The aim of this study was to investigate the mechanism underlying autophagy deficiency during hepatic carcinogenesis. For this purpose, we used choline-deficient, amino acid-defined (CDAA) hepatocarcinogenesis model in mice. miRNA microarrays combined with computational target predictions and GO analysis were used to identify molecular processes involved in carcinogenesis. PCR profiler array was employed to detect the dysregulated autophagy-related genes during carcinogenesis. We observed induction of hepatic tumours with increased inflammation, DNA damage, and cell death. These cellular processes were particularly detected upon oncogenic transformation of hepatocytes in which ER stress was excessively induced. Microarray combined with GO analysis showed that transformation of hepatocytes resulted in dysregulated events associated with cytoplasmic vesicle formation, which, in turn, was related to ER stress-induced autophagy. Defects of autophagy were observed in livers harbouring tumours and suffered a loss of expression of autophagy-related protein 9b (Atg9b). Hepatocytes lacking Atg9b were vulnerable to cell death induced by ER stress stimulus mainly caused by accumulation of ubiquitinated proteins. Loss of Atg9b also blocked recruitment of p62-associated ubiquitinated protein for autophagosome-lysosome degradation as Atg9b-driven phagophores may facilitate docking of both LC3 and p62 to initiate autophagy-associated degradation. miR-3091-3p from tumour-derived exosomes, which were internalised by hepatocytes, could suppress Atg9b expression. Observations from this study advance our knowledge about the regulation of autophagy during hepatocarcinogenesis.

Cancer stem cell-like cells (CSCL) are responsible for tumor recurrence associated with conventional therapy (e.g. surgery, radiation, and chemotherapy). Here, we developed a novel multifunctional nucleus-targeting nanoparticle-based gene delivery system which is capable of targeting and eradicating CSCL. These nanoparticles can facilitate efficient endosomal escape and spontaneously penetrate into nucleus without additional nuclear localization signal. They also induced extremely high gene transfection efficiency (>95%) even in culture medium containing 30% serum, which significantly surpassed that of some commercial transfection reagents, such as Lipofectamine 2000 and Lipofectamine 3000 etc. Especially, when loaded with the TRAIL gene, this system mediated remarkable depletion of CSCL. Upon systemic administration, the nanoparticles accumulated in tumor sites while sparing the non-cancer tissues and significantly inhibited the growth of tumors with no evident systemic toxicity. Taken together, our results suggest that these novel multifunctional, nucleus-targeting nanoparticles are a very promising in vivo gene delivery system capable of targeting CSCL and represent a new treatment candidate for improving the survival of cancer patients.

Despite promising progress of cancer gene therapy made, these therapeutics were still limited by the diversity of gene sizes and types. CRISPR/dCas9 mediated activation of tumor endogenous gene has shown great potential to surmount hinders of genetic varieties during the process of cancer gene therapy. However, the blood interference along with complicated tumor extra/intracellular microenvironment substantially compromise the performance of CRISPR/dCas9-based therapeutics in vivo. Methods: In this study, we constructed a programmable hierarchical-responsive nanoCRISPR (PICASSO) that can achieve sequential responses to the multiple physiological barriers in vivo. The core-shell structure endows PICASSO with long blood circulation capacity and tumor target accumulation as well as efficient cellular uptake and lysosomal escape, leading to high-performance of CRISPR/dCas9-mediated gene activation, which favors the antitumor efficacy. Results: Owing to these properties, PICASSO facilitated CRISPR/dCas9 mediated efficient transcriptional activation of various types of endogenous gene, and long non-protein-coding genes (LncRNA) containing targets ranging in size from ~1 kb to ~2000 kb in tumor cells. Intravenous administration of PICASSO to the tumor-bearing mice can achieve effective transcriptional activation of therapeutic endogenous gene, resulting in remarkable CRISPR/dCas9-mediate tumor inhibition with minimal adverse effect. Conclusions: Taken together, these characteristics allow PICASSO to unleash the potential of CRISPR/dCas9-based therapeutics in oncological treatment. The study provides a simple and versatile strategy to break through the restriction of sizes and types against cancer by utilization of tumor endogenous gene.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

