LncRNA HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression.

LncRNA HOX antisense intergenic RNA (HOTAIR) can regulate cancer-related gene expression and promote stem cell and tumor cell proliferation via mechanisms including the competing endogenous RNA (ceRNA) mechanism. HOTAIR is abundantly expressed in the genital tubercle of E11.5, E12.5, and E13.5 embryos, whereas it became barely detectable at E13.5 and expressed again in adult mouse testis. However, the underlying function and mechanism of HOTAIR in spermatogenesis have not been elucidated. Interestingly, other researchers reported that the function of gene Nanos C2HC-Type Zinc Finger 2 (nanos2) includes the maintenance of both the primordial germ cells (PGCs) and germline stem cells, and Nanos2 protein and transcripts (NANOS2) were detected only in PGCs from day E11.5 and undifferentiated spermatogonia in spermatogenesis. We therefore investigated the relationship between HOTAIR and NANOS2 in maintaining spermatogonial stem cell population. We found that, compared to the adult mouse, the expression levels of HOTAIR and NANOS2 in embryo mouse were significantly higher and miR-761expression level was lower. In mouse GC-1 spermatogonia cells, overexpression of miRNA-761 significantly inhibited the expression of NANOS2 and HOTAIR, suppressed the proliferation, and promotes apoptosis of cells. Knock down and overexpression of HOTAIR indicated that HOTAIR expression was positively correlated with NANOS2 expression; overexpressed HOTAIR could promote proliferation and suppresses apoptosis of GC-1 cells. By a rescue experiment and dual luciferase reporter assay, miR-761 was identified as a direct target of HOTAIR, and NANOS2 was identified as the direct target of miR-761. The above results indicate that HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression. Our findings elucidate one of possible mechanisms and importance of HOTAIR in maintaining spermatogonial stem cell population, and provide new candidate genes and possible pathogenesis for male infertility.

Association Between Mitochondrial DNA Copy Number and Head and Neck Squamous Cell Carcinoma: A Systematic Review and Dose-Response Meta-Analysis.

BACKGROUND The association between mitochondrial DNA (mtDNA) copy number and head and neck squamous cell carcinoma (HNSCC) risk remains unclear. Therefore, we aimed to evaluate the relationship between mtDNA copy number and HNSCC risk. MATERIAL AND METHODS We searched PubMed, Web of Science, and EMBASE until August 2020. Studies that assessed the association between mtDNA copy number and HNSCC as the outcome of interest were included. We performed a 2-class and dose-response meta-analysis to assess the association between cancer risk and mtDNA. RESULTS Eight articles (2 cohort studies and 6 case-control studies) with a total of 3913 patients were included in our meta-analysis. The overall results showed that mean mtDNA copy number level from 9 studies was 0.71 higher in patients with cancer than in non-cancer controls (the standardized mean differences (SMD) 0.71, 95% CI: 0.28-1.15, P<0.001). However, when 4 studies were pooled by dichotomizing mtDNA copy number at the median value into high- and low-content groups, no significant association between mtDNA content and overall cancer risk was found (odds ratio (OR)=0.87, 95% CI: 0.52-1.44, P=0.584). Furthermore, we observed a non-linear association from 3 studies between increased mtDNA copy number levels (P for nonlinearity <0.001). CONCLUSIONS The elevated mtDNA copy number could predict the risk of HNSCC as a biomarker. Moreover, there was non-linear relationship of risk between HNSCC and mtDNA copy number.

Methadone Inhibits Viral Restriction Factors and Facilitates HIV Infection in Macrophages.

Opioid abuse alters the functions of immune cells in both in vitro and in vivo systems, including macrophages. Here, we investigated the effects of methadone, a widely used opioid receptor agonist for treatment of opiate addiction, on the expression of intracellular viral restriction factors and HIV replication in primary human macrophages. We showed that methadone enhanced the HIV infectivity in primary human macrophages. Mechanistically, methadone treatment of macrophages reduced the expression of interferons (IFN-ß and IFN-λ2) and the IFN-stimulated anti-HIV genes (APOBEC3F/G and MxB). In addition, methadone-treated macrophages showed lower levels of several anti-HIV microRNAs (miRNA-28, miR-125b, miR-150, and miR-155) compared to untreated cells. Exogenous IFN-ß treatment restored the methadone-induced reduction in the expression of the above genes. These effects of methadone on HIV and the antiviral factors were antagonized by pretreatment of cells with naltrexone. These findings provide additional evidence to support further studies on the role of opiates, including methadone, in the immunopathogenesis of HIV disease.

