Peripheral Blood Genes Crosstalk between COVID-19 and Sepsis.

Severe coronavirus disease 2019 (COVID-19) has led to a rapid increase in death rates all over the world. Sepsis is a life-threatening disease associated with a dysregulated host immune response. It has been shown that COVID-19 shares many similarities with sepsis in many aspects. However, the molecular mechanisms underlying sepsis and COVID-19 are not well understood. The aim of this study was to identify common transcriptional signatures, regulators, and pathways between COVID-19 and sepsis, which may provide a new direction for the treatment of COVID-19 and sepsis. First, COVID-19 blood gene expression profile (GSE179850) data and sepsis blood expression profile (GSE134347) data were obtained from GEO. Then, we intersected the differentially expressed genes (DEG) from these two datasets to obtain common DEGs. Finally, the common DEGs were used for functional enrichment analysis, transcription factor and miRNA prediction, pathway analysis, and candidate drug analysis. A total of 307 common DEGs were identified between the sepsis and COVID-19 datasets. Protein-protein interactions (PPIs) were constructed using the STRING database. Subsequently, hub genes were identified based on PPI networks. In addition, we performed GO functional analysis and KEGG pathway analysis of common DEGs, and found a common association between sepsis and COVID-19. Finally, we identified transcription factor-gene interaction, DEGs-miRNA co-regulatory networks, and protein-drug interaction, respectively. Through ROC analysis, we identified 10 central hub genes as potential biomarkers. In this study, we identified SARS-CoV-2 infection as a high risk factor for sepsis. Our study may provide a potential therapeutic direction for the treatment of COVID-19 patients suffering from sepsis.

Bifidobacterium infantis-Mediated Herpes Simplex Virus-TK/Ganciclovir Treatment Inhibits Cancer Metastasis in Mouse Model.

Previous studies have found that Bifidobacterium infantis-mediated herpes simplex virus-TK/ganciclovir (BF-TK/GCV) reduces the expression of VEGF and CD146, implying tumor metastasis inhibition. However, the mechanism by which BF-TK/GCV inhibits tumor metastasis is not fully studied. Here, we comprehensively identified and quantified protein expression profiling for the first time in gastric cancer (GC) cells MKN-45 upon BF-TK/GCV treatment using quantitative proteomics. A total of 159 and 72 differential expression proteins (DEPs) were significantly changed in the BF-TK/GCV/BF-TK and BF-TK/GCV/BF/GCV comparative analysis. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis enriched some metastasis-related pathways such as gap junction and cell adhesion molecules pathways. Moreover, the transwell assay proved that BF-TK/GCV inhibited the invasion and migration of tumor cells. Furthermore, immunohistochemistry (IHC) demonstrated that BF-TK/GCV reduced the expression of HIF-1α, mTOR, NF-κB1-p105, VCAM1, MMP13, CXCL12, ATG16, and CEBPB, which were associated with tumor metastasis. In summary, BF-TK/GCV inhibited tumor metastasis, which deepened and expanded the understanding of the antitumor mechanism of BF-TK/GCV.

Bifidobacterial recombinant thymidine kinase-ganciclovir gene therapy system induces FasL and TNFR2 mediated antitumor apoptosis in solid tumors.

BACKGROUND: Directly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms. METHODS: Bifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression. RESULTS: The results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy. CONCLUSIONS: BF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim without inducing necroptosis and autophagy. Furthermore, BF-rTK + GCV showed to repress the inflammation of tumor through downregulating TNF-α expression. Survival analysis results of multiple cancer models confirmed that BF-rTK + GCV system has a wide field of application in solid tumor gene therapy.

Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1.

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

Intravenous Administration Is an Effective and Safe Route for Cancer Gene Therapy Using the Bifidobacterium-Mediated Recombinant HSV-1 Thymidine Kinase and Ganciclovir.