Rationale: Cisplatin-based chemotherapy is the first-line treatment for late-stage head and neck squamous cell carcinoma (HNSCC). However, resistance to cisplatin has become a major obstacle for effective therapy. Cancer stem cells (CSCs) are critical for tumor initiation, growth, metastasis, and chemoresistance. How to effectively eliminate CSCs and overcome chemoresistance remains a key challenge. Herein, we confirmed that MYC plays critical roles in chemoresistance, and explored targeting MYC to overcome cisplatin resistance in preclinical models. Methods: The roles of MYC in HNSCC cisplatin resistance and cancer stemness were tested in vitro and in vivo. The combined therapeutic efficiency of MYC targeting using the small molecule MYC inhibitor MYCi975 and cisplatin was assessed in a 4‑nitroquinoline 1-oxide-induced model and in a patient-derived xenograft model. Results: MYC was highly-expressed in cisplatin-resistant HNSCC. Targeting MYC using MYCi975 eliminated CSCs, prevented metastasis, and overcame cisplatin resistance. MYCi975 also induced tumor cell-intrinsic immune responses, and promoted CD8+ T cell infiltration. Mechanistically, MYCi975 induced the DNA damage response and activated the cGAS-STING-IRF3 signaling pathway to increase CD8+ T cell-recruiting chemokines. Conclusions: Our findings suggested that targeting MYC might eliminate CSCs, prevent metastasis, and activate antitumor immunity to overcome cisplatin resistance in HNSCC.

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by ATP7B pathogenic mutations. The symptoms of WD can be effectively prevented if the affected individuals are identified and intervened early. However, clinical utility of this molecular analysis is challenging due to hundreds of variants with various clinical effects in the gene. Here, we aim to describe the spectrum of ATP7B variants and assess their clinical effects in the Han Chinese population.

Background: Imaging amyloid-beta (Aβ) deposits with high fidelity in naturally aging brains is crucial for the early diagnosis of Alzheimer's disease (AD). However, this is impeded by the lack of highly sensitive probes. Methods: By conducting computational modelling to quantitatively fine-tune the twisted intramolecular charge transfer (TICT) tendency of Thioflavin T (ThT) analogues, we developed an ultrasensitive probe AH-2. AH-2 retained the binding affinity and binding mode of ThT towards Aβ deposits, and exhibited ca 10-fold less background fluorescence and 5-10 folds of improved signal-to-background contrast upon binding Aβ deposits. These desirable features endowed AH-2 the sensitivity to detect Aβ deposition in naturally aging wild-type mice. Results: AH-2 imaging revealed that Aβ puncta signals appeared near the nuclei in young mice and spread through the intracellular and extracellular compartments in older mice. Moreover, Aβ deposits were observed to emerge earlier in mice cerebral cortex than in the hippocampus region. Given this desirable sensitivity and good spatiotemporal resolution, AH-2 was successfully applied in the preclinical evaluation of Aβ-targeted treatment by melatonin. Conclusions: We expect that AH-2 is promising for early diagnosis of AD and will serve as a sensitive tool for studying Aβ-related AD pathology.

Myocardial infarction (MI) is identified as one of the major causes of mortality and disability worldwide. For severe myocardial infarction, even advanced forms of clinical intervention often lead to unsatisfactory therapeutic results. Thus, alternative strategies for MI treatment are still desirable. Previously studies reported the capacity of degradative fragment of h-HA (high molecular weight hyaluronic acid), hyaluronan oligosaccharides (<10 disaccharides units, o-HA), for wound healing by influence on angiogenesis, inspiring us to study its potential for myocardial functional recovery against MI. However, there are few reports about o-HA in MI therapy. Methods: In our study, we synthesized o-HA with 6~10 disaccharides (4-5 kDa) by enzymatic degradation and investigated its therapeutic effects on MI. Results: We found that o-HA could reduce infarct size and apoptosis in MI region, also promote myocardial angiogenesis and myocardial function reconstruction in MI mouse model. Furthermore, our results also indicated that o-HA in cardiac improved polarization of M2 type macrophage, removed the inflammatory response caused by neutrophil for accelerating myocardial function reconstruction in vivo. The transcriptomic analyses revealed that o-HA could activate expression of chemokines Ccl2 and Cxcl5 for promoting macrophage polarization and stimulate MAPK and JAK/STAT signaling pathway for compensatory response of myocardial function. Conclusion: Collectively, our results suggested o-HA with 6~10 disaccharides might be a potential agent for reconstruction of cardiac function against MI.

