LncRNA KCNQ1OT1/miR-496/HMGB1 Signaling Axis Promotes Invasion and Migration of Non-small Cell Lung Cancer Cells.

Enhanced invasion and migration of non-small cell lung cancer (NSCLC) cells is the major cause of metastasis and poor prognosis in NSCLC. This study was conducted to investigate the role and mechanism of lncRNA KCNQ1OT1 in the proliferation, invasion, and migration of NSCLC cells. The expression of KCNQ1OT1 in NSCLC was analyzed in the StarBase database, and the target miRNA of KCNQ1OT1 as well as the target genes of the miRNA was predicted. Then, the mRNA expression levels of KCNQ1OT1, miR-496, and HMGB1 were detected in clinical tissue samples and cells by qRT-PCR assay. Besides, the protein levels of HMGB1 were detected by Western blot. MTT assay, transwell assay, and scratch assay were used to determine the proliferation, invasion, and migration ability of NSCLC cells, respectively. Correlation analysis was performed to assess the correlation between the expression of KCNQ1OT1, miR-496, and HMGB1 in clinical NSCLC samples. Dual-luciferase reporter gene assay was conducted to analyze the interaction between KCNQ1OT1 and miR-496 and between miR-496 and HMGB1. The database results showed that KCNQ1OT1 was highly expressed in NSCLC. Similarly, we found that the expression level of KCNQ1OT1 was significantly higher in NSCLC tissues and cells than that in the corresponding normal tissues and cells. The results of MTT assay, transwell assay, and scratch assay demonstrated that KCNQ1OT1 significantly enhanced the proliferation, invasion, and migration of NSCLC cells. Further mechanism exploration revealed that KCNQ1OT1 could sponge miR-496, and miR-496 directly targeted and regulated the expression of HMGB1. The expression of miR-496 and either KCNQ1OT1 or HMGB1 were negatively correlated in NSCLC, while the expression of KCNQ1OT1 and HMGB1 were positively correlated. Compared with normal paracancer tissues, miR-496 was much lower and HMGB1 was much higher expressed in NSCLC tissues. The results of cotransfection also further demonstrated that miR-496 inhibitor or sh-HMGB1 cotransfected with sh-KCNQ1OT1 could significantly decrease or increase the ability of sh-KCNQ1OT1 to inhibit the proliferation, invasion, and migration of H1299 cells, respectively. In conclusion, lncRNA KCNQ1OT1 promotes the invasion and migration of NSCLC cells through miR-496/HMGB1 signaling axis.

[A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system].

Hemophilia B, or the Christmas disease, is a common human disease caused by coagulation factor Ⅸ (FⅨ) deficiency. It is an X-linked recessive hereditary disease. Here we obtained FⅨ-knockout mouse strains with phenotype of hemophilia B with the CRISPR/Cas system efficiently. We chose the 8th exon as the target locus, and co-injected codon-optimized Cas9 mRNA with sgRNA of FⅨ into C57BL/6 mice zygotes. We obtained 60 mice in total and genotyped them by high resolution melting (HRM) and sequencing. The results showed the mutation rate was 85.0% in total, and 79.5% and 95.2% in males and females, respectively. No off-targets were detected in the similar locus by HRM. We future measured the FⅨ activity of each mice. The FⅨ: C of mutant mice were significantly below the normal level and reduced to 6.82% of wild-type mice. The activity assay demonstrated that all the mutant mice were lack of FⅨ. In summary, we have generated hemophilia B model mice with extreme efficiency, using the RNA-guided Cas9 nuclease gene editing system.

Overexpression of STAT3 potentiates growth, survival, and radioresistance of non-small-cell lung cancer (NSCLC) cells.

