Hepatic COX1 loss leads to impaired autophagic flux and exacerbates nonalcoholic steatohepatitis

The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis (NASH) remain largely unknown. We aimed to elucidate the roles of hepatic cyclooxygenase 1 (COX1) in autophagy and the pathogenesis of diet-induced steatohepatitis in mice. Human nonalcoholic fatty liver disease (NAFLD) liver samples were used to examine the protein expression of COX1 and the level of autophagy. Cox1Δhepa mice and their wildtype littermates were generated and fed with 3 different NASH models. We found that hepatic COX1 expression was increased in patients with NASH and diet-induced NASH mice models accompanied by impaired autophagy. COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy. Mechanistically, COX1 directly interacted with WD repeat domain, phosphoinositide interacting 2 (WIPI2), which was crucial for autophagosome maturation. Adeno-associated virus (AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1Δhepa mice, indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy. In conclusion, we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2. Targeting the COX1-WIPI2 axis may be a novel therapeutic strategy for NASH.

Biallelic mutations in WDR12 are associated with male infertility with tapered-head sperm / 亚洲男科学杂志(英文版)

Teratozoospermia is a rare disease associated with male infertility. Several recurrent genetic mutations have been reported to be associated with abnormal sperm morphology, but the genetic basis of tapered-head sperm is not well understood. In this study, whole-exome sequencing (WES) identified a homozygous WD repeat domain 12 (WDR12; p.Ser162Ala/c.484T>G) variant in an infertile patient with tapered-head spermatozoa from a consanguineous Chinese family. Bioinformatic analysis predicted this mutation to be a pathogenic variant. To verify the effect of this variant, we analyzed WDR12 protein expression in spermatozoa of the patient and a control individual, as well as in the 293T cell line, by Western blot analysis, and found that WDR12 expression was significantly downregulated. To understand the role of normal WDR12, we evaluated its mRNA and protein expression in mice at different ages. We observed that WDR12 expression was increased in pachytene spermatocytes, with intense staining visible in round spermatid nuclei. Based on these results, the data suggest that the rare biallelic pathogenic missense variant (p.Ser162Ala/c.484T>G) in the WDR12 gene is associated with tapered-head spermatozoa. In addition, after intracytoplasmic sperm injection (ICSI), a successful pregnancy was achieved. This finding indicates that infertility associated with this WDR12 homozygous mutation can be overcome by ICSI. The present results may provide novel insights into understanding the molecular mechanisms of male infertility.

Knockdown of WDR1 Inhibits Nuclear Translocation of STAT3 to Regulate Smooth Muscle Cell Proliferation and Migration / 中国生物化学与分子生物学报

Nuclear transport of signal transducer and activator of transcription 3 (STAT3) is a prerequisite for its biological function. WD Repeat domain 1 (WDR1), a regulator of the cytoskeleton factors, may affect STAT3 nuclear translocation. However, the molecular mechanism regarding the effect of WDR1 on STAT3 nuclear translocation is still unknown. To investigate the effect of WDR1 on STAT3 nuclear translocation in smooth muscle cells, a human aortic vascular smooth muscle cells (HAVSMCs) model with stable knockdown of WDR1 was constructed. The results of RT-qPCR and Western blot showed that the activation and expression of STAT3 were not significantly altered after knockdown of Wdr1 (P>0. 05); the results of nucleoplasm isolation showed that the nucleoplasm distribution of STAT3 was significantly affected after knockdown of WDR1 compared with the control group. Subsequent results showed that the expression of STAT3 nuclear input-associated protein β (importin β) was inhibited (P < 0. 05) and the nucleoplasmic ratio of Ras-associated nuclear protein (Ran) was significantly decreased compared to the control group. Results from CCK8 and Transwell assays indicated that overexpression of importin β was able to rescue the inhibition of proliferation and migration of HAVSMCs caused by WDR1 knockdown. Further results showed that knockdown of WDR1 resulted in a significant decrease in the expression of nuclear transport factor 2 (NTF2) associated with the Ran nucleotide cycle (P<0. 05). After overexpression of NTF2, the results of CCK8 and Transwell experiments showed that the proliferation and migration ability of HAVSMCs were significantly enhanced (P < 0. 05). Summarizing the above results, knockdown of WDR1, by inhibiting the expression of importin β and NTF2, alters the nucleoplasmic distribution of Ran and decreases the nuclear translocation of STAT3, thus regulating the proliferation and migration of smooth muscle cells.

