第7卷，第3期

Ferroptosis is a novel form of iron-dependent cell death characterized by lipid peroxidation. While the importance and disease relevance of ferroptosis is gaining recognition, much remains unknown about various genetic and non-genetic determinants of ferroptosis. Hippo signaling pathway is an evolutionarily conserved pathway that responds to various environmental cues and controls organ size, cell proliferation, death, and self-renewal capacity. In cancer biology, Hippo pathway is a potent tumor suppressing mechanism and its dysregulation contributes to apoptosis evasion, cancer development, metastasis, and treatment resistance. Hippo dysregulation leads to aberrant activation of YAP and TAZ, the two major transcription co-activators of TEADs, that induce the expression of genes triggering tumor-promoting phenotypes, including enhanced cell proliferation, self-renewal and apoptosis inhibition. The Hippo pathway is regulated by the cell-cell contact and cellular density/confluence. Recently, ferroptosis has also been found being regulated by the cellular contact and density. The YAP/TAZ activation under low density, while confers apoptosis resistance, renders cancer cells sensitivity to ferroptosis. These findings establish YAP/TAZ and Hippo pathways as novel determinants of ferroptosis. Therefore, inducing ferroptosis may have therapeutic potential for YAP/TAZ-activated chemo-resistant and metastatic tumor cells. Reciprocally, various YAP/TAZ-targeting treatments under clinical development may confer ferroptosis resistance, limiting the therapeutic efficacy.

1488

Aquaporins (AQPs) are highly conserved small transmembrane proteins, which are responsible for the water transport across the cell membrane. AQPs are abundantly expressed in numerous types of cells such as epithelial and endothelial cells. The expression of AQP-1, -3, -4, -5, -8 and-9 were found in the digestive system, where these six AQP isoforms serve essential roles including mediating the transmembrane water transport and regulating the secretion of gastrointestinal (GI) fluids, consequently facilitating the digestion and absorption of GI contents. In addition, the expression levels of AQPs are controlled by various factors, and AQPs can stimulate numerous signaling pathways; however, aberrant expression of AQPs in the GI tracts are associated with the initiation and development of numerous diseases. Thus, this review provides an overview of the expression and functions of AQPs in the digestive system.

1595

With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques, is less than 1 nM. During the last 3 decades, the implication of these endogenous CTS in the pathogenesis of hypertension and other volume-expanded disorders is widely disputed. The threshold for inhibition by CTS of human and rodent α1-Na, K-ATPase is ~1 and 1000 nM, respectively, that rules out the functioning of endogenous CTS (ECTS) as natriuretic hormones and regulators of cell adhesion, cell-to-cell communication, gene transcription and translation, which are mediated by dissipation of the transmembrane gradients of monovalent cations. In several types of cells ouabain and marinobufagenin at concentrations corresponding to its plasma level activate Na, K-ATPase, decrease the[Na⁺]_i/[K⁺]_i-ratio and increase cell proliferation. Possible physiological significance and mechanism of non-canonical -dependent and -independent cell responses to CTS are discussed.

1451

Inwardly rectifying potassium (Kir) channels make it easier for K⁺ to enter into a cell and subsequently regulate cellular biological functions. Kir5.1 (encoded by KCNJ16) alone can form a homotetramer and can form heterotetramers with Kir4.1 (encoded by KCNJ10) or Kir4.2 (encoded by KCNJ15). In most cases, homomeric Kir5.1 is non-functional, while heteromeric Kir5.1 on the cell membrane contributes to the inward flow of K⁺ions, which can be regulated by intracellular pH and a variety of signaling mechanisms. In the form of a heterotetramer, Kir5.1 regulates Kir4.1/4.2 activity and is involved in the maintenance of nephron function. Actually, homomeric Kir5.1 may also play a very important role in diseases, including in the ventilatory response to hypoxia and hypercapnia, hearing impairment, cardiovascular disease and cancer. With an increase in the number of studies into the roles of Kir channels, researchers are paying more attention to the pathophysiological functions of Kir5.1. This minireview provides an overview regarding these Kir5.1 roles.

1506

Epigenetic mechanisms play an important part in the regulation of gene expression and these alterations may induce long-term changes in gene function and metabolism. They have received extensive attention in bridging the gap between environmental exposures and disease development via their influence on gene expression. DNA methylation is the earliest discovered epigenetic alteration. In this review, we try to examine the role of DNA methylation and histone modification in Age related macular degeneration (AMD) and Diabetic Retinopathy (DR), its vascular complications and recent progress. Given the complex nature of AMD and DR, it is crucial to improve therapeutics which will greatly enhance the quality of life and reduce the burden for millions of patients living with these potentially blinding conditions.

