Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.

The sustained release of profibrotic cytokines, mainly transforming growth factor-ß (TGF-ß), leads to the occurrence of kidney fibrosis and chronic kidney disease (CKD). Connective tissue growth factor (CTGF) appears to be an alternative target to TGF-ß for antifibrotic therapy in CKD. In this study, we found that long noncoding RNA AI662270 was significantly increased in various renal fibrosis models. In vivo, ectopic expression of AI662270 alone was sufficient to activate interstitial fibroblasts and drive kidney fibrosis, whereas inhibition of AI662270 blocked the activation of interstitial fibroblasts and ameliorated kidney fibrosis in various murine models. Mechanistic studies revealed that overexpression of AI662270 significantly increased CTGF product, which was required for the role of AI662270 in driving kidney fibrosis. Furthermore, AI662270 binds to the CTGF promoter and directly interacts with METTL3, the methyltransferase of RNA N6 -methyladenosine (m6 A) modification. Functionally, AI662270-mediated recruitment of METTL3 increased the m6 A methylation of CTGF mRNA and consequently enhanced CTGF mRNA stability. In conclusion, our results support that AI662270 promotes CTGF expression at the posttranscriptional stage by recruiting METTL3 to the CTGF promoter and depositing m6 A modifications on the nascent mRNA, thereby, uncovering a novel regulatory mechanism of CTGF in the pathogenesis of kidney fibrosis.

Tubule-derived INHBB promotes interstitial fibroblast activation and renal fibrosis.

Upstream stimuli for myofibroblast activation are of considerable interest for understanding the mechanisms underlying renal fibrosis. Activin B, a member of the TGF-ß family, exists as a homodimer of inhibin subunit beta B (INHBB), but its role in renal fibrosis remains unknown. We found that INHBB expression was significantly increased in various renal fibrosis models and human chronic kidney disease specimens with renal fibrosis. Notably, the increase of INHBB occurred mainly in the tubular epithelial cells (TECs). In vivo, inhibiting INHBB blocked the activation of interstitial fibroblasts and ameliorated the renal fibrosis induced by unilateral ureteral obstruction or ischemia-reperfusion injury, while ectopic expression of INHBB in the TECs was able to activate interstitial fibroblasts and initiate interstitial fibrosis. In vitro, overexpression of INHBB in TECs led to the secretion of activin B, thereby promoting the proliferation and activation of interstitial fibroblasts through activin B/Smad signaling. Furthermore, inhibition of activin B/Smad signaling attenuated the fibrotic response caused by tubular INHBB. Mechanistically, the upregulation of INHBB depended on the transcription factor Sox9 in the injured TECs. Clinical analyses also identified a positive correlation between Sox9 and INHBB expression in human specimens, suggesting the Sox9/INHBB axis as a positive regulator of renal fibrosis. In conclusion, tubule-derived INHBB is implicated in the pathogenesis of renal fibrosis by activating the surrounding fibroblasts in a paracrine manner, thereby exhibiting as a potential therapeutic target. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Correlation of the detection rate of upper GI cancer with artificial intelligence score: results from a multicenter trial (with video).

BACKGROUND AND AIMS: The quality of EGD is a prerequisite for a high detection rate of upper GI lesions, especially early gastric cancer. Our previous study showed that an artificial intelligence system, named intelligent detection endoscopic assistant (IDEA), could help to monitor blind spots and provide an operation score during EGD. Here, we verified the effectiveness of IDEA to help evaluate the quality of EGD in a large-scale multicenter trial. METHODS: Patients undergoing EGD in 12 hospitals were consecutively enrolled. All hospitals were equipped with IDEA developed using deep convolutional neural networks and long short-term memory. Patients were examined by EGD, and the results were recorded by IDEA. The primary outcome was the detection rate of upper GI cancer. Secondary outcomes were part scores, total scores, and endoscopic procedure time, which were analyzed by IDEA. RESULTS: A total of 17,787 patients were recruited. The total detection rate of cancer-positive cases was 1.50%, ranging from .60% to 3.94% in each hospital. The total detection rate of early cancer-positive cases was .36%, ranging from .00% to 1.58% in each hospital. The average total score analyzed by IDEA ranged from 64.87 ± 16.87 to 83.50 ± 9.57 in each hospital. The cancer detection rate in each hospital was positively correlated with total score (r = .775, P = .003). Similarly, the early cancer detection rate was positively correlated with total score (r = .756, P = .004). CONCLUSIONS: This multicenter trial confirmed that the quality of the EGD result is positively correlated with the detection rate of cancer, which can be monitored by IDEA. (Clinical trial registration number: ChiCTR2000029001.).