Expression and mechanism of microRNA-27a in cervical cancer / 解剖学报

Objective To investigate the effects of microRNA (miR)-27a on proliferation, apoptosis and invasion of cervical cancer cells by targeting F-box and WD repeat domain containing protein 7 (FBXW7) expression. Methods Thirty cases of cervical cancer and paracancerous tissues and 30 cases of normal tissues were used in the experiment. The expression of miR-27a in cervical cancer, paracancerous tissue, normal cervical tissue, cervical cancer cells (SiHa, Caski, HeLa, HCC94) and cervical squamous epithelial immortalized cell H8 were detected by Real-time PCR. MiR-27a inhibitor and its negative control were transfected into SiHa cells by liposome transfection. CCK-8 assay, flow cytometry and Transwell assay were used to detect the effects of miR-27a on the proliferation, cell cycle, apoptotic rate and invasive ability of SiHa cells. Bioinformatics was used to predict the targeting gene of miR-27a. Double luciferase reporter gene assay combined with Western blotting was used to verify the targeting regulation of miR-27a on FBXW7. Results Compared with the normal cervical tissues and the adjacent tissues, the expression of miR-27a was higher in cervical cancer tissues (P<0.05) ; Compared with the cervical squamous epithelial immortalized cells H8, the expression of miR-27a in cervical cancer cells SiHa, Caski, HeLa and HCC94 was higher (P<0.05). Inhibiting the expression of miR-27a in SiHa cells could significantly reduce the proliferation activity of cells (P<0.05), increase the proportion of G

Effect of BRWD3 on lymph node metastasis in breast cancer / 解放军医学杂志

Objective: To explore the effect of the bromodomain and WD repeat domain containing 3 (BRWD3) on lymph node metastasis in breast cancer and its mechanism. Methods: In vitro cell invasion experiments were used to explore the effect of BRWD3 on the invasion phenotype of breast cancer cell lines. Mouse lymph node metastasis model and lung metastasis model were used to investigate the role of BRWD3 in regulating breast cancer lymph node metastasis and lung metastasis in BALB/c nude mice. The BRWD3 co-expressed genes were searched through cBioPortal databases to analyze the biological functions and pathways of BRWD3, constructed a BRWD3 molecular regulatory network, which is examined with Western blotting analysis partly. The public breast cancer dataset and the KMPLOT analysis platform was used to analyze the expression of BRWD3 in breast cancer and the relationship between the expression of BRWD3 and breast cancer prognosis. Results: In vitro experiments showed that knockdown of BRWD3 significantly enhanced the invasion and migration of breast cancer cells (P<0.01), but did not promote their proliferation. The lymph node metastasis model demonstrated that knockdown of BRWD3 dramatically enhanced lymph node metastasis of breast cancer. Interestingly, the lung metastasis model showed that BRWD3 did not affect the mouse lung metastasis ability. Further functional clustering GO analysis of the co-expressed genes of BRWD3 suggested that they are mainly involved in gene expression regulation, DNA damage repair, chromosome organization and modification, ubiquitination, etc. Meanwhile, KEGG enrichment analysis showed that they were involved in signaling pathways such as ubiquitination, oxidative phosphorylation, MAPK, etc. Besides, via the Western blotting experiment, it was found that knockdown of BRWD3 increased the phosphorylation of ERK1/2. Moreover, BRWD3 expression in breast cancer with lymph node metastasis is significantly lower than in patients without lymph node metastasis. Further, the survival analysis in KMPLOT found that the prognosis of patients with low expression of BRWD3 was poor, which was significantly lower than that of patients with high expression of BRWD3. Conclusions: BRWD3 can regulate breast cancer invasion in vitro and lymph node metastasis in vivo. Afterward, the prognosis of patients with low expression of BRWD3 is poor. Meanwhile, ubiquination, oxidative phosphorylation, MAPK pathway, etc. were the possible regulation pathways of BRWD3, which provide a new theoretical basis for the research and application of molecular markers related to breast cancer lymph node metastasis.

