MicroRNA Analysis Paired with Novel Cell Health Assays

Promega CorporationPromega Corporation©2016 Promega Corporation

MicroRNA Analysis Paired with Novel Cell

Health Assays: A Complete Workflow

Brad Hook, Ph.D

Manager, NA Scientific Applications

Promega Corporation

2Promega CorporationPromega Corporation©2016 Promega Corporation.

Presentation Outline

• From cells to RNA – a seemingly easy, yet complex

path

• Study 1: Differential expression of miRNA in 2D and

3D human colon cancer cell cultures after therapeutic

compound treatment

• Study 2: MicroRNA analysis paired with a novel live

cell viability assay: A complete epigenetic workflow in

human cancer cell lines


From Cells to RNA Profiling – Complete Workflow

Cells Compound

Incubation

Total RNAPurificationViability

Cytotoxicity

Quantitation Amplification

Other Cell-Based Tests:• Enzyme activity – HDAC• P450 assay• Caspase activity• Many others


Study 1: miRNAs Associated with Cancer


Study 1: Experimental Workflow

Differential expression of miRNA in 2D and 3D human colon cancer

cell cultures after therapeutic compound treatment

Compound

Treatment

(5-Fluorouracil)

Caspase activity

Live cells Dead cells

Amplification

Live cell 2° Necrosis

Apoptotic cells

mRNA/microRNA profiling


Cells in Culture – 2D vs 3D

Why 3D cell culture?

• Cells growing on surfaces are artificial and unnatural

• Extra-cellular matrix plays an important role in regulating

cellular behavior by influencing cells with biochemical

signals and topographical cues

• In 3D culture, we can control scaffold morphology,

architecture and components

Therefore cells behave and respond more like they would in

vivo to stimuli.


Cells in Culture – 2D vs 3D

Many 3D models exist. In this experiment we used:

~700µm

3D Spheroids Matrigel®


Study 1: Experimental Setup

Part 1: Determine compound potency at multiple time-points in two

human colon cancer cell lines, HCT116 and HT-29.

Compound

Treatment

(5-Fluorouracil)

48hr

Cell Health



Study 1: Cell Viability – RealTime-Glo™



Compound

Treatment

(5-Fluorouracil)

48hr

Cell Health


RealTime-Glo™ MT Cell Viability Assay


Study 1: Cytotoxicity – CellTox™ Green



Compound

Treatment

(5-Fluorouracil)

48hr

Cell Health


CellTox™ Green Cytotoxicity Assay


Why Use “Real-Time Assays”?

Benefits of multiplexing and kinetic monitoring:

• Saves $, time, plastics

• Fewer cells

• More data from a single sample

• Data normalization


Essential Equipment – Plate Reader

GloMax® Discover Multimode Detection System

• Luminescence, Fluorescence, and Absorbance (UV-Visible)

• 6-, 12-, 24-, 48-, 96- and 384-well formats

• Filtered Luminescence, BRET, FRET

• Easy to use software

• Integrated protocols

• Customizable protocols


Study 1: Potency Over Time

0.90

1.10

1.30

1.50

1.70

1.90

2.10

2.30

2.50

0

20

40

60

80

100

120

140

-6.5 -6 -5.5 -5 -4.5 -4 -3.5

Fold

ch

ange

in c

yto

toxi

city

Per

cen

t vi

abili

ty

Log[5-Fluorouracil], M

HCT116

1hr Viability 24hr Viability 48hr Viability 48hr Cytotoxicity

Two human colon cancer cell lines (2D cultures) were treated with

5-Fluorouracil and monitored for viability and cytotoxicity over a 48 hour

time period using the RealTime-Glo™ MT Cell Viability and CellTox™

Green Cytotoxicity Assays.

0.90

1.10

1.30

1.50

1.70

1.90

2.10

2.30

2.50

0

20

40

60

80

100

120

140

-6.5 -6 -5.5 -5 -4.5 -4 -3.5

Fold

ch

ange

in c

yto

toxi

city

Per

cen

t vi

abili

ty


HT-29

1hr Viability 24hr Viability 48hr Viability 48hr Cytotoxicity



Part 1: Conclusion

• A dose-dependent response to 5-Fluorouracil was observed in the

HCT116 cells. As compound dose increased, viability decreased and

cytotoxicity increased.