The herpes simplex virus thymidine kinase/ganciclovir (HSV TK/GCV) system is one of the best studied cancer suicide gene therapy systems. Our previous study showed that caspase 3 expression was upregulated and bladder tumor growth was significantly reduced in rats treated with a combination of Bifidobacterium (BF) and HSV TK/GCV (BF-rTK/GCV). However, it was raised whether the BF-mediated recombinant thymidine kinase combined with ganciclovir (BF-rTK/GCV) was safe to administer via venous for cancer gene therapy. To answer this question, the antitumor effects of BF-rTK/GCV were mainly evaluated in a xenograft nude mouse model bearing MKN-45 gastric tumor cells. The immune response, including analysis of cytokine profiles, was analyzed to evaluate the safety of intramuscular and intravenous injection of BF-rTK in BALB/c mice. The results suggested that gastric tumor growth was significantly inhibited in vivo by BF-rTK/GCV. However, the BF-rTK/GCV had no effect on mouse body weight, indicating that the treatment was safe for the host. The results of cytokine profile analysis indicated that intravenous injection of a low dose of BF-rTK resulted in a weaker cytokine response than that obtained with intramuscular injection. Furthermore, immunohistochemical analysis showed that intravenous administration did not affect the expression of immune-associated TLR2 and TLR4. Finally, the BF-rTK/GCV inhibited vascular endothelial growth factor (VEGF) expression in mouse model, which is helpful for inhibiting of tumor angiogenesis. That meant intravenous administration of BF-rTK/GCV was an effective and safe way for cancer gene therapy.

[Clinical efficacy of different doses of gamma globulin combined with glucocorticoid in treatment of moderate/severe acute Guillain-Barré syndrome in children: a comparative analysis].

OBJECTIVE: To investigate the clinical efficacy and safety of intravenous injection of low-dose versus high-dose gamma globulin combined with glucocorticoid pulse therapy in the treatment of children with moderate/severe acute Guillain-Barré syndrome (GBS). METHODS: A total of 100 children with moderate/severe acute GBS were randomly assigned to low-dose group (n=48) and high-dose group (n=52). The children in the low-dose and high-dose groups were treated with 0.2 g/(kg · d) and 0.4 g/(kg · d) gamma globulin respectively combined with methylprednisolone. The two groups were compared in terms of the time to improvements of symptoms after treatment, serum levels of inflammatory factors, proportion of children undergoing invasive ventilation, treatment response rate, and adverse events. RESULTS: After 5 days of treatment, the low- and high-dose groups had significant reductions in serum levels of tumor necrosis factor-α, interleukin-6, and C-reactive protein, and there were no significant differences in the reductions of these markers between the two groups. There were no significant differences between the two groups in the time to recovery of respiratory muscle paralysis, time to an improvement in muscle strength of one grade, time to recovery of sensory disturbance, and length of hospital stay. There was no significant difference in the treatment response rate between the low- and high-dose groups (90% vs 92%). There were also no significant differences in the incidence rates of pyrexia, headache, nausea, and palpitation between the two groups. CONCLUSIONS: Low-dose versus high-dose gamma globulin combined with methylprednisolone pulse therapy have comparable clinical efficacy and safety in the treatment of children with moderate/severe acute GBS.

Sequencing analysis and characterization of the plasmid pBIF10 isolated from Bifidobacterium longum.

A resident plasmid, pBIF10, was isolated from Bifidobacterium longum B200304, and the full-length sequence of pBIF10 was analyzed. In this sequence, we identified at least 17 major open reading frames longer than 200 bp. A tetracycline resistance gene, tetQ, was identified and verified to confer antibiotic resistance to tetracycline. The plasmid replicon with replication protein B gene (repB) and a typical iteron was identified in pBIF10. An artificial clone vector was constructed with the replicon of pBIF10; the results showed that repB controlled plasmid replication in other bifidobacteria host cells at low transformation frequency. Taken together, the analysis and characterization of pBIF10 provided necessary information for the understanding of antibiotic resistance mediated by a plasmid in a Bifidobacterium strain. GC% and repB sequence analyses indicated that pBIF10 was a molecular hybrid of at least 2 other bacterial genera plasmids.

Escherichia coli LTB26 mutant enhances immune responses to rotavirus antigen VP8 in a mouse model.

Adjuvant is a major supplementary component of vaccines to boost adaptive immune responses. To select an efficient adjuvant from the heat-labile toxin B subunit (LTB) of E. coli, four LTB mutants (numbered LTB26, LTB34, LTB57, and LTB85) were generated by multi-amino acid random replacement. Mice have been intranasally vaccinated with human rotavirus VP8 admixed. Among the four mutants, enzyme-linked immunosorbent assay (ELISA) revealed that LTB26 had enhanced mucosal immune adjuvanticity compared to LTB, showing significantly enhanced immune responses in both serum IgG and mucosal sIgA levels. The 3D modeling analysis suggested that the enhanced immune adjuvanticity of LTB26 might be due to the change of the first LTB α-helix to a ß-sheet. The molecular mechanism was studied using transcriptomic and flow cytometric (FCM) analysis. The transcriptomic data demonstrated that LTB26 enhanced immune response by enhancing B cell receptor (BCR) and major histocompatibility complex (MHC) II+-related pathways. Furthermore, LTB26 promoted Th1 and Th2-type immune responses which were confirmed by detecting IFN-γ and IL-4 expression levels. Immunohistochemical analysis demonstrated that LTB26 enhanced both Th1 and Th2 type immunity. Therefore, LTB26 was a potent mucosal immune adjuvant meeting the requirement for use in human clinics in the future.