F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

In mouse fertilized eggs, correct assembly and distribution of the actin cytoskeleton are intimately related to cleavage in early-stage embryos. However, in mouse fertilized eggs, mechanisms and involved factors responsible for regulating the actin cytoskeleton are poorly defined. In this study, mTORC2, PKB/Akt and Girdin were found to modulate division of mouse fertilized eggs by regulating distribution of the actin cytoskeleton. RNA interference (RNAi)-mediated depletion of mTORC2, Akt1 or Girdin disrupted F-actin rearrangement and strongly inhibited egg development. PKB/Akt has been proven to be a downstream target of the mTORC2 signalling pathway. Girdin, a newly found actin cross-linker, has been proven to be a downstream target of the Akt signalling pathway. Furthermore, phosphorylation of both Akt1 and girdin was affected by knockdown of mTORC2. Akt1 positively regulated development of the mouse fertilized eggs by girdin-mediated F-actin rearrangement. Thus it seems that girdin could be a downstream target of the Akt1-mediated signalling pathway. Collectively, this study aimed to prove participation of mTORC2/Akt in F-actin assembly in early-stage cleavage of mouse fertilized eggs via the function of girdin. OBJECTIVES: In mouse fertilized eggs, the proper assembly and distribution of actin cytoskeleton is intimately related with the cleavage of early-stage embryo. However, in mammals, especially in mouse fertilized eggs, the mechanisms and involved factors responsible for regulating the actin cytoskeleton are poorly defined. The aim of this study was to determine the role of mTORC2,PKB/Akt and Girdin in early development of fertilized mouse eggs, via regulating the distribution of actin cytoskeleton. MATERIALS AND METHODS: Changes of F-actin after treatting with mTORC2 shRNA, Akt siRNA or Girdin siRNA were observed by Immunofluorescence staining and laser-scanning confocal microscopy. Levels of phosphorylated Girdin at Se1417 were detected by Western immunoblotting. Percentages of cells undergoing division were determined by counting, using a dissecting microscope. RESULTS: RNA interference (RNAi)-mediated depletion of mTORC2, Akt1 or Girdin disrupts F-actin rearrangement, and remarkably inhibited the development of mouse-fertilized eggs. PKB/Akt has been proved to be a downstream target of the mTORC2 signaling pathway. Girdin, the newly found actin-cross linker, has been proved to be a downstream target of the Akt signaling pathway. Furthermore phosphorylation of both Akt1 and Girdin were affected by knockdown of mTORC2. Akt1 positively regulates the development of mouse-fertilized eggs by Girdin mediated F-actin rearrangement. Girdin could be a downstream target of the Akt1-mediated signaling pathway. CONCLUSIONS: Collectively, this study aimed to prove the participation of mTORC2/Akt in F-actin assembling in early-stage cleavage of mouse fertilized eggs via the function of Girdin.

MicroRNA-873 mediates multidrug resistance in ovarian cancer cells by targeting ABCB1.

Ovarian cancer is commonly treated with cisplatin and paclitaxel combination chemotherapy; however, ovarian cancer cells often develop resistance to these drugs. Increasingly, microRNAs (miRNAs) including miR-873 have been implicated in drug resistance in many cancers, but the role of miR-873 in ovarian cancer remains unknown. MTT cell viability assays revealed that the sensitivities of ovarian cancer lines to cisplatin and paclitaxel increased following transfection with miR-873 (P < 0.05). After predicting the miR-873 binding region in the 3'-untranslated region of ABCB1, dual-luciferase reporter assay confirmed this prediction. RT-PCR and Western blotting revealed that MDR1 expression was significantly downregulated after transfection with miR-873 and upregulated after transfection with anti-miR-873 at both mRNA and protein levels compared to negative controls (P < 0.05). Experiments in a mouse xenograft model confirmed that intratumoral administration of miR-873 could enhance the efficacy of cisplatin in inhibiting tumor growth in ovarian cancer in vivo (P < 0.05). ABCB1 overexpression reduced sensitivities of ovarian cancer lines OVCAR3 and A2780 to cisplatin and paclitaxel, which can be reversed by miR-873 mimic transfection (P < 0.05). In summary, we demonstrated that overexpression of miR-873 increased the sensitivity of ovarian cancer cells to cisplatin and paclitaxel by targeting MDR1 expression. Our findings suggest that combination therapies with chemotherapy agents and miR-873 may suppress drug resistance in ovarian cancer.

