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Current guidelines recommend adjuvant chemotherapy (ACT) for stage II colorectal cancer (CRC) patients by using clinical high risk factors, with which circulating tumor cells (CTCs) were not considered. Here, an assessment to detect CTCs based on subtraction enrichment mediated by magnetic beads conjugated with CD45, immunofluorescence staining of CK, and fluorescence in situ hybridization of CEP8 is established. Both CEP8- and CK-positive CTCs have the potential to improve the risk stratification of stage II CRC patients. Patients with preoperative CTCs of ≥4 had a significantly higher recurrence risk than those with preoperative CTCs of4 in two external validation cohorts (

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0–4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease. Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23a∼27a∼24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment. Significance: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.

Rationale: Cancer stem cells (CSCs) are considered to be essential for tumorigenesis, recurrence, and metastasis and therefore serve as a biomarker for tumor progression in diverse cancers. Recent studies have illustrated that specific miRNAs exhibit novel therapeutic potential by controlling CSC properties. miR-1275 is upregulated in lung adenocarcinoma (LUAD) and enhances its stemness. However, the underlying mechanisms have not been elucidated. Methods: miRNA expression microarray of LUAD and adjacent nontumor tissues was used to identify miRNAs involved in LUAD malignant progression. miR-1275 expression level was determined using quantitative real-time PCR (RT-qPCR) and in situ hybridization (ISH), and its correlation with clinicopathological characteristics was analyzed in LUAD specimens. The upstream regulator of miR-1275 was validated by chromatin immunoprecipitation (ChIP). The biological functions and underlying mechanisms of miR-1275 were investigated both in vitro and in vivo. Results: MiR-1275 was highly upregulated in lung cancer cell lines and LUAD tissues. Overexpression of miR-1275 in lung cancer patients was associated with shorter overall- and recurrence-free-survival. Proto-oncogene HIF-1ɑ was identified as the transcription mediator of miR-1275. Activation of Wnt/β-catenin and Notch signaling by miR-1275 was found to enhance the stemness of LUAD cells, while antagonizing miR-1275 or suppressing Wnt/β-catenin and Notch pathways potently reversed miR-1275-induced pathway co-activation and stemness. Enhanced stemness dramatically promoted tumorigenicity, recurrence, and metastasis. miR-1275 directly targeted multiple antagonists of Wnt/β-catenin and Notch pathways, including DKK3, SFRP1, GSK3β, RUNX3, and NUMB, respectively, which resulted in signaling activation. Conclusions: Our findings identified miR-1275 as a potential oncogene in LUAD that exerts its tumorigenic effect through co-activating Wnt/β-catenin and Notch signaling pathways. Thus, HIF-1ɑ-regulated miR-1275 might be a potential therapeutic target for LUAD.

Currently, the most commonly applied method for the determination of breast cancer subtypes is to test estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 by immunohistochemistry (IHC). However, the IHC method has substantial intraobserver and interobserver variability. ESR1, PGR, ERBB2, and MKi67 mRNA tests by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay may improve the diagnostic objectivity and efficiency. Here, we compared the concordance between RT-qPCR and IHC for assessment of the same biomarkers and evaluated the subtypes.A total of 265 eligible cases were divided into a training cohort and a validation cohort, and the expressions of ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 were tested by IHC and RT-qPCR. Then, the appropriate cutoff of RT-qPCR was calculated in the training cohort. The concordance between RT-qPCR and IHC was calculated for individual marker. In addition, we investigated the subtypes based on the RT-qPCR results.The Spearman correlation coefficients between ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 by IHC and RT-qPCR were 0.768, 0.699, 0.762, and 0.387, respectively. The cutoff values for the RT-qPCR assay of ESR1 (1%), PGR (1%), ERBB2, and MKi67 (14%) were 35.539, 32.139, 36.398, and 29.176, respectively. The overall percent agreement (OPA) between ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 by IHC and RT-qPCR was 92.48%, 73.68%, 92.80%, and 74.44%, respectively. A total of 224 (84.53%) specimens were concordant for the breast cancer subtypes (IHC-based type) by RT-qPCR.Evaluation of breast cancer biomarker status by RT-qPCR was highly concordant with IHC. RT-qPCR can be used as a supplementary method to detect molecular markers of breast cancer.