A novel PAX9 variant in a Chinese family with non-syndromic oligodontia and genotype-phenotype analysis of PAX9variants.

OBJECTIVES: This study aimed to identifyPAX9variants in non-syndromic tooth agenesis families of China, as well as to analyze the genotype⁃phenotype of non-syndromic tooth agenesis caused by PAX9variants, which can provide a basis for the genetic diagnosis of tooth agenesis. METHODS: We collected the data of 44 patients with non-syndromic oligodontia who underwent treatment at Stomatological Hospital of Hebei Medical University between 2018 and 2023. Whole-exome sequencing was performed on the peripheral blood of the proband and its core family members, and the variants were verified by Sanger sequencing. Pathogenicity analysis and function prediction of the variants were performed using bioinformatics tools. The correlation between the genotype of PAX9 variant and its corresponding phenotype was examined by reviewing 55 publications retrieved from PubMed. The studies involved 232 tooth agenesis patients with PAX9 variants. RESULTS: A novel PAX9 c.447delG (p.Pro150Argfs*62) and a reported PAX9 c.406C>T (p.Gln136*) were identified in two Chinese families. Through bioinformatics analysis and three-dimensional structural modeling, we postulated that the frameshift variant was pathogenic. The outcome was the premature cessation of PAX9 protein, which caused severe structural and functional deficiencies. Summarizing the PAX9 genotype-phenotype relationship revealed that patients carrying the PAX9 variant commonly led to loss of the second molars. CONCLUSIONS: We identified the novel PAX9 c.447delG (p.Pro150Argfs*62) in a Chinese family of non-syndromic oligodontia, expanding the known variant spectrum of PAX9. The most susceptible tooth position for PAX9 variants of tooth agenesis was the second molars and the deciduous molars during the deciduous dentition.

Prognosis Risk Model Based on Pyroptosis-Related lncRNAs for Gastric Cancer.

Gastric cancer (GC) is a malignant tumor with a low survival rate, high recurrence rate, and poor prognosis. With respect to this, pyroptosis is a type of programmed cell death that can affect the occurrence and development of tumors. Indeed, long non-coding RNAs (lncRNAs) were broadly applied for the purposes of early diagnosis, treatment, and prognostic analysis in regard to cancer. Based on the association of these three purposes, we developed a novel prognosis risk model based on pyroptosis-related lncRNAs (PRlncRNAs) for GC. The PRlncRNAs were obtained via univariate and multivariate Cox regression in order to build the predictive signatures. The Kaplan-Meier and gene set enrichment analysis (GSEA) methods were used to evaluate the overall survival (OS) and functional differences between the high- and low-risk groups. Moreover, the correlation of the signatures with immune cell infiltration was determined through single-sample gene set enrichment analysis (ssGSEA). Finally, we analyzed this correlation with the treatment responses in the GC patients; then, we performed quantitative reverse transcription polymerase chain reactions (qRT-PCRs) in order to verify the risk model. The high-risk group received a worse performance in terms of prognosis and OS when compared to the low-risk group. With respect to this, the area under the receiver operating characteristic curve (ROC) was found to be 0.808. Through conducting the GSEA, it was found that the high-risk groups possessed a significant enrichment in terms of tumor-immunity pathways. Furthermore, the ssGSEA revealed that the predictive features possessed strong associations with immune cell infiltration in regard to GC. In addition, we highlighted that anti-immune checkpoint therapy, combined with conventional chemotherapy drugs, may be more suitable for high-risk patients. The expression levels of LINC01315, AP003392.1, AP000695.2, and HAGLR were significantly different between the GC cell lines and the normal cell lines. As such, the six PRlncRNAs could be regarded as important prognostic biomarkers for the purposes of subsequent diagnoses, treatments, prognostic predictions, and the mechanism research of GC.

Deciphering the rule of antigen-antibody amino acid interaction.

