[Genetic analysis of a child with Generalized arterial calcification of infancy due to variant of ABCC6 gene].

OBJECTIVE: To explore the clinical manifestations and genetic basis for a rare case of Generalized arterial calcification of infancy (GACI). METHODS: A 44-day-old female infant who was treated at Baoding Hospital of Beijing Children's Hospital Affiliated to Capital Medical University on August 26, 2022 was selected as the study subject. Clinical data of the child was collected, and Trio-whole exome sequencing (Trio-WES), whole genome copy number variation sequencing (CNV-seq) and minigene splicing assay were carried out to analyze the pathogenicity of the variants. RESULTS: The child had presented with fever and high inflammatory indicators, for which treatment with various antibiotics was ineffective. Ultrasound had revealed extensive arterial calcification and arterial wall thickening. The child was suspected for GACI with arteritis related to the primary disease. Her fever was relieved by treatment with glucocorticoid and biological agents. Trio-WES revealed that she has harbored compound heterozygous variants of the ABCC6 gene, namely c.4404-1G>A and c.4041+5G>T, for which the latter was unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the variants were classified as likely pathogenic (PVS1+PM2_Supporting) and variant of unknown significance (PM2_Supporting+PM3+PP3), respectively. The result of CNV-seq was negative. And the minigene splicing assay has further verified that both variants can result in alternative splicing. CONCLUSION: For pyrexia with unknown causes and refractory to conventional treatment, it is necessary to recommend early genetic testing to avoid missed diagnosis of GACI.

Prognostic factors and constructing a nomogram in tracheal cancer patients treated with surgical intervention: A study based on SEER database.

Although surgery is considered the first choice of treatment for patients diagnosed with tracheal cancer, the prediction of overall survival (OS) in patients undergoing surgical intervention is poor. To address this issue, we developed a nomogram that combined a risk classification system to estimate the OS of patients with tracheal cancer who underwent surgical intervention. A total of 525 qualified patients were selected from the surveillance, epidemiology, and end results database between 1975 and 2018 and were randomly divided into training and validation cohorts (7:3). The parameters of independent prognostic ability were determined using Cox regression analysis, and a nomogram was formed. The predictive ability of the nomogram was tested using the area under the curve of receiver operating characteristic curves and calibration curves. Survival curves were assessed between the different risk classification groups using the Kaplan-Meier method. The results indicated that Age, stage, histology, and tumor size were independent prognostic factors and were included in the predictive model. The calibration plots demonstrated good agreement between the nomogram prediction and actual observation for 24- and 36-month OS. The receiver operating characteristic curves suggested that the predictive model had good discrimination ability, with the area under the curves (training group 0.817, 0.785, and 0.801, respectively) and validation group (0.744, 0.794, and 0.822, respectively). Furthermore, the low-risk group had a better prognosis than the high-risk group in the total, training, and validation cohorts (all P < .001). This study established a novel nomogram system to predict OS and identify independent prognostic factors in patients with tracheal cancer who underwent surgical intervention. This model has the potential to assist doctors in making decisions regarding treatment options.

Companion responses to diagnosis in Chinese outpatient clinical interaction.

Patients regularly attend clinical consultations with companions in Chinese outpatient clinics. Despite companions' significant influence on clinical consultations, how companions respond to diagnosis and their contributions to the activity of diagnosis in Chinese outpatient clinical interaction remain under-researched. The present study, by adopting the method of conversation analysis, investigated clinicians' diagnostic deliveries and companions' subsequent responses in Chinese outpatient clinical interaction. The data for this study consisted of 79 video recordings of clinical consultations in the Chinese orthopedic outpatient clinic, approximately lasting 12 h and involving three male clinicians, 79 patients (37 male/42 female), and 91 companions (51 male/40 female). Three basic categories of companion responses to diagnosis were identified: minimal verbal responses, embodied responses, and extended responses. It was demonstrated that these distinct responses allowed companions to challenge clinicians' medical authority in the activity of diagnosis by delivering their own diagnostic judgments, resisting clinicians' diagnoses, and orienting to clinicians' accounts for their diagnostic statements and reasoning, thus displaying companion agency in the Chinese outpatient clinical decision-making and indicating a transition from a paternalistic model to a family-centered model of the doctor-patient relationship in the Chinese orthopedic outpatient interaction. This study furthers current knowledge of companion involvement across healthcare contexts and contributes to raising clinicians' awareness of the significance of companions' contributions in Chinese outpatient clinical interaction.