Dual-Functionalized MSCs that Express CX3CR1 and IL-25 Exhibit Enhanced Therapeutic Effects on Inflammatory Bowel Disease.

Mesenchymal stem cells (MSCs) have shown great promise in inflammatory bowel disease (IBD) treatment, owing to their immunosuppressive capabilities, but their therapeutic effectiveness is sometimes thwarted by their low efficiency in entering the inflamed colon and variable immunomodulatory ability in vivo. Here, we demonstrated a new methodology to manipulate MSCs to express CX3C chemokine receptor 1 (CX3CR1) and interleukin-25 (IL-25) to promote their delivery to the inflamed colon and enhance their immunosuppressive capability. Compared to MSCs without treatment, MSCs infected with a lentivirus (LV) encoding CX3CR1 and IL-25 (CX3CR1&IL-25-LV-MSCs) exhibited enhanced targeting to the inflamed colon and could further move into extravascular space of the colon tissues via trans-endothelial migration in dextran sodium sulfate (DSS)-challenged mice after MSC intravenous injection. The administration of the CX3CR1&IL-25-LV-MSCs achieved a better therapeutic effect than that of the untreated MSCs, as indicated by pathological indices and inflammatory markers. Antibody-blocking studies indicated that the enhanced therapeutic effects of dual-functionalized MSCs were dependent on CX3CR1 and IL-25 function. Overall, this strategy, which is based on enhancing the homing and immunosuppressive abilities of MSCs, represents a promising therapeutic approach that may be valuable in IBD therapy.

Upregulation of HER2 in tubular epithelial cell drives fibroblast activation and renal fibrosis.

Tubular epithelial cell-derived profibrotic factors are known as the driving force in renal fibrosis for their roles in activating the surrounding fibroblast. However, the mechanisms driving their expressions remain undefined. Here, we find that kidney human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, significantly increased in unilateral ureteral obstruction-induced renal fibrosis, in type 1 and type 2 diabetic nephropathy, and in kidney biopsies from patients with renal fibrosis. Notably, the upregulation of HER2 mainly occurred in proximal tubular epithelial cells (PTECs). In vivo, the ectopic expression of HER2 in these cells was sufficient to activate the interstitial fibroblast and initiate interstitial fibrosis, whereas inhibiting HER2 reduced the accumulation of myofibroblasts and the extent of renal fibrosis in the mouse obstruction model and in streptozotocin (STZ)-induced diabetic mice. We also generated a tubular epithelial cell subline stably expressing HER2 and performed transcriptome RNA sequence analysis. This showed that sustained HER2 expression significantly induced the expression of profibrotic connective tissue growth factor (CTGF). Mechanistically, the induction of CTGF depended on the HER2-mediated activation of Stat3 in the tubular epithelium. In vitro, the incubation of kidney fibroblasts with culture medium from HER2-overexpressed tubular epithelial cells promoted fibroblast proliferation and activation, whereas silencing CTGF impeded the profibrotic effects of the tubular epithelial cell preconditioned media. Thus, our results highlight the significance of HER2 in tubular injury and characterize its role in promoting surrounding fibroblast activation and renal fibrosis in a paracrine manner.

TGF-ß-mediated upregulation of Sox9 in fibroblast promotes renal fibrosis.

