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The present study compared the efficacy and safety of regorafenib plus programmed death-1 inhibitors (R-P) with regorafenib monotherapy as second-line therapies for advanced hepatocellular carcinoma (HCC). A systematic search of relevant literature published in PubMed, Embase, Web of Science and Cochrane Library databases until October 2023 was conducted. Two authors independently performed data extraction and screening using standardized protocols. Stata/MP 17.0 was used for the meta-analysis to evaluate the impact of R-P treatment on major outcome indicators, including overall survival, progression-free survival (PFS), tumor response and adverse reactions, in patients with advanced HCC. The results indicated that five cohort studies involving 444 patients with advanced HCC were included. The results revealed that R-P treatment improved overall survival [hazard ratio (HR), 0.61; 95% confidence interval (CI) 0.48-0.77; I 2 =0.0%; P=0.663] and PFS (HR, 0.51; 95% CI 0.41-0.63; I 2 =17.5%; P=0.303). Additionally, it increased the objective response rate (risk ratio, 2.33; 95% CI, 1.49-3.64; I 2 =0.0%; P=0.994) and disease control rate (HR, 1.40; 95% CI, 1.20-1.63; I 2 =0.0%; P=0.892) compared with those of regorafenib. However, R-P treatment was associated with an increased incidence of adverse events, such as hypothyroidism, thrombocytopenia and rash, compared with that in regorafenib. In conclusion, R-P is superior to regorafenib monotherapy in terms of survival benefits and tumor response., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Li et al.)

To compare the clinical efficacy and safety of robot-assisted resection and open surgery for cholangiocarcinoma (CCA). We conducted a comprehensive search of PubMed, the Cochrane Library, and Embase databases for studies comparing treatment for CCA, covering the period from database inception to January 30, 2024. Two researchers will independently screen literature and extract data, followed by meta-analysis using Review Manager 5.3 software. A total of 5 articles with 513 patients were finally included. Among them, 231 in the robotic group, and 282 in the open group. The Meta-analysis revealed that the robotic group had a significant advantage in terms of intraoperative blood loss (MD =  - 101.44, 95% CI - 135.73 to - 67.15, P < 0.05), lymph node harvest(MD = 1.03, 95% CI 0.30- 1.76, P < 0.05) and length of hospital stay(MD =  - 1.92, 95% CI - 2.87 to- 0.97, P < 0.05). However, there were no statistically significant differences between the two groups in terms of transfusion rate (OR = 0.62, 95% CI 0.31-1.23, P > 0.05), R0 resection (OR = 1.49, 95% CI 0.89- 2.50, P > 0.05), 30-day mortality (OR = 1.68, 95% CI 0.43-6.65, P > 0.05) and complications (OR = 0.76, 95% CI 0.30- 1.95, P > 0.05). Robotic-assisted radical resection for CCA is feasible and safe, and its long-term efficacy and oncological outcomes need to be confirmed by further studies., (© 2024. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Background: Isothermal amplification is considered to be one of the most promising tools for point-of-care testing molecular diagnosis. However, its clinical application is severely hindered by nonspecific amplification. Thus, it is important to investigate the exact mechanism of nonspecific amplification and develop a high-specific isothermal amplification assay., Methods: Four sets of primer pairs were incubated with Bst DNA polymerase to produce nonspecific amplification. Gel electrophoresis, DNA sequencing, and sequence function analysis were used to investigate the mechanism of nonspecific product generation, which was discovered to be nonspecific tailing and replication slippage mediated tandem repeats generation (NT&RS). Using this knowledge, a novel isothermal amplification technology, bridging primer assisted slippage isothermal amplification (BASIS), was developed., Results: During NT&RS, the Bst DNA polymerase triggers nonspecific tailing on the 3'-ends of DNAs, thereby producing sticky-end DNAs over time. The hybridization and extension between these sticky DNAs generate repetitive DNAs, which can trigger self-extension via replication slippage, thereby leading to nonspecific tandem repeats (TRs) generation and nonspecific amplification. Based on the NT&RS, we developed the BASIS assay. The BASIS is carried out by using a well-designed bridging primer, which can form hybrids with primer-based amplicons, thereby generating specific repetitive DNA and triggering specific amplification. The BASIS can detect 10 copies of target DNA, resist interfering DNA disruption, and provide genotyping ability, thereby offering 100% accuracy for type 16 human papillomavirus detection., Conclusion: We discovered the mechanism for Bst-mediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity., (© Crown copyright 2023.)

