Decreased expression of elastin and lysyl oxidase family genes in urogenital tissues of aging mice.

AIM: This study aims to investigate the expression levels of elastin and lysyl oxidase (LOX) family members in the urogenital tissues of natural aging mice and accelerated ovarian aging mice. METHOD: Uteri, vaginas and bladders were harvested from 18-month-old female mice and accelerated ovarian aging mice developed by chemotherapeutic agents. Untreated 3-month-old female mice were used as controls. The expression levels of elastin and LOX family members were determined by real-time polymerase chain reaction and western blot. RESULTS: Compared with untreated young female mice, the expression of elastin and LOX family members significantly decreased both in natural aging mice and accelerated ovarian aging mice. CONCLUSION: Aging is a high-risk factor for pelvic floor disorders. The failure of elastic fiber synthesis and assembly due to the decline in expression levels of elastin and LOX family members during aging may explain the molecular mechanism causing pelvic floor disorders.

A Potential Role of NFIL3 in Atherosclerosis.

Nuclear factor interleukin-3 (NFIL3), a proline- and acidic-residue-rich (PAR) bZIP transcription factor, is called the E4 binding protein 4 (E4BP4) as well, which is relevant to regulate the circadian rhythms and the viability of cells. More and more evidence has shown that NFIL3 is associated with different cardiovascular diseases. In recent years, it has been found that NFIL3 has significant functions in the progression of atherosclerosis (AS) via the regulation of inflammatory response, macrophage polarization, some immune cells and lipid metabolism. In this overview, we sum up the function of NFIL3 during the development of AS and offer meaningful views how to treat cardiovascular disease related to AS.

Alternative splicing of VEGFA is regulated by RBM10 in endometrial cancer.

Vascular endothelial growth factor A (VEGFA) gene has three alternative exons which results in multiple isoforms. VEGFA has been found overexpressed in patients with endometrial cancer, but the VEGFA expression pattern and how it is regulated are still unknown. The level of VEGFA transcripts and protein isoforms were detected by semi-quantitative Polymerase chain reaction (PCR) and immunoblotting in 29 paired endometrial tumor and adjacent nontumor control tissues. The level of three alternative splicing related proteins: RBM5, RBM6, and RBM10 was determined by immunoblotting. The H3K27Ac level in RBM10 promoter region was detected by ChIP-PCR. The RBM10 promoter region methylation level were quantified by methylation-sensitive high resolution melting. VEGFA165a was overexpressed and VEGFA165b level was reduced in tumors. RBM10 level was reduced in tumors. RBM10 level was negatively correlated with VEGFA165a level and positively correlated with VEGFA165b level in tumors. Using HEC-1-A and RL95-2 cells, we confirmed that VEGFA165a/b expressed pattern was controlled by RBM10. MALAT1 level was increased in tumors but not involved in VEGFA alternative splicing. Reduced H3K27Ac level and increased DNA methylation in the promoter region controlled RBM10 expression in tumors. VEGFA alternative splicing in endometrial cancer was regulated by RBM10, the expression of which was controlled by histone acetylation and DNA methylation.

miR-335 modulates Numb alternative splicing via targeting RBM10 in endometrial cancer.

Numb is a conserved protein plays important roles in the development of cancer. Two Numb isoforms have been found produced by alternative splicing and play contrast roles in regulating cellular functions. It is reported that the expression of Numb long isoform (Numb-L) was increased in various kinds of cancers, but in endometrial cancer, the condition is still unknown. The level of two Numb transcripts and protein isoforms were detected by semiquantitative polymerase chain reaction and immunoblotting in 47 paired endometrial tumor and adjacent non-tumor control tissues. The level of three alternative splicing related proteins: RBM5, RBM6, and RBM10 was determined by immunoblotting. MiRNAs targeting RBM10 were predicted by bioinformatics tools and their interaction with RBM10 was confirmed by luciferase assay and immunoblotting. The function of miR-335 in endometrial cancer was examined in xenograft mouse model. Numb-L level was increased in tumors and negatively correlated with RBM10 protein level. RBM10 mRNA level was not significantly altered in endometrial tumors suggesting its expression may regulated by post transcriptional regulators such as miRNAs. We identified miR-133a, miR-133b, and miR-335 directly target RBM10, but only miR-335 level increased in tumors and negatively correlated with RBM10 protein level. miR-335 overexpression promoted tumor growth by downregulating RBM10 and upregulating Numb-L level in xenograft mouse model. miR-335 overexpression promoted Numb-L expression via targeting RBM10 in endometrial cancer, which may provide new biomarkers for EC diagnosis.