1577

The nuclear factor kappa B (NF-κB) family of transcription factors plays an essential role as stressors in the cellular environment, and controls the expression of important regulatory genes such as immunity, inflammation, death, and cell proliferation. NF-κB protein is located in the cytoplasm, and can be activated by various cellular stimuli. There are two pathways for NF-κB activation, as the canonical and non-canonical pathways, which require complex molecular interactions with adapter proteins and phosphorylation and ubiquitinase enzymes. Accordingly, this increases NF-κB translocation in the nucleus and regulates gene expression. In this study, the concepts that emerge in different cellular systems allow the design of NF-κB function in humans. This would not only allow the development for rare diseases associated with NF-κB, but would also be used as a source of useful information to eliminate widespread consequences such as cancer or inflammatory/immune diseases.

1414

Despite the tremendous progresses toward our understanding of the mechanisms of how liver cancer was developed, the therapeutic outcomes of liver cancer in the clinic have very limited improvement within the past three decades or so. In addition, both the incidence and mortality of liver cancer worldwide are not dropping, but increasing steadily, in the last decade. Thus, it is time for us to rethink what has been wrong and how could we do better in the upcoming years, in order to achieve our goal of improving the therapeutic outcomes of patients with liver cancer in the clinic, and at the meantime, effectively reducing the incidence of liver cancer by blocking malignant transformation of hepatocytes from chronic viral infection. This is also one of the main reasons why we try to organize this special issue on primary liver cancer in the journal of Genes & Diseases. In this perspective, I will summarize the major obstacles confronted with in the prevention and management of patients with chronic hepatitis B infection and subsequent development of liver cirrhosis and liver cancer. Next, I will delineate the pitfalls and underlying mechanisms of why the current anti-viral strategies and therapeutic agents are not as effective as one expected in terms of successful reduction or prevention chronic hepatitis B infection associated liver cirrhosis and liver cancer. I will then provide my personal perspectives on potential approaches and strategies for effective prevention and management of hepatitis B-related liver cancer.

1895

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, puri fication of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Magbind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

1278

Metastasis and metabolism reprogramming are two major hallmarks of cancer. In the initiation and progression of cancer, tumor cells are known to undergo fundamental metabolic changes to sustain their development and progression. In recent years, much more attentions have been drawn to their important roles in facilitating cancer metastasis through regulating the biological properties. In this review, we summarized the recent progresses in the studies of metabolism reprogramming of cancer metastasis, particularly of primary liver cancer, and highlight their potential applications.

2017

The presence of VPS33B in tumors has rarely been reported. Downregulated VPS33B protein expression is an unfavorable factor that promotes the pathogenesis of lung adenocarcinoma (LUAD). Overexpressed VPS33B was shown to reduce the migration, invasion, metastasis, and chemoresistance of LUAD cells to cisplatin (DDP) in vivo and in vitro. Mechanistic analyses have indicated that VPS33B first suppresses epidermal growth factor receptor (EGFR) Ras/ERK signaling, which further reduces the expression of the oncogenic factor c-Myc. Downregulated c-Myc expression reduces the rate at which it binds the p53 promoter and weakens its transcription inhibition; therefore, decreased c-Myc stimulates p53 expression, leading to decreased epithelial-to-mesenchymal transition (EMT) signal. NESG1 has been shown to be an unfavorable indicator of non-small-cell lung cancer (NSCLC). Here, NESG1 was identified as an interactive protein of VPS33B. In addition, NESG1 was found to exhibit mutual stimulation with VPS33B via reduced RAS/ERK/c-Jun-mediated transcription repression. Knockdown of NESG1 activated EGFR/Ras/ERK/c-Myc signaling and further downregulated p53 expression, which thus activated EMT signaling and promoted LUAD migration and invasion. Finally, we observed that nicotine suppressed VPS33B expression by inducing PI3K/AKT/c-Jun-mediated transcription suppression. Our study demonstrates that VPS33B as a tumor suppressor is significantly involved in the pathogenesis of LUAD.

1500

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers globally. In contrast to the declining death rates observed for all other common cancers such as breast, lung, and prostate cancers, the death rates for HCC continue to increase by~2-3% per year because HCC is frequently diagnosed late and there is no curative therapy for an advanced HCC. The early diagnosis of HCC is truly a big challenge. Over the past years, the early diagnosis of HCC has relied on surveillance with ultrasonography (US) and serological assessments of alpha-fetoprotein (AFP). However, the specificity and sensitivity of US/AFP is not satisfactory enough to detect early onset HCC. Recent technological advancements offer hope for early HCC diagnosis. Herein, we review the progress made in HCC diagnostics, with a focus on emerging imaging techniques and biomarkers for early disease diagnosis.

