Cucurbitacins mitigate vascular neointimal hyperplasia by suppressing cyclin A2 expression and inhibiting VSMC proliferation.

BACKGROUND: Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health. Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug-eluting stent is an effective approach to improve restenosis. Cucurbitacins have been demonstrated to exert an anti-proliferation effect in various tumors and a hypotensive effect. This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L. (CuECs) and cucurbitacin B (CuB) on restenosis. METHODS: C57BL/6 mice were subjected to left carotid artery ligation and subcutaneously injected with CuECs or CuB for 4 weeks. Hematoxylin-Eosin, immunofluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia. Western blot, real-time PCR, flow cytometry analysis, EdU staining and cellular immunofluorescence assay were employed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro. The potential interactions of CuECs with cyclin A2 were performed by molecular docking. RESULTS: The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells. Furthermore, CuECs and CuB mediated cell cycle arrest at the S phase. Autodocking analysis demonstrated that CuB, CuD, CuE and CuI had high binding energy for cyclin A2. Our study also showed that CuECs and CuB dramatically inhibited FBS-induced cyclin A2 expression. Moreover, the expression of cyclin A2 in CuEC- and CuB-treated neointima was downregulated. CONCLUSIONS: CuECs, especially CuB, exert an anti-proliferation effect in VSMCs and may be potential drugs to prevent restenosis.

The effect of miR-381 on proliferation and prognosis of breast cancer by altering CCNA2 expression.

BACKGROUND: MiR-381 can regulate the expression of cyclin A2 (CCNA2) to inhibit the proliferation and migration of bladder cancer cells, but whether miR-381 has the same function in breast cancer is not well know. METHODS: The over express or silence miR-381 expressing cell lines were constructed by lentivirus infection to reveal the biological functions of miR-381 in vitro. The expression of miR-381 and CCNA2 in 162 breast cancer patients were detected to further reveal their impact and predictive value on progression-free survival (PFS) and overall survival (OS). RESULTS: After transfection of MDA-MB-231 and MCF-7 cells with miR-381 mimics, the expression of miR-381 was effectively up-regulated and CCNA2 was effectively down-regulated, while the opposite results were observed in tumour cell which transfected with miR-381 inhibitors. After transfection of cell lines with miR-381 mimics, tumour cell activity was significantly reduced, while the opposite results were observed in tumour cell which transfected with miR-381 inhibitors. The area under curves (AUCs) of miRNA-381 and CCNA2 for predicting PFS and OS were 0.711, 0.695, 0.694 and 0.675 respectively. Cox regression analysis showed that miRNA-381 ≥ 1.65 2-ΔΔCt and CCNA ≥ 2.95 2-ΔΔCt were the influence factors of PFS and OS, the hazard ratio (HR) values were 0.553, 2.075, 0.462 and 2.089, respectively. CONCLUSION: miR-381 inhibitors breast cancer cells proliferation and migration by down-regulating the expression of CCNA2, both of them can predict the prognosis of breast cancer.

miR-381 can regulate the expression of cyclin A2 and inhibit the proliferation and migration of bladder cancer cells, but whether miR-381 has the same function in breast cancer is not well know. We analysed the levels of miR-381 and cyclin A2 in breast cancer patients and breast cancer cells to reveal the mechanism of miR-381 affecting the expression of cyclin A2. We found miRNA-381 affects the proliferation and migration of breast cancer cells by down-regulating the expression of cyclin A2. The expression of serum miR-381 and cyclin A2 have important values in predicting the prognosis of breast cancer. Our findings provide mechanistic insights into how miR-381 regulates the proliferation and migration of breast cancer, as well as a new target for clinical treatment. Future research may focus on how to improve patient prognosis by up-regulating expression of miR-381 and down-regulating the expression of cyclin A2.

Clinicopathological role of Cyclin A2 in uterine corpus endometrial carcinoma: Integration of tissue microarrays and ScRNA-Seq.