How to efficiently obtain high-purity cancer stem cells (CSCs) has been the basis of CSC research, but the optimal conditions for serum-free suspension culture of CSCs are still unclear. The present study aimed to define the optimal culture medium composition and culture time for the enrichment of colon CSCs via suspension culture. Suspension cell cultures of colon cancer DLD-1 cells were prepared using serum-free medium (SFM) containing variable concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce spheroids. Culture times were set at 10, 20 and 30 days. A total of nine different concentrations of EGF and bFGF were added to SFM to generate nine experimental groups. The proportions of CD44 + , CD133 + , and CD44 + CD133 + double-positive spheroid cells were detected via flow cytometry. mRNA expression of stemness-, epithelial-mesenchymal transition- and Wnt/β-catenin pathway-associated genes was determined via reverse transcription-quantitative PCR. Self-renewal ability was evaluated by a sphere-forming assay. Tumorigenesis was studied in vitro using a colony formation assay and in vivo via subcutaneous cell injection in nude mice. It was found that the highest expression proportions of CD133 + and CD44 + spheroid cells were observed in group (G)9 (20 ng/ml EGF + 20 ng/ml bFGF) at 30 days (F=123.554 and 99.528, respectively, P<0.001), CD133 + CD44 + cells were also observed in G9 at 30 days (and at 10 days in G3 and 20 days in G6; F=57.897, P<0.001). G9 at 30 days also displayed the highest expression of Krüppel-like factor 4, leucine-rich repeat-containing G protein-coupled receptor 5, CD44, CD133, Vimentin and Wnt-3a (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001) and the lowest expression of E-cadherin (F=10.851, P<0.001). G9 at 30 days produced the highest yield of cell spheroids, as determined by a sphere forming assay (F=19.147, P<0.001); colony formation assays also exhibited the greatest number of colonies derived from G9 spheroids at 30 days (F=60.767, P<0.01), which also generated the largest mean tumor volume in the subcutaneous tumorigenesis xenograft model (F=12.539, P<0.01). In conclusion, 20 ng/ml EGF + 20 ng/ml bFGF effectively enriched colon CSCs when added to suspension culture for 30 days, and conferred the highest efficiency compared with other combinations., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Zhou et al.)

The exploration of an old drug for new biomedical applications has an absolute predominance in shortening the clinical conversion time of drugs for clinical application. In this work, carbon nanoparticles suspension injection (CNSI), the first clinically approved carbon nanoparticles in China, is explored as a new nano-radioprotective agent for potent intestinal radioprotection. CNSI shows powerful radioprotective performance in the intestine under oral administration, including efficient free radical scavenging ability, good biosafety, high chemical stability, and relatively long retention time. For example, CNSI shows high reactive oxygen species (ROS) scavenging activities, which effectively alleviates the mitochondrial dysfunction and DNA double-strand breaks to protect the cells against radiation-induced damage. Most importantly, this efficient ROS scavenging ability greatly helps restrain the apoptosis of the small intestinal epithelial and crypt stem cells, which decreases the damage of the mechanical barrier and thus relieves radiation enteritis. Moreover, CNSI helps remove the free radicals in the intestinal microenvironment and thus maintain the balance of intestinal flora so as to mitigate the radiation enteritis. The finding suggests a new application of clinically approved carbon nanoparticles, which not only promotes the development of new intestinal radioprotector, but also has a great potential for clinical transformation., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To investigate the effect and mechanism of SB525334 on self-renewal, migration and invasion of ovarian cancer stem cells., Methods: ALDHhigh-expressing cancer stem cells (CSCs) were isolated from human ovarian cancer cell line SKOV-3 by flow cytometry and treated with 2μg/mL SB525334 for 6h. The sphere forming assay was used to detect the ability of self-renewal of CSCs and the colony formation assay was used to detect the tumorigenicity in vitro. Transwell migration and invasion assay were used to detect the migration and invasion ability of CSCs. To further explore the mechanism, real-time quantitative PCR and flow cytometry were used to detect the mRNA and protein expression of TGF-β, Smad2, Smad3, phosphorylated Smad2, phosphorylated Smad3 and Smad4, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes E-cadherin, Snail, Vimentin were also assessed., Results: The self-renewal ability, tumorigenicity in vitro, migration and invasion ability of CSCs were significantly attenuated after SB525334 treatment. The expressions of TGF-β, phosphorylated Smad2, phosphorylated Smad3, Snail, and Vimentin were decreased, while Smad4 and E-cadherin expressions were increased., Conclusion: SB525334 may inhibit the self-renewal, invasion and migration of ovarian CSCs by blocking the TGF-β/Smad/EMT pathway., Competing Interests: The authors have declared that no competing interests exist.

