Screening, expression and anti-tumor functional identification of anti-LAG-3 nanobodies.

OBJECTIVE: To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells. METHODS: Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjµgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 µg/mL. CONCLUSION: The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.

LncRNA DANCR restrained the survival of mycobacterium tuberculosis H37Ra by sponging miR-1301-3p/miR-5194.

Tuberculosis is a worldwide contagion caused by Mycobacterium tuberculosis (MTB). MTB is characterized by intracellular parasitism and is semi-dormant inside host cells. The persistent inflammation caused by MTB can form a granuloma in lesion regions and intensify the latency of bacteria. In recent years, several studies have proven that long non-coding RNAs (lncRNAs) play critical roles in modulating autophagy. In our study, the Gene Expression Omnibus (GEO) databases were searched for lncRNAs that are associated with tuberculosis. We found that lncRNA differentiation antagonizing non-protein coding RNA (DANCR) increased in the peripheral blood samples collected from 54 pulmonary tuberculosis patients compared to 23 healthy donors. By constructing DANCR overexpression cells, we analyzed the possible cellular function of DANCR. After analyzing our experiments, it was found that the data revealed that upregulation of DANCR facilitated the expression of signal transducer and activator of transcription 3, autophagy-related 4D cysteine peptides, autophagy-related 5, Ras homolog enriched in the brain, and microtubule-associated protein 1A/1B light chain 3 (STAT3, ATG4D, ATG5, RHEB, and LC3, respectively) by sponging miR-1301-3p and miR-5194. Immunofluorescence analysis indicated that DANCR played a positive role in both autophagosome formation and fusion of autolysosomes in macrophages. The colony-forming unit (CFU) assay data also showed that the cells overexpressing DANCR were more efficient in eliminating the intracellular H37Ra strain. Consequently, these data suggest that DANCR restrained intracellular survival of M. tuberculosis by promoting autophagy via miR-1301-3p and miR-5194.

Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.

Background: The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Methods: Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Results: Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3'-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. Conclusion: mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.

Screening and identification of an anti-PD-1 nanobody with antitumor activity.

Blocking of PD-1 or PD-L1 with corresponding antibody to enhance T cell response and mediate antitumor activity has been successfully applied in clinical practice. Several immune checkpoint inhibitors including monoclonal antibodies targeting PD-1 have been approved by the Food and Drug Administration (FDA) in cancer immunotherapy. However, the application of traditional antibodies has limited due to their drawbacks of large molecular weight and low tissue penetration. As the high specificity and strong tissue penetration of nanobodies (Nbs), efforts have been taken to develop Nbs for cancer therapy. Herein, we aim to screen a specific Nb against human PD-1 derived from a naïve camel Nb phage display library and further study its biological characteristic and anti-tumor activity. Finally, an anti-PD-1 Nb with high specificity and affinity was screened and generated, its cytotoxicity and antitumor effect was also confirmed in vitro and vivo. All of these indicate that the anti-PD-1 Nb may provide an alternative and appealing therapeutic agent for cancer immunotherapy.

Spermidine Promotes Nb CAR-T Mediated Cytotoxicity to Lymphoma Cells Through Elevating Proliferation and Memory.

Purpose: Due to the natural advantages of spermidine in immunity, we investigated the effects of spermidine pretreatment on nanobody-based CAR-T cells (Nb CAR-T) mediated cytotoxicity and potential mechanism. Patients and Methods: The optimal concentration of spermidine was determined by detecting its impact on viability and proliferation of T cells. The phenotypic characteristic of CAR-T cells, which were treated with spermidine for 4 days, was examined by flow cytometry. The expansion ability of CAR-T cells was monitored in being cocultured with tumor cells. Additionally, CAR-T cells were stimulated by lymphoma cells to test its cytotoxicity in vitro, and the supernatant in co-culture models were collected to test the cytokine production. Furthermore, xenograft models were constructed to detect the anti-tumor activity of CAR-T cells in vivo. Results: The optimal concentration of spermidine acting on T cells was 5µM. The antigen-dependent proliferation of spermidine pretreatment CD19 CAR-T cells or Nb CAR-T cells was increased compared to control. Central memory T cells(TCM) dominated the CAR-T cell population in the presence of spermidine. When spermidine pretreatment CAR-T cells were stimulated with Daudi cells, the secretion of IL-2 and IFN-γ has been significantly enhanced. The ability of CAR-T cells to lysis Daudi cells was enhanced with the help of spermidine, even at higher tumor loads. Pre-treated Nb CAR-T cells with spermidine were able to control tumor cells in vivo, and therefore prolong mice survival. Conclusion: Our results revealed that spermidine could promote Nb CAR-T mediated cytotoxicity to lymphomas cells through enhancing memory and proliferation, and provided a meaningful approach to strengthen the anti-tumor effect of CAR-T cells.

