPK invasion of bladder cancer cells were detected by Transwell assay. As shown in figure3-6,when silencing MAPK in cells compared to control cells, its invasive ability was significantly reduced. The percentage of invasive cells in MAPK-Ri-3 cells was 44%. (WT,121+14; negative control,118+13; MAPK-Ri-3,44+7).Conclusion:Smoking, occupation and education background may be the risk factors influencing the incidence of Han patients with bladder cancer; high expression of MAPK in peripheral blood of patients with bladder cancer and carcinoma in elderly and non elderly patients, laparoscopic radical cystectomy for two equally safe and effective. MAPK may regulate the proliferation, apoptosis and cell cycle of bladder cancer cells by Bax/Bcl-2. MAPK is expected to be an effective target for the treatment and diagnosis of bladder cancer. |
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