[Hydroxysafflor yellow A inhibits proliferation, migration, and chemoresistance of colorectal cancer cells through Akt/mTOR-autophagy pathway].

In recent years, the clinical treatment of colorectal cancer(CRC) has made great progress, but chemoresistance is still one of the main reasons for reducing the survival rate of patients with colorectal cancer. Therefore, ameliorating chemotherapy resis-tance is an urgent problem to be solved. The purpose of this study was to investigate the regulatory role and related molecular mechanisms of hydroxysafflor yellow A(HSYA) in colorectal cancer cell proliferation, migration, and 5-fluorouracil(5-FU) chemoresistance. In this study, HCT116 and HT-29 cells were used as research subjects. Firstly, methyl thiazolyl tetrazolium(MTT) assay and colony formation assay were used to detect and analyze the effect of HSYA on the proliferation of CRC cells. Secondly, the effect of HSYA on the cell cycle in CRC cells was analyzed by cell cycle assay. Furthermore, the effect of HSYA on the migration of CRC cells was analyzed by wound-healing assay and Transwell assay. Based on the above, the influences of HSYA on 5-FU chemoresistance of CRC cells and related molecular mechanisms were explored and analyzed. The results showed that HSYA significantly inhibited the proliferation and migration of CRC cells, and arrested the cell cycle in G_0/G_1 phase. In addition, HSYA significantly ameliorated the chemoresistance of CRC cells to 5-FU. The results of acridine orange staining and Western blot showed that the autophagy activity of CRC cells in the HSYA and 5-FU combined treatment group was significantly higher than that in the 5-FU single drug treatment group. As compared with the 5-FU single drug treatment group, the phosphorylation levels of protein kinase B(Akt) and mammalian target of rapamycin(mTOR) in the HSYA and 5-FU combined treatment group were significantly reduced, indicating that the Akt/mTOR signaling pathway in the combined treatment group was down-regulated in CRC cells. In conclusion, HSYA may upregulate autophagy activity through the Akt/mTOR signaling pathway, thereby inhibiting the proliferation and migration of CRC cells and ameliorating the chemoresistance to 5-FU.

Hypo-Expression of Tuberin Promotes Adenomyosis via the mTOR1-Autophagy Axis.

Adenomyosis (AM) is a disease in which endometrial tissue invades the myometrium and has a 10-60% prevalence in reproductive-aged women. TSC2 regulates autophagy via mTOR1 signalling in colorectal cancer and endometrial carcinoma. Dysregulation of autophagy is implicated in adenomyosis pathogenesis. However, whether TSC2 participates in adenomyosis via autophagy remains obscure. Here, we found that the expression of TSC2 in adenomyosis was significantly decreased than that in normal endometrium during the secretory phase. Moreover, TSC2 and autophagy marker expression was significantly lower in ectopic lesions than in eutopic samples. TSC2 downregulation inhibited autophagy through mTOR1 signalling pathway activation in endometrial cells, leading to excessive proliferation, migration, and EMT; TSC2 overexpression induced the opposite effects. Rapamycin treatment suppressed cell proliferation, migration and EMT in the absence of TSC2. In parallel, an autophagy-specific inhibitor (SAR-405) restored migration and EMT under rapamycin treatment in TSC2-knockdown Ishikawa cells. Finally, SAR-405 treatment promoted EMT and migration of overexpressing cells. Collectively, our results suggest that TSC2 controls endometrial epithelial cell migration and EMT by regulating mTOR1-autophagy axis activation and that hypo-expression of TSC2 in the endometrium might promote adenomyosis.

Long non-coding RNA KCNQ1OT1 promotes the progression of gastric cancer via the miR-145-5p/ARF6 axis.

