RPLP1 Is Up-Regulated in Human Adenomyosis and Endometrial Adenocarcinoma Epithelial Cells and Is Essential for Cell Survival and Migration In Vitro.

Adenomyosis is defined as the development of endometrial epithelial glands and stroma within the myometrial layer of the uterus. These "ectopic" lesions share many cellular characteristics with endometriotic epithelial cells as well as endometrial adenocarcinoma cells, including enhanced proliferation, migration, invasion and progesterone resistance. We recently reported that the 60S acidic ribosomal protein P1, RPLP1, is up-regulated in endometriotic epithelial cells and lesion tissue where it plays a role in cell survival. To evaluate if a similar pattern of expression and function for RPLP1 exists in adenomyosis and endometrial cancer, we examined RPLP1 expression in adenomyosis and endometrial cancer tissue specimens and assessed its function in vitro using well-characterized cell lines. A total of 12 control endometrial biopsies and 20 eutopic endometrial and matched adenomyosis biopsies as well as 103 endometrial adenocarcinoma biopsies were evaluated for RPLP1 localization by immunohistochemistry. Endometrial adenocarcinoma cell lines, Ishikawa, HEC1A, HEC1B and AN3 were evaluated for RPLP1 protein and transcript expression, while in vitro function was evaluated by knocking down RPLP1 expression and assessing cell survival and migration. RPLP1 protein was up-regulated in eutopic epithelia as well as in adenomyosis lesions compared to eutopic endometria from control subjects. RPLP1 was also significantly up-regulated in endometrial adenocarcinoma tissue. Knockdown of RPLP1 in endometrial adenocarcinoma cell lines was associated with reduced cell survival and migration. RPLP1 expression is up-regulated in eutopic and ectopic adenomyotic epithelia as well as in the epithelia of endometrial cancer specimens. In vitro studies support an essential role for RPLP1 in mediating cell survival and migration, processes which are all involved in pathophysiology associated with both diseases.

Identification of Hub Genes Associated with the Development of Acute Kidney Injury by Weighted Gene Co-Expression Network Analysis.

BACKGROUND: Acute kidney injury (AKI) is a severe clinical syndrome, causing a profound medical and socioeconomic burden worldwide. This study aimed to explore underlying molecular targets related to the progression of AKI. METHODS: A public database originated from the NCBI GEO database (serial number: GSE121190) and a well-established and unbiased method of weighted gene co-expression network analysis (WGCNA) to identify hub genes and potential pathways were used. Furthermore, the unbiased hub genes were validated in 2 classic models of AKI in a rodent model: chemically established AKI by cisplatin- and ischemia reperfusion-induced AKI. RESULTS: A total of 17 modules were finally obtained by the unbiased method of WGCNA, where the genes in turquoise module displayed strong correlation with the development of AKI. In addition, the results of gene ontology revealed that the genes in turquoise module were involved in renal injury and renal fibrosis. Thus, the hub genes were further validated by experimental methods and primarily obtained Rplp1 and Lgals1 as key candidate genes related to the progression of AKI by the advantage of quantitative PCR, Western blotting, and in situ tissue fluorescence. Importantly, the expression of Rplp1 and Lgals1 at the protein level showed positive correlation with renal function, including serum Cr and BUN. CONCLUSIONS: By the advantage of unbiased bioinformatic method and consequent experimental verification, this study lays the foundation basis for the pathogenesis and therapeutic agent development of AKI.

60S acidic ribosomal protein P1 (RPLP1) is elevated in human endometriotic tissue and in a murine model of endometriosis and is essential for endometriotic epithelial cell survival in vitro.

