Anti-cancer Effects Of E-cadherin Transcriptional Activation Induced By Small RNAs In Prostate Cancer Cell And Investigation Of Related Molecular Mechanism

Part I. The expression of E-cadherin in prostatic carcinoma and its relationship with the pathological gradingObjective:To investigate the expression of E-cadherin in the matched prostatic carcinoma (Pca) and adjacent tissues as well as their relationship with the pathological grading.Methods:(1) The tissue microarray collecting 29 cases of prostatic carcinoma and the adjacent tissues were analyzed by immuno-histochemical staining to detect the expression of E-cadherin. (2) Quantitative real-time PCR and Western blotting was used to detect the expression of E-cadherin in prostate cancer cell lines PC-3 and DU145.Results:(1) The positive rates of E-cadherin in PCa and the adjacent tussues were 93.1% and 100%; there is no significant difference in the expression of E-cadherin between PCa and the adjacent tussues; The strong positive expression rates of E-cadherin on poorly differentiated prostate cancer specimens(12.5%) is obviously lower than well-differentiated group(50%). (2) Quantitative PCR and Western blot confirmed that the expression level of E-cadherin in PC-3 were significantly lower than in DU145.Conclusion:(1) There is no significant difference in the expression of E-cadherin between PCa and the adjacent tussues; (2) The expression of E-cadherin is closely related to pathological grading of PCa, suggesting that the abnormal expression of E-cadherin may affect the progress and the metastasis of prostate cancer.Part?. Screening functional dsRNAs which can up-regulate the expression of E-cadherinObjective:To design and screen a relatively more efficient candidate dsRNAs molecules that can up-regulate the expression of E-cadherin.Methods:(1) Analysis of the sequence of E-cadherin gene, especially the sequence of the E-cadherin gene promoter region. Based on the design rules of siRNA and saRNA provided by the literatures, we design 7 candidate dsRNA moleculars among the promoter region of E-cadherin(avoiding the high CpG region). The control dsRNA were designed to be non-homologous to the whole human genome. (2) Those candidate dsRNAs were synthesized and then transfected into PC-3 and DU 145 cells, and MOCK and dsControl transfection were used in the control groups. (3) Functional transcriptional regulating dsRNAs were defined as to increase 2.5 times of the traget gene expression on the mRNA level by Quantitative real-time PCR and Western blotting was used to confirm the effect.Results:To our surprise, among 7 pairs of dsRNA moleculars,1 pair can up-regulate E-cadherin,4 pairs can knock-down the expression of E-cadherin, the other 2 pairs have no effect.Conclusion:There are many targets on the promoter region of E-cadherin which can be regulated by dsRNA, and transcriptional regulation of gene expression by small RNA may be a universal phenomenon.Part?. The related molecular mechanism of functional dsRNA in transcriptional modulating the expression of E-cadherinObjective:To investigate the related molecular mechanism of functional dsRNA in regulating the expression of E-cadherin. Methods:(1) Laser confocal microscope was used to detect the localization of fluorescence labeled dsRNAs; (2) RT-PCR using gene specific primers was used to detect the non-coding sense or antisense RNA transcript on the E-cadherin promoter. (3) Asymmetric small RNAs were designed according to the verified functional transcription-gene-modulating dsRNA and then the E-cadherin expression level was detected to evaluate the regulation capacity of Asymmetric small RNAs (4) ShRNA target to Ago-1 or Ago-2 and a scramble shRNA were constructed into the pLKO.1 vector individually, and then packaged the lentivirus in 293T cell; PC-3 were infected and puromycin were added to screen the stable cell lines. Then, those verified functional transcription-gene-modulating dsRNA were transfected into the stable cell lines.Results:(1) Functional dsRNA can enter into the nuleus. (2) Both non-coding sense and antisense RNA transcripts were detected. (3) The antisense strand