Long non-coding RNAs (lncRNAs) play crucial roles in human diseases. However, the detailed role of lncRNAs in hypertrophic scar fibroblasts (HSFs) is inadequately understood. This study aimed to investigate the potential role of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in hypertrophic scarring. Expression of lncRNAs, miRNAs, and genes were detected by polymerase chain reaction; protein expression was evaluated using western blotting. Cellular function was determined using the CCK-8 assay. The interaction between microRNA (miR)-29-3p and NEAT1 or fibroblast growth factor receptor substrate 2 (FRS2) was verified by luciferase and RNA pull-down assays. The results showed that NEAT1 was overexpressed in the hypertrophic dermis and in HSFs. However, knockdown of NEAT1 suppressed the proliferation and extracellular matrix (ECM) production of HSFs. Moreover, NEAT1 functioned as a competing endogenous RNA to upregulate FRS2 by sponging miR-29-3p. Downregulation of miR-29-3p or overexpression of FRS2 antagonized the effects of NEAT1 knockdown and promoted HSF proliferation and ECM release. In conclusion, NEAT1 knockdown protected against hypertrophic scarring by modulating the miR-29-3p/FRS2 axis, which is a viable target in scar treatment.

To effectively inhibit the growth of breast cancer cells (MDA-MB-231 cells) by the combination method of chemotherapy and magnetic hyperthermia, we fabricated a biomimetic drug delivery (CSiFePNs) system composed of mesoporous silica nanoparticles (MSNs) containing superparamagnetic ferroferric oxide and Paclitaxel (PTX) coated with MDA-MB-231 cell membranes (CMs). In the in vitro cytotoxicity tests, the MDA-MB-231 cells incubated with CSiFePNs obtained IC50 value of 0.8 μgL−1, 3.5-fold higher than that of SiFePNs. The combination method of chemotherapy and magnetic hyperthermia can effectively inhibit the growth of MDA-MB-231 cells.

Aim To evaluate the diagnostic value of interleukin-18 (IL-18) and troponin (TnI) in sepsis. Methods This retrospective analysis included 117 patients with sepsis (patient group) and 92 subjects who attended regular physical examinations (control group). We compared IL-18 and TnI expressions before treatment (T1) and on day 5 (T2), day 10 (T3) and day 15 (T4) of treatment. Acute Physiology and Chronic Health Evaluation II (APACHE II) guidelines were used to analyse the correlation between IL-18, TnI and APACHE II scores. Results At T1, T2, T3 and T4, the IL-18 and TnI levels were all higher in the patient group than in the control group (p Conclusions Monitoring TnI and IL-18 levels can effectively evaluate the severity and recovery of patients with sepsis.

Esophageal cancer is one of the most common type of malignancies. Telomerase activity, which is absent or weakly detected in the majority of human somatic cells, is elevated in esophageal cancer. Although azidothymidine (AZT), a reverse transcriptase inhibitor, has been utilized as a treatment for tumors, its role in treating esophageal cancer has not been confirmed. The aim of the present study was to determine the effect of AZT on telomerase activity and the proliferation of the human esophageal cancer cell line TE-11. A telomeric repeat amplification assay was utilized to detect telomerase activity following treatment of TE-11 cells with AZT. The effect of AZT on TE-11 cell cycle distribution was determined by flow cytometry. Cellular DNA damage was evaluated by a comet assay and an MTT assay demonstrated that AZT significantly inhibited the viability of TE-11 cells, in a time-and dose-dependent manner. In addition, TE-11 cells treated with various concentrations of AZT exhibited a significant reduction in telomerase activity and percentage of cells in the G1/G0 phase, and an increase in the percentage of cells in the S phase. High doses of AZT caused DNA damage, and enhanced the expression levels of γ-H2A histone family member X and phosphorylated checkpoint kinase 2 in TE-11 cells. These results demonstrated that AZT effectively inhibits proliferation of the TE-11 human esophageal cancer cell line in vitro. The growth inhibitory effects were associated with a reduction in telomerase activity, S and G2/M phase cell cycle arrest, and enhanced DNA damage, suggesting that AZT may be utilized in the clinic for the treatment of esophageal cancer.