Endometriosis is a female disease which is defined as the presence of ectopic endometrial tissue and is dependent on estrogen for its survival in these ectopic locations. Expression of the ribosomal protein large P1 (RPLP1) is associated with cell proliferation and invasion in several pathologies, but a role in the pathophysiology of endometriosis has not been explored. In this study, we aimed to evaluate the expression and function of RPLP1 with respect to endometriosis pathophysiology. RPLP1 protein was localised by immunohistochemistry (IHC) in eutopic and ectopic tissue from 28 subjects with confirmed endometriosis and from 20 women without signs or symptoms of the disease, while transcript levels were evaluated by qRT-PCR in 77 endometriotic lesions and 55 matched eutopic endometrial biopsies, and protein expression was evaluated using western blotting in 20 of these matched samples. To evaluate the mechanism for enhanced lesion expression of RPLP1, an experimental murine model of endometriosis was used and RPLP1 expression was localized using IHC. In vitro studies using an endometriosis cell line coupled with shRNA knockdown was used to demonstrate its role in cell survival. Expression of RPLP1 mRNA and protein were significantly higher in ectopic lesion tissue compared to paired eutopic endometrium and immunohistochemical localisation revealed predominant localisation to epithelial cells. This pattern of lesion RPLP1 was recapitulated in mice with experimentally induced endometriosis. Stable knockdown of RPLP1 protein resulted in a significant decrease in cell survival in vitro. These studies reveal that RPLP1 is associated with cell proliferation and/or survival and may play a role in the pathophysiology of endometriosis.

RPLP1 promotes tumor metastasis and is associated with a poor prognosis in triple-negative breast cancer patients.

BACKGROUND: Cancer metastasis is the major reason for cancer related deaths, and the mechanism of cancer metastasis still unclear. RPLP1, a member of a group of proteins known as ribosomal proteins, is associated with tumorigenesis and primary cell immortalization and is involved in cellular transformation. However, the expression and potential function of RPLP1 in TNBC remain unclear. METHODS: The expression of RPLP1 in TNBC tissues and cell lines were detected with Real-Time PCR and western blotting. 81 cases of TNBC tissue samples and adjacent non-tumor tissue samples were tested by immunochemistry to determine the correlation between the RPLP1 expression and clinicopathological characteristics. In vitro, we determined the role and mechanistic pathways of RPLP1 in tumor metastasis in TNBC cell lines. RESULTS: In this study, we detected high levels of RPLP1 expression in TNBC samples and cell lines. RPLP1 is upregulated in triple-negative breast cancer (TNBC) tissues and cells, and high expression levels correlate with an increased risk of recurrence and metastasis. Furthermore, high RPLP1 expression was associated with a poor prognosis and was an independent prognostic marker for TNBC. In RPLP1-induced cancer metastasis, RPLP1 may increase cancer cell invasion, which is likely the result of its effect on the cancer cell epithelial-mesenchymal transition. CONCLUSIONS: Altogether, our findings indicate RPLP1 is a poor prognostic potential biomarker and anti-metastasis candidate therapeutic target in triple-negative breast cancer.

A germline mutation of CDKN2A and a novel RPLP1-C19MC fusion detected in a rare melanotic neuroectodermal tumor of infancy: a case report.

BACKGROUND: Melanotic neuroectodermal tumor of infancy (MNTI) is exceptionally rare and occurs predominantly in the head and neck (92.8 % cases). The patient reported here is only the eighth case of MNTI presenting in an extremity, and the first reported in the fibula. CASE PRESENTATION: A 2-month-old female presented with a mass arising in the fibula. Exhaustive genomic, transcriptomic, epigenetic and pathological characterization was performed on the excised primary tumor and a derived cell line. Whole-exome analysis of genomic DNA from both the tumor and blood indicated no somatic, non-synonymous coding mutations within the tumor, but a heterozygous, unique germline, loss of function mutation in CDKN2A (p16(INK4A), D74A). SNP-array CGH on DNA samples revealed the tumor to be euploid, with no detectable gene copy number variants. Multiple chromosomal translocations were identified by RNA-Seq, and fusion genes included RPLP1-C19MC, potentially deregulating the C19MC cluster, an imprinted locus containing microRNA genes reactivated by gene fusion in embryonal tumors with multilayered rosettes. Since the presumed cell of origin of MNTI is from the neural crest, we also compared gene expression with a dataset from human neural crest cells and identified 185 genes with significantly different expression. Consistent with the melanotic phenotype of the tumor, elevated expression of tyrosinase was observed. Other highly expressed genes encoded muscle proteins and modulators of the extracellular matrix. A derived MNTI cell line was sensitive to inhibitors of lysine demethylase, but not to compounds targeting other epigenetic regulators. CONCLUSIONS: In the absence of somatic copy number variations or mutations, the fully transformed phenotype of the MNTI may have arisen in infancy because of the combined effects of a germline CDKN2A mutation, tumor promoting somatic fusion genes and epigenetic deregulation. Very little is known about the etiology of MNTI and this report advances knowledge of these rare tumors by providing the first comprehensive genomic, transcriptomic and epigenetic characterization of a case.