of small activating RNA (dsE-661, dsE-640 and dsE-215) is the guide strand, suggesting that the DNA sense strand or the sense RNA on the E-cadherin promoter may be the target of those functional activating dsRNA; (4) The sense strand of small interfering RNA (dsE-604) is the guide strand, suggesting that the DNA antisense strand or the antisense RNA on the E-cadherin promoter may be the target of those functional interfering dsRNA;(5) Ago-2 is required in the transcriptional regulation of E-cadherin by functional dsRNA, and Ago-1 may play an important role in the TGS.Conclusion:(1). Functional dsRNA can work in the nucleus; (2) The promoter-associated sense or antisense RNA transcripts may serve as a scalfold for the functional dsRNA in transcriptional regulating the expression of E-cadherin.(3) Small activated RNA (dsE-661 and dsE-640) and small interfering RNA (dsE-604) have total different guide sense and corresponding target, resulting in completely opposite activity; (4) Both Ago-1 and Ago-2 may play an an important role in the transcriptional gene regulation.Part IV. The therapeutic effects of E-cadherin activation induced by the asymmetric activating small RNA on prostate cancer cells and related molecular mechanism1. The therapeutic effects of E-cadherin activation by aaE-217 on prostate cancer cell Objective:To evaluate the anti-cancer effects of E-cadherin transcriptional activation induced by aaE-217 in prostate cancer cell PC-3.Methods:(1)Asymmetric RNA�aaE-217 were designed according to the reported activating RNA�dsE-215 in Li's literature; and then were synthesized. The control dsRNA were designed to be non-homologous to the whole human genome. (2) PC-3 cells were treated with aaE-217 (50 nM), dsControl (50 nM), or mock transfection. Then quantitative PCR and western blotting were employed to detect the expression of E-cadherin. (3)At various times following transfection, cell viability assay, apoptosis analysis, clonogenic survival assay, scratch test and transwell chamber assay were used to determine the anti-tumor effect of aaE-217 on PC-3.Results:(1) aaE-217 transfection significantly inhibited the viability and clone formation capacity of prostate cancer cells. Flow-cytometric analysis of aaE-217-treated cells labeled with PI and annexin V revealed that aaE-217 caused a significant increase in PC-3 cell apoptosis, whereas, the migration capacity and invasive ability of PC-3 cells were inhibited significantly.Conclusion:Activation of E-cadherin gene expression by RNAa based on asymmetric activating RNA moleculars may have significant therapeutic potential for the treatment of prostatic carcinoma and other cancers.2. The related molecular mechanism of therapeutic effects on prostate cancer cells induced by aaE-217Objective:To investigate the related molecular mechanism of the anticancer effects on prostate cancer cells of E-cadherin activation induced by aaE-217.Methods:(1) 72hr following transfection, Elisa assay were used to test uPA?MMP2?MMP7 and OPN in the cell culture medium. And Western blots were also performed to detect the expression of P53, cleaved PARP, MMP2 and MMP7. (2) The re-distribution of beta-catenin was determined by measuring its expression in the nucleus, cytoplasm and membrane of PC-3 cells after aaE-217 transfection, and then confirmed by immunofluorescence assay.Results:(1)Elisa assays have shown that uPA?MMP2?MMP7 and OPN declined in the aaE-217 transfected PC-3 cells; (2) The increase in P53?Cleaved-PARP, and the loss of MMP2 and MMP7 were found in the aaE-217 treated PC-3 cells by Western blot assay; (3) Both Western blot and immunofluorescence results demonstrated that the up-regulation of E-cadherin by aaE-217 induced relocation of beta-catenin from cytoplasm and nucleus to the membrane in PC-3 cells.Conclusion:The re-distribution of beta-catenin from cytoplasm and nucleus to the membrane in PC-3 cells caused by E-cadherin activation may reduce the amount of beta-catenin in nucleus as transcription factor, thus inhibitting the expression of downstream tumor metastasis factors or indirectly activate the expression of apoptosis-related factors.