• As exposure to 5-Fluorouracil increased, cell viability decreased.

• HT-29 cells were more resistant to 5-Fluorouracil compared to HCT116

cells.

Compound Treatment (30µM)

HCT116

48hr

Cell Health




0.90

1.40

1.90

2.40

0

20

40

60

80

100

120

140

-6.5 -6 -5.5 -5 -4.5 -4 -3.5

Fold

ch

ange

in c

yto

toxi

city

Perc

ent

viab

ility


HCT116

1hr Viability 24hr Viability

48hr Viability 48hr Cytotoxicity

Why 30µM 5-Fluorouracil?



Differential expression of miRNA in 2D and 3D human colon cancer

cell cultures after therapeutic compound treatment

HCT116

5-Fluorouracil - 30µM

48hr

Caspase activity


Amplification


Apoptotic cells


Used to determine potencyand timing


Study 1: Apoptosis

Part 2: Determine mode of death – are cells starting to become

apoptotic with 30µM treatment of 5-Fluorouracil at 48hr?

HCT116


48hr

Caspase activity


Apoptotic cellsUsed to determine mode of death

Caspase-Glo® 3/7 Assay


Study 1: Apoptosis



0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

3D Matrigel 3D Spheroid 2D

Fold

ch

ange

fro

m D

SMO

co

ntr

ol

HCT116-Caspase-Glo® 3/7

Conclusion: Cells are not going through apoptosis. Why not?


Study 1: Apoptosis



0.90

1.40

1.90

2.40

0

20

40

60

80

100

120

140

-6.5 -6 -5.5 -5 -4.5 -4 -3.5

Fold

ch

ange

in c

yto

toxi

city

Perc

ent

viab

ility


HCT116

1hr Viability 24hr Viability

48hr Viability 48hr Cytotoxicity

Cells are not dying at this time point with this dose of 5-Fluorouracil. We might

observe apoptosis at longer time points and higher doses of compound.





HCT116

5-Fluorouracil – 30µM

48hr

Caspase activity


Amplification


Apoptotic cells


Cells not going through apoptosis



Part 3: mRNA/microRNA profiling. Determine changes in mRNA and

miRNA transcript levels after 30µM treatment of 5-Fluorouracil at 48hr.

HCT116


48hr

Caspase activity


Amplification


Apoptotic cells



Study 1: mRNA/microRNA Analysis



HCT116


48hr

Total RNA isolation Amplification


ReliaPrep™ miRNA Cell and Tissue Miniprep System





ReliaPrep™ miRNA Cell and Tissue

Miniprep System

• Fast and simple – less than 40min

• Isolates total RNA including miRNA

• No phenol:chloroform

• No ethanol precipitation





Before RT-qPCR, it is important to quantify the RNA levels in the sample eluates.

The QuantiFluor® RNA system contains a fluorescent RNA-binding dye that enables

sensitive quantitation of small amounts of RNA in solution. The Quantus™ fluorometer

is a small, dual-channel fluorometer for providing highly sensitive fluorescent detection

when quantifying nucleic acids.





HCT116


48hr

Total RNA isolation Amplification

mRNA/microRNA

profiling

Taqman® miRNA Reverse Transcription kit+

GoTaq® Probe qPCR Master Mix





Taqman® miRNA Reverse Transcription kit+

GoTaq® Probe qPCR Master Mix

miR-302a-3P miR-323-3p miR-320c miR-30b-5p RNU6B

Expected results based on literature


Study 1: Pulling It All Together



HCT116


48hr


Amplification




0

0.2

0.4

0.6

0.8

1

1.2

1.4

Viability Cytotoxicity

Fold

Ch

ange

fro

m D

MSO

co

ntr

ol

Cell Health Assays

Control 3D Matrigel 3D Spheroid 2D

Cells (all cultures) treated with 30µM 5-Fluorouracil showed a decrease in viability but no significant increase in cytotoxicity, indicating an anti-proliferative effect.