NHERF4 hijacks Mas-mediated PLC/AKT signaling to suppress the invasive potential of clear cell renal cell carcinoma cells.

The Mas receptor has been reported to promote migration and invasion of clear cell renal cell carcinoma (ccRCC) cells via Ang-(1-7)-dependent AKT signaling. However, the mechanism underlying the regulation of Mas function remains unknown. Here, eight PDZ domain-containing proteins were identified as Mas interactors using surface plasmon resonance (SPR) coupled to mass spectrometry (MS). NHERF4 was the only downregulated gene across multiple independent ccRCC datasets. GST pull-down and co-immunoprecipitation assays confirmed physical interaction between NHERF4 and Mas. Using NHERF4 overexpression and knockdown assays, we found that NHERF4 inhibited Mas-induced migration, invasion and in vivo metastasis of ccRCC cells. Mechanistically, NHERF4 suppressed Mas-stimulated AKT phosphorylation and the PLC/Ca2+ response. We further demonstrated that NHERF4 compromised Mas-mediated migration and invasion of ccRCC cells via regulation of the PLC/AKT signaling axis. Analysis of the ccRCC dataset revealed that low levels of NHERF4 expression were correlated with higher TNM stage, and independently predicted poor prognosis of ccRCC patients. Overall, our study identified NHERF4 as a novel regulator of ccRCC invasiveness, and a prognostic biomarker, which may be beneficial for determining optimal therapeutic strategies for ccRCC patients.

Patient resistance towards clinicians' diagnostic test-taking advice and its management in Chinese outpatient clinic interaction.

Performing diagnostic tests is a fundamental information-gathering activity in diagnostic process. However, little attention has been paid to the interactional process where a diagnostic test is advised and received, especially in Chinese medical settings. Decision making over prescribing diagnostic tests consists of clinicians' advice and patients' acceptance or resistance/rejection. Drawing on audio-recordings of clinician-patient encounters in Chinese outpatient clinics as data and conversation analysis as a method, we discuss how patient resistance to clinicians' diagnostic test-taking advice is displayed and managed over sequences of interaction. Two types of advice deliveries have been identified: advice either with no diagnostic utterances or with indeterminate diagnostic utterances. We find that patients demonstrate their resistance towards the former type of advice in two ways: questioning clinicians' decisions and proposing an alternative plan. Displaying resistance to the latter type of advice, patients have been found to recurrently resort to one way: proffering additional information about personal experience. Confronted with resistance, clinicians generally proceed to justify decisions by either asserting their epistemic primacy in determining a test or lowering certainty in the original speculative diagnosis. Towards persistent resistance, clinicians mainly employ two techniques to impose acceptance onto patients: repeating the initial advice and terminating forcefully current sequence. This study adds to a growing body of research on resistance in medical settings and contributes to our understanding of the decision making over medical investigations in Chinese outpatient clinic interaction.

Loss of PDZK1 expression activates PI3K/AKT signaling via PTEN phosphorylation in gastric cancer.