Background and Objectives: Whether post-mastectomy radiotherapy (PMRT) could improve prognosis for T1-2 breast cancer patients with one to three lymph node metastases remains controversial. The present study aimed to determine the significance of PMRT in patients with T1-2N1M0 breast cancer. Methods: Data of 45,646 patients from the Surveillance, Epidemiology, and End Results (SEER) database were analyzed; 12,585 matched patients were divided into a PMRT group and non-radiotherapy group (no-PMRT), respectively, using the propensity score matching method. Univariate and multivariate analyses were performed to determine the prognostic factors of breast cancer, and subgroup analysis was performed according to the number of lymph node metastases. Results: With the median follow-up of 62 months, 5-year cancer-specific survival was 91.48% in the PMRT group and 91.88% in the no-PMRT group ( P = 0.405). PMRT did not improve the breast cancer-specific survival (BCSS) in patients with stage T1-2N1M0 (HR = 0.99, 95% CI = 0.92-1.06, P = 0.715). In subgroup analysis, radiotherapy improved the BCSS in patients with three nodes positive, with the 5-year BCSS at 88.5% in the radiation group and 86.6% in the no-radiation group (HR = 0.78, 95% CI = 0.65-0.90, P < 0.001). In patients with two nodes positive, 5-year BCSS was 90.3% in the PMRT group and 89.5% in the no-PMRT group, with no significant difference between the two groups (HR = 0.96, 95% CI = 0.85-1.09, P = 0.552). In patients with one node positive, 5-year BCSS was higher in the no-PMRT group (92.1%) than that in the PMRT group (90.8%); radiotherapy increased the cancer-related death compared with those who did not receive it (HR = 1.21, 95% CI = 1.08-1.36, P = 0.002). Conclusion: The benefit of PMRT in T1-2N1M0 patients was obviously different, and the recommendation of PMRT for this population should be individualized. PMRT should be considered for patients with three nodes positive, should be suggested cautiously in those with two nodes positive, and could be omitted in those with one node positive., (Copyright © 2020 Chen, Huang, Leng, Yang, Li, Yang and Hou.)

Although melatonin is necessary for circadian regulation of sleep, the mechanisms underlying this effect of melatonin are still unclear. In the present study, we showed that melatonin suppressed the activity of GABAergic neurons in the lateral hypothalamus, which has been reported to play a crucial role in maintaining wakefulness. The inhibitory effect of the melatonin was mediated by activation of melatonin 1 receptors and depended on the inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels. At behavioral levels, infusion of melatonin into the lateral hypothalamus significantly decreased the locomotor and exploratory activities and increased the time of immobility in open filed. Additionally, using electroencephalogram (EEG) and electromyogram (EMG) recordings, we found that infusion of melatonin into the lateral hypothalamus decreased the time spent in wakefulness and increased the amount of sleep. Overall, these results suggest that melatonin inhibits GABAergic neurons in the lateral hypothalamus via melatonin 1 receptor-dependent inhibition of the HCN channels, which is consistent with a decrease in wakefulness. These findings provide a new mechanism underlying the hypnotic effect of the melatonin.