The success of checkpoint inhibitors in cancer treatment is associated with the infiltration of tissue-resident memory T (Trm) cells. In this study, we found that about 30% of tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment of gastric adenocarcinoma were CD69

Aims Circular RNAs (circRNAs) have been emerged as novel regulators in multiple tumorigenesis, including melanoma. CircRNA_0084043 was recently demonstrated to be deregulated in human melanoma cells. Nevertheless, its role and mechanism are largely unrevealed in melanoma. Materials and methods Expression of circ_0084043, miRNA (miR)-429 and tribbles homolog 2 (TRIB2) was detected using reverse transcription-quantitative PCR quantitative PCR (RT-qPCR) and western blotting. Cell proliferation, apoptosis, migration and invasion were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and transwell assays, respectively. The activation of Wnt/β-catenin pathway was evaluated by western blotting. The target binding among circ_0084043, miR-429 and TRIB2 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. In vivo, mice xenograft model was generated to investigate tumor growth. Key findings Expression of circ_0084043 and TRIB2 was upregulated in human melanoma tissues and cell lines. Both circ_0084043 knockdown and TRIB2 silencing could decrease cell proliferation, migration and invasion, but facilitate apoptosis in A375 and SK-MEL-28 cells. Furthermore, TRIB2 restoration partially abrogated the tumor-suppressive role of circ_0084043 knockdown in melanoma cells in vitro. Then, we verified that circ_0084043 positively and physically controlled TRIB2 expression through sponging miR-429. Besides, expression of β-catenin, c-Myc and cyclinD1 was inhibited in A375 and SK-MEL-28 cells when circ_0084043 was knocked down, accompanied with increased miR-429 and decreased TRIB2. Notably, circ_0084043 downregulation impeded tumor growth of A375 cells in vivo. Significance Knockdown of circ_0084043 suppressed the malignant development of melanoma presumably through modulating miR429/TRIB2 axis and inactivating Wnt/β-catenin signaling pathway.

Purpose: Examining the role of developmental signaling pathways in “driver gene–negative” lung adenocarcinoma (patients with lung adenocarcinoma negative for EGFR, KRAS, BRAF, HER2, MET, ALK, RET, and ROS1 were identified as “driver gene–negative”) may shed light on the clinical research and treatment for this lung adenocarcinoma subgroup. We aimed to investigate whether developmental signaling pathways activation can stratify the risk of “driver gene–negative” lung adenocarcinoma. Experimental Design: In the discovery phase, we profiled the mRNA expression of each candidate gene using genome-wide microarrays in 52 paired lung adenocarcinoma and adjacent normal tissues. In the training phase, tissue microarrays and LASSO Cox regression analysis were applied to further screen candidate molecules in 189 patients, and we developed a predictive signature. In the validation phase, one internal cohort and two external cohorts were used to validate our novel prognostic signature. Results: Kyoto Encyclopedia of Genes and Genomes pathway analysis based on whole-genome microarrays indicated that the Wnt/β-catenin pathway was activated in “driver gene–negative” lung adenocarcinoma. Furthermore, the Wnt/β-catenin pathway–based gene expression profiles revealed 39 transcripts differentially expressed. Finally, a Wnt/β-catenin pathway–based CSDW signature comprising 4 genes (CTNNB1 or β-catenin, SOX9, DVL3, and Wnt2b) was developed to classify patients into high-risk and low-risk groups in the training cohort. Patients with high-risk scores in the training cohort had shorter overall survival [HR, 10.42; 6.46–16.79; P < 0.001) than patients with low-risk scores. Conclusions: The CSDW signature is a reliable prognostic tool and may represent genes that are potential drug targets for “driver gene–negative” lung adenocarcinoma.

Lethal-7 (let-7) microRNA (miRNA) serves a pivotal role in a number of physiological processes and is associated with the occurrence and development of multiple disorders such as cancer. The present study aimed to use a newly developed stem-loop strategy for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to distinguish let-7 miRNA family members that differ by as little as a single nucleotide. For the miRNAs comprising 16 identical nucleotides at the 5′-end, different stem-loop RT primers were designed and used in RT-qPCR to assess the expression profiles of a panel of let-7 family member miRNAs in human glioblastoma U87 cells. Amplification efficiency was evaluated through correlation analysis between total RNA input and the quantification threshold values. Melting curve profiles were measured to estimate the amplification specificity of the improved stem-loop RT-qPCR compared with those of the poly(A)-tailing method. In addition, the discrimination ability of the modified stem-loop method was examined. Compared with poly(A) tailing, the modified stem-loop RT method was able to specifically reverse transcribe the diverse let-7 miRNA family members followed by accurate quantification, with a theoretical amplification efficiency of ~100%. This modified stem-loop method was able to distinguish miRNAs with a single base difference. This innovative method may be used in the clinical detection of let-7 expression levels in a variety of tumour samples, and may provide valuable data for disease diagnosis and prognostic evaluation. In addition, this method may offer a new avenue for developing particular stem-loop approaches in measuring other miRNAs with little discrepancy.