BACKGROUND: Long non-coding RNA KCNQ1 opposite strand/antisense transcript one gene (KCNQ1OT1) has been reported to be involved in the progression of many types of human cancer, whereas its role in gastric cancer (GC) remains unknown. The present study aimed to investigate the role of KCNQ1OT1 in GC. METHODS: In total, 25 GC tissues and adjacent normal tissues were collected. The expression of KCNQ1OT1, miR-145-5p and ARF6 in GC tissues and cell lines was detected by quantitative reverse transcriptase-polymerase chain reaction or western blotting. Bioinformatics analysis and a dual luciferase reporter assay were performed to determine the relationship between KCNQ1OT1 and miR-145-5p or miR-145-5p and ARF6. Gain- and loss-of function of KCNQ1OT1 and miR-145-5p were achieved to confirm their roles in GC cells. Cell counting kit-8, colony formation and flow cytometry assays were used to evaluate cell viability, proliferation and apoptosis. A xenograft tumor model was established with BGC803 tumor cells transfected with sh-KCNQ1OT1 or empty vector to determine the role of LINC01089 in vivo. RESULTS: The expression levels of KCNQ1OT1 were markedly elevated in GC tissues and cells. Knockdown of KCNQ1OT1 inhibited GC tumor growth, reduced GC cell viability and colony formation, and induced GC cell apoptosis. The expression levels of miR-145-5p were significantly decreased in GC cells and correlated with the expression of KCNQ1OT1 in GC tumors. Moreover, KCNQ1OT1 directly binds with miR-145-5p, which is targeting ARF6. Knockdown of KCNQ1OT1 increased the expression levels of miR-145-5p. Inhibition of miR-145-5p increased the expression levels of KCNQ1OT1 and also attenuated the effects of knockdown of KCNQ1OT1 on the viability, proliferation and apoptosis of GC cells. In addition, overexpression of miR-145-5p reduced GC cell viability and colony formation and induced GC cell apoptosis, whereas overexpression of ARF6 attenuated the effects of overexpression of miR-145-5p on GC cell viability, colony formation and apoptosis. CONCLUSIONS: KCNQ1OT1 can promote GC progression through the miR-145-5p/ARF6 axis. KCNQ1OT1 may serve as a therapeutic target and a diagnostic biomarker of GC.

[RHNO1: A novel noncoding RNA highly expressed in the testis and involved in DNA double-strand break repair].

OBJECTIVE: To investigate the role of a long noncoding RNA (lncRNA) transcribed from the RHNO1 gene we newly identified in DNA double-strand break (DSB) repair. METHODS: The transcription and translation of the RHNO1 gene were validated by Western blot, real-time PCR and liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on the overexpressed RHNO1 plasmid. The transcription level of RHNO1 in the mouse tissue was detected by real-time PCR and its expression in the spermatogenic cycle determined by in situ hybridization. The role of RHNO1 in the DNA DSB repair was further verified using the DSB model established by exposing the germ cells to ultraviolent radiation. RESULTS: The full-length RHNO1 gene could be transcribed as a novel lncRNA in vitro, highly expressed in the mouse testis tissue, and mainly located in spermatocytes and round spermatids. RHNO1 was involved in DNA DSB repair in the spermatogenic cells. CONCLUSIONS: We identified a novel lncRNA, RHNO1, which is highly expressed in the mouse testis and participates in DNA damage repair in the germ cell line.

Autism-like behaviors and abnormality of glucose metabolism in offspring derived from aging males with epigenetically modified sperm.

Accumulating evidence from epidemiological studies of humans and genetic models in rodents has shown that offspring from males of advanced paternal age (APA) are susceptible to metabolic and neurological disorders. However, knowledge of molecular mechanism(s) underlying these metabolic and behavioral changes at the intergeneration and trans-generation levels from APA is limited. Here, we characterized changes on glucose and cholesterol metabolism, and also autism spectrum disorders (ASD)-like behaviors in 1st and 2nd generations from 12- and 18-month-old male mice, respectively. Whole Genome Bisulfite Sequencing (WGBS) of sperm from APA mice identified differentially methylated regions (DMRs) within the whole genome, and DMRs within promoter regions, suggesting that specific genes and relevant pathways might be associated with autism and aberrant glucose metabolism in the offspring from APA males. These results strongly suggest that epigenetic reprogramming induced by aging in male sperm may lead to high risks of aberrant glucose metabolism and the development of ASD behaviors in intergenerational and transgenerational offspring.

Activation of the LINC00242/miR-141/FOXC1 axis underpins the development of gastric cancer.

BACKGROUND: Long non-coding RNAs (LncRNAs) are a class of newly identified transcripts recognized as critical governors of gene expression during human carcinogenesis, whereas their tumor-suppressive or tumor-promoting effects on gastric cancer (GC) are required for further investigation. In the study, we identify the expression pattern of a novel lncRNA LINC00242 in GC and its possible permissive role in the development of GC. METHODS: The study included 68 pairs of GC and adjacent normal gastric tissue samples. The viability, migration, and invasion of cultured human GC cells HGC27 were evaluated by CCK-8 and Transwell chamber assays. In vitro tube formation of human brain microvascular endothelial cells (HBMVECs) in HGC27 cell coculture was detected. The regulatory network of LINC00242/miR-141/FOXC1 was verified using dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. Subcutaneous xenografts of HGC27 cells were performed in nude mice. RESULTS: LINC00242 was highly expressed in GC tissues and cells and contributed to poor prognosis. LINC00242 knockdown inhibited HGC27 cell viability, migration and invasion, and tube formation of HBMVECs. LINC00242 interacted with miR-141 and positively regulated FOXC1, a target gene of miR-141. LINC00242 knockdown was partially lost in HGC27 cells upon miR-141 inhibition or FOXC1 overexpression. The tumor-promoting effect of LINC00242 on GC was demonstrated in nude mice. CONCLUSION: Taken together, the present study demonstrates the oncogenic role of the LINC00242/miR-141/FOXC1 axis in GC, highlighting a theoretical basis for GC treatment.

