Functional genomics in a mammalian model such as mice is fundamental for understanding human biology. The CRISPR/Cas9 system dramatically changed the tempo of obtaining genetic mouse models due to high efficiency. However, experimental evidence for the establishment of sgRNA knock-in animals and analyses of their value in functional genomics are still not sufficient, particularly in mammalian models. In this study, we demonstrate that the establishment of sgRNA knock-in mice is feasible, and more importantly, crosses between sgRNA knock-in mice and the Cas9 constitutively expressing mice result in complete deletion of the target gene. Such sgRNA knock-in provides an alternative approach for in vivo genetic modification and can be useful in multiple circumstances, such as maintenance of genetically modified animals, which are difficult to breed as homozygotes, and cross of such mice to diverse genomic backgrounds to obtain genetically modified animals.

Ultraviolet (UV) radiation is a major environmental factor contributing skin damage. As UV exposure is inevitable, it is necessary to pay attention to the underlying molecular mechanisms of UV-induced skin damage to develop effective therapies. tRNA-derived stress-induced RNAs (tiRNAs) and tRNA-derived fragments (tRFs) are tRNA-derived small RNAs (tsRNAs) that are a novel class of short, non-coding RNAs. However, the functions behind tRFs & tiRNAs in UV-induced skin injury are not yet clear. Firstly, the animal model of ultraviolet irradiation induced skin damage was established. Then the skin samples were preserved for the follow-up experiment. Sequencing was used to screen expression profiles and predict target genes. Compared with normal skin, a total of 31 differentially expressed tRFs & tiRNAs were screened. Among these, 10 tRFs & tiRNAs were shown to be significantly different in expression levels, where there were 4 up-regulated and 6 down-regulated target genes. Bioinformatics analyses revealed potential up-regulated tsRNAs (tRF-Val-AAC-012, tRF-Pro-AGG-012, tRF-Val-CAC-018, tRF-Val-AAC-031) and down-regulated tsRNAs (tRF-Arg-CCT-002, tRF-Trp-TCA-001, tiRNA-Ser-GCT-001, tRF-Gly-CCC-019, tRF-Ala-TGC-001, tRF-Ala-TGC-002). In summary, it was speculated that tRF-Gly-CCC-019 plays an important role in acute skin injury induced by UVB radiation by regulating the ras-related C3 botulinum toxin substrate 1 (Rac1) gene in the WNT signaling pathway. This study provides new insights into the mechanisms and therapeutic targets of UV-induced skin injury.