3258

Sulindac has shown significant clinical benefit in preventing colorectal cancer progression, but its mechanism of action has not been fully elucidated. We have found that sulindac sulfide (SS) is able to inhibit cell cycle progression in human colorectal cancer cells, particularly through G1 arrest. To understand the underlying mechanisms of sulindac inhibitory activity, we have demonstrated that Cyclin G2 up-regulation upon SS treatment can substantially delay cell cycle progression by enhancing the transcriptional activity of FOXO3a in human colorectal tumor cells. MiR-182, an oncogenic microRNA known to inhibit FOXO3a gene expression, is also involved in the suppressive effect of SS on cell cycle progression. This process begins with the down-regulation of miR-182, followed by the enhancement of FOXO3a transcriptional activity and the up-regulation of Cyclin G2. To further determine the clinical utility of this axis, we analyzed the expression of miR-182/FOXO3a/Cyclin G2 in human colorectal tumor samples. Our results show not only that there are significant differences in miR-182/FOXO3a/Cyclin G2 between tumors and normal tissues, but also that the synergetic effect of miR-182 and FOXO3a is associated with predicting tumor progression. Our study demonstrates a novel mechanistic axis consisting of miR-182/FOXO3a/Cyclin G2 that mediates sulindac inhibition of cell cycle progression.

1675

The global incidence of liver cancer continues to grow. Liver cancer, especially hepatocellular carcinoma, has high recurrence and mortality rates. Here, we review the past decade's diagnostic, therapeutic, and management strategies for hepatocellular carcinoma, and summarize new patient management approaches, including enhanced recovery after surgery, targeted therapy, and immunotherapy. We compare traditional and innovative management methods, which comprise developments in precision medicine, and consider their limitations. Ongoing innovation and technological advances enable surgeons to gain deeper understandings of the multidimensionality of hepatocellular carcinoma, thereby promoting the continuous development of precision therapy.

1974

Liver cancer, mostly hepatocellular carcinoma (HCC), is the second leading cause of cancer mortality globally. Most patients were diagnosed at an advanced stage, and systemic therapy is the standard of care. All the approved systemic therapies for HCC are molecular targeted therapies with anti-angiogenic effects targeting the vascular endothelial growth factor signaling pathway. Sorafenib and lenvatinib are the first-line treatment, and regorafenib, ramucirumab, and cabozantinib are second-line treatment options. Although anti-PD-1 antibodies, including nivolumab and pembrolizumab, demonstrated promising anti-tumor effects as monotherapy for advanced HCC in phase II clinical trials, both failed in phase III studies. Anti-angiogenic treatment remains the backbone of systemic therapy for HCC. In this review, we summarized the approved anti-angiogenic medicines and discussed the potential strategies to improve the efficacy of anti-angiogenic therapy, including combination therapy with other treatments, and discussed the approaches to overcome the drawbacks of anti-angiogenic therapies.

1973

The transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) can promote hypoxic-ischemic brain damage (HIBD) nerve repair, but finding suitable seed cells to optimize transplantation and improve treatment efficiency is an urgent problem to be solved. In this study, we induced hUC-MSCs into dedifferentiated hUC-MSCs (De-hUC-MSCs), and the morphology, stem cell surface markers, proliferation and tri-directional differentiation ability of the De-hUC-MSCs and hUC-MSCs were detected. A whole-gene chip was utilized for genome cluster, gene ontology and KEGG pathway analyses of differentially expressed genes. De-hUC-MSCs were transplanted into HIBD rats, and behavioral experiments and immunofluorescence assays were used to assess the therapeutic effect. A lentivirus vector for human stromal cell-derived factor-1 (hSDF-1α) was constructed, and the role of hSDF-1α in the neuroprotective effect and mechanism of De-hUC-MSCs was verified. De-hUC-MSCs displayed similar cell morphology, stem cell surface marker expression, cell proliferation and even three-dimensional differentiation ability as hUC-MSCs but exhibited greater treatment potential in vivo. The reprogramming mechanism of hSDF-1α participated in the dedifferentiation process. By successfully constructing a stable hSDF-1α cell line, we found that De-hUC-MSCs might participate in nerve repair through the hSDF-1α/CXCR4/PI3K/Akt pathway. De-hUC-MSCs reprogramming of endogenous hSDF-1α expression may mediate the hSDF-1α/CXCR4/PI3K/Akt pathway involved in nerve repair in HIBD rats.