BACKGROUND: The comprehensive expression level and potential molecular role of Cyclin A2 (CCNA2) in uterine corpus endometrial carcinoma (UCEC) remains undiscovered. METHODS: UCEC and normal endometrium tissues from in-house and public databases were collected for investigating protein and messenger RNA expression of CCNA2. The transcription factors of CCNA2 were identified by the Cistrome database. The prognostic significance of CCNA2 in UCEC was evaluated through univariate and multivariate Cox regression as well as Kaplan-Meier curve analysis. Single-cell RNA-sequencing (scRNA-seq) analysis was performed to explore cell types in UCEC, and the AUCell algorithm was used to investigate the activity of CCNA2 in different cell types. RESULTS: A total of 32 in-house UCEC and 30 normal endometrial tissues as well as 720 UCEC and 165 control samples from public databases were eligible and collected. Integrated calculation showed that the CCNA2 expression was up-regulated in the UCEC tissues (SMD = 2.43, 95% confidence interval 2.23∼2.64). E2F1 and FOXM1 were identified as transcription factors due to the presence of binding peaks on transcription site of CCNA2. CCNA2 predicted worse prognosis in UCEC. However, CCNA2 was not an independent prognostic factor in UCEC. The scRNA-seq analysis disclosed five cell types: B cells, T cells, monocytes, natural killer cells, and epithelial cells in UCEC. The expression of CCNA2 was mainly located in B cells and T cells. Moreover, CCNA2 was active in T cells and B cells using the AUCell algorithm. CONCLUSION: CCNA2 was up-regulated and mainly located in T cells and B cells in UCEC. Overexpression of CCNA2 predicted unfavorable prognosis of UCEC.

CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle.

Glioblastoma (GBM) is characterized by significant heterogeneity, leading to poor survival outcomes for patients, despite the implementation of comprehensive treatment strategies. The roles of cyclin A2 (CCNA2) and NIMA related kinase 2 (NEK2) have been extensively studied in numerous cancers, but their specific functions in GBM remain to be elucidated. The present study aimed to investigate the potential molecular mechanisms of CCNA2 and NEK2 in GBM. CCNA2 and NEK2 expression and prognosis in glioma were evaluated by bioinformatics methods. In addition, the distribution of CCNA2 and NEK2 expression in GBM subsets was determined using pseudo-time analysis and tricycle position of single-cell sequencing. Gene Expression Omnibus and Kyoto Encyclopedia of Genes and Genome databases were employed and enrichment analyses were conducted to investigate potential signaling pathways in GBM subsets and a nomogram was established to predict 1-, 2- and 3-year overall survival probability in GBM. CCNA2 and NEK2 expression levels were further validated by western blot analysis and immunohistochemical staining in GBM samples. High expression of CCNA2 and NEK2 in glioma indicates poor clinical outcomes. Single-cell sequencing of GBM revealed that these genes were upregulated in a subset of positive neural progenitor cells (P-NPCs), which showed significant proliferation and progression properties and may activate G2M checkpoint pathways. A comprehensive nomogram predicts 1-, 2- and 3-year overall survival probability in GBM by considering P-NPCs, age, chemotherapy and radiotherapy scores. CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle, thus indicating the potential of novel therapy directed to CCNA2 and NEK2 in GBM.

Phosphorylation of Tau Protein by CDK2/cyclin A and GSK3ß Recombinant Kinases: Analysis of Phosphorylation Patterns by Nuclear Magnetic Resonance Spectroscopy.

Posttranslational modifications (PTMs) of proteins can be investigated by Nuclear Magnetic Resonance (NMR) spectroscopy as a powerful analytical tool to define modification sites, their relative stoichiometry, and crosstalk between modifications. As a Structural Biology method, NMR provides important additional information on changes in protein conformation and dynamics upon modification as well as a mapping of binding sites upon biomolecular interactions. Indeed, PTMs not only mediate functional modulation in protein-protein interactions, but can also induce diverse structural responses with different biological outcomes. Here we present protocols that have been developed for the production and phosphorylation of the neuronal tau protein. Under its aggregated form, tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases named tauopathies involving tau dysfunction and/or mutations. As a common feature shared by various tauopathies, tau aggregates are found into a form displaying an increased, abnormal phosphorylation, also referred to hyperphosphorylation. We have used NMR to investigate the phosphorylation patterns of tau induced by several kinases or cell extracts, how phosphorylation affects the local and overall conformation of tau, its interactions with partners (proteins, DNA, small-molecules, etc.) including tubulin and microtubules, and its capacity to form insoluble fibrillar aggregates. We present here detailed protocols for in vitro phosphorylation of tau by the recombinant kinases CDK2/cyclin A and GSK3ß, the production of the recombinant kinases thereof, as well as the analytical characterization of phosphorylated tau by NMR spectroscopy.

