Molecular and Cellular Probes最新文献

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.

Background

Aberrant expression of circRNAs is involved in the progression of hepatocellular carcinoma (HCC). This study aimed at screening the pro-tumorigenic circular RNAs (circRNAs) in HCC and the mechanisms of circCPSF6 expression influencing HCC characteristics.

Method

circCPSF6 was identified in HCC tissues using high-throughput sequencing data, and its expression was verified in both HCC tissues and cell lines using quantitative real-time PCR (qRT-PCR). CCK-8 and Transwell assays were used to evaluate the effects of circCPSF6 on HCC proliferation and migration. A xenograft mouse model was used to investigate the effects of circCPSF6 on HCC progression in vivo, and the significance of circCPSF6 in HCC was verified both in vivo and in vitro. circCPSF6-associated miRNAs and mRNAs were identified using bioinformatic analyses. Luciferase reporter, RNA pull-down, Fluorescence in situ hybridization, and RNA immunoprecipitation assays were performed to elucidate the circCPSF6 regulatory axis in HCC.

Result

CircCPSF6 expression was increased in HCC cell lines and tissues, and the expression of its parental mRNA was positively correlated with tumor severity and negatively correlated with survival. Mechanistic analyses of HCC cell lines showed that tumorigenesis was inhibited by circCPSF6 knockdown and promoted by its overexpression. Functional analyses revealed that circCPSF6 mediated HCC development by sponging miR-145-5p as a competing endogenous RNA. Furthermore, this sponging upregulated the miR-145-5p target gene MAP4K4, a classical pro-tumorigenic gene.

Conclusion

Our findings reveal a regulatory network that includes the circCPSF6–miR-145-5p–MAP4K4 axis. Elements of this axis are potential HCC biomarkers, as well as targets for HCC treatment.

Lung cancer is one of the most common malignant tumors and has a poor prognosis and a low survival rate. Traditional treatments, such as radiotherapy and chemotherapy, still face some challenges because of high drug resistance and toxicity. Therefore, it is necessary to discover a new kind of targeted drug with low toxicity and high efficiency. CDK12 is a cell cycle-dependent kinase whose main function is to activate RNA polymerase II (RNAPII) and promote the transcriptional extension of RNA. However, the role and molecular mechanism of CDK12 in lung cancer are still unclear.

In this study, the mutation and RNA-Seq data of CDK12 in lung adenocarcinoma and squamous cell carcinoma were downloaded from The Cancer Genome Atlas (TCGA) database and analyzed with the custom scripts. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and cell colony formation assays. A subcutaneous tumor experiment in nude mice was used to examine the effects of CDK12 knockdown on the in vivo tumor growth of NSCLC cells. The cell cycle distribution and the apoptosis rate of lung cancer cells were assessed by flow cytometry. Regulation of TANK-binding kinase 1 (TBK1) by CDK12 was evaluated by quantitative PCR, immunoprecipitation and Western blot analysis.

In this study we have analyzed the mutation and expression data of The Cancer Genome Atlas (TCGA) database and found that CDK12 is highly expressed in lung cancer tissues. Clinical correlation analysis showed that high expression of CDK12 in NSCLC reduces patient survival, but its high expression is only related to early tumor progression and has no significant correlation with late tumor progression and metastasis. Furthermore, we present evidence that CDK12 depletion in lung cancer cell lines not only leads to the inhibition of cell growth and induces apoptosis but also inhibits tumor growth of NSCLC cells in vivo. CDK12 positively regulates the expression of the oncogene TBK1 in lung cancer cells. These results revealed that CDK12 affects the progression of non-small cell lung cancer through positive regulation of TBK1 expression, suggesting that CDK12 might be a potential molecular target for the treatment of non-small cell lung cancer.

Malignant melanoma is the most lethal form of skin cancer, and its incidence rates are increasing in Europe, America, and Oceania countries. Despite immune checkpoint inhibitors, such as PD-1 inhibitors, have been shown to have significant therapeutic effects on malignant melanoma, many patients are unresponsive to these treatments, even emerged resistance. There is an urgent need to discover novel biomarkers that might distinguish resistant patients from responders. In this study, we used a series of bioinformatics analyses and experimental validation. The GSE65041 was used for differential expression analysis. Kaplan-Meier was used to assess the prognostic value. ESTIMATE, ssGSEA, EPIC, TIMER, quanTiseq and MCPcounter for estimation of immune infiltration in the tumor microenvironment. We eventually identified that CD3ζ was significantly down-regulated in IHC PD-L1(−) melanoma patients. Low level of CD3ζ expression possessed a poor prognosis. CD3ζ low expression population is significantly associated with lower immune infiltration. In vivo experiment, CD3ζ expression was significantly down-regulated in mice melanoma after intradermally injected with B16–F10R cells. Compared to their wildtype counterparts, melanoma resistant mice treated with nivolumab showed significant reductions in tumor volume and weight when adding CD3ζ. In vitro experiment, the addition of CD3ζ increased nivolumab effection on inhibiting B16–F10R cell viability. Our findings indicated that CD3ζ could be a novel predictive biomarker of PD-1 inhibitor resistance in melanoma.

