Yilu Lu

A microarray for microRNA profiling in mouse testis tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Recent studies indicate that miRNAs are mechanistically involved in the development of mammalian spermatogenesis. However, little work has been done to compare the miRNA expression patterns between immature and mature mouse testes. Here, we employed a miRNA microarray to detect 892 miRNAs in order to evaluate the expression patterns of miRNA. The expression of 19 miRNAs was significantly different between immature and mature individuals. Fourteen miRNAs were significantly upregulated and five miRNAs were downregulated in immature mice and this result was further confirmed by a quantitative real-time RT-PCR assay. Many target genes involved in spermatogenesis are predicted by MiRscan performing miRNA target scanning. Our data indicated specific miRNAs expression in immature mouse testis and suggested that miRNAs have a role in regulating spermatogenesis.

Microarray profiling of microRNAs expressed in testis tissues of developing primates

PurposeMicroRNAs (miRNAs) are small non-coding RNA molecules that have been identified as potent regulators of gene expression. Recent studies indicate that miRNAs are involved in mammalian spermatogenesis but the mechanism of regulation is largely unknown.MethodsmiRNA microarray was employed to compare miRNA expression profiles of testis tissues from immature rhesus monkey (Sample IR), mature rhesus monkey (Sample MR), and mature human (Sample MH). Real-time RT-PCR was uesd to confirm the changed miRNAs.ResultsTwenty-six miRNAs were shared by samples IR/MR and IR/MH with differential expression patterns greater than three-fold difference. PicTar and TargetScan prediction tools predicted a number of target mRNAs, and some of these target genes predicted by miRNAs have been shown to associate with spermatogenesis.ConclusionsOur results indicate that miRNAs are extensively involved in spermatogenesis and provide additional information for further studies of spermatogenetic mechanisms.

Piwil2 suppresses p53 by inducing phosphorylation of signal transducer and activator of transcription 3 in tumor cells.

Piwi proteins have been implicated in germ cell proliferation, differentiation, germline stem cell maintenance and transposon control in germline from Drosophila to mammals. The Piwi-like2 (piwil2) gene is mainly expressed in testis or embryonic cells among normal tissues but widely expressed in tumors. However, it remains to be fully determined through which mechanism piwil2 is involved in tumorigenesis. Here we report that Human piwil2, or Hili represses the tumor suppressor P53 in human cancer cells. Immunoprecipitation analysis shows that Piwil2 can directly associate with Signal Transducer and Activator of Transcription 3 (STAT3) protein via its PAZ domain and form a Piwil2/STAT3/c-Src triple protein-protein complex. Furthermore, STAT3 is phosphorylated by c-Src and translocated to nucleus, then binds to P53 promoter and represses its transcription. The present study demonstrated that Piwil2 plays a role in anti-apoptosis in tumor cells possessing P53 as a positive regulator of STAT3 signaling pathway, providing novel sights into roles of Piwil2 in tumorigenesis.

Identification of piRNAs in Hela cells by massive parallel sequencing

Piwi proteins and Piwi-interacting RNAs (piRNAs) have been implicated in transposon control in germ line from Drosophila to mammals. To examine the profile of small RNA transcriptome and explore the potential roles of Human Piwi-like 2 gene (alias HILI) and its associated piRNAs in human cancer cells, small RNA libraries prepared from HILI-overexpressed, HILI-knockdown, and control HeLa cells were respectively sequenced using Solexa, a next-generation massive parallel sequencing technology. A set of piRNAs and other repeat-associated small RNAs were observed in HeLa cells. By using in situ hybridization, piR-49322 was localized in the nucleolus and around the periphery of nuclear membrane in HeLa cells. Following the overexpression of HILI, the retrotransposon element LINE1 was significantly repressed, while LINE1-associated small RNAs decreased in abundance. The present study demonstrated that HILI along with piRNAs plays a role in LINE1 suppression in HeLa cancer cell line.

HILI Inhibits TGF-β Signaling by Interacting with Hsp90 and Promoting TβR Degradation

PIWIL2, called HILI in humans, is a member of the PIWI subfamily. This subfamily has highly conserved PAZ and Piwi domains and is implicated in several critical functions, including embryonic development, stem-cell self-renewal, RNA silencing, and translational control. However, the underlying molecular mechanism remains largely unknown. Transforming growth factor-β (TGF-β) is a secreted multifunctional protein that controls several developmental processes and the pathogenesis of many diseases. TGF-β signaling is activated by phosphorylation of transmembrane serine/threonine kinase receptors, TGF-β type II (TβRII), and type I (TβRI), which are stabilized by Hsp90 via specific interactions with this molecular chaperone. Here, we present evidence that HILI suppresses TGF-β signaling by physically associating with Hsp90 in human embryonic kidney cells (HEK-293). Our research shows that HILI mediates the loss of TGF-β-induced Smad2/3 phosphorylation. We also demonstrate that HILI interacts with Hsp90 to prevent formation of Hsp90-TβR heteromeric complexes, and improves ubiquitination and degradation of TβRs dependent on the ubiquitin E3 ligase Smurf2. This work reveals a critical negative regulation level of TGF-β signaling mediated by HILI (human PIWIL2) by its ability to interact with Hsp90 and promote TβR degradation.

