SARS-CoV-2 N protein-induced Dicer, XPO5, SRSF3, and hnRNPA3 downregulation causes pneumonia.

Though RNAi and RNA-splicing machineries are involved in regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, their precise roles in coronavirus disease 2019 (COVID-19) pathogenesis remain unclear. Herein, we show that decreased RNAi component (Dicer and XPO5) and splicing factor (SRSF3 and hnRNPA3) expression correlate with increased COVID-19 severity. SARS-CoV-2 N protein induces the autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, inhibiting miRNA biogenesis and RNA splicing and triggering DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 knockdown increases, while their overexpression decreases, N protein-induced pneumonia's severity. Older mice show lower expression of Dicer, XPO5, SRSF3, and hnRNPA3 in their lung tissues and exhibit more severe N protein-induced pneumonia than younger mice. PJ34, a poly(ADP-ribose) polymerase inhibitor, or anastrozole, an aromatase inhibitor, ameliorates N protein- or SARS-CoV-2-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.

Protocol for isolating small cytosolic dsDNA from cultured murine cells.

We recently identified a class of small cytosolic double-stranded DNA (scDNA) approximately 20-40 bp in size in human and mouse cells. Here, we present a protocol for scDNA isolation from cultured murine cells. We describe steps for cytosolic compartment separation, DNA isolation in the cytosolic fraction using phenol-chloroform extraction, and ethanol precipitation. We then detail procedures for denaturing purified cytosolic DNA through urea polyacrylamide gel electrophoresis and obtaining scDNA in the cytosolic DNA fraction via gel purification. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Upregulation of CELSR1 expression promotes ovarian cancer cell proliferation, migration, and invasion.

Cadherin epidermal growth factor and laminin-G seven-pass G-type receptor 1 (CELSR1) is a planar cell polarity protein involved in the transmission of directional cues to align either individual cells within an epithelial sheet or multicellular clusters. CELSR1 has been suggested to play a role in glioma, breast cancer, and chronic lymphocytic leukemia development; however, whether it has a role in the pathogenesis of ovarian cancer remains unknown. The aim of this study was to determine the role of CELSR1 in ovarian cancer and elucidate its underlying molecular mechanisms. By analyzing gene expression data downloaded from the Cancer Genome Atlas database, we found that CELSR1 expression was upregulated in ovarian cancer tissues compared to that in normal ovarian tissues. High CELSR1 expression levels were associated with poor prognosis in patients with ovarian cancer. Cell proliferation, scratch, and transwell assays revealed that CELSR1 promoted the proliferation, migration, and invasion of ovarian cancer cells in vitro. In addition, transcriptome sequencing analysis revealed that CELSR1 knockdown in T29H cells resulted in the dysregulation of the expression of 1320 genes. Further analysis revealed that genes involved in proliferation- and migration-associated signaling pathways were regulated by CELSR1. Our study demonstrates that CELSR1 is highly expressed in ovarian cancer cells and regulates their proliferation and migration, suggesting its potential as a diagnostic marker and therapeutic target.

Mutual communication between radiosensitive and radioresistant esophageal cancer cells modulates their radiosensitivity.

Radiotherapy is an important treatment modality for patients with esophageal cancer; however, the response to radiation varies among different tumor subpopulations due to tumor heterogeneity. Cancer cells that survive radiotherapy (i.e., radioresistant) may proliferate, ultimately resulting in cancer relapse. However, the interaction between radiosensitive and radioresistant cancer cells remains to be elucidated. In this study, we found that the mutual communication between radiosensitive and radioresistant esophageal cancer cells modulated their radiosensitivity. Radiosensitive cells secreted more exosomal let-7a and less interleukin-6 (IL-6) than radioresistant cells. Exosomal let-7a secreted by radiosensitive cells increased the radiosensitivity of radioresistant cells, whereas IL-6 secreted by radioresistant cells decreased the radiosensitivity of radiosensitive cells. Although the serum levels of let-7a and IL-6 before radiotherapy did not vary significantly between patients with radioresistant and radiosensitive diseases, radiotherapy induced a more pronounced decrease in serum let-7a levels and a greater increase in serum IL-6 levels in patients with radioresistant cancer compared to those with radiosensitive cancer. The percentage decrease in serum let-7a and the percentage increase in serum IL-6 levels at the early stage of radiotherapy were inversely associated with tumor regression after radiotherapy. Our findings suggest that early changes in serum let-7a and IL-6 levels may be used as a biomarker to predict the response to radiotherapy in patients with esophageal cancer and provide new insights into subsequent treatments.

