Circ_0081723 enhances cervical cancer progression and modulates CREBRF via sponging miR-545-3p.

Circular RNAs (circRNAs) have been confirmed to be an important modulator and therapeutic target of cervical cancer (CC). The aim of this study is to explore the role and mechanism of circ_0081723 in CC progression. Circ_0081723, microRNA-545-3p (miR-545-3p), and CREB3 regulatory factor (CREBRF) levels were detected using quantitative real-time PCR (qRT-PCR) assay. CREBRF, ki-67, Bcl-2 related X protein (Bax), and E-cadherin expression levels were determined using western blot (WB) and immunohistochemistry (IHC) assays. Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8), cell colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Flow cytometry was used to measure cell apoptosis.  Cell migration and invasion were examined using Transwell assay. Interaction between miR-545-3p and circ_0081723 or CREBRF was verified using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assays. The biological role of circ_0081723 on CC growth was examined using the xenograft tumor model in vivo. Circ_0081723 and CREBRF were increased, and miR-545-3p was decreased in CC tissues and cells. Circ_0081723 silencing suppressed CC cell growth and motility whereas boosted CC cell apoptosis. Besides, circ_0081723 acted as a molecular sponge for miR-545-3p, and circ_0081723 knockdown-induced effects were largely reversed by miR-545-3p downregulation in CC cells. Moreover, miR-545-3p repressed CC progression by targeting CREBRF.  Circ_0081723 absence blocked xenograft tumor growth in vivo. Circ_0081723 stimulated CC cell malignant behaviors by regulating the miR-545-3p/CREBRF pathway, providing a possible circRNA-targeted therapy for CC.

Changes in the tight junctions of the testis during aging: Role of the p38 MAPK/MMP9 pathway and autophagy in Sertoli cells.

Impaired tight junction (TJ) function and autophagy and the activated p38 mitogen-activated protein kinase (MAPK)/matrix metalloproteinase 9 (MMP9) pathway in Sertoli cells cause spermatogenic disorders. However, it is unclear whether reduced TJ barrier function and autophagy and the activated p38 MAPK/MMP9 pathway in Sertoli cells are closely associated with age-related testicular dysfunction. Thus, we evaluated these changes in Sertoli cells using 6-, 12-, 18-, and 24-month-old Sprague-Dawley rats. The results showed that testicular morphology gradually degenerated, as evidenced by increased exfoliated germ cells, decreased seminiferous tubule diameter and seminiferous epithelium height, and reduced the numbers of spermatogonia, primary spermatocytes and spermatids during the process of aging. In addition, the TJs formed by adjacent Sertoli cells were progressively destroyed accompanied by an abnormal ultrastructure and decreased expression of the TJ proteins zonula occludens-1 (ZO-1), occludin, and claudin-11 with aging. Furthermore, the expression of phosphorylated p38MAPK and MMP-9 in Sertoli cells and testis gradually increased, and the expression of occludin co-localizated with MMP-9 progressively decreased. Meanwhile, autophagy levels also gradually decreased, including decreased autophagic vacuole formation and weak expression of light chain 3 (LC3) and autophagy-related 5 (Atg5) in Sertoli cells. Taken together, our results indicate that aging causes impaired TJ barrier function and degeneration of seminiferous tubules. The mechanism might be related to the activated p38MAPK/MMP9 pathway and inactivated autophagy in Sertoli cells.

Icariin Improves Age-Related Testicular Dysfunction by Alleviating Sertoli Cell Injury via Upregulation of the ERα/Nrf2-Signaling Pathway.