Chemoresistance in breast cancer has been of great interest in past studies. However, the development of rational therapeutic strategies targeting chemoresistant cells is still a challenge in clinical oncology. By integrating data from global differences of gene expression and phospho-receptor tyrosine kinases between sensitive parental cells (MCF-7) and doxorubicin-resistant cells (MCF-7/ADR), we identified Axl as a potential target for chemoresistance and metastasis in multidrug resistant breast cancer cells. We analyzed Axl expression in 57 breast cancer cell lines and detected a dramatic increase in its expression level in mesenchymal breast cancer cell lines. Axl silencing suppressed invasive and metastatic potentials of chemoresistant breast cancer cells as well as increased elimination of cancer cells when combined with doxorubicin. Furthermore, in preclinical assays, an Axl inhibitor R428 showed increased cell death upon doxorubicin treatment. Additionally, using phospho-kinase array based proteomic analysis, we identified that Akt/GSK-3β/β-catenin cascade was responsible for Axl-induced cell invasion. Nuclear translocation of β-catenin then induced transcriptional upregulation of ZEB1, which in turn regulated DNA damage repair and doxorubicin-resistance in breast cancer cells. Most importantly, Axl was correlated with its downstream targets in tumor samples and was associated with poor prognosis in breast cancer patients. These results demonstrate that Gas6/Axl axis confers aggressiveness in breast cancer and may represent a therapeutic target for chemoresistance and metastasis.

Recently, near-infrared (NIR) light-based photothermal therapy (PTT) has been widely applied in cancer treatment. However, in most cases, the tissue penetration depth of NIR light is not sufficient and thus photothermal therapy is unable to completely eradicate deep, seated tumors inevitably leading to recurrence of the tumor. Due to this significant limitation of NIR, improved therapeutic strategies are urgently needed. Methods: We developed an endogenous vaccine based on a novel nanoparticle platform for combinatorial photothermal ablation and immunotherapy. The design was based on fluorophore-loaded liposomes (IR-7-lipo) coated with a multivalent immunoadjuvant (HA-CpG). In vitro PTT potency was assessed in cells by LIVE/DEAD and Annexin V-FITC/PI assays. The effect on bone marrow-derived dendritic cells (BMDC) maturation and antigen presentation was evaluated by flow cytometry (FCM) with specific antibodies. After treatment, the immune cell populations in tumor micro-environment and the cytokines in the serum were detected by FCM and Elisa assay, respectively. Finally, the therapeutic outcome was investigated in an animal model. Results: Upon irradiation with 808 nm laser, IR-7-lipo induced tumor cell necrosis and released tumor-associated antigens, while the multivalent immunoadjuvant improved the expression of co-stimulatory molecules on BMDC and promoted antigen presentation. The combination therapy of PTT and immunotherapy regulated the tumor micro-environment, decreased immunosuppression, and potentiated host antitumor immunity. Most significantly, due to an enhanced antitumor immune response, combined photothermal immunotherapy was effective in eradicating tumors in mice and inhibiting tumor metastasis. Conclusion: This endogenous vaccination strategy based on synergistic photothermal and immunotherapy may provide a potentially effective approach for treatment of cancers, especially those difficult to be surgically removed.