Krüppel-like factor 4 (KLF4) was closely associated with epithelial-mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)-enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5 + CD44 + EpCAM + colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF-β1 pathway, whereas enforced KLF4 overexpression activated TGF-β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF-β1 pathway inhibition invalidated KLF4-facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF-β1/Smad/Snail pathway in Lgr5 + CD44 + EpCAM + colorectal CSCs., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background/aims: Although EpCAM+CD44+ cells exhibit more stem-like properties than did EpCAM-CD44- cells, the specificity of EpCAM combined with CD44 in defining CSCs needs further improvement. Lgr5 is used as a biomarker to isolate cancer stem cells (CSCs) in colorectal cancer. However, it remains unclear whether Lgr5, along with EpCAM and CD44, can further identify and define CSCs in colorectal cancer., Methods: Lgr5+CD44+EpCAM+, Lgr5+CD44+EpCAM-, Lgr5+CD44-EpCAM+, Lgr5-CD44+EpCAM+, and Lgr5-CD44-EpCAM-cells were separately isolated using fluorescence-activated cell sorting (FACS). Colony formation, self-renewal, differentiation, and tumorigenic properties of these cells were investigated through in vitro experiments and in vivo tumor xenograft models. The expression of stemness genes and CSC- and epithelial-mesenchymal transition (EMT)-related genes, such as KLF4, Oct4, Sox2, Nanog, CD133, CD44, CD166, ALDH1, Lgr5, E-cadherin, ZO-1, Vimentin, Snail, Slug, and Twist, was examined using real-time PCR., Results: Lgr5-positive subpopulations exhibited higher capacities for colony formation, self-renewal, differentiation, and tumorigenicity as well as higher expression of stemness genes and mesenchymal genes and lower expression of epithelial genes than did Lgr5-negative subpopulations., Conclusion: Our data revealed that tumorigenic cells were highly restricted to Lgr5-positive subpopulations. Most importantly, Lgr5+CD44+EpCAM+ cells exhibited more pronounced CSC-like traits than did any other subpopulation, indicating that Lgr5 combined with CD44 and EpCAM can further improve the stem-like traits of CSCs in colorectal cancer., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: MicroRNAs (miRNAs) play a crucial role in erythropoiesis. MiR-23a∼27a∼24-2 clusters have been proven to take part in erythropoiesis via some proteins. CDC25B (cell division control Cdc2 phosphostase B) is also the target of mir-27a; whether it regulates erythropoiesis and its mechanism are unknown., Methods: To evaluate the potential role of miR-27a during erythroid differentiation, we performed miR-27a gain- and loss-of-function experiments on hemin-induced K562 cells. We detected miR-27a expression after hemin stimulation at different time points. At the same time, the γ-globin gene also was measured via real-time PCR. According to the results of the chips, we screened the target protein of miR-27a through a dual-luciferase reporter assay and identified it via Western blot analyses. To evaluate the function of CDC25B, benzidine staining and flow cytometry were employed to detect the cell differentiation and cell cycle., Results: We found that miR-27a promotes hemin-induced erythroid differentiation of human K562 cells by targeting cell division cycle 25 B (CDC25B). Overexpression of miR-27a promotes the differentiation of hemin-induced K562 cells, as demonstrated by γ-globin overexpression. The inhibition of miR-27a expression suppresses erythroid differentiation, thus leading to a reduction in the γ-globin gene. CDC25B was identified as a new target of miR-27a during erythroid differentiation. Overexpression of miR-27a led to decreased CDC25B expression after hemin treatment, and CDC25B was up-regulated when miR-27a expression was inhibited. Moreover, the inhibition of CDC25B affected erythroid differentiation, as assessed by γ-globin expression., Conclusion: This study is the first report of the interaction between miR-27a and CDC25B, and it improves the understanding of miRNA functions during erythroid differentiation., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Previous studies demonstrated that immune dysregulation is an important cause of hepatitis B virus (HBV)‑mediated liver damage. Co‑stimulators including programmed cell death protein 1 (PD‑1) and inducible T cell co‑stimulator (ICOS) are involved in the pathogenesis of HBV. In the present study, the serum levels of soluble (s)PD‑1 and sICOS in patients with chronic HBV infections, were investigated, and the association between sPD‑1 and sICOS levels and liver injury degree was investigated. Serum sPD‑1 and sICOS levels were increased in the HBV‑patient group particularly in the HBV external core antigen positive group. In the immune clearance group, sPD‑1 and sICOS were increased compared with the tolerance group. Furthermore, the relative mRNA expression levels were also increased in patients with HBV. However there was no correlation between sPD‑1 and sICOS levels and HBV antibodies or PD‑1/ICOS mRNA copies. The altered sPD‑1 and sICOS serum levels in the different HBV groups may reflect the dysregulation of T cell activation, and may be associated with the HBV pathological process.

