Construction Of Recombinant Plasmid Vector Of Beclin-1 And Study On Autophagy Gene Beclin-1 In Prostate Cancer

In generally, Prostate cancer (prostate cancer, PCa) is a age-related diseases.As society developing, PCa has been already become another important cancer threatening the health of men. However, the early detection of prostate cancer is not easy with the current medical standards, and PCa was often misdiagnosed as benign prostate disease.When made the finally diagnosis, the PCa was alaway advanced, then there wasn't effective treatment for it . With the increased level of cancer treatment, there have been used many new treatment method, in which autophagy is one of a hotspot. Beclin-1 is an autophagy gene, and are closely related the autophagy of tumor cells. Then, we constructed the combinant plasmid vector of Beclin-1, and initially identified the expression of Beclin-1 in prostate cancer cells and tissue. The results is foundational for the future experimental work.1 Construction of recombinant Plasmid vector of beclin-1Beclin-1 gene DNA sequence retrieved from GenBank, using primer design software Primer 5.0 designed the special primers of the coding region of Beclin-1 including BamHⅠand HindIII restriction sites. Then we extracted the total RNA using the LNCaP cell, and the total RNA was reversed transcription to cDNA.As a template, cDNA was amplified by PCR, then the product was the coding region of Beclin-1 ,which length is 1353bp. And then was cloned into the E.coli vector pQE-80. The recombinant plasmid vector of pQE-8O-Beclin-1 was identified by the digestion of restriction endonuclease.The DNA sequence of the coding region of Beclin-1 was compared with that in GenBank for homology.The homology was 100% in nucleotide acid.The recombinant plasmid vector pQE-80-Beclin-1 was constructed successfully.2 The expression of autophay gene Beclin-1 in Prostate cancerBeclin-1 gene DNA sequence retrieved from GenBank, using primer design software Primer 5.0 designed the special primers of of Beclin-1 and the control gene GADPH, which length is 750bp and 250bp respectively. We collected the covered bottom of the prostate cancer cell lines and control cell line QZG, and extracted the total RNA, then RNA was reversed transcription to cDNA, -20℃preservation. After the cDNA quantification, take the same amount of cDNA as a template and GAPDH as a control gene, the expression of Beclinl mRNA in PCa cell, Hela cell and QZG cell were measured by PCR. Comparing with the QZG,the expression of Beclin-1 was significantly down-regulated in PCa cell, A549 cell and Hela cell.We collected the covered bottom of the prostate cancer cell lines and other cell lines,extracted the total protein. After the protein quantification, with taking the same amount of protein, we got the result that Beclin-1 protein was remarkable down- regulated using Western-blot compared with QZG cell.With the cell climbing sheets and PCa tissue chip using immunocytochemical staining method, we observed the Beclin-1 protein expression in cell climbing sheets and PCa tissue chip, the results showed that Beclin-1 protein expression decreased in PCa cell and tissue.Then we concluded that Beclin-1 was down- regulated in prostate cancer. The abnormal expression of Beclin-l is closely associated with the autophagy attenuation,and may play an important role in prostate cancer process. By up-regulated the expression of Beclin-1 in prostate cancer maybe a new method in cancer treatment.