CTC1 is a component of the mammalian CST (CTC1-STN1-TEN1) complex which plays essential roles in resolving replication problems to facilitate telomeric DNA and genomic DNA replication. We previously reported that the depletion of CTC1 leads to stalled replication fork restart defects. Moreover, the mutation in CTC1 caused cancer-prone diseases including Coats plus (CP) or dyskeratosis congenita (DC). To better understand the CTC1 regulatory axis, the microRNAs (miRNAs) targeting to CTC1 were predicted by a bioinformatics tool, and the selected candidates were further confirmed by a dual-luciferase reporter assay. Here, our current results revealed that miR-376a significantly reduced CTC1 expression at the transcription level by recognizing CTC1 3'-UTR. In addition, the overexpression of miR-376a induced telomere replication defection and resulted in direct replicative telomere damage, which could be rescued by adding back CTC1. Telomere shortening was also observed upon miR-376a treatment. Furthermore, for the clinical patient samples, the high expression of miR-376a was associated with the deregulation of CTC1 and a poor outcome for the rectum adenocarcinoma patients. Together, our results uncovered a novel role of miR-376a in stimulating rectum adenocarcinoma progression via CTC1 downregulating induced telomere dysfunction.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. The activation of the toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-κB (TLR4/MyD88/NF-κB) pathway contributes to the development and progression of HCC. The ubiquitin-proteasome system regulates TLR4 expression. However, whether ubiquitin specific peptidase 13 (USP13) stabilizes TLR4 and facilitates HCC progression remains unclear. Here, quantitative real-time PCR (qRT-PCR) and immunohistochemistry analysis revealed that USP13 expression in HCC tissues was higher than in non-tumor liver tissues. Moreover, the elevated expression of USP13 was detected in HCC cells (SK-HEP-1, HepG2, Huh7, and Hep3B) compared to LO2 cells. Interestingly, the positive staining of USP13 was closely correlated with tumor size ≥ 5 cm and advanced tumor stage and conferred to significantly lower survival of HCC patients. Next, USP13 knockdown prominently reduced the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of Hep3B and Huh7 cells, while USP13 overexpression enhanced these biological behaviors of HepG2 and LO2 cells. The silencing of USP13 significantly restrained the growth and lung metastasis of HCC cells in vivo. Mechanistically, the USP13 depletion markedly inhibited the TLR4/MyD88/NF-κB pathway in HCC cells. USP13 interacted with TLR4 and inhibited the ubiquitin-mediated degradation of TLR4. Significantly, TLR4 re-expression remarkably reversed the effects of USP13 knockdown on HCC cells. USP13 expression was markedly upregulated in HCC cells under hypoxia conditions. Notably, USP13 knockdown repressed hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC cells. In conclusion, our study uncovered that hypoxia-induced USP13 facilitated HCC progression via enhancing TLR4 deubiquitination and subsequently activating the TLR4/MyD88/NF-κB pathway.

RNA N6-methyladenosine (m6A) modification in tumorigenesis and progression has been highlighted and discovered in recent years. However, the molecular and clinical implications of m6A modification in melanoma tumor microenvironment (TME) and immune infiltration remain largely unknown. Here, we utilized consensus molecular clustering with nonnegative matrix factorization based on the melanoma transcriptomic profiles of 23 m6A regulators to determine the m6A modification clusters and m6A-related gene signature. Three distinct m6A modification patterns (m6A-C1, C2, and C3), which are characterized by specific m6A regulator expression, survival outcomes, and biological pathways, were identified in more than 1,000 melanoma samples. The immune profile analyses showed that these three m6A modification subtypes were highly consistent with the three known immune phenotypes: immune-desert (C1), immune-excluded (C2), and immune-inflamed (C3). Tumor digital cytometry (CIBERSORT, ssGSEA) algorithm revealed an upregulated infiltration of CD8+ T cell and NK cell in m6A-C3 subtype. An m6A scoring scheme calculated by principal component of m6A signatures stratified melanoma patients into high- and low-m6sig score subgroups; a high score was significantly associated with prolonged survival and enhanced immune infiltration. Furthermore, fewer somatic copy number alternations (SCNA) and PD-L1 expression were found in patients with high m6Sig score. In addition, patients with high m6Sig score demonstrated marked immune responses and durable clinical benefits in two independent immunotherapy cohorts. Overall, this study indicated that m6A modification is involved in melanoma tumor microenvironment immune regulation and contributes to formation of tumor immunogenicity. Comprehensive evaluation of the m6A modification pattern of individual tumors will provide more insights into molecular mechanisms of TME characterization and promote more effective personalized biotherapy strategies.

Fetal growth restriction (FGR) is a worldwide problem, and a major cause of perinatal morbidity. The precise molecular mechanisms involved in placental development and function during FGR remain poorly understood. Circular RNAs (circRNAs) are important biological molecules associated with disease pathogenesis. However, the role of circRNAs in FGR has not been well studied.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is one of the most severe complications of systemic sclerosis (SSc) and is the leading cause of SSc-related deaths. However, the precise pathogenesis of pulmonary fibrosis in SSc-ILD remains unknown. This study aimed to evaluate the competing endogenous RNA (ceRNA) regulatory network and immune cell infiltration patterns in SSc-ILD.

