Exosomes derived from bone marrow mesenchymal stem cells promote proliferation and migration via upregulation yes-associated protein/transcriptional coactivator with PDZ binding motif expression in breast cancer cells.

Bone marrow mesenchymal stem cells (BM-MSCs), with the properties of self-renewal and pluripotency, can migrate to the tumor sites and exert complex effects on tumor progression and communications by releasing exosomes. However, to our knowledge, only a few studies have reported the effects of BM-MSCs exosomes on breast cancer cells development. Here, utilizing exosomes isolated from in vitro BM-MSCs, we systematically investigated this issue in a breast cancer cell line. In this study, we found that BM-MSCs exosomes are actively incorporated by breast cancer cell MDA-MB-231 cells and subsequently promote MDA-MB-231 cells proliferation and migration. Mechanistically, we further found Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) which are Hippo signaling components were involved in this promoting progress. Consistently, YAP and TAZ knockdown could significantly reverse breast cancer cells proliferation and migration improved by BM-MSCs exosomes. Taken together, our findings demonstrated a new mechanism through which BM-MSCs-derived exosomes may contribute to breast cancer cells proliferation and migration, which might provide an evidence for novel drug discovery based on exosomes and Hippo signaling.

Identification of long noncoding RNAs in silkworm larvae infected with Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the most important pathogens causing severe disease to silkworm. Emerging evidence indicates that long noncoding RNAs (lncRNAs) play importantly regulatory roles in virus infection and host immune response. To better understand the interaction between silkworm, Bombyx mori and BmCPV, we performed a comparative transcriptome analysis on lncRNAs and mRNAs between the virus-infected and noninfected silkworm larvae midgut at two time points postinoculation. A total of 16,753 genes and 1845 candidate lncRNAs were identified, among which 356 messenger RNA (mRNAs) and 41 lncRNAs were differentially expressed (DE). Target gene prediction revealed that most of DEmRNAs (123) were coexpressed with 28 DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans- regulation by BmCPV-induced lncRNAs, and a regulatory network of DElncRNAs and DEmRNAs was then constructed. According to the network, many genes involved in apoptosis, autophagy, and antiviral response, such as ATG3, PDCD6, IBP2, and MFB1, could be targeted by different DElncRNAs, implying the essential roles of these genes and lncRNAs in BmCPV infection. In all, our studies revealed for the first time the alteration of lncRNA expression in BmCPV-infected larvae and its potential influence on BmCPV replication, providing a new perspective for host-cypovirus interaction studies.

Bombyx mori Apolipophorin-III inhibits Beauveria bassiana directly and through regulating expression of genes relevant to immune signaling pathways.

Insect Apolipophorin-III is a multifunctional protein and also plays an important role in insect innate immunity. Early transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-III (BmApoLp-III) in silkworm larvae infected with Beauveria bassiana was significantly up-regulated. In this study, BmApoLp-III gene was cloned, its expression patterns in different larval tissues investigated, the BmApoLp-III protein was successfully expressed with prokaryotic expression system and its antifungal effect was verified. The results showed that the BmApoLp-III gene was expressed in all the tested tissues of the 5th instar larvae infected by B. bassiana, with the highest expression in fat body. The fungistatic zone test showed that the recombinant BmApoLp-III has a significant antifungal effect on B. bassiana. Injecting purified BmApoLp-III to the larvae delayed the onset and death of the infected larvae. Conversely, silencing BmApoLp-III gene by RNAi resulted in early morbidity and death of the infected larvae. At the same time, injecting BmApoLp-III up-regulated the expression of genes including BmßGRP4 and BmMyd88 in the Toll signaling pathway, BmCTL5 and BmHOP in the Jak/STAT signaling pathway, serine proteinase inhibitor BmSerpin5, and antimicrobial peptide BmCecA, but down-regulated the expression of BmTak1 of Imd signaling pathway; while silencing BmApoLp-III gene down-regulated the expression of BmßGRP1 and BmSpaetzle, BmCTL5 and BmHOP, BmSerpin2 and BmSerpin5, BmBAEE and BmPPO2 of relevant pathways and BmCecA, but up-regulated the expression of BmPGRP-Lc and BmTak1 of Imd pathway. These results indicate that the BmApoLp-III could not only directly inhibit B. bassiana, but also participate in regulation of the expression of immune signaling pathway related genes, promote the expression of immune effectors, and indirectly inhibit the reproduction of B. bassiana in the silkworm.

In vitro Function Study of Different Negative Charge Pullulan Nanoparticles for Sentinel Lymph Node Angiography.