Spectrophotometric Detection of the BRCA1 Gene via Exponential Isothermal Amplification and Hybridization Chain Reaction of Surface-Bound Probes.

In this work, we demonstrated an ultrasensitive approach with a dual-amplification strategy for DNA assay based on isothermal exponential amplification (EXPAR) and the hybridization chain reaction (HCR). In the presence of target DNA, the hairpin probe DNA (HP1) recognized and partially hybridized with the target DNA to form double-stranded structures containing the full recognition sequences for nicking endonuclease and then initiated EXPAR. Under the reaction of EXPAR, a large number of single-stranded DNA (ssDNA) was produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA can bind to the surface-bound probe on the well of the microplate and trigger the hybridization chain reaction, resulting in the production of numerous double-stranded DNA concatamers with biotin labeling. In the presence of streptavidin-conjugated horseradish peroxidase (HRP), the amplified signal can be detected by a spectrophotometer via HRP-catalyzed substrate 3,3'5,5'-tetramethylbenzidine (TMB). This proposed dual-amplification method provides a detection limit of 74.48 aM, which also exhibits good linearity ranging from 0.1 fM to 100 pM.

CCL3 Promotes Proliferation of Colorectal Cancer Related with TRAF6/NF-κB Molecular Pathway.

Chemokine C-C motif chemokine ligand 3 (CCL3) plays an important role in the invasion and metastasis of malignant tumors. For developing new therapeutic targets and antitumor drugs, the effect of chemokine CCL3 and the related cytokine network on colorectal cancer should be investigated. This study used cell, tissue, and animal experiments to prove that CCL3 is highly expressed in colorectal cancer and confirmed that CCL3 can promote the proliferation of cancer cells, and its expression is closely related to TRAF6/NF-κB molecular pathway. In addition, protein chip technology was used to examine colorectal cancer tissue samples and identify the key factors of chemokine CCL3 and the toll-like receptors/nuclear factor-κB (TLR/NF-κB) pathway in cancer and metastatic lymph nodes. Furthermore, the lentiviral vector technology was employed for transfection to construct interference and overexpression cell lines. The experimental results reveal the mechanism of CCL3 and TNF receptor-associated factor 6 (TRAF6)/NF-κB pathway-related factors and their effects on the proliferation of colon cancer cells. Finally, the expression and significance of CCL3 in colorectal cancer tissues and its correlation with clinical pathology were studied by immunohistochemistry. Also, the results confirmed that CCL3 and C-C motif chemokine receptor 5 (CCR5) were expressed in adjacent tissues, colorectal cancer tissues, and metastatic cancer. The expression level was correlated with the clinical stage and nerve invasion. The expression of chemokine CCL3 and receptor CCR5 was positively correlated with the expression of TRAF6 and NF-κB and could promote the proliferation, invasion, and migration of colorectal cancer cells through TRAF6 and NF-κB.

Chemokines in progression, chemoresistance, diagnosis, and prognosis of colorectal cancer.

Plenty of factors affect the oncogenesis and progression of colorectal cancer in the tumor microenvironment, including various immune cells, stromal cells, cytokines, and other factors. Chemokine is a member of the cytokine superfamily. It is an indispensable component in the tumor microenvironment. Chemokines play an antitumor or pro-tumor role by recruitment or polarization of recruiting immune cells. Meanwhile, chemokines, as signal molecules, participate in the formation of a cross talk among signaling pathways and non-coding RNAs, which may be involved in promoting tumor progression. In addition, they also function in immune escape. Chemokines are related to drug resistance of tumor cells and may even provide reference for the diagnosis, therapy, and prognosis of patients with colorectal cancer.

Potential and functional prediction of six circular RNAs as diagnostic markers for colorectal cancer.