Background: Activation of the thermogenic program in white and brown adipocytes presents a promising avenue for increasing energy expenditure during the treatment of obesity. The endogenous mechanism for promoting thermogenesis in brown adipocytes or browning in white adipocytes has indicated that the gut microbiota is a crucial regulator of the host energy balance. However, whether the effects of the therapeutic intervention-induced modulation of the gut microbiota on adipocyte browning involved the regulation of leptin remains unclear. Method: The adipose features were analyzed by body composition analysis, infrared camera observations, transmission electron microscopy and H&E staining. The gene and protein expression in adipose tissue were detected by qRT-PCR, immunoblotting, immunohistochemistry and immunofluorescence staining. The gut microbiome signature was identified by 16S rRNA gene amplicon sequencing, and both mice with high-fat diet-induced obesity (DIO) and mice with antibiotics-induced microbiome depletion were subjected to fecal microbiota transplantation. Results: Treatment with Panax notoginseng saponins (PNS) shaped the murine gut microbiome by increasing the abundances of Akkermansia muciniphila and Parabacteroides distasonis, and as a result, DIO mice harbored a distal gut microbiota with a significantly increased capacity to reduce host adiposity. The PNS-induced modulation of the gut microbiota in DIO mice could increase brown adipose tissue (BAT) thermogenesis and beige adipocyte reconstruction by activating the leptin-AMPK/STAT3 signaling pathway, which results in the promotion of energy expenditure. Leptin has an essential influence on the anti-obesity effects of PNS. In cases of leptin deficiency, the PNS-induced modulation of the gut microbiota exerts negative effects on thermogenesis and browning in white adipose tissue (WAT), which indicates that PNS fail to reduce obesity in leptin gene-deficient mice. The PNS-induced modulation of the gut microbiota exerted a minimal effect on DIO mice with antibiotic-induced microbiome depletion, which confirmed the correlation between altered gut microbiota and the remodeling of adipose tissues in DIO mice. The direct influence of leptin on browning via the AMPKα/STAT3 signaling pathway in C3H101/2 cells supported our in vivo results that signalling through the leptin-AMPK/STAT3 pathway induced by the PNS-modulated gut microbiota was involved in beige adipocyte reconstruction. Conclusion: Our results revealed that leptin signaling is critical for alterations in microbiota-fat crosstalk and provide promising avenues for therapeutic intervention in the treatment of obesity.

Rationale: The rennin-angiotensin-aldosterone system (RAAS) plays a critical role in the pathogenesis of diabetic cardiomyopathy, but the role of a member of RAAS, angiotensin IV (Ang IV), in this disease and its underlying mechanism are unclear. This study was aimed to clarify the effects of Ang IV and its downstream mediator forkhead box protein O1 (FoxO1) on diabetic cardiomyopathy. Methods:In vivo, diabetic mice were treated with low-, medium- and high-dose Ang IV, AT4R antagonist divalinal, FoxO1 inhibitor AS1842856 (AS), or their combinations. In vitro, H9C2 cardiomyocytes and cardiac fibroblasts were treated with different concentrations of glucose, low-, medium- and high-dose Ang IV, divalinal, FoxO1-overexpression plasmid (FoxO1-OE), AS, or their combinations. Results: Ang IV treatment dose-dependently attenuated left ventricular dysfunction, fibrosis, and myocyte apoptosis in diabetic mice. Besides, enhanced autophagy and FoxO1 protein expression by diabetes were dose-dependently suppressed by Ang IV treatment. However, these cardioprotective effects of Ang IV were completely abolished by divalinal administration. Bioinformatics analysis revealed that the differentially expressed genes were enriched in autophagy, apoptosis, and FoxO signaling pathways among control, diabetes, and diabetes+high-dose Ang IV groups. Similar to Ang IV, AS treatment ameliorated diabetic cardiomyopathy in mice. In vitro, high glucose stimulation increased collagen expression, apoptosis, overactive autophagy flux and FoxO1 nuclear translocation in cardiomyocytes, and upregulated collagen and FoxO1 expression in cardiac fibroblasts, which were substantially attenuated by Ang IV treatment. However, these protective effects of Ang IV were completely blocked by the use of divalinal or FoxO1-OE, and these detrimental effects were reversed by the additional administration of AS. Conclusions: Ang IV treatment dose-dependently attenuated left ventricular dysfunction and remodeling in a mouse model of diabetic cardiomyopathy, and the mechanisms involved stimulation of AT4R and suppression of FoxO1-mediated fibrosis, apoptosis, and overactive autophagy.

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis.