TGF-ß signaling plays a principal role in renal fibrosis, but the precise mechanisms and the downstream factors are still largely unknown. Sox9 exhibits diverse roles in regulating the production of extracellular matrix proteins. Here we found that Sox9 was induced by TGF-ß in the kidney fibroblast and acted as an important downstream mediator of TGF-ß signaling in promoting renal fibrosis. TGF-ß/Smad signaling mediated the upregulation of Sox9 in kidney fibroblast by binding to a conserved enhancer. In different mouse models of renal fibrosis, as well as in the kidney biopsy tissue from patients with renal fibrosis, Sox9 expression significantly increased. Immunostaining confirmed the upregulation of Sox9 in the kidney fibroblast during renal fibrosis. Delivery of Sox9 knockdown plasmid to the kidney by ultrasound microbubble-mediated gene transfer suppressed the unilateral ureteral obstruction (UUO) or folic acid-induced mouse renal fibrosis, whereas ectopic expression of Sox9 aggravated renal fibrosis. In addition, we identified Sox9 as a direct target of miR-30. Notably, miR-30 expression was significantly inhibited by TGF-ß1 in the kidney fibroblast and the downregulation of miR-30 was observed in renal fibrosis. Mechanistically, inhibition of miR-30 independently strengthened the effect of TGF-ß/Smad signaling on Sox9 upregulation. Adenovirus-mediated ectopic expression of miR-30 in kidney fibroblast greatly reduced UUO-induced renal fibrosis by targeting Sox9. These findings link Sox9 to intrinsic mechanisms of TGF-ß signaling in renal fibrosis and may have therapeutic potential for tissue fibrosis.

MicroRNA-30b Suppresses Epithelial-Mesenchymal Transition and Metastasis of Hepatoma Cells.

Epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis in HCC. Growing evidence indicates that upregulation of Snail, the major EMT inducer, significantly correlates with the metastasis and poor prognosis of HCC. Here, we investigate the underlying mechanism of miR-30b in suppressing metastasis of hepatoma cells by targeting Snail. In this study, we found that miR-30b was significantly downregulated and negatively associated with Snail production in HCC cell lines with higher metastatic potentials. Gain- and loss-of-function studies revealed that miR-30b could dramatically inhibit in vitro HCC cell migration and invasion. In vivo orthotopic liver xenograft model further demonstrated that stable over-expression of miR-30b significantly repressed the local invasion and lung metastasis of hepatoma cells. Meanwhile, the restoration of miR-30b expression suppressed the distant colonization of hepatoma cells. Both gain- and loss-of-function studies showed that miR-30b suppressed the EMT of hepatoma cells as indicated by the morphology changes and deregulation of epithelial and mesenchymal markers. Using RNAi, we further investigated the role of Snail in HCC cell EMT and demonstrated that knockdown of Snail significantly inhibited the EMT and cancer cell metastasis. Additionally, miR-30b exhibited inhibitory effects on HCC cell proliferation in vitro and in vivo. In conclusion, our findings highlight the significance of miR-30b downregulation in HCC tumor metastasis and invasiveness, and implicate a new potential therapeutic target for HCC metastasis. J. Cell. Physiol. 232: 625-634, 2017. © 2016 Wiley Periodicals, Inc.

MicroRNA-125b-5p modulates the inflammatory state of macrophages via targeting B7-H4.

As a newly identified negative costimulatory molecule of B7 family, B7-H4 suppresses T cell function via inhibiting cell proliferation and cytokine secretion. Although B7-H4 mRNA is widely distributed in various tissues, its protein expression is strongly limited, suggesting B7-H4 may be regulated post-transcriptionally. However, the mechanism underlying the inducement of B7-H4 expression remains unclear. In the present study, we identified specific targeting sites for miR-125b-5p in the 3'-UTR of B7-H4 gene, and showed that overexpression of miR-125b-5p downregulated B7-H4 expression in macrophages. We also demonstrated that in the activated macrophages, B7-H4 expression could be significantly induced by dexamethasone treatment post-transcriptionally, and that the induction of B7-H4 expression was accomplished by inversely correlated alteration of miR-125b-5p level. Additionally, our data showed that the inflammatory state of macrophages was enhanced by miR-125b-5p at least partially via targeting B7-H4. Taken together, our results demonstrated for the first time that miR-125b-5p could regulate the inflammatory state of macrophages via directly targeting B7-H4.