[PKB/Akt regulates the aggregation of actin by Girdin in mouse fertilized eggs].

The purpose of this study is to reveal the role of Girdin in regulating the aggregation of actin filaments by studying the relationship between PKB/Akt and Girdin. First we used Scansite software (http://scansite.mit.edu) to predict relevant target sites of PKB/Akt on mouse Girdin. To gain insight into the role of phosphorylation of Girdin by PKB/Akt, we assessed the location of phosphorylated Girdin in fertilized eggs by staining with anti-P-Girdin 1 417 Ab. We detected a distinct increase in the fluorescence signal of F-actin and P-Girdin 1 417 after microinjection of Akt WT and myr-Akt. The addition of myr-Akt induced phosphorylation of Girdin in mouse fertilized eggs. In addition, siRNA-mediated Akt-knockdown blocked phosphorylation of Girdin. The distribution of actin filaments was obviously scattered. These results strongly suggest that PKB/Akt could directly phosphorylate Girdin on Ser1 417 and promote its function in mouse fertilized eggs.

Successive recruitment of p-CDC25B-Ser351 and p-cyclin B1-Ser123 to centrosomes contributes to the release of mouse oocytes from prophase I arrest.

BACKGROUND: The molecular mechanism that controls the activation of Cyclin B1-CDK1 complex has been widely investigated. It is generally believed that CDC25B acts as a "starter phosphatase" of mitosis. In this study, we investigate the sequential regulation of meiotic resumption by CDC25B and Cyclin B1 in mouse oocytes. RESULTS: Injection of mRNAs coding for CDC25B-Ser351A and/or Cyclin B1-Ser123A shows a more potent maturation-inhibiting ability than their respective wild type. Co-injection of mRNAs coding for phosphor-mimic CDC25B-Ser351D and Cyclin B1-Ser123D can rescue this prophase I arrest induced by CDC25B-Ser351A or Cyclin B1-Ser123A. In addition, p-CDC25B-Ser351 is co-localized at the microtubule-organizing centers (MTOCs) with Aurora kinase A (AURKA) during maturation and p-Cyclin B1-Ser123 is only captured on MTOCs shortly before germinal vesicle breakdown (GVBD). Depletion of AURKA not only resulted in metaphase I (MI) spindle defects and anaphase I (AI) abnormal chromosomes separation but also prevented the phosphorylation of CDC25B-Ser351 at centrosomes. AURKA depletion induced deficiencies of spindle assembly and progression to MII can be rescued by CDC25B-Ser351D mRNA injection. CONCLUSIONS: AURKA induced phosphorylation and recruitment of CDC25B to MTOCs prior to p-Cyclin B1-Ser123, and this sequential regulation is essential for the commitment of the oocytes to resume meiosis.

Glucosamine, a naturally occurring amino monosaccharide, inhibits A549 and H446 cell proliferation by blocking G1/S transition.

Uncontrolled proliferation is important in tumorigenesis. In the present study, the effects of glucosamine on lung cancer cell proliferation were investigated. The expression of cyclin E, one of the key cyclins in the G1/S transition, and Skp2, the ubiquitin ligase subunit that targets the negative cell cycle regulator, p27Kip1, were also assessed. Moreover, the underlying mechanisms of action of glucosamine were investigated in lung cancer cells. A549 and H446 cells were synchronized using thymidine in the presence or absence of glucosamine. The effect of glucosamine on lung cancer cell proliferation was determined by MTT assay. Cyclin E and p27Kip1 proteins and their phosphorylation levels were detected by western blot analysis. Furthermore, the effect of glucosamine on the cell cycle was evaluated by flow cytometry. Glucosamine was found to inhibit lung cancer cell proliferation and to suppress Skp2 and cyclin E expression. Notably, the phosphorylation levels of cyclin E (Thr62) and p27Kip1 (Thr187) were downregulated by glucosamine, and negatively correlated with degradation. Glucosamine was also found to arrest lung cancer cells in the G1/S phase. Thus, glucosamine may inhibit lung cancer cell proliferation by blocking G1/S transition through the inhibition of cyclin E and Skp2 protein expression.

Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos.

