第8卷，第2期

Protein arginine methyltransferase 5 (PRMT5) deregulation has emerged as an important prognostic indicator in human cancers. Through aberrant methylation-mediated epigenetic modification of signaling molecules, PRMT5 overexpression contributes to dysregulation of a variety of cellular processes related to cancer progression. However, themechanismsgoverningPRMT5expressionlevelsincancer remain largely unknown. In this study, we examined factors that regulate PRMT5 expression at multiple levels. We mapped three regions of the proximal promoter of PRMT5 and identified NF-Ya, SMAD3, and ZNF143 as part of a key signature network node regulating PRMT5 expression in HT29 colorectal cancer (CRC) cells. Importantly, we provide evidence that knockdown or ligand induced activation of SMAD3, and ZNF143 led to changes in PRMT5 transcript and protein levels, respectively. We showed that PRMT5 expression positively correlates with both TGF-β2 and ZNF143 expression, suggesting that activation and/or upregulation of these proteins may be partly responsible for PRMT5 overexpression in a subset of CRC patients. Collectively, our data present a complex model that involves cell-autonomous induction of PRMT5 in CRC cells by transcriptional mechanisms and upregulation of PRMT5 mRNA and protein, which encompasses processes involving gene amplification and increased transcription and protein turnover rates.

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Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disorder characterized by mucocutaneous pigmentation and gastrointestinal (GI) hamartomatous polyposis and is associated with an increased risk of gastrointestinal, breast, gynecologic and other extra-GI malignancies. The serine/threonine kinase 11 (STK11) gene has been identified as a pathogenic factor in PJS. STK11 is a tumor suppressor gene located on chromosome 19p13.3 and includes 9 coding exons. The STK11 protein is composed of 433 amino acids (aa) and comprises a kinase catalytic region (aa 49-309) as well as N-and C-terminal regulatory domains. The catalytic region is further divided into 3 functional domains: an ATP-binding and orientation domain (aa 49-106), the catalytic site (aa 123-148), and a substratebinding domain (aa 171-225). STK11 plays an important role in tumorigenesis since it is implicated in numerous key biological processes involving cell metabolism, cell cycle regulation, cell polarity and motility, and angiogenesis. STK11 germline mutations have a wide genetic heterogeneity, and genotype-phenotype associations are not yet clearly established. Here we report a novel frameshift mutation involving the STK11 gene (c.388dupG, p. Glu130-Glyfs*33) in an Italian family, in which this mutation is associated with gastric lesions. In light of this finding, we performed a meta-analysis to ascertain whether STK11 nonsense and frameshift germline mutations may affect gastric cancer (GC) susceptibility in PJS patients based on their location. These data revealed a trend toward a higher risk of developing GC in PJS patients with STK11 truncating mutations in region aa 107-170.

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Lynch syndrome (LS) is a highly penetrant inherited cancer predisposition syndrome, characterized by autosomal dominant inheritance and germline mutations in DNA mismatch repair (MMR) genes, including MLH1, MSH2, MSH6 and PMS2. This study aimed to analyze the molecular defects and clinical manifestations of three LS families and propose individual prevention strategies suitable for mutation carriers in different families. The pathogenic gene mutations in each family were identified based on immunohistochemistry results combined with wholeexome sequencing, and the above gene mutations in the other family members were detected using Sanger sequencing. Two novel mutations (c.1927_1936del, p.I643fs and c.1668-2A > G) in the MLH1 gene and a known mutation (c.C2228T, p.S743L) in the MSH2 gene were identified, as pathogenic mutations for LS based on the sequencing data and pattern of tumorigenesis in the family members. The three mutations caused frameshift mutation and abnormal splicing of MLH1, as well as base replacement of MSH2, resulting in reduced mismatch repair activity of the MLH1 and MSH2 proteins. The findings expand the spectrum of known germline mutations of the MMR gene in the Chinese population, and reaffirm the importance of genetic testing for LS.