Sertoli cells play crucial roles in spermatogenesis and are impaired by aging. Icariin, a flavonoid from Epimedium, has been reported to exhibit anti-aging effects and improve testicular dysfunction in the clinical setting. However, whether icariin improves age-related degeneration of testicular function via protection from Sertoli cell injury remains unclear. In the present study, we evaluated the protective effect of icariin on Sertoli cell injury and explored the possible mechanism(s) in vivo and in vitro. Dietary administration of icariin for 4 months significantly ameliorated the age-related decline in testicular function by increasing testicular and epididymal weights and indices, sperm count and sperm viability, testicular testosterone and estradiol concentrations, and seminiferous tubule diameters and heights. In addition, icariin protected age-related Sertoli cells from injury as evidenced by an analysis of Sertoli cell number, ultrastructure, and function. Such changes were accompanied by upregulation of ERα and Nrf2 signaling in Sertoli cells. Parallel in vitro studies also demonstrated that icariin inhibited untoward effects on the TM4 mouse Sertoli cell line with concomitant upregulation of ERα and Nrf2 signaling. Conversely, ERα siRNA reversed icariin-mediated protection of Sertoli cell injury. Our data suggest that icariin effectively ameliorates age-related degeneration of testicular function by alleviating Sertoli cell injury via the ERα/Nrf2 signal-transduction pathway. Thus, mitigating Sertoli cell damage via the ERα/Nrf2 signaling pathway likely represents a promising strategy for the prevention of age-related testicular dysfunction.

[Protective effect of Wuzi Yanzong recipe on testicular germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress].

To study the protective effects of Wuzi Yanzong recipe on testis germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress (ERS), 16-month-old male SPF grade SD rats were randomly divided into three groups: ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1 and 4 g·kg⁻¹), with 10 rats in each group. In addition, 2-month-old SD male rats were used as adult control group. The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given the medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately collected. The change of testicular tissue morphology was observed by HE staining. The expression levels of ER stress-related proteins GRP78, p-PERK, p-eif2α, ATF4, p-IRE1, XBP1, ATF6 and apoptosis-related proteins CHOP, caspase12 and p-JNK in testes were detected by Western blot. Compared with the ageing model group, Wuzi Yanzong recipe alleviated the morphological changes of testicular tissue. Western blot results showed that Wuzi Yanzong recipe significantly increased the expression levels of endoplasmic reticulum stress-related proteins GRP78, p-PERK, p-eif2α, ATF4, p-IRE1, XBP1, ATF6 and significantly decreased the expression levels of endoplasmic reticulum-induced apoptosis-related proteins CHOP, caspase 12 and p-JNK. In conclusion, Wuzi Yanzong recipe can alleviate the ageing-related apoptosis of testicular germ cells in natural ageing rats by regulating endoplasmic reticulum stress.

[Protective effect of Wuzi Yanzong recipe on testicular DNA oxidative damage in natural ageing rats].

To study the protective effects of Wuzi Yanzong recipe on DNA oxidative damage of testis germ cells in natural ageing rats based on Nrf2/HO-1 signaling pathway and base excision repair (BER). In the study, 16-month-old SPF grade male SD rats were randomly divided into three groups, namely ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1, 4 g·kg⁻¹). In addition, 2-month-old SD rats were used as adult control group (10 rats in each group). The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately removed. The vitality of superoxide dismutase (SOD) and malondialdehyde (MDA) content in testis were detected by xanthine oxidase method and thiobarbituric acid (TBA) method. The levels of Nrf2 and 8-OHdG were detected by immunofluorescence. The protein expression levels of Nrf2, HO-1, NQO1, APE1, OGG1 and XRCC1 were detected by Western blot. Compared with the ageing model group, WZ significantly increased the SOD vitality and decreased MDA content of testis. In addition, immunofluorescence results showed that WZ significantly attenuated testicular DNA oxidative damage and improved antioxidant capacity. Such changes were accompanied by the down-regulation of DNA oxidative damage response protein 8-OHdG levels and the up-regulation of Nrf2 levels. Moreover, Western blot results showed that WZ significantly increased the protein expression levels of Nrf2, HO-1 and NQO1 of the testis germ cells, when compared with ageing model group. In parallel, the protein expression levels of APE1, OGG1 and XRCC1 were significantly decreased. In conclusion, WZ improves ageing-related DNA oxidative damage via Nrf2/HO-1 and BER pathways.

[Decline of secretory function of TM4 Sertoli cells stimulated by D-galactose in mice and its mechanism].