Cervical cancer is the fourth most prevalent cancer in women, which decreases quality of life of the patients. Traditional interventions have failed to improve the overall survival period of patients due to high tumor recurrence after treatment or late diagnosis. Fortunately, preliminary evidence suggests that anti-angiogenic and immunotherapy can efficiently treat against cervical cancer. However, there is no clear evidence on the efficacy of immunotherapy in cervical cancer. Therefore, in this study, we classified cervical cancers in the TCGA dataset using various algorithms and explored the relationship between the immune profile and corresponding sensitivity of the tumors to immunotherapy. Results showed that patients with tumors had higher expression of immunocytes and longer overall survival time. In addition, we build a scoring system based on the immune landscape of the tumor microenvironment of cervical cancer. Tumors with higher scores exhibited better survival outcomes and were more sensitive to immunotherapy. In this study, the immune landscape of cervical cancer was analyzed, and the subtype of cervical cancer based on that difference was proposed. Besides, the subtype of cervical cancer showed different sensitivity to immunotherapeutic response which further confirmed its relationship with tumor immune landscape.

SCL/TAL1 Interrupting locus (STIL) is a ciliary-related gene involved in regulating the cell cycle and duplication of centrioles in dividing cells. STIL has been found disordered in multiple cancers and driven carcinogenesis. However, the molecular mechanisms and biological functions of STIL in cancers remain ambiguous. Here, we systematically analyzed the genetic alterations, molecular mechanisms, and clinical relevance of STIL across >10,000 samples representing 33 cancer types in The Cancer Genome Atlas (TCGA) dataset. We found that STIL expression is up-regulated in most cancer types compared with their adjacent normal tissues. The expression dysregulation of STIL was affected by copy number variation, mutation, and DNA methylation. High STIL expression was associated with worse outcomes and promoted the progression of cancers. Gene Ontology (GO) enrichment analysis and Gene Set Variation Analysis (GSVA) further revealed that STIL is involved in cell cycle progression, Mitotic spindle, G2M checkpoint, and E2F targets pathways across cancer types. STIL expression was negatively correlated with multiple genes taking part in ciliogenesis and was positively correlated with several genes which participated with centrosomal duplication or cilia degradation. Moreover, STIL silencing could promote primary cilia formation and inhibit cell cycle protein expression in prostate and kidney cancer cell lines. The phenotype and protein expression alteration due to STIL silencing could be reversed by IFT88 silencing in cancer cells. These results revealed that STIL could regulate the cell cycle through primary cilia in tumor cells. In summary, our results revealed the importance of STIL in cancers. Targeting STIL might be a novel therapeutic approach for cancers.

Proper differentiation of odontoblasts is crucial for the development of tooth roots. Previous studies have reported the osteogenic/odontogenic potential of pre-odontoblasts during root odontoblast differentiation. However, the underlying molecular pathway that orchestrates these processes remains largely unclear. In this study, ablation of transforming growth factor-β receptor type 2 (Tgfbr2) in root pre-odontoblasts resulted in abnormal formation of root osteodentin, which was associated with ectopic osteogenic differentiation of root odontoblasts. Disrupting TGF-β signaling caused upregulation of Wnt signaling characterized by increased Wnt6, Wnt10a, Tcf-1, and Axin2 expression. Interestingly, inhibiting Wnt signaling by deleting Wntless (wls) in Osteocalcin (Ocn)-Cre; Tgfbr2 fl/fl ; Wls fl/fl mice or overexpressing the Wnt antagonist Dkk1 in Ocn-Cre; Tgfbr2 fl/fl ; ROSA26 Dkk1 mice decreased ectopic osteogenic differentiation and arrested odontoblast differentiation. Our results suggest that TGF-β signaling acts with Wnt signaling to regulate root odontogenic differentiation.