Clinical implications of increased lymph vessel density in the lymphatic metastasis of early-stage invasive cervical carcinoma: a clinical immunohistochemical method study.

BACKGROUND: Cervical cancer is the most common malignant gynecological cancer, and lymphatic metastasis can occur in the early stage of tumor growth. Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), a marker for lymphatic endothelium, provides powerful tools for studying tumor lymphangiogenesis. The purpose of this study is to investigate the clinical implications of lymphangiogenesis in the metastasis of early-stage invasive cervical carcinoma. METHODS: We used immunohistochemical (IHC) staining with the antibody against LYVE-1 to measure lymph vessel density in 41 cases of early-stage invasive cervical carcinoma and 12 cases of normal cervical samples. We then analyzed the correlation between lymph vessel density and clinicopathological features of the tumors. RESULTS: (1) The majority of peritumoral lymphatics were enlarged, dilated, and irregular. In contrast, intratumoral lymph vessels were small and collapsed. The peritumoral lymphatic vessel density (PLVD) was significantly higher than the intratumoral lymphatic vessel density (ILVD) (P < 0.01). (2) Both ILVD and PLVD were significantly higher than the LVD of the control cervixes (P < 0.01). (3) Both ILVD and PLVD were significantly associated with lymph node metastasis (ILVD, P < 0.05; PLVD, P < 0.01) and lymphatic vessel invasion (ILVD, P < 0.05; PLVD, P < 0.01). Both the ILVD and PLVD in patients with histological grade HG2 and HG3 were significantly higher than those with HG1 (P < 0.05). CONCLUSION: Tumor lymphangiogenesis in early-stage invasive cervical carcinoma may play an important role in the process of lymphatic metastasis.

[Expression of elastin, lysyl oxidase and elafin in the cardinal ligament of women with pelvic organ prolapse].

OBJECTIVE: To investigate expression of elastin, lysyl oxidase (LOX) and elafin in cardinal ligament of women with pelvic organ prolapse (POP) so as to determine their contributions to POP. METHODS: The cardinal ligament samples were obtained from 60 POP subjects and 60 non-POP control women undergoing hysterectomy. RT-PCR was used to verify the mRNA level of elastin, LOX and elafin. The protein concentration of the three genes was determined by western blotting technique, electrophoretic separation and quantification. RESULTS: The premenopausal and postmenopausal POP groups demonstrated significantly decreased expressions of elastin in cardinal ligament both in mRNA and protein levels than control group (mRNA 0.42 +/- 0.22, 0.26 +/- 0.20 versus 0.79 +/- 0.30, 0.63 +/- 0.23; protein 0.44 +/- 0.32, 0.20 +/- 0.19 versus 0.89 +/- 0.27, 0.78 +/- 0.25; P < 0.05). There was an identical tendency in the expression of LOX (mRNA 0.37 +/- 0.18, 0.20 +/- 0.14 versus 0.65 +/- 0.22, 0.53 +/- 0.20; protein 0.45 +/- 0.27, 0.26 +/- 0.21 versus 0.85 +/- 0.39, 0.69 +/- 0.31; P < 0.05). In POP group, the mRNA and protein levels of elastin and LOX in postmenopausal patients were significantly lower than premenopausal patients (P < 0.05). Inversely, POP group demonstrated an increased expression of elafin in cardinal ligament both in mRNA and protein levels than corresponding control group (mRNA 1.33 +/- 0.35, 1.47 +/- 0.37 versus 0.62 +/- 0.25, 0.55 +/- 0.24; protein 0.85 +/- 0.30, 0.76 +/- 0.35 versus 0.21 +/- 0.15, 0.29 +/- 0.22; P < 0.05). There was no significant difference in the expression of elafin between premenopausal and postmenopausal POP groups either in mRNA or protein levels (P > 0.05). There was a positive correlation between elastin and LOX both in mRNA and protein levels in POP group(r = 0.9959, 0.9708; P < 0.05), but there was no correlation between elastin and elafin (r = -0.0402, -0.0365; P > 0.05). CONCLUSIONS: The results suggest that the decreased expression of elastin and LOX and the increased expression of elafin in the cardinal ligaments may contribute to POP.