MicroRNAs (miRNAs) are small, non-coding RNAs which regulate gene expression at the post-transcriptional level. Abnormal expression of miRNAs occurs frequently in human tumors. Despite the fact that reduced expression of miR-128 has been observed in glioma tissues and cells, the role of miR-128 in tumors has not been fully characterized. In the present study, cell adhesion assays indicated that overexpression of miR-128 can promote cell-cell adhesion. Target site prediction algorithms indicated that miR-128 binds the 3'-untranslated regions of erythropoietin-producing hepatocellular receptor (Eph)B1 and EphB2 mRNAs. Luciferase reporter assays confirmed that miR-128 binds and regulates EphB1 and EphB2 mRNAs. Overexpression of EphB2 reduced the ability of miR-128 to promote cell-cell adhesion. The wound-healing assay indicated that miR-128 significantly inhibited cell migration via EphB2. This study revealed the novel functions of miR-128 in cell-cell adhesion and cell migration in glioma cells through the regulation of EphB2, and identified EphB1 and EphB2 as novel miR-128 targets.

Gliomas are the most common and aggressive primary tumors in the central nervous system. Recently, Max interactor-1 (MXI1), an antagonist of c-Myc that is involved in brain tumor progression, has been reported to be deregulated in a variety of tumors including glioma. However, the mechanism of MXI1 deregulation in gliomas remains unclear. In this study, we show that the relative expression level of MXI1 is markedly down-regulated in glioma cell lines. Using integrated bioinformatic analysis and experimental confirmation, we identified several miRNAs by screening a panel of predicted miRNAs that may regulate the MXI1 3'UTR. The strongest inhibitory miRNA, miR-155, can attenuate the activity of a luciferase reporter gene that is fused with the MXI1 3'UTR and decrease the expression levels of MXI1 mRNA and protein in U87 glioma cells. The potential role of miR-155 in promoting glioma cell proliferation by targeting MXI1 was confirmed in various glioma cell lines by rescue experiments using MTT assays, EdU incorporation assay, and cell counting experiments. In addition, we determined that the level of MXI1 mRNA was inversely correlated with the expression of miR-155 in 18 sets of glioblastoma multiforme specimens. These findings reveal for the first time that the targeting of MXI1 by miR-155 may result in a reduction in MXI1 expression and promote glioma cell proliferation; this result suggests a novel function of miR-155 in targeting MXI1 in glioma-genesis.

Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3' untranslated regions [3'UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3'UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3'UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3' end targeted regions, as well as the 5' end (seed region) binding sites., published_or_final_version

MicroRNAs (miRNAs) are small, non‑coding RNAs which regulate gene expression at the post-transcriptional level. Abnormal expression of miRNAs occurs frequently in tumors. Although the two miRNAs miR‑24‑3p and miR‑27a‑3p come from two duplicated gene clusters of miR‑23a~27a~24‑2 and miR‑23b~27b~24‑1 which are found to be deregulated in a variety of cancers, the role of cooperation of the two clusters and the function of the two miRNAs in tumors have not been completely characterized. Here, we show that overexpression of miR‑24‑3p and miR‑27a‑3p could promote cell proliferation using the MTT assay. By integrated bioinformatic analysis and experimental confirmation, we identified MXI1, which has been found to act as a tumor suppressor gene by affecting c‑Myc, as a direct target of miR‑24‑3p and miR‑27a‑3p. While targeting the MXI1 3' untranslated region by miR‑24‑3p or miR‑27a‑3p, luciferase activity was attenuated. The two miRNAs promote glioma cell proliferation via targeting MXI1 and the experiment was confirmed by the rescue experiments. Furthermore, our results show that two clusters of miR-23a~27a~24-2 and miR‑23b~27b~24‑1 regulate MXI1 synergistically. These findings reveal, for the first time, the novel functions of cooperation of miR‑24‑3p and miR‑27a‑3p from two clusters in promoting cell proliferation through MXI1. Additionally, we observed that miR‑27a‑3p is upregulated in glioma tissues.