Background: To evaluate the effect of no drainage in patients who underwent thyroidectomy and neck lymph node dissection., Methods: We followed the methodological standard expected by Cochrane. We searched the following databases by March 23, 2017: PubMed, The Cochrane Library, EMBASE via Ovid SP, and Medline via Ovid SP. Two reviewers screened the studies and extracted the data. Randomized controlled trials (RCTs) or nonrandomized interventional studies assessing the effect of no drainage following thyroidectomy with lymph node dissection were included., Results: Three studies with 387 participants were included. There was no statistical difference between groups for the overall perioperative complications (2 RCTs, n = 234, RR 1.56, 95% CI 0.53-4.64), or specific complications such as seroma (2 RCTs, n = 234, RR 1.81, 95% CI 0.46-7.07), hematoma (2 RCTs, n = 234, RR 0.72, 95% CI 0.11-4.83) or hemorrhage (1 RCT, n = 69, RR 0.29, 95% CI 0.01-6.87). One case required reoperation due to hemorrhage in the drainage group was reported in 1 study (n = 32). No mortality was reported. Two studies (n = 234) stated a longer hospital stay in the drainage group than that in the group without drainage. There was moderate or serious bias for the risk of bias of included studies., Conclusion: The effect of no-drainage in patients with thyroid cancer who received thyroidectomy with neck dissections remains uncertain, since there are very few studies that addressed the question. Drainage may lead to longer hospital stay than nondrainage. More randomized or nonrandmized studies are required to address this issue., (Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Cancer stem cells (CSCs) are a rare tumorigenic population of cells found in multiple types of cancer. It has been suggested that CSCs are responsible for cancer drug resistance, metastasis and recurrence. Therefore, it is important to develop techniques to correctly sort and identify CSCs. In the current study, the sorting and identification of aldehyde dehydrogenase high (ALDH high ) CSCs was performed using flow cytometry. Cells from three colon cancer cell lines were cultured in serum-free medium to obtain CSCs-enriched spheroid cells. Subsequently, two subpopulations of ALDH high CSCs were isolated by flow cytometry either with the use of propidium iodide (PI) or not, respectively. The two subpopulations of ALDH high CSCs exhibited distinct characteristics, including stem cell related gene expression, self-renewal capacity and tumorigenicity in vitro and in vivo . Key regulators of the epithelial-mesenchymal transition (EMT), including vimentin, snail and slug were highly expressed in ALDH high CSCs. Therefore, the current study indicates that PI staining prior to the sorting of ALDH high CSCs by flow cytometry is an appropriate system for the study of CSCs. The current study also demonstrated that there was partial overlap between the transcriptional programs underlying the EMT and CSCs.

Purpose: MicroRNAs (miRs) have been frequently reported dysregulating in tumors and playing a crucial role in tumor development and progression. However, the expression of miR-155 and its role in gastric cancer (GC) are still obscure., Methods: qRT-PCR was applied to detect miR-155 expression in 60 matched GC samples and four GC cell lines, and the relationship between miR-155 levels and clinicopathological features of GC was analyzed. Next, the effects of miR-155 on GC cell growth were evaluated by gain- and loss-of-function analysis. Finally, the target gene(s) of miR-155 in GC cells were explored., Results: Our results revealed that miR-155 levels were significantly lower in both GC tissues and GC cell lines than in their normal controls, and its expression inversely correlated with tumor size and the pathologic stage. Moreover, our study showed that enforced expression of miR-155 impaired GC cell proliferation, promoted G1 phase arrest and induced apoptosis in vitro. In addition, we identified cyclin D1 as the direct target of miR-155, and knockdown of cyclin D1 partially phenocopied the role of miR-155 in GC cells., Conclusions: Our findings suggest that miR-155 may act as a potential diagnostic marker for early-stage GC and may represent a novel therapeutic target for GC treatment.

Cancer stem cells (CSCs), a rare population in any type of cancers, including colon cancer, are tumorigenic. It has been thought that CSCs are responsible for cancer recurrence, metastasis, and drug resistance. Isolating CSCs in colon cancers is challenging, and thus the molecular mechanism regulating the self-renewing and differentiation of CSCs remains unknown. We cultured DLD-1 cells, one of types of cells derived from colon cancers, in serum-free medium to obtain spheroid cells. These cells possessed the characteristics of CSCs, with the expression of CD133, CD166, Lgr5, and ALDH1, higher capacities of chemo-resistance, migration, invasion, and tumorigenicity in vitro and in vivo than the adherent DLD-1 cells. Krüppel-like factor 4 (KLF4) is essential factor for maintaining self-renewal of adult and embryonic stem cells. It has been used to induce pluripotent stem cells (iPS) from somatic cells. Since KLF4 is expressed in colon cancer cells, we investigated its role in spheroid cells isolated from DLD-1 cells and found that KLF4 was overexpressed only in spheroid cells and reducing the expression of KLF4 by short-hairpin RNA significantly decreased the capacities of these cells to resist the chemicals, migrate, invade, and generate tumors in vitro and in vivo. The spheroid cells with reduced KLF4 expression also had decreased expression of CSCs markers and mesenchymal markers. Taken together, culturing DLD-1 cells in serum-free medium enriches CSCs and the expression of KLF4 is essential for the characteristics of CSCs in DLD-1; thus KLF4 can be a potential therapeutic target for treating colon cancer.