The long non-coding RPPH1 is decreased in leukocytes and increased in plasma from women developing pre-eclampsia†.

Previous studies show differentially expressed long non-coding RNA present in the placenta from women with pre-eclampsia, potentially playing a vital role in the pathogenesis of the complication. In a published microarray study, Ribonuclease P RNA component H1 was decreased in leukocytes from women that later developed pre-eclampsia. We hypothesized that Ribonuclease P RNA component H1 decreased during pregnancy in women developing pre-eclampsia and important for the development of the complication. We isolated RNA from extracellular vesicles, leukocytes and plasma using blood samples taken at weeks 22-24 and 36-38 in women who subsequently developed pre-eclampsia and from healthy pregnancy. The expression of Ribonuclease P RNA component H1 was quantified using qPCR. Expression of Ribonuclease P RNA component H1 at 22-24 weeks was further examined to investigate its discriminatory potential of subsequent pre-eclampsia and association with clinical markers. We found lower expression of Ribonuclease P RNA component H1 in leukocytes at 22-24 and 36-38 weeks amongst women who subsequent developed pre-eclampsia compared with those who did not, while increased Ribonuclease P RNA component H1 expression was found in plasma at 36-38 weeks. Pre-eclampsia risk factors could not account for this difference in the Ribonuclease P RNA component H1 expression. Prediction of pre-eclampsia at 22-24 weeks using Ribonuclease P RNA component H1 expression in leukocytes in addition to the screening algorithm used today had a significantly better performance. In conclusion, Ribonuclease P RNA component H1 expression in leukocytes was significantly decreased in women with pre-eclampsia, and the expression at 22-24 weeks associated with the subsequent development of pre-eclampsia. Ribonuclease P RNA component H1 in leukocytes may be a useful biomarker for prediction and/or early detection of pre-eclampsia and an unknown regulator of the signaling affecting immune cells.

Recent insights into noncanonical 5' capping and decapping of RNA.

The 5' N7-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5' capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as "no-cap decay." It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.

LncRNA RPPH1 acts as a molecular sponge for miR-122 to regulate Wnt1/ß-catenin signaling in hepatocellular carcinoma.

This study aimed to explore the role of lncRNA RPPH1 in hepatocellular carcinoma. The expression of RPPH1 and miR-122 was determined by Real-time PCR. Cell proliferation and colony formation assays were employed to monitor cell growth in vitro. Wound healing and Transwell assays were applied to detect cell migration and invasion. A dual-luciferase reporter assay was used to verify the interaction between RPPH1 and miR-122. The in vivo function of RPPH1 was illustrated by xenograft tumor models. The results showed that the expression of RPPH1 was markedly upregulated in human HCC specimens and cell lines compared to normal controls. However, the trend of miR-122 was the opposite. RPPH1 facilitates the proliferation, migration, and invasion of HCC cells and synchronously suppresses cell apoptosis. The dual-luciferase assay confirmed the relationship between RPPH1 and miR-122. Rescue experiments showed that RPPH1 acted as a competing endogenous RNA (ceRNA) by sponging miR-122 in HCC cells. Moreover, RPPH1 positively regulated the expression of Wnt1 and its downstream targets through miR-122. Our study demonstrates for the first time that RPPH1 promotes HCC progression via the miR-122/Wnt1/ß-catenin axis, which may represent a valuable therapeutic target for patients with HCC.

RNA Structure Protects the 5' End of an Uncapped Tombusvirus RNA Genome from Xrn Digestion.

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5' termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5' access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5' terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5' access, we have termed this 5'-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5' terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5' capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5' end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5'-end-attacking cellular enzymes.

Circ_RPPH1 regulates glioma cell malignancy by binding to miR-627-5p/miR-663a to induce SDC1 expression.

