Mechanism for regulation of trophoblast invasion through piR-3127964 via PIWIL-3/sialophorin pathway / 中华围产医学杂志

Objective:To investigate the molecular mechanism for regulation of trophoblast invasion by piR-3127964, which is differentially expressed in placental tissues of preeclamptic and healthy pregnant women.Methods:Placenta samples of healthy (control group, n=12) and preeclamptic pregnant (PE group, n=10) women who delivered by caesarean section and chorionic villi specimens of patients undergoing artificial abortion were collected in the Department of Obstetrics of the First Affiliated Hospital of Chongqing Medical University during November 2020 to August 2021. Total RNA was extracted from placenta samples and sequenced and the expression of piR-3127964 in different tissues was determined by real-time quantitative-polymerase chain reaction (qRT-PCR). The expressions of PIWI proteins including PIWIL-1, PIWIL-2 and PIWIL-3 in different tissues were detected by Western blot. The expressions of two candidate targets, guanine nucleotide-binding protein-like 3-like (GNL3L) mRNA and sialophorin (SPN) mRNA were evaluated by qRT-PCR after exogenous treating HTR-8/SVneo cells with mimics, inhibitor or negative control of piR-3127964, respectively. qRT-PCR was also used to detect the relative expression of GNL3L and SPN at mRNA level in placentas of all women. The interactions between GNL3L/SPN and piR-3127964 were analyzed by double luciferase reporter gene detection. The localization of piR-3127964 and SPN in chorionic villi was detected by fluorescence in situ hybridization and immunofluorescence. Transwell assay was performed to analyze the influence of piR-3127964 on the invasion of HTR-8/SVneo cells and the possible mechanism. Independent sample t-test, analysis of variance, and LSD post test were used for analysis Results:(1) Enrichment pathways of candidate targets predicted by differentially expressed piR-3127964 were associated with cell motility. There were statistically significant differences in piR-3127964 expression in villi, healthy and preeclamptic placentas (2.950±0.853 vs 1.036±0.303 vs 0.254±0.155, F=27.35, P<0.05), and piR-3127964 was predominantly expressed in extravillous cytotrophoblasts (EVTs). (2) The expression of PIWIL-3 protein in placentas of preeclamptic patients was significantly lower than that in healthy placentas and villi (0.810±0.400 vs 3.175±0.429 and 6.843±1.379, F=49.36, P<0.05). (3) Compared with the control group, exogenous piR-3127964 mimics (piR-mimics) and inhibitors (piR-inhibitor) significantly affected the expression of SPN mRNA (0.971±0.045 vs 0.732±0.010, F=6.50; 1.076±0.073 vs 1.293±0.092, F=7.58; both P<0.05), while the expression of GNL3L mRNA had no significant correlation with piR-3127964 level. (4) The luciferase activity of wild-type SPN (SPN-WT) plasmids was significantly affected by piR-mimics (1.010±0.049 vs 0.645±0.047, t=9.34, P<0.05) and piR-inhibitor (1.035±0.058 vs 1.397±0.015, t=－10.60, P<0.05). (5) SPN mRNA was significantly upregulated in placentas of preeclamptic patients than in healthy placentas (2.097±0.239 vs 1.305±0.290, t=－4.22, P<0.05), but no significant difference in the expression of GNL3L mRNA was observed. Immunofluorescence experiment showed that SPN was expressed in EVTs. (6) The invasive potential of HTR8/SVneo cells treated with piR-inhibitor was significantly inhibited, but this effect could be reversed by SPN knockdown (160.714±53.860 vs 371.667±103.061 and 344.333±120.267, F=9.76, both P<0.05). Conclusions:piR-3127964 expression is abnormally downregulated in placentas of preeclamptic patients, resulting in inhibition of trophoblasts invasion through upregulation of SPN expression, which may be related to the pathogenesis of preeclampsia.

