| Esophageal cancer is a heckneyed malignant tumor of alimentary canal,the sixth most common cause of cancer death,and the global incidence is eighth.The incidence of esophageal cancer in China ranks first in the world,with about half of the deaths in 400,000/year of esophageal cancer worldwide.Henan is one of the high vulnerable areas of esophageal cancer in China,especially in Linzhou,Henan province.The etiology of esophageal cancer may be related to dietary habits,living environment,heredity and many other factors.It,The pathogenesis of esophageal cancer which is a complicated and gradual process involving multi-stage,multi-factor and multi-genes is not yet clear.Although the treatment methods such as surgery,chemotherapy and radiotherapy were improved,the 5-year survival rate was19%,the advanced esophageal cancer was only 0.9%and the prognosis was very poor what had Seriously threaten people's survival and health.It is imperative to seek new alternative therapies.gene targeting therapy has become a hot research topic in the treatment of esophageal squamous cell carcinoma.PPM1D(WIP1)is a oncogene Which has the activity of silk/threonine protein phosphatase.It is a wild type of p53-induced phosphatase which plays an important role in a variety of tumor cell growth,the regulation of cell cycle,cell stress,DNA damage repair.The study showed that PPM1D was expressed in high-level expression in esophageal carcinoma,but low in normal esophageal epithelium tissue.Come so far,domestic and foreign scholars have made huge strides in the reserch of PPM1D gene in the field of ovarian Clear Cell carcinoma,neuroblastoma,medulloblastoma,breast cancer,hepatocellular carcinoma,prostate cancer,bladder urothelial carcinoma,pancreatic cancer,thyroid cancer,stomach cancer,colorectal cancinoma etc.It is implying that the development and of occurrence tumors is involve with PPM1D intimately.At present,the relationship between PPM1D and the development of esophageal squamous cell carcinoma has not been reported in the world.At first,In the present study,the authors used immunohistochemical method to test the expression of PPM1D in esophageal squamous cell cancer,and the relationship of PPM1D and the clinical biological behavior was analyzed in esophageal squamous cell carcinoma.The technology of RNA interfering down-regulated expressions of PPM1D protein in ESCC EC1 cells,Western blotting methods investigated expressions of PPM1D protein with effect of PPM1D siRNA.Flow cytometry and TUNEL technology analyzed cell apoptosis of EC-1cells with effection of down-regulation of PPM1D expression.The ability of invasion and metastasis in esophageal cell EC1 cells were detected by scratch experiment and transwell invasion.Finally,after transfection with PPM1D siRNA,Western blotting methods detected the expressions of cell apoptosis related protein bcl-2 and bax and invasive metastasis-associated protein mmp2 and mmp9.The aim of this study was to explore the possible role of PPM1D gene in the pathogenesis of esophageal squamous cell carcinoma,and to provide a theoretical basis for finding new sites for targeted treatment of esophageal squamous carcinoma.Methods(1)Immunohistochemistry was used to detect the expression of PPM1D protein in 91 cases of ESCC tissues and corresponding normal esophageal epithelial tissues.Investigate the associations of PPM1D expression with clinicopathological features as well as prognosis of the patients with ESCC.(2)Using Western blotting to detect the interference effect of PPM1DsiRNA on PPM1D in all groups of esophageal squamous cell carcinoma.(3)The effect of PPM1D down-regulated on apoptosis of esophageal squamous cell carcinoma using Flow cytometry and TUNEL technique after transfection with PPM1D siRNA and control siRNA.(4)The ability of invasion and metastasis of esophageal cells in each group was detected by scratch experiment and Transwell invasion experiment.(5)The expressions of cell apoptosis related proteins Bcl-2 and Bax and cell invasion and metastasis related proteins MMP2 and MMP9 were analyzed byWestern blotting methods after transfection with PPM1D siRNA and control siRNA.Statistics analysis:SPSS version 17.0 analyses the experiment data by One-way analysis of variance.The means±standard deviations expresses summary statistics.Inspection level was expressed as?=0.05 in all statistical analysis.Results(1)Positive expression ratio of PPM1D in ESCC tissues(57/91,62.6%)was significantly higher than that in normal esophageal epithelial tissues(16/91,17.6%),and significant differences were found between two groups(?~2=38.449,P<0.05).Expression of PPM1D protein wasn't related to the gender(?~2=0.359,P>0.05),age(?~2=1.655,P>0.05)and histological differentiation(?~2=2.904,P>0.05)of the patients with ESCC,but closely correlated with clinical staging(?~2=5.705,P<0.05),invasive depth(?~2=15.796,P<0.05)and lymph node metastasis(?~2=8.340,P<0.05).(2)After PPM1D siRNA transfection,the protein expression of EC-1 cell PPM1D gene decreased to about 1/3 of the blank control group and control siRNA group,and significant differences were found between two groups(F=577.8,P<0.05).There was no difference in the expression of PPM1D between the blank control group and control siRNA group(P>0.05).(3)The cell apoptosis results demonstrated that after 48h Transfection,the early apoptosis rate of EC1 cells in PPM1D siRNA group was 6.1%,significantly higher than that in the blank control group and control siRNA group(1.3%and 0.7%)(F=103.6,P<0.05).nevertheless the number of live cells in the blank control group and the control siRNA group of EC-1 cell were 97.4%and 97.9%,which was significantly higher than the number of live cells in the PPM1D siRNA group(77.6%)(F=102.5,P<0.05).(4)The results of TUNEL showed that there was no significant difference in the apoptosis of the two groups(P>0.05)between the blank control group and the control siRNA group.However,compared with the control siRNA group and the blank control group,the apoptosis of PPM1D siRNA group was significantly increased after 48h transfection(F=34,P<0.05).(5)The results of scratch test showed that there was no significant difference in the migration rate between the two groups in the blank control group and the control siRNA group(P>0.05).However,the migration rate of the PPM1D siRNA group after 48h Transfection was significantly inhibited compared with the blank control group and the control siRNA group(F=126.8,P<0.05).(6)The Transwell experimental results demonstrated that the transmembrane cell number of EC-1 cells in PPM1D siRNA group was(43.6±20.17)significantly lower than that in the blank control group of esophageal squamous cell carcinoma EC-1 cells(129.4±26.87)and the control siRNA group of esophageal squamous cell carcinoma EC-1 cells(119.2±24.85),The difference was statistically significant(F=114.37,P<0.05).(7)Compared with the blank control group and the control siRNA group,the results of Western blotting showed that the expression level of Bcl-2 protein in PPM1D group reduced significantly(F=105.1,P<0.05),but the expression level of Bax protein increased markedly and the difference was statistically significant(F=298.9,P<0.05).Furthermore there was no difference between the blank control group and the control siRNA group(P<0.05).(8)Compared with the blank control group and the control siRNA group,the results of Western blotting showed that the expression level of MMP2 protein and MMP9 protein in PPM1D group was decreased significantly,the difference was statistically significant(F=183,P<0.05;F=6.460,P<0.05).Furthermore there was no difference between the blank control group and the control siRNA group(P>0.05).Conclusion(1)PPM1D may be involved in the occurrence,development,invasion and metastasis of esophageal squamous cell carcinoma.(2)In esophageal squamous cell carcinoma EC-1 cells,the expressions of PPM1D protein is down-regulated by PPM1D siRNA effectively.(3)PPM1D mediated cell apopotosis and cell invasion may be tightly associated with the alterations of Bcl-2,Bax,MMP2 and MMP-9 expression in ESCC. |
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