DAZL binds to 3′UTR of Tex19.1 mRNAs and regulates Tex19.1 expression

Spermatogenesis is a complex process subject to strict controls at both levels of transcription and translation. It has been proposed that DAZL protein binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. In male mice, loss of Dazl results in numerous defects throughout the mitotic and meiotic process of germ cell development. Tex19.1 also plays an important role during spermatogenesis and Tex19.1−/− knockout males exhibit impaired spermatogenesis. Mouse DAZL protein can bind to 3′UTR of mTex19.1 mRNAs and may repress mTex19.1 expression at the translational level. These have been confirmed by both electrophoretic mobility shift assay and translation assay in Zebrafish embryo detecting the luciferase activity. Taken together these data suggest that mDazl may regulate mTex19.1 expression through binding to 3′UTR of mTex19.1 mRNAs in germ cells.

Piwil2 Inhibits Keratin 8 Degradation through Promoting p38-Induced Phosphorylation To Resist Fas-Mediated Apoptosis

ABSTRACT The piwi-like 2 (piwil2) gene is widely expressed in tumors and protects cells from apoptosis induced by a variety of stress stimuli. However, the role of Piwil2 in Fas-mediated apoptosis remains unknown. Here, we present evidence that Piwil2 inhibits Fas-mediated apoptosis. By a bacterial two-hybrid screening, we identify a new Piwil2-interacting partner, keratin 8 (K8), a major intermediate filament protein protecting the cell from Fas-mediated apoptosis. Our results show that Piwil2 binds to K8 and p38 through its PIWI domain and forms a Piwil2/K8/P38 triple protein-protein complex. Thus, Piwil2 increases the phosphorylation level of K8 Ser-73 and then inhibits ubiquitin-mediated degradation of K8. As a result, the knockdown of Piwil2 increases the Fas protein level at the membrane. In addition to our previous finding that Piwil2 inhibits the expression of p53 through the Src/STAT3 pathway, here we demonstrate that Piwil2 represses p53 phosphorylation through p38. Our present study indicates that Piwil2 plays a role in Fas-mediated apoptosis for the first time and also can affect p53 phosphorylation in tumor cells, revealing a novel mechanism of Piwil2 in apoptosis, and supports that Piwil2 plays an active role in tumorigenesis.

A TALEN‐based specific transcript knock‐down of PIWIL2 suppresses cell growth in HepG2 tumor cell

PIWIL2 is widely expressed in various tumours and implicated in playing a role in tumourigenesis. For a more thorough study of PIWIL2 functions in tumour cells, we aimed to establish PIWIL2‐specific transcript knock‐down/knockout HepG2 cell lines using transcription activator‐like effector nuclease (TALEN) technology. Furthermore, we proposed to use the cell models to explore PIWIL2 functions in TGF‐β signalling in HepG2 cells. HepG2s are human hepatocellular carcinoma cells.

PIWIL1 destabilizes microtubule by suppressing phosphorylation at Ser16 and RLIM-mediated degradation of stathmin1

Human PIWIL1, alias HIWI, is a member of Piwi protein family and expressed in various tumors. However, the underlying mechanism of PIWIL1 in tumorigenesis remains largely unknown. Stathmin1 is a cytosolic phosphoprotein which has a critical role in regulating microtubule dynamics and is overexpressed in many cancers. Here we report that PIWIL1 can directly bind to Stathmin1. Meanwhile, PIWIL1 can up-regulate the expression of Stathmin1 through inhibiting ubiquitin-mediated degradation induced by an E3 ubiquitin ligase RLIM. Furthermore, PIWIL1 can also reduce phosphorylation level of Stathmin1 at Ser-16 through inhibiting the interaction between CaMKII and Stathmin1. Our results showed that PIWIL1 suppresses microtubule polymerization, and promotes cell proliferation and migration via Stathmin1 for the first time. Our study reveals a novel mechanism for PIWIL1 in tumorigenesis.

HILI destabilizes microtubules by suppressing phosphorylation and Gigaxonin-mediated degradation of TBCB.

Human PIWIL2, aka HILI, is a member of PIWI protein family and overexpresses in various tumors. However, the underlying mechanisms of HILI in tumorigenesis remain largely unknown. TBCB has a critical role in regulating microtubule dynamics and is overexpressed in many cancers. Here we report that HILI inhibits Gigaxonin-mediated TBCB ubiquitination and degradation by interacting with TBCB, promoting the binding between HSP90 and TBCB, and suppressing the interaction between Gigaxonin and TBCB. Meanwhile, HILI can also reduce phosphorylation level of TBCB induced by PAK1. Our results showed that HILI suppresses microtubule polymerization and promotes cell proliferation, migration and invasion via TBCB for the first time, revealing a novel mechanism for HILI in tumorigenesis.