OBJECTIVE: Activation of signal transducer and activator of transcription 3 (STAT3) play important roles in tumorigenesis and tumor progression. The overexpression of STAT3 has been found in various malignancies including non-small-cell lung carcinoma (NSCLC). The purpose of this study was to explore the correlation between overexpression of STAT3 gene and growth, survival, and radiosensitivity of NSCLC cells. METHODS: Subclones using vector-based short hairpin RNA (shRNA) were established. RT-PCR and Western blot assays were performed to detect the expression of STAT3 mRNA and protein in untransfected or stably transfected NSCLC cells. Then, MTT and soft agar colony assays were performed to determine the effect of STAT3 inhibition on in vitro growth of NSCLC cells. Hoechst staining assay was performed to analyze the effect of STAT3 inhibition on apoptosis of NSCLC cells. Additionally, clonogenic survival assays were performed to detect the effect of STAT3 inhibition on in vitro radiosensitivity of NSCLC cells. Finally, to examine the effect of pSUPER-shSTAT3 on proliferation and radiosensitivity in vivo, a subcutaneous (s.c.) tumor formation assay in nude mice was performed. RESULTS: We successfully established two stable transfected cell lines (A549/shSTAT3 and SK-MES-1/shSTAT3) in which the expression of STAT3 mRNA and protein was down-regulated. Those two stable subclones showed a significantly dramatic reduction in colony-forming ability and proliferation not only in vitro but also in vivo. The apoptotic rates of A549/shSTAT3 and SK-MES-1/shSTAT3 cells increased to 19.2% and 16.4%, respectively. Moreover, shRNA-mediated STAT3 inhibition could also significantly enhance radiosensitivity of NSCLC cells both in vitro and in vivo. CONCLUSION: Together, the overexpression of STAT3 is correlated with growth, survival, and radioresistance of NSCLC cells, and STAT3 might be a molecular therapeutic target for gene therapy or radiosensitization of NSCLC.

[The application of combined interventional procedures for the management of intractable central airway stenosis].

OBJECTIVE: to evaluate the application of combined five interventional procedures in the management of intractable central airway stenosis. METHODS: clinical manifestations and pulmonary functions of 138 patients with intractable central airway stenosis were evaluated. Five interventional procedures, including high-frequency electrotome, argon plasma coagulation (APC), cryotherapy, stent placement and high-pressure balloon dilatation, were used in this study. Among them, two or more procedures were combined to manage complicated airway stenosis according to stenosis causes, types, position, degree and duration, or functions of distal lung tissue and airway. Forty-two cases were treated with high-frequency electrotome and APC, 54 cases with high-frequency electrotome, cryotherapy and APC, 29 cases with high-frequency electrotome, cryotherapy, APC and stent placement, and 13 cases with cryotherapy, APC and high-pressure balloon dilatation. Airways opening, short-term therapeutic effects and improvement of pulmonary functions were evaluated when ideal curative effects were achieved in the first month after intervention. RESULTS: the total short-term effective rate in the 138 patients was 100%. The airway diameter was increased from (2.6 ± 1.5) mm before operation to (6.2 ± 1.7) mm after operation (P < 0.05). Dyspnea score was decreased from (2.4 ± 0.8) before operation to (0.7 ± 0.6) after operation (P < 0.05). FEV(1) was increased from (1.8 ± 0.6) L before operation to (3.1 ± 0.7) L after operation (P < 0.05). Among 23 cases benign disease, including 4 benign tumor, 15 tuberculosis and 4 other granulomatosis, 5 cases with various degrees of restenosis needed further interventional therapy after 3 months of follow up and effective rate was 78.3% (18/23). After 6 months of follow-up, 3 cases with restenosis needed re-intervention, and the effective rate was 86.9% (20/23). All of the 23 cases did not experience stenosis after 12 months of follow-up. Patients with malignant tumor were not followed up for long term. CONCLUSION: combination of five interventional procedures, including high-frequency electrotome, APC, cryotherapy, stent placement and high-pressure balloon dilatation, has fewer complications and favorable clinical effects in management of intractable central airway stenosis.