Phosphorylation of PTEN plays an important role in carcinogenesis and progression of gastric cancer. However, the underlying mechanism of PTEN phosphorylation regulation remains largely elusive. In the present study, PDZK1 was identified as a novel binding protein of PTEN by association of PTEN through its carboxyl terminus and PDZ domains of PDZK1. By direct interaction with PTEN, PDZK1 inhibited the phosphorylation of PTEN at S380/T382/T383 cluster and further enhanced the capacity of PTEN to suppress PI3K/AKT activation. PDZK1 suppressed gastric cancer cell proliferation by diminishing PI3K/AKT activation via inhibition of PTEN phosphorylation in vitro and in vivo. The expression of PDZK1 was frequently downregulated in gastric cancer specimens and correlated with progression and poor prognosis of gastric cancer patients. Downregulation of PDZK1 was associated with PTEN inactivation, AKT signaling and cell proliferation activation in clinical specimens. Thus, low levels of PDZK1 in gastric cancer specimens lead to increase proliferation of gastric cancer cells via phosphorylation of PTEN at the S380/T382/T383 cluster and constitutively activation of PI3K/AKT signaling, which results in poor prognosis of gastric cancer patients.

HPV16 E6 promotes cervical cancer cell migration and invasion by downregulation of NHERF1.

HPV16 is the predominant type of HPV causing invasive cervical cancer. However, the underlying molecular mechanism of the unparalleled carcinogenic power of HPV16 compared to other types of high-risk (HR)-HPV including HPV18 remains elusive. The PDZ binding motif (PBM) of high-risk HPV E6 plays an important role in neoplasia and progression of cervical cancer. HPV16 E6 rather than HPV18 E6, interacted with NHERF1 by its PBM region, and induced degradation of NHERF1. NHERF1 retarded the assembly of cytoskeleton by downregulation of ACTN4, thereby inhibited the migration and invasion of cervical cancer cells in both cell and mouse model. HPV16 E6 was confirmed to enhance actin polymerization with increased ACTN4 level by downregulation of NHERF1, and result in enhanced migration and invasion of cervical cancer cells. GSEA analysis of cervical cancer specimens also showed that HPV16 E6 rather than HPV18 E6, was significantly associated with actin cytoskeleton assembly. That downregulation of NHERF1 by HPV16 E6 promoted cytoskeleton assembly and cell invasion, was an important cause in cervical cancer carcinogenesis. These findings provided the differential mechanism between HPV16 E6 and HPV18 E6 in the development and progression of cervical cancer, which may partially explain the differences of carcinogenic power between these two types of HR-HPVs.

NHERF1 inhibits beta-catenin-mediated proliferation of cervical cancer cells through suppression of alpha-actinin-4 expression.

Cervical cancer is one of the most lethal types of cancer in female. Aberrant activation of Wnt/ß-catenin signaling pathway has been found to be involved in cervical cancer development and progression, whereas the underlying molecular mechanisms remain poorly understood. The present study showed that NHERF1 was a novel gene associated with both cell proliferation and Wnt signaling pathway in cervical cancer by analysis of differential gene expression and gene cluster for the cervical cancer specimens from GEO data sets. It was further demonstrated in cellular study that NHERF1 inhibition of cervical cancer cell proliferation through Wnt/ß-catenin signaling was dependent on α-actinin-4 (ACTN4) expression. A negative association between NHERF1 expression and levels of ACTN4 and ß-catenin was found in mouse xenograft model and cervical cancer specimens. Low levels of NHERF1 in cervical cancer specimens were found to associate with activation of cell proliferation and Wnt/ß-catenin signaling by gene set enrichment analysis, and also were an independent predictive factor for worse prognosis of cervical cancer patients by Cox regression analysis. These findings demonstrate that NHERF1 inhibits Wnt signaling-mediated proliferation of cervical cancer via suppression of ACTN4, and NHERF1 downregulation may contribute to the progression of cervical cancer. These findings may also shed some lights for understanding the underlying mechanisms of cisplatin resistance and worse prognosis of HPV-inactive cervical cancer patients.

NHERF1 inhibits proliferation of triple-negative breast cancer cells by suppressing GPER signaling.