Background: Circular RNAs (circRNAs) have been discovered in colorectal cancer (CRC), but there are few reports on the expression distribution and functional mining analysis of circRNAs. Methods: Differentially expressed circRNAs in CRC tissues and adjacent normal tissues were screened and identified by microarray and qRT-PCR. ROC curves of the six circRNAs were analyzed. A series of bioinformatics analyses on differentially expressed circRNAs were performed. Results: A total of 207 up-regulated and 357 down-regulated circRNAs in CRC were screened, and three top up-regulated and down-regulated circRNAs were chosen to be verified in 33 pairs of CRCs by qRT-PCR. 6 circRNAs showed high diagnostic values (AUC = 0.6860, AUC = 0.8127, AUC = 0.7502, AUC = 0.9945, AUC = 0.9642, AUC = 0.9486 for hsa_circRNA_100833, hsa_circRNA_103828, hsa_circRNA_103831 and hsa_circRNA_103752, hsa_circRNA_071106, hsa_circRNA_102293). A circRNA-miRNA-mRNA regulatory network (cirReNET) including six candidate circRNAs, 19 miRNAs and 210 mRNA was constructed, and the functions of the cirReNET were predicted and displayed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on these mRNAs and protein-protein interaction (PPI) network of the hub genes acquired by string and CytoHubba. Conclusion: A cirReNET containing potential diagnostic and predictive indicators of CRCs and several critical circRNA-miRNA-mRNA regulatory axes (cirReAXEs) in CRC were mined, and may provide a novel route to study the mechanism and clinical targets of CRC.

Tumor-infiltrating lymphocyte: features and prognosis of lymphocytes infiltration on colorectal cancer.

Tumor-infiltrating lymphocytes (TILs) are vital elements of the tumor microenvironment (TME), and the anti-tumor activity of TILs on colorectal cancer (CRC) has been a topic of concern. However, the characteristics and prognosis of the various types of lymphocyte infiltration in CRC have not been fully explained. Our study aimed to identify distinct features and prognosis of TILs. We integrated multiple-cohort databases to illustrate the features, proportions, and prognosis of TILs on CRC. We found that macrophages were significantly enriched in CRC. When we used the scRNA-seq database to further evaluate the proportion of TILs, we noticed markedly higher numbers of CD4 + T cell, B cell, and CD8 + T cell in four Gene Expression Omnibus Series (GSE) CRC cohorts. Interestingly, we found that the infiltrating level of TIL subgroups from highest to lowest is always dendritic cells, CD8 + T cells, CD4 + T cells, neutrophils, B cells, and macrophages; the proportion of infiltration is largely constant regardless of mutations in specific genes or somatic copy number variation (sCNV). In addition, the data corroborated that CD4+ TILs and CD8+ TILs have certain application values in the prognosis of CRCs, and age negatively related to CD8+ TILs and B plasma infiltration. Finally, patients with CRC who are older than 70 years have a better response to immune-checkpoint blockade.

Long­chain non­coding RNA GAS5 promotes cell autophagy by modulating the miR­181c­5p/ATG5 and miR­1192/ATG12 axes.

The main aim of the present study was to explore the role of long­chain non­coding RNA (lncRNA) growth arrest­specific transcript 5 (GAS5) in macrophage autophagy. Firstly, the expression of lncRNA GAS5 during cell starvation or following treatment with 3­methyladenine was determined using reverse transcription­quantitative PCR (RT­qPCR). Additionally, fluorescent in situ hybridization (FISH) assay was utilized to determine the localization of the expression of lncRNA GAS5 in RAW264.7 cells. In vitro cell models were established through the transfection of LV5­lncRNA GAS5 (LV5­GAS5) or LV3­shRNA­lnc GAS5 (sh­GAS5), in order to overexpress or knockdown lncRNA GAS5 expression in RAW264.7 cells. The potential target microRNAs (miRNAs/miRs) of lncRNA GAS5 were analyzed using bioinformatics. The formation of autophagic bodies was detected with the use of laser confocal and transmission electron microscopy. Dual­luciferase reporter assay was performed to determine the target specificities of miR­181c­5p or miR­1192 to lncRNA GAS5 and autophagy­related gene (ATG) or ATG12. The mRNA levels of miR181c­5p, miR­1192, as well as ATG5 and ATG12 were detected using RT­qPCR. The protein levels of microtubule­associated proteins 1A/1B light chain 3B (LC3), p62, ATG5 and ATG12 were measured using western blot analysis. It was revealed that lncRNA GAS5 expression in RAW264.7 macrophages increased significantly during starvation­induced autophagy, and that lncRNA GAS5 overexpression was able to markedly promote the formation of autophagic bodies. Bioinformatics analysis demonstrated that miR­181c­5p and miR­1192 were potential targets of lncRNA GAS5, which was further confirmed by RT­qPCR, western blot analysis and the dual­luciferase reporter assay. Finally, it was confirmed that lncRNA GAS5 promoted autophagy by sponging miR­181c­5p and miR­1192, and upregulating the expression levels of the key autophagic regulators, ATG5 and ATG12. On the whole, the present study demonstrates that total, lncRNA GAS5 promotes macrophage autophagy by targeting the miR­181c­5p/ATG5 and miR­1192/ATG12 axes.

