Exosomes from uterine fluid promote capacitation of human sperm.

Background: Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. Methods: EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). Results: The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30-200 nm) by NTA. CD9 and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Conclusion: Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus.

Th17 cells promote tumor growth in an immunocompetent orthotopic mouse model of prostate cancer.

Interleukin-17 (IL-17) has been demonstrated to promote development of a variety of cancers including prostate cancer in genetically modified mouse models. IL-17 is the main product secreted by T helper 17 (Th17) cells. A recent study has shown that Th17 cells and related genes are upregulated in human prostate cancers. However, there is no direct experimental evidence to demonstrate Th17's role in prostate cancer. In the present study, we co-implanted mouse prostate cancer MPC3-luc cells with Th17-polarized mouse splenocytes in the prostate of immunocompetent C57BL/6J male mice. We found that Th17-polarized splenocytes promoted orthotopic allograft prostate tumor growth compared to the control splenocytes. The numbers of IL-17-positive lymphocytes and macrophages were higher in the prostate tumors grown from co-implantation of MPC3-luc cells and Th17-polarized splenocytes, compared to the prostate tumors grown from co-implantation of MPC3-luc cells and control splenocytes. Our findings provide the first direct experimental evidence that Th17 cells may promote prostate cancer growth.

MiR-361-5p decreases the tumorigenicity of epithelial ovarian cancer cells by targeting at RPL22L1 and c-Met signaling.

A large number of studies have shown that miRNAs are important regulators of epithelial-to-mesenchymal transition (EMT) and are associated with metastasis in epithelial ovarian cancer (EOC). MiR-361-5p has been shown to play pivotal roles in tumorigenesis and metastasis; however, a role for miR-361-5p in EOC has not been reported. In this study, we found that miR-361-5p was significantly down-regulated in EOC tissues and cell lines. In addition, over-expression of miR-361-5p inhibited the migration and invasion of EOC cells in vitro. MiR-361-5p influenced the expression of the EMT-associated proteins by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal markers, N-cadherin and vimentin. Further studies identify miR-361-5p directly targeted Ribosomal L22-like1 (RPL22L1) and c-Met. Moreover, miR-361-5p repressed the Akt/mTOR pathway after c-Met inhibition. Reintroduction of RPL22L1 and c-Met reversed miR-361-5p-induced EMT suppression. Consistently, inverse correlations were also observed between the expression of miR-361-5p and RPL22L1 or c-Met in human EOC tissue samples. Taken together, miR-361-5p inhibited the EMT progression in EOC cells by targeting RPL22L1 and c-Met/Akt/mTOR signaling.

[Effect and mechanism of an autophagy gene beclin 1 on cervical cancer HeLa cells].

OBJECTIVE: To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. METHODS: The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3.1(+)-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1(+) group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. RESULTS: (1) The mRNA expression of beclin 1 and caspase-9: pcDNA3.1(+)-beclin 1 group were 994.72 ± 468.76 and 12.88 ± 2.71, pSUPER-beclin 1 group were 0.18 ± 0.63 and 0.11 ± 0.08, pcDNA3.1(+) group were 0.57 ± 0.12 and 4.28 ± 3.25, pSUPER group were 0.67 ± 0.29 and 2.77 ± 1.27, and HeLa group were 0.74 ± 0.25 and 3.67 ± 3.78, respectively. The eukaryotic expression vector pcDNA3.1(+)-beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells (P < 0.05), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9(P < 0.05). (2) The cell proliferations: pcDNA3.1(+)-beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells (P < 0.05). (3) The rate of apoptosis: pcDNA3.1(+)-beclin 1 group was (28.2 ± 2.3)%, pcDNA3.1(+) group was (14.6 ± 4.6)%, pSUPER-beclin 1 group was (5.7 ± 2.0)%, pSUPER group wa (16.2 ± 3.1)%, and HeLa group was (11.2 ± 3.0)%. The pcDNA3.1(+)-beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate (P < 0.05). (4) The activity of autophagy: more autophagy cells were identified in pcDNA3.1(+)-beclin 1 group; the rate of autophagy of five group were (10.3 ± 1.5)% in pcDNA3.1(+)-beclin 1 group, (3.6 ± 0.8)% in pcDNA3.1(+) group, (1.2 ± 0.3)% in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1.1)% in HeLa group, there was statistical significances between test groups and control groups (P < 0.05). (5) Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1(+)-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups (P < 0.05). The tumor size was smaller from 21st day after injection in pcDNA3.1(+)-beclin 1 group than in control groups (P < 0.05). From 28th day after injection, the tumor weigh was (0.52 ± 0.08) g in pSUPER-beclin 1 group, apparently more than HeLa group (0.37 ± 0.12) g and pSUPER group (0.34 ± 0.24) g (P < 0.05). While in pcDNA3.1(+)-beclin 1 group the tumor weighed (0.18 ± 0.12)g, which was lower than HeLa group and pcDNA3.1(+) group(0.34 ± 0.18) g (P < 0.05). CONCLUSIONS: Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.