Purpose: Antigenic drift is the biggest challenge for mutagenic RNA virus vaccine development. The primary purpose is to determine the IEMM (immune escape mutation map) of 20 amino acids' replacement to reveal the rule of the viral immune escape. Methods: To determine the relationship between epitope mutation and immune escape, we use universal protein tags as a linear epitope model. To describe and draw amino acid linkage diagrams, mutations of protein tags are classified into four categories: IEM (immune escape mutation), ADERM (antibody-dependent enhancement risk mutation), EQM (equivalent mutation), and IVM (invalid mutation). To overcome the data limitation, a general antigen-antibody (Ag-Ab) interaction map was constructed by analyzing the published three-dimensional (3D) Ag-Ab interaction patterns. Results: (i) One residue interacts with multiple amino acids in antigen-antibody interaction. (ii) Most amino acid replacements are IVM and EQM. (iii) Once aromatic amino acids replace non-aromatic amino acids, the mutation is often IEM. (iv) Substituting residues with the same physical and chemical properties easily leads to IVM. Therefore, this study has important theoretical significance for future research on antigenic drift, antibody rescue, and vaccine renewal design. Conclusion: The antigenic epitope mutations were typed into IEM, ADERM, EQM, and IVM types to describe and quantify the results of antigenic mutations. The antigen-antibody interaction rule was summarized as a one-to-many interaction rule. To sum up, the epitope mutation rules were defined as IVM and EQM predomination rules and the aryl mutation escape rule.

The DACH1 Gene Transcriptional Activation and Protein Degradation Mediated by Transactivator Tas of Prototype Foamy Virus.

Foamy viruses are members of the Retroviridae family's Spumaretrovirinae subfamily. They induce cell vacuolation and exhibit a foamy pathogenic impact after infecting cells. DACH1 (dachshund family transcription factor 1) is a crucial cytokine linked to tumor development, and is associated with the growth of many different malignant tumor cells. Additionally, DACH1 suppresses pancreatic cell proliferation and is involved in diabetes insulin signaling. Prototype foamy viruses (PFVs) were used for the investigation of the regulatory mechanism of FVs on cellular DACH1 expression. The results show that DACH1 expression in PFV-infected cells was inconsistent at both the transcriptional and protein levels. At the transcriptional level, DACH1 was significantly activated by PFV transactivator Tas, and dual-luciferase reporter gene tests, EMSA, and ChIP assays found a Tas response element of 21 nucleotides in the DACH1 promoter. PFV and Tas did not boost the levels of DACH1 protein in a manner consistent with the high levels of DACH1 transcription expression. It was noted that Tas increased the expression of the Ser/Thr protein phosphatase PPM1E, causing PPM1E-mediated post-translational SUMOylation alterations of DACH1 to prompt DACH1 to degrade. The reason for DACH1 protein degradation is that DACH1 inhibits PFV replication. To sum up, these findings show that PFV upregulated the transcription of DACH1, while urging its protein into PPM1E-mediated SUMOylation, to eliminate the adverse effect of DACH1 overexpression of host cells on viral replication and promote virus survival.

A robust high selectivity fluorescence turn-on nanoprobe for peroxynitrite detection in inflammatory cells and mice.

Changed levels of intracellular peroxynitrite anion (ONOO-) are closely related to the occurrence and development of inflammation. Specific imaging of ONOO- at sites of inflammation can be of great significance not only for inflammation diagnosis but also for obtaining a deeper understanding of the role of ONOO- in inflammation. Therefore, there is an urgent need for constructing some reliable tools to study the relationship between ONOO- and inflammation in biosystems. In this work, we developed a robust high selectivity fluorescence turn-on nanoprobe (Rhb-ONOO) for inflammation-targeted imaging of ONOO-. The Rhb-ONOO was obtained by self-assembly of amphiphilic Rhb-ONOO, which was constructed by the condensation reaction of the hydrophobic, ONOO--response and deep red-emitting fluorophore (Rhb) with hydrophilic biopolymer glycol chitosan (GC). Rhb-ONOO showed rapid response towards ONOO- during 60 s, high sensitivity with 19-fold enhancement of fluorescence intensity ratio (I628/I0), and excellent selectivity towards ONOO- over other analytes as well as a good linear relationship was observed between the I628/I0 and the ONOO- concentration range 0-1 µM, with an excellent limit of detection (LOD) of 33 nM. Impressively, it was successfully employed Rhb-ONOO for ONOO- imaging in living inflammatory cells and drug-induced inflammatory mice, illustrating nanoprobe Rhb-ONOO has excellent potential for further study ONOO--related inflammatory diseases.