MicroRNA-142-3p inhibits autophagy and promotes intracellular survival of Mycobacterium tuberculosis by targeting ATG16L1 and ATG4c.

BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis), which parasitizes host macrophages and lead to cellular immunologic responses, such as autophagy and apoptosis. Several studies had indicated that autophagy played important roles in alleviating intracellular survival of M. tuberculosis by accelerating the maturation of phagosome. Previously, we found miR-142-3p was significantly down-regulated in the macrophages after infection with M. tuberculosis. However, the role of miR-142-3p in the regulation of autophagy and M. tuberculosis survival is elusive. METHODS: Bioinformatics analysis was used to obtain target genes of miR-142-3p; the binding sites of ATG16L1 and ATG4c were further confirmed with dual luciferase reporter assay; RAW264.7 cells were infected with H37Ra and the expression of miR-142-3p was measured by qRT-PCR; the autophagic marker protein was detected by western blot as well as immunofluorescence microscopy and transmission electron microscopes analysis. RESULTS: Overexpression of miR-142-3p significantly inhibited H37Ra-induced activation of autophagy, blocked the maturation of phagosome in macrophages and promoted M. tuberculosis survival in macrophages. Furthermore, miR-142-3p negatively-regulated expression of ATG16L1 and ATG4c by directly targeting its 3'-UTR, and meaningfully abated the level of autophagy. CONCLUSION: These findings suggested that miR-142-3p inhibited M. tuberculosis-induced activation of autophagy and promoted H37Ra survival in RAW264.7 cells by targeting ATG16L1 and ATG4c.

Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells.

BACKGROUND: Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. METHODS: CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function. RESULTS: The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%. CONCLUSIONS: Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

The underlying molecular mechanisms and prognostic factors of RNA binding protein in colorectal cancer: a study based on multiple online databases.

BACKGROUND: RNA binding protein (RBP) is an active factor involved in the occurrence and development of colorectal cancer (CRC). Therefore, the potential mechanism of RBP in CRC needs to be clarified by dry-lab analyses or wet-lab experiments. METHODS: The differential RBP gene obtained from the GEPIA 2 (Gene Expression Profiling Interactive Analysis 2) were performed functional enrichment analysis. Then, the alternative splicing (AS) events related to survival were acquired by univariate regression analysis, and the correlation between RBP and AS was analyzed by R software. The online databases were conducted to analyze the mutation and methylation of RBPs in CRC. Moreover, 5 key RBP signatures were obtained through univariate and multivariate Cox regression analysis and established as RBP prognosis model. Subsequently, the above model was verified through another randomized group of TCGA CRC cohorts. Finally, multiple online databases and qRT-PCR analysis were carried to further confirm the expression of the above 5 RBP signatures in CRC. RESULTS: Through a comprehensive bioinformatics analysis, it was revealed that RBPs had genetic and epigenetic changes in CRC. We obtained 300 differentially expressed RBPs in CRC samples. The functional analysis suggested that they mainly participated in spliceosome. Then, a regulatory network for RBP was established to participate in AS and DDX39B was detected to act as a potentially essential factor in the regulation of AS in CRC. Our analysis discovered that 11 differentially expressed RBPs with a mutation frequency higher than 5%. Furthermore, we found that 10 differentially expressed RBPs had methylation sites related to the prognosis of CRC, and a prognostic model was constructed by the 5 RBP signatures. In another randomized group of TCGA CRC cohorts, the prognostic performance of the 5 RBP signatures was verified. CONCLUSION: The potential mechanisms that regulate the aberrant expression of RBPs in the development of CRC was explored, a network that regulated AS was established, and the RBP-related prognosis model was constructed and verified, which could improve the individualized prognosis prediction of CRC.

