Modern Topics in Biology

 

RASSF1A Site-Specific Methylation Hotspots in Cancer and Correlation with RASSF1C and MOAP-1

1-    What has been known about RASSF1A methylation and cancer?

 

RASSF1A, a tumor suppressor, promoter methylation gene silencing occurs in several solid cancers with recent data suggesting a high susceptibility of the RASSF1A promoter to epigenetic modifications due to the predicted 75CpG sites which are very prone to methylation. So, in the absence of RASFF1A improper sister chromatid separation ensues leading to inheritable aneuploidy and malignancy, as well as an unrestricted NF_kB activation and uncontrolled inflammation in intestinal cells.

Also studies have shown that, RASSF1A-/- mice after 12-16 months of age have increased tumor incidence accompanied with increased cytokine production.

2-    What has this paper added to scientific community?

This paper explored detailed CpG methylation of RASSF1A and linked it to RASSF1C and MOAP-1 expression.

 

3-      What is its significance?

It has confirmed that the RASSF1A promoter is robustly methylated within a 32-CpG region in solid tumors and results in lower mRNA expression. However, the 19 predicted CpG sites in MOAP-1 promoter were not found to be methylated in cancer cell lines. Although, MOAP-1 mRNA expression positively correlated with RASFF1A expression in numerous cancers, whereas RASSF1C expression remained the same or was increased in cell lines or tissues with epigenetic loss of RASSF1A.

 

4-      What are the major findings?

This study has revealed the importance of investigating individual CpGs for RASSF1A methylation analysis where it has revealed CpG methylation hotspots within the RASSF1A promoter in both patient tumor tissues and cancer cell lines. In addition, a positive correlation between RASSF1A and MOAP-1 in breast, colorectal and thyroid cancers was observed suggesting an intimate connection beyond biological function. Also, in breast thyroid and colorectal cancers, the ratio of RASSF1A to RASSF1C changed in the malignant state to suggest caution when obtaining mRNA expression levels from microarray studies in genomic databases.

 

5-      What techniques did the authors use?
Choose one technique and describe the steps in your own words.

 

Techniques used: Quantitative Real-Time PCR and Pyrosequencing for Methylation Analysis.

      Pyrosequencing is a method of DNA sequencing based on the “sequencing by synthesis” principle and based on detecting DNA polymerase activity and another chemoluminescent enzyme.

       It involves synthesizing a new complementary strand to a single DNA strand of interest after the addition of dNTPs. (dATPs, dGTPs, dCTPs, dTTPs)

      DNA polymerase incorporates the correct complementary dNTPs onto the template releasing pyrophosphate (PPi).

       This released Pyrophosphate with adenylyl sulfate can be converted into ATP by the enzyme ATP-sulfurylase.  (PPi+APS à ATP+Sulfate)

      ATP can be utilized by luciferase enzyme to generate light (enzyme that generates light in fireflies) in the presence of oxygen and luciferin.

       Produced light is detected by camera and analyzed in a program.

 

6-      YOUR CRITIQUE on how things could of been done different.

 

Performing two Pyrosequencing assays covering more than 19CPG sites out of the 120CpG sites predicted to be methylated in the MOAP-1 promoter to ensure the non-methylation of the MOAP-1 promoter in cancer cell lines.