0

100200300

400

500600700

800900

1000

5-FU DMSO 5-FU DMSO 5-FU DMSO

3D Matrigel 3D spheroid 2D

Yiel

d (

ng)

Total RNA Yield

Total RNA was isolated from all samples and quantitated using the QuantiFluor® RNA

System on the Quantus™ Fluorometer. As expected RNA yields were lower with

treated samples as the number of viable cells were lower than the DMSO control.



0.1 1 10

miR-30b-5p

miR-320c

miR-323-3p

miR-302a-3p

Fold Change in miRNA expression

2D 3D spheroid 3D Matrigel

With all four miRNAs tested, the fold change in miRNA expression was lowest with

3D spheroids, highest with 2D cultures and moderate with 3D Matrigel. This could be

due to the inability of the compound to penetrate to the center of larger 3D complexes

or the difference in culturing format effects on cellular responsiveness.



Breast Adenocarcinoma Cell Lines Tested:

MCF7: HER low, ERα+, PR+

MDA-MB-231: “Triple Negative”

Compare two common breast cancer cell lines for responses to HDAC inhibitor treatment

HDAC Inhibitors

TMP269: HDAC class IIa Inhibitor

Sodium Butyrate: Pan HDAC

Inhibitor



MicroRNA Analysis

Paired with a Novel

Live Cell Viability

Assay: A Complete

Epigenetic Workflow

in Human Cancer

Cell Lines



MicroRNA Analysis

Paired with a Novel

Live Cell Viability

Assay: A Complete

Epigenetic Workflow

in Human Cancer

Cell Lines


Study 2: HDAC Activity

Part 1: Test HDAC activity in MCF7 and MDA-MB-231 cells when treated

with Sodium Butyrate and TMP269 for 48hr.

Sodium Butyrate and TMP269 are known HDAC inhibitors. Based on data

from previous studies, we chose a single dosing concentration of each

compound (2mM Sodium Butyrate and 1µM TM269) known to effect HDAC

activity.

Promega offers multiple HDAC assays:

• HDAC-Glo™ I/II Assay

• HDAC-Glo™ Class IIa

• HDAC-Glo™ 2

• SIRT-Glo™ Assays


Study 2: HDAC Activity

Part 1: Test HDAC activity in MCF7 and MDA-MB-231 cells when treated

with Sodium Butyrate and TMP269 for 48hrs.

0

0.5

1

1.5

1µM TMP269 2mM SodiumButyrate

DMSORel

ativ

e A

ctiv

ity

to C

on

tro

l

MCF7 MDA MB 231

HDAC-Glo™ I/II Assay

0

0.5

1

1.5

2


DMSOR

elat

ive

Act

ivit

y to

DM

SO

MCF7 MDA MB 231

HDAC-Glo™ Class IIa Assay

HDAC activity decreases with TMP269 and Sodium Butyrate treatment.



MicroRNA Analysis

Paired with a Novel

Live Cell Viability

Assay: A Complete

Epigenetic Workflow

in Human Cancer

Cell Lines


Part 2: Determine viability and toxicity of MCF7 and MDA-MB-231 cells

when treated with Sodium Butyrate and TMP269 for 48hr.

Cell viability monitored during HDAC inhibitor treatment

0

20

40

60

80

100

120


% V

iab

ility

Rel

ativ

e to

DM

SO

4 24 48

MCF7

0

20

40

60

80

100

120

140

1µM TMP269 2mM Sodium Butyrate

% V

iab

ility

Rel

ativ

e to

DM

SO

4 24 48

MDA-MB-231

Cell viability was measured with RealTime-Glo™ MT Cell Viability Assay at 4, 24, and 48 hours after compound treatment. N=8 for each condition.

Study 2: Cell Health Assays




when treated with Sodium Butyrate and TMP269 for 48hrs.

Cytotoxicity monitored during HDAC inhibitor treatment

Cell death was measured with CellTox™ Green Cytotoxicity Assay at 4, 24, and 48 hours after compound treatment. N=8 for each condition.

0

50

100

150

1µM TMP269 2mM Sodium Butyrate

% S

ign

al R

elat

ive

to D

MSO

4 24 48

MDA-MB-231

0

50

100

150

1µM TMP269 2mM Sodium Butyrate% S

ign

al R

elat

ive

to D

MSO

4 24 48

MCF7




when treated with Sodium Butyrate and TMP269 for 48hr.