[Study of reduction rat T-lymphocytes activation and proliferation by inhibition inducible co-stimulator gene expression].

OBJECTIVE: To observe the change of activation and proliferation ability of rat T-lymphocytes after suppress ICOS gene expression by RNA interference. METHODS: Four interference sites targeting at rat ICOS gene were designed and four pairs of oligonucleotide fragments were cloned into the pSilencer 4.1-CMV neo plasmid vectors then transfected into rat lymphocytes with cationic liposome. The expression of mRNA and protein of ICOS was detected by RT-PCR and flow cytometry. The alteration of lymphocyte proliferation ability was evaluated by mix lymphocyte reaction, and the secretion levels of IFN-gamma and IL-4 were measured by ELISA procedure. RESULTS: After transfection, the expression of mRNA and protein of ICOS in test groups were lower than that in control groups (P < 0.05). The ability of T-lymphocytes in proliferation was poor and the levels of IFN-gamma and IL-4 were reduced with ICOS gene shut down. CONCLUSIONS: RNA interference plasmid vector can suppress ICOS expression in rat T-lymphocytes significantly, and it may be useful for further study on transplantation immunity tolerance.

Peripheral platelet/lymphocyte ratio predicts lymph node metastasis and acts as a superior prognostic factor for cervical cancer when combined with neutrophil: Lymphocyte.

Inflammation-based indicators such as neutrophil/lymphocyte ratio (NLR), derived NLR (dNLR), and platelet/lymphocyte ratio (PLR) have been reported to possess significant predictive value for several types of cancer. We investigated the predictive value of these 3 biomarkers on lymph node metastasis (LNM) and clinical outcome in patients with stage Ib1-IIa cervical cancer undergoing radical surgery.A total of 407 patients with FIGO stage Ib1-IIa cervical cancer, who underwent radical surgery between January 2006 and December 2009 at the Department of Gynecological and Oncology of Shandong Cancer Hospital Affiliated to Shandong University were recruited. Binary logistic regression analysis was performed to determine the relationship between PLR, NLR, dNLR, and LNM. Multivariate Cox regression analysis was performed to determine the association between the 3 indices and recurrence-free survival (RFS) and overall survival (OS).Optimal cut-off values for the 3 indices were determined by applying receiver operating curve (ROC) analysis. Univariate and binary logistic regression analyses both indicate that PLR was significantly associated with increased LNM (P < 0.05). In the multivariate survival analysis, increased preoperative PLR and NLR were significantly associated with reduced RFS (P = 0.001 and P = 0.002, respectively), whereas a combination of both PLR and NLR revealed a more significant association with reduced RFS (P < 0.001). Furthermore, increased preoperative PLR and NLR were significantly associated with reduced OS (P = 0.007 and P = 0.009, respectively), whereas the combined use of PLR and NLR revealed a more significant association with reduced OS (P = 0.003).PLR is an independent risk factor for increased LNM and clinical outcome in patients with stage Ib1-IIa cervical cancer. A combination of PLR and NLR may enable better risk stratification for predicting survival.