Although there are several treatments available for gastric cancer (GC), the prognosis of the disease is still poor due to many factors, such as late diagnosis and tumor heterogeneity. To identify potential therapeutic targets, bioinformatics techniques and clinical sample validation were employed and prostate transmembrane protein androgen induced 1 (PMEPA1) was selected for further study. In the present study, we found that elevated PMEPA1 expression correlates with a worse prognosis and weaker anti-tumor immunity in GC patients. Moreover, our study showed that PMEPA1 not only influences cell proliferation, clone formation, invasion, and migration in vitro, but also plays an important role in GC progression in vivo. Mechanically, PMEPA1 exerts its oncogenic effects through activating the Wnt/β-catenin signaling pathway. Therefore, PMEPA1 is a potential target for treating GC effectively.

Introduction

Hemoglobin A1c (HbA1c) is used to monitor glucose homeostasis and to identify risk for diabetes. As diabetic patients are frequently present with dyslipidaemia, low-grade inflammation and hyperuricemia, we tested whether HbA1c levels can be estimated having the information about lipid profile, uric acid (UA) and C-reactive protein (CRP) levels. We developed formulas to describe the association of these parameters with HbA1c levels.

Methods

Data of 9599 male and 10,817 female patients, measured between 2008 and 2018, were analysed. Patients represented a general hospital patient population with overrepresentation of those with elevated HbA1c over 5.6%. The impact of gender, age, CRP, lipid profile and UA levels on HbA1c % on HbA1c levels was tested with multiple linear regression model. The magnitude of effects of individual factors was used to develop formulas to describe the association between HbA1c and other cardiometabolic parameters. With these formulas we estimated median HbA1c values in each age in both gender and compared them to measured HbA1c levels.

Results

The developed formulas are as follow: HbA1c (estimated) in women = 0.752 + 0.237*log10(HDL/cholesterol) + 0.156*log10 (cholesterol) + 0.077*log10 (triglyceride) + 0.025*log10(CRP) +0.001*log10 (age) −0.026*log10(HDL/LDL) −0.063*log10 (uric acid)-0.075*log10 (LDL)-0.199*log10(HDL); HbA1c (estimated) in men = 1.146 + 0.08*log10 (triglyceride) + 0.046*log10(CRP) + 0.01*log10 (cholesterol) + 0.001*log10 (age) −0.014*log10(HDL)-0.018*log10(HDL/LDL)-0.025*log10(HDL/cholesterol) −0.068*log10 (LDL)-0.159*log10 (uric acid)

Between 20 and 70 years of age, estimated HbA1c matched perfectly to measured HbA1c in.

Conclusion

At population level, HbA1c levels can be estimated almost exactly based on lipid profile, CRP and uric acid levels in female patients between 20 and 70 years.

Lung cancer (LC) is the primary reason for cancer-associated fatalities globally. Due to both tumor-suppressing and tumor-promoting activities, the TGF-β family of growth factors is extremely essential to tumorigenesis. A non-coding single-stranded short RNA called microRNA (miRNA), which is made up of about 22 nt and is encoded by endogenous genes, can control normal and pathological pathways in various kinds of cancer, including LC. Recent research demonstrated that the TGF-β signaling directly can affect the synthesis of miRNAs through suppressor of mothers against decapentaplegic (SMAD)-dependent activity or other unidentified pathways, which could generate allostatic feedback as a result of TGF-β signaling stimulation and ultimately affect the destiny of cancer tissues. In this review, we emphasize the critical functions of miRNAs in lung cancer progression and, more critically, how they affect the TGF-β signaling pathway, and explore the role of both the TGF-β signaling pathway and miRNAs as potential therapeutic targets for improving the treatments of LC patients.