Increases in cyclin A/Cdk activity and in PP2A-B55 inhibition by FAM122A are key mitosis-inducing events.

Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.

Cyclin A2 Expression as Predictive Biomarker in Muscle-Invasive Upper Tract Urothelial Carcinoma.

INTRODUCTION: The aim was to evaluate the prognostic value of altered Cyclin A2 (CCNA2) gene expression in upper tract urothelial carcinoma (UTUC) and to assess its predictive potential as a prognostic factor for overall survival (OS) and disease-free survival. METHODS: 62 patients who underwent surgical treatment for UTUC were included. Gene expression of CCNA2, MKI67, and p53 was analyzed by quantitative reverse transcriptase polymerase chain reaction. Survival analyses were performed using the Kaplan-Meier method and the log-rank test. For Cox regression analyses, uni- and multivariable hazard ratios were calculated. Spearman correlation was used to analyze correlation of CCNA2 expression with MKI67 and p53. RESULTS: The median age of the cohort was 73 years, and it consisted of 48 males (77.4%) and 14 females (22.6%). Patients with high CCNA2 expression levels showed longer OS (HR 0.33; 95% CI: 0.15-0.74; p = 0.0073). Multivariable Cox regression analyses identified CCNA2 overexpression (HR 0.37; 95% CI: 0.16-0.85; p = 0.0189) and grading G2 (vs. G3) (HR 0.39; 95% CI: 0.17-0.87; p = 0.0168) to be independent predictors for longer OS. CCNA2 expression correlated positively with MKI67 expression (Rho = 0.4376, p = 0.0005). CONCLUSION: Low CCNA2 expression is significantly associated with worse OS. Thus, CCNA2 might serve as a potential biomarker in muscle-invasive UTUC and may be used to characterize a subset of patients having an unfavorable outcome and for future risk assessment scores.

Nanoparticles of a Pyrazolo-Pyridazine Derivative as Potential EGFR and CDK-2 Inhibitors: Design, Structure Determination, Anticancer Evaluation and In Silico Studies.

The strategic planning of this study is based upon using the nanoformulation method to prepare nanoparticles 4-SLNs and 4-LPHNPs of the previously prepared 4,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-amine (4) after confirming its structure with single crystal X-ray analysis. These nanoparticles exhibited promising cytotoxic activity against HepG-2, HCT-116 and MCF-7 cancer cell lines in comparison with the reference doxorubicin and the original derivative 4. Moreover, their inhibitory assessment against EGFR and CDK-2/cyclin A2 displayed improved and more favorable impact than the parent 4 and the references. Detection of their influence upon cancer biomarkers revealed upregulation of Bax, p53 and caspase-3 levels and downregulation of Bcl-2 levels. The docking simulation demonstrated that the presence of the pyrazolo[3,4-c]pyridazin-3-amine scaffold is amenable to enclosure and binding well within EGFR and CDK-2 receptors through different hydrophilic interactions. The pharmacokinetic and physicochemical properties of target 4 were also assessed with ADME investigation, and the outcome indicated good drug-like characteristics.

[Cyclin A1 affects the invasion, metastasis, and prognosis of hepatocellular carcinoma].