Synergistic Anti-cancer Effect and Mechanism of FZKA Decoction Combined with Gefitinib on A549 Cells / 中国实验方剂学杂志

Objective::To observe the effect of Fuzheng Kangai (FZKA) decoction combined with gefitinib on the cells proliferation, apoptosis, invasion and metastasis of human lung adenocarcinoma A549 cells in vitro and in vivo, and relevant mechanisms. Method::The A549 cell proliferation of the control group, FZKA decoction groups (0.2, 0.4, 0.8, 1.6, 3.2 g·L-1), Gefitinib groups (10, 20, 40, 60, 80, 100 μmol·L-1) for 24, 48, 72 hours, and FZKA decoction (2 g·L-1) combined with Gefitinib (10 μmol·L-1) groups for 24 hours was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The changes of cell apoptosis, invasion and metastasis abilities of A549 cells were analyzed by flow cytometry, Wound Healing, transwell invasion assay. Western blot assay was used to examine the protein expressions of cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2 associated X (Bax), F-box and WD repeat domain-containing (FBW7) and myeloid cell leukemia-1 (MCL-1) in vitro. Result::Compared with control group, FZKA decoction group and Gefitinib group inhibited the cell proliferation, cell apoptosis, cell invasion and metastasis abilities in a dose-dependent and time-dependent manner, and improve the protein expressions of Bax, Caspase-3, FBW7, but decreased the protein expressions of Bcl-2, MCL-1 (P<0.05). Compared with treatment with Gefitinib alone, FZKA combined with Gefitinib inhibited the proliferation of A549 cells, and induced apoptosis more significantly (P<0.05). Compared with treatment with Gefitinib alone, the cell scratch healing and invasion abilities were significantly reduced after combined treatment (P<0.05). FZKA decoction combined with Gefitinib up-regulated Bax, Caspase-3 and FBW7 protein expressions, and down-regulated Bcl-2 and MCL-1 protein expressions compared with treatment with Gifitinib alone (P<0.05). Conclusion::FZKA decoction combined with Gefitinib can inhibit the proliferation, invasion and metastasis, and induce apoptosis on A549 cells. The mechanism may be associated with the FBW7/MCL-1 pathway.

Morphological analysis of autophagy / 药学学报

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional subcellular organelles and proteins in all living cells. The process of autophagy can be divided into three relatively independent steps: the initiation of phagophore, the formation of autophagosome and the maturation/degradation stage. Different morphological characteristics and molecular marker changes can be observed at these stages. Morphological approaches are useful to produce novel knowledge that would not be achieved through other experimental methods. Here we summarize the morphological methods in monitoring autophagy, the principles in data interpretation and the cautions that should be considered in the study of autophagy.

Expression and clinical significance of MCL-1 and FBW7 proteins in breast cancer polyploid induced by spindle poisons / 天津医药

Objective To investigate the expression and clinical significance of myeloid cell leukemia-1 (MCL-1) and F-box and WD repeat domain-containing 7 (FBW7) in breast cancer polyploid induced by spindle poisons. Methods (1) Nocodazole spindle poison was used to treat breast cancer cell MDA-MB-231. The morphological changes of cells were ob?served under microscope, and cells were harvested in 0, 6, 12, 24, 48 and 72 h. The cell cycle and DNA-ploidy changes were examined by flow cytometry. The expressions of FBW7 and MCL-1 proteins were detected by Western blot assay. (2) A multikinase inhibitor (Sorafenib) with Nocodazole or Taxol was used to treat MDA-MB-231 cells. MCL-1 protein expression was detected by Western blot assay after 48 h treatment. The cell cycle and DNA-ploidy changes were examined by flow cy?tometry after 48 h treatment. MTT method was used to observe cell proliferation after 48 and 72 h treatment. Results (1)Af?ter treatment by Nocodazole, polyploid characteristics of large cell size and nucleus were appeared. The percentages of octa?ploid were (0.8±0.2)%, (8.5±2.3)%, (7.8±2.0)%, (9.9±0.9)%, (28.2±0.8)%and (35.1±4.9)%after 0, 6, 12, 24, 48 and 72 h treatment, showing the increasing trend in turn (P<0.001). The number of polyploidy (tetraploid and octaploid) cells was as high as (97.6±0.7)%after 48 h treatment. The expression level of FBW7 protein was decreased significantly but the expres?sion of MCL-1 protein was increased significantly after 48 h treatment. (2) After 48 h treatment, the expression level of MCL-1 protein, polyploidy percentage and cell proliferation decreased significantly in Nocodazole+Sorafenib group and Taxol+Sorafenib group compared with those of Nocodazole group and Taxol group (P<0.05). Conclusion The lower expression of FBW7 protein and over-expression of MCL-1 protein are correlated with the formation of breast cancer polyploidy. Sorafenib can reduce polyploid tumor cells by inhibiting MCL-1 protein expression.