Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3.

OBJECTIVE: Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear. METHODS: The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells. RESULTS: HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well. CONCLUSIONS: Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

Enhancer RNA IRS2e is essential for IRS2 expression and the oncogenic properties in oral squamous cell carcinoma.

The crucial roles of enhancer RNAs (eRNAs) in the regulation of gene expression in human diseases have drawn wider and wider attention in recent years. However, the specific expression profile and function of eRNAs are still rarely discussed in oral squamous cell carcinoma (OSCC), the most common subtype of head and neck squamous cell carcinoma (HNSC). In this study, we aimed to investigate the expression and function of an uncharacterized eRNA, insulin receptor substrate 2 enhancer RNA (IRS2e), in OSCC. We found that IRS2e was overexpressed in HNSC and its overexpression was positively correlated with a poor prognosis. The downregulation of IRS2e by short hairpin RNA significantly inhibited cell growth and induced cellular apoptosis and cell-cycle arrest in OSCC cells. Furthermore, the ablation of IRS2e inhibited tumor growth in vivo. Mechanically, IRS2e is essential for the expression of insulin receptor substrate 2 (IRS2), an oncogene nearby IRS2e in chromosome 13. Altogether, our study demonstrated that IRS2e is a novel oncogenic eRNA required for oncogene IRS2 expression in OSCC.

Treg plasticity and human diseases.

INTRODUCTION: As a subset of CD4+ T cells, regulatory T cells (Tregs) with the characteristic expression of transcription factor FOXP3 play a key role in maintaining self-tolerance and regulating immune responses. However, in some inflammatory circumstances, Tregs can express cytokines of other T help (Th) cells by internal reprogramming, which is called Treg plasticity. These reprogrammed Tregs with impaired suppressive ability contribute to the progression of diseases by secreting pro-inflammatory cytokines. However, in the tumor microenvironment (TME), such changes in phenotype rarely occur in Tregs, on the contrary, Tregs usually display a stronger suppressive function and inhibit anti-tumor immunity. It is important to understand the mechanisms of Treg plasticity in inflammatory diseases and cancers. OBJECTIVES: In this review, we summarize the characteristics of different Th-like Tregs and discuss the potential mechanisms of these changes in phenotype. Furthermore, we summarize the Treg plasticity in human diseases and discuss the effects of these changes in phenotype on disease progression, as well as the potential application of drugs or reagents that regulate Treg plasticity in human diseases. CONCLUSIONS: Treg plasticity is associated with inflammatory diseases and cancers. Regulating Treg plasticity is a promising direction for the treatment of inflammatory diseases and cancers.

The N6-methyladenosine demethylase FTO is required for odontoblast differentiation in vitro and dentine formation in mice by promoting RUNX2 exon 5 inclusion through RBM4.

AIM: Fat mass and obesity-associated (FTO) protein, the first discovered N6-methyladenine (m6A) demethylase, played positive roles in bone formation. In this study, the aim was to investigate the function and potential mechanism of Fto in dentine formation. METHODOLOGY: In vivo model, postnatal 12-day (PN12), 4-week-old (4 wk), 6-week-old (6 wk) healthy male C57BL/6J were randomly divided into Fto knockout (Fto-/- ) mice and wild-type (WT) littermates according to their genotypes, with 3-5 mice in each group. The mandibles of Fto-/- mice and WT control littermates were isolated for analysis by micro-computed tomography (micro-CT), 3-dimensional reconstruction and Haematoxylin-eosin (HE) staining. In vitro, mouse dental papilla cells (mDPCs) and human dental stem pulp cells (hDPSCs) were cultured with odontogenetic medium to evaluate differentiation capacity; expression levels of odontoblastic related genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). The inclusion levels of Runt-related transcription factor 2 (RUNX2) exon 5 in mDPCs and hDPSCs were detected by semiquantitative real-time polymerase chain reaction (RT-PCR). The RNA binding motif protein 4 (RBM4) m6A site was verified through m6A methylated RNA immunoprecipitation (MeRIP) and the stability of RBM4 mRNA influenced by FTO knockdown was measured by mRNA stability assay. Differences with p values < .05 were regarded as statistically significant. RESULTS: We discovered that Fto-/- mice showed significant dentine formation defects characterized by widened pulp cavity, enlarged pulp-tooth volume ratio, thinned dentine and pre-dentine layer of root (p < .05). Fto-/- mDPCs and FTO-silencing hDPSCs not only exhibited insufficient mineralization ability and decreased expression levels of odontoblastic mineralization related genes (p < .05), but showed significantly reduced Runx2 exon 5 inclusion level (p < .05). FTO knockdown increased the m6A level of RBM4 and destabilized the mRNA of RBM4, thus contributing to the reduced RBM4 expression level. Moreover, Rbm4 overexpression in Fto-/- mDPCs can partly restore Runx2 exon 5 inclusion level and the differentiation ability disrupted by Fto knockout. CONCLUSION: Thus, within the limitations of this study, the data suggest that FTO promotes odontoblastic differentiation during dentine formation by stabilizing RBM4 mRNA to promote RUNX2 exon 5 inclusion.

