Promoter Methylation Status Modulate the Expression of Tumor Suppressor (RbL2/p130) Gene in Breast Cancer

<div><p>Background</p><p>Aberrant expression of tumor suppressor genes may correspond to the abnormal cell development and tumorigenesis. Rbl2/p130, a member of retinoblastoma family of proteins, has growth suppressive properties. Numerous studies reported de-regulation of Rbl2/p130 in various types of cancer as a consequence of a number of genetic alterations. However, role of epigenetic mechanisms like DNA methylation in Rbl2/p130 expression remains elusive.</p><p>Methods</p><p>In the current study, 76 breast cancer tumors along with normal tissues (n = 76), blood (n = 76) of respective individuals and control blood (n = 50) were analyzed. Rbl2/p130 expression was analyzed by quantitative real time PCR (syber green method). Promoter methylation status was studied through methylation specific PCR of bisulfite converted genomic DNA. Data was analyzed using various statistical tests.</p><p>Results</p><p>We report significantly reduced Rbl2/p130 expression (P = 0.001) in tumors tissues as compared to control samples. Similarly, Rbl2/p130 expression varies with age and disease stages (P = 0.022), which suggest its involvement in tumor progression. Aberrant promoter methylation (Δmeth) was found in almost all the diseased samples and that was significantly different (P<0.001) with control samples. Similarly, methylation status varies significantly with tumor progression stages (P = 0.022). Hyper-methylation was observed at -1, +3, +15 and +75 of Rbl2/p130 promoter flanking around the TSS. Statistical analysis revealed that Rbl2/p130 expression negatively correlates to its promoter methylation (r = -0.412) in tumor tissues. Our results reflect an epigenetic regulation of Rbl2/p130 expression in breast cancer. This highlights the importance of Rbl2/p130 promoter methylation in breast cancer pathogenesis.</p></div

Map of Rbl2/p130 promoter showing methylated and un-methylated CpG’s cytosine ranging from −10 to +80 with reference to TSS (transcription start site).

<p>Map of Rbl2/p130 promoter showing methylated and un-methylated CpG’s cytosine ranging from −10 to +80 with reference to TSS (transcription start site).</p

Relative expression profiling of Rbl2/p130 gene in different age groups and tumor stages.

<p>(<b>A)</b> Changes in relative Rbl2/p130 expression (ΔCT values) with age, both in normal and tumor tissues; (<b>B)</b> Observed changes in Rbl2/p130 in tumor and control tissues; (<b>C)</b> Differences in relative expression of Rbl2/p130 transcript as observed among control and different disease stages (<b>D)</b> Showing Rbl2 expression in control and disease samples, in both the study cohort (<45 and ≥45 years).</p

Rbl2/p130 promoter methylation and transcript expression analysis among various histological types of breast cancer.

<p><b>(A)</b> Relative expression of Rbl2/p130 in control and tumor tissues among various histological types of breast cancer. <b>(B)</b> Change in promoter methylation (ΔMeth) status in control and diseased tissues among different histological different types of breast cancer. <b>DCI;</b> ductal carcinoma in situ. <b>IDC;</b> invasive ductal carcinoma. <b>ILC;</b> invasive lobular carcinoma.</p

Statistical relation of Rbl2/p130 expression and its promoter methylation.

<p>(<b>A)</b> Differences in Rbl2/p130 promoter methylation status of tumor and normal tissues in study cohort I (<45 years) and study cohort II (≥45 years); (<b>B)</b> Observed differences in Rbl2/p130 promoter methylation status of various tumor tissues at different disease stages; (<b>C & D)</b> Correlation of relative expression (ΔCT values) and promoter methylation status as observed. Unmeth (C panel) and Meth (D panel) values are plotted separately. Adjusted R-values are shown in respective panels.</p

Mean comparison of Rbl2/p130 promoter methylation status using tukey test.

<p>Mean comparison of Rbl2/p130 promoter methylation status using tukey test.</p

Promoter methylation analysis of Rbl2/p130 gene in normal individuals and breast cancer patients.

<p><b>(A)</b> 2% agarose gel showing amplified products of methyl specific PCR (MSP). “M” represents methylation, “UM” represents un-methylation and L represents DNA size ladder. (<b>B)</b> Upper panel shows methylation of CpG “C” in tumors, whereas lower panel highlights un-methylation in control individuals. <u><b>T</b></u> shows position of non CpG “C” converted into <b>T</b> as a results of bisulfite conversion while arrows indicates the position of methylated cytosine at CpGs sites.</p

Table_1_A novel dual-task paradigm with story recall shows significant differences in the gait kinematics in older adults with cognitive impairment: A cross-sectional study.XLS

ObjectiveCognitive and motor dysfunctions in older people become more evident while dual-tasking. Several dual-task paradigms have been used to identify older individuals at the risk of developing Alzheimer’s disease and dementia. This study evaluated gait kinematic parameters for dual-task (DT) conditions in older adults with mild cognitive impairment (MCI), subjective cognitive decline (SCD), and normal cognition (NC).MethodThis is a cross-sectional, clinical-based study carried out at the Zhongshan Rehabilitation Branch of First Affiliated Hospital of Nanjing Medical University, China.ParticipantsWe recruited 83 community-dwelling participants and sorted them into MCI (n = 24), SCD (n = 33), and NC (n = 26) groups based on neuropsychological tests. Their mean age was 72.0 (5.55) years, and male–female ratio was 42/41 (p = 0.112). Each participant performed one single-task walk and four DT walks: DT calculation with subtracting serial sevens; DT naming animals; DT story recall; and DT words recall.Outcome and measuresKinematic gait parameters of speed, knee peak extension angle, and dual-task cost (DTC) were obtained using the Vicon Nexus motion capture system and calculated by Visual 3D software. A mixed-effect linear regression model was used to analyze the data.ResultsThe difference in gait speed under DT story recall and DT calculation was −0.099 m/s and − 0.119 m/s (p = 0.04, p = 0.013) between MCI and SCD, respectively. Knee peak extension angle under DT story recall, words recall, and single task was bigger in the MCI group compared to the NC group, respectively (p = 0.001, p = 0.001, p = 0.004). DTC was higher in the DT story recall test than all other DT conditions (p ConclusionKinematic gait parameters of knee peak extension angle for the DT story recall were found to be sensitive enough to discriminate MCI individuals from NC group. DTC under DT story recall was higher than the other DT conditions.</p