Communication between small cytosolic dsDNA and autophagy inhibits CGAS (cyclic GMP-AMP synthase) activation.

The CGAS (cyclic GMP-AMP synthase)-STING1 (stimulator of interferon response cGAMP interactor 1) pathway is an important innate immune pathway that induces proinflammatory cytokine production following stimulation with dsDNA > 45 bp. We recently identified a class of ~ 20-40 bp small cytosolic dsDNA (scDNA) that blocks CGAS-STING1 activation. In this punctum, we discuss the mechanism underlying the inhibition of CGAS-STING1 activation via scDNA. scDNA binds to CGAS but cannot activate its enzymatic activity. It competes with dsDNA > 45 bp for binding with CGAS to inhibit CGAS-STING1 activation. Moreover, scDNA activates macroautophagy/autophagy and induces the autophagic degradation of STING1 and long dsDNA. Autophagy then increases scDNA levels, driving a feedback loop that accelerates the degradation of STING1 and long cytosolic dsDNA. These findings reveal that mutual communication between scDNA and autophagy inhibits CGAS-STING1 activation following stimulation with dsDNA > 45 bp.

Upregulation of FAM50A promotes cancer development.

FAM50A encodes a nuclear protein involved in mRNA processing; however, its role in cancer development remains unclear. Herein, we conducted an integrative pan-cancer analysis using The Cancer Genome Atlas, Genotype-Tissue Expression, and the Clinical Proteomic Tumor Analysis Consortium databases. Based on the gene expression data from TCGA and GTEx databases, we compared FAM50A mRNA levels in 33 types of human cancer tissues to those in corresponding normal tissues and found that FAM50A mRNA level was upregulated in 20 of the 33 types of common cancer tissues. Then, we compared the DNA methylation status of the FAM50A promoter in tumor tissues to that in corresponding normal tissues. FAM50A upregulation was accompanied by promoter hypomethylation in 8 of the 20 types of tumor tissues, suggesting that promoter hypomethylation contributes to the upregulation of FAM50A in these cancer tissues. Elevated FAM50A expression in 10 types of cancer tissues was associated with poor prognosis in patients with cancer. FAM50A expression was positively correlated with CD4+ T-lymphocyte and dendritic cell infiltration in cancer tissues but was negatively correlated with CD8+ T-cell infiltration in cancer tissues. FAM50A knockdown caused DNA damage, induced interferon beta and interleukin-6 expression, and repressed the proliferation, invasion, and migration of cancer cells. Our findings indicate that FAM50A might be useful in cancer detection, reveal insights into its role in cancer development, and may contribute to the development of cancer diagnostics and treatments.

Small cytosolic double-stranded DNA represses cyclic GMP-AMP synthase activation and induces autophagy.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a major mediator of inflammation following stimulation with >45 bp double-stranded DNA (dsDNA). Herein, we identify a class of ∼20-40 bp small cytosolic dsDNA (scDNA) molecules that compete with long dsDNA (200-1,500 bp herring testis [HT]-DNA) for binding to cGAS, thus repressing HT-DNA-induced cGAS activation. The scDNA promotes cGAS and Beclin-1 interaction, releasing Rubicon, a negative regulator of phosphatidylinositol 3-kinase class III (PI3KC3), from the Beclin-1-PI3KC3 complex. This leads to PI3KC3 activation and induces autophagy, causing degradation of STING and long cytosolic dsDNA. Moreover, DNA damage decreases, and autophagy inducers increase scDNA levels. scDNA transfection and treatment with autophagy inducers attenuate DNA damage-induced cGAS activation. Thus, scDNA molecules serve as effective brakes for cGAS activation, preventing excessive inflammatory cytokine production following DNA damage. Our findings may have therapeutic implications for cytosolic DNA-associated inflammatory diseases.

Upregulation of TUBG1 expression promotes hepatocellular carcinoma development.