Myocardial infarction (MI), a main cause of heart failure, leads to irreversible cardiomyocytes loss and cardiac function impairment. Current clinical treatments for MI are largely ineffective as they mostly aim to alleviate symptoms rather than repairing the injured myocardium. Thus, development of more effective therapies is compelling. This study aims to investigate whether the extracellular vesicles (EVs) carrying specific anti-apoptotic miRNA can be efficiently internalized into myocardium to achieve desired therapeutic outcomes. Methods: EVs were isolated from HEK293T cells overexpressing miRNA-21 (miR21-EVs) and identified. The RNase resistant rate of miR21-EVs was calculated by real-time PCR and compared with liposomes and polyethylenimine (PEI). Confocal laser scanning microscopy was used for visualizing the cellular internalization of miR21-EVs in primary cultured mouse neonatal cardiomyocytes (CMs), H9c2 rat cardiomyoblasts, and human umbilical vein endothelial cells (HUVECs). The effect of miR21-EVs on the expression of PDCD4, a pro-apoptotic protein that plays an important role in regulating myocardial apoptosis, was also evaluated in these three cell types by real-time PCR and Western blot analysis. In vivo, miR21-EVs was directly injected into the infarct zone following ligation of the left anterior descending of coronary artery in mice. The miR21-EVs distribution and blood vessel (capillary and arteriole) density were evaluated by immunofluorescence staining. Fluorescence in situ hybridization of miRNA-21 was also carried out to confirm the miR21-EVs distribution in vitro and in vivo. The protein level of PDCD4 in myocardium was assessed by immunohistochemical staining. The anti-apoptotic effect of miR21-EVs in cardiomyocytes and endothelial cells were measured using TUNEL staining. Four weeks after injection, the cardiac histological and functional recovery was evaluated by histochemistry staining and echocardiography, respectively. Results: In contrast to liposomes and PEI, EVs significantly inhibited miRNA-21 degradation. MiR21-EVs efficiently delivered miRNA-21 into cardiomyocytes and endothelial cells within 4 hours. Exogenous miRNA-21 in turn significantly reduced PDCD4 expression and attenuated cell apoptosis in vitro. Consistently and importantly, in a preclinical MI animal model, miRNA-21-loaded EVs effectively sent miRNA-21 into cardiomyocytes and endothelial cells, drastically inhibited cell apoptosis and led to significant cardiac function improvement. Conclusion: Our results suggest the cell-derived, genetically engineered EVs may be used therapeutically for the delivery of miRNAs for the rescue of MI and may benefit patients in the future.

Organ ischemia reperfusion injury (IRI), associated with acute hepatocyte death, remains an unresolved problem in clinical orthotopic liver transplantation (OLT). Autophagy, an intracellular self-digesting progress, is responsible for cell reprograming required to regain post-stress homeostasis. Methods: Here, we analyzed the cytoprotective mechanism of pituitary adenylate cyclase-activating polypeptide (PACAP)-promoted hepatocellular autophagy in a clinically relevant mouse model of extended hepatic cold storage (4 °C UW solution for 20 h) followed by syngeneic OLT. Results: In contrast to 41.7% of liver graft failure by day 7 post-transplant in control group, PACAP treatment significantly improved graft survival (91.7% by day 14), and promoted autophagy-associated regeneration programs in OLT. In parallel in vitro studies, PACAP-enhanced autophagy ameliorated cellular damage (LDH/ALT levels), and diminished necrosis in H2O2-stressed primary hepatocytes. Interestingly, PACAP not only induced nuclear cAMP response element-binding protein (CREB), but also triggered reprogramming factor Kruppel-like factor 4 (KLF4) expression in IR-stressed OLT. Indeed, CREB inhibition attenuated hepatic autophagy and recreated hepatocellular injury in otherwise PACAP-protected livers. Furthermore, CREB inhibition suppressed PACAP-induced KLF4 expression, whereas KLF4 blockade abolished PACAP-promoted autophagy and neutralized PACAP-mediated hepatoprotection both in vivo and in vitro. Conclusion: Current study documents the essential neural regulation of PACAP-promoted autophagy in hepatocellular homeostasis in OLT, which provides the emerging therapeutic principle to combat hepatic IRI in OLT.