Import of human miRNA-RISC complex into Plasmodium falciparum and regulation of the parasite gene expression

During its life cycle, the malarial parasite Plasmodium goes through different asexual stages in human blood, and asexualand sexual stage in mosquito. Expression of stage-specific proteins is important for successful completion of its life cycleand requires tight gene regulation. In case of Plasmodium, due to relative paucity of the transcription factors, it is postulatedthat post-transcriptional regulation plays an important role in stage-specific gene expression. Although miRNA-mediatedgene regulation has been well-established to function in post-transcriptional regulation in many eukaryotes, existence ofsuch a phenomenon or the presence of miRNA-associated factors in Plasmodium remains unclear. A number of miRNAsare shown to be imported into Plasmodium falciparum from erythrocytes and role of these miRNAs is not understood. Herewe show that human Argonaute 2 (hAgo2) a component of the miRISC complex is imported by P. falciparum. In theparasite hAgo2 exists as in a complex with specific human miRNAs like let-7a and miR15a which can potentially target thePlasmodium genes Rad54 and Lipid/sterol:H? symporter respectively. We show that hAgo2 associates with Rad54, Lipid/sterol:H? symporter and other P. falciparum transcripts. These results highlight the existence of a mechanism by whichmalarial parasite imports hAgo2-miRNA complex from the host cells to regulate its gene expression.

Downregulation of OsAGO17 by artificial microRNA causes pollen abortion resulting in the reduction of grain yield in rice

Background: Pollen development is an important reproductive process that directly affects pollen fertility and grain yield in rice. Argonaute (AGO) proteins, the core effectors of RNA-mediated silencing, play important roles in regulating plant growth and development. However, few AGO proteins in rice were reported to be involved in pollen development. In this study, artificial microRNA technology was used to assess the function of OsAGO17 in pollen development. Results: In this study, OsAGO17, a rice-specific gene, was specifically expressed in rice pollen grains, with the highest expression in uninucleate microspores. Downregulation of OsAGO17 by artificial microRNA technology based on the endogenous osa-miRNA319a precursor was successfully achieved. It is found that downregulation of OsAGO17 could significantly affect pollen fertility and cause pollen abortion, thus suggesting that OsAGO17 functions in rice pollen development. In addition, the downregulation of OsAGO17 mainly caused a low seed-setting rate, thereby resulting in the reduction of grain yield, whereas the downregulation of OsAGO17 did not significantly affect rice vegetative growth and other agricultural traits including number of florets per panicle, number of primary branch per panicle, and 100-grain weight. Furthermore, the result of subcellular localization analysis indicated that the OsAGO17 protein was localized to both the nucleus and the cytoplasm. Conclusion: These results represent the first report of the biological function for OsAGO17 in rice and indicate that OsAGO17 may possibly play crucial regulatory roles in rice pollen development. It helps us to better understand the mechanism of pollen development in rice.

Translational control in plant antiviral immunity

Abstract Due to the limited coding capacity of viral genomes, plant viruses depend extensively on the host cell machinery to support the viral life cycle and, thereby, interact with a large number of host proteins during infection. Within this context, as plant viruses do not harbor translation-required components, they have developed several strategies to subvert the host protein synthesis machinery to produce rapidly and efficiently the viral proteins. As a countermeasure against infection, plants have evolved defense mechanisms that impair viral infections. Among them, the host-mediated translational suppression has been characterized as an efficient mean to restrict infection. To specifically suppress translation of viral mRNAs, plants can deploy susceptible recessive resistance genes, which encode translation initiation factors from the eIF4E and eIF4G family and are required for viral mRNA translation and multiplication. Additionally, recent evidence has demonstrated that, alternatively to the cleavage of viral RNA targets, host cells can suppress viral protein translation to silence viral RNA. Finally, a novel strategy of plant antiviral defense based on suppression of host global translation, which is mediated by the transmembrane immune receptor NIK1 (nuclear shuttle protein (NSP)-Interacting Kinase1), is discussed in this review.

