Change from a very linear view of how genetic information is processed to a complex network that proceeds in various directions to create the necessary degree of regulatory specificity.
-Make sure to point out the ones that are most widely used. Northern blotting, microarry, RT-PCR, and surface enhance Raman spectrometry.
Round about method is what I am doing in my lab, so talk about it then. Luciferase assay is the only concrete way of establishing a direct relationship between a certain miRNA and mRNA.
Heat map of the expression of miRNAs enriched in BAT compared to WAT & Skeletal muscleRelative quantitation of each mRNA in various mouse tissues, miR-223 is the controlExpression changes during differentiation
Test of miRNA inhibitors effectivenessShows accumulation of lipid droplets in BAT at day 4Mimics increase expressionrtPCR of adipogenesis markers
-In the microarray we wanted to find changes in expression that were similar in both the mouse and human tissue samples.-qPCR, obviously can’t look at changes in expression for everything so we focused on genes important for glucose homeostasis and adipocyte differentiation-
Result was I then had ~15 miRNAs that were of potential interest. So I then ran them through Target Scan, online database/algorithm to help identify targets. miRNAs that regulate a certain protein.
Predictions are ranked based on:-site type contribution-3’pairing-local AU relationships-position
Just targets related to glucose homeostasis.miR-425#, miR-501-5p, miR-574-3p, miR-10b#, miR-339-3p, miR-17, miR-107, miR-32, miR-191#, miR-708, miR-346, miR-494, miR-145#, miR-489, miR-449bmiR-194, miR-302a, miR-200a, miR-181c, miR-346
Mir193b–365 is essential for brown fat differentiation Lei Sun, Huangming Xie, Marcelo A. Mori, Ryan Alexander, Bingbing Yuan, Shilpa M. Hattangadi, Qingqing Liu, C. Ronald Kahn and Harvey F. Lodish
History of miRNA Research• Their importance was first recognized in 1993 in C. Elegans when a mutation caused loss of function in lin-4 and let-7.• Caused disruption of developmental timing and revealed a previously unknown level of complexity in genetic regulation.
Biogenesis of a miRNA• miRNA gene is transcribed• Pri-miRNAPre-miRNAmature miRNA• Mature miRNA Duplex is split in two and the single stranded molecule is incorporated into the RISC (Ribosome Induced Silencing Complex) assembly
Profiling miRNAs• Reverse transcription of miRNAS and total RNAs for cDNA.• Helps to use a variety of tissues.• With the cDNA you can then analyze expression via microarray or real time PCR.• Important to note that this only tells you if something is there.
Function Analysis of miRNAs• If you want to see a miRNA:mRNA relationship and/or establish a miRNA’s role in a certain phenotype, there are really 2 different methods• Reporter Assay• “Round-a-bout” Method
Diabetes and Obesity• According to the NIDDK diabetes is a chronic lifelong disease which is characterized by chronic high levels of sugar in the blood.• In reality the picture is much, much bigger
Their Method• Identified lineage rich miRNAs by comparing genome wide miRNA expression patterns in WAT, BAT, and skeletal muscle microarrays• Looked for miRNAs expressed in at least three samples with different expression in all 3 tissues.• CAGE analysis which measures expression levels of transcription starting (start points)
• Use LNA modified inhibitors to block miR-193 expression and see what happens.• Check brown fat markers with Western Blotting which pointed to miR-193 being critical to certain steps in development
Conclusions• Determine that miR-193b-365 complex is necessary for BAT differentiation• Determine that its relationship to Prdm16 and Pparα helps upregulate this microRNA during differentiation, inducing adipogenic factors, while suppressing myogenic ones.• miR-193b also associates closely with the Runx1t1 mRNA, and other mRNAs
The Future• Examine function within a mouse model using adenoviruses to specifically introduce inhibitors and mimics• Examine their therapeutic potential
MiRNA Profiling and FunctionAnalysis in a Diet Induced Mouse Model of Obesity and Diabetes
What my lab does• Diabetes and Obesity focus in addition to regenerative biology• Specifically the crucial roles of Cdk4, Smad3, and TGF-β in cell cycle regulation and metabolic function.• The miRNA component comes in when considering why certain genes are turned on/off in the obese and overweight.
My Experiment• Microarray profiling of human and mouse miRNAs in Liver, Adipose, and Muscle tissue• qPCR of total cDNA from samples looking for changes in expression of genes related to glucose homeostasis• Narrow down list of miRNAs using additional qPCR and target scan software• Look for phenotypic affects.
Real Time and a Microarray• Microarray Cards were • Real time was then used to profile used to look for genes everything we could important in look for in muscle and WAT/BAT, metabolism, a adipose tissue. nd glucose homeostasis• Over 500 hits. • PC, Lxr, Akt2, Creb3, Lca• Narrowed down the list d, IR, FAS, PEPCK, Scd1, to things that behaved Ggpale, Srebp2, Pgc1a, similarly in mice and Pgc1b, Ppara, etc. human tissues.
Target Scan• Online database for predicting miRNA targets.• Looks at 8mer and 7mer sites of mRNA which match up with “Seed region” of miRNA• There is a lot of overlap.
Changes in Gene Expression during differen a on for BAT 16 14 12 pparalpha prdm16 10 smad3 cox8bFold Change 8 UCP1 ppargamma 6 pgc1a FAS 4 Serpin3AK. resis n 2 ap2 0 0 1 2 3 4 5 6 7 8 9 -2 Day No.
Results?• miRNAs: 425, 501, 574, 10b, 339, 17, 107, 32, 191, 708, 346, 494, 145, 489, 449b, 194, 302a, 200a, 181c, 346• Gene expression is clearly different between tissue types and normal, overweight, and obese models.• miRNAs are heavily regulated
Future Experiments• Looking at miRNA expression in first 24hrs.• Want to examine expression in various tissues: liver, muscle, hypothalamus, pancreas• Use lento/adeno viruses to insert LNA- miRNAs into the mice and see what happens