G protein-coupled estrogen receptor (GPER) signaling is activated in triple-negative breast cancer (TNBC); however, the detailed mechanisms of its regulation remain unclear. The present study aimed to elucidate the molecular mechanisms involved in GPER activation in TNBC. In MDA-MB-231 cells, a TNBC cell line, NHERF1 interaction with GPER was verified by co-immunoprecipitation and immunofluorescent staining assays. Overexpression of NHERF1 in MDA-MB-231 cells inhibited GPER-mediated proliferation and phosphorylation of ERK1/2 and Akt. Furthermore, NHERF1 expression levels were negatively correlated with the gene signatures of GPER activation, ERK1/2 and Akt signaling, and cell proliferation in early stage of TNBC tumors from the TCGA data set. Taken together, NHERF1 inhibited the activation of GPER-mediated signaling and suppressed the proliferation of triple-negative breast cancer cells. Loss of NHERF1 expression may play a pivotal role in the early stage of TNBC carcinogenesis.

Physcion induces apoptosis in hepatocellular carcinoma by modulating miR-370.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies. The current study is designed to explore the role of physcion, a major active ingredient in several traditional herbal medicinal plants, for the treatment of HCC. HCC cell lines, SMMC7721 and HepG2, were treated with physcion and its apoptosis-inducing effect was examined. Both in vitro and in vivo results from the present study demonstrated that physcion treatment resulted in apoptotic cell death in HCC cells via upregulation of miR-370. Furthermore, our findings showed that the physcion modulated the level of miR-370 through AMPK/Sp1/DNMT1 signaling. Taken together, these results showed that physcion exerts anti-tumor effect against HCC, which may be a potential agent for the adjunct chemotherapy.

MAGI3 negatively regulates Wnt/ß-catenin signaling and suppresses malignant phenotypes of glioma cells.

Gliomas are the most common primary brain malignancies and are associated with a poor prognosis. Here, we showed that the PDZ domain-containing protein membrane-associated guanylate kinase inverted 3 (MAGI3) was downregulated at the both mRNA and protein levels in human glioma samples. MAGI3 inhibited proliferation, migration, and cell cycle progression of glioma cells in its overexpression and knockdown studies. By using GST pull-down and co-immunoprecipitation assays, we found that MAGI3 bound to ß-catenin through its PDZ domains and the PDZ-binding motif of ß-catenin. MAGI3 overexpression inhibited ß-catenin transcriptional activity via its interaction with ß-catenin. Consistently, MAGI3 overexpression in glioma cells C6 suppressed expression of ß-catenin target genes including Cyclin D1 and Axin2, whereas MAGI3 knockdown in glioma cells U373 and LN229 enhanced their expression. MAGI3 overexpression decreased growth of C6 subcutaneous tumors in mice, and inhibited expression of ß-catenin target genes in xenograft tumors. Furthermore, analysis based on the Gene Expression Omnibus (GEO) glioma dataset showed association of MAGI3 expression with overall survival and tumor grade. Finally, we demonstrated negative correlation between MAGI3 expression and activity of Wnt/ß-catenin signaling through GSEA of three public glioma datasets and immunohistochemical staining of clinical glioma samples. Taken together, these results identify MAGI3 as a novel tumor suppressor and provide insight into the pathogenesis of glioma.

Tumor suppressive microRNA-200a inhibits renal cell carcinoma development by directly targeting TGFB2.

A large body of evidence indicates that microRNAs play a critical role in tumor initiation and progression by negatively regulating oncogenes or tumor suppressor genes. Here, we report that the expression of miR-200a was notably downregulated in 45 renal cell carcinoma (RCC) samples. Restoration of miR-200a suppressed cell proliferation, migration, and invasion in two RCC cell lines. Furthermore, we used an epithelial-to-mesenchymal transition PCR array to explore the putative target genes of miR-200a. By performing quantitative real-time PCR, ELISA, and luciferase reporter assays, transforming growth factor beta2 (TGFB2) was validated as a direct target gene of miR-200a. Moreover, siRNA-mediated knockdown of TGFB2 partially phenocopied the effect of miR-200a overexpression. These results suggest that miR-200a suppresses RCC development via directly targeting TGFB2, indicating that miR-200a may present a novel target for diagnostic and therapeutic strategies in RCC.

