RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

Modulation of miR-145-5p and miR-146b-5p levels is linked to reduced parasite load in H9C2 Trypanosoma cruzi infected cardiomyoblasts.

In the heart tissue of acutely Trypanosoma cruzi-infected mice miR-145-5p and miR-146b-5p are, respectively, downregulated and upregulated. Here, we used the H9C2 rat cardiomyoblast cell line infected with the Colombian T. cruzi strain to investigate the parasite-host cell interplay, focusing on the regulation of miR-145-5p and miR-146b-5p expression. Next, we explored the effects of interventions with the trypanosomicidal drug Benznidazole (Bz) alone or combined with Pentoxifylline (PTX), a methylxanthine derivative shown to modulate immunological and cardiac abnormalities in a model of chronic chagasic cardiomyopathy, on parasite load and expression of miR-145-5p and miR-146b-5p. The infection of H9C2 cells with trypomastigote forms allowed parasite cycle with intracellular forms multiplication and trypomastigote release. After 48 and 144 h of infection, upregulation of miR-145-5p (24 h: 2.38 ± 0.26; 48 h: 3.15 ± 0.9-fold change) and miR-146b-5b (24 h: 2.60 ± 0.46; 48 h: 2.97 ± 0.23-fold change) was detected. The peak of both miRNA levels paralleled with release of trypomastigote forms. Addition of 3 µM and 10 µM of Bz 48 h after infection reduced parasite load but did not interfere with miR-145-5p and miR-146b-5p levels. Addition of PTX did not interfere with Bz-induced parasite control efficacy. Conversely, combined Bz + PTX treatment decreased the levels of both microRNAs, resembling the expression levels detected in non-infected H9C2 cells. Moreover, the use of miR-145-5p and miR-146b-5p mimic/inhibitor systems before infection of H9C2 cells decreased parasite load, 72 h postinfection. When H9C2 cells were treated with miR-145-5p and miR-146b-5p mimic/inhibitor 48 h after infection, all the used systems, except the miR-146b-5p inhibitor, reduced parasite load. Altogether, our data indicate that these microRNAs putatively control signaling pathways crucial for parasite-host cell interaction. Thus, miR-145-5p and miR-146b-5p deserve to be further investigated as biomarkers of parasite control and tools to identify therapeutic adjuvants to etiological treatment in Chagas disease.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Micro-RNA-338-3p Promotes the Development of Atherosclerosis by Targeting Desmin and Promoting Proliferation.

Atherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.

MicroRNA miR-29a Inhibits Colon Cancer Progression by Downregulating B7-H3 Expression: Potential Molecular Targets for Colon Cancer Therapy.

MiR-29a belongs to one of the subtypes of miRNAs known as non-coding single-stranded RNAs and is preferentially expressed in normal tissues. B7-H3, a member of the B7/CD28 immunoglobulin superfamily, was shown to be overexpressed in several solid malignant tumors, including colon cancer. In addition, it is associated with tumor progression and poor prognosis. We used immunohistochemical and Western blotting to assess B7-H3 protein expression levels in colon cancer and adjacent normal tissues and then compared their relationships with clinicopathological factors. Quantitative real-time reverse-transcription PCR was used to assess B7-H3 and miRNA-29a mRNA expression levels, and then their relationship and clinical significance were evaluated. In addition, colon cancer Caco-2 cells, which constitutively overexpress B7-H3, were transfected with lentivirus particles for miR-29a upregulation. Invasion and migration assays were carried out in vitro along with the establishment of a subcutaneous xenograft model in vivo to determine the role of miRNA-29a in colon cancer progression. The B7-H3 protein showed elevated expression in colon carcinoma and was relevant to TNM staging, lymph node metastasis, and reduced survival. Meanwhile, miR-29a was preferentially expressed in normal colon tissues, while B7-H3 transcript levels had no marked differences between tumor and normal tissue specimens. In vitro, miR-29a upregulation resulted in reduced B7-H3 expression. Furthermore, miR-29a upregulation reduced the invasive and migratory abilities of colon carcinoma cells. In animal models, upregulation of miR-29a slowed down the growth of subcutaneous xenotransplanted tumors and resulted in prolonged survival time. MiR-29a downregulates B7-H3 expression and accordingly inhibits colon cancer progression, invasion, and migration, indicating miR-29a and B7-H3 might represent novel molecular targets for advanced immunotherapy in colon cancer.

