RNA interference (RNAi), a highly conserved evolutionary process of post-transcriptional gene silencing, can be triggered by small interfering RNAs (siRNAs) that mediate sequence-specific mRNA degradation. The article summarized some aspects of the mechanism of RNAi, siRNA design and delivery of siRNAs to mammalian somatic cells. And some hurdles in practice were also discussed.
Animals ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Animals ; Humans ; MicroRNAs ; genetics ; Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Neoplasm ; genetics ; RNA, Small Interfering ; genetics

The small RNAs include siRNA and miRNA. SiRNA is the splicing product of exogenous dsRNA by which to keep genome stability, while miRNA are processed from endogenous genome and work as a post-transcriptional regulator for gene expression. The small RNAs act in two ways: mediate degradation of of target RNA and inhibit translation of protein. The former requires the accurate complementation between small RNA and target RNA, while the latter requires only partial complementation. Which mechanism used depends on complementation degree, but not their origins. The RNAi as a technique for down-regulating target gene expression has been widely used in functional genomic studies and hematologic studies, especially for translocation-related fusion gene, apoptosis-related gene and MDR gene in leukemia. The results show that RNAi technique not only is the powerful tool for study mechanism but also has therapeutic potentials in clinic. Some studies reveal that changes of miRNA expression exist in many hematological malignancies and relate to known oncogenes, which indicates the miRNA is involved in pathogenesis of these diseases. This article reviews the discovery and effect of RNAi and small RNAs, as well as the similarities and differences between siRNA and miRNA, and focuses on the research of small RNAs in hematological malignancies.
Hematologic Neoplasms ; genetics ; Humans ; MicroRNAs ; genetics ; RNA ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVE: To summarize the regulatory effect of non-coding RNA (ncRNA) on type H vessels angiogenesis of bone. METHODS: Recent domestic and foreign related literature about the regulation of ncRNA in type H vessels angiogenesis was widely reviewed and summarized. RESULTS: Type H vessels is a special subtype of bone vessels with the ability to couple bone formation. At present, the research on ncRNA regulating type H vessels angiogenesis in bone diseases mainly focuses on microRNA, long ncRNA, and small interfering RNA, which can affect the expressions of hypoxia inducible factor 1α, platelet derived growth factor BB, slit guidance ligand 3, and other factors through their own unique ways of action, thus regulating type H vessels angiogenesis and participating in the occurrence and development of bone diseases. CONCLUSION At present, the mechanism of ncRNA regulating bone type H vessels angiogenesis has been preliminarily explored. With the deepening of research, ncRNA is expected to be a new target for the diagnosis and treatment of vascular related bone diseases.
Humans ; RNA, Untranslated/genetics* ; RNA, Long Noncoding ; Bone Diseases/genetics* ; MicroRNAs/genetics* ; RNA, Small Interfering

OBJECTIVETo construct a lentiviral RNA interference system targeting heparanase (HPSE) based on miR30 and to test its silencing effect.

METHODSThree heparanase-shRNA structures were designed based miR30. The targeting fragments were obtained by PCR, then inserted into the vector LV PP-GFP to construct the recombinant lentiviral vector LV PP-GFP/miR-HPSE-shRNA, which was identified by PCR and sequencing. The 293T cells were co-transfect with LV PP-GFP/miR-HPSE-shRNA, pHelper 1.0 vector and pHelper 2.0 vector to produce lentiviruses, with which A375 cells were infected. Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein.

RESULTSThe lentiviral miR30-based RNAi vector targeting heparanase was constructed and confirmed by PCR and sequencing. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of both heparanase mRNA and protein in infected A375 cells were decreased significantly than those in control group.

CONCLUSIONThe lentiviral miR30-based RNAi vector targeting heparanase was been constructed successfully, which can be used for further study on RNAi-mediated oncolytic viruses.

Genetic Vectors ; Glucuronidase ; genetics ; Lentivirus ; genetics ; MicroRNAs ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P < 0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P < 0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P < 0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P < 0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P < 0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P < 0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.
Bees/genetics* ; Animals ; Larva/metabolism* ; RNA, Circular/genetics* ; RNA, Small Interfering/genetics* ; MicroRNAs/genetics*

RNA interference (RNAi) is one of the important mechanisms to regulate gene expression in eukaryotes. One of the original functions of RNAi is to facilitate the antiviral strategy of host. Early studies reveal that invertebrates can use RNAi to resist viruses. However, if this mechanism exists in mammals is still controversial. The latest studies confirm that mammals do have the RNAi-based immunity, and researchers believe that RNAi-based antiviral immunity is a brand-new immunological mechanism that was neglected in the past. It is worthy to note that virus can also use RNAi to enhance its infectivity and immune escape in host cells. This review introduces the research history of RNAi-based antiviral immunity in animals and summarizes the main findings in this field. Last but not least, we indicate a series of unresolved questions about RNAi-based antiviral immunity, and explore the relationship between RNAi-based antiviral immunity and other innate immunological pathways. The virus-mediated RNAi pathway in animal is not only an interesting basic biology question, but also has important guiding roles in the development of antiviral drugs.
Animals ; Antiviral Agents ; Immunity, Innate/genetics* ; Mammals ; RNA Interference ; RNA, Small Interfering/genetics* ; RNA, Viral

OBJECTIVETo construct and identification of a lentiviral vector for RNA interference (RNAi) targeting STUB1 gene.

METHODSA pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting STUB1 gene was designed, synthesized and inserted into linearized pMagic 4.0 vector. The recombinant plasmid was identified by double restriction digestion with Age I/EcoR I and DNA sequencing.

RESULTPCR and DNA sequencing showed that the shRNA sequence was successfully inserted into pMagic 4.0 vector. The pMagic 4.0 vector was successfully packaged into lentivirus particles.

CONCLUSIONA lentiviral shRNA expression vector and particles targeting STUB1 gene has been successfully constructed for the further study of the STUB1 gene.

Gene Targeting ; Genetic Vectors ; Lentivirus ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Ubiquitin-Protein Ligases ; genetics

OBJECTIVETo construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.

METHODSA plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.

RESULTSCotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.

CONCLUSIONSThe study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Animals ; Fibrinogen ; biosynthesis ; genetics ; Gene Expression ; Mice ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

Eukaryotes produce various types of 19-30 nt small RNAs, which act as guides to the regulation of gene expressions, such as mRNA degradation and translational repression. The Argonaute family members related to small RNA functions fall into 2 subfamilies. One is the AGO subfamily, whose 4 members distribute widely, confirmedly bind to miRNAs and siRNAs and inhibit the expression of target mRNAs through a pathway like RNA interference. The other is the PIWI subfamily, including PIWI, Aubgine (AUB) and AGO3, exclusively expressed in the testis. Recently, four research groups have isolated a new class of small RNAs from the mammalian testis, which interacts with the PIWI subfamily, hence named piwi-interfering RNAs (piRNAs), and is suggestive of an important role in spermatogenesis.
Animals ; Male ; MicroRNAs ; classification ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Spermatogenesis ; genetics ; Testis ; metabolism