[Idenfication of microRNAs profiles in nasopharyngeal carcinoma].

OBJECTIVE: To filtrate and prove the different microRNAs (miRs) profiles in nasopharyngeal carcinoma. METHOD: Screening the different expressions of miRs between nasopharyngeal carcinoma and the inflammatory tissues by the application of expression profiling of chip high-throughput and large-scale microarray analysis. Then we used RT-QPCR technology to prove the accuracy of screening results. RESULT: There were significant expression differences of miRs between nasopharyngeal carcinoma and the control tissues, 144 human miRs had 2 or more fold the difference ratio. Compared with the inflammatory tissues, we have found that miRs-34b, miRs-449b and miRs-7-1 significantly low expressed in nasopharyngeal carcinoma, yet miRs-125b, miRs-184, miRs-196b, miRs-205 and miRs-24-1 expressed high. The results were consistent with the microarray analysis. CONCLUSION: The difference expressed miRs might be closely related to the process of nasopharyngeal carcinoma, and the research on miRs profiles maybe provide a powerful target basis for early diagnosis and therapy of nasopharyngeal carcinoma.

miR-145 functions as tumor suppressor and targets two oncogenes, ANGPT2 and NEDD9, in renal cell carcinoma.

PURPOSE: Abnormal expression of miRNAs is closely related to a variety of human cancers. The purpose of this study is to identify new tumor suppressor miRNA and elucidate its physiological function and mechanism in renal cell carcinoma (RCC). METHODS: The expression of miR-145 in 45 RCC and adjacent normal tissues was performed by quantitative RT-PCR. Cell proliferation, migration, invasion, apoptosis and cycle assays were carried out for functional analysis after miR-145 transfection. Two target genes of miR-145 were identified by luciferase reporter assay. The altered expression of 84 epithelial to mesenchymal transition (EMT)-related genes after miR-145 transfection was detected by RT(2) Profiler EMT PCR array. RESULTS: The expression of miR-145 was downregulated in RCC compared to their normal adjacent tissues. Restoring miR-145 expression in RCC cell lines dramatically suppressed cell proliferation, migration and invasion, and induced cell apoptosis and G2-phase arrest. We further validated those miR-145 targets two oncogenes, ANGPT2 and NEDD9 in RCC. In addition, miR-145 was found to regulate numerous genes involved in the EMT. CONCLUSIONS: These findings demonstrate that miR-145 functions as tumor suppressor in RCC, suggesting that miR-145 may be a potential therapeutic target for RCC.

Identification of miR-508-3p and miR-509-3p that are associated with cell invasion and migration and involved in the apoptosis of renal cell carcinoma.

MicroRNAs (miRNAs) have emerged as powerful regulators of multiple processes linked to human cancer, including cell apoptosis, proliferation and migration, suggesting that the regulation of miRNA function could play a critical role in cancer progression. Recent studies have found that human serum/plasma contains stably expressed miRNAs. If they prove indicative of disease states, miRNAs measured from peripheral blood samples may be a source for routine clinical detection of cancer. Our studies showed that both miR-508-3p and miR-509-3p were down-regulated in renal cancer tissues. The level of miR-508-3p but not miR-509-3p in renal cell carcinoma (RCC) patient plasma demonstrated significant differences from that in control plasma. In addition, the overexpression of miR-508-3p and miR-509-3p suppressed the proliferation of RCC cells (786-0), induced cell apoptosis and inhibited cell migration in vitro. Our data demonstrated that miR-508-3p and miR-509-3p played an important role as tumor suppressor genes during tumor formation and that they may serve as novel diagnostic markers for RCC.