The Study On Downregulation Of GRP78and Upregulation Of KLF4Against Malignant Biological Behaviors In Prostate Cancer Cells Induced By Small RNA

BackgroundProstate cancer is one of the most common malignant tumors of urinary system. In china, incidence of prostate cancer in the male has been on gradually rising. In recent years, the gene therapy of CRPC has become a research hotspot, in which, seeking an effective gene targets for therapy of CRPC is particularly important. As a key factor, glucose regulation protein78KD (GRP78) plays an important role in the development, survival and progression of tumor cell. In recent years, many studies found that downregulation of GRP78expression in different tumor cells show the anti-tumor effect on these tumors. However, downregulation of GRP78expression in prostate cancer cells has been little investigated. Furthermore, some researches show that the asymmetric small interference RNA (asiRNA) show more potential on the efficiency and durable of downregulation of target gene than the symmetric small interference RNA. Therefore, we design a series of asiRNA and try to downregulate the expression of GRP78in prostate cancer cells by using the asiRNAs, and try to investigate its anti-tumor effect on prostate cancer cells and its possible mechanisms.MethodsThe expressions of GRP78in three prostate cells have been investigated by RT-PCR and Western Blotting. A series of asiRNAs, which specific target on the mRNA sequence of GRP78gene, have been designed and synthesized. Then, the downregulation efficiency of GRP78has been evaluated in the PC-3cells by using real-time PCR, RT-PCR and Western Blotting. Furthermore, the anti-tumor effect on the growth rate, apoptosis and migration in PC-3cells was investigated by using MTT assay, FCM and transwell chambers. Finally, the apoptosis-relative protein and migration-relative proteins was evaluated by using Western Blotting to elucidate the possible mechanisms under the anti-tumor effect of asiRNA.ResultsThe15bp GRP78-specific asiRNA (asiGRP78-3) show more potential on efficiency and durable in downregulation of GRP78expression in PC-3cells compared with the symmetric small interference RNA. The asiGRP78also show the potential on the inhibition of growth rate and migration and induction of apoptosis in PC-3cells. Mechanistic studies show that downregulation of GRP78by asiGRP78-3inhibited the phosphorylation of AKT and decreased the expressions of pro-caspase9, pro-caspase3and vimentin in PC-3cells which may be possible mechanisms under the anti-tumor effect of asiRNA.ConclusionsThe GRP78-specific asiRNA can effective down regulate the expression of GRP78in prostate cancer cells. Downregulation of GRP78expression with asiRNA show the anti-tumor effect in prostate cancer cells, and the GRP78could be the target in gene therapy of prostate cancer. BackgroundProstate cancer is one of the most common malignant tumors of urinary system, but the treatment of castration-resistant prostate cancer (CRPC) is still poor. In recent years, the gene therapy of CRPC has become a research hotspot. Kruppel-like factor4(KLF4) is a very important eukaryotic transcription factor, and the knockdown of the KLF4expression could promote the proliferation and migration in cancer cells. The recent studies show that small RNAs can not only downregulate the expressions of target gene but also upregulate the expressions of target gene. Compare with the synthetic dsRNA, the short hairpin RNA (shRNA) expressed by plasmid vector has more capability in the effects of continuous and could be conjunct to the target proteins. In our study, we tried to construct the pENTR/CMV-EGFP-U6-saKLF4-496ukaryotic expression plasmid vector. The shRNA expressed by the pENTR/CMV-EGFP-U6-saKLF4-496plasmid can bind with the sequence in the promoter of KLF4gene, and then we transfect the plasmid into PC-3cells to investigate its effect in upregulation of KLF4expression. Furthermore, we investigated the effect of pENTR/CMV-EGFP-U6-saKLF4-496plasmid on the biological behaviour in PCa cells.MethodsThe shRNA of saKLF4-496sequence was designed to bind with the-496site in KLF4promoter. The oligo DNA of shRNA designed by saKLF4-496sequence was linked with pENTR/CMV-EGFP-U6vector, and then the recombinant plasmid pENTR/CMV-EGFP-U6-saKLF4-496was constructed. The PC-3cells were transfected with the pENTR/CMV-EGFP-U6-saKLF4-496plasmids which was identified by sequence analysis. The expression levels of KLF4in PC-3cells were deteced by RT-PCR and Western Blotting. The migration capacity of PC-3cells after transfection was assayed by transwell chamber. ResultsThe pENTR/CMV-EGFP-U6-saKLF4-496plasmid was successfully constructed. The mRNA and protein levels of KLF4in PC-3cells were upregulation in96hours after transfection with pENTR/CMV-EGFP-U6-saKLF4-496. The migration of PC-3cells was inhibited by transfection with pENTR/CMV-EGFP-U6-saKLF4-496plasmids.ConclusionsThe pENTR/CMV-EGFP-U6-saKLF4-496plasmid can be successfully constructed. The expression of KLF4in PCa cells can be unregulated by pENTR/CMV-EGFP-U6-saKLF4-496plasmid, and the migration capacity of PC-3cells could be inhibited by the pENTR/CMV-EGFP-U6-saKLF4-496plasmid.