Tubulin γ-1 (TUBG1) is a highly conserved component of the centrosome and its deregulation is involved in the development of several types of cancer. However, the role of TUBG1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we found that TUBG1 was upregulated in human HCC cells and tissues and that TUBG1 upregulation was associated with promoter hypomethylation in HCC tissues. TUBG1 knockdown suppressed the proliferation, invasion, and migration of HCC cells. While TUBG1 expression was positively correlated with CD4 + memory T lymphocyte infiltration, it was negatively correlated with CD4 + regulatory T-cell infiltration in human HCC tissues. Furthermore, TUBG1 expression was positively correlated with the expression of genes involved in cell division. Noticeably, high expression of TUBG1 was associated with poor prognosis in patients with HCC. Overall, our findings revealed that TUBG1 promotes hepatocarcinogenesis by increasing proliferation, invasion, and migration of HCC cells and may regulate T lymphocyte infiltration. The current findings provide important insights into TUBG1 regulation in HCC, which could provide new therapeutic targets for hepatocarcinoma which has a very high incidence and mortality rate worldwide.

Tryptophan 2, 3­dioxygenase promotes proliferation, migration and invasion of ovarian cancer cells.

Tryptophan 2,3­dioxygenase (TDO2) is a key rate­limiting enzyme in the kynurenine pathway and promotes tumor growth and escape from immune surveillance in different types of cancer. The present study aimed to investigate whether TDO2 serves a role in the development of ovarian cancer. Reverse transcription­quantitative PCR and western blotting were used to detect the expression of TDO2 in different cell lines. The effects of TDO2 overexpression, TDO2 knockdown and TDO2 inhibitor on ovarian cancer cell proliferation, migration and invasion were determined by MTS, colony formation and Transwell assays. The expression of TDO2 in ovarian cancer tissues, normal ovarian tissues and fallopian tube tissues were analyzed using the gene expression data from The Cancer Genome Atlas and Genotype­Tissue Expression project. Immune cell infiltration in cancer tissues was evaluated using the single sample gene set enrichment analysis algorithm. The present study found that RasV12­mediated oncogenic transformation was accompanied by the upregulation of TDO2. In addition, it was demonstrated that TDO2 was upregulated in ovarian cancer tissues compared with normal ovarian tissues. TDO2 overexpression promoted proliferation, migration and invasion of ovarian cancer cells, whereas TDO2 knockdown repressed these phenotypes. Treatment with LM10, a TDO2 inhibitor, also repressed the proliferation, migration and invasion of ovarian cancer cells. The present study indicated that TDO2 can be used as a new target for the treatment of ovarian cancer.

Luteolin mediated targeting of protein network and microRNAs in different cancers: Focus on JAK-STAT, NOTCH, mTOR and TRAIL-mediated signaling pathways.

There has always been a keen interest of basic and clinical researchers to search for cancer therapeutics having minimum off-target effects and maximum anticancer activities. In accordance with this approach, there has been an explosion in the field of natural products research in the past few decades because of extra-ordinary list of natural extracts and their biologically and pharmacologically active constituents having significant medicinal properties. Apparently, luteolin-mediated anticancer effects have been investigated in different cancers but there is superfluousness of superficial data. Generalized scientific evidence encompassing apoptosis, DNA damage and anti-inflammatory effects has been reported extensively. However, how luteolin modulates deregulated oncogenic pathways in different cancers has not been comprehensively uncovered. In this review we have attempted to focus on cutting-edge research which has unveiled remarkable abilities of luteolin to modulate deregulated oncogenic pathways in different cancers. We have partitioned the review into various sections to separately discuss advancements in therapeutic targeting of oncogenic protein networks. We have provided detailed mechanistic insights related to JAK-STAT signaling and summarized how luteolin inhibited STAT proteins to inhibit STAT-driven gene network. We have also individually analyzed Wnt/ß-catenin and NOTCH pathway and how luteolin effectively targeted these pathways. Mapping of the signaling landscape has revealed that NOTCH pathway can be targeted therapeutically. NOTCH pathway was noted to be targeted by luteolin. We have also conceptually analyzed how luteolin restored TRAIL-induced apoptosis in resistant cancers. Luteolin induced an increase in pro-apoptotic proteins and efficiently inhibited anti-apoptotic proteins to induce apoptosis. Luteolin mediated regulation of non-coding RNAs is an exciting and emerging facet. Excitingly, there is sequential and systematic accumulation of clues which have started to shed light on intricate regulation of microRNAs by luteolin in different cancers. Collectively, sophisticated information will enable us to develop a refined understanding of the multi-layered regulation of signaling pathways and non-coding RNAs by luteolin in different cancers.