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Liver cancer presents divergent clinical behaviors. There remain opportunities for molecular markers to improve liver cancer diagnosis and prognosis, especially since tRNA-derived small RNAs (tsRNA) have rarely been studied. In this study, a random forests (RF) diagnostic model was built based upon tsRNA profiling of paired tumor and adjacent normal samples and validated by independent validation (IV). A LASSO model was used to developed a seven-tsRNA-based risk score signature for liver cancer prognosis. Model performance was evaluated by a receiver operating characteristic curve (ROC curve) and Precision-Recall curve (PR curve). The five-tsRNA-based RF diagnosis model had area under the receiver operating characteristic curve (AUROC) 88% and area under the precision-recall curve (AUPR) 87% in the discovery cohort and 87% and 86% in IV-AUROC and IV-AUPR, respectively. The seven-tsRNA-based prognostic model predicts the overall survival of liver cancer patients (Hazard Ratio 2.02, 95% CI 1.36-3.00, P< 0.001), independent of standard clinicopathological prognostic factors. Moreover, the model successfully categorizes patients into high-low risk groups. Diagnostic and prognostic modeling can be reliably utilized in the diagnosis of liver cancer and high- low risk classification of patients based upon tsRNA characterization.

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Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Knock-in mice carrying a CAG repeat-expanded Htt will develop HD phenotypes. Previous studies suggested dysregulated molecular networks in a CAG length genotype-and the age-dependent manner in brain tissues from knock-in mice carrying expanded Htt CAG repeats. Furthermore, a large-scale phenome analysis defined a behavioral signature for HD genotype in knock-in mice carrying expanded Htt CAG repeats. However, an integrated analysis correlating phenotype features with genotypes (CAG repeat expansions) was not conducted previously. In this study, we revealed the landscape of the behavioral features and gene expression correlations based on 445 mRNA samples and 445 microRNA samples, together with behavioral features (396 PhenoCube behaviors and 111 NeuroCube behaviors) in Htt CAG-knock-in mice. We identified 37 behavioral features that were significantly associated with CAG repeat length including the number of steps and hind limb stand duration. The behavioral features were associated with several gene coexpression groups involved in neuronal dysfunctions, which were also supported by the single-cell RNA sequencing data in the striatum and the spatial gene expression in the brain. We also identified 15 chemicals with significant responses for genes with enriched behavioral features, most of them are agonist or antagonist for dopamine receptors and serotonin receptors used for neurology/psychiatry. Our study provides further evidence that abnormal neuronal signal transduction in the striatum plays an important role in causing HD-related phenotypic behaviors and provided rich information for the further pharmacotherapeutic intervention possibility for HD.

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This study aimed to investigate the role of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) in chemosensitivity of osteosarcoma (OS), and to reveal the possible underlying mechanisms. In this study, we found that the expression of lncRNA MEG3 was significantly lower in OS tissues and cell lines. Furthermore, lncRNA MEG3 overexpression enhanced chemosensitivity of OS by inhibiting cell proliferation, migration, autophagy, and promoting antitumor immunity. LncRNA MEG3 functioned as miR-21-5 sponge to regulate p53 expression in OS. Mechanically, lncRNA MEG3 promoted OS chemosensitivity by regulating antitumor immunity via miR-21-5p/p53 pathway and autophagy. Collectively, this study provided the evidence that lncRNA MEG3 might be a promising therapeutic target for OS chemoresistance.

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Proximal tubule (PT) transports most of the renal Ca²⁺, which was usually described as paracellular (passive). We found a regulated Ca²⁺ entry pathway in PT cells via the apical transient receptor potential canonical 3 (TRPC3) channel, which initiates transcellular Ca²⁺ transport. Although TRPC3 knockout (-/-) mice were mildly hypercalciuric and displayed luminal calcium phosphate (CaP) crystals at Loop of Henle (LOH), no CaP + calcium oxalate (CaOx) mixed urine crystals were spotted, which are mostly found in calcium nephrolithiasis (CaNL). Thus, we used oral calcium gluconate (CaG; 2%) to raise the PT luminal[Ca²⁺]_o further in TRPC3^-/- mice for developing such mixed stones to understand the mechanistic role of PT-Ca²⁺ signaling in CaNL. Expectedly, CaG-treated mice urine samples presented with numerous mixed crystals with remains of PT cells, which were pronounced in TRPC3^-/- mice, indicating PT cell damage. Notably, PT cells from CaG-treated groups switched their mode of Ca²⁺ entry from receptor-operated to store-operated pathway with a sustained rise in intracellular[Ca²⁺] ([Ca²⁺]_i), indicating the stagnation in PT Ca²⁺ transport. Moreover, those PT cells from CaG-treated groups demonstrated an upregulation of calcification, inflammation, fibrotic, oxidative stress, and apoptotic genes; effects of which were more robust in TRPC3 ablated condition. Furthermore, kidneys from CaG-treated groups exhibited fibrosis, tubular injury and calcifications with significant reactive oxygen species generation in the urine, thus, indicating in vivo CaNL. Taken together, excess PT luminal Ca²⁺ due to escalation of hypercalciuria in TRPC3 ablated mice induced surplus CaP crystal formation and caused stagnation of PT[Ca²⁺]_i, invoking PT cell injury, hence mixed stone formation.