BACKGROUND: Many dyes or radioactive markers used for sentinel lymph node (SLN) have the shortcomings of false positive and radiation injury. Indocyanine green (ICG) seems to have a lower false positive rate and tissue damage, without a clear field of vision during the operation. METHODS: For the shortcomings, we successfully synthesized three anionic pullulan materials, changed the degree of hydrophobic for size controlling (< 50nm) to prepare CHP nanoparticles (NPs) and changed the succinyl degree to prepare CHPC NPs with different negative surface potential. RESULTS: The size of those NPs were less than 50nm under (transmission electron microscope) TEM, with hydrodynamic size of 90.67 ± 2.2 nm of CHP, 105.8 ± 1.7 nm of CHPC1 and 115.9 ± 2.3 nm of CHPC2. Moreover, the Zeta potential of CHP, CHPC1 and CHPC2 were -1.9 ± 0.2 mV, -9.6 ± 0.3 mV and -19.4 ± 0.7 mV. The size of ICG-loading CHP, CHPC1 and CHPC2 NPs increased to 109.4 ± 2.7 nm, 113.8 ± 1.2 nm and 30.6 ± 3.5 nm, as the zeta potential decreased to -2.7 ± 0.4 mV, -12.5 ± 1.6 mV and -23.1 ± 1.2 mV. With the increasing degree of succinyl, the size increased and the zeta potential decreased. At the same time, the higher degree of succinyl drug-loading NPs have lower release and have increased the stability of ICG. We found that the blank-NPs had no significant toxicity to normal cells (HSF), as the ICG@CHP group had larger toxicity than the CHPCs and control. Moreover, the cellular uptake was decreased with the increased degree of succinyl. CONCLUSION: In this study, we successfully prepared CHPC2 carriers with the maximum negative surface charge, for follow-up research and providing new ideas for SLN.

Circ_0001777 Affects Triple-negative Breast Cancer Progression Through the miR-95-3p/AKAP12 Axis.

BACKGROUND: Triple Negative Breast Cancer (TNBC) is 1 of the most serious cancer. Circular RNA_0001777 (circ_0001777) expression was decreased in TNBC tissues. However, the molecular mechanism of circ_0001777 remains unknown. METHODS: The expression of circ_0001777, microRNA-95-3p (miR-95-3p) and A-kinase anchor protein 12 (AKAP12) were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). A series of in vitro experiments were designed to explore the function of circ_0001777 in TNBC cells and the regulatory mechanism between circ_0001777 and miR-95-3p and AKAP12 in TNBC cells. Western blot examined the relative protein levels in TNBC cells. Bioinformatics prediction site predicted the relationship between miR-95-3p and circ_0001777 or AKAP12 and was verified by Dual-luciferase reporter assays. The xenotransplantation model was established to study the role of circ_0001777 in vivo. RESULTS: The expression of circ_0001777 and AKAP12 was decreased in TNBC tissues, while the expression of miR-95-3p was increased. Circ_0001777 can sponge miR-95-3p, and AKAP12 is the target of miR-95-3p. In vitro complement experiments, overexpression of circ_0001777 significantly decreased the malignant behavior of TNBC, while co-transfection of miR-95-3p partially up-regulated this change. In addition, AKAP12 knockdown increased the proliferation, migration, and invasion of TNBC cells inhibited by overexpression of circ_0001777. Mechanically, circ_0001777 regulates AKAP12 expression in TNBC cells by sponge miR-95-3p. In addition, in vivo studies have shown that overexpression of circ_0001777 inhibits tumor growth. CONCLUSION: Overexpression of circ_0001777 decreased proliferation, migration, and invasion of TNBC cells by regulating the miR-95-3p/AKAP12 axis, suggesting that circ_0001777/miR-95-3p/AKAP12 axis may be a potential regulatory mechanism for the treatment of TNBC.

Circ_0108942 Regulates the Progression of Breast Cancer by Regulating the MiR-1178-3p/TMED3 Axis.