Argonaute binding within human nuclear RNA and its impact on alternative splicing.

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

Knockdown of HCG18 Inhibits Cell Viability, Migration and Invasion in Pediatric Osteosarcoma by Targeting miR-188-5p/FOXC1 Axis.

Understanding the underlying mechanisms of pediatric osteosarcoma (OS) migration and invasion is important for prognosis and treatment. We tried to measure the expression of long non-coding RNA HLA complex group 18 (HCG18) in OS and reveal its function in the malignant behaviors of OS cells. This study detected the expression of HCG18, miR-188-5p and forkhead box C1 (FOXC1) in OS tissues and cell lines by quantitative real-time PCR (qRT-PCR). The relevance between miR-188-5p and HCG18 or FOXC1 was affirmed by dual-luciferase reporter (DLR) assay. Cell viability was analyzed by MTT assay. Transwell assay was utilized to test cell invasion and migration. FOXC1 protein expression was detected by western blot. HCG18 expression was elevated in OS tissues, and enhanced HCG18 expression was related to metastasis. HCG18 silencing repressed the viability, migration and invasion of OS cells. Moreover, HCG18 interacted with miR-188-5p. MiR-188-5p up-regulation repressed cell viability, invasion and migration in OS cells. FOXC1, a known target of miR-188-5p, was negatively modulated by miR-188-5p. Furthermore, miR-188-5p inhibition or FOXC1 over-expression partially abolished the reduced of cell viability, invasion and migration mediated by HCG18 silencing in OS cell lines. This study revealed that HCG18 knockdown repressed the viability, invasion and migration of OS cells by targeting miR-188-5p and regulating FOXC1 expression. Thus, HCG18/ miR-188-5p/FOX may be a hopeful target for OS therapy.

Genetic predisposition in the 2'-5'A pathway in the development of type 1 diabetes: potential contribution to dysregulation of innate antiviral immunity.

AIMS/HYPOTHESIS: The incidence of type 1 diabetes is increasing more rapidly than can be explained by genetic drift. Viruses may play an important role in the disease, as they seem to activate the 2'-5'-linked oligoadenylate (2'-5'A) pathway of the innate antiviral immune system. Our aim was to investigate this possibility. METHODS: Innate antiviral immune pathways were searched for type 1 diabetes-associated polymorphisms using genome-wide association study data. SNPs within ±250kb flanking regions of the transcription start site of 64 genes were examined. These pathways were also investigated for type 1 diabetes-associated RNA expression profiles using laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection (DiViD) study and the network of Pancreatic Organ Donors (nPOD). RESULTS: We found 27 novel SNPs in genes nominally associated with type 1 diabetes. Three of those SNPs were located upstream of the 2'-5'A pathway, namely SNP rs4767000 (p = 1.03 × 10-9, OR 1.123), rs1034687 (p = 2.16 × 10-7, OR 0.869) and rs739744 (p = 1.03 × 10-9, OR 1.123). We also identified a large group of dysregulated islet genes in relation to type 1 diabetes, of which two were novel. The most aberrant genes were a group of IFN-stimulated genes. Of those, the following distinct pathways were targeted by the dysregulation (compared with the non-diabetic control group): OAS1 increased by 111% (p < 1.00 × 10-4, 95% CI -0.43, -0.15); MX1 increased by 142% (p < 1.00 × 10-4, 95% CI -0.52, -0.22); and ISG15 increased by 197% (p = 2.00 × 10-4, 95% CI -0.68, -0.18). CONCLUSIONS/INTERPRETATION: We identified a genetic predisposition in the 2'-5'A pathway that potentially contributes to dysregulation of the innate antiviral immune system in type 1 diabetes. This study describes a potential role for the 2'-5'A pathway and other components of the innate antiviral immune system in beta cell autoimmunity.