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Periodontitis is an oral chronic inflammatory disease. Inhibiting tissue destruction and promoting tissue regeneration are important means for the treatment of periodontitis. Metformin not only has hypoglycemic effect but also has anti-inflammatory effect. Metformin has been shown to inhibit oxidative stress and activate autophagy through AMPK/mTOR pathway. High mobility group box 1 (HMGB1) has been implicated in the pathogenesis of many inflammatory diseases including periodontitis, it can participate in the induction of oxidative stress. HMGB1 is an autophagy regulator under oxidative stress, which can activate mTOR pathway. However, it is not clear whether metformin is related to HMGB1 and its mechanism in the process of periodontitis. Cell viability and expression of inflammatory cytokines were clarified by Cell Counting Kit-8, real-time PCR and enzyme-linked immunosorbent assay. Western blot and immunofluorescence were conducted to determine HMGB1 intracellular localization and expression of autophagy-associated proteins in vitro. Experimental periodontitis mice model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression and localization of HMGB1 in vivo. The results of CCK-8, real-time PCR, enzyme-linked immunosorbent assay, Western blot and immunofluorescence showed lipopolysaccharide (LPS) treatment inhibited cell viability, and increased HMGB1 expression at a dose-independent manner. Metformin can reduce the effect of LPS. It also improves autophagy pathway inhibited by LPS and down-regulates mTOR expression. In addition, metformin attenuated alveolar bone resorption induced by ligation. This study provides new evidence for that metformin is a potential drug for the treatment of periodontitis and HMGB1 may be a potential target for periodontal intervention.

1315

Autophagy has been extensively studied and occurs in many biological settings. However, a question remains as to whether ischemia enhances Beclin-1/LC3-II-dependent macroautophagy in vascular endothelial cells, as has been previously thought. Furthermore, the effect of the level of autophagy on cell or skin flap survival still requires elucidation. We created a lethal ischemia model in human umbilical vascular endothelial cells (HUVECs), performed quantitative proteomics and bioinformatics analyses, and verified the autophagic status and effect both in vitro and in vivo. The significantly upregulated proteins encoded by autophagy-related genes (ATGs) included ATG2A, ATG3, ATG4B, ATG5, ATG7, ATG9A, ATG12, ATG16, and ATG101. The significantly enhanced lysosomal proteins were cathepsin B, cathepsin D, lysosome-associated membrane protein 1 (LAMP1), and LAMP2. However, the differentially expressed proteins excluded Beclin-1, microtubule-associated protein light chain 3 (LC3) -I, and LC3-II. Western blot analyses verified that the protein expression levels of Beclin-1, LC3-I, and LC3-II were neither upregulated nor downregulated in ischemia- challenged HUVECs. The autophagic status was not enhanced by rapamycin in ischemic HUVECs but appeared to be inhibited by chloroquine. Our in vivo study on rats showed that a downregulation in autophagic status jeopardized skin flap survival. In conclusion, Ischemia neither enhanced nor inhibited Beclin-1/LC3-II-dependent canonical macroautophagy both in vitro and in vivo, in contradiction to previous studies. An appropriate autophagic homeostasis can minimize cell or skin flap damage.

1207

Regulatory T (T_reg) cells constitute a dynamic population that is critical in autoimmunity. T_reg cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities. However, recent studies demonstrated that certain inflammatory conditions induce T_reg cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells. Therefore, the identification of novel targets crucial to both T_reg cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity. In this study, we found that conditional Pp6 knockout (cKO) in T_reg cells led to spontaneous autoinflammation, immune cell activation, and diminished levels of FoxP3 in CD4⁺ T cells in mice. Loss of Pp6 in T_reg cells exacerbated two classical mouse models of T_reg related autoinflammation. Mechanistically, Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression in T_reg cells. In summary, our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling, thus maintaining T_reg cell stability and preventing autoimmune diseases.