Upregulation of TIGIT and PD-1 in Colorectal Cancer with Mismatch-repair Deficiency.

Background: The role of T cell Ig and ITIM domain (TIGIT) and programmed cell death-1 (PD-1) in colorectal cancer (CRC) with mismatch repair deficiency is unknown.Methods: This was a study of 60 CRC patients with mismatch repair deficiency and 30 healthy controls between June 2015 and October 2015.Results: The expression of Foxp3, PD-1, and TIGIT was higher in cancer tissues compared with adjacent mucosa (all P < .05). Patients with advanced TNM stage had a significantly higher expression of TIGIT (P = .025) and PD-1 (P = .020) than patients with early-stage CRC. The disease-free survival (DFS) of patients with high TIGIT (HR = 3.96, 95%CI: 1.34-11.69, P = .013) or PD-1 (HR = 214.8, 95%CI: 49.88-925.2, P < .001) expression were better. The overall survival (OS) of the patients with CRC and high expression of PD-1 was worse than those with low expression (HR = 4.01, 95%CI:1.26-12.69, P = .019).Conclusion: TIGIT and PD-1 are upregulated in CRC with mismatch repair deficiency and associated with TNM stage and DFS.

Establishment of a Colorectal Cancer-Related MicroRNA-mRNA Regulatory Network by Microarray and Bioinformatics.

Colorectal cancer (CRC) is one of the most malignant cancers with high morbidity and mortality. MicroRNAs (miRNAs) are small non-coding RNAs that affect biological processes by binding to mRNAs and regulating their expression, and epigenetic alterations including miRNA dysregulation are significantly involved in CRC development. Determining the effect of the miRNA-mRNA network on CRC could be helpful for developing novel therapeutic targets and prognostic biomarkers, and even improving survival. In this study, microarray assays were used to screen differentially expressed miRNAs (DE miRNAs) and mRNAs (DE mRNAs) in CRC and the adjacent normal tissues. Among the detected genes, 42 miRNAs and 142 mRNAs were significantly upregulated in CRC, while 23 miRNAs and 279 mRNAs were significantly downregulated. Through overlapping of predicted targets of DE miRNAs and anti-expressed DE mRNAs, networks of DE miRNAs and DE mRNAs in CRC were established. Additionally, the formation of a protein-protein interaction network of DE mRNAs possibly targeted by DE miRNAs, functional annotation and pathway analysis, stable subnetwork mining, and determination of hub genes provided the probable mechanism used by DE miRNAs and DE mRNAs to regulate CRC growth. Finally, validation of expression and prognostic potential of hub genes provided further support for the results above and indicated that CCL-28, GPR15, PNOC, NUSAP1, and their interacted miRNAs may be a potential signature for prognosis of CRC patients. In sum, we successfully established miRNA-mRNA regulatory networks based on microarray results targeting CRC, and these findings may elucidate the mechanisms used for CRC growth and identify miRNA-related signatures for prognosis and treatment of CRC.

[Integrin α5 silencing inhibits proliferation, invasion and metastasis of human liver cancer Bel-7404 cells in vitro].

OBJECTIVE: To analyze the association of integrinα5 (ITGA5) with grading of liver cancer and the overall patient survival and investigate the effects of integrin α5 (ITGA5) silencing on the proliferation, invasion and migration abilities of human liver cancer Bel-7404 cells. METHODS: UALCAN was used to analyze the expression of ITGA5 in liver cancer tissues and normal tissues, and expression in different grades of liver cancer tissues. GEPIA was used to analyze the relationship between ITGA5 expression and the survival of liver cancer patients through.The ITGA5 shRNA lentiviral vector was used to infect Bel-7404 cells to establish a cell line with stable ITGA5 silencing verified by Western blotting. Plate clone formation assay and Transwell assay were used to detect the proliferation, invasion and migration of Bel-7404 cells. The correlation between ITGA5 and PI3K in liver cancer tissues and control tissues was analyzed using Oncomine cancer specimen database. RESULTS: The expression of ITGA5 was significantly higher in liver cancer than in normal tissues (P < 0.05). The expression of ITGA5 was significantly lower in grade 1 than in grade 2 liver cancer, and also lower in grade 2 than in grade 3 liver cancer (P < 0.05). The patients with high ITGA5 expression had a significantly lower overall survival rate than those with low ITGA5 expression (P < 0.05). Plate clone formation assay showed that the clone formation rate was significantly lowered in Bel-7404 cells with ITGA5 silencing compared with the blank and negative control cells (P < 0.05). ITGA5 silencing significantly attenuated the migration of Bel-7404 cells as shown by Transwell assay (P < 0.05). ITGA5 and PI3K were both highly expressed and showed a positive correlation in liver cancer tissues (P < 0.05). CONCLUSIONS: ITGA5 is closely related to the progression of liver cancer and the patients' prognosis. ITGA5 silencing inhibits the proliferation, invasion and migration of liver cancer cells. ITGA5 promotes the liver cancer growth and metastasis possibly by regulating the PI3K signaling pathway.