MicroRNA-139-5p inhibits inflammatory and oxidative stress responses of Salmonella-infected macrophages through modulating TRAF6.

Evidence indicates that macrophages play an important role in the immune system. Therefore, research involving inflammatory and oxidative stress responses in macrophages is of great significance. Many factors contribute to inflammation and oxidative stress, including Salmonella. We investigated the effect of the miR-139-5p/TRAF6 axis on the inflammatory and oxidative stress responses of Salmonella -infected macrophages. Our findings revealed that miR-139-5p decreased IL-1ß and TNF-α levels to inhibit Salmonella-induced inflammatory responses in the RAW264.7 macrophage cell line. Furthermore, miR-139-5p inhibited Salmonella-induced oxidative stress by strengthening SOD, CAT and GSH-PX activity, as well as lowering the malondialdehyde level in the RAW264.7 macrophages cell line. Subsequently, it was verified that TRAF6 was a downstream target of miR-139-5p in RAW264.7 cells. Rescue assays indicated that the over-expression of miR-139-5p inhibits the effects of TRAF6 on inflammatory and oxidative stress responses including Salmonella infection in RAW264.7 cells. To our knowledge, this study is the first to verify that miR-139-5p inhibits inflammatory and oxidative stress responses of Salmonella-infected macrophages through regulating TRAF6. This discovery may offer new insights on inflammatory and oxidative stress responses in macrophages.

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage.

Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA-binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2'-hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase-mediated general acid-base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small-molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme.

MicroRNA-520c-3p targeting of RelA/p65 suppresses atherosclerotic plaque formation.

Atherosclerosis is a chronic inflammatory disease, and it's the leading cause of death worldwide. Dysregulation of microRNAs (miRNAs) has been found to be associated with atherosclerosis. miR-520c-3p has been implicated in several types of cancer. However, little is known about the role of miR-520c-3p in atherosclerosis. In this study, we found that miR-520c-3p agomir decreased atherosclerotic plaque size, collagen content, the quantity of PCNA-positive cell and RelA/p65 expression of vascular smooth muscle cells (VSMCs) in the aortic valve of apoE-/- mice in vivo. The possible mechanisms of the protective effects of miR-520c-3p on atherosclerotic mice were then investigated in VSMCs. in vitro experiments showed that miR-520c-3p expressions were significantly reduced in human aortic vascular smooth muscle cell (HASMCs) treated with platelet-derived growth factor (PDGF-BB). miR-520c-3p mimics repress PDGF-BB-mediated the proliferation, migration and decrease in the percentage of cells in G2/M phase, which was associated with downregulation of RelA/p65. Mechanistically, miRNA pull-down, luciferase reporter and mRNA stability assays confirmed miR-520c-3p mimics was able to directly target 3'-UTR of RelA/p65 mRNA and decreased half-life of RelA/p65 mRNA in HASMCs. Overexpression of RelA/p65 reversed the inhibition of cell proliferation induced by miR-520c-3p mimics in HASMCs. In conclusion, our findings suggest that miR-520c-3p inhibits PDGF-BB-mediated the proliferation and migration of HASMCs by targeting RelA/p65, which may provide potential therapeutic strategies in atherosclerosis treatment.

MiR-149 attenuates the proliferation and migration of TGF-ß1-induced airway smooth muscle cells by targeting TRPM7 and affecting downstream MAPK signal pathway.