The novel testicular enrichment protein Cfap58 is required for Notch-associated ciliogenesis.

Cilia and flagella are critical organelles with conserved internal structures and diverse developmental and physiological processes according to cell type. Although the core components of structures are shared with thousands of associated proteins involved in cilia or flagella formation, we hypothesized that some unknown proteins, such as outer dense fiber 2 (Odf2/Cenexin) perform distinct functions in these organelles. In the present study, we identified several uncharacterized proteins through mass spectrometry interactome analysis of Odf2/Cenexin proteins. We further examined the expression patterns and functions of a protein named cilia and flagella associated protein 58 (Cfap58) in cultured astrocytes and sperm flagella. The results of a combination of biochemical analyses and drug administration studies reveal that Cfap58 is a testis-enrichment protein that exhibits similar localization to Odf2/Cenexin proteins and is required for the elongation of the primary cilium and sperm midpiece via modulation of the Notch signaling pathway. However, the cell cycle-related functions and localization of Odf2/Cenexin in the mother centriole were not altered in Cfap58 knockdown cells. These findings indicate that Cfap58 may be partially recruited by Odf2/Cenexin proteins and is indispensable for the cilia and flagellar assembly. These data provide us with a better understanding of ciliogenesis and flagellar elongation and may aid in identifying new targets for diseases caused by Notch-mediated ciliopathies and flagellar abnormalities.

Comparative analysis of mammalian sperm ultrastructure reveals relationships between sperm morphology, mitochondrial functions and motility.

BACKGROUND: Sperm morphology mainly refers to the shape of the head, the length of the flagellar segments, including the midpiece, principal piece and end piece, and the size of the accessory structures, including axonemes, outer dense fibers (ODFs), mitochondrial sheath (MS) and fibrous sheath (FS). Across species, there is considerable diversity in morphology. An established theory posits that the length of the sperm flagellum, especially the length of the midpiece, is a critical factor influencing sperm metabolism and velocity. However, our understanding of the relationships between sperm ultrastructures and the sperm flagellar length is incomplete. METHODS: The morphologies of sperm from 10 mammalian species, human, mouse, rat, dog, rabbit, goat, pig, bull, guinea pig and golden hamster, were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). According to the SEM and TME images, the length of sperm heads and flagellar segments, the cross-sectional areas of the accessory structures and flagella and the width of sperm heads were measured using Image J software. The variation tendencies (referred to as slope) of the accessory structures along flagella were calculated by the linear regression method. Mitochondrial functions were measured using commercial kits. The velocities of sperm were measured using CASA software. RESULTS: The three-dimensional morphologies of sperm from 10 species and the slopes of internal accessory structures along flagella were obtained. The width of the axoneme tapered slightly from the base to the tip of the sperm flagellum, and slopes of the axonemes correlated negatively with the variability in flagellar length across species. Additionally, the cross-sectional areas of the ODFs and/or the MS were positively correlated with the lengths of the midpiece, principal piece, and total flagellum, as well as with sperm velocities. Mitochondrial volumes were positively correlated with ATP content and sperm swimming velocities. CONCLUSIONS: Our results not only show the relationship between sperm internal structures, flagellar length and sperm physiology but also provide sizes of mitochondria and ODFs as new targets with which to study the regulation of sperm length and velocity.

Correlation study on chromogranin A genetic polymorphism and prognosis of critically ill patients.