Endoplasmic reticulum stress-mediated inflammatory signaling pathways within the osteolytic periosteum and interface membrane in particle-induced osteolysis.

Aseptic loosening secondary to periprosthetic inflammatory osteolysis results from the biological response to wear particles and is a leading cause of arthroplasty failure. The origin of this inflammatory response remains unclear. We aim to validate the definite link between endoplasmic reticulum (ER) stress and particle-induced inflammatory signaling pathways in periprosthetic osteolysis. We examine the histopathologic changes of osteolysis and the expression of specific biomarkers for ER-stress-mediated inflammatory signaling pathways (IRE1α, GRP78/Bip, c-Fos, NF-κB, ROS and Ca(2+)). Moreover, pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) and osteoclastogenic molecules (VEGF, OPG, RANKL and M-CSF) were assessed in clinical interface membranes and murine periosteum tissues. We found wear particles to be capable of inducing ER stress in macrophages within clinical osteolytic interface membranes and murine osteolytic periosteum tissues and to be associated with the inflammatory response and osteoclastogenesis. Blocking ER stress with sodium 4-phenylbutyrate (4-PBA) results in a dramatic amelioration of particle-induced osteolysis and a significant reduction of ER-stress intensity. Simultaneously, this ER-stress blocker also lessens inflammatory cell infiltration, diminishes the capability of osteoclastogenesis and reduces the inflammatory response by lowering IRE1α, GRP78/Bip, c-Fos, NF-κB, ROS and Ca(2+) levels. Thus, ER stress plays an important role in particle-induced inflammatory osteolysis and osteoclastogenic reactions. The pharmacological targeting of ER-stress-mediated inflammatory signaling pathways might be an appealing approach for alleviating or preventing particle-induced osteolysis in at-risk patients.

miR-124-3p functions as a tumor suppressor in breast cancer by targeting CBL.

BACKGROUND: The origin and development of breast cancer remain complex and obscure. Recently, microRNA (miRNA) has been identified as an important regulator of the initiation and progression of breast cancer, and some studies have shown the essential role of miR-124-3p as a tumor suppressor in breast tumorigenesis. However, the detailed role of miR-124-3p in breast cancer remains poorly understood. METHODS: Quantitative RT-PCR and western blotting assays were used to measure miR-124-3p and CBL expression levels in breast cancer tissues, respectively. Luciferase reporter assay was employed to validate the direct targeting of CBL by miR-124-3p. Cell proliferation and invasion assays were performed to analyze the biological functions of miR-124-3p and CBL in breast cancer cells. RESULTS: In the present study, we found that miR-124-3p was consistently downregulated in breast cancer tissues. Moreover, we showed that miR-124-3p significantly suppressed the proliferation and invasion of breast cancer cells. In addition, we investigated the molecular mechanism through which miR-124-3p contributes to breast cancer tumorigenesis and identified CBL (Cbl proto-oncogene, E3 ubiquitin protein ligase) as a direct target gene of miR-124-3p. Moreover, we found that ectopic expression of CBL can attenuate the inhibitory effect of miR-124-3p on cell proliferation and invasion in breast cancer cells. CONCLUSIONS: This study identified a new regulatory axis in which miR-124-3p and CBL regulate the proliferation and invasion of breast cancer cells.

Dual TNF-α/IL-12p40 Interference as a Strategy to Protect Against Colitis Based on miR-16 Precursors With Macrophage Targeting Vectors.

