| Prostate cancer is one of the common malignancy in genitourinary systemof man.The molecular modulation of Prostate cancer is still known. Theabnormal expression of HER-2/neu was found another key factor in theoccurrence of cancer in rescent years' research. HER-2/neu has a low expression in many secretory epithelium normally.HER-2/neu takes a part in signal transmittion through many intracellularmolecules. It is basically thought that the overexpression of HER-2/neu cancause tumor through transactivating androgen receptor. In human cancers, the molecular alteration of HER-2/neu is generally theoverexpression of normal gene outcome. Since the HER-2/neu protein locates inchorion and the diffussion is confined bidirectionally, even middle-levelHER-2/neu overexpression could cause the activation. The HER-2/neu proteinlevel of mamary cancer cell is 10-100 times higher than the adjacent normalepithelium through the immunohistochemical pigmentation. Craf. etc. castrated SCID male mice that had male-hormone-dependent typeprostate cancer and got the male-hormone-independent prostate cancer derivedfrom the originally male-hormone-dependent LAPC-4 type. HER-2/neu proteinexpression level enhanced 2-25 times. The overexpression of HER-2/neu has thefunction of promoting the male hormone independent-growth in vitro and invivo. All these proved that the augmentation and overexpression of HER-2/neuis a very important factor that causes the protate cancer.Prostate cancer cells were selected as the test object in this research. Theoverexpression of HER-2/neu protein prostate cancer cell PC-3M was provedthrough immunohistochemical pigmentation while HER-2/neu protein could notbe found in normal prostate tissue, which was coincident with upper data.In conclusion, the overexpression of HER-2/neu protein play a importantrole in the occurrence and development of prostate cancer, and might become aindex to judge the outcome. Thus, the selection of HER-2/neu as the target genein this research has sufficient lab base.HER-2/neu has very important physiological function which involvessignal transfer intracellular, cell cycle regulatory, apoptosis and hermal creationin tumor etc.. HER-2/neu-induced intracellular signal pathway includesmitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase(PI-3K)/AKT pathway. MAPK is the main signal pathway for activating HER-2and other tyrosine kinase. Another pathway induced by HER-2 is PI-3K/AKTpathway which plays a important role in the cell survival. HER-3 receptor couldcombine PI-3K motif, while HER-2 lack this. But HER-2 can induce HER-3phosphorylation and then combines PI-3K, and the PI-3K/AKT kinase becomesactivity. BAD and the caspase-9 were phosphorylated by the kinase as thetargets. The phosphorylated AKT inhibits the glycogen synthesis kinase-Ⅲ,andthe latter increases the cyclin D1 which starts the cell cycle. The transcript factornamed Forkhead and Raf are also the direct/indirect targets of AKT. The lastresearch work shows that over expression of HER-2/neu activate AKT and NF-κB which might resist the apoptosis and the cancer cells might be able to resistTNFαand might decrease the defense ability of the host to the tumor.Activated HER-2 can induce several separated pass ways activation, eg.PLC-γ/PI3-K,STAT/JAK,Ras/MAPK,stress activated protein kinase,Src,etc. and then the early gene-fos and jun were activated later. CyclinD and p27might be the key factors which mediate the karyokinesis that induced by HER-2and theG1/S transfer signal pass way. CyclinD1 was up regulated by activatedHER-2 through Ras/MAPK and PI3-K/AKT, then lowered the stability of p21,accelerated the transformation of G1/S and resisted apoptosis. While CyclinD1was down regulated,p27 was up regulated and Cdk2 was restricted by Herceptinwhich block the cell cycle. These results show that HER-2 might be a essentialtarget for the tumor therapy.Nowadays, the technique of RNAi becomes a new heated point of genetherapy. RNA interference is a new credible method for inhibition of geneexpression. The short double strands RNA interferences the target geneexpression specifically and the mRNA were degraded. A unique phenotypewhich lack of a unique gene was induced in the cell. Due to the inhibition of thegene expression at the RNA grade, the method was named RNA interference.RNA interference, higher transfection efficacy, stronger gene block, highstability, easy taking to the cell, was used as a new gene therapy which target onthe HER-2/neu. Plasmid pSUPER was used in this research work to construct acombined plasmid contained HER-2/neuRNA which could express hereditarilyin eukaryote. pSUPER siRNA systerm is a plasmid vector which was constructby using the RNA III polymeraseH1 RNA promoter and could transcript shortRNA(<400nt) quickly with more copies. Similar to the universally usedpromoter U6, the promoter H1 has a clear start transcript code, and the transcriptended by five continuous thymines. The transcript product, the short RNA hadtwo uracils as the 3' tail. pSUPER siRNA systerm could induce gene close downspecifically and effectively and then lead gene dysfunction. Foreign DNA wasinserted between the sites of HindIII 和 BglII to construct the specificHER-2/neu interfering vector. It was transfected into Prostate cancer cells inorder to observe how the combined plasmid affected the expression of HER-2/neu and how the over expression of HER-2/neu was blocked affected thecell proliferation. The final aims were to understand the function of HER-2/neu in inducing the prostate cancer and to know the relationship between HER-2/neu and the existent of prostate cancer. RT-PCR and Western bolt were usedto confirm this plasmid could inhibit the HER-2/neu expression remarkably atthe mRNA and protein level. We also found when the prostate tumor cellsproliferation was inhibited and the HER-2/neu expression was lower at thesame time. These results clued on that HER-2/neu over expression was anearly phenomenon of prostate cancer and had a close relativity to the cancer.HER-2/neu was an important target molecular of gene therapy to prostatecancer. This research work used promoter H1 to transcript dsRNA which hadhair pin structure. dsRNA inhibited the expression of HER-2/neu in theprostate cancer cells successfully. We also verified that the proliferation of thetumor cells was due to the inhibition of the HER-2/neu expression and tumorcell apoptosis was also induced by HER-2/neu.Through the integrated analysis, conclusion could be drawn: ⑴HER-2protein overexpresses in prostate cancer cells PC-2M, while HER-2/neu proteincould be found in normal prostate through immunohistochemical technique. ⑵Transfering dsRNA tip structure with H1 promoter, inserting the outside DNAsegment into Hind III and BglII site of pSUPER plasmid, interferencevector of pSUPER-HER-2/neu successful combination was proved throughenzyme digestion, PCR, sequenceing.⑶ pSUPER-HER-2/neu plasmid couldinhibit HER-2 expression notably in mRNA level using RT-PCR technique.⑷Western bolt, FCM proved pSUPER-HER-2/neu plasmid could inhibitHER-2/neu expression notably in protein level.⑸ HER-2/neu expression'sdown-regulation combined with growth inhibition of prostate cancer cell couldinduce apoptosis of tumor cell through the time change of cell apoptosis testedby FCM.For the first time we report the usage of RNAi technique could induce thegrowth inhibition and apoptosis of prostate cancer cell.This experiment's resultalso prove that with the inhibition of HER-2 genetic expression, the growth ofprostate cancer cell could be inhibited and the apoptosis could be induced.All these results indict that the siRNA-HER-2/neu recombinant plasmid issuccessful, thus a new target point is set up for the prostate cancer genetictherapy.
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