MicroRNA-106a Inhibits Autophagy Process and Antimicrobial Responses by Targeting ULK1, ATG7, and ATG16L1 During Mycobacterial Infection.

Autophagy is a key element of innate immune response against invading pathogens including Mycobacterium tuberculosis (M. tuberculosis). The emerging roles of microRNAs in regulating host antimicrobial responses against M. tuberculosis have gained widespread attention. However, the process by which miRNAs specifically influence antibacterial autophagy during mycobacterial infection is largely uncharacterized. In this study, we demonstrate a novel role of miR-106a in regulating macrophage autophagy against M. tuberculosis. H37Ra infection leads to downregulation of miR-106a in a time- and dose-dependent manner and concomitant upregulation of its three targets (ULK1, ATG7, and ATG16L1) in THP-1 macrophages. MiR-106a could inhibit autophagy activation and antimicrobial responses to M. tuberculosis by targeting ULK1, ATG7, and ATG16L1. Overexpression of miR-106a dramatically inhibited H37Ra-induced activation of autophagy in human THP-1 macrophages, whereas inhibitors of miR-106a remarkably promoted H37Ra-induced autophagy. The inhibitory effect of miR-106a on autophagy process during mycobacterial infection was also confirmed by Transmission Electron Microscope (TEM) observation. More importantly, forced expression of miR-106a increased mycobacterial survival, while transfection with miR-106a inhibitors attenuated the survival of intracellular mycobacteria. Taken together, these data demonstrated that miR-106a functioned as a negative regulator in autophagy and antimicrobial effects by targeting ULK1, ATG7, and ATG16L1 during M. tuberculosis infection, which may provide a potential target for developing diagnostic reagents or antibacterials against tuberculosis.

LncRNA expression profile during autophagy and Malat1 function in macrophages.

Long noncoding RNAs (lncRNAs) are a class of functional non-coding transcripts that are longer than 200 nt and regulate gene expression via diverse mechanisms in eukaryotes. In fact, they have emerged as critical epigenetic and transcriptional regulators of autophagy in mammals in response to various stressors. Autophagy not only plays a crucial role in maintaining cellular homeostasis, but it is also essential to immunity, targets intracellular pathogens for degradation, modulates inflammation, and participates in adaptive immune responses. However, the expression profile of lncRNA and its role in regulating autophagy in macrophages have been poorly defined. Here, we used transcriptomic and bioinformatics to analysis LncRNA expression profile during autophagy and functional studies to evaluate the function of the metastasis-associated lung adenocarcinoma transcript-1 (Malat1) lncRNA in macrophages. A total of 1112 putative lncRNAs (240 novel lncRNAs) were identified, including 831 large intergenic, 129 intronic, and 152 anti-sense lncRNA, of which 59 differentially expressed transcripts exhibited a greater than 1.5-fold change under different conditions. The interaction of Malat1 lncRNA with microRNA (mir)-23-3p and lysosomal-associated membrane protein 1 (Lamp1) was found, Malat1 releases inhibition of Lamp1 expression in macrophages through competitive adsorption of mir-23-3p. The results of this study provide a better understanding of lncRNA function in macrophages and a basis for further investigation into the roles and mechanisms of ncRNA in immunology, particularly the functions of Malat1 and mir-23-3p in the pathogenesis of macrophages.

Novel miR-1958 Promotes Mycobacterium tuberculosis Survival in RAW264.7 Cells by Inhibiting Autophagy Via Atg5.

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR- 1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.