BACKGROUND: Recent studies revealed the key role of circular RNA (circRNA) in glioma progression. However, the effect of circ_0000520, also named as circRNA ribonuclease P RNA component H1 (circ_RPPH1), in glioma development was unknown. The study aimed to reveal the role of circ_RPPH1 in glioma cell malignancy. METHODS: Human astrocytes (NHA) and glioma cell lines (A172 and U251) were employed in this study. Quantitative real-time polymerase chain reaction and western blot were used to check the expression of circ_RPPH1, microRNA-627-5p (miR-627-5p), miR-663a and syndecan 1 (SDC1). Immunohistochemistry assay was conducted to assess the protein expression of nuclear proliferation marker ki67 and matrix metalloprotein 9 (MMP9). Cell viability was assessed by 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation and apoptosis were investigated by flow cytometry analysis, 5-Ethynyl-29-deoxyuridine, or cell colony formation assay. Cell migration and invasion were evaluated by transwell assays. The interaction between miRNAs (miR-627-5p and miR-663a) and circ_RPPH1 or SDC1 was identified by a dual-luciferase reporter assay. A mouse model assay was performed to reveal the impact of circ_RPPH1 knockdown on glioma cell malignancy in vivo by analyzing neoplasm volume and weight. RESULTS: Circ_RPPH1 and SDC1 expression were significantly increased, whereas miR-627-5p and miR-663a expression were decreased in glioma tissues and cells in comparison with healthy brain tissues or human astrocytes. Circ_RPPH1 depletion led to the decreased cell proliferation, migration and invasion, and the increased cell apoptosis. Additionally, circ_RPPH1 bound to miR-627-5p/miR-663a and mediated glioma cell processes by interacting with them. SDC1 overexpression attenuated miR-627-5p/miR-663a-mediated actions. Moreover, circ_RPPH1 regulated SDC1 expression through interaction with miR-627-5p and/or miR-663a. Furthermore, circ_RPPH1 knockdown inhibited glioma cell malignancy in vivo, accompanied by the decreases of ki67 and MMP9 expression. CONCLUSION: Circ_RPPH1 knockdown inhibited glioma tumorigenesis by downregulating SDC1 by binding to miR-627-5p/miR-663a, showing that circ_RPPH1 might be an effective therapeutic target for glioma.

cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3' cyclic phosphates and 5' hydroxyl ends.

Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate and a 3'-fragment with a 5'-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5'-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. [Figure: see text].

LncRNA RPPH1 attenuates Aß25-35-induced endoplasmic reticulum stress and apoptosis in SH-SY5Y cells via miR-326/PKM2.

BACKGROUND: The durative endoplasmic reticulum stress (ERS) and subsequent apoptosis contributes to the development and progression of Alzheimer's disease (AD). MiR-326 can reduce pyruvate kinase M2 (PKM2) expression, leading to ERS. Whereas, lncRNA RPPH1 is able to increase dendritic spine density and protect hippocampal pyramidal neurons through targeting miR-326. Our study aims to investigate the regulation of lncRNA RPPH1 and miR-326/PKM2 on ERS and related apoptosis in AD. METHODS: SH-SY5Y cells treated with Aß25-35 were selected as an in vitro AD model. RPPH1 and miR-326 overexpression and silencing cells were established by transforming vectors. The expression of RPPH1 and miR-326 were detected by qRT-PCR. MTT, flow cytometric, intracellular calcium assay and Western blot were used to test the functions of RPPH1 and miR-326 in SH-SY5Y cell proliferation, apoptosis and ERS. Dual-luciferase assay was used to detect the interaction among RPPH1, miR-326 and PKM2. RESULTS: RPPH1 overexpression enhanced the viability of SH-SY5Y cells, and attenuated the apoptosis of of SH-SY5Y cells. Moreover, RPPH1 overexpression down-regulated ER stress related proteins such as GRP78, CHOP and cleaved caspase-12. Mechanistically, RPPH1 directly targeted miR-326, thereby counteracting its inhibitory effect on PKM2 expression, contributing to attenuation of apoptosis and ERS induced by Aß25-35. CONCLUSION: Aß25-35-induced ERS and apoptosis in SH-SY5Y cells can be attenuated by lncRNA RPPH1 through regulating miR-326/PKM2 axis. This study provided therapeutic options for AD patients.

Searching for Biological Function of the Mysterious PA2504 Protein from Pseudomonas aeruginosa.