[Effect of autophagy gene Beclin 1 on the growth of cervical cancer HeLa cells in vitro and vivo].

OBJECTIVE: To investigate the effects of autophagy gene Beclin 1 on growth of cervical cancer HeLa cells in vitro and vivo. METHODS: The eukaryotic expression vector of Beclin1 was constructed and transfected via lipofectamine into HeLa cells. The experimental cells were classified into 3 groups: pcDNA3.1(+)-Beclin1 group,pcDNA3.1(+) group and HeLa group. Real time-ploymerase chain reaction and Western blot were used for detecting expression of Beclin1 mRNA and protein in the transfected cells. Flow cytometry (FCM) was employed to observe the effect of transfection on the apoptosis of HeLa cells, and proliferation was analyzed by MTT assay. The formation of autophagic vacuoles was measured by MDC staining. HeLa cells transfected with plasmid pcDNA3.1(+)-Beclin1 and pcDNA3.1(+) were inoculated subcutaneously in nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed. RESULTS: Eukaryotic expression vector pcDNA3.1(+)-Beclin1 was constructed successfully. It significantly improved the expression of Beclin1 mRNA and protein in HeLa cells. The proliferation of HeLa cells was inhibited, and the inhibition rate was 58.7%. FCM investigation showed that the apoptotic rate was (28.22 ± 2.34)% of pcDNA3.1(+)-Beclin1 group, significantly higher than the (14.6 ± 4.6)% in the pcDNA3.1(+) group and (11.2 ± 3.0)% in the HeLa group (P < 0.05). The monodansylcadaverin (MDC) staining showed significantly more autophagic vacuoles in the pcDNA3.1(+)-Beclin1 group (10.9%) than that in the pcDNA3.1(+) group (3.1%) and HeLa group (2.5%) (P < 0.05). After transfected with vector pcDNA3.1(+)-Beclin1, the carcinogenic activity of HeLa cells was decreased in nude mice, and the inhibition rate of tumor growth was 52.2%. CONCLUSIONS: Autophagy gene Beclin 1 overexpression can inhibit the proliferation and growth of HeLa cells in vitro and vivo,while promote autophagy and apoptosis of HeLa cells. So it might be one of new gene therapy strategies for cervical carcinoma.

[Growth inhibitory effects of Beclin1 gene on epithelial ovarian cancer SKOV3 cells].

OBJECTIVE: To explore the effect of Beclin1 overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro and in vivo. METHODS: The recombinant plasmid pcDNA3.1/Beclin1 was constructed and transfected into SKOV3 cells via lipofectamine 2000. MTT assay was used to evaluate the effect of Beclin1 overexpression on the proliferation and growth of the transfected cells, whose apoptosis and autophagy were analyzed by flow cytometry. SKOV3 cells transfected with the plasmids pcDNA3.1/Beclin1 or pcDNA3.1 were inoculated subcutaneously in nude mice, and their carcinogenic and growth activities in vivo were evaluated. RESULTS: MTT assay showed that transfection with pcDNA3.1/Beclin1 significantly inhibited the proliferations of SKOV3 cells, with a cell inhibition rate of 58.68% (P<0.05). The transfection also resulted in a cell apoptosis rate of (21.26-/+3.89)%, significantly higher than that of pcDNA3.1 trasnfection (P<0.05). Flow cytomerty showed that pcDNA3.1/Beclin1 transfection of SKOV3 cells produced a significantly higher MDC fluorescent intensity than pcDNA3.1 transfection. The SKOV3 cells transfected with vector pcDNA3.1/Beclin1 also showed decreased carcinogenic activity in nude mice, with a growth inhibition rate of 50.27%. CONCLUSION: Beclin1 overexpression can inhibit the proliferation and growth of SKOV3 cells in vitro and vivo, suggesting its potential role in gene therapy of ovarian carcinoma.

[Expression of THY1 gene in epithelial ovarian cancer].