Objective: To investigate the effect of cyclin A1 on the invasion, metastasis, and prognosis of hepatocellular carcinoma (HCC). Methods: Immunohistochemistry (IHC) was used to detect the expressional condition of cyclin A1 in HCC and paraffin-embedded non-tumor adjacent tissues. Kaplan-Meier method was used for the survival analysis of patients with HCC. Western blot (WB) was used to detect the expression of cyclin A1 in HCCLM3 and QGY-7703 cells. Scratch wound healing assay, transwell migration, and invasion assay were used to detect the effect of cyclin A1 overexpression on cell migration and invasion ability. WB was used to detect changes in the expression of matrix metalloproteinase (MMP) 2, MMP9, and vascular endothelial growth factor (VEGF) after overexpression of cyclin A1. Measurement data were compared using a t-test and analysis of variance. Count data was measured using χ (2) test and the Log-rank method was performed for survival analysis. Results: Cyclin A1 expression rates were higher in the tissues of HCC patients with recurrent metastasis than in the tissues of patients without recurrent metastasis (60.42% vs. 46.81%, χ (2) = 4.711, P < 0.05). The overall postoperative survival time (OS) and disease-free survival (DFS) were shorter in patients with high cyclin A1 expression than those with low cyclin A1 expression (45.9 months vs. 53.1 months; 42.9 months vs. 51.3 months, and P < 0.01). The postoperative OS and DFS were shorter in patients with high cyclin A1 expression and recurrent metastasis than those with low cyclin A1 expression without recurrent metastasis (31.7 months vs. 43.9 months; 18.0 months vs. 31.5 months, and P < 0.05). HCCLM3 and QGY-7703 cells were higher in the cyclin A1-pEX group than in the empty vector (vector) group (1.56 ± 0.06 vs. 0.18 ± 0.01, t = 18.75, P < 0.001; 1.31 ± 0.05 vs.0.37 ± 0.02, t = 15.17, P < 0.001). The migrated distances of HCCLM3 cells in the cyclin A1-pEX group and the vector group were (536.7 ± 14.5) µm and (327.3 ± 9.3) µm, t = 11.84, P < 0.05, respectively, while the migrated distances of QGY-7703 cells in the two groups were (916.7 ± 35.3) µm and (320.0 ± 20.8) µm, t = 13.54, P < 0.01. The migrated numbers of HCCLM3 cells in the cyclin A1-pEX group and vector group were (37.3 ± 2.4) and (7.0 ± 1.2), t = 12.67, P < 0.001, and the number of invasive cells was (73.7 ± 4.1) and (12.6 ± 1.5), t = 12.36, P < 0.001, respectively. The migrated numbers of QGY-7703 cells in the two groups were (153.3 ± 6.0) and (17.7 ± 3.7), t = 17.59, P < 0.001, and the number of invasive cells was (45.0 ± 2.9) and (9.3 ± 1.5), t = 10.66, P < 0.001, respectively. The expression levels of MMP2, MMP9, and VEGF in HCCLM3 and QGY-7703 cells were significantly higher in the cyclin A1-pEX group than those in the vector group (P < 0.05). Conclusion: Cyclin A1 plays an important role in HCC invasion and metastasis, but HCC patients with high cyclin A1 expression have a poor prognosis. Hence, cyclin A1 has high guiding significance for evaluating patient prognosis.

Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

1,6-Hexanediol regulates angiogenesis via suppression of cyclin A1-mediated endothelial function.

BACKGROUND: Angiogenesis plays important roles in physiological and pathologic conditions, but the mechanisms underlying this complex process often remain to be elucidated. In recent years, liquid-liquid phase separation (LLPS) has emerged as a new concept to explain many cellular functions and diseases. However, whether LLPS is involved in angiogenesis has not been studied until now. Here, we investigated the potential role of LLPS in angiogenesis and endothelial function. RESULTS: We found 1,6-hexanediol (1,6-HD), an inhibitor of LLPS, but not 2,5-hexanediol (2,5-HD) dramatically decreases neovascularization of Matrigel plug and angiogenesis response of murine corneal in vivo. Moreover, 1,6-HD but not 2,5-HD inhibits microvessel outgrowth of aortic ring and endothelial network formation. The endothelial function of migration, proliferation, and cell growth is suppressed by 1,6-HD. Global transcriptional analysis by RNA-sequencing reveals that 1,6-HD specifically blocks cell cycle and downregulates cell cycle-related genes including cyclin A1. Further experimental data show that 1,6-HD treatment greatly reduces the expression of cyclin A1 but with minimal effect on cyclin D1, cyclin E1, CDK2, and CDK4. The inhibitory effect of 1,6-HD on cyclin A1 is mainly through transcriptional regulation because proteasome inhibitors fail to rescue its expression. Furthermore, overexpression of cyclin A1 in HUVECs largely rescues the dysregulated tube formation upon 1,6-HD treatment. CONCLUSIONS: Our data reveal a critical role of LLPS inhibitor 1,6-HD in angiogenesis and endothelial function, which specifically affects endothelial G1/S transition through transcriptional suppression of CCNA1, implying LLPS as a possible novel player to modulate angiogenesis, and thus, it might represent an interesting therapeutic target to be investigated in clinic angiogenesis-related diseases in future.

Identification and Validation of Cyclin A2 and Cyclin E2 as Potential Biomarkers in Small Cell Lung Cancer.