In Silico Study of miRNA Based Gene Regulation, Involved in Solid Cancer, by the Assistance of Argonaute Protein

Solid tumor is generally observed in tissues of epithelial or endothelial cells of lung, breast, prostate, pancreases, colorectal, stomach, and bladder, where several genes transcription is regulated by the microRNAs (miRNAs). Argonaute (AGO) protein is a family of protein which assists in miRNAs to bind with mRNAs of the target genes. Hence, study of the binding mechanism between AGO protein and miRNAs, and also with miRNAs-mRNAs duplex is crucial for understanding the RNA silencing mechanism. In the current work, 64 genes and 23 miRNAs have been selected from literatures, whose deregulation is well established in seven types of solid cancer like lung, breast, prostate, pancreases, colorectal, stomach, and bladder cancer. In silico study reveals, miRNAs namely, miR-106a, miR-21, and miR-29b-2 have a strong binding affinity towards PTEN, TGFBR2, and VEGFA genes, respectively, suggested as important factors in RNA silencing mechanism. Furthermore, interaction between AGO protein (PDB ID-3F73, chain A) with selected miRNAs and with miRNAs-mRNAs duplex were studied computationally to understand their binding at molecular level. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding miRNAs based gene silencing mechanism in solid cancer.

Elucidation of the Molecular Interaction between miRNAs and the HOXA9 Gene, Involved in Acute Myeloid Leukemia, by the Assistance of Argonaute Protein through a Computational Approach

Acute myeloid leukemia is a well characterized blood cancer in which the unnatural growth of immature white blood cell takes place, where several genes transcription is regulated by the micro RNAs (miRNAs). Argonaute (AGO) protein is a protein family that binds to the miRNAs and mRNA complex where a strong binding affinity is crucial for its RNA silencing function. By understanding pattern recognition between the miRNAs-mRNA complex and its binding affinity with AGO protein, one can decipher the regulation of a particular gene and develop suitable siRNA for the same in disease condition. In the current work, HOXA9 gene has been selected from literature, whose deregulation is well-established in acute myeloid leukemia. Four miRNAs (mir-145, mir-126, let-7a, and mir-196b) have been selected to target mRNA of HOXA9 (NCBI accession No. NM_152739.3). The binding interaction between mRNAs and mRNA of HOXA9 gene was studied computationally. From result, it was observed mir-145 has highest affinity for HOXA9 gene. Furthermore, the interaction between miRNAs-mRNA duplex of all chosen miRNAs are docked with AGO protein (PDB ID: 3F73, chain A) to study their interaction at molecular level through an in silico approach. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding of AGO-assisted miRNA based gene silencing mechanism in HOXA9 gene associated in acute myeloid leukemia computationally.

Emerging role of microRNAs in lipid metabolism

microRNAs (miRNAs or miRs) are small non-coding RNAs that are involved in post-transcriptional regulation of their target genes in a sequence-specific manner. Emerging evidence demonstrates that miRNAs are critical regulators of lipid synthesis, fatty acid oxidation and lipoprotein formation and secretion. Dysregulation of miRNAs disrupts gene regulatory network, leading to metabolic syndrome and its related diseases. In this review, we introduced epigenetic and transcriptional regulation of miRNAs expression. We emphasized on several representative miRNAs that are functionally involved into lipid metabolism, including miR-33/33(⁎), miR122, miR27a/b, miR378/378(⁎), miR-34a and miR-21. Understanding the function of miRNAs in lipid homeostasis may provide potential therapeutic strategies for fatty liver disease.

The Expression of AGO2 and DGCR8 in Idiopathic Sudden Sensorineural Hearing Loss

OBJECTIVES: The microRNAs have been implicated in the development and function of the inner ear, especially in contribution to hearing. However, the impact of idiopathic sudden sensorineural hearing loss (SSNHL) on expression of miRNA biogenesis-related components has not been established. To investigate the regulations of microRNA (miRNA) biogenesis-related components, argonaute 2 (AGO2) and DiGeorge syndrome critical region gene 8 (DGCR8) mRNA expression in SSNHL and to evaluate the value of clinical parameters on their expression. METHODS: Thirty-seven patients diagnosed with SSNHL and fifty-one healthy volunteers were included in this study. We measured mRNA expression levels of AGO2 and DGCR8 in whole blood cells but erythrocytes of patients with SSNHL and controls, using reverse transcription and real-time polymerase chain reaction analysis. RESULTS: The mRNA expression level of AGO2 is upregulated in SSNHL. The expression level of AGO2 was significantly correlated with that of DGCR8 in both patients with SSNHL and controls. Expression level of AGO2 in SSNHL was correlated with white blood cell counts. CONCLUSION: This study demonstrated for the first time that the AGO2 mRNA expression level was upregulated in SSNHL, suggesting its important role in pathobiology of SSNHL development.