Cryotherapy Attenuates Inflammation via the lncRNA SNHG1/miR-9-5p/NFKB1 Regulatory Axis in Periodontal Ligament Cells.

Cryotherapy is a common non-pharmacological method to relieve pain and inflammation. Clinical studies have shown that cryotherapy can reduce postoperative pain after root canal therapy, but the mechanism remains unclear. In this study, we aimed to investigate the underlying molecular mechanisms by which cryotherapy reduces inflammation in lipopolysaccharide (LPS)-stimulated periodontal ligament cells through transcriptome sequencing analysis. We found that cryotherapy significantly reduced the expression of multiple proinflammatory cytokines and chemokines, and NFKB1 was the key regulator down-regulated by cryotherapy. Importantly, we discovered that lncRNA SNHG1 expression level significantly decreased after cold treatment. SNHG1 expression was positively related to NFKB1 while negatively correlated with miR-9-5p, which formed a novel ceRNA regulatory pathway. Knockdown of SNHG1 significantly reduced the expression of NFKB1, IL1B, and IL6, while overexpression of SNHG1 significantly increased the expression of these genes. In conclusion, our study demonstrated that cryotherapy can effectively reduce inflammation in LPS-induced periodontal ligament cells by suppressing the lncRNA SNHG1/miR-9-5p/NFKB1 axis.

PD-L1 Exon 3 Is a Hidden Switch of Its Expression and Function in Oral Cancer Cells.

The interaction between programmed cell death 1 ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) protects tumor cells from immune surveillance. PD-L1 exon 3 is a potential alternative exon and encodes an Ig variable (IgV) domain. Here, we found that a lack of exon 3 leads to the significant loss of cellular membrane locations and the dramatically reduced protein expression of PD-L1, indicating that PD-L1 exon 3 is essential for its protein expression and translocation to the cell membrane. Notably, oral cancer cells show almost no exon 3 skipping to ensure the expression of the full-length, functional PD-L1 protein. We discovered two key exonic splicing enhancers (ESEs) for exon 3 inclusion. Two efficient antisense oligonucleotides (ASOs) were identified to block these two ESEs, which can significantly trigger exon 3 skipping and decrease the production of full-length, functional PD-L1 on the surface of cancer cells. Treatment of oral cancer cells with these ASOs significantly enhanced immune cells' suppression of cancer cell proliferation. Surprisingly, these two ASOs also significantly inhibited cell growth and induced cell pyroptosis in oral cancer cells. Altogether, the results of our study demonstrate the pivotal roles of exon 3 in PD-L1 expression and provide a novel anti-PD-L1 method.

Salivary Extracellular Vesicles: Biomarkers and Beyond in Human Diseases.

Extracellular vesicles, as bioactive molecules, have been extensively studied. There are abundant studies in the literature on their biogenesis, secretion, structure, and content, and their roles in pathophysiological processes. Extracellular vesicles have been reviewed as biomarkers for use in diagnostic tools. Saliva contains many extracellular vesicles, and compared with other body fluids, it is easier to obtain in a non-invasive way, making its acquisition more easily accepted by patients. In recent years, there have been numerous new studies investigating the role of salivary extracellular vesicles as biomarkers. These studies have significant implications for future clinical diagnosis. Therefore, in this paper, we summarize and review the potential applications of salivary extracellular vesicles as biomarkers, and we also describe their other functions (e.g., hemostasis, innate immune defense) in both oral and non-oral diseases.

SRSF3-Mediated Ki67 Exon 7-Inclusion Promotes Head and Neck Squamous Cell Carcinoma Progression via Repressing AKR1C2.