Estrogen receptor-related receptor (Esrra) induces ribosomal protein Rplp1-mediated adaptive hepatic translation during prolonged starvation.

Protein translation is an energy-intensive ribosome-driven process that is reduced during nutrient scarcity to conserve cellular resources. During prolonged starvation, cells selectively translate specific proteins to enhance their survival (adaptive translation); however, this process is poorly understood. Accordingly, we analyzed protein translation and mRNA transcription by multiple methods in vitro and in vivo to investigate adaptive hepatic translation during starvation. While acute starvation suppressed protein translation in general, proteomic analysis showed that prolonged starvation selectively induced translation of lysosome and autolysosome proteins. Significantly, the expression of the orphan nuclear receptor, estrogen-related receptor alpha (Esrra) increased during prolonged starvation and served as a master regulator of this adaptive translation by transcriptionally stimulating 60S acidic ribosomal protein P1 (Rplp1) gene expression. Overexpression or siRNA knockdown of Esrra expression in vitro or in vivo led to parallel changes in Rplp1 gene expression, lysosome/autophagy protein translation, and autophagy. Remarkably, we have found that Esrra had dual functions by not only regulating transcription but also controling adaptive translation via the Esrra/Rplp1/lysosome/autophagy pathway during prolonged starvation.

Unveiling the prognostic implications of RPLP1 upregulation in osteosarcoma.

Osteosarcoma, a malignant bone tumor characterized by a high rate of metastasis and poor survival, presents a critical need for identifying novel biomarkers associated with metastasis. In this study, we conducted an extensive analysis utilizing transcriptional and clinical data sourced from databases such as GEO, TCGA, CCLE, R2, and Xena. And we discovered that Ribosomal protein LP1 (RPLP1) ranked among the top upregulated genes in relation to osteosarcoma metastasis. Notably, RPLP1 exhibited significant expression in both osteosarcoma cell lines and patient samples. Moreover, multiple osteosarcoma studies revealed a strong correlation between RPLP1 overexpression and worse metastasis-free survival as well as overall survival. Additionally, we observed a consistent association between dysregulation of RPLP1 and reduced overall survival across various tumor types. Knocking down of RPLP1 led to the down-regulation of MYL5 and functional enrichment toward cell cycle and cellular interaction. Based on these findings, we propose that RPLP1 has the potential to serve as a prognostic biomarker, indicating increased metastasis and worse survival outcomes in osteosarcoma. These insights contribute to a better understanding of the disease and may pave the way for future research and therapeutic approaches.

RPLP1, an NS4B-interacting protein, enhances production of CSFV through promoting translation of viral genome.