INTRODUCTION: Small cell lung cancer (SCLC) is a special type of lung cancer sensitive to radiotherapy and chemotherapy but is prone to drug resistance and recurrence and has a very poor prognosis. This study aimed to explore the potential biomarkers and therapeutic targets for SCLC. METHODS: After batch normalization of GSE40275, GSE1037, and GSE44447 datasets, R was used to screen SCLC's differentially expressed genes (DEGs) and hub genes. We used immunohistochemistry (IHC) to assess the tissue's expression level of the hub gene. The clinical value of the hub gene was further evaluated based on the collected clinical-pathological data. RESULTS: In this study, a total of 230 DEGs (133 upregulated and 97 downregulated) were screened by the R package. The IHC showed that the expression of CCNA2 and CCNE2 in SCLC tissues was significantly higher than that in normal tissues (p < 0.01). Overexpression of CCNA2 was closely associated with the extensive period of NCCN (p = 0.004), tumor position (p = 0.046), and clinical stage (p = 0.002). The high expression levels of CCNE2 were related to high survival in chemotherapy patients (p = 0.019). CONCLUSION: CCNA2 and CCNE2 may serve as potential biomarkers of diagnosis and treatment for SCLC.

Cyclin A participates in the TSO1-MYB3R1 regulatory module to maintain shoot meristem size and fertility in Arabidopsis.

The stem cell pools at the shoot apex and root tip give rise to all the above- and below-ground tissues of a plant. Previous studies in Arabidopsis identified a TSO1-MYB3R1 transcriptional module that controls the number and size of the stem cell pools at the shoot apex and root tip. As TSO1 and MYB3R1 are homologous to components of an animal cell cycle regulatory complex, DREAM, Arabidopsis mutants of TSO1 and MYB3R1 provide valuable tools for investigations into the link between cell cycle regulation and stem cell maintenance in plants. In this study, an Arabidopsis cyclin A gene, CYCA3;4, was identified as a member of the TSO1-MYB3R1 regulatory module and cyca3;4 mutations suppressed the tso1-1 mutant phenotype specifically in the shoot. The work reveals how the TSO1-MYB3R1 module is integrated with the cell cycle machinery to control cell division at the shoot meristem.

Target Selection for T-Cell Therapy in Epithelial Ovarian Cancer: Systematic Prioritization of Self-Antigens.

Adoptive T cell-receptor therapy (ACT) could represent a promising approach in the targeted treatment of epithelial ovarian cancer (EOC). However, the identification of suitable tumor-associated antigens (TAAs) as targets is challenging. We identified and prioritized TAAs for ACT and other immunotherapeutic interventions in EOC. A comprehensive list of pre-described TAAs was created and candidates were prioritized, using predefined weighted criteria. Highly ranked TAAs were immunohistochemically stained in a tissue microarray of 58 EOC samples to identify associations of TAA expression with grade, stage, response to platinum, and prognosis. Preselection based on expression data resulted in 38 TAAs, which were prioritized. Along with already published Cyclin A1, the TAAs KIF20A, CT45, and LY6K emerged as most promising targets, with high expression in EOC samples and several identified peptides in ligandome analysis. Expression of these TAAs showed prognostic relevance independent of molecular subtypes. By using a systematic vetting algorithm, we identified KIF20A, CT45, and LY6K to be promising candidates for immunotherapy in EOC. Results are supported by IHC and HLA-ligandome data. The described method might be helpful for the prioritization of TAAs in other tumor entities.

FBXL6 is dysregulated in keloids and promotes keloid fibroblast growth by inducing c-Myc expression.

C-MYC-mediated keloid fibroblasts proliferation and collagen deposit may contribute to the development of keloids. F-box and leucine-rich repeat protein 6 (FBXL6) is reported to be involved in tumour progression, while the role of FBXL6 in keloid fibroblasts is not deciphered. Normal control skins, hypertrophic scars and keloid tissues were collected and prepared for FBXL6 detection. FBXL6 short hairpin RNAs (shRNAs) or FBXL6 over-expression plasmids were transfected into keloid fibroblasts, and then c-MYC plasmids were further transfected. Cell viability was assayed with a Cell-Counting Kit-8 kit. The relative expression of FBXL6, Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I was detected with real-time PCR and Western blot. Elevated FBXL6 expression could be observed in keloid tissues and hypertrophic scars. FBXL6 shRNAs transfection could inhibit the viability of keloid fibroblasts with diminished c-MYC expression and down-regulated Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I expression. At the same time, overexpressed FBXL6 could promote the proliferation of keloid fibroblasts. Overexpression of c-MYC could promote the proliferation of keloid fibroblasts reduced by FBXL6 shRNAs with up-regulated Cyclin A1 and Collagen I expression. FBXL6 could promote the growth of keloid fibroblasts by inducing c-MYC expression, which could be targeted in keloids treatment.

