The Study Of The Regulation Of Psamrna And Pca3mrna Fene Expression With Cyclopamine Block Hh Singaling Pathways In Prostate Cancer Lncap Cells

Prostate cancer is the most commonly diagnosed tumor, it is also thesecond-leading reason of cancer death in men in the United States.Now,the incidence of prostate cancer, even if far lower than western national, butshowed a momentous increase trend in China with shifting lifestyles,longer life expectancy and the upgrading of diagnosis. Patients with newlydiagnosed prostate cancer frequently have advanced, loss of opportunity forsurgery, poor prognosis with compared to the West countries patient.Castration therapy plus antiandrogen therapy is the standard methd forprostate cancer. Unforiunately,most of,men ultimately develop castrate–resistant prostate caneers(CRPC),unresponsive to hormonal manipulations.Alfully high mortality rate of CRPC has become a key health risk for oldermen. Currently on the pathogenesis of prostate cancer is not clear. Throughactive intervention to prevent growth of androgen-independent prostatecancer cells obtained, or block progress is the best treatment for prostatecancer. Current research focuses mainly on the androgen receptor,apoptosis, and cell cycle regulation of tumor angiogenesis, gene-targetedtherapy, and many other aspects. In recent years the development ofmolecular biology techniques such as inhibiting signal transduction, cancergene-targeted therapy provides new ways for treatment of prostate cancer. The study found Hh signaling pathway plays an important role in thegrowth and metastasis of advanced prostatic cancer, Hh signaling pathwayplayed a key role in the deterioration of prostate cancer, treatmentresistance and transfer, increased channel activity continued will lead towidespread metastasis of prostate cancer, controls the activity of thepathway can inhibit tumor metastasis and invasion force. Hh signalingpathway inhibitors attracted widespread attention at home and abroad andresearch in recent years. Cyclopamine is the antagonist of the Hh pathway,it can make Smo conformational changes in the Hh signaling pathways,inhibits the activity of Smo, which inhibits signaling pathway, reduces cellinvasion forces, inducing cell apoptosis. It induced apoptosis of cancercells at the same time, no effect on normal cell growth, was a true sense oftargeted therapy, as an anti-cancer drug enters phase I clinical;PCA3mRNA genes of prostate cancer was discovered in recent years oneof the most-specific cancer gene high expression in prostate cancer cells. Innormal prostate, or benign prostatic hyperplasia tissue expression of verylow or no expression, in prostate cancer tissues and organs other than thefree expression, analysis of expression of PCA3can differentiate betweenbenign and malignant tissue, expression in prostate cancer can accuratelyquantify the advantage, so apply it to enormous potential for targeting genetherapy for advanced prostate cancer. Prostate-specific antigen genesmRNA(PSAmRNA) is the indicator of prostate cancer is more specific,initial findings have been confirmed on the international PSAmRNAindicator in the staging of prostate cancer, clinical monitoring andprognosis, there are of great significance, description detected in peripheralblood PSAmRNA blood exists in prostate cancer cells, Means that prostatetumor cells in micro-metastasis. If through some sort of therapy can makePSAmRNA expression down-or even disappear, meaning that valid for the treatment of metastatic prostate cancer. Research of relationshipbetween Hh signal pathway blocking and prostate cancer gene-targetedtherapy currently both at home and abroad has not been reported, thisresearch discussion the effect of PCA3mRNA and the PSAmRNA geneexpression of prostate cancer LNCaP cell. Revealing the Hh signalingpathway blockade on regulation of target gene expression in prostate cancerand impact conditions, further understanding the mechanism of regulationof target gene expression and biological function, provide furthertheoretical basis for targeted therapy for prostate cancer, Foundation forfuture combined use and animal experimental study on the theoreticalbasis.Main contents:1. Observation of different concentrations of Cyclopamine (1,5,10,15Mu mol/L) at a different time (24,48,72h) on proliferation andapoptosis of prostate cancer cell line LNCaP effect: By MTT colorimetricmethod for observation of effects of Cyclopamine on growth andproliferation of LNCaP cells in vitro; Hoechst33,258dyeing methods ofobserving the morphology of LNCaP cells apoptosis; PI staining and flowcytometric detection of cell apoptosis.2. Observation of different concentrations of Cyclopamine (1,5,10,15Mu mol/L) at a different time (24,48,72h) effects on the expression ofPSAmRNA gene in LNCaP prostate cancer cells: Real-time fluorescencequantitative reverse transcriptase polymerase chain reaction (FQ-RT-PCR)was used to clear effect of Cyclopamine on expression of PSAmRNAgene and its relationship with time, concentration.3.Observation of different concentrations of Cyclopamine (1,5,10,15Mu mol/L) at