Transcription factor E2F8 activates PDK1-mediated DNA damage repair to enhance cisplatin resistance in lung adenocarcinoma.

INTRODUCTION: Cisplatin (DDP) is the commonest chemo drug in lung adenocarcinoma (LUAD) treatment, and DDP resistance is a significant barrier to therapeutic therapy. This study attempted to elucidate the impact of PDK1 on DDP resistance in LUAD and its mechanism. METHODS: Bioinformatics analysis was used to determine the expression and enriched pathways of PDK1 in LUAD tissue. Subsequently, E2F8, the upstream transcription factor of PDK1 was predicted, and the binding relationship between the two was analyzed using dual-luciferase and ChIP experiments. PDK1 and E2F8 levels in LUAD tissues and cells were detected via qPCR. Cell viability, proliferation, and apoptosis levels were assayed by CCK-8, EdU, and flow cytometry experiments, respectively. Comet assay was used to assess DNA damage, and immunofluorescence was used to assess the expression of γ-H2AX. NHEJ reporter assay was to assess DNA repair efficiency. Western blot tested levels of DNA damage repair (DDR)-related proteins. Immunohistochemistry assessed the expression of relevant genes. Finally, an animal model was constructed to investigate the influence of PDK1 expression on LUAD growth. RESULTS: PDK1 was found to be upregulated in LUAD and enhanced DDP resistance by mediating DDR. E2F8 was identified as an upstream transcription factor of PDK1 and was highly expressed in LUAD. Rescue experiments presented that knocking down E2F8 could weaken the promotion of PDK1 overexpression on DDR-mediated DDP resistance in LUAD. In vivo experiments showed that knocking down PDK1 plus DDP significantly reduced the growth of xenograft tumors. CONCLUSION: Our results indicated that the E2F8/PDK1 axis mediated DDR to promote DDP resistance in LUAD. Our findings lead to an improved treatment strategy after drug resistance.

Evolution of the thermostability of actin-depolymerizing factors enhances the adaptation of pollen germination to high temperature.

Double fertilization in many flowering plants (angiosperms) often occurs during the hot summer season, but the mechanisms that enable angiosperms to adapt specifically to high temperatures are largely unknown. The actin cytoskeleton is essential for pollen germination and the polarized growth of pollen tubes, yet how this process responds to high temperatures remains unclear. Here, we reveal that the high thermal stability of 11 Arabidopsis (Arabidopsis thaliana) actin-depolymerizing factors (ADFs) is significantly different: ADFs that specifically accumulate in tip-growing cells (pollen and root hairs) exhibit high thermal stability. Through ancestral protein reconstruction, we found that subclass II ADFs (expressed specifically in pollen) have undergone a dynamic wave-like evolution of the retention, loss, and regeneration of thermostable sites. Additionally, the sites of AtADF7 with high thermal stability are conserved in ADFs specific to angiosperm pollen. Moreover, the high thermal stability of ADFs is required to regulate actin dynamics and turnover at high temperatures to promote pollen germination. Collectively, these findings suggest strategies for the adaptation of sexual reproduction to high temperature in angiosperms at the cell biology level.

Long Noncoding RNA LINC00473 Ameliorates Depression-Like Behaviors in Female Mice by Acting as a Molecular Sponge to Regulate miR-497-5p/BDNF Axis.

Background: Depression was a common life-threatening psychiatric disorder and occurs more frequently in women than in men. Long noncoding RNAs (lncRNAs), such as LINC00473, had been reported to be involved in the progression of depression. Methods: Chronic unpredictable moderate stress in mice (CUMS) was applied to construct a depression model. Subsequently, RT-qPCR was applied to check the level of LINC00473 and microRNA-497-5p (miR-497-5p) in the hippocampal region of the mice induced by CUMS. CUMS mice were injected with lentiviral vectors of LINC00473 (LV-LINC00473), miR-497-5p inhibitor, short hairpin- (sh-) brain-derived neurotrophic factor (sh-BDNF), or miR-497-5p mimic to evaluate depressive behaviors, including sucrose preference test, forced swim test, elevated plus maze, and tail suspension test. Moreover, the production of hypothalamic neurotransmitters was assessed with the usage of ELISA kits. Dual-luciferase reporter assay, RNA pull-down, and RIP analysis were performed to measure the relationship between miR-497-5p and LINC00473 or BDNF. Further, western blot was employed to determine the protein level of BDNF. Results: We discovered that LINC00473 level was downregulated in the female mice with depression, but not in male mice. Besides, the depressive behaviors induced by CUMS in mice, including the decrease of sucrose preference and time in open arm, as well as the increase of immobility time and swimming resting time were all ameliorated by LINC00473 overexpression. Moreover, the concentration of neurotransmitters was decreased in CUMS-induced mouse hypothalamus, which was blocked by LV-LINC00473 lentiviral vector administration. Mechanistically, LINC00473 directly targeted miR-497-5p. Absence of miR-497-5p revealed the antidepression effects on CUMS-induced mice, and miR-497-5p upregulation could counter the antidepressive impacts of LINC00473 upregulation on CUMS-induced mice. Furthermore, LINC00473 could target miR-497-5p to modulate BDNF level. Knockdown of BDNF could abrogate the improving influences of miR-497-5p suppression on CUMS-induced depression. Conclusions: LINC00473 ameliorated CUMS-caused depression by encouraging BDNF expression via binding to miR-497-5p, which might provide a potential therapeutic target for depression in females.