The expression and clinical significance of microRNA-574-3p in colon cancer / 中国医师杂志

Objective:To study the expression and clinical significance of microRNA-574-3p (miR-574-3p) in colon cancer.Methods:A total of 106 colon cancer patients who were admitted to the First Hospital of Qinhuangdao and Shijiazhuang Hospital of Traditional Chinese Medicine from June 2012 to June 2015 were selected as the research objects. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-574-3p in colon cancer tissues and normal adjacent tissues. The relationship between the expression of miR-574-3p and the clinicopathological characteristics and prognosis of patients with colon cancer was analyzed. Immunohistochemical staining was used to detect the relationship between the expression of miR-574-3p and the expression of CyclinA2 or E-cadherin.Results:Compared with normal tissues adjacent to cancer, the expression level of miR-574-3p in 106 cases of colon cancer was significantly lower ( P<0.01). The decreased expression of miR-574-3p was related to tumor diameter, Dukes stage, histological grade and lymph node metastasis (all P<0.05), but not to age and tumor location (all P>0.05). The patients with low expression of miR-574-3p, high Dukes stage and histological grade, and lymph node metastasis had poor survival (all P<0.05). The 5-year overall survival rate of patients with decreased miR-574-3p expression in cancer tissue was significantly lower than that of patients without decreased miR-574-3p expression ( P=0.007 6). Compared with patients with no decreased miR-574-3p expression, patients with decreased miR-574-3p expression had higher CyclinA2 protein integrated optical density (IOD) value and lower E-cadherin protein IOD value in colon cancer tissues (all P<0.05). Conclusions:The decreased expression of miR-574-3p is related to the poor prognosis of colon cancer patients, which may affect tumor recurrence and metastasis by regulating the expression of CyclinA2 and E-cadherin proteins.

RNA interference analysis of potential functions of cyclin A in the reproductive development of male oriental river prawns (Macrobrachium nipponense).

Cyclin A (CycA) plays essential roles in regulating multiple steps of the cell cycle, and it affects gonad development in mammals and invertebrates. Previous RNA interference (RNAi) analysis revealed that knocking-down the expression of CycA in female oriental river prawns (Macrobrachium nipponense) inhibited ovarian development. CycA was also predicted to have regulatory roles in reproductive development of male M. nipponense based on significant changes of Mn-CycA expression after eyestalk ablation. The goal of this study was to investigate the potential functions of CycA in the reproductive development of male M. nipponense using RNAi and histological observations. Quantitative real-time PCR analysis revealed that both single-side and double-side eyestalk ablation stimulated the expressions of Mn-CycA, and the expression was higher in prawns with double-side eyestalk ablation (p < 0.05). Mn-CycA expression was significantly higher in the testis and androgenic gland during the reproductive season than during the non-reproductive season (p < 0.05). In the RNAi analysis, Mn-CycA expression significantly decreased after prawns were injected with dsCycA, and the expression of insulin-like androgenic gland hormone (Mn-IAG) also decreased as Mn-CycA expression decreased. This result indicated that CycA positively regulated the expression of IAG in M. nipponense. Histological observations revealed that the number of sperm decreased dramatically to <5% of the total cells in the testis of the dsCycA-treated group compared to that of control group on day 14, indicating that knockdown of Mn-CycA expression inhibited testis development by affecting the expression of Mn-IAG in M. nipponense. These results highlighted the functions of CycA in male reproductive development of M. nipponense, which can be applied to future studies of male reproduction in other crustacean species.

Novel benzylidene benzofuranone analogues as potential anticancer agents: design, synthesis and in vitro evaluation based on CDK2 inhibition assays.