BACKGROUND: Breast cancer (BC) has posed a fatal threat to women's lives and the search for new methods of diagnosis and treatment is an important way to break the bottleneck of high mortality in BC. Circular RNAs (circRNAs) have been confirmed to be aberrantly expressed in several types of cancers, and this study is intended to elucidate the role and mechanism of circ_0108942 in BC. MATERIALS AND METHODS: The levels of circ_0108942, microRNA-1178-3p (miR-1178-3p), and transmembrane p24 trafficking protein 3 (TMED3) were measured using real-time quantitative polymerase chain reaction (RT-qPCR) or western blot. Meanwhile, the cell proliferation, migration, invasion, angiopoiesis, and apoptosis were analyzed using 5-ethynyl-2'-deoxyuridine (EdU), transwell, tubule formation, and flow cytometry assays. Protein levels were determined by western blot. In addition, we used dual-luciferase reporter and RNA pull-down assays to identify the interplay between miR-1178-3p and circ_0108942 or TMED3. Lastly, the impact of circ_0108942 on the growth of BC tumors in vivo was analyzed by xenograft models. RESULTS: Circ_0108942 and TMED3 were notably upregulated in BC, and the miR-1178-3p was downregulated. Functionally, silencing circ_0108942 suppressed cell proliferation, migration, invasion and promoted apoptosis in BC cells. In mechanism, circ_0108942 regulated TMED3 expression by sponging miR-1178-3p. Meanwhile, circ_0108942 knockdown also greatly constrained tumor growth in vivo. CONCLUSION: Circ_0108942 boosted BC progression by regulating miR-1178-3p and thus upregulating TMED3.

Development of a model for predicting mortality of breast cancer admitted to Intensive Care Unit.

Background: There is still not a mortality prediction model built for breast cancer admitted to intensive care unit (ICU). Objectives: We aimed to build a prognostic model with comprehensive data achieved from eICU database. Methods: Outcome was defined as all-cause in-hospital mortality. Least absolute shrinkage and selection operator (LASSO) was conducted to select important variables which were then taken into logistic regression to build the model. Bootstrap method was then conducted for internal validation. Results: 448 patients were included in this study and 79 (17.6%) died in hospital. Only 5 items were included in the model and the area under the curve (AUC) was 0.844 (95% confidence interval [CI]: 0.804-0.884). Calibration curve and Brier score (0.111, 95% CI: 0.090-0.127) showed good calibration of the model. After internal validation, corrected AUC and Brier score were 0.834 and 0.116. Decision curve analysis (DCA) also showed effective clinical use of the model. The model can be easily assessed on website of https://breastcancer123.shinyapps.io/BreastCancerICU/. Conclusions: The model derived in this study can provide an accurate prognosis for breast cancer admitted to ICU easily, which can help better clinical management.

A cypovirus encoded microRNA negatively regulates the NF-κB pathway to enhance viral multiplication in Silkworm, Bombyx mori.

MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double-stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPV-encoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRT-PCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKKß), via binding to the target mRNA 3'-untranslated region, which fine-tuned the host NF-κB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.

Two Putative Cypovirus-Encoded miRNAs Co-regulate the Host Gene of GTP-Binding Nuclear Protein Ran and Facilitate Virus Replication.

microRNA (miRNA) plays important roles in regulating various biological processes, including host-pathogen interaction. Recent studies have demonstrated that virus-encoded miRNAs can manipulate host gene expression to ensure viral effective multiplication. Bombyx mori cypovirus (BmCPV), a double-stranded RNA virus with a segmented genome, is one of the important pathogens for the economically important insect silkworm. Our present study indicated that two putative miRNAs encoded by BmCPV could promote viral replication by inhibiting the gene expression of B. mori GTP-binding nuclear protein Ran (BmRan), an essential component of the exportin-5-mediated nucleocytoplasmic transport of small RNAs. BmCPV-miR-1 and BmCPV-miR-3 are two of the BmCPV-encoded miRNAs identified in our previous studies. BmRan is a common target gene of them with binding sites all located in the 3'-untranslated region (3'-UTR) of its mRNA. The expression levels of the two miRNAs in the midgut of larvae infected with BmCPV gradually increased with the advance of infection, while the expression of the target gene BmRan decreased gradually. The miRNAs and the recombinant target gene consisting of reporter gene mCherry and 3'-UTR of BmRan mRNA were expressed in HEK293T cells for validating the interaction between the miRNAs and the target gene. qRT-PCR results revealed that BmCPV-miR-1 and BmCPV-miR-3 negatively regulate target gene expression not only separately but also cooperatively by binding to the 3'-UTR of BmRan mRNA. By transfecting miRNA mimics into BmN cells and injecting the mimics into the body of silkworm larvae, it was indicated that both BmCPV-miR-1 and BmCPV-miR-3 could repress the expression of BmRan in BmN cells and in the silkworm, and the cooperative action of the two miRNAs could enhance the repression of BmRan expression. Furthermore, the repression of BmRan could facilitate the replication of BmCPV genomic RNAs. It is speculated that BmCPV-miR-1 and BmCPV-miR-3 might reduce the generation of host miRNAs by inhibiting expression of BmRan, thus creating a favorable intracellular environment for virus replication. Our results are helpful to better understand the pathogenic mechanism of BmCPV to the silkworm, and provide insights into one of the evasion strategies used by viruses to counter the host defense for their effective multiplication.