Structural difference analysis of adult's intestinal flora basing on the 16S rDNA gene sequencing technology.

OBJECTIVE: Through 16S rDNA technology, we aimed at separating adults aging 20-50 years old into a few groups and processing the high-throughput sequencing analysis, in order to explore the features and differences of intestinal flora in each age group in a microcosmic perspective. PATIENTS AND METHODS: 120 stool specimens were collected strictly in accordance with acceptance criteria and exclusion criteria. 49 subjects aging 20-29 years old (Group AGE1), 51 subjects aging 30-39 years old (Group AGE2), and 20 subjects aging 40-49 years old (Group AGE3) were divided into 3 groups. Bacteria DNA from fresh stool specimens of 3 groups were abstracted. Illumina MiSeq high-throughput sequencing platform was applied to process 16S rDNA sequencing in Area 338F_806R for intestinal flora detection. I-Sanger Bio-cloud platform was applied for the analysis of intestinal flora structure changes in phylum level and genus level. RESULTS: Among the age of 20-50, with older age, the abundance of intestinal flora decreased among healthy adults more than 40 years old. In addition, the diversity and sample dispersion of intestinal flora is significantly different from people among 20-40 years old. The decrease ratio of Firmicutes/Bacteroidetes indicated that as the age grows, glucose tolerance might decrease. Comparing with people among 20-40 years old, the amount of Bifidobacterium and Eubacterium in people over 40 years old have significantly decreased. The decrease of Bifidobacterium and Eubacterium may increase the risks of cognitive impairment and lower the anti-inflammation and anti-cancer efficacy in human body, respectively. Subdoligranulum relates to poor metabolism and chronic inflammation and it happens more in people aged over 40 than young people who are among 20-40 years old. CONCLUSIONS: There are differences in the intestinal flora of healthy adults aged 20-50. Effective intervention of the intestinal flora may play a role in delaying aging and preventing diseases.

Effect of specific silencing of EMMPRIN on the growth and cell cycle distribution of MCF-7 breast cancer cells.

The extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin family and shows increased expression in tumor cells. We examined the effect of RNAi-mediated EMMPRIN gene silencing induced by lentiviral on the growth and cycle distribution of MCF-7 breast cancer cells. Lentiviral expressing EMMPRIN-short hairpin RNA were packaged to infect MCF-7 cells. The inhibition efficiency of EMMPRIN was validated by real-time fluorescent quantitation polymerase chain reaction and western blotting. The effect of EMMPRIN on cell proliferation ability was detected using the MTT assay and clone formation experiments. Changes in cell cycle were detected by flow cytometry. EMMPRIN-short hairpin RNA-packaged lentiviral significantly down-regulated EMMPRIN mRNA and protein expression, significantly inhibited cell proliferation and in vitro tumorigenicity, and induced cell cycle abnormalities. Cells in the G0/G1 and G2/M phases were increased, while cells in the S phase were decreased after infection of MCF-7 cells for 3 days. The EMMPRIN gene facilitates breast cancer cell malignant proliferation by regulating cell cycle distribution and may be a molecular target for breast cancer gene therapy.

Differential expression and signaling activation of insulin receptor isoforms A and B: A link between breast cancer and diabetes.

We showed that when insulin-like growth factor II (IGF-II) is highly expressed in breast tissues and cell lines, the IGF-I receptor signaling pathway is highly activated. Since IGF-II activates the insulin receptor (INSR), we propose that the INSR signaling is also activated in this system. We examined the expression of both INSR isoforms, insulin receptor A (INSR-A) and insulin receptor B (INSR-B), and the downstream signaling pathways in breast cancer (BC) cells and in paired (normal/tumor) breast tissues from 100 patients. Analysis was performed by real-time PCR, Western blot, immunohistochemistry, and phospho-ELISA techniques. Tumor tissues and cell lines from African-American patients expressed higher levels of INSR-A, but lower levels of INSR-B. Accordingly, insulin receptor substrate 1 and focal adhesion kinase activation were significantly increased in these women. We conclude that higher INSR-A and lower INSR-B contribute to higher proliferation and lower metabolic response. Thus, differential expression of INSR isoforms represents a potential biological link between BC and diabetes.

Insulin-like growth factors I and II receptors in the breast cancer survival disparity among African-American women.

OBJECTIVE: African-American (AA) women with breast cancer are more likely to have advanced disease at diagnosis, higher risk of recurrence and poorer prognosis than Caucasian (CA) women. We have recently shown higher insulin-like growth factor II (IGF-II) expression in paired breast tissue samples from AA women as compared to CA women. IGF-II is a potent mitogen that induces cell proliferation and survival signals through activation of the IGF-I and Insulin receptors (IGF-IR, IR) while IGF-II circulating levels are regulated by cellular uptake through the IGF2 receptor. We hypothesize that differential expression of the IGF1R and IGF2R among AA and CA women potentiates IGF-II mitogenic effects, thus contributing to the health disparity observed between these ethnic groups. DESIGN: We examined IGF-IR and IGF2R mRNA, protein expression and IGF1R phosphorylation in paired breast tissue samples from AA and CA women by Real Time-PCR, Western blot analysis, immunohistochemistry and ELISA techniques. RESULTS: Our results showed significantly increased expression of IGF1R in AA normal tissues as compared to CA normal tissues. IGF1R expression was similar between AA normal and malignant tissues, while IGF1R, IRS-1 and Shc phosphorylation was significantly higher in AA tumor samples. Significantly higher levels of IGF2R were found in CA tumor samples as compared to AA tumor samples. CONCLUSIONS: We conclude that IGF1R and IGF2R differential expression may contribute to the increased risk of malignant transformation in young AA women and to the more aggressive breast cancer phenotype observed among AA breast cancer patients and represent, along with IGF-II, potential therapeutic targets in breast cancer.

Differential insulin-like growth factor II (IGF-II) expression: A potential role for breast cancer survival disparity.

OBJECTIVE: Increased risk of cancer and other adult diseases have been associated with perinatal exposure to adverse conditions such as stress and famine. Recently, Insulin-like growth factor II (IGF-II) was identified as the first gene associated with altered expression caused by fetal exposure to poor nutrition. IGF-II regulates fetal development and breast cancer cell survival, in part, by regulating anti-apoptotic proteins through activation of the IGF-I and insulin receptors. African-American (AA) women have a lower overall breast cancer (BC) incidence, however, they present with advanced disease at diagnosis, poorer prognosis and lower survival than Caucasian (CA) women. The reasons for the BC survival disparity are not well understood. We hypothesize that IGF-II plays a role in the survival disparity observed among AA breast cancer patients by stimulating rapid tumor growth, inhibiting apoptosis, and promoting metastasis. DESIGN: This study examines IGF-II expression and regulation of the anti-apoptotic proteins Bcl-2, Bcl-X(L), and survivin in Hs578t (ER-), CRL 2335 (ER-), and CRL 2329 (ER+) breast cancer cells and compares with the expression of these proteins in paired breast tissue samples from AA and CA women by qRT-PCR and Western blotting. RESULTS: IGF-II expression was significantly higher in AA cell lines and tissue samples when compared to Caucasians. IGF-II siRNA treatment decreased anti-apoptotic protein levels in all cell lines (regardless of ER status). These effects were blocked by the addition of recombinant IGF-II. Of significance, IGF-II expression and regulation of Bcl-X(L) and survivin in cell lines correlated with their expression in paired breast tissues. CONCLUSIONS: IGF-II and the anti-apoptotic proteins differential expression among AA and CA patients may contribute to the breast cancer survival disparities observed between these ethnic groups.