1255

KRAS is one of the most commonly mutated oncogenes in cancers and therapeutics directly targeting the KRas have been challenging. Among the different known mutants, KRas^G12C has been proved to be successfully targeted recently. Several covalent inhibitors selectively targeting KRas^G12C have shown promising efficacy against cancers harboring KRAS^G12Cmutation in clinical trials and AMG510 (sotorasib) has been approved for the treatment of KRAS^G12C-mutated locally advanced or metastatic non-small cell lung cancer. However, the overall responsive rate of KRas^G12C inhibitors was around 50% in patients with non-small cell lung cancer and the efficacy in patients with colorectal cancer or appendiceal cancer appears to be less desirable. It is of great importance to discover biomarkers to distinguish patients who are likely benefitted. Moreover, adaptive resistance would occur inevitably with the persistent administration like other molecularly targeted therapies. Several combinatorial regimens have been studied in an effort to potentiate the efficacy of KRas^G12C inhibitors in preclinical settings. This review summarized the recent progress of covalent KRas^G12C inhibitors with a focus on identifying biomarkers to predict or monitor the efficacy and proposing rational drug combinations based on elucidation of the mechanisms of drug resistance.

1264

Long non-coding RNAs (lncRNAs) are a novel class of non-coding RNA (ncRNA), that have been studied extensively in the field of tumor research in recent years. In the case of tumor-associated lncRNAs, lncRNA cytoskeleton regulator RNA (CYTOR) displays extensive functions in tumorigenesis, including invasion, metastasis, malignant proliferation, glycolysis, and inflammatory response. Moreover, the dysregulation of CYTOR is closely related to clinicopathological characteristics, such as tumor stage, lymph node metastasis and infiltration, and poor prognosis of tumor patients. In this review, we provide a novel strategy to summarize the biological functions and clinical value of CYTOR in tumors through an overview of the literature combined with gene set enrichment analysis. A deeper understanding of the role of CYTOR in tumorigenesis may provide new diagnostic, prognostic and therapeutic markers for human tumors.

1254

Breast cancer, an unceasingly occurring neoplasm, is one of the major determinants of mortality in women. Several ineffective attempts have been pursued using with conventional therapies against breast cancer. Resistance to existing therapies and their respective debilitating adverse effects have led research toward a new era of cancer treatment using viruses. Virotherapy constitutes a developing treatment modality with multiple mechanisms of therapeutic activity in which the viruses can be directly oncolyticand can express transgenes or induce host immune response against tumor cells. Several different DNA-and RNA-containing viruses have been considered for virotherapy of breast cancer including adenovirus, herpes virus, vaccinia, reovirus, Newcastle Disease virus, measles virus and vesicular stomatitis virus. This review aims to summarize the viro-therapeutical agents against breast malignancies. Key Scientific Concepts of Review: In this review paper, we proposed a new strategy to virus’s combinatorial treatments using several kinds of transgenes and drugs. These recombinant viruses have provided evidence of treatment efficacy against human breast cancer.

1241

Autophagy, as a special programmed cell death, is a critical degradative process that eliminates intracellular abnormal proteins or damage organelles to balance cell energy and favor cell metabolism with autophagy-related (ATG) proteins. Autophagy activation is being increasingly recognized as an essential hallmark in tumorigenesis through influencing the metabolism of stromal cells in the tumor microenvironment (TME) which comprises of tumor cells, cancer-associated fibroblasts (CAFs), cancer-associated endothelial cells (CAEs), immune cells and adipocytes. Tumor cells can reuse autophagy-involved recycling to maintain mitochondrial function and energy supply to meet the metabolic demand of their growth and proliferation. However, the mechanism through which autophagy can promote a crosstalk between tumor and stroma cells is not clear. Reprogramed metabolism is one of the main characteristics of TME leading to higher adaptability of tumor cells with diverse mechanisms. The activation of autophagy has expanded our understanding on the interaction between tumor metabolism and TME. The aim of this review is to report recent advances on the metabolic cross-talk between stromal cells and solid tumor cells induced by autophagy in TME and revealed potential therapeutic targets.

1317

We regret that two errors were made in "Lipopolysaccharide-induced podocyte injury is regulated by calcineurin/NFAT and TLR4/MyD88/NF-κB signaling pathways through Angiopoietin-like protein 4" (Genes & Disease DOI: 10.1016/j.gendis.2020.07.005). One is the strain of the mice described in the materials and methods, it should be C57BL/6 mice, but we erroneously wrote BALB/c mice which we didn't notice during the submission of the original manuscript. Besides, In Fig. S3a of the original manuscript, it should be the mRNA expression of CD14, but we erroneously showed the mRNA expression of Angptl4. The corrected Fig. S3a is as follows:

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