| Prostate carcer is the third leading cause of incidence rate for male urogenitalsystem malignant tumor in China. The prostate cancer is insidious without typicalsymptoms,so it is always in the late stage that we can do nothing.We don’t know how totreat the difficult disease. The early diagnosis of patients mainly depends on PSA lever inserum and Digital Rectal Exam is also a main auxiliary mean.Based on these criteria tocarried out the diagnosis, treatment measures are generally divided into two parts whichare conservative treatment and active treatment alertly.The active treatment alertly forprostate cancer patients includes prostate cancer radical prostatectomy, radiotherapy,experimental prostate cancer Local therapy and endocrine therapy. Because of lackingeffective treatment methods for later period prostate cancer, Now, more and moreattentions have been paid to the gene therapy in prostate cancer. There will be a greatbreakthough that it is reported that gene therapy play a important cole in prostate cancertreatment. Programmed cell death factor4(PDCD4) is a new anti-oncogene,which can be tested in many malignancies such as liver cancer,lung cancer, breast carcinoma,colorectal cancer and ovarian cancer. We discovered that the expression of mRNA andprotein in PDCD4is reduced and lacks in these tumors. Besides, it is related to thepathological stage and prognosis. Therefore, we have reasons to believe that thetumor-targeting gene therapy aim at PDCD4is likely to become a new breakout to genetherapy of prostate cancer.Objective:1、To observe the expression pattern and distribution of Programmed cell deathfactor PDCD4in the normal prostate tissue and different classifications prostatecancer,so as to identify that if they can provide the evidence of prognosis and diseaseprogress for the tumor patients.2、To structure and pack the lentivirus expression system that could overexpressPDCD4molecular. Next, we build a prostate cancer cell subline that could highly expressPDCD4using this system. Then, we observe its influence on proliferation ability ofprostate cancer cell and monitor its sensitive regulating effect on therapeutic.3、To explore the post-transcriptional control mechanism of PDCD4after itsexpression, and to know the negative effect of the possible upstream regulatory genesmiR-155.Methods:1、An immunohistochemical staining technique was utilized in the surgical resectionspecimens of prostate cancer patients which contained42cases of cancerous tissue and12cases of para-carcinoma tissue. According to the WHO pathology grade of prostatecancer and Gleason scoring system,the tumor is classified as cases of well-differentiatedcarcinoma with Gleason scores1-4、14cases of moderately differentiated carcinoma withGleason scores5-7and19cases of poorly differentiated carcinoma with Gleason scores8-10.2、Coding region segments were amplified by PCR，then were cloned into thepENTRA-3C vector. Purpose gene was transferred into pLenti6.3lentiviral expressionvector by LR clonase Ⅱ recombinase and packaged. Compared with red fluorescent protein mCherry pLenti-6.3-mCherry lentiviral expression vector, we infected huma nprostate cancer DU145cells then screened stable expression PDCD4protein cellsubstrain by Blasticidin. Using Western blot method to detect the expression of PDCD4protein, using MTT method and plate clone formation test to detect the cell proliferationcapacity. Using TRAIL to detect the drug sensitive to chemotherapy in high expressionPDCD4prostate cancer cells.3、Cloning the promoter fragment amplified by PCR into the T vector andsequencing the gene segments. If the sequencing was correct, we would clone it into thereporter gene vector. Choosing Wild-type sea cucumber luciferase report gene vectorpRL as internal reference and using it with lipidosome2000vector to transfect mammals.After48hours, we would collect the samples and do the fluorescence analysis.Totransfect the DU145cells with inhibitor and mimics which are the upstream regulatoryelements of PDCD4in the third expriment. After that,we would observe its affects onthe prostate cancer cell and drug sensitive to TRAIL in the prostate cancer cell.Results:1、Immunohistochemistry: The positive expression rate in the42prostate cancertissues is30.95%(14/42) which is much lower than75%(8/12) in benign prostatehyperplasia tissue. The expression of PDCD4in48cases of prostate tissue wasanalysised statistically.The result is that the expression of PDCD4is closely related toclinical TNM stage, Gleason score, histological type, histological grade (P<0.05).2、We have found that miR-155which lies in upstream of PDCD4is a negativeregulator. Inhibition of miR-155induced spontaneous apoptosis and increasedsensitivity to TRAIL induced apoptosis in DU145cells.3、We have constructed the recombinant lentivirus vector pLenti6.3-PDCD4. Usingrecombinant lentivirus expression system screened cell substrain which can over-expressPDCD4protein stably.Then, we use Western blot method to verify this. Determined byMTT and plate clone formation experiment showed that the proliferation capacity of highexpression PDCD4is restrained in DU145cell substrain,and improved the sensitivity toTRAIL induced apoptosis in prostate cancer cells. Conclusion:The expression of PDCD4in prostate cancer tissue is obvious lower thanthat in prostate hyperplasia the tissue,the differences between the two are significantlystatistically significant (P<0.05).We found that high differentiation positive rate was63.6%;Moderately differentiated positive rate is82.4%; Low positive rate was85.0%byusing Gleason score system,the relationship between expression of PDCD4anddifferentiation degree is negative correlation, after statistics analysis (P<0.05),we foundthat the difference is significant and have a statistically significance. It is showed that theexpression of PDCD4is decreased along with malignant degree of tumor increased, theresults suggest PDCD4play a certain role in the process of tumor development, and hasimportant reference value in guiding clinical treatment. We have successfully built aPDCD4protein lentiviral expression vector, selected stable expression of PDCD4DU145cell line, found that overexpression of PDCD4protein can significantly inhibitproliferation in DU145cell. We have found that miR-155which lies in upstream ofPDCD4is a negative regulator. Inhibited miR-155can induce spontaneous apoptosisand improve its sensitivity to the TRAIL induced apoptosis in DU145cell,which can laya solid foundation to further prove the presence of miR-155-PDCD4imaginary axis. |
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