Rationale: Abnormal autophagic death of endothelial cells is detrimental to plaque structure as endothelial loss promotes lesional thrombosis. As emerging functional biomarkers, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular. This study is aimed to determine the role of hsa_circ_0030042 in abnormal endothelial cell autophagy and plaque stability. Methods: circRNA sequencing and quantitative polymerase chain reaction were performed to detect hsa_circ_0030042 expression in coronary heart disease (CHD) and human umbilical vein endothelial cells (HUVECs). Transfection of stubRFP-sensGFP-LC3 adenovirus, flow cytometry, and electron microscopy were used to identify the role of hsa_circ_0030042 in ox-LDL‒induced abnormal autophagy in vitro. Bioinformatic analysis, RNA immunoprecipitation, immunofluorescence assay and other in vitro experiments were performed to elucidate the mechanism underlying hsa_circ_0030042-mediated regulation of autophagy. To evaluate the role of hsa_circ_0030042 in atherosclerotic plaques and endothelial function, we measured the carotid artery tension and performed histopathology and immunohistochemistry analysis. Results: hsa_circ_0030042 was significantly downregulated in CHD, while upon overexpression, it acted as an endogenous eukaryotic initiation factor 4A-III (eIF4A3) sponge to inhibit ox-LDL-induced abnormal autophagy of HUVECs and maintain plaque stability in vivo. Furthermore, hsa_circ_0030042 influenced autophagy by sponging eIF4A3 and blocking its recruitment to beclin1 and forkhead box O1 (FOXO1) mRNA, while hsa_circ_0030042-induced inhibition of beclin1 and FOXO1 was counteracted by eIF4A3 overexpression or decreased hsa_circ_0030042 binding. In high-fat-diet fed ApoE-/- mice, hsa_circ_0030042 also ameliorated plaque stability and counteracted eIF4A3-induced plaque instability. Conclusions: These results demonstrate a novel pathway involving hsa_circ_0030042, eIF4A3, FOXO1, and beclin1; hence, modulating their levels may be a potential therapeutic strategy against CHD.

Background: Lung cancer has the highest mortality rate among cancers worldwide, with non-small cell lung cancer (NSCLC) the most common type. Increasing evidence shows that PHB2 is highly expressed in other cancer types; however, the effects of PHB2 in NSCLC are currently poorly understood. Method: PHB2 expression and its clinical relevance in NSCLC tumor tissues were analyzed using a tissue microarray. The biological role of PHB2 in NSCLC was investigated in vitro and in vivo using immunohistochemistry and immunofluorescence staining, gene expression knockdown and overexpression, cell proliferation assay, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, wound healing assay, Transwell assay, western blot analysis, qRT-PCR, coimmunoprecipitation, and mass spectrometry analysis. Results: Our major finding is that PHB2 facilitates tumorigenesis in NSCLC by interacting with and stabilizing RACK1, which further induces activation of downstream tumor-promoting effectors. PHB2 was found to be overexpressed in NSCLC tumor tissues, and its expression was correlated with clinicopathological features. Furthermore, PHB2 overexpression promoted proliferation, migration, and invasion, whereas PHB2 knockdown enhanced apoptosis in NSCLC cells. The stimulating effect of PHB2 on tumorigenesis was also verified in vivo. In addition, PHB2 interacted with RACK1 and increased its expression through posttranslational modification, which further induced activation of the Akt and FAK pathways. Conclusions: Our results reveal the effects of PHB2 on tumorigenesis and its regulation of RACK1 and RACK1-associated proteins and downstream signaling in NSCLC. We believe that the crosstalk between PHB2 and RACK1 provides us with a great opportunity to design and develop novel therapeutic strategies for NSCLC.

CRISPR-Cas9 is a Nobel Prize-winning robust gene-editing tool developed in the last decade. This technique enables a stable genetic engineering method with high precision on the genomes of all organisms. The latest advances in the technology include a genome library screening approach, which can detect survival-essential and drug resistance genes via gain or loss of function. The versatile machinery allows genomic screening for gene activation or inhibition, and targets non-coding sequences, such as promoters, miRNAs, and lncRNAs. In this review, we introduce the emerging high-throughput CRISPR-Cas9 library genome screening technology and its working principles to detect survival and drug resistance genes through positive and negative selection. The technology is compared with other existing approaches while focusing on the advantages of its variable applications in anti-cancer drug discovery, including functions and target identification, non-coding RNA information, actions of small molecules, and drug target discoveries. The combination of the CRISPR-Cas9 system with multi-omic platforms represents a dynamic field expected to advance anti-cancer drug discovery and precision medicine in the clinic.