PFKFB4 modulated by miR-195-5p can boost the malignant progression of cervical cancer cells.

PFKFB4 is dysregulated in varying tumors and has the biological function of regulating tumor progression. However, its biological function in cervical cancer is poorly understood. We obtained the upstream regulatory gene (miR-195-5p) of PFKFB4 through bioinformatics analysis. Then, experiments were introduced to measure expression and targeting relationship of miR-195-5p and PFKFB4 in cervical cancer cells, in order to evaluate their influence on proliferation, migration, invasion and angiogenesis of cervical cancer cells. As expressed in results, PFKFB4 was abnormally increased and boosted malignant progression of cervical cancer cells. Besides, miR-195-5p was markedly decreased and restrained PFKFB4 in cervical cancer. While tumor-suppressive effect of miR-195-5p was partially restored by overexpressing PFKFB4, indicating that miR-195-5p and PFKFB4 may be new therapeutic targets for cervical cancer patients.

In vitro inhibitory effect of obtusofolin on the activity of CYP3A4, 2C9, and 2E1.

BACKGROUND: Obtusofolin is the major active ingredient of Catsia tora L., which possesses the activity of improving eyesight and protecting the optic nerve. Investigation on the interaction of obtusofolin with cytochrome P450 enzymes (CYP450s) could provide a reference for the clinical application of obtusofolin. METHODS: The effect of obtusofolin on the activity of CYP450s was investigated in the presence of 100 µM obtusofolin in pooled human liver microsomes (HLMs) and fitted with the Lineweaver-Burk plots to characterize the specific inhibition model and kinetic parameters. RESULTS: Obtusofolin was found to significantly inhibited the activity of CYP3A4, 2C9, and 2E1. In the presence of 0, 2.5, 5, 10, 25, 50, and 100 µM obtusofolin, the inhibition of these CYP450s showed a dose-dependent manner with the IC50 values of 17.1 ± 0.25, 10.8 ± 0.13, and 15.5 ± 0.16 µM, respectively. The inhibition of CYP3A4 was best fitted with the non-competitive inhibition model with the Ki value of 8.82 µM. While the inhibition of CYP2C9 and 2E1 was competitive with the Ki values of 5.54 and 7.79 µM, respectively. After incubating for 0, 5, 10, 15, and 30 min, the inhibition of CYP3A4 was revealed to be time-dependent with the KI value of 4.87 µM- 1 and the Kinact value of 0.0515 min- 1. CONCLUSIONS: The in vitro inhibitory effect of obtusofolin implying the potential drug-drug interaction between obtusofolin and corresponding substrates, which needs further in vivo validations.

miR­146a reduces depressive behavior by inhibiting microglial activation.

Depression is one of the major psychiatric diseases affecting the quality of life for individuals worldwide. Numerous reports have investigated depression, although its etiology remains to be elucidated. microRNA (miR)­146a is suggested to regulate innate immune and inflammatory responses. However, it is unclear whether miR­146a is involved in depression. Depression model mice were established using lipopolysaccharide­induced depression and chronic unpredictable mild stress, separately. miR­146a mimic and short interfering RNA were used to treat depressed mice. Depression­like behaviors and levels of pro­inflammatory cytokines were measured, while ionized calcium binding adapter molecule 1 (Iba­1) expression in hippocampus was quantified by immunohistochemistry. Neuroinflammatory factor levels in hippocampus were measured by western blotting. BV­2 cells were used to confirm that miR­146a suppressed microglia activation. Compared with control mice, the two depressed mouse models showed clearly decreased sucrose preference and significantly increased immobility time in the forced swimming test and tail suspension test (P<0.05). miR­146a overexpression significantly increased sucrose preference and reduced immobility time in depressed mice (P<0.05). However, total distance traveled in the locomotor activity test did not differ among groups. Compared with controls, expression levels of Iba­1, inducible nitric oxide, IL­1ß, TNF­α, interleukin 1 receptor associated kinase 1 (IRAK1), TNF receptor­associated factor 6 (TRAF6) and phosphorylated NF­κB p65 were significantly increased in depressed mice (P<0.05). miR­146a overexpression effectively inhibited expression of these neuroinflammatory proteins, while miR­146a silencing significantly upregulated their expression (P<0.05). Consistent with these in vivo results, miR­146a mimic treatment inhibited TNF­α, IL­1ß, IRAK1 and TRAF6 expression in BV­2 cells. miR­146a improved depressive behaviors in depressed model mice by inhibiting microglial activation and neuroinflammatory factor expression.