Objective To establish a model of decline in secretion of senescent TM4 cells in vitro induced by D-galactose (D-gal). Methods Different concentrations of D-Gal (25, 50, 100, 150, 200 and 250 mmol/L) were used to induce TM4 cell senescence. The viability of TM4 cells was detected by MTT assay. The cell cycle was analyzed by flow cytometry. The cell morphology was observed by light microscopy and the percentage of senescent cells was observed by senescence-associated ß-galactosidase (SA-ß-Gal) staining. The mRNA expression levels of P21, P16, glial-derived neurotrophic factor (GDNF) and stem cell factor (SCF) were detected by reverse transcription PCR. The protein expression levels of GDNF, SCF, nuclear factor erythroid 2 like 2 (NRF2), NAD(P)H dehydrogenase [quinone] 1 (NQO-1) and heme oxygenase-1 (HO-1) were detected by Western blot analysis. Results Compared with normal control group, D-Gal stimulation significantly decreased the cell viability in a concentration-dependent manner. The arrest of D-Gal-treated cells in G1 phase of cell cycle significantly increased, while it significantly decreased in S phase, and D-Gal induced cell cycle arrest at G0/G1 phase in TM4 cells. The percentages of SA-ß-Gal positive cells increased significantly. The expression levels of P21 and P16 mRNAs were significantly up-regulated. The mRNA and protein levels of GDNF and SCF-1 were significantly down-regulated. Furthermore, the expression levels of oxidative stress-related protein NRF2, HO-1 and NQO-1 were significantly reduced. Conclusion The model of declined secretion function of senescent TM4 cells induced by D-Gal we established is stable and reliable. Its mechanism may be related to the down-regulation of NRF2 signaling pathway.

[Involvement of matrix metalloproteinase-2, -9, and tissue inhibitors of metalloproteinase-1, 2 in occurrence of the accrete placenta].

OBJECTIVE: To investigate the roles of matrix metalloproteinase-9, -2 (MMP-9, 2), and tissue inhibitors of metalloproteinase-1, 2 (TIMP-1, 2) in pathogenesis of the accretio placenta. METHODS: The women with the placenta accrete were recruited and the placenta (23) and deciduas tissues (9) after labor were obtained, and the placenta (28) and deciduas (11) from women without the placenta accreta were obtained as control to get, too. The expressions of MMP-9, -2, TIMP-1, 2 in the placental and decidual tissues were analyzed by real-time PCR. RESULTS: mRNA expression of MMP-9 in the placenta accreta was (3.21 +/- 0.76) copies/microg total RNA, significantly higher (P < 0.05) than that of normal placenta [(3.84 +/- 0.24) copies/microg total RNA)]. MMP-9 transcription in the decidua accreta was (2.50 +/- 0.49) copies/microg total RNA, significantly higher (P < 0.05) than that of normal decidua [(3.81 +/- 0.66) copies/microg total RNA]. mRNA expression of TIMP-1 in normal placenta and placenta accreta was (5.91 +/- 0.56) and (5.92 +/- 0.46) copies/microg total RNA, respectively, with no significant difference between the two groups. mRNA expression of TIMP-1 in the accrete deciduas was (6.63 +/- 0.51) copies/microg total RNA, significantly lower (P < 0.05) than that of normal decidua (7.09 +/- 0.55) copies/microg. mRNA expression of MMP-2 in the accrete placenta was (4.55 +/- 1.13) copies/microg total RNA, significantly higher (P < 0.05) than that of normal placenta (5.53 +/- 0.59) copies/microg. mRNA expression of MMP-2 in the accrete decidua and normal decidua was (6.07 +/- 0.83) and (5.97 +/- 0.76) copies/microg total RNA, respectively, with no significant difference between the two groups. mRNA expression of TIMP-2 in the accrete placenta was (4.69 +/- 0.60) copies/microg total RNA, significantly higher (P < 0.05) than that of normal placenta (3.79 +/- 1.06) copies/microg. mRNA expression of TIMP-2 in the accrete decidua was (5.06 +/- 0.33) copies/microg total RNA, higher significantly (P < 0.05) than that of normal decidua (3.98 +/- 0.60) copies/microg. CONCLUSIONS: The upregulation of MMP-9, MMP-2 in placenta and downregulation of TIMP-1 in decidua were involved in occurrence of the placental accreta, and the roles of TIMP-2 in occurrence of the placental accreta need to elucidated.