MicroRNA-106a regulates phosphatase and tensin homologue expression and promotes the proliferation and invasion of ovarian cancer cells.

Ovarian cancer is a leading cause of malignant gynecological tumor-related mortality among women. The treatment of ovarian cancer patients continues to be challenging. MicroRNA­106a (miR­106a) is widely expressed in diverse human tumors. In the present study, we investigated the biological and pathological roles of miR-106a in ovarian cancers. We found that miR-106a expression was significantly increased in primary ovarian cancer tissues and ovarian cancer cells compared with the level in normal tissues. Ectopic expression of an miR-106a inhibitor attenuated ovarian cancer cell proliferation and invasion. miR-106a promoted the growth and invasion of SKOV3 cells by targeting phosphatase and tensin homolog (PTEN). Furthermore, the present study revealed that IL-6 inhibited miR-106a expression by activating STAT3. Tocilizumab, a humanized anti-human IL-6R antibody, that competitively inhibits IL-6/IL-6R signaling, did not inhibit the proliferation and invasion of SKOV3 cells. In conclusion, our studies revealed that miR-106a was significantly increased in the ovarian cancer tissues and cell lines. Downregulation of the expression of miR-106a inhibited cell growth and metastasis of ovarian cancer cells. Together, the present study suggests that miR­106a acts as an oncogene in ovarian cancers.

Decreased pretreatment lymphocyte/monocyte ratio is associated with poor prognosis in stage Ib1-IIa cervical cancer patients who undergo radical surgery.

BACKGROUND: Recently, pretreatment monocyte counts and the lymphocyte/monocyte ratio (LMR) have been proven to be significantly associated with the clinical outcomes of several types of cancer. In this study, we analyzed the prognostic significance of the LMR in stage Ib1-IIa cervical cancer patients who underwent a radical operation. METHODS: A total of 485 patients with stage Ib1-IIa cervical cancer were included in this retrospective study. We evaluated the prognostic values of the absolute lymphocyte count, absolute monocyte count, and LMR by applying receiver operating characteristic curves. Kaplan-Meier curves and multivariate Cox proportional analyses were used to determine the recurrence-free survival (RFS) and overall survival (OS). RESULTS: The area under the curve was 0.640 for the RFS and 0.647 for the OS using the LMR. In the univariate analysis, an elevated preoperative LMR was significantly associated with an increased RFS (hazard ratio [HR], 0.373; 95% confidence interval [CI]: 0.247-0.563; P<0.001), and this result remained significant in the multivariate analysis (HR, 0.439; 95% CI: 0.279-0.693; P<0.001). In the univariate analysis, an elevated LMR was also significantly associated with an increased OS (HR, 0.381; 95% CI: 0.233-0.622; P<0.001), and the significance persisted in the multivariate analysis (HR, 0.417; 95% CI: 0.244-0.714; P=0.001). CONCLUSION: A decreased pretreatment LMR is associated with a poor prognosis in stage Ib1-IIa cervical cancer patients who undergo a radical operation. A prospective study is warranted for further validation of our findings.

[Analysis on the relationship between p57 and cholesterol in cellular membrane].

AIM: To investigate the relationship between p57 and cholesterol in cellular membrane under physiological conditions. METHODS: Different concentrations of mbetaCD were used to extract cholesterol from cellular membrane of U937 cells. The cellular membrane was prepared by ultracentrifugation and the content change of p57 in cellular membrane component was measured by Western blot analysis. Discontinuous sucrose density gradient centrifugation was used to prepare lipid rafts and the content of p57 in them was measured by Western blot analysis. RESULTS: The degradation of cholesterol level in cellular membrane didn't lead to obvious content change of p57 in cellular membrane component. Furthermore, no p57 protein was found in lipid rafts. CONCLUSION: No relationship between p57 and cholesterol in cellular membrane was found in the physiological condition, which is obviously different from that in pathological condition caused by the invasion of pathogenic mycobacteria.