OBJECTIVE: To detect the expession of THY1 in ovarian serous cystadenocarcinoma tissues. METHODS: Immunohistochemistry was performed to detect the expression of THY1 gene in formalin-fixed, paraffin-embedded specimens of normal ovaries (n = 25), ovarian serous cystadenoma (n = 25), and serous cystadenocarcinoma (n = 53). The correlation of THY1 expression with clinicopathological parameters was statistically analyzed. RESULTS: The positive expression rates of THY1 protein in normal ovaries, ovarian serous cystadenomas and ovarian serous cystadenocarcinomas were 60.0% (15/25), 72.0% (18/25) and 34.0% (18/53), respectively. The values of IOD of THY1 protein expression were 288,449.2 +/- 60,087.3, 271,655.6 +/- 66,588.7 and 252,087.6 +/- 45,559.4, respectively. The expression of THY1 protein was significantly down-regulated in ovarian serous cystadenocarcinoma tissues compared with that in normal ovarian tissues and ovarian serous cystadenoma tissues (P < 0.05). THY1 expression was negatively correlated with surgical-pathological staging, histological differentiation and lymph node involvement (P < 0.05). CONCLUSION: The decreased level of THY1 expression may be related with the occurrence and development of ovarian serous cystadenocarcinoma.

Beclin 1-mediated macroautophagy involves regulation of caspase-9 expression in cervical cancer HeLa cells.

OBJECTIVE: To investigate the role of Beclin 1 in HeLa cells and to obtain further insight into the relationship between autophagy and apoptosis. METHODS: Beclin 1 silencing was achieved using RNA interference. The expression of gene was measured using quantitative real time RT-PCR and Western blotting. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. The ultrastructural analysis was under the electron microscope. RESULTS: In pSUPER-Bec transfectants (Beclin 1 gene partially silenced) the expression of mRNA and protein of Beclin 1 were significantly suppressed in comparison to pSUPER-non (scramble RNA control) or untreated cells in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in pSUPER-Bec transfectants compared with pSUPER-non transfectants. Meanwhile pcDNA3.1-Bec transfectants (Beclin 1 gene overexpressed) showed reduction of cell proliferation but augmentation of cell programmed death compared with vector vehicle. The autophagy-promoting activity of beclin 1 in HeLa cells is associated with inhibition of HeLa cellular proliferation, in vivo tumorigenesis in nude mice. The expression pattern of caspase-9 was extraordinarily similar to that of Beclin 1in siRNA against Beclin 1 transfectants and constructive expression of Beclin 1transfectants. CONCLUSION: siRNA against Beclin 1 transfectants promoted the cell proliferation but overexpression of Beclin 1 promoted the autophagy cell death, and in the process of autophagy triggered by Beclin 1 expression followed accordingly the regulation of the expression of caspase-9. We conjecture that the autophagy gene Beclin 1 may be the critical molecular switch that plays an important role in fine tuning the autophagy and apoptosis through caspase-9, and defection of autophagy or apoptosis may be an important mechanism in tumorigenesis.

[Construction of shRNA expression vectors for autophagy gene beclin 1 and their downregulation effect on caspase-9].

The shRNA expression vectors were constructed and transfected via lipofectamine into HeLa cells. Real time-ploymerase chain reaction (RT-PCR) and Western blot were used for detecting the expression of mRNA and protein of Beclin1 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis and cell cycle of HeLa, and proliferation was analyzed by MTT assay. The expression of caspase-9 in transfection cells was also detected by RT-PCR and Western blot. The constructed vectors significantly inhibited the expressin of mRNA and the protein of Beclin1 in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in these cells. After transfection of the constructed vectors into HeLa cells, the expression of caspase-9 was effectively inhibited. All of these indicate that autophagy and apoptosis are two types of programmed cell death, that autophagy gene Beclin 1 plays an important role in these two types, and that defect of autophagy and apoptosis may be important in tumor genesis.

[Construction of shRNA expression vectors for autophagy gene Beclin 1 and effect of the vectors on transfected HeLa cells in vitro].

OBJECTIVE: Autophagy gene Beclin 1 plays an important role in several types of human cancer. In this study, RNA interference (RNAi) technique was employed to determine the effect of inhibiting Beclin 1 on the growth of tumor cells. METHODS: According to the encoding sequence of mRNA of Beclin 1, the target site for the RNAi technique was designed and the vector for shRNA (short hairpin RNA) expression in tumor cells was constructed. The HeLa cell line was transfected with the sfRNA to inhibit the expression of Beclin 1. RESULTS: The constructed vector significantly inhibited the expression of the mRNA and protein of Beclin 1 in the HeLa cells. The growth of the transfected cells was promoted, and less apoptosis cells were identified in these cells. CONCLUSIONS: The shRNA expression vector can effectively inhibit the expression of Beclin 1 in the HeLa cells, and promote the growth of HeLa cells.

[Expression and involved signal transduction pathway of autophagy gene Beclin 1 in epithelial ovarian cancer].

