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AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2.METHODS:RT-PCR and Western blot was used to determine the expres-sion of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29.HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay.ELISPOT as-say was used to investigate the levels of IFN-γ.The cytotoxicity assay in vitro was also used to determine the ability of indu-cing T cell response by the peptides.RESULTS:The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule.ELISPOT assay showed P485 and P965 induced CTLs of IFN-γrelease form CTLs.The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION:The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.
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Objective To investigate the gene expression of PIWIL2 in the bladder urothelial carcinoma (BTCC) and siRNA interact on PIWIL2 gene expression in human bladder cancer cell line BIU-87.Methods Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to detect the PIWIL2 expressions in tissues of BTCC (46 cases),cystitis glandularis(21 cases),adjacent non-cancerous tissues (17 cases) and normal bladder tissues (7 cases). 3 specific siRNA targeted PIWIL2 gene were synthesized after designed and transferred. After siRNA was transferred into BIU-87 cells, MTI and TUNEL methods were applied to detect the proliferation inhibitory rate (IR) and apoptosis index (AI) in BIU-87 cells,qRT-PCR and Western blot were used to examine effects of siRNA on the expressions of the PIWIL2 gene and protein,respectively.Results The expression rate of PIWIL2 mRNA in BTCC tissues was 76.08 ％(35/46) and significantly higher than those in the cystitis glandularis tissues (42.86 ％,9/21),adjacent non-cancerous tissues (41.17 ％,7/17) and normal tissues (7.14 ％,1/14) (P =0.008,P =0.010,P =0.000).The IR [(37.52±8.84) ％,(64.36±9.64)％] and (62.94±8.43) ％] and AI [(26.18±5.42) ％,(38.75±6.19) ％ and (40.02±5.64) ％] of BIU-87 cells in the siRNA 1～3 groups were respectively significantly higher than those [(1.97±0.02) ％ and (3.35±0.47) ％] in the control group(P=0.000),and expressions of PIWIL2 mRNA and protein in the siRNA groups were both lower than those in the control group. Moreover, the effects of siRNA 2 group and siRNA 3 group on inhibiting PIWIL2 expression, IR and AI of BIU-87 cells were stronger than siRNA 1 group. Conclusion The over-expression of PIWIL2 suggested that it played an important role in the mechanism of development and malignant progression of BTCC. The siRNA of transcription can significantly inhibit its expression, induce cell apoptosis and inhibit the growth of BIU-87 cells which might provide the experimental evidence for the gene targeting therapy of bladder tumor.
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BACKGROUND: There are no established reports about the expression of the Piwil gene, a subfamily of the Piwi gene involved in RNA silencing and self-renewal, in colorectal carcinomas. It is known that the degree of PIWIL2 expression is higher in colorectal carcinomas. But its clinicopathologic significance remains undetermined. This study reassessed the relationship between PIWIL2 expression and the clinicopathologic parameters in colorectal carcinomas. METHODS: An immunohistochemistry of PIWIL2 expression was done in 60 cases of colorectal carcinoma. This was followed by an analysis of the correlation between PIWIL2 expression and clinicopathologic features and a survival analysis. RESULTS: There were 44 cases (73.3%) where the degree of PIWIL2 expression was relatively higher. The high degree of PIWIL2 expression was significantly correlated with the lower degree of differentiation (p=0.039), deep invasion (p=0.019) and perineural invasion (p=0.027). The overall survival was longer in patients with the lower degree of PIWIL2 expression than in those with the higher degree of PIWIL2 expression. CONCLUSIONS: Our results showed that the degree of PIWIL2 expression was relatively higher in colorectal carcinomas and it was significantly correlated with variable clinicopathologic indicators for a poor prognosis. This indicates that PIWIL2-positive cells contribute to the progression of colorectal cancer.
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Humans , Colorectal Neoplasms , Genes, vif , Immunohistochemistry , Prognosis , RNA InterferenceABSTRACT
Objective: To prepare rabbit polyclonal antibody against human P-element-induced wimpy testis like 2 (HIWI2) protein, identify its properties and investigate its distribution in normal and tumor tissues by means of tissue chip. Methods: PIWIL2 peptide was synthesized chemically and conjugated to Keyhole limpet hemocyanin (KLH) as an immunogen. Then the PIWIL2-KLH conjugation was injected into rabbits subcutaneously to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blotting after purification by affinity chromatography. PIWIL2 was then immuno-stained on the tissue chip to study its distribution in the normal and tumor tissues. Results: Rabbit's antibodies against human PIWIL2 were prepared after the injection of PIWIL2-KLH conjugation subcutaneously. These antibodies were identified as PIWIL2 peptides by ELISA and Western blotting assay. PIWIL2 protein expression was tissue-specific in tumor tissues, with PIWIL2 protein found in the cytoplasm of smooth muscle cells of most normal and tumor tissues. Conclusion: The polyclonal antibodies against human PIWIL2 protein have been successfully prepared, which provides a basis for further study on the role of PIWIL2 in the pathway of miRNA/RNA.