The classical anticancer agents do not have their efficacy on inhibiting the G2 phase of the cell cycle. There are a very few reports available on drugs that work at G2 phase. Flavopiridol is one such drug candidate. In the current study, we sought to make analogues of flavopiridol. Still, the conditions used during their synthesis were unfavourable for the formation of flavopiridol and led to the generation of benzofuranones. In the present work, a new series of benzylidene benzofuranones were designed, synthesized and evaluated for their antioxidant, anti-colorectal cancer activity. Molecular docking, MMGBSA and molecular dynamics studies were conducted to assess their binding affinity at the active site of CDK2. Based on the cytotoxicity exhibited by test compounds, the compound NISOA4 (from isopropyl series) was further selected for mechanistic anticancer studies on HCT 116 cell lines. The compound selected was evaluated by comet assay, DNA fragmentation assay, cell cycle analysis, apoptosis detection by annexin FITC, semi-quantitative RTPCR based gene expression studies and FRET assay on the target CDK2/Cyclin A. Compound NISOA4 exhibited marked olive moments in comet assay studies. The apoptotic DNA fragmentation for compound NISOA4 demonstrated a marked change in the DNA fragmentation. The compound exhibited cell cycle arrest at G2/M phase at both the test concentrations. Apoptosis induction was observed at both the test concentrations and the compound was found to be a potent proapoptotic agent. It exhibited marked inhibition for the CDK2 gene expression and did not show any effect on CyclinA gene expression. However, the compound NISOA4 along with other analogues showed appreciable inhibition for the CDK2/Cyclin A target enzyme. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03312-1.

Lidocaine Inhibited Tendon Cell Proliferation and Extracellular Matrix Production by Down Regulation of Cyclin A, CDK2, Type I and Type III Collagen Expression.

Lidocaine injection is a common treatment for tendon injuries. However, the evidence suggests that lidocaine is toxic to tendon cells. This study investigated the effects of lidocaine on cultured tendon cells, focusing on the molecular mechanisms underlying cell proliferation and extracellular matrix (ECM) production. Tendon cells cultured from rat Achilles tendons were treated with 0.5, 1.0, or 1.5 mg/mL lidocaine for 24 h. Cell proliferation was evaluated by Cell Counting Kit 8 (CCK-8) assay and bromodeoxyuridine (BrdU) assay. Cell apoptosis was assessed by Annexin V and propidium iodide (PI) stain. Cell cycle progression and cell mitosis were assessed through flow cytometry and immunofluorescence staining, respectively. The expression of cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2), p21, p27, p53, matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), type I collagen, and type III collagen were examined through Western blotting, and the enzymatic activity of MMP-9 was determined through gelatin zymography. Lidocaine reduced cell proliferation and reduced G1/S transition and cell mitosis. Lidocaine did not have a significant negative effect on cell apoptosis. Lidocaine significantly inhibited cyclin A and CDK2 expression but promoted p21, p27, and p53 expression. Furthermore, the expression of MMP-2 and MMP-9 increased, whereas that of type I and type III collagen decreased. Lidocaine also increased the enzymatic activity of MMP-9. Our findings support the premise that lidocaine inhibits tendon cell proliferation by changing the expression of cell-cycle-related proteins and reduces ECM production by altering levels of MMPs and collagens.

The Therapeutic Role of PNU-74654 in Hepatocellular Carcinoma May Involve Suppression of NF-κB Signaling.

Background and Objectives: PNU-74654, a Wnt/ß-catenin inhibitor, has reported antitumor activities; however, the therapeutic potential of PNU-74654 in hepatocellular carcinoma (HCC) has not been investigated in detail. The aim of this study was to clarify the cytotoxic effects of PNU-74654 against HCC and to uncover its molecular mechanism. Materials and Methods: HepG2 and Huh7 liver cancer cell lines were selected to determine the antitumor properties of PNU-74654. Survival of the liver cancer cells in response to PNU-74654 was assessed by cell viability assays, and the apoptosis effect of PNU-74654 was analyzed by flow cytometry and visualized by Hoechst staining. An oncology array was used to explore the underlying molecular routes of PNU-74654 action in the cells. The migration properties were examined with a wound healing assay, and western blotting was conducted to evaluate protein expression. Results: Treatment with PNU-74654 decreased cell viability and inhibited cell migration. The cell cycle analysis and Hoechst staining revealed an increase in the population of cells at the sub-G1 stage and apoptotic morphological changes in the nucleus. The oncology array identified 84 oncology-related proteins and a suppressed expression of Bcl-xL and survivin. Western blotting showed that PNU-74654 could interfere with cell cycle-related proteins through the NF-κB pathway. Conclusions: PNU-74654 shows antiproliferative and antimigration effects against HepG2 and Huh7 cells, and its antitumor activity may be attributable to its interference in cell cycle regulation and the NF-κB pathway.