1243

Hepatocellular carcinoma (HCC) is one of the most common causes of cancerrelated death around the world due to advanced clinical stage at diagnosis, high incidence of recurrence and metastasis after surgical treatment. It is in urgent need to create appropriate animal models to explore the mechanism, patterns, risk factors, and therapeutic strategies of HCC metastasis and recurrence. However, most of the established models lack the phenotype of invasion and metastasis in patient, or have unstable phenotype. To establish HCC models with stable metastasis phenotype requires profound understanding in cancer metastasis biology and scientific methodology. Over the past 3 decades, HCC models with stable metastasis have been extensively studied. This paper reviewed the history and development of HCC animal models and cell models, focusing on the screening and maintaining of metastatic potential and phenotype. In-depth studies using these models vastly promote the understanding of cellular and molecular mechanisms and development of therapeutic strategies on HCC metastasis.

2027

Ubiquitin-specific peptidase 18 (USP18) plays an important role in the development of CD11b⁺ dendritic cells (DCs) and Th17 cells, however, its role in the differentiation of other T cell subsets, especially in regulatory T (Treg) cells, is unknown. In our study, we used Usp18 KO mice to study the loss of USP18 on the impact of Treg cell differentiation and function. We found that USP18 deficiency upregulates the differentiation of Treg cells, which may lead to disrupted homeostasis of peripheral T cells, and downregulates INF-γ, IL-2, IL-17A producing CD4⁺T cells and INF-g producing CD8⁺T cells. Mechanistically, we also found that the upregulation of Tregs is due to elevated expression of CD25 in Usp18 KO mice. Finally, we found that the suppressive function of Usp18 KO Tregs is downregulated. Altogether, our study was the first to identify the role of USP18 in Tregs differentiation and its suppressive function, which may provide a new reference for the treatment of Treg function in many autoimmune diseases, and USP18 can be used as a new therapeutic target for precise medical treatment.

1549

The phenomenon of enhanced invasion and metastasis of residual tumor cells has been observed in an increasing number of patients receiving chemoradiotherapy recently, and tumor metastasis will undoubtedly limit patient prognosis. However, the key mechanism by which chemoradiotherapy affects the invasion and metastasis of tumor cells remains unclear. Studies have shown that chemoradiotherapy may directly act on tumor cells and alter the tumor microenvironment, or induce cell apoptosis and autophagy to promote tumor cell survival and metastasis. In this review, we summarize the potential mechanisms by which chemoradiotherapy may affect the biological behavior of tumor cells and open up new avenues for reducing tumor recurrence and metastasis after treatment. These insights will improve the efficacy of chemoradiotherapy.

1898

Preeclampsia is a pregnancy complication which threatens the survival of mothers and fetuses. It originates from abnormal placentation, especially insufficient fusion of the cytotrophoblast cells to form the syncytiotrophoblast. In this study, we found that THBS1, a matricellular protein that mediates cell-to-cell and cell-to-matrix interactions, is downregulated during the fusion of primary cytotrophoblast and BeWo cells, but upregulated in the placenta of pregnancies complicated by preeclampsia. Also, THBS1 was observed to interact with CD36, a membrane signal receptor and activator of the cAMP signaling pathway, to regulate the fusion of cytotrophoblast cells. Overexpression of THBS1 inhibited the cAMP signaling pathway and reduced the BeWo cells fusion ratio, while the effects of THBS1 were abolished by a CD36-blocking antibody. Our results suggest that THBS1 signals through a CD36-mediated cAMP pathway to regulate syncytialization of the cytotrophoblast cells, and that its upregulation impairs placental formation to cause preeclampsia. Thus, THBS1 can serve as a therapeutic target regarding the mitigation of abnormal syncytialization and preeclampsia.

1475

Tumor recurrence rate after surgery or ablation of hepatocellular carcinoma (HCC) is as high as 70%. However, there are no widely accepted adjuvant therapies; therefore, no treatment has been recommended by guidelines from the American Association for the Study of Liver Disease or the European Association for the Study of the Liver. All the registered trials failed to find any treatment to prolong recurrence-free survival, which is the primary outcome in most studies, including sorafenib. Some investigator-initiated studies revealed that antihepatitis B virus agents, interferon-a, transcatheter chemoembolization, chemokine-induced killer cells, and other treatments prolonged patient recurrence-free survival or overall survival after curative therapies. In this review, we summarize the current status of adjuvant treatments for HCC and explain the challenges associated with designing a clinical trial for adjuvant therapy. Promising new treatments being used as adjuvant therapy, especially anti-PD-1 antibodies, are also discussed.