For nearly half of the proteome of an important pathogen, Pseudomonas aeruginosa, the function has not yet been recognised. Here, we characterise one such mysterious protein PA2504, originally isolated by us as a sole partner of the RppH RNA hydrolase involved in transcription regulation of multiple genes. This study aims at elucidating details of PA2504 function and discussing its implications for bacterial biology. We show that PA2504 forms homodimers and is evenly distributed in the cytoplasm of bacterial cells. Molecular modelling identified the presence of a Tudor-like domain in PA2504. Transcriptomic analysis of a ΔPA2504 mutant showed that 42 transcripts, mainly coding for proteins involved in sulphur metabolism, were affected by the lack of PA2504. In vivo crosslinking of cellular proteins in the exponential and stationary phase of growth revealed several polypeptides that bound to PA2504 exclusively in the stationary phase. Mass spectrometry analysis identified them as the 30S ribosomal protein S4, the translation elongation factor TufA, and the global response regulator GacA. These results indicate that PA2504 may function as a tether for several important cellular factors.

Application of a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme for transcription start site mapping.

Changes in the 5' leader of an mRNA can have profound effects on its translational efficiency with little effect on abundance. Sequencing-based methods to accurately map the 5' leader by identifying the first transcribed nucleotide rely on enzymatic removal of the 5' eukaryotic cap structure by tobacco acid pyrophosphatase (TAP). However, commercial TAP production has been problematic and has now been discontinued. RppH, a bacterial enzyme that can also cleave the 5' cap, and Cap-Clip, a plant-derived enzyme, have been marketed as TAP replacements. We have engineered a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme that functions as a superior TAP replacement. It can be purified from E. coli overexpression in high yields using standard biochemical methods. This constitutively active enzyme is four orders of magnitude more catalytically efficient than RppH at 5' cap removal, compares favorably to Cap-Clip, and the 5' monophosphorylated RNA product is suitable for standard RNA cloning methods. This engineered enzyme is a better replacement for TAP treatment than the current marketed use of RppH and can be produced cost-effectively in a general laboratory setting, unlike Cap-Clip.

LncRNA Rpph1 protects amyloid-ß induced neuronal injury in SK-N-SH cells via miR-122/Wnt1 axis.

Objective: To investigate the role of lncRNA Rpph1 on amyloid-ß induced neuronal injury in SK-N-SH cells and underlying mechanism.Methods: In vitro Alzheimer's disease (AD) model was established using the SK-N-SH cells treated with Aß25-35 peptide. APPswe/PS1ΔE9 double transgenic mice were used as AD animal model. Rpph1 was over-expressed and miR-122 was inhibited or overexpressed in SK-N-SH cells via transfection with pcDNA3.1-oe Rpph1 vector, miR-122 inhibitor or miR-122 mimic, respectively. Cell viabilities and apoptosis were evaluated using MTT or flow cytometry assay, respectively. Quantitative real-time PCR (RT-qPCR) was used to determine expression of Rpph1 and miR-122. Western blotting was used to determine the expression of apoptosis related proteins as well as Wnt/ß-catenin signaling related proteins. Dual luciferase reporter assay was conducted to confirm the binding of miR-122 with predictive binding site in 3' UTR of Rpph1 and Wnt1.Results: Both lncRNA Rpph1 and miR-122 were up-regulated in AD mouse. Either over-expression of Rpph1 or inhibition of miR-122 restored the cell viability or decreased cell apoptosis rate in Aß induced SK-N-SH cells. Overexpression of miR-122 inhibited the cell viability while did not influence the Aß level in SK-N-SH cells. Furthermore, over-expression of Rpph1, as well as inhibition of miR-122, elevated Bcl-2, c-myc, Survivin and decreased Bax expression via activating Wnt/ß-catenin signaling. Dual luciferase reporter assay showed that miR-122 could directly target to 3'UTR of Rpph1 and Wnt1.Conclusion: Both lncRNA Rpph1 and miR-122 were up-regulated in AD mouse and Rpph1 activated Wnt/ß-catenin signaling to ameliorate amyloid-ß induced neuronal apoptosis in SK-N-SH cells via direct targeting miR-122.

NAD in RNA: unconventional headgear.

Although widely assumed to bear a 5'-terminal triphosphate or monophosphate, recent evidence suggests that the 5' end of bacterial RNA can sometimes bear a modification reminiscent of a eukaryotic cap. A new study has now identified Escherichia coli RNAs that begin with a noncanonical cap resembling the redox cofactor nicotinamide adenine dinucleotide (NAD), as well as a cellular enzyme that can remove it. The biological function of such caps remains to be determined.