Ki67 is a well-known proliferation marker with a large size of around 350 kDa, but its biological function remains largely unknown. The roles of Ki67 in tumor prognosis are still controversial. Ki67 has two isoforms generated by alternative splicing of exon 7. The roles and regulatory mechanisms of Ki67 isoforms in tumor progression are not clear. In the present study, we surprisingly find that the increased inclusion of Ki67 exon 7, not total Ki67 expression level, was significantly associated with poor prognosis in multiple cancer types, including head and neck squamous cell carcinoma (HNSCC). Importantly, the Ki67 exon 7-included isoform is required for HNSCC cell proliferation, cell cycle progression, cell migration, and tumorigenesis. Unexpectedly, Ki67 exon 7-included isoform is positively associated with intracellular reactive oxygen species (ROS) level. Mechanically, splicing factor SRSF3 could promote exon 7 inclusion via its two exonic splicing enhancers. RNA-seq revealed that aldo-keto reductase AKR1C2 is a novel tumor-suppressive gene targeted by Ki67 exon 7-included isoform in HNSCC cells. Our study illuminates that the inclusion of Ki67 exon 7 has important prognostic value in cancers and is essential for tumorigenesis. Our study also suggested a new SRSF3/Ki67/AKR1C2 regulatory axis during HNSCC tumor progression.

[Guideline for clinical comprehensive evaluation of Chinese patent medicine (2022 version)].

Currently,the research or publications related to the clinical comprehensive evaluation of Chinese patent medicine are increasing,which attracts the broad attention of all circles. According to the completed clinical evaluation report on Chinese patent medicine,there are still practical problems and technical difficulties such as unclear responsibility of the evaluation organization,unclear evaluation subject,miscellaneous evaluation objects,and incomplete and nonstandard evaluation process. In terms of evaluation standards and specifications,there are different types of specifications or guidelines with different emphases issued by different academic groups or relevant institutions. The professional guideline is required to guide the standardized and efficient clinical comprehensive evaluation of Chinese patent medicine and further improve the authority and quality of evaluation. In combination with the characteristics of Chinese patent medicine and the latest research achievement at home and abroad,the detailed specifications were formulated from six aspects including design,theme selection,content and index,outcome,application and appraisal,and quality control. The guideline was developed based on the guideline development requirements of China Assoication of Chinese medicine. After several rounds of expert consensus and public consultation,the current version of the guideline has been developed.

RAN and YBX1 are required for cell proliferation and IL-4 expression and linked to poor prognosis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world, with a high mortality rate. RAN is a member of the Ras GTPase family and is overexpressed in a range of cancers, however, the relationship between RAN and OSCC is rarely reported. In this study, we found that RAN is overexpressed in OSCC tissues. RAN inhibition retarded OSCC cell proliferation and led to apoptosis and cell cycle arrest. Knockdown of RAN inhibited tumor growth in vivo. Strikingly, we found that RAN and oncogene Y-box binding protein-1 (YBX1) are positively associated with the immune infiltrates of CD4+ Th2 cells in multiple types of cancer, and can promote IL-4 expression. IL-4 treatment can partially rescue RAN knockdown-induced cell apoptosis in OSCC cells. Moreover, overexpression of RAN could rescue cell growth inhibition caused by knockdown of YBX1. Furthermore, patients with low expression of both RAN and YBX1 had better overall survival than others. Collectively, these findings indicate that RAN is a target of YBX1. RAN and YBX1 are required for cell proliferation and IL-4 expression. RAN and YBX1 are co-expressed and can serve as potential co-biomarkers for poor prognosis in OSCC.

Roles of Regulatory T Cell-Derived Extracellular Vesicles in Human Diseases.

Regulatory T (Treg) cells play crucial roles in maintaining immune self-tolerance and immune homeostasis, and closely associated with many human diseases. Recently, Treg cells-derived extracellular vesicles (Treg-EVs) have been demonstrated as a novel cell-contact independent inhibitory mechanism of Treg cells. Treg-EVs contain many specific biological molecules, which are delivered to target cells and modulate immune responses by inhibiting T cell proliferation, inducing T cell apoptosis, and changing the cytokine expression profiles of target cells. The abnormal quantity or function of Treg-EVs is associated with several types of human diseases or conditions, such as transplant rejection, inflammatory diseases, autoimmune diseases, and cancers. Treg-EVs are promising novel potential targets for disease diagnosis, therapy, and drug transport. Moreover, Treg-EVs possess distinct advantages over Treg cell-based immunotherapies. However, the therapeutic potential of Treg-EVs is limited by some factors, such as the standardized protocol for isolation and purification, large scale production, and drug loading efficiency. In this review, we systematically describe the structure, components, functions, and basic mechanisms of action of Treg-EVs and discuss the emerging roles in pathogenesis and the potential application of Treg-EVs in human diseases.

Oncogene SRSF3 suppresses autophagy via inhibiting BECN1 expression.