Construction of Bifidobacterium infantis as a live oral vaccine that expresses antigens of the major fimbrial subunit (CfaB) and the B subunit of heat-labile enterotoxin (LTB) from enterotoxigenic Escherichia coli.

We sought to develop Bifidobacterium infantis (BI) as a vehicle for the expression of heterologous antigens. Two proteins of enterotoxigenic Escherichia coli (ETEC) were expressed in BI: CfaB, a major fimbrial subunit protein, and LTB, the B subunit of heat-labile enterotoxin. The expression of CfaB and LTB in BI was verified by electrophoretic analysis. Sprague-Dawley rats were then subjected to intragastric immunization with BI-CfaB and BI-LTB systems both separately and together. ELISA was used to characterize the serum and mucosal immune responses against ETEC antigens. The immunized rats were intraperitoneally challenged with wild-type ETEC H10407 to study the immune response in vivo. The serum titres of IgG and faecal IgA antibodies in the BI-CfaB plus BI-LTB mixed vaccination group were significantly greater than those in the other two groups, which were immunized with a single vaccine (P<0.05). However, no significant difference was seen between the two groups that received a single immunization. These results suggest that expressing CfaB and LTB in BI provides a probiotic system with immunogenic properties. Furthermore, the expression of LTB in BI preserved its mucosal adjuvant effect. So this study confirms that BI can be used as a novel oral vaccine expression system for a heterologous antigen and BI-LTB can provide mucosal adjuvant properties.

Analysis of Risk Factors for Pneumonia Death in ICU Environment Based on Logistic Regression.

Objective: To explore the risk and protect factors for death of pneumonia patients in intensive care unit (ICU), we conducted this logistic regression research. Methods: We collected demographic and nursing care data for 80 patients form Wuhan fourth hospital, in which 40 patients were dead and the other 40 patients were alive. Difference analysis, Pearson's correlation matrix, and logistic regression were conducted to explore the risk and protective factors for living status of pneumonia patients in ICU. Results: A total of 40 individuals were dead from pneumonia in ICU. The demographic and nursing information had no difference between death and living groups except age. After that, correlation analysis showed that there were correlations between living status, age, and marriage. Logistic regression showed that age (odds ratio (OR) = 0.94, P < 0.05) and no education (OR = 0.21, P < 0.05) may be harmful for pneumonia patients living status while high-quality nursing (OR = 2.72, P < 0.05) may be helpful for pneumonia patients living status. Conclusion: High-quality care plays an important role in protecting the survival of patients with pneumonia, and old age and uneducated may be the risk factors for the death of patients with pneumonia.

Porphyromonas gingivalis Lipopolysaccharide-Induced B Cell Differentiation by Toll-like Receptors 2 and 4.

BACKGROUND: Porphyromonas gingivalis (P. gingivalis) is a pathogenic bacterium widely present in subgingival plaques of patients with periodontitis. It induces periodontitis with bone loss as its main feature by changing the number and composition of symbiotic microorganisms, as well as inducing the natural immune response of the host. However, the mechanism of the latter remains unclear. OBJECTIVE: This study aims to investigate the effect of P. gingivalis lipopolysaccharide (LPS) on regulatory B cells (Breg) in the occurrence and development of periodontitis. METHODS: We detected the mRNA levels of IL-10 in B cells under the stimulation of P. gingivalis LPS and/or E. coli LPS, distinguished IL-10-producing cells from different B cell subgroups using flow cytometry. Through toll-like receptor (TLR) knockout mice, the role of TLR2 and TLR4 in this process was also evaluated. RESULTS: Results showed that P. gingivalis stimulated B cells to produce IL-10 via TLR2/4. CD5+B1 subset is the main source of IL-10+Breg cell. Under P. gingivalis LPS stimulation, CD5+IgM+CD93-IL-10+B cell subset increased significantly, which was regulated through TLR2/ 4. CONCLUSION: The results of this study provides new insights into the immunopathogenic mechanism of P. gingivalis, preliminarily discussed the effect of P. gingivalis on the production of Breg, and present a theoretical foundation for subsequent investigations on the occurrence and development of periodontitis.

Suppression of epidermal growth factor receptor (EGFR) expression by small hairpin RNA inhibits the growth of human nonsmall cell lung cancers bearing wild-type and mutant EGFR.