Caloric restriction (CR) is a novel dietary therapy that has a protective effect on myocardial ischemia. However, the mechanisms underlying the therapeutic effect of CR remain unclear. Transfer RNA-derived small RNAs (tsRNAs) are a novel type of short non-coding RNAs that have potential regulatory functions in various physiological and pathological processes. In this study, we explored new therapeutic targets of CR through tsRNA sequencing. Rats were randomly divided into three groups: a normal control group (norm group), isoproterenol (ISO)-induced myocardial ischemic group (MI group), and CR pretreatment plus ISO-induced myocardial ischemic group (CR + MI group). Triphenyl tetrazolium chloride staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, serum creatine kinase (CK) and lactic acid dehydrogenase activity detection kits, and creatine kinase isoenzyme 1 levels were used to measure the degree of myocardial ischemic injury. These indicators of myocardial ischemia were significantly improved in the CR + MI group compared with those in the MI group. In the ischemic myocardial tissue of the MI group, a total of 708 precisely matched tsRNAs were identified, and 302 tsRNAs (fold change >1.5, P < 0.05) were significantly changed when compared with those in the norm group. Furthermore, 55 tsRNAs were significantly regulated by CR pretreatment, among which five tsRNAs (tiRNA-His-GTG-004, tRF-Gly-TCC-018, tRF-Cys-GCA-022, tRF-Lys-CTT-026, tRF-Met-CAT-008) were randomly selected and verified by quantitative real-time polymerase chain reaction. In addition, predictions of target genes and bioinformatics analysis indicated that these tsRNAs may play a therapeutic role through the regulation of macromolecular metabolism. In conclusion, our findings reveal that tsRNAs are potential therapeutic targets for CR pre-pretreatment to improve myocardial ischemic injury. This study provides new ideas for future research on elucidating the mechanisms of CR pretreatment in ameliorating myocardial ischemic injury.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignant tumors worldwide and has poor prognosis. DEAD box proteins31 (DDX31) participate in cellular processes involving RNA secondary structure changes. However, the functions of DDX31 in PDAC remain to be elucidated. Methods: The key gene DDX31 was identified using a combination of a risk model and weighted gene co-expression network analysis (WGCNA) with R software. The biological functions of DDX31 in PDAC were investigated through bioinformatics analysis and in vitro experiments. Results: Combining with WGCNA and risk model, DDX31 was identified as a potential factor of the invasive metastasis properties of PDAC, and its expression was closely related to the malignant differentiation of PDAC. The results of gene set enrichment analysis (GSEA) showed that DDX31 was correlated with cell invasive metastasis and proliferation by activating MAPK signaling pathway. The inhibition of DDX31 inhibited the invasion and migration of PDAC cells. Survival analysis showed that DDX31 expression was negatively associated with the poor prognosis in patients with PDAC. Interpretation: DDX31 may be a potential factor for PDAC. The inhibition of DDX31 may be a potential way to treat PDAC.

Immunotherapy and sorafenib exert anti-tumor effects via ferroptosis, but reliable biomarkers for the individual treatment and prognosis prediction of hepatocellular carcinoma (HCC) based on the ferroptosis and immune status remain lacking.

Aberrant regulation of angiogenesis involves in the growth and metastasis of tumors, but angiogenesis inhibitors fail to improve overall survival of pancreatic cancer patients in previous phase III clinical trials. A comprehensive knowledge of the mechanism of angiogenesis inhibitors against pancreatic cancer is helpful for clinical purpose and for the selection of patients who might benefit from the inhibitors. In this work, multi-omics analyses (transcriptomics, proteomics, and phosphoproteomics profiling) were carried out to delineate the mechanism of anlotinib, a novel angiogenesis inhibitor, against pancreatic cancer cells. The results showed that anlotinib exerted noteworthy cytotoxicity on pancreatic cancer cells. Multi-omics analyses revealed that anlotinib had a profound inhibitory effect on ribosome, and regulated cell cycle, RNA metabolism and lysosome. Based on the multi-omics results and available data deposited in public databases, an anlotinib-related gene signature was further constructed to identify a subgroup of pancreatic cancer patients who had a dismal prognosis and might be responsive to anlotinib.