siRNA-mediated DNA methylation and H3K9 dimethylation in plants

Heterochromatic siRNAs regulate transcriptional gene silencing by inducing DNA methylation and histone H3K9 dimethylation. Recent advances have revealed the distinct phases involved in siRNA mediated silencing pathway, although the precise functions of a number of factors remain undesignated, putative mechanisms for the connection between DNA and histone methylation have been investigated, and much effort has been invested to understand the biological functions of siRNA-mediated epigenetic modification. In this review, we summarize the mechanism of siRNA-mediated epigenetic modification, which involves the production of siRNA and the recruitments of DNA and histone methytransferases to the target sequences assisted by complementary pairing between 24-nt siRNAs and nascent scaffold RNAs, the roles of siRNA-mediated epigenetic modification in maintaining genome stability and regulating gene expression have been discussed, newly identified players of the siRNA mediated silencing pathway have also been introduced.

Argonaute-2-null embryonic stem cells are retarded in self-renewal and differentiation.

RNA interference (RNAi) pathways regulate self-renewal and differentiation of embryonic stem (ES) cells. Argonaute 2 (Ago2) is a vital component of RNA-induced silencing complex (RISC) and the only Ago protein with slicer activity. We generated Ago2-deficient ES cells by conditional gene targeting. Ago2-deficient ES cells are defective in the small-RNA-mediated gene silencing and are significantly compromised in biogenesis of mature microRNA. The self-renewal rate of Ago2-deficient ES cells is affected due to failure of silencing of Cdkn1a by EScell- specific microRNAs (miRNA) in the absence of Ago2. Interestingly, unlike Dicer- and Dgcr8-deficient ES cells, they differentiate to all three germ layers both in vivo and in vitro. However, early differentiation of Ago2-deficient ES cells is delayed by 2–4 days as indicated by persistence of higher levels of self-renewal/ pluripotency markers during differentiation. Further, appearance of morphological and differentiation markers is also delayed during the differentiation. In this study we show that Ago2 is essential for normal self-renewal and differentiation. Also, our data suggest that self-renewal and differentiation of ES cells are regulated by both siRNA and miRNA pathways.

Modeling and analysis of Schistosoma Argonaute protein molecular spatial conformation

<p><b>OBJECTIVE</b>To analyze the amino acid sequence composition, secondary structure, the spatial conformation of its domain and other characteristics of Argonaute protein.</p><p><b>METHODS</b>Bioinformatics tools and the internet server were used. Firstly, the amino acid sequence composition features of the Argonaute protein were analyzed, and the phylogenetic tree was constructed. Secondly, Argonaute protein's distribution of secondary structure and its physicochemical properties were predicted. Lastly, the protein functional expression form of the domain group was established through the Phyre-based analysis on the spatial conformation of Argonaute protein domains.</p><p><b>RESULTS</b>593 amino acids were encoded by Argonaute protein, the phylogenetic tree was constructed, and Argonaute protein's distribution of secondary structure and its physicochemical properties were obtained through analysis. In addition, the functional expression form which comprised the N-terminal PAZ domain and C-terminal Piwi domain for the Argonaute protein was obtained with Phyre.</p><p><b>CONCLUSIONS</b>The information relationship between the structure and function of the Argonaute protein can be initially established with bioinformatics tools and the internet server, and this provides the theoretical basis for further clarifying the function of Schistosoma Argonaute protein.</p>

Modeling and analysis of Schistosoma Argonaute protein molecular spatial conformation

Objective: To analyze the amino acid sequence composition, secondary structure, the spatial conformation of its domain and other characteristics of Argonaute protein. Methods:Bioinformatics tools and the internet server were used. Firstly, the amino acid sequence composition features of the Argonaute protein were analyzed, and the phylogenetic tree was constructed. Secondly, Argonaute protein’s distribution of secondary structure and its physicochemical properties were predicted. Lastly, the protein functional expression form of the domain group was established through the Phyre-based analysis on the spatial conformation of Argonaute protein domains. Results: 593 amino acids were encoded by Argonaute protein, the phylogenetic tree was constructed, and Argonaute protein’s distribution of secondary structure and its physicochemical properties were obtained through analysis. In addition, the functional expression form which comprised the N-terminal PAZ domain and C-terminal Piwi domain for the Argonaute protein was obtained with Phyre. Conclusions: The information relationship between the structure and function of the Argonaute protein can be initially established with bioinformatics tools and the internet server, and this provides the theoretical basis for further clarifying the function of Schistosoma Argonaute protein.