Rescuing Dicer expression in inflamed colon tissues alleviates colitis and prevents colitis-associated tumorigenesis.

Chronic inflammation is known to promote carcinogenesis; Dicer heterozygous mice are more likely to develop colitis-associated tumors. This study investigates whether Dicer is downregulated in inflamed colon tissues before malignancy occurs and whether increasing Dicer expression in inflamed colon tissues can alleviate colitis and prevent colitis-associated tumorigenesis. Methods: Gene expression in colon tissues was analyzed by immunohistochemistry, immunoblots, and real-time RT-PCR. Hydrogen peroxide or N-acetyl-L-cysteine was used to induce or alleviate oxidative stress, respectively. Mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors. Berberine, anastrozole, or pranoprofen was used to rescue Dicer expression in inflammatory colon tissues. Results: Oxidative stress repressed Dicer expression in inflamed colon tissues by inducing miR-215 expression. Decreased Dicer expression increased DNA damage and cytosolic DNA and promoted interleukin-6 expression upon hydrogen peroxide treatment. Dicer overexpression in inflamed colon tissues alleviated inflammation and repressed colitis-associated carcinogenesis. Furthermore, we found that anastrozole, berberine, and pranoprofen could promote Dicer expression and protect cells from hydrogen peroxide-induced DNA damage, thereby reducing cytosolic DNA and partially repressing interleukin-6 expression upon hydrogen peroxide treatment. Rescuing Dicer expression using anastrozole, berberine, or pranoprofen in inflamed colon tissues alleviated colitis and prevented colitis-associated tumorigenesis. Conclusions: Dicer was downregulated in inflamed colon tissues before malignancy occurred. Decreased Dicer expression further exaggerated inflammation, which may promote carcinogenesis. Anastrozole, berberine, and pranoprofen alleviated colitis and colitis-associated tumorigenesis by promoting Dicer expression. Our study provides insight into potential colitis treatment and colitis-associated colon cancer prevention strategies.

Degradation of endogenous proteins and generation of a null-like phenotype in zebrafish using Trim-Away technology.

Trim-Away is a recent technique to rapidly deplete a protein from any cell type. Guided by antibodies, TRIM21 selects proteins for destruction. However, the applicability of this method in model organisms has not been investigated. Here, we show that Trim-Away can degrade proteins in zebrafish embryos. Trim-Away depletes proteins faster than morpholinos, which enables analysis of protein function during early embryogenesis. Furthermore, Trim-Away can be applied to evaluate the role of maternally contributed proteins in zebrafish embryos. Our findings indicate that Trim-Away is a powerful tool to perform functional analysis of proteins during zebrafish development.

The signaling pathways that mediate the anti-cancer effects of caloric restriction.

Caloric restriction (CR) has been shown to promote longevity and ameliorate aging-associated diseases, including cancer. Extensive research over recent decades has revealed that CR reduces IGF-1/PI3K/AKT signaling and increases sirtuin signaling. We recently found that CR also enhances ALDOA/DNA-PK/p53 signaling. In the present review, we summarize the molecular mechanisms underlying the modulation of the IGF-1/PI3K/AKT pathway, sirtuin signaling, and the ALDOA/DNA-PK/p53 pathway by CR. We also summarize the evidence concerning the roles of these signaling pathways in carcinogenesis, and discuss how they are regulated by CR. Finally, we discuss the crosstalk between these signaling pathways.

MicroRNA-34a promotes MICB expression in hepatocytes.

MicroRNA-34a (miR-34a) behaves as a tumor suppressor by decreasing the expression of oncogenes involved in multiple carcinogenic pathways. Intravenous delivery of miR-34a mimics has been investigated in clinical trials as a potential treatment for advanced cancers; however, the effect of miR-34a on cancer immune surveillance is controversial. In the current study, we found that miR-34a plays a dual role in the regulation of major histocompatibility complex class I-related sequence B (MICB) protein, a ligand of the NKG2D receptor. MiR-34a could both induce and reduce MICB expression by upregulating ataxia telangiectasia and Rad3-related (ATR) protein kinase and downregulating the transcription factor E2F1, respectively. The net effect of miR-34a on MICB expression depended on endogenous E2F1 levels. Overexpression of miR-34a promoted MICB expression in hepatocytes and hepatocellular carcinoma (HCC) cells that have low E2F1 levels but not in HCC cells that have high E2F1 levels. In HCC patients, the expression of miR-34a and MICB showed positive correlation in paratumor liver tissues, which have low E2F1 levels, but not in HCC tissues, which have high E2F1 levels. We showed that miR-34a overexpression in non-transformed liver cells enhanced cytolysis and interferon-γ production by NK-92MI cells. Furthermore, higher miR-34a expression in tumor and paratumor tissues was associated with positive and negative outcomes, respectively, in HCC patients. Our findings suggest that miR-34a induces MICB expression in paratumor liver tissues, which may cause liver damage and serious cytokine release syndrome, thus disclosing potential side effects of systemic administration of miR-34a in anticancer therapy.