Asthma is considered as a general term for various chronic inflammatory diseases of the respiratory tract. Growing evidences have supported that microRNAs were involved in mediating cell proliferation, migration, and other cellular functions. MiR-149 has been found to take part in the development of various cancers. However, whether miR-149 participated in the proliferation and migration of transforming growth factor beta 1 (TGF-ß1)-induced airway smooth muscle cells was still unknown. In this study, the expression level of miR-149 in human airway smooth muscle cells (ASMCs) was decreased after TGF-ß1 treatment in vitro. Additionally, the over-expression of miR-149 obviously suppressed proliferation and migration in human ASMCs. Besides, we found that overexpression of miR-149 could inhibit the expression of transient receptor potential melastatin 7 (TRPM7) both in protein and gene levels. Furthermore, we demonstrated that miR-149 could inhibit the cell proliferation and migration in human ASMCs by targeting TRPM7 through modulating mitogen-activated protein kinases (MAPKs) signaling pathway. Taken together, we strongly supported that miR-149 might be a key inhibitor of asthma by targeting TRMP7. Therefore, our finding suggests a promising biomarker for the development of further targeted therapies for asthma.

miR-590-3p Alleviates diabetic peripheral neuropathic pain by targeting RAP1A and suppressing infiltration by the T cells.

BACKGROUND: MicroRNAs play a crucial role in diabetic peripheral neuropathic pain (DPNP). miR-590-3p is a novel miRNA and involved in multiple diseases. However, the pathological mechanism of miR-590-3p in DPNP needs to be elucidated. MATERIALS AND METHODS: The db/db mice and db/m mice were selected to mimic diabetes and control, respectively, for in vivo studies. The miR-590-3p agomir was injected into db/db mice and pain-related behavioral tests were performed. The interaction of miR-590-3p with target gene was confirmed by dual-luciferase reporter assay. The expression of target gene was determined by qRT-PCR and western blot assay. The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: miR-590-3p was down-regulated in diabetic peripheral neuropathy mice. More importantly, miR-590-3p agomir alleviated pain-related behavior, reduced TNF-α, IL-1ß and IL-6 concentrations, and inhibited neural infiltration by immune cells in db/db mice. Interestingly, RAP1A was predicted to be the target of miR-590-3p by Targetscan, and was actually regulated by miR-590-3p. Finally, the rescue experiments proved that overexpression of RAP1A partially abrogated the suppressive impact of miR-590-3p on T cells proliferation and migration. CONCLUSION: miR-590-3p ameliorates DPNP via targeting RAP1A and inhibiting T cells infiltration, indicating that exogenous miR-590-3p may be a potential candidate for clinical treatment of DPNP.

Exosomal lncRNA HEIH promotes cisplatin resistance in tongue squamous cell carcinoma via targeting miR-3619-5p/HDGF axis.

BACKGROUND: Accumulating evidence has suggested that long noncoding RNAs (lncRNAs) are involved in the progression of types of human cancers. It has been known that exosomes can mediate cell-cell crosstalk by transferring lncRNAs in tumor progression. This study aimed to investigate the role of exosomal lncRNA HEIH on cisplatin (DDP) resistance in tongue squamous cell carcinoma (TSCC). METHODS: The expression of HEIH in human oral keratinocytes cell line (HOK), DDP-sensitive TSCC cell line (SCC4/S) and DDP-resistant TSCC cell line (SCC4/DDP) was measured. SCC4/S and SCC4/DDP cells were transfected with sh-HEIH to examine TSCC cell proliferation and apoptosis. The DDP-resistant exosomes were extracted and identified. The expression of miR­3619-5p and TDGF in DDP-sensitive recipient cells was determined. The binding capacity between HEIH and miR­3619-5p, along with miR­3619-5p and TDGF was verified. RESULTS: HEIH expression was significantly upregulated in SCC4/DDP cells. Downregulation of HEIH inhibited DDP resistance and cell proliferation and promoted cell apoptosis. HEIH acted as a competing endogenous RNA (ceRNA) for miR­3619-5p to upregulate HDGF expression. Exosomal HEIH promoted cell proliferation and drug resistance and inhibited cell apoptosis by sponging miR­3169-5p and upregulating HDGF. CONCLUSION: Exosomal HEIH acted as a ceRNA for miR­3619-5p to upregulate HDGF, thereby promoting DDP resistance in TSCC cells.

Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture.

BACKGROUND: We recently identified 39 human microRNAs, which effectively suppress hepatitis B virus (HBV) replication in hepatocytes. Chronic HBV infection often results in active, hepatitis-related liver fibrosis; hence, we assessed whether any of these microRNAs have anti-fibrotic potential and predicted that miR-6133-5p may target several fibrosis-related genes. METHODS: The hepatic stellate cell line LX-2 was transfected with an miR-6133-5p mimic and subsequently treated with Transforming growth factor (TGF)-ß. The mRNA and protein products of the COL1A1 gene, encoding collagen, and the ACTA2 gene, an activation marker of hepatic stellate cells, were quantified. RESULTS: The expression of COL1A1 and ACTA2 was markedly reduced in LX-2 cells treated with miR-6133-5p. Interestingly, phosphorylation of c-Jun N-terminal kinase (JNK) was also significantly decreased by miR-6133-5p treatment. The expression of several predicted target genes of miR-6133-5p, including TGFBR2 (which encodes Transforming Growth Factor Beta Receptor 2) and FGFR1 (which encodes Fibroblast Growth Factor Receptor 1), was also reduced in miR-6133-5p-treated cells. The knockdown of TGFBR2 by the corresponding small interfering RNA greatly suppressed the expression of COL1A1 and ACTA2. Treatment with the JNK inhibitor, SP600125, also suppressed COL1A1 and ACTA2 expression, indicating that TGFBR2 and JNK mediate the anti-fibrotic effect of miR-6133-5p. The downregulation of FGFR1 may result in a decrease of phosphorylated Akt, ERK (extracellular signal-regulated kinase), and JNK. CONCLUSION: miR-6133-5p has a strong anti-fibrotic effect, mediated by inactivation of TGFBR2, Akt, and JNK.

Dysregulation of LncRNA ANRIL mediated by miR-411-3p inhibits the malignant proliferation and tumor stem cell like property of multiple myeloma via hypoxia-inducible factor 1α.

Long non-coding RNA (lncRNA) ANRIL has been reported to be closely related to the relapse of multiple myeloma patients. However, the functional role and underlying mechanism of lncRNA ANRIL in multiple myeloma are not known. This study aims to investigate the biological function of lncRNA ANRIL in multiple myeloma. In this study, compared with normal tissues from healthy donors, lncRNA ANRIL and HIF-1α expressions were up-regulated in tumor tissues from multiple myeloma patients. miR-411-3p expression was down-regulated in tumor tissues from multiple myeloma patients. Besides, lncRNA ANRIL can interact with miR-411-3p. HIF-1α was confirmed to be a target of miR-411-3p. Correlation analysis showed that lncRNA ANRIL expression was negatively correlated with miR-411-3p expression. HIF-1α expression was negatively correlated with miR-411-3p expression. Further transfection experiments showed that knockdown of ANRIL or overexpression of miR-411-3p significantly inhibited cell proliferation, tumor formation ability and tumor stem cell like property, promoted cell apoptosis in vitro. Finally, miR-411-3p mimic reduced tumor volume, improved survival rate, suppressed malignant proliferation and tumor stem cell like property in U266 xenograft model. Our results demonstrate that lncRNA ANRIL mediated by miR-411-3p promotes the malignant proliferation and tumor stem cell like property of multiple myeloma through regulating HIF-1α.

MicroRNA-302 represses epithelial-mesenchymal transition and cisplatin resistance by regulating ATAD2 in ovarian carcinoma.