Autophagy is an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been demonstrated to be associated with many human diseases, including cancer. Alternative splicing of pre-mRNA is also an evolutionarily conserved regulatory mechanism of gene expression. Dysregulation of alternative splicing is increasingly linked to cancer. However, the association between these two cellular conserved processes is unclear. Splicing factors are critical players in the regulation of alternative splicing of pre-mRNA. We analyzed the expression of 28 splicing factors during hypoxia-induced autophagy in three oral squamous cell carcinoma (OSCC) cell lines. We discovered that oncogenes SRSF3 and SRSF1 are significantly downregulated in all three cell lines. Moreover, knockdown of SRSF3 increased autophagic activity, whereas overexpression of SRSF3 inhibited hypoxia-induced autophagy. Loss-of-function and gain-of-function assays also showed that SRSF3 inhibits the expression of p65 and FoxO1 and their downstream target gene BECN1, a key regulator of autophagy. Our results demonstrated that splicing factor SRSF3 is an autophagy suppressor.

Enhanced transfection efficiency by using a novel semi-attachment method in cell line and primary cells.

DNA transfection in cells is a key technique in biological studies. Cationic liposomes can form nanoparticle complexes with DNA and are widely used for gene delivery in mammalian cells. However, the major drawback of cationic liposomes is their low transfection efficiency in hard-to-transfect cells, such as primary cultured cells. In this study, we established a novel semi-attachment transfection method that showed remarkably improved transfection efficiency compared with traditional forward transfection method.

Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor.

A lot of evidence has been found on the link between tumorigenesis and the aberrant expression of splicing factors. A number of splicing factors have been reported to be either oncogenic or overexpressed in cancer cells. However, splicing factors can also play negative roles in tumorigenesis. In the current review, we focus on splicing factor poly(rC)-binding protein 1 (PCBP1), a novel tumor suppressor that is characterized by downregulation in many cancer types and shows inhibition of tumor formation and metastasis. Notably, the messenger RNA levels of PCBP1 are not significantly decreased in most cancer types. In fact, PCBP1 protein is often degraded or shows a loss-of-function through phosphorylation in cancer cells. PCBP1 is highly homologous to its family member, PCBP2. Interestingly, PCBP2 appears to be an oncogenic splicing factor. A growing body of evidence has shown that PCBP1 regulates alternative splicing, translation, and RNA stability of many cancer-related genes. Taking together, PCBP1 has distinctive tumor suppressive functions, and increasing PCBP1 expression may represent a new approach for cancer treatment.

Oral squamous cancer cell exploits hnRNP A1 to regulate cell cycle and proliferation.

Oral squamous cell carcinoma (OSCC) is a common human malignant tumor with high mortality. So far, the molecular pathogenesis of OSCC remains largely unclear. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an important multi-function splicing factor and closely related to tumorigenesis. hnRNP A1 is overexpressed in various tumors, and promotes aerobic glycolysis and elongation of telomere, but the function of hnRNP A1 in cell cycle and proliferation remains unclear. We found that hnRNP A1 was overexpressed in OSCC tissues, and was required for the growth of OSCC cells. Moreover, hnRNP A1 was highly expressed in the G2/M cell cycle phase. Knockdown of hnRNP A1 induced G2/M arrest. DNA microarray assay result showed that hnRNP A1 regulated the expression of a number of target genes associated with G2/M phase. Moreover, hnRNP A1 controlled the alternative splicing of CDK2 exon 5. These findings suggested that hnRNP A1 plays key roles in the regulation of cell cycle progression and pathogenesis of OSCC.

[Research on symmetrical optical waveguide based surface plasmon resonance sensing with spectral interrogation].

Surface plasmon resonance (SPR) sensors with spectral interrogation can adopt fiber to transmit light signals, thus leaving the sensing part separated, which is very convenient for miniaturization, remote-sensing and on-site analysis. Symmetrical optical waveguide (SOW) SPR has the same refractive index of the-two buffer media layers adjacent to the metal film, resulting in longer propagation distance, deeper penetration depth and better performance compared to conventional SPR In the present paper, we developed a symmetrical optical, waveguide (SOW) SPR sensor with wavelength interrogation. In the system, MgF2-Au-MgF2 film was used as SOW module for glucose sensing, and a fiber based light source and detection was used in the spectral interrogation. In the experiment, a refractive index resolution of 2.8 x 10(-7) RIU in fluid protocol was acquired. This technique provides advantages of high resolution and could have potential use in compact design, on-site analysis and remote sensing.

A novel inducible expression system for the functional study of toxic gene in bacteria.