The WEE1 kinase family has been shown to be the major kinase family responsible for phosphorylating Tyr 15 on cyclin-dependent kinase 1 (CDK1). WEE1 homolog 2 (WEE2, also known as WEE1B) was first identified in Xenopus laevis and more recently in humans and mice, and is responsible for phosphorylating the CDK1 inhibitory site and maintaining meiotic arrest in oocytes. However, the mechanism by which WEE1B is regulated in one-cell stage mouse embryos remains to be elucidated. In the present study, we examined the role of WEE1B-Ser 15 in G2/M transition of one-cell stage mouse embryos. WEE1B-Ser 15 was phosphorylated during the G1 and S phases, whereas Ser 15 was dephosphorylated during G2 and M phases in vivo. Overexpression of the phosphor-mimic Wee1B-S15D mutant delayed the re-entry of embryos into mitosis more efficiently than Wee1B-wild type (Wee1B-WT) by direct phosphorylation of CDK1-Tyr 15. The results of the present study suggested that WEE1B acts as a direct downstream substrate of protein kinase A (PKA) and that Ser 15 of WEE1B is a potential PKA phosphorylation target in the G2/M transition of mouse embryos. In addition, WEE1B inhibits mitosis by negatively regulating M phase promoting factor activity in one-cell stage mouse embryos.

Protein kinase B/Akt may regulate G2/M transition in the fertilized mouse egg by changing the localization of p21(Cip1/WAF1).

Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21(Cip1/WAF1) has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21(Cip1/WAF1) after injecting fertilized mouse eggs with Akt mRNA. In one-cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr-Akt), p21(Cip1/WAF1) was retained in the cytoplasm. Microinjection of mRNA of kinase-deficient Akt(Akt-KD) resulted in nuclear localization of p21(Cip1/WAF1) . Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21(Cip1/WAF1) . However, there was no obvious difference in the protein expression of p21(Cip1/WAF1) . Therefore, Akt controls the cell cycle by changing the subcellular localization of p21(Cip1/WAF1) , most likely by affecting the phosphorylation status of p21(Cip1/WAF1) .

Ser149 is another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs.

It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser(149), Ser(229), and Ser(321) of Cdc25B, and explored the role of Ser(149) in G(2)/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr(15), resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser(149) was phosphorylated at G(1) and S phases, whereas dephosphorylated at G(2) and M phases, and the phosphorylation of Cdc25B-Ser(149) was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G(1) and S phases and translocated to the nucleus at the G(2) phase. Collectively, our findings provide evidence that Ser(149) may be another potential PKA phosphorylation target of Cdc25B in G(2)/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.

[The effect of protein kinase B on the expression and location of p21 in early development of mouse fertilized eggs].

To investigate the effect of Protein kinase B on the expression and location of p21 in mouse early development. Immunopreciptation technology was used to detect the localization of p21 and Western blotting was used to analyze the expression of p21 after microinjecting mRNA of WT-PKB, myt-PKB and PKB-KD to mouse eggs. There was no obvious difference between the three kinds of mRNA in the expression of p21. But the cell localization altered. The p21 retain in cytoplasm after microjecting myt-PKB. In mouse fertilized egg PKB/Akt controls the cell cycle by changing the cell localization of p21.

Protein kinase A regulates cell cycle progression of mouse fertilized eggs by means of MPF.

Cell cycle of one-cell stage mouse fertilized eggs was accompanied by fluctuation in the concentration of adenosine 3'5'-monophosphate (cAMP) and in the activity of free catalytic subunit of cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of free catalytic subunit of PKA decreased at the onset of mitosis and increased at the transition between mitosis and G1 phase. Stimulation of PKA by microinjection of cAMP into one-cell stage mouse embryos at G2 phase induced interphase arrest and prevented the activation of M-phase promoting factor (MPF). Upon blockage of the activation of PKA by microinjecting a thermostable PKA inhibitor (PKI) into one-cell stage mouse embryos at G2 phase, the increase in the MPF activity occurred 30 min earlier than in control group. When a high dose of PKI was microinjected, a transition into interphase was prevented, and the activity of MPF remained high. Western blot analysis showed that Cdc2 remained phosphorylated in cAMP microinjected embryos by the time when control embryos were at metaphase and showed dephosphorylated Cdc2; conversely, Cdc2 dephosphorylation was accelerated in PKI-microinjected embryos. At the same time, Cdc2 was phosphorylated at Tyr15 at G2 phase and even at M phase when cAMP was microinjected but was dephosphorylated when PKI was microinjected. PKI microinjection also prevented cyclin B degradation and sustained MPF activity, thus delaying the transition from metaphase to anaphase. Our results show that PKA, by inhibiting MPF, regulates cell cycle progression of fertilized eggs.