Cytokines are central components of the mucosal inflammatory responses that take place during the development of Crohn's disease. Cell-specific combination therapies against cytokines may lead to increased efficacy and even reduced side effects. Therefore, a colonic macrophage-specific therapy using miR-16 precursors that can target both TNF-α and IL-12p40 was tested for its efficacy in experimental colitic mice. Galactosylated low molecular weight chitosan (G-LMWC) associated with miR-16 precursors were intracolonically injected into mice. The cellular localization of miR-16 precursors was determined. The therapeutic effects and possible mechanism were further studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. The results show that specific upregulation of miR-16 level in colonic macrophages significantly reduces TNF-α and IL-12p40 expression, which could suppress the associated mucosal inflammation and ultimately result in the relief of colitic symptoms. This strategy, based on the dual silencing of colonic macrophage-specific cytokines, represents a potential therapeutic approach that may be valuable for colitis therapy.

Loss of MicroRNA-101 Promotes Epithelial to Mesenchymal Transition in Hepatocytes.

Epithelial-to-mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. However, the involvement of microRNA in the process of EMT remains to be fully defined in hepatocyte. ZEB1 is a well-known transcriptional repressor of E-cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis. Computational microRNA target predictions detect a conserved sequence matching to miR-101 in the 3'UTR of ZEB1 mRNA. Our results confirm that miR-101 suppresses ZEB1 expression by targeting the predicted site of ZEB1 3'UTR. Subsequent investigations show that miR-101 is significantly downregulated in the cultured hepatocytes undergoing EMT and in the hepatocytes isolated from fibrotic liver. Along with the loss of miR-101, the ZEB1 expression increases simultaneously in hepatocytes. In addition, miR-101 levels in HCC cell lines are negatively associated with the ZEB1 productions and the metastatic potentials of tumor cells. Mechanistically, we demonstrate that miR-101 significantly inhibits the TGF-ß1-induced EMT in hepatocytes, whereas inhibition of miR-101 promotes the EMT process as indicated by the changes of morphology, cell migration, and the expression profiles of EMT markers. In the fibrotic liver, ectopic expression of miR-101 can significantly downregulate ZEB1 in the hepatocyte and thereby reduces the mesenchymal marker expression. Moreover, miR-101 significantly inhibits the proliferation and migration of HCC cell. Our results demonstrate that miR-101 regulates HCC cell phenotype by upregulating the epithelial marker genes and suppressing the mesenchymal ones.

Identification of miR-199a-5p in serum as noninvasive biomarkers for detecting and monitoring osteosarcoma.

Emerging evidence has suggested that circulating microRNAs (miRNAs) in serum/plasma can serve as noninvasive biomarkers for cancer detection; however, little is known about circulating miRNA profiles in osteosarcoma, a primary malignant bone tumor with high morbidity in young adults and adolescents. The objective of this study was to investigate whether circulating miRNAs in serum could be a useful biomarker for detecting osteosarcoma and monitoring tumor dynamics. Serum samples were obtained from 60 patients before surgery, 28 patients after 1 month of surgery, and 60 healthy individuals. The study was divided into three steps: (1) initial screening of the profiles of circulating miRNAs in pooled serum samples from both healthy controls and pre- and postoperative osteosarcoma patients using a TaqMan low-density qPCR array (TLDA); (2) evaluation of miRNA concentration in individual serum samples from 60 preoperative osteosarcoma patients and 60 healthy controls by a quantitative RT-PCR assay; and (3) evaluation of miRNA concentration in paired serum samples from 28 pre- and postoperative osteosarcoma patients by a quantitative RT-PCR assay. The initial analysis showed that concentrations of serum miRNAs were significantly altered between preoperative osteosarcoma patients and healthy controls and between pre- and postoperative osteosarcoma patients. The quantitative RT-PCR assay showed that serum miR-199a-5p concentrations were significantly higher in osteosarcoma patients than in controls. The value of the area under the ROC curve was 0.8606. Serum levels of miR-199a-5p were significantly lower in post- than preoperative samples. The results indicated the potential of circulating miRNAs as novel noninvasive biomarkers for detecting and monitoring osteosarcoma.