Exploration of the Synergy Between 2D Nanosheets and a Non-2D Filler in Mixed Matrix Membranes for Gas Separation.

Dual-filler MMMs have attracted special interests in recent years because of the possibility of producing synergetic effect. This study is aimed at exploring the underlying synergy between two-dimensional (2D) nanosheets and a non-2D filler in mixed matrix membranes for gas separation. MXene or graphene oxide (GO) as typical nanosheet filler is selected to be in pair with a non-2D filler, SiO2 or halloysite nanotubes (HNTs), with Pebax as the polymer matrix. In this way, four pairs of binary fillers are designed and the corresponding four groups of MMMs are fabricated. By tuning the mass ratio of binary fillers, synergetic effect is found for each group of MMMs. However, the two 2D fillers found different preferential non-2D partners. GO works better with HNTs than SiO2, while MXene prefers SiO2 to HNTs. To be noted, GO/HNTs renders the membranes the maximum enhancement of CO2 permeability (153%) and CO2/N2 selectivity (72%) compared to Pebax control membrane, while each of them as single filler only brought about very limited enhancement of CO2 separation performance. The possible mechanisms are thoroughly discussed in terms of filler dispersion, nanosheet flexibility, and the tortuosity and connectivity of the surface diffusion pathways along nanosheets.

A randomized controlled study of single-agent cisplatin and radiotherapy versus docetaxel/cisplatin and radiotherapy in high-risk early-stage cervical cancer after radical surgery.

BACKGROUND: This study explored whether docetaxel/cisplatin and radiotherapy (TP-R) increases overall survival (OS) and recurrence-free survival (RFS) compared to single-agent cisplatin and radiotherapy (C-R) in patients with high-risk early-stage cervical cancer post surgery. METHODS: Patients with clinical stage IB and IIA carcinoma of the cervix, initially treated with radical hysterectomy and pelvic lymphadenectomy, and who had positive pelvic lymph nodes and/or positive margins and/or the diameter of the primary tumor ≥4 cm and/or depth of interstitial infiltration ≥1/2 and/or lymphovascular space invasion were eligible for this study. Patients were randomized to receive C-R or TP-R. Radiotherapy in both groups was external radiation (46-54 Gy) followed by high-dose rate brachytherapy (12-24 Gy). Patients were given cisplatin (40 mg/m(2)) every week for five cycles (C-R group) or docetaxel (30 mg/m(2)) and cisplatin (30 mg/m(2)) every week for five cycles (TP-R group). RESULTS: Between 2003 and 2008, 320 patients were entered onto the study. Final analyses included 285 patients. One hundred and forty patients comprised the C-R group and 145 were in the TP-R group. The 5-year OS were 74.3 % in the C-R group and 82.8 % in the TP-R group. The hazard ratio (HR) for death was 0.65 in the TP-R group (95 % CI: 0.39-1.09, P = 0.098). The RFS were 69.3 % in the C-R group and 79.3 % in the TP-R group, and the HR for recurrence was 0.64 in the TP-R group (95 % CI: 0.40-1.03, P = 0.061). Recurrence rates were similar in both groups (27 in the C-R group and 18 in the TP-R group, P = 0.112). The seriousness of late side effects was similar in the two groups, with a higher rate of reversible hematological effects in the TP-R group. CONCLUSIONS: Compared with single-agent cisplatin and radiotherapy, docetaxel/cisplatin in combination with radiotherapy does not increase OS but has the trend of increasing RFS in patients with high-risk early-stage cervical cancer. However, docetaxel/cisplatin in combination with radiotherapy is associated with a higher incidence of side effects, this effect was reversible, and the incidence of late side effects was similar in the two treatment groups.