1900

Circular RNAs are a large class of noncoding RNAs. Smad5 functions in cell differentiation, cell proliferation and metastasis. It has been reported that lnc-Smad5 can inhibit the proliferation of diffuse large B cell lymphoma. However, the function of circ-Smad5 has not yet been reported. Lentivirus vectors were constructed to establish circ-Smad5 upregulated and circ-Smad5 downregulated cell models. A CCK-8 assay was used to detect the proliferation of JB6 cells. FACS was used to analyze the cell cycle in the cell models. Western blot, immunofluorescence staining and TOP/FOP flash dual luciferase activity assays were used to determine the activity of the Wnt signaling pathway. The results revealed that the expression level of circ-Smad5 in JB6 cells was significantly lower than the expression level of linearized-Smad5. Compared with the control group, the percentage of S phase cells and the expression level of cyclin D1 protein were significantly higher in the sh-circ-Smad5 group. In the sh-circ-Smad5 group, β-catenin and LEF-1 were significantly increased, p-b-catenin was significantly decreased, and the relative activity of the TOP/FOP reporter gene was higher compared to the control group levels. These phenomena could be reversed by treating with Wnt signaling inhibitor PNU-74654. We conclude that the circ-Smad5 retards the proliferation and the cell cycle progression of JB6 cells. Thus, circ-Smad5 may function by inhibiting the activation of Wnt/β-catenin/Lef 1 signaling, which inhibits the expression of cyclin D1. To the best of our knowledge, we are the first to report the function of circ-Smad5.

1586

In order to explore the genomic basis for liver cancer metastasis, whole-exome sequencing (WES) was performed on patient-derived hepatocellular carcinoma (HCC) cell lines with differential metastatic potentials and analyzed their clonal evolution relationships. An evolutionary tree based on genomic single nucleotide polymorphism (SNP) was constructed in MegaX software. The WES data showed that the average percentage of heterogeneous mutations in each HCC cell lines was 16.55% (range, 15.38%-18.17%). C: G > T: A and T: A > C: G somatic transitions were the two most frequent substitutions. In these metastatic HCC cell lines, non-silent gene mutations were found in 21.88% of known driver genes and 10 classical signaling pathways. The protein interaction network was constructed by STRING, and hub genes were found in the shared trunk mutation genes and the heterogeneous branch mutations respectively. In cBioPortal database, some of the selected hub genes were found to be associated with poor overall survival (OS) of HCC patients. Among the mutated HCC driver genes, a novel KEAP1 mutation with a homozygous frameshift truncation at the c-terminal Nrf2 binding region was detected and verified in MHCC97-H and HCC97LM3 cells. In conclusion, WES data demonstrate that HCC cell lines from tumor biopsy specimens of the same patient have obtained different metastatic potentials through repeated selection in rodents in vivo, and they do indeed have a genetic relationship at the genomic level.

1908

Ocular cells like, retinal pigment epithelium (RPE) is a highly specialized pigmented monolayer of post-mitotic cells, which is located in the posterior segment of the eye between neuro sensory retina and vascular choroid. It functions as a selective barrier and nourishes retinal visual cells. As a result of high-level oxygen consumption of retinal cells, RPE cells are vulnerable to chronic oxidative stress and an increased level of reactive oxygen species (ROS) generated from mitochondria. These oxidative stress and ROS generation in retinal cells lead to RPE degeneration. Various sources including mtDNA damage could be an important factor of oxidative stress in RPE. Gene therapy and mitochondrial transfer studies are emerging fields in ocular disease research. For retinal degenerative diseases stem cell-based transplantation methods are developed from basic research to preclinical and clinical trials. Translational research contributions of gene and cell therapy would be a new strategy to prevent, treat and cure various ocular diseases. This review focuses on the effect of oxidative stress in ocular cell degeneration and recent translational researches on retinal degenerative diseases to cure blindness.

1937

MiR-4295, located on chromosome 10q25.2, is a unique miRNA with a wide range of biological functions. miR-4295 is widely expressed in vivo, participating in the biological processes of multiple cancers. Although miR-4295 is dysregulated in various cancers, it has also been found to have the function of inhibiting cancer. At the same time, the expression of miR-4295 is related to prognosis and can be affected by numerous factors connecting to the therapeutic effects of various drugs. This article is to better summarize the role of miR-4295 in cancer and review the potential diagnostic, prognostic, and therapeutic value of miR-4295, which may provide insight into subsequent research.

