Stem Cell Protein Piwil2 Variant PL2V60 Regulates Precancerous Stem Cell Proliferation
R Shen, Y Yin, D-T Yin, Q-T Yan, L Chen, G He, SH Basrsky, J-X Gao. Ohio State University, Columbus, OH; The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
Background: During the tumor development, a germline stem cell protein piwil2 appears to play an important role, because it is silenced normally in adult tissues except for in the GS cells but activated ectopically in various types of tumor. Recently, we have reported that piwil2 is stably expressed in pCSCs derived from lymphoma. Knockdown of piwil2 mRNA using piwil2-specific siRNA reduced pCSC expansion in vitro. However, transduction of piwil2 gene into stem/progenitor cells induced proliferation-associated cell death (PACD), which is strikingly contradictory to its role in pCSCs. Therefore, we hypothesized that the proliferation of pCSCs might be regulated by piwil2 variants rather than whole piwil2.
Design: First, we determined whether the transcripts of piwil2 or its variants were expressed in pCSCs, using piwil2-specific Gene Exon Array (GEA) RT-PCR. Then, the piwil2 variants expressed in pCSCs were quantitatively defined by Western-blot, using a novel antibody to the piwil2 peptide common for its variants, which is generated by us. Finally, the function of piwil2 variants expressed in pCSCs was verified using RNAi and biochemistry approaches.
Results: GEA RT-PCR analysis of 3 clones of pCSCs reveal that whole piwil2 did not express in pCSCs but its potential variants, which appeared to be truncated at 5'-end of transcripts. The peptide-affinity purified polyclonal antibody to a common peptide of piwil2 variants could react with piwil2 and several variants, designated as PL2V80, 65, 60, 52, and 42, which were expressed in both murine testis and human tumor cell lines. Western-blot analysis revealed that only PL2V60 was strongly expressed in pCSCs (3 6 units; normalized against -actin of the same samples), and piwil2 and other variants were not significantly detectable except for PLV50, which was detected in trace amount in pCSCs. As control, the PL2V60 was almost not detectable in normal splenocytes (0.05 units). Knockdown of PL2V60 mRNA and protein significantly inhibited pCSC proliferation in vitro. Moreover, PL2V60 expression was not limited to hematopoietic tumor cells; it was broadly detected in various types of human tumors such as breast and cervical cancer in a high level.
Conclusions: Our study indicates that PL2V60 is ectopically expressed in pCSCs, regulating their proliferation, and that it has the potential to be used as a novel biomarker for early detection and intervention of cancer.
Tuesday, March 10, 2009 1:00 PM
Poster Session IV # 209, Tuesday Afternoon