Classical swine fever virus (CSFV), the etiological agent of classical swine fever (CSF), causes serious financial losses to the pig industry. Using yeast two-hybrid screening, we have previously identified ribosomal protein RPLP1 as a potential binding partner of CSFV NS4B. In this study, the interaction between host RPLP1 and CSFV NS4B was further characterized by co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and confocal microscopy. In addition, lentivirus-mediated shRNA knockdown of RPLP1 drastically attenuated CSFV growth, while stable overexpression of RPLP1 markedly enhanced CSFV production. Moreover, cellular RPLP1 expression was found to be significantly up-regulated along with CSFV infection. Dual-luciferase reporter assay showed that depletion of RPLP1 had no effects on the activity of CSFV internal ribosome entry site (IRES). In the first life cycle of CSFV, further studies revealed that RPLP1 depletion did not influence the intracellular viral RNA abundance but diminished the intracellular and extracellular progeny virus titers as well as the viral E2 protein expression, which indicates that RPLP1 is crucial for CSFV genome translation. In summary, this study demonstrated that RPLP1 interacts with CSFV NS4B and enhances virus production via promoting translation of viral genome.

Mechanism of action of hepatitis B virus S antigen transport-inhibiting oligonucleotide polymer, STOPS, molecules.

A functional cure of chronic hepatitis B requires eliminating the hepatitis B virus (HBV)-encoded surface antigen (HBsAg), which can suppress immune responses. STOPS are phosphorothioated single-stranded oligonucleotides containing novel chemistries that significantly reduce HBsAgs produced by HBV-infected liver cells. The STOPS molecule ALG-10000 functions inside cells to reduce the levels of multiple HBV-encoded molecules. However, it does not bind HBV molecules. An affinity resin coupled with ALG-10000 was found to bind several proteins from liver cells harboring replicating HBV. Silencing RNAs targeting host factors SRSF1, HNRNPA2B1, GRP78 (HspA5), RPLP1, and RPLP2 reduced HBsAg levels and other HBV molecules that are concomitantly reduced by STOPS. Host proteins RPLP1/RPLP2 and GRP78 function in the translation of membrane proteins, protein folding, and degradation. ALG-10000 and the knockdowns of RPLP1/2 and GRP78 decreased the levels of HBsAg and increased their ubiquitination and proteasome degradation. GRP78, RPLP1, and RPLP2 affected HBsAg production only when HBsAg was expressed with HBV regulatory sequences, suggesting that HBV has evolved to engage with these STOPS-interacting molecules. The STOPS inhibition of HBsAg levels in HBV-infected cells occurs by sequestering cellular proteins needed for proper expression and folding of HBsAg.

Disruption of the ribosomal P complex leads to stress-induced autophagy.

The human ribosomal P complex, which consists of the acidic ribosomal P proteins RPLP0, RPLP1, and RPLP2 (RPLP proteins), recruits translational factors, facilitating protein synthesis. Recently, we showed that overexpression of RPLP1 immortalizes primary cells and contributes to transformation. Moreover, RPLP proteins are overexpressed in human cancer, with the highest incidence in breast carcinomas. It is thought that disruption of the P complex would directly affect protein synthesis, causing cell growth arrest and eventually apoptosis. Here, we report a distinct mechanism by which cancer cells undergo cell cycle arrest and induced autophagy when RPLP proteins are downregulated. We found that absence of RPLP0, RPLP1, or RPLP2 resulted in reactive oxygen species (ROS) accumulation and MAPK1/ERK2 signaling pathway activation. Moreover, ROS generation led to endoplasmic reticulum (ER) stress that involved the EIF2AK3/PERK-EIF2S1/eIF2α-EIF2S2-EIF2S3-ATF4/ATF-4- and ATF6/ATF-6-dependent arms of the unfolded protein response (UPR). RPLP protein-deficient cells treated with autophagy inhibitors experienced apoptotic cell death as an alternative to autophagy. Strikingly, antioxidant treatment prevented UPR activation and autophagy while restoring the proliferative capacity of these cells. Our results indicate that ROS are a critical signal generated by disruption of the P complex that causes a cellular response that follows a sequential order: first ROS, then ER stress/UPR activation, and finally autophagy. Importantly, inhibition of the first step alone is able to restore the proliferative capacity of the cells, preventing UPR activation and autophagy. Overall, our results support a role for autophagy as a survival mechanism in response to stress due to RPLP protein deficiency.

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda.

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.