1308

This review provides an updated account on the current methods, principles and mechanism of action of therapies for the detection of molecular markers of therapeutic importance in the prognosis of breast cancer progression and recurrence, which includes estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor2 (HER2). Indeed, hormone-receptors namely, ER, PR, proto-oncogene HER2 are the basic molecular markers that are recognized and established prognostic factors and predictors of response, for therapeutic practice. These markers can be detected by using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), which are established, faster and cost effective detection methods. These molecular markers along with clinicopathological prognostic parameters give the best prediction of the prognosis of cancer recurrence and progress. Finally, hormone receptors and HER2 as molecular markers are of prime therapeutic importance and have the capability to take part in future drug development techniques.

1334

Among the gynecological cancers, ovarian cancer is the most lethal. Its therapeutic options include a combination of chemotherapy with platinum-based compounds and cytoreductive surgery. Most ovarian cancer patients exhibit an initial response to platinum-based therapy, however, platinum resistance has led to up to 80% of this responsive cohort becoming refractory. Ovarian cancer recurrence and drug resistance to current chemotherapeutic options is a global challenge. Chemo-resistance is a complex phenomenon that involves multiple genes and signal transduction pathways. Therefore, it is important to elucidate on the underlying molecular mechanisms involved in chemo-resistance. This inform decisions regarding therapeutic management and help in the identification of novel and effective drug targets. Studies have documented the individual biomarkers of platinum-resistance in ovarian cancer that are potential therapeutic targets. This review summarizes the molecular mechanisms of platinum resistance in ovarian cancer, novel drug targets, and clinical outcomes.

1252

This study investigated the role of N6-methyladenosine RNA methylation in liver regeneration following partial hepatectomy in mice. We created a liver-specific knockout mouse model by the deletion of Mettl3, a key component of the N6-methyladenosine methyltransferase complex, using the albumin-Cre system. Mettl3 liver-specific knockout mice and their wild-type littermates were subjected to 2/3 partial hepatectomy. Transcriptomic changes in liver tissue at 48 h after partial hepatectomy were detected by RNA-seq. Immunohistochemistry and immunofluorescence were used to determine protein expression levels of Ki67, hepatocyte nuclear factor 4 alpha, and cytokeratin 19. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling was also performed. Liver weight/body weight ratios after partial hepatectomy were significantly lower in Mettl3 liverspecific knockout mice than in wild-type mice at 48 h after 2/3 partial hepatectomy (3.1%±0.11% vs.2.7%±0.03%). Compared with wild-type littermates, Mettl3 liver-specific knockout mice showed reduced bromodeoxyuridine staining and reduced Ki-67 expression at 48 h after 2/3 partial hepatectomy. RNA-seq analysis showed that Mettl3 liver-specific knockout delayed the cell cycle progression in murine liver by downregulating the expression levels of genes encoding cyclins D1, A2, B1, and B2. Loss of Mettl3-mediated N6-methyladenosine function led to attenuated liver regeneration by altering the mRNA decay of suppressor of cytokine signaling 6, thereby inhibiting the phosphorylation of signal transducer and activator of transcription 3 during early liver regeneration. These results demonstrated the importance of N6-methyladenosine mRNA modification in liver regeneration and suggest that Mettl3 targeting might facilitate liver regeneration.

1317

The deltex family protein DTX3 is believed to possess E3 ubiquitin ligase activity, as it contains a classic RING finger domain. However, its biological role and the underlying mechanism in cancer remain largely elusive. Here, we identified DTX3 as a novel mutant p53-interacting protein in ovarian carcinoma. Mechanistically, DTX3 mediated mutant p53 ubiquitination and stabilization by perturbing the MDM2-mutant p53 interaction, consequently leading to activation of diverse mutant p53 target genes. Importantly, a positive correlation between the expression of DTX3 and mutant p53 target genes was further validated in ovarian carcinomas. Ectopic DTX3 promoted, while depletion of DTX3 suppressed, ovarian cancer cell proliferation and invasion. Remarkably, the pro-tumorigenic effect of DTX3 is dependent on mutant p53, because ablation of mutant p53 significantly impaired DTX3-induced gene expression and ovarian cancer cell growth and propagation. Furthermore, DTX3 elevated the expression of mutant p53 target genes and boosted ovarian tumor growth in vivo. Finally, DTX3 was amplified and overexpressed in ovarian carcinomas, which is significantly associated with unfavorable prognosis. Altogether, our findings unveil the oncogenic role of DTX3 in ovarian cancer development by bolstering mutant p53 activity.