Dicer regulates non-homologous end joining and is associated with chemosensitivity in colon cancer patients.

DNA double-strand break (DSB) repair is an important mechanism underlying chemotherapy resistance in human cancers. Dicer participates in DSB repair by facilitating homologous recombination. However, whether Dicer is involved in non-homologous end joining (NHEJ) remains unknown. Here, we addressed whether Dicer regulates NHEJ and chemosensitivity in colon cancer cells. Using our recently developed NHEJ assay, we found that DSB introduction by I-SceI cleavage leads to Dicer upregulation. Dicer knockdown increased SIRT7 binding and decreased the level of H3K18Ac (acetylated lysine 18 of histone H3) at DSB sites, thereby repressing the recruitment of NHEJ factors to DSB sites and inhibiting NHEJ. Dicer overexpression reduced SIRT7 binding and increased the level of H3K18Ac at DSB sites, promoting the recruitment of NHEJ factors to DSBs and moderately enhancing NHEJ. Dicer knockdown and overexpression increased and decreased, respectively, the chemosensitivity of colon cancer cells. Dicer protein expression in colon cancer tissues of patients was directly correlated with chemoresistance. Our findings revealed a function of Dicer in NHEJ-mediated DSB repair and the association of Dicer expression with chemoresistance in colon cancer patients.

Clinical value of integrated-signature miRNAs in esophageal cancer.

MicroRNAs (miRNAs) are crucial regulators of gene expression in tumorigenesis and are of great interest to researchers, but miRNA profiles are often inconsistent between studies. The aim of this study was to confirm candidate miRNA biomarkers for esophageal cancer from integrated-miRNA expression profiling data and TCGA (The Cancer Genome Atlas) data in tissues. Here, we identify five significant miRNAs by a comprehensive analysis in esophageal cancer, and two of them (hsa-miR-100-5p and hsa-miR-133b) show better prognoses with significant difference for both 3-year and 5-year survival. Additionally, they participate in esophageal cancer occurrence and development according to KEGG and Panther enrichment analyses. Therefore, these five miRNAs may serve as miRNA biomarkers in esophageal cancer. Analysis of differential expression for target genes of these miRNAs may also provide new therapeutic alternatives in esophageal cancer.

The Fra-1-miR-134-SDS22 feedback loop amplifies ERK/JNK signaling and reduces chemosensitivity in ovarian cancer cells.

The Fra-1 transcription factor is frequently upregulated in multiple types of tumors. Here we found that Fra-1 promotes miR-134 expression. miR-134 activates JNK and ERK by targeting SDS22, which in turn induces Fra-1 expression and leads to miR-134 upregulation. In addition, miR-134 augmented H2AX S139 phosphorylation by activating JNK and promoted non-homologous end joining (NHEJ)-mediated DNA repair. Therefore, ectopic miR-134 expression reduced chemosensitivity in ovarian cancer cells. Furthermore, miR-134 promotes cell proliferation, migration and invasion of ovarian cancer cells, and enhances tumor growth in vivo. Of particular significance, both Fra-1 and miR-134 are upregulated in ovarian cancer tissues, and Fra-1 and miR-134 expression is positively correlated. High levels of miR-134 expression were associated with a reduced median survival of ovarian cancer patients. Our study revealed that a Fra-1-miR-134 axis drives a positive feedback loop that amplifies ERK/JNK signaling and reduces chemosensitivity in ovarian cancer cells.

The ID1-CULLIN3 Axis Regulates Intracellular SHH and WNT Signaling in Glioblastoma Stem Cells.

Inhibitor of differentiation 1 (ID1) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. Here, we report that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase. ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 and DVL2 and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and ß-CATENIN effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.