a different time (24,48,72h) effects on the expression ofPCA3mRNA gene in LNCaP prostate cancer cells: Real-time fluorescence quantitative reverse transcriptase polymerase chain reaction(FQ-RT-PCR) was used to clear effect of Cyclopamine on expression ofPCA3mRNA gene and its relationship with time, concentration.4. PSAmRNA and PCA3mRNA genes expression of curves as theextension of time and concentration increases while differentconcentrations of Cyclopamine and LNCaP cells joint training,5.Data of MTT, flow cytometry detection, Hoechst33258dyeing areanalysis using SPSS10.0statistical software, number of samples arecompared using variance analysis, P<0.05is difference, P<0.01issignificant differences. FQ-RT-PCR results tested with2-△△C tmethod ofcomparative quantitative, then take on average, the value of relativeexpression of each sample is equal to the mean value of housekeeping geneexpression values divided by the target gene expression values, using SPSSmedical statistical analysis software for analysis. P<0.05is a significantdifference.Main results:1.MTT assay showed Cyclopamine Group1μmol/L inhibition onproliferation of LNCaP cells no obvious effect, no significant differencecompared with the control group (P>0.0).5,10and15μmol/L Group havesignificant inhibitory effects on proliferation of LNCaP cells, Inhibition isenhanced as the concentration increases,10μmol/L Group at48h is IC50.As time extended, Cell growth has decreased significantly, Inhibitory effectenhanced as time extended, and there are very significant differencescompared to the control group (P<0.01).2. Hoechst33258dyeing, under the fluorescent microscopeobservation, Normal cells of the nucleus has a normal blue, Apoptosis cellnuclei are tight and concentrated dye, or pieces of dense concentrated dye,color white. When the concentration of1u mol/L, no significant changes of apoptosis, as the increase of the concentration, cell apoptosismorphological become more evident, and at the same time for the sameconcentration, time extension, change of apoptosis is also growing.3.Flowcytometry analysis result:1μ mol/L Group24,48,72hapoptosis rate, respectively,7.69%,5.73%,5.73%；5μmol/L Groupapoptosis rate of7.81%,7.37%,8.27%; Apoptosis rate of10μ mol/Lgroup,37.21%,57.98%,57.98%；Apoptosis rate of15μ mol/L group21.16%,71.31%,72.90%。There are very significant differencescompared with the control group (P<0.01), there are no significantdifference of apoptosis rate between1and5μ mol/L Group on24,48,72h(P>0.05); there are more significant difference of apoptosis rate comparedwith10and15μ μ mol/L Group (P<0.01); the rate of apoptosis shows asignificant difference of10and15μmol/L Group between24h and48,72h(P<0.05). AV-PI double dye fluorescence microscopic observation showscell apoptosis morphology changes as concentration increases and timeprolonged.4. The results of PSAmRNA gene expression by FQ-RT-PCRdetection: Four different concentration,1,5,10,15u mol/L,after24,48,72h stimulation, expressions levels of PSA mRNA gene showsdecreasing trend, differences are significant compared with the controlgroup (P<0.01). Detection of three time period displays the expression ofPSAmRNA gene with a difference (P<0.05), It is worth noting that theexpression of PSAmRNA gene is extremely low at10and15u mol/Lconcentration5.1,5,10,15u mol/L four different concentrations of cyclopaminestimulating the prostate cancer cell line LNCaP, After24,48,72hstimulation and expression level of PCA3mRNA gene is significantly lower than blank control group (P<0.01), As the concentration increases, PCA3gene expression shows diminishing trend, difference significant. Worthy ofnote is the expression of PCA3gene is very low at the concentration of10μmol/L in different time periods.Main conclusions:1. Cyclopamine significantly inhibits proliferation of LNCaP cells andinduces apoptosis. Apoptosis rate increases significantly with increasingconcentration and duration of the time is time-concentration dependency.Hh signaling pathway is closely contacted with prostate cancer, regulatingactivity of Hh signaling pathway can provide new ideas and direction fortreatment of advanced prostate cancer2. Cyclopamine dramatically reduced PSAmRNA genes expressionlevel of LNCaP cells. The expression level of PSAmRNA genesignificantly reduced as Cyclopamine concentration increases, timeextension on the expression of not very notable. That shows Cyclopamineis effective for treatment of metastatic prostate cancer.10,15μmol/Lconcentration shows highly obvious results.3. Cyclopamine dramatically reduced PCA3mRNA genes expressionlevel of LNCaP cells. The expression level of PCA3mRNA genesignificantly reduced as Cyclopamine concentration increases and timeprolonged, Cyclopamine is effective for treatment of advanced prostatecancer.10μmol/L concentration shows highly obvious results.4. Cyclopamine block Hh signaling pathways can significantlydecrease PCA3mRNA and PSAmRNA genes expression of LNCaP cells,especially at concentration of10μmol/L. Hh signaling pathways blockedwill provide new ideas for treatment of advanced prostate cancer.