Suspected SARS-CoV-2 infection with fever and coronary heart disease: A case report.

BACKGROUND: The coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Suspected cases accounted for a large proportion in the early stage of the COVID-19 outbreak. The deviation of the nucleic acid test by throat swab (the current gold standard of COVID-19) caused by variation in sampling techniques and reagent kits and coupled with nonspecific clinical manifestations make confirmation of the suspected cases difficult. Proper management of the suspected cases of COVID-19 is crucial for disease control. CASE SUMMARY: A 65-year-old male presented with fever, lymphopenia, and chest computed tomography (CT) images similar to COVID-19 after percutaneous coronary intervention. The patient was diagnosed as having bacterial pneumonia with cardiogenic pulmonary edema instead of COVID-19. This was based on four negative results for throat swab detection of SARS-CoV-2 nucleic acid using reverse transcriptase-polymerase chain reaction assay and one negative result for serological antibody of SARS-CoV-2 with the serological assay. Additionally, the distribution of ground-glass opacities and thickened blood vessels from the CT images differed from COVID-19 features, which further supported the exclusion of COVID-19. CONCLUSION: Distinguishing COVID-19 patients from those with bacterial pneumonia with cardiogenic pulmonary edema can be difficult. Therefore, it requires serious identification.

Inhibition of viral replication downregulates CD4(+)CD25(high) regulatory T cells and programmed death-ligand 1 in chronic hepatitis B.

Chronic hepatitis B is characterized by an impaired immune response to hepatitis B virus (HBV). Telbivudine treatment has significantly improved the clinical outcome of chronic HBV infection. However, the underlying mechanism behind the antiviral response of patients treated with nucleoside analogs remains unclear. To gather more evidence about the mechanism responsible for the weak immune response, in this study we analyzed the effects on HBV viral load of treatment with the nucleoside analogue telbivudine and the percentage of Tregs, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) expression, and related cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum of 28 patients with chronic hepatitis B were collected at baseline, and 3 mo and 6 mo after therapy was begun. In parallel with the decline in viral load and serum ALT normalization, we found a decline in circulating CD4(+)CD25(high) Tregs, PD-L1 on CD4(+) T cells, and IL-9 production. The expression of PD-1 on CD4(+) T cells and the production of IFN-γ did not increase during therapy. Our findings suggest that the antiviral effect of the nucleoside analogs may be attributable not only to their direct effect on virus suppression, but also to their immunoregulatory capabilities.

Circulating CD4+CD25high regulatory T cells and expression of PD-1 and BTLA on CD4+ T cells in patients with chronic hepatitis B virus infection.

The roles of regulatory T cells (Tregs) and PD-1 in hepatitis B have not been clearly described. Also, the role of B and T lymphocyte attenuator (BTLA), which serves as a negative regulator of T-cell activation, is still unknown in hepatitis B. In this study, we analyzed the frequency of circulating CD4(+)CD25(high) Tregs in patients with chronic hepatitis B (CHB), and subsequently investigated expression of PD-1 and BTLA on CD4(+) T cells, as well as their relationships with the clinical index of CHB patients. A total of 39 CHB patients and 19 healthy persons as controls were enrolled in the study. We found that the frequency of CD4(+)CD25(high) Tregs and PD-1 expression on CD4(+) T cells was significantly increased in CHB patients compared with normal controls. However, BTLA expression on CD4(+) T cells showed no significant difference between the two groups. The frequency of Tregs was significantly higher in patients with HBV DNA titers >or=10(8) than in those with HBV DNA titers <10(8). Circulating CD4(+)CD25(high) Treg frequency and PD-1 expression on CD4(+) T cells correlated positively with serum HBV DNA load in CHB patients. Our findings suggest that the increased frequency of CD4(+)CD25(high) Tregs and PD-1 expression on CD4(+) T lymphocytes may inhibit the cellular immune response against HBV and affect viral clearance, leading to the persistence of chronic HBV infection.