Analysis of differentially upregulated proteins in ptsHIcrr- and rppH- mutants in Escherichia coli during an adaptive laboratory evolution experiment.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS- derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH-) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH- mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS- and rppH- mutations in the deep of a proteomic approach to understanding the relevance of rppH- phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH-, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH- were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS- strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS- derivatives during the ALE experiment. This result suggests that the evolving PTS- population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH- mutation in the PTS- background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

Inhibition of breast cancer cell proliferation and tumorigenesis by long non-coding RNA RPPH1 down-regulation of miR-122 expression.

BACKGROUND: Recent studies showed that long non-coding RNA (lncRNA) plays an important role in many life activities. RPPH1 is one of the lncRNA genes that are expressed differently between breast cancer and normal tissues by the lncRNA gene chip. Our study was conducted to examine the regulation of lncRNA RPPH1 in breast cancer. METHODS: Two cell lines, MCF-7 and MDA-MB-231, were selected to be the research objects in this study; RPPH1 overexpression and knockdown models were established by transforming vectors. Real-time polymerase chain reaction, MTT assay, clone formation and cell flow cytometer assay were used to test the function of RPPH1. Dual-luciferase assay was used to detect a target relationship between RPPH1 and miR-122. RESULTS: RPPH1 overexpression promoted cell cycle and proliferation and increased colony formation. In the RPPH1 overexpression model, there was a target relationship between RPPH1 and miR-122, and some of the downstream genes of miR-122, including ADAM10, PKM2, NOD2 and IGF1R, were increased. Moreover, we found that lentivirus-mediated interference of lncRNA RPPH1 inhibited tumour growth in nude mice. CONCLUSION: Breast cancer progression can be promoted by directly targeting miR-122 through lncRNA RPPH1. This study provided evidence that can serve as the molecular basis for improving treatment options for patients.

ß-Asarone Protects PC12 Cells Against Hypoxia-Induced Injury Via Negatively Regulating RPPH1/MiR-542-3p/DEDD2 Axis.

Hypoxic injury to the brain is very intricate under the control of biochemical reactions induced by various factors and mechanisms. Long non-coding RNAs (lncRNAs) have already been revealed to affect pathological processes in the nervous system of different degrees. This research aimed to investigate the mechanisms implicated in hypoxic brain injury. ß-Asarone mitigated the decrease of cell viability, superoxide dismutase activity, and mitochondrial membrane potential, as well as the increase of cell apoptosis, lactate dehydrogenase release, malondialdehyde content, and reactive oxidative species production by cobalt chloride. LncRNA ribonuclease P RNA component H1 (RPPH1) was discovered to be highly expressed in hypoxia-induced PC12 cells, and ß-Asarone addition led to a decline in RPPH1 expression. RPPH1 overexpression reversed the effect of ß-Asarone on hypoxia-induced injury in PC12 cells. Furthermore, we proved that RPPH1 could sponge miR-542-3p. Subsequently, death effector domain containing 2 (DEDD2) was proven as the downstream gene of RPPH1/miR-542-3p axis. Eventually, the whole regulation mechanism of RPPH1/miR-542-3p/DEDD2 axis was testified through rescue assays. The impacts of ß-Asarone on hypoxia-induced PC12 cells could be countervailed by RPPH1 augment, which was also discovered to be neutralized in response to miR-542-3p overexpression or DEDD2 depletion. These findings offered a novel perspective for understanding neuroprotection.

circRNA RPPH1 Facilitates the Aggravation of Breast Cancer Development by Regulating miR-542-3p/ARHGAP1 Pathway.