1311

The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.

1954

Osteosarcoma is the most common malignant bone tumour, and the metastasis of osteosarcoma is an important cause of death. Evidence has shown that the mevalonate pathway is highly activated and is expected to be a new target for tumour therapy. In this study, we investigated the effect of mevalonate signalling on osteosarcoma metastasis and its molecular mechanism. First, we found that the key rate-limiting enzyme of mevalonate signalling, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), was highly expressed in osteosarcoma cells, and inhibition of HMGCR with simvastatin significantly inhibited the motility of 143B cells. Next, we found that YAP1 activity was significantly upregulated in osteosarcoma cells and that YAP1 knockdown inhibited the motility of 143B cells. We also found that the mevalonate pathway regulated the motility of 143B cells by modulating YAP1 phosphorylation and cellular localization. Moreover, we found that the activity of YAP1 was regulated by the mevalonate pathway by modulating the cell membrane localization of RhoA. Finally, we demonstrated that inhibition of the mevalonate pathway notably reduced the lung metastasis of 143B cells, as reflected by the decreased incidence and number of metastatic nodules and the increased survival time of the nude mice. Taken together, our findings suggest that the mevalonate pathway can promote the metastasis of osteosarcoma by activating YAP1 via RhoA. Inhibition of the mevalonate pathway may be a promising therapeutic strategy for osteosarcoma metastasis.

1365

Excessive oxidative stress is a major causative factor of endothelial dysfunction in hypertension. As an endogenous pro-oxidant, thioredoxin-interacting protein (TXNIP) contributes to oxidative damage in various tissues. The present study aimed to investigate the role of TXNIP in mediating endothelial dysfunction in hypertension. In vivo, an experimental model of acquired hypertension was established with two-kidney, one-clip (2K1C) surgery. The expression of TXNIP in the vascular endothelial cells of multiple vessels was significantly increased in hypertensive rats compared with sham-operated rats. Resveratrol, a TXNIP inhibitor, suppressed vascular oxidative damage and increased the expression and activity of eNOS in the aorta of hypertensive rats. Notably, impaired endothelium-dependent vasodilation was effectively improved by TXNIP inhibition in hypertensive rats. In vitro, we observed that Ang II increased the expression of TXNIP in primary human aortic endothelial cells (HAECs) and that TXNIP knockdown by RNA interference alleviated cellular oxidative stress damage and mitigated the impaired eNOS activation and intracellular nitric oxide (NO) production observed in Ang II-treated HAECs. However, inhibiting thioredoxin (TRX) with PX-12 completely blunted the protective effect of silencing TXNIP. In addition, TXNIP knockdown facilitated TRX expression and promoted TRX nuclear translocation to further activate AP1 and REF1.TRX overexpression exhibited favorable effects on eNOS/NO homeostasis in Ang II-treated HAECs. Thus, TXNIP contributes to oxidative stress and endothelial dysfunction in hypertension, and these effects are dependent on the antioxidant capacity of TRX, suggesting that targeting TXNIP may be a novel strategy for antihypertensive therapy.

1257

A substantial number of male infertility is caused by azoospermia. However, the underlying etiology and the molecular basis remain largely unknown. Through single-cell (sc) RNA sequencing, we had analyzed testis biopsy samples from two patients with obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). We found only somatic cells in the NOA samples and explored the transcriptional changes in Sertoli cells in response to a loss of interactions with germ cells. Moreover, we observed a germ cell population discrepancy between an OA (postvasectomy) patient and a healthy individual. We confirmed this observation in a secondary study with two datasets at GSM3526588 and GSE124263 for detailed analysis wherein the regulatory mechanisms at the transcriptional level were identified. These findings thus provide valuable information on human spermatogenesis, and we also identified insightful information for further research on reproduction-related diseases.

1353

SIRT6 belongs to class III sirtuin family with NAD+-dependent histone deacetylase activities and controls multiple processes including aging, metabolism and inflammation. In recent years, increasing studies showed tumor suppressor role of SIRT6 in HCC development. We established a two-stage DEN followed CCl4 induced liver carcinogenesis in the hepatic-specific SIRT6 HKO mice models and found that hepatic SIRT6 deficit significantly promotes liver injury and liver cancer through inhibition of the ERK1/2 pathway. SIRT6 was compensatory upregulated in mice tumor tissues and human HCC cells and overexpressed SIRT6 inhibits tumor growth both in vitro and in vivo. Taken together, we provide a useful mouse model for delineating the molecular pathways involved in chronic liver diseases and primary liver cancer and suggest that SIRT6 can be a promising target for HCC therapies.