Epithelial-mesenchymal transition (EMT) is an important contributor to drug resistance in ovarian cancer. The aims of this study were to explore the potential role of the miR-302 cluster in modulating EMT and cisplatin resistance in ovarian cancer. We used qRT-PCR and western blotting to show that miR-302 expression was lower in chemoresistant than in chemosensitive cells, and miR-302 was upregulated in chemosensitive, but not chemoresistant ovarian cancer cells in response to cisplatin treatment. We identified ATAD2 as a target of miR-302 and showed that ectopic expression of miR-302 increased cisplatin sensitivity and inhibited EMT and the invasiveness of cisplatin-resistant cells in vitro by targeting ATAD2. Knockdown of ATAD2 restored cisplatin sensitivity and reversed EMT/metastasis in cisplatin-resistant cells, as shown by western blotting and invasion/migration assays. The effect of miR-302 overexpression on EMT and invasiveness was mediated by the modulation of ß-catenin nuclear expression. Immunofluorescence analysis showed that ATAD2 overexpression reversed the miR-302-induced downregulation of nuclear ß-catenin in cisplatin resistant cells. A xenograft tumor model was used to show that miR-302 increases the antitumor effect of cisplatin in vivo. Taken together, these results identify a potential regulatory axis involving miR-302 and ATAD2 with a role in chemoresistance, indicating that activation of miR-302 or inactivation of ATAD2 could serve as a novel approach to reverse cisplatin resistance in ovarian cancer.

miR-29a-3p suppresses hepatic fibrosis pathogenesis by modulating hepatic stellate cell proliferation via targeting PIK3R3 gene expression.

OBJECTIVE: Hepatic stellate cells (HSC) activation and proliferation mediated the pathogenic development of hepatic fibrosis (HF). However, the underlying mechanisms remain poorly understood. In this study, we aimed to investigate the miR-29a-3p and its effects on PIK3R3 expression in HF pathogenesis. METHODS: LX-2 cells treated with TGF-ß1 was used as the in vitro HF model. The expression of microRNAs and proteins in LX-2 cells were detected by quantitative RT-PCR and western blotting. Then, miR-29a-3p expression in LX-2 cells were altered via transfection with specific mimics or inhibitors, followed by cell proliferation measured through CCK-8, Edu staining and colony formation. The dual luciferase reporter assay was done to assess binding of miR-29a-3p with PIK3R3 gene sequences. Moreover, PIK3R3 gene overexpression in LX-2 cell was realized through transfection with recombinant pcDNA3.0-PIK3R3 plasmids. RESULTS: Successful establishment of cellular HF model was validated through the increased Col-I and a-SMA expression in TGF-ß1-treated LX-2 cells shown by qRT-PCR and Western blot. In such model, miR-29a-3p expression in LX-2 cells showed the greatest decrease among four candidate microRNAs in response to TGF-ß1 treatment. Also, miR-29a-3p directly binds with the 3' UTR region of the PIK3R3 gene to suppress its expression in LX-2 cells. Furthermore, PIK3R3 gene overexpression effectively abrogated the changes of LX-2 cell proliferation, AKT phosphorylation and Col-I and a-SMA expression caused by miR-29a-3p mimics. CONCLUSION: MiR-29a-3p regulates hepatic stellate cell proliferation and hepatic fibrosis pathogenesis by targeting PIK3R3 expression and modulating the PI-3K/AKT signaling.

Inhibiting microRNA-301b suppresses cell growth and targets RNF38 in cervical carcinoma.

It has been reported microRNA-301b (miR-301b) was involved in the tumorigenesis of some cancers, but it has not been investigated in cervical carcinoma yet. In this study, miR-301b was found significantly upregulated in cervical carcinoma, and patients with high miR-301b expression had a shorter overall survival. When miR-301b was knocked down in cervical carcinoma cells, the cell growth could be significantly abolished. Our further studies showed miR-301b targeted RNF38, and inhibited its expression in cervical carcinoma cells. Moreover, RNF38 was found downregulated in cervical carcinoma, and miR-301b expression in cervical tissues was found negatively correlated with RNF38 expression. In addition, overexpression of RNF38 significantly inhibited cervical carcinoma cell growth, but overexpression of miR-301b suppressed RNF38-induced cell growth inhibition in cervical carcinoma. Collectively, this study suggested miR-301b could be a novel target for cervical carcinoma treatment.