Background: Circular RNAs (circRNAs) have important roles in human malignancies, including breast cancer (BC). In this study, we explored the function of circRNA ribonuclease P RNA component H1 (circ_RPPH1) in BC development and clarify the mechanistic pathway. Materials and Methods: Expression of circ_RPPH1, microRNA-542-3p (miR-542-3p), and Rho GTPase-activating protein 1 (ARHGAP1) in BC tissues and cells was determined by quantitative real-time polymerase chain reaction or Western blot assay. The stability of circ_RPPH1 was confirmed by RNase R and actinomycin D treatment. Cell viability and colony formation ability were measured by methyl thiazolyl tetrazolium (MTT) assay and colony formation assay, respectively. Western blot analysis was also used to detect proliferation biomarker (Ki67) and epithelial-mesenchymal transition (EMT) biomarkers (E-cadherin, N-cadherin, and vimentin). Flow cytometry and Transwell assays were performed to monitor cell apoptosis, migration, and invasion. The binding potency between miR-542-3p and circ_RPPH1 or ARHGAP1 was validated by dual-luciferase reporter assay. Functional role of circ_RPPH1 in vivo was investigated by xenograft tumor reporter assay. Results: Upregulation of circ_RPPH1 and ARHGAP1, and downregulation of miR-542-3p were detected in BC tissues and cells. circ_RPPH1 knockdown or miR-542-3p introduction inhibited BC cell proliferation and metastasis, while promoted apoptosis in vitro. circ_RPPH1 sponged miR-542-3p to upregulate ARHGAP1 expression, thereby affecting BC progression. Moreover, depletion of circ_RPPH1 suppressed tumor growth in vivo. Conclusions: circ_RPPH1 contributed to BC tumorigenesis by sponging miR-542-3p and upregulating ARHGAP1, affording a novel mechanistic pathway in BC development.

Identification of Circular RNA-Based Immunomodulatory Networks in Colorectal Cancer.

BACKGROUND: Circular RNAs (circRNAs) have been recently proposed as hub molecules in various diseases, especially in tumours. We found that circRNAs derived from ribonuclease P RNA component H1 (RPPH1) were highly expressed in colorectal cancer (CRC) samples from Gene Expression Omnibus (GEO) datasets. OBJECTIVE: We sought to identify new circRNAs derived from RPPH1 and investigate their regulation of the competing endogenous RNA (ceRNA) and RNA binding protein (RBP) networks of CRC immune infiltration. METHODS: The circRNA expression profiles miRNA and mRNA data were extracted from the GEO and The Cancer Genome Atlas (TCGA) datasets, respectively. The differentially expressed (DE) RNAs were identified using R software and online server tools, and the circRNA-miRNA-mRNA and circRNA-protein networks were constructed using Cytoscape. The relationship between targeted genes and immune infiltration was identified using the GEPIA2 and TIMER2 online server tools. RESULTS: A ceRNA network, including eight circRNAs, five miRNAs, and six mRNAs, was revealed. Moreover, a circRNA-protein network, including eight circRNAs and 49 proteins, was established. The targeted genes, ENOX1, NCAM1, SAMD4A, and ZC3H10, are closely related to CRC tumour-infiltrating macrophages. CONCLUSIONS: We analysed the characteristics of circRNA from RPPH1 as competing for endogenous RNA binding miRNA or protein in CRC macrophage infiltration. The results point towards the development of a new diagnostic and therapeutic paradigm for CRC.

Mediation of circ_RPPH1 on miR-146b-3p/E2F2 pathway to hinder the growth and metastasis of breast carcinoma cells.

BACKGROUND: Nova Circular RNA (circRNA) of non-coding RNA has gradually become an important regulatory factor, and it has made people attach great concern over the occurrence and development of many diseases, particularly carcinomas. circ_RPPH1 is a newly discovered circRNA. Gene Expression Omnibus (GEO) analysis showed that there are high contents of circ_RPPH1 in breast cancer (BC), but the mechanism of circRNA in BC remains unclear. METHODS: Real-time quantitative PCR (qRT-PCR) was applied to test the role of circ_RPPH1 in BC patients, and functional experiments were applied to test the role of circ_RPPH1 on BC tumor. Fluorescence in situ hybridization, double luciferase reporter gene analysis, RNA pull-down and RNA immunoprecipitation experiments were performed to explore the correlation of circ_RPPH1 with miR-146b-3p/E2F2 in BC. RESULTS: circ_RPPH1 was evidently enhanced in BC, and its content was related to the clinical stage and pathological grade. circ_RPPH1 can accelerate the proliferation, migration and invasion, and promote tumorigenesis and metastasis. Mechanism exploration indicated that circ_RPPH1 acted as ceRNA (competing endogenous RNA) of miR-146b-3p, so as to reduce the inhibitory role of miR-146b-3p on its target E2F2. CONCLUSION: Circ_RPPH1/miR-146b-3p/E2F2 axis can promote the progression of BC, and it might be a latent therapeutic target for clinical BC.