MicroRNA-101 suppresses liver fibrosis by targeting the TGFß signalling pathway.

Transforming growth factor-ß (TGFß) is crucial for liver fibrogenesis and the blunting of TGFß signalling in hepatic stellate cells (HSCs) or hepatocytes can effectively inhibit liver fibrosis. microRNAs (miRNAs) have emerged as key regulators in modulating TGFß signalling and liver fibrogenesis. However, the regulation of TGFß receptor I (TßRI) production by miRNA remains poorly understood. Here we demonstrate that the miR-101 family members act as suppressors of TGFß signalling by targeting TßRI and its transcriptional activator Kruppel-like factor 6 (KLF6) during liver fibrogenesis. Using a mouse model of carbon tetrachloride (CCl4 )-induced liver fibrosis, we conducted a time-course experiment and observed significant down-regulation of miR-101 in the fibrotic liver as well as in the activated HSCs and injured hepatocytes in the process of liver fibrosis. Meanwhile, up-regulation of TßRI/KLF6 was observed in the fibrotic liver. Subsequent investigations validated that TßRI and KLF6 were direct targets of miR-101. Lentivirus-mediated ectopic expression of miR-101 in liver greatly reduced CCl4 -induced liver fibrosis, whereas intravenous administration of antisense miR-101 oligonucleotides aggravated hepatic fibrogenesis. Mechanistic studies revealed that miR-101 inhibited profibrogenic TGFß signalling by suppressing TßRI expression in both HSCs and hepatocytes. Additionally, miR-101 promoted the reversal of activated HSCs to a quiescent state, as indicated by suppression of proliferation and migration, loss of activation markers and gain of quiescent HSC-specific markers. In hepatocytes, miR-101 attenuated profibrogenic TGFß signalling and suppressed the consequent up-regulation of profibrogenic cytokines, as well as TGFß-induced hepatocyte apoptosis and the inhibition of cell proliferation. The pleiotropic roles of miR-101 in hepatic fibrogenesis suggest that it could be a potential therapeutic target for liver fibrosis.

miR-141 Regulates colonic leukocytic trafficking by targeting CXCL12ß during murine colitis and human Crohn's disease.

OBJECTIVE: Emerging evidence suggests that microRNA (miRNA)-mediated gene regulation influences a variety of autoimmune disease processes, including Crohn's disease (CD), but the biological function of miRNAs in CD remains unclear. We examine miRNA level in colon tissues and study the potential functions of miRNAs that regulate pathological genes during the inflammation process. DESIGN: miRNA levels were assayed in the inflamed colon of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced and IL-10 knockout (KO) chronic colitis mice and CD patients by microarray or qRT-PCR. The influence of differently expressed miR-141 on its putative target genes, CXCL12ß, and leukocyte migration was investigated in colonic epithelia cells, colitis models and CD patients. The role of miR-141 was further studied in the experimental colitis mice by intracolonic administration of miR-141 precursors or inhibitors. RESULTS: An inverse correlation between miR-141 and CXCL12ß/total-CXCL12 was observed predominantly in the epithelial cells of the inflamed colons from colitic mice and CD patients. Further study demonstrated that miR-141 directly regulated CXCL12ß expression and CXCL12ß-mediated leukocyte migration. Upregulation or downregulation of miR-141 in the TNBS-induced or IL-10 KO colitic colon regulated leukocyte infiltration and alleviated or aggravated experimental colitis, respectively. Additionally, colonic overexpression of CXCL12ß abolished the therapeutic effect of miR-141 in TNBS-induced colitis. CONCLUSIONS: This study showed that the pathway of miR-141 targeting CXCL12ß is a possible mechanism underlying inflammatory cell trafficking during colonic inflammation process. Inhibiting colonic CXCL12ß expression and blocking colonic immune cell recruitment by using miRNA precursors represents a promising approach that may be valuable for CD treatment.