OBJECTIVE: To investigate the relationship between the autophagy gene Beclin 1 involving the PI3K/PKB signaling pathway and the occurrence, development of epithelial ovarian carcinoma. METHODS: The expression of Beclin 1, Class I PI3K (p110alpha), Class III PI3K (hvps34) or p-PKB was, by immunohistochemistry, detected respectively in 25 normal ovarian tissues, 25 benign neoplasia tissues, 19 borderline tissues, and 69 epithelial ovarian carcinoma tissues. RESULTS: The higher expressions of Beclin 1 and hvps34 were found in normal and benign ovarian neoplasia tissues; the expressions were reduced in the borderline lesion tissue; and the lowest level of expressions could be detected in the ovarian carcinoma tissue (P < 0.05). The expressions of p110alpha and p-PKB increased slightly in borderline ovarian tissue, and were higher in the epithelial ovarian carcinoma tissue than other three groups (P < 0.05). Beclin 1 expressions in the epithelial ovarian cancer tissues with stage I - II, high-middle grade differentiation and negative lymph node metastasis were higher than those with stage III - IV, low grade differentiation and positive lymph node metastasis, but the expressions of p110alpha and p-PKB in were lower (P < 0.05). CONCLUSION: Beclin 1 expression is down-regulated in epithelial ovarian cancer tissue while the p110alpha, hvps34 and p-PKB are having the abnormal expressions on PI3K/PKB signaling pathway, which may be correlated with the occurrence and development of epithelial ovarian carcinoma.

[Correlation of autophagy gene Beclin1 to tumorigenesis and development of epithelial ovarian cancer].

BACKGROUND & OBJECTIVE: Some studies have showed that autophagy suppression may result in malignant transformation, and the inactivation of autophagy gene Beclin1 induces malignancy. This study was to investigate the role of Beclin1 in the tumorigenesis and development of epithelial ovarian carcinoma, and to explore the effect of Beclin1 overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro. METHODS: The expression of Beclin1 in 25 specimens of normal ovarian tissue, 25 specimens of benign ovarian neoplasia, 19 specimens of borderline ovarian tissue, and 69 specimens of epithelial ovarian carcinoma was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3.1/Beclin1 was constructed and transfected into SKOV3 cellsû plasmid pcDNA3.1 was used as control. The effect of Beclin1 overexpression on the proliferation of SKOV3 cells was evaluated by MTT assay. Cell apoptosis was measured by flow cytometry (FCM). RESULTS: The expression of Beclin1 was high in normal and benign ovarian neoplasia tissues, and there was no significant difference between the 2 groups (P>0.05). Reduced Beclin1 expression was observed in borderline lesions, and the lowest level was detected in ovarian carcinoma tissues (P<0.05). The inhibition rate was significantly higher in pcDNA3.1/Beclin1-SKOV3 cells than in pcDNA3.1-SKOV3 cells [(68.75+/-5.10)% vs. (10.91+/-4.20)%, P<0.05]. At 72 h after transfection, the apoptosis rate was significantly higher in pcDNA3.1/Beclin1-SKOV3 cells than in pcDNA3.1-SKOV3 cells and SKOV3 cells [(19.07+/-0.65)% vs. (4.30+/-0.50)% and (3.87+/-0.84)%, P<0.05]. CONCLUSIONS: Beclin1 expression is down-regulated in epithelial ovarian cancer tissues, which may relate to tumorigenesis and development of epithelial ovarian cancer. Beclin1 overexpression can inhibit proliferation and induce apoptosis of SKOV3 cells.

[Expression and clinical significance of autophagy gene Beclin 1 in cervical squamous cell carcinoma].

OBJECTIVE: Autophagy gene Beclin 1 plays an important role in several types of human cancers. So we, in this study, employed the immunohistochemical manner to detect the Beclin 1 gene expression and explore its clinical significance in cervical aquamous cell carcinoma. METHODS: SP immunohistochemistry technique was used to detect the expression of Beclin 1 gene in specimens of 81 cervical squamous cell carcinoma, 20 cervical intraepithelial neoplasm (CIN, II - III), and 20 normal cervix. Correlations between the expressions of Beclin 1 gene and the clinicopathologic factors of cervical squamous cell carcinoma were statistically analyzed. RESULTS: The rates of negative, weak, strong expression of Beclin 1 in cervical squamous cell carcinoma were 43.2% (35/81), 34.6% (28/81) or 22.2% (18/81) respectively, and the significantly lower than those in normal cervix and CIN II - III (P = 0.011). The Beclin 1 expressions did not associate with FIGO stage, age, depth of cervical infiltration, tumor size, and gross type of cervical lesion (P < 0.05), but were related to pelvic lymph node metastases and histological tumor grade (P < 0.05). CONCLUSION: s Expression of autophagy gene Beclin 1 decreases in cervical aquamous cell carcinoma, and is closely related to pelvic lymph node metastases and histological tumor grade.