In the present study, we have used plasmid-based RNA interference (RNAi) strategy to downregulate the expression of epidermal growth factor receptor (EGFR) in EGFR wild-type (H292) and mutant (H1975) lung tumor models. The targeted knockdown of EGFR by small hairpin RNA not only inhibited growth of H292 xenograft but also inhibited H1975 lung cancer cell and xenograft, which bore L858R/T790M EGFR and was resistant to EGFR tyrosine kinase inhibitors. These data demonstrated that small hairpin RNA was an effective therapy against mutant EGFR-expressing cancer cells and thus considered to be a promising strategy in the treatment of lung cancers.

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice.

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.

Protein Tyrosine Kinase 7 Regulates EGFR/Akt Signaling Pathway and Correlates With Malignant Progression in Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, is associated with high invasiveness, high metastatic occurrence and poor prognosis. Protein tyrosine kinase 7 (PTK7) plays an important role in multiple cancers. However, the role of PTK7 in TNBC has not been well addressed. This study was performed to evaluate the role of PTK7 in the progression of TNBC. METHODS: Correlation of PTK7 expression with clinicopathological parameters was assessed using tissue microarray immunohistochemistry (IHC) staining in 280 patients with breast cancer. PTK7 expression in TNBC (MDA-MB-468, MDA-MB-436 and MDA-MB-231) and non-TNBC (MCF7 and SK-BR-3) breast cancer cell lines were examined using immunoblotting assay. PTK7 correlated genes in invasive breast carcinoma were analyzed using cBioPortal breast cancer datasets including 1,904 patients. PTK7 overexpressed or knockdown TNBC cell lines (MDA-MB-468 and MDA-MB-436) were used to analyze the potential roles of PTK7 in TNBC metastasis and tumor progression. A TNBC tumor bearing mouse model was established to further analyze the role of PTK7 in TNBC tumorigenicity in vivo. RESULTS: PTK7 is highly expressed in breast cancer and correlates with worse prognosis and associates with tumor metastasis and progression in TNBC. Co-expression analysis and gain- or loss-of-function of PTK7 in TNBC cell lines revealed that PTK7 participates in EGFR/Akt signaling regulation and associated with extracellular matrix organization and migration genes in breast cancer, including COL1A1, FN1, WNT5B, MMP11, MMP14 and SDC1. Gain- or loss-of-function experiments of PTK7 suggested that PTK7 promotes proliferation and migration in TNBC cell lines. PTK7 knockdown MDA-MB-468 cell bearing mouse model further demonstrated that PTK7-deficiency inhibits TNBC tumor progression in vivo. CONCLUSION: This study identified PTK7 as a potential marker of worse prognosis in TNBC and revealed PTK7 promotes TNBC metastasis and progression via EGFR/Akt signaling pathway.

FAM83A as a Potential Biological Marker Is Regulated by miR-206 to Promote Cervical Cancer Progression Through PI3K/AKT/mTOR Pathway.

Background and Objective: Chemotherapy and radiotherapy are effective treatment options for cervical cancer (CC), but their efficacy is limited by short survival rate of about 5 years particularly for advance stage CC. Bioinformatics analysis combined with experimental in vivo and in vitro data can identify potential markers of tumorigenesis and cancer progression to improve CC prognosis and survival rate of the patients. This study aims to investigate the prognostic value of family with sequence similarity 83, member A (FAM83A) gene and miR-206 in promoting CC progression and the involved genetic signaling pathways. Method: This was a bioinformatic analysis study based on RNA sequencing data of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and verification by in vivo and in vitro experimental data. It was designed to evaluate whether the aberrantly expressed gene signatures could serve as new potential biomarker to improve prognosis prediction in CC. The TCGA RNA sequencing data [306 cervical squamous cell carcinoma (SCC) and endocervical adenocarcinoma samples and 13 adjacent samples] and GEO data (GSE9750 and GSE52903 datasets) were integrated and performed a bioinformatics analysis. Results: The results showed that CC-associated FAM83A gene serves as a key regulator of CC development and progression. Functionally, we observed that FAM83A is significantly overexpressed in CC, which is linked to poor overall survival as well as disease-free survival in CC patients. The in-vitro and in-vivo assessments performed after silencing FAM83A revealed that cell proliferation was significantly inhibited and the S-phase cell cycle arrest was induced. Mechanistically, FAM83A plays a role in PI3K/AKT signaling, and its downstream molecules could promote CC cell proliferation. Furthermore, functionality assessments by in-vitro luciferase reporter system and immunoblot analysis showed that miR-206 was the upstream of FAM83A and negatively correlated with FAM83A. Conclusion: The miR-206/FAM83A/PI3K/AKT signaling pathway possibly serves as a critical effector in CC progression indicating the potential prognostic value of FAM83A gene as a novel biomarker for CC progression.