OBJECTIVE: The objective was to investigate the correlation between single nucleotide polymorphism (SNP) of chromogranin A (CHGA) and prognosis of critically ill patients. METHODS: We screened 357 critically ill patients consecutively admitted to our intensive care unit. The -89/-415/-462 SNP locus in the promoter region and the +9559/+9578/+9590/+9611 SNP locus in exon 7 coding of CHGA were genotyped by polymerase chain reaction and DNA sequencing technology. Subsequently, the correlation between genotype and prognosis of patients was analyzed. RESULTS: (1) Three hundred critically ill Chinese Han patients were enrolled in the study. CHGA-415/-462/+9559/+9611 SNPs were polymorphically distributed. Phenotypes of the 4 SNPs were shown not to be in linkage disequilibrium, and there were no significant differences in the minor allele frequencies (MAFs) of the 4 SNPs between participants of this study and healthy people in Asia. (2) The CHGA-415 T/C MAF of the nonsurvival group was significantly higher than that of the survival group (MAF 0.3813 and 0.2864, respectively; P=.026). Survival analysis showed that there were significant differences between the CHGA-415 T/C mutation group (including TC and CC genotypes) and the wild-type group (TT genotype) (log rank=8.887, P=.003). The mortality in the mutant group was significantly higher than that in the wild-type group (0.3333 and 0.1852, respectively; P=.004). (3) Binary logistic analysis showed that CHGA-415 T/C polymorphism was an independent risk factor for the mortality of critically ill patients (odds ratio, 2.286; 95% confidence interval, 1.165-4.484; P=.016). CONCLUSIONS: Critically ill patients with CHGA-415 T/C mutant genotype display higher 30-day mortality than those with the wild-type group. CHGA-415 T/C polymorphism is an independent risk factor of poor prognosis in critically ill Chinese Han patients.

Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4.

Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (15 mg/kg), omeprazole (15 mg/kg), and midazolam (10 mg/kg), was given orally to rats treated for 7 d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug-CAP interactions.

[A study of inhibitory effect of chromogranin A derived peptide CGA(47 _ 66) on hyper - permeability of endothelium induced by serum of septic patient].

OBJECTIVE: To explore the role of chromogranin A ( CGA) derived peptide CGA47~ ( Chromfungin, CHR) on septic serum induced high permeability of vascular endothelial cells. METHODS: Human umbilical venous endothelial cell line (EA.hy926 cells) was exposed to CHR, serum of septic shock patient, and tumor necrosis factor-a (TNF-a) respectively. Methyl thiazolyl tetrazolium (MTT) method, Transwell assay and immunofluorescence were performed to determine cell viability (absorbance (A) value J, permeability of monolayer endothelial cells (A value) , and the morphological characteristic and distribution ofF -actin respectively. RESULTS: Compared with the blank control group, when EA.hy926 were exposed to CHR with 1, 10, 100 nmol/L the cell activity was not significantly affected (A value: 1.219 ± 0.253, 1.179 ± 0.065, 1.179 ± 0.062 vs. 1.306 ± 0.162, all P>0.05), while when the cells was exposed to CHR in 1 000 nmol/L the cell activity was significantly inhibited (A value: 1.049 ± 0.256 vs. 1.306 ± 0.162, t=-2.390, P=0.031 ). Compared with blank control group, when the cells were exposed to CHR of 1, 10, 100 nmol/L a significant decrease in permeability in EA.hy926 cells was observed (A value: 1.619 ± 0.324, 1.496 ± 0.356, 1.132 ± 0.280 vs. 2.315 ± 0.440, P<0.05 or P<0.01 ). Treatment of septic shock patient's serum or TNF-a to EA. hy926 produced an obvious increase in its permeability (septic serum group A value: 1.204 ± 0.248 vs. 0.277 ± 0.017, P<0.01; TNF-a group A value: 2.485 ± 0.113 vs. 1.602 ± 0.679, P<0.05). High-permeability induced by TNF-a or septic shock patient's serum was alleviated hy CHR in the concentration of 1, 10, 100 nmol/L in a dose-dependent manner (septic serum + CHR group A value: 0.299 ± 0.065, 0.224 ± 0.028, 0.131 ± 0.015 vs. 1.204 ± 0.248; TNF -a + CHR group A value: 1.995 ± 0.394, 1.920 ± 0.096, 1.744 ± 0.475 vs. 2.485 ± 0.113, P<0.05 or P<0.01 ). Under a laser scanning confocal microscope, it was found that the F-actin cytoskeleton of EA.hy926 cells was redistributed, and more stress fibers were found in the septic shock patient's serum group and TNF-α group, while CHR obviously alleviated the above effects induced by septic shock patient's serum or TNF-α. CONCLUSION: In a dose-dependent manner, CHR may inhibit increased permeability of vascular endothelial cells induced by septic shock patient's serum, its underlying mechanism may be related to inhibition of the effect of TNF-α.