Ischaemic stroke (IS) and venous thromboembolism (VTE) are two forms of thromboembolism that, although distinct, seem to share numerous risk factors. Concerning genetic risk factors, while many VTE genetic markers have been reported, inclusively by genome-wide association studies (GWAS), the identification and validation of genetic determinants underlying IS pathogenesis have been challenging. Considering that IS and VTE shared biological pathways and aetiological factors, the severity of IS might be also influenced by VTE-related genetic variants. Thus, the present study was designed to analyse the impact of six VTE GWAS-identified genetic variants on the clinical outcome of 363 acute IS patients. Results revealed that the single-nucleotide polymorphism (SNP) F11 rs4253417 was an independent predictor of the 5-year risk of death among patients with total anterior circulation infarct (TACI). Namely, the ones carrying the SNP C allele presented a fourfold increase in the 5-year risk of death compared to TT genotype carriers (CC/CT vs. TT; adjusted HR, 4.240; 95% CI, 1.260–14.270; P = 0.020). This SNP is known to be associated with coagulation factor XI (FXI) levels, thus with implications in haemostasis and inflammation. As such, F11 rs4253417 might be a promising prognostic biomarker among TACI patients to aid in clinical decision-making. However, additional investigation is required to confirm the study's results and dissect the underlying mechanisms.

Introduction

Oral squamous cell carcinoma (OSCC) is the most common cancers arising from the head and neck region. There is growing evidence that lncRNAs play an important role in OSCC progression. The study aims to investigate correlations between the expression levels of LncRNAs of PARROT, MYCNUT, DANCR, and KTN1-AS1 with clinicopathological characteristics and finding suitable biomarkers for OSCC.

Material and method

Total lncRNAs related to cancers and HNSC trascriptomics data were downloaded from lncRNADisease v2.0 database and xenabrowser, respectively. Then, ACO was perfomed on shared of LncRNAs between two databases. Finally, some lncRNAs were proposed as potential biomarkers.

Thirty biopsies samples from patients with the OSCC and 30 healthy subjects were collected by the surgery. Questionnaires including clinical and demographic data were filled for all cases. Using Real-time PCR, the expression levels of PARROT, MYCNUT, DANCR, and KTN1-AS1 lncRNAs were quantified.

Result

According to the results,17 novel gene symbol was identified.All the candidate lncRNAs the expression levels of PARROT, MYCNUT, DANCR, and KTN1-AS1 were remarkably upregulated in OSCC tumors in comparison with control group (RQ: 10.00 (P < 0.0001), RQ: 2.920 (P < 0.0001), RQ: 1.623 (P = 0.002), and 4.467 (P < 0.0001), respectively). Also, we found significant associations between tumor lncRNAs expression of PARRPT and DANCER and tumor metastasis (P = 0.009, and P = 0.005, respectively). Additionally, lncRNA KTN1-AS1 expression level was significantly higher in the patients with tumor size more than 3 cm, in comparison with tumor less than 3 cm (P = 0.005). According ROC analysis, all these candidate lncRNAs can be a significant predictor for OSCC (AUC of PARROT lncRNA = 69.72%, AUC of MYCNUT = 98.22%, AUC of DANCR = 74.83%, and AUC of KTN1-AS1 = 99.22%).

Conclusion

we found that overexpression levels of PARROT, MYCNUT, DANCR, and KTN1-AS1 lncRNAs were correlated with poor clinicopathological characteristics in patients with OSCC. Also, PARROT, MYCNUT, DANCR, and KTN1-AS1 are novel biomarker for the detection of OSCC.

Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific position in the genome. Until now, 585 million SNPs have been identified in the human genome, and therefore, a widely applicable method is desirable to detect a specific SNP. Herein we report a simple and reliable genotyping assay, which seems to be suitable for medium and small size laboratories, as well, to easily genotype most of the SNPs. In our study, all of the possible base variations (A-T, A-G, A-C, T-G, T-C, G-C) were tested to prove the general feasibility of our technique. The basis of the assay is a fluorescent PCR, in which both allele-specific primers, differing only at the 3′ end according to the sequence of the SNP, were present, and the length of one of them was modified with 3 bp by adding an adapter sequence to the 5’ end of that primer. The competitive presence of both allele-specific primers excludes the false amplification of the absent allele (which can happen in simple allele-specific PCR (AS-PCR)) and ensures the amplification of the proper allele(s). Unlike other complicated genotyping methods that use of manipulation of fluorescent dyes for genotyping, we apply an approach based on the length of amplicons from different alleles to differentiate between them. In our experiment (named variable fragment length allele-specific polymerase chain reaction (VFLASP)), the investigated six SNPs, containing the six available base variations, gave clear and reliable results after detecting the amplicons by capillary electrophoresis.