The autoregulatory feedback loop of microRNA-21/programmed cell death protein 4/activation protein-1 (MiR-21/PDCD4/AP-1) as a driving force for hepatic fibrosis development.

Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-ß (TGF-ß) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.

MicroRNA-31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1).

MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3'-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

A 3-D artificial colon tissue mimic for the evaluation of nanoparticle-based drug delivery system.

Functional engineered nanoparticles are promising drug delivery carriers. As the construction of a functional nanocarrier always needs the optimization of multiple technical variables, efficient in vitro high-throughput evaluation methods would help to shorten the development cycle. In the present study, we generated a tissue mimic of the colon of inflammatory bowel disease (IBD) patients. Generally, Caco-2 cells and THP-1 cells were grown in a 3-D matrix with different number, spatial distribution and specific extracellular cell matrix (ECM) composition according to real healthy and inflamed animal colon tissues. After interlerukin-1ß/lipopolysaccharide (LPS) stimulation, the artificial model closely resembled the pathological features of IBD patient's colon, including massive cytokines and mucus production, epithelium defect and leukocytic infiltration. The tissue and cellular uptake of three different nanoparticles in the artificial model was similar to that in 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitic mice. Most importantly, our artificial tissue can be placed into 96-well plates for high-throughput screening of drug delivery carriers for the treatment of IBD. Our study suggested a readily achievable way to improve current methodologies for the development of colon targeted drug delivery systems.

Infliximab inhibits TNF-α-dependent activation of the NLRP3/IL-1ß pathway in acne inversa.

Background: Acne inversa (AI) is a refractory inflammatory skin disease, and TNF-α plays an important role in the pathogenesis of AI. By blocking TNF-α, infliximab (IFX) has been proven to be a promising method. Objectives: To explore the underlying mechanisms of IFX treatment in AI patients. Methods: In this research, we integrated transcriptome sequencing data from the samples of our patients with AI and the GEO database. Ex vivo skin culture of AI patients was conducted to evaluate the efficacy of IFX treatment. Animal studies and cell experiments were used to explore the therapeutic effect and mechanism of IFX treatment. Results: Both TNF-α and NLRP3 inflammasome-related pathways were enriched in skin lesions of AI patients and murine AI models. After IFX treatment, the NLRP3 inflammasome-related pathway was effectively blocked, and the IL-1ß level was normalized in ex vivo AI skin explants and murine AI models. Mechanistically, IFX suppressed the NF-κB signaling pathway to lower the expression of NLRP3 and IL-1ß in keratinocytes. Conclusions: IFX treatment alleviated skin lesions in murine AI models and downregulated NLRP3 and IL-1ß expression levels by inhibiting the NF-κB signaling pathway, which was helpful for understanding the mechanism of IFX therapy.

Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy.

The CRISPR-Cas9 system offers substantial potential for cancer therapy by enabling precise manipulation of key genes involved in tumorigenesis and immune response. Despite its promise, the system faces critical challenges, including the preservation of cell viability post-editing and ensuring safe in vivo delivery. To address these issues, this study develops an in vivo CRISPR-Cas9 system targeting tumor-associated macrophages (TAMs). We employ bacterial protoplast-derived nanovesicles (NVs) modified with pH-responsive PEG-conjugated phospholipid derivatives and galactosamine-conjugated phospholipid derivatives tailored for TAM targeting. Utilizing plasmid-transformed E. coli protoplasts as production platforms, we successfully load NVs with two key components: a Cas9-sgRNA ribonucleoprotein targeting Pik3cg, a pivotal molecular switch of macrophage polarization, and bacterial CpG-rich DNA fragments, acting as potent TLR9 ligands. This NV-based, self-assembly approach shows promise for scalable clinical production. Our strategy remodels the tumor microenvironment by stabilizing an M1-like phenotype in TAMs, thus inhibiting tumor growth in female mice. This in vivo CRISPR-Cas9 technology opens avenues for cancer immunotherapy, overcoming challenges related to cell viability and safe, precise in vivo delivery.