The cloning and expression of toxic proteins in bacteria have posed a great challenge because of the leaky expression in inducible expression systems. Using artificial gene synthesis and clone screening methods, we identified a mutant T5 promoter, which significantly reduced leaky expression of lac operator. The mutant T5 promoter contains two T deletions at -35 region and may reduce promoter activity. A bacterial lethal gene, Φ174 lytic gene E, was successfully cloned in this system and expressed in the presence of isopropyl ß-D-1-thiogalactopyranoside. The system is compatible with existing T5 inducible expression systems and can be used for the controlled expression of toxic proteins.

An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3.

PD-1 is a key immune checkpoint molecule. Anti-PD-1 immunotherapy is encouraging in cancer treatment. However, it still needs to be improved. PD-1 has at least five isoforms generated by alternative splicing. An isoform without exon 3 encoding soluble PD-1 (sPD-1) can activate anti-tumor immunity by inhibiting the interaction between cellular surface full-length PD-1 (flPD-1) and PD-L1. However, the regulatory mechanism of exon 3 splicing remains largely unknown. Here, we screened the exon 3 sequence by mutation and searched corresponding splicing factors by SpliceAid database and pulldown assay. The alternative splicing of PD-1 exon 3 was analyzed by RT-PCR. The expression levels of flPD-1 and sPD-1 were analyzed by Western blot, flow cytometry, and ELISA. We discovered that an exonic splicing enhancer (ESE) of exon 3 is essential for its inclusion. Moreover, SRSF3 can bind to this ESE and enhance exon 3 inclusion and flPD-1 expression. We designed and screened out an antisense oligonucleotide (ASO) targeting PD-1 to block the interaction between SRSF3 and ESE, and significantly increase exon 3 skipping and sPD-1 expression, which was verified in various tumor cells in addition to oral cancer cells. Altogether, our results uncovered the regulatory mechanism of human PD-1 exon 3 splicing and sPD-1 expression and further designed a novel anti-PD-1 ASO, which are useful for developing a new method of anti-cancer immunotherapy.

Multi-channel hyperspectral fluorescence detection excited by coupled plasmon-waveguide resonance.

We propose in this paper a biosensor scheme based on coupled plasmon-waveguide resonance (CPWR) excited fluorescence spectroscopy. A symmetrical structure that offers higher surface electric field strengths, longer surface propagation lengths and depths is developed to support guided waveguide modes for the efficient excitation of fluorescence. The optimal parameters for the sensor films are theoretically and experimentally investigated, leading to a detection limit of 0.1 nM (for a Cy5 solution). Multiplex analysis possible with the fluorescence detection is further advanced by employing the hyperspectral fluorescence technique to record the full spectra for every pixel on the sample plane. We demonstrate experimentally that highly overlapping fluorescence (Cy5 and Dylight680) can be distinguished and ratios of different emission sources can be determined accurately. This biosensor shows great potential for multiplex detections of fluorescence analytes.

Epigenetic Control of Cancer Cell Proliferation and Cell Cycle Progression by HNRNPK via Promoting Exon 4 Inclusion of Histone Code Reader SPIN1.

Heterogeneous nuclear ribonucleoprotein K (HNRNPK, hnRNP K), a multifunctional RNA/DNA binding protein, mainly regulates transcription, translation and RNA splicing, and then plays oncogenic roles in many cancers. However, the related mechanisms remain largely unknown. Here, we found that HNRNPK can partially epigenetically regulate cancer cell proliferation via increasing transcription and exon 4-inclusion of SPIN1, an important oncogenic histone code reader. This exon 4 skipping event of SPIN1 generates a long non-coding RNA, followed by the downregulation of SPIN1 protein. SPIN1 is one of the most significantly co-expressed genes of HNRNPK in thirteen TCGA cancers. Our further studies revealed HNRNPK knockdown significantly inhibited cell growth and cell cycle progression in oral squamous cell carcinoma (OSCC) cells and promoted cell apoptosis. Overexpression of SPIN1 was able to partially rescue the growth inhibition triggered by HNRNPK knockdown. Moreover, CCND1 (Cyclin D1), a key cell cycle regulator and oncogene, epigenetically up-regulated by SPIN1, was also positively regulated by HNRNPK. In addition, we discovered that HNRNPK promoted SPIN1 exon 4 inclusion by interacting with an intronic splicing enhancer in intron 4. Collectively, our study suggests a novel epigenetic regulatory pathway of HNRNPK in OSCC, mediated by controlling the transcription activity and alternative splicing of SPIN1 gene.