Value of long non-coding RNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy.

BACKGROUND: Esophageal cancer is a common digestive tract tumor that is generally treated with radiotherapy. Poor responses to radiotherapy in most patients generally result in local radiotherapy failure, so it is essential to find new radiosensitizers that can enhance the response of cancer cells to radiotherapy and improve the survival of esophageal cancer patients with radiation resistance. The long non-coding RNA (lncRNA) Rpph1 is highly expressed in human gastric cancer tissues, and represses breast cancer cell proliferation and tumorigenesis. However, the expression of lncRNA Rpph1 in esophageal cancer and its relationship with radio-sensitivity has not been studied. AIM: To explore the value of lncRNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy. METHODS: Eighty-three patients with esophageal cancer admitted to Qilu Hospital of Shandong University and 90 healthy participants who received physical examinations were collected as research participants. The expression of Rpph1 was determined by qRT-PCR. siRNA-NC and siRNA-Rpph1 were transfected into esophageal cancer cell lines, and cells without transfection were designated as the blank control group. Cell survival was tested by colony formation assays, and the levels of proteins related to apoptosis and epithelial-mesenchymal transitions were determined by Western blot assays. Cell proliferation was assessed by MTT assays, cell apoptosis by flow cytometry, and cell migration by wound-healing assays. Changes in cell cycle distribution were monitored. RESULTS: Rpph1 was highly expressed in esophageal carcinoma, making it a promising marker for the diagnosis of esophageal cancer. Rpph1 could also be used to distinguish different short-term responses, T stages, N stages, and clinical stages of esophageal cancer patients. The results of 3-year overall survival favored patients with lower Rpph1 expression over patients with higher Rpph1 expression (P < 0.05). In vitro and in vivo experiments showed that silencing Rpph1 expression led to higher sensitivity of esophageal cancer cells to radiotherapy, stronger apoptosis in esophageal cancer cells induced by radiotherapy, higher expression of Bax and caspase-3, and lower expression of Bcl-2 (Bax, caspase-3, and Bcl-2 are apoptosis-related proteins). Additionally, silencing Rpph1 attenuated radiation-induced G2/M phase arrest, and significantly inhibited the expression of proteins involved in cell proliferation, migration, and epithelial-mesenchymal transition regulation in esophageal cancer cells. CONCLUSION: Rpph1 is highly expressed in esophageal cancer. Silencing Rpph1 expression can promote cell apoptosis, inhibit cell proliferation and migration, and increase radio-sensitivity.

Long non-coding RNA RPPH1 promotes the proliferation, invasion and migration of human acute myeloid leukemia cells through down-regulating miR-330-5p expression.

Multiple studies have revealed that the long non-coding RNA RPPH1 (Ribonuclease P RNA Component H1) is involved in disease progression of solid tumors and neurodegenerative diseases. We aimed to explore the functions of RPPH1 in the pathogenesis of acute myeloid leukemia (AML) and the underlying molecular mechanisms. The expression of RPPH1 was examined in blood samples of AML patients and human AML cell lines including THP-1 and HL-60. The microRNAs (miRNAs) targets of RPPH1 were predicted with online tools and validated with the dual luciferase reporter assay. The malignant behaviors of AML cells with lentivirus medicated knockdown of RPPH1 and/or administration of miR-330-5p inhibitor were assessed. Cell proliferation was determined by the CCK-8 and EdU incorporation methods, and cell invasion and migration were assayed with transwell experiments. The effects of RPPH1 knockdown on in vivo tumor growth were evaluated in nude mice with xenografted THP-1 cells. RPPH1 was expressed in the AML tissues and cell lines and its high expression predicted worse overall survival in AML patients. miR-330-5p was validated to be a direct target of RPPH1. Knockdown of RPPH1 suppressed the proliferation, invasion and migration ability of human AML cells, which was partially reversed by additional administration with miR-330-5p inhibitor. RPPH1 knockdown significantly inhibited the growth of xenografted THP-1 tumor in nude mice. Our work highlights the contributions of RPPH1 in promoting AML progression through targeting miR-330-5p, and suggests that the RPPH1/miR-330-5p axis is a potential target for AML treatments.

RppH can faithfully replace TAP to allow cloning of 5'-triphosphate carrying small RNAs.

RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. •We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.