1384

Human idiopathic hypercalciuria (IH) is the most common cause of calcium oxalate nephrolithiasis with perturbed calcium metabolism with increased bone resorption and decreased renal calcium reabsorption, which can be phenotype-copied in the genetic hypercalciuric stone-forming (GHS) rat model. We previously demonstrated that high VDR expression plays important roles in the development of hypercalciuria in the GHS rats. However, the underlying mechanism through which VDR impact hypercalciuria development remains to be fully understood. Here, we sought to determine how VDR regulated its target genes that are implicated in calcium homeostasis and potentially hypercalciuria. We found that VDR expression in the GHS rats was elevated in the calcium transporting tissues, as well as in the thymus and prostate, but not in lung, brain, heart, liver and spleen, when compared with control SD rats. Snail expression in the GHS rats was significantly downregulated in kidney, intestine, thymus and testis. Intraperitoneal injection of 1, 25 (OH) ₂D₃ significantly upregulated the expression of renal calcium sensing receptor (CaSR), intestinal calcium transporters transient receptor potential vanilloid type 6 (TRPV6), and VDR in GHS rats, compared with that in control SD rats. ChIP assays revealed that VDR specifically bound to the proximal promoters of target genes, followed by histone H3 hyperacetylation or hypermethylation. Collectively, our results suggest that elevated VDR expression may contribute to the development of hypercalciuria by sensitizing VDR target genes to 1, 25 (OH) ₂D₃ through histone modifications at their promoter regions in a genetic hypercalciuric stone-forming (GHS) rat model.

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The BRCA1-PALB2-BRCA2 axis, or the BRCA pathway, plays key roles in genome stability maintenance and suppression of breast and several other cancers. Due to frequent p53 mutations in human BRCA1 breast cancers and mouse mammary tumors from Brca1, Brca2 and Palb2 conditional knockout models, it is often thought that p53 inactivation accelerates BRCA1/2 and PALB2-associated tumorigenesis. Here, we studied tumor development in mice with a mutation in Palb2 that disengages the PALB2-BRCA1 interaction in different Trp53 backgrounds. Rather than mammary tumors, Palb2 and Trp53 compound mutant mice developed, with greatly reduced latencies, lymphomas and sarcomas that are typically associated with germline Trp53 inactivation. Whole exome sequencing failed to identify any significant differences in genomic features between the same tumor types of Trp53 single mutant and Palb2; Trp53 compound mutant mice. These results suggest that loss of the BRCA pathway accelerates p53-associated tumor development, possibly without altering the fundamental tumorigenic processes.

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DLC1 is a focal adhesion molecule that regulates cell polarity, proliferation, migration, and survival. DLC1 functions as a tumor suppressor and its expression is often downregulated in various malignant neoplasms of epithelial origin. Recent studies have suggested that lack of DLC1 in endothelial cells may contribute to the development of angiosarcoma, and that DLC1 mutations have been identified in patients with nephrotic syndrome, a disease mainly due to leaky glomerular filtration barriers. To demonstrate whether lack of endothelial DLC1 induces angiosarcoma and/or damages glomerular capillaries leading to nephrotic syndrome, we have extended our analyses on endothelial cell-specific DLC1 knockout mice with focuses on their liver and kidney function. Mice were monitored up to 24 months of age. However, no histological or clinical difference was found between DLC1 knockout and wild type mice, indicating that lack of endothelial DLC1 alone does not compromise kidney and liver function in mice.

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In order to assess Stathmin as an immunohistochemical (IHC) indicator of phosphatidylinositol 3-kinase (PI3K) pathway activity in HPV-negative head & neck squamous cell carcinoma (HNSCC), we compared Stathmin IHC to expression of other pathway components. We also evaluated the relationship between Stathmin IHC and the mutational status of four key pathway genes. Finally, we ascertained whether Stathmin IHC correlates with tumor grade or primary site. Correlation exists between high Stathmin expression and high pAKT1 expression, indicating a role for Stathmin IHC as a marker of pathway activity. Our analysis did not show correlation between Stathmin IHC and mutation of the four genes evaluated. We also observed an association between high Stathmin expression and oropharyngeal primary site. Our results suggest utility of Stathmin IHC as an indicator of PI3K pathway activity, and thereby demonstrate potential relevance of Stathmin IHC in the context of HNSCC.

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