MCF7 :

• TMP269 • No Effect on Viability• No Effect on Cell Death• No Effect on HDAC I/II activity• Decrease in HDAC Class IIa Activity

• Sodium Butyrate • Decrease in Cell Growth• Minimal increase in Cell Death• Decrease in HDAC I/II activity• Increase in HDAC Class IIa Activity

MDA-MB-231 :

• TMP269 • No Effect on Viability• No Effect on Cell Death• No Effect on HDAC I/II activity• Decrease in HDAC Class IIa Activity

• Sodium Butyrate • Minimal Decrease in Growth• Minimal increase in Cell Death• Decrease in HDAC I/II activity• No Effect on HDAC Class IIa Activity



MicroRNA Analysis

Paired with a Novel

Live Cell Viability

Assay: A Complete

Epigenetic Workflow

in Human Cancer

Cell Lines

ReliaPrep™ miRNA Cell and Tissue Miniprep System


Part 3: mRNA and miRNA expression levels in MCF7 and MDA-MB-231

cells when treated with Sodium Butyrate and TMP269 for 48hr.

Study 2: mRNA/miRNA Analysis

00.20.40.60.8

11.2

1µMTMP269

2mMSodiumButyrate

DMSORel

ativ

e Ex

pre

ssio

n

MCF7 MDA MB 231

-1

0

1

2

3

4

5

miR

31

miR

14

1

miR

20

a

miR

31

miR

14

1

miR

20

a

1µM TMP269 2 mM SodiumButyrate

Rel

ativ

e Ex

pre

ssio

n

MCF7 MDA MB 231

mRNA Expression (CDK2) miRNA Expression

• CDK2 mRNA expression is decreased with sodium butyrate treatment in both cell lines

• miR31 expression is increased with sodium butyrate treatment in MCF7 cells


Study 2: Conclusions

MCF7 : • TMP269

• No Effect on Viability• No Effect on Cell Death• No Effect on HDAC I/II activity• Decrease in HDAC Class IIa Activity

• Sodium Butyrate • Decrease in Cell Growth• Minimal increase in Cell Death• Decrease in HDAC I/II activity• Increase in HDAC Class IIa Activity• Decrease in CDK2 mRNA• Decrease in miR31

MDA-MB-231 : • TMP269

• No Effect on Viability• No Effect on Cell Death• No Effect on HDAC I/II activity• Decrease in HDAC Class IIa Activity

• Sodium Butyrate • Minimal Decrease in Growth• Minimal increase in Cell Death• Decrease in HDAC I/II activity• No Effect on HDAC Class IIa Activity• Decrease in CDK2 mRNA


From Cells to RNA Profiling – Complete Workflow

Cells Compound

Incubation

Total RNAPurificationViability

Cytotoxicity

Quantitation Amplification

Other Cell-Based Tests:• Enzyme activity – HDAC• P450 assay• Caspase activity• Many others


Technical Services

Scientists Ready to Help


Thank you for attending and we would be happy to answer any questions you may have in the chat window.

Learn more about the Promega products used in this webinar:• RealTime-Glo™ MT Cell Viability Assay• CellTox™ Green Cytotoxicity Assay• Caspase-Glo® 3/7 Assay • HDAC-Glo™ I/II Assay• HDAC-Glo™ Class IIa Assay• QuantiFluor® RNA System• Quantus™ Fluorometer• GloMax® Discover• ReliaPrep™ miRNA Cell and Tissue Miniprep System• GoTaq® Probe qPCR Master Mix• GoTaq® Probe 1-Step RT-qPCR System

http://www.promega.com/products/cell-health-and-metabolism/cell-viability-assays/realtime-glo-monitor-cell-viability-in-real-time/realtime_glo-mt-cell-viability-assay/

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http://www.promega.com/products/cell-signaling/histone-deacetylase-assays/predictive-flexible-hdac-and-sirt-activity-assays/hdac_glo-i_ii-assays-and-screening-systems/

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MicroRNA Analysis Paired with Novel Cell Health Assays

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