Generation and preliminary application of polyclonal antibodies against human Argonaute2 / 中国免疫学杂志

Objective:To generate rabbit polyclonal antibody against human Argonaute2 (Ago2) protein and to identify its functional characterization for determination of differential expression and cellular localization of Ago2 protein in various cell lines.Methods:DNAstar software was applied for searching the high antigenicity region of Ago2 gene sequence termed k-Ago2.Prokaryotic expressing plasmid was constructed and transformed to E.coli BL21 (DE3) to induce expression by IPTG.The fusion protein was injected into rabbits subcutaneously to produce polyclonal antibodies after purification by gel regaining.ELISA was operated to detect antibody titer.Western blot was used to identify the specificity and sensitivity of the antibodies and detect the differential expression of Ago2 protein in various cell lines.Meanwhile,immunofluorescence experiments were arranged to show cellular localization of Ago2 protein.Results:The prokaryotic expressing plasmid was constructed correctly.K-Ago2 protein was expressed and purified,and then rabbit polyclonal antibodies against Ago2 were generated after immunization with k-Ago2 protein.The titer detected by ELISA was 1∶19 000.Western blot results demonstrated the high specificity of the antibodies.Finally,we successfully observed the differential expression and cellular localization of Ago2 protein in various cell lines.Conclusion:The polyclonal antibody against Ago2 protein has been achieved successfully.It will be propitious for the intensive study of the RNAi mechanism and even profound clinical application.

Preparation,identification and tissue localization of PIWIL proteins / 基础医学与临床

Objective To prepare polyclonal antibodies against human PIWIL and to identify their property and tissue distribution of PIWIL.Methods PIWIL polypeptide was synthesized and conjugated to Keyhole limpet hemocyanin(KLH) as an immunogen.Then PIWIL-KLH conjugations were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification by affinity chromatography.PIWIL were then stained on the tissue chip to study their distribution.Results Rabbit antibodies against PIWIL were prepared after injection of PIWIL-KLH conjugation.These antibodies specially recognized PIWIL peptides.Expression of PIWIL was found in the cytoplasm of epithelia cells of varied normal tissues and tumor tissues.Conclusion The successful preparation of the polyclonal antibody against PIWIL will provide an efficient reagent for further study of its role in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.

PIWI and tumor / 医学研究生学报

The PIWI family members are defined as conserved PAZ and PIWI domains and play important roles in stem-cell self-renewal,spermatogenesis,RNA interference and translational regulation.Recent researches have showen that some PIWI are specifically expressed in tumors and associated with tumor development and growth.PIWI is likely to be a new significante index for tumor diagnose and prognosis,the feasibility of PIWI acting in tumor gene theropy could be in vestigated by studying the mechanism of PIWI expression and regulation.

Human argonaute3 protein:preparation and characterization of polyclonal antibody and the antigen distribution in tumor tissues by tissue array / 中国免疫学杂志

Objective:To prepare rabbit polyclonal antibody against human argonaute 3(AGO3) protein,to identify its properties and investigate the tissue distribution of AGO3 using tissue array.Methods:AGO3 peptide was synthesized using chemical method,and then conjugated to Keyhole limpet hemocyanin(KLH) as immunogen.The AGO3-KLH conjugate were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification using affinity chromatography.Then the distribution of AGO3 in tissues was examined through immuno-stainning by tissue array.Results:Rabbit polyclonal antibodies against AGO3 were raised after immunization with AGO3-KLH conjugates.The anti-serum titer after the last inoculation was up to 1∶20 000.The preparations of the antibody were confirmed to raised recognize AGO3 peptides specially by ELISA and Western blot.AGO3 protein was stained positively in the cytoplasm of tumor cells and epithelial cells in many normal tissues.Conclusion:The polyclonal antibody against AGO3 protein has been achieved successfully,and it provides an efficient tool for further studying the roles of AGO3 in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.