COMSATS University Islamabad

Islamabad Campus

Synopsis for the degree of M.S./M.Phil. Ph.D

PART-1

Name of Student Ummara Javed

Department Biosciences
Registration No. SP18-RMG-008
Name of Research Supervisor Dr. Muhammad Jawad Khan

Name of Research Co-Supervisor Dr. Afrose Liaquat

Title of Research Proposal Expression Analysis of miR182-5p and its Target Genes
in Obese Diabetic Individuals of Pakistan

Summary of the Research Proposal

The incidence of obesity has significantly accelerated the rate of insulin resistance and Type 2
Diabetes Mellitus (T2D) in developed and developing nations. Diabetes is a drawn out sickness
portrayed as raised glucose levels because of either lack of insulin production or resistance to
insulin. Reported by the American Diabetes Association (ADA), the ailment is the seventh
prominent reason of death in the United States. Previously, it was considered a fairly separate
entity of the disease, but in fact Type 2 Diabetes is often a manifestation of a much wider
underlying disorder known as Metabolic Syndrome (MetS). It involves the group of insulin
resistance, abnormal cholesterol levels, excessive or low levels of blood lipids, elevated blood
pressure, and surplus body fat around the waist. Over the previous two decades, there has been a
dramatic rise in the amount of individuals with (MetS) and this rise is linked to the worldwide
obesity and incidence of T2D. Studies in humans and animals show that Obesity changes the
expression of microRNAs (miRNAs) in metabolically significant organs, engaging in
modification of normal physiology thus acting as disease mediators. miRNAs have recently
developed a worldwide interest in onset of complicated processes such as MetS. Deep sequencing
in one of the research evaluated the miRNA expression profile in whole blood, filtering overall
24 miRNAs with differential expression. Out of differentially expressed miRNAs, miR-182
related to abnormal glucose metabolism had the maximum copy numbers in the blood. Decrease
of miR-182 in T2D is fully consistent with former researches. So in this study we will measure
the expression of miR-182 in obese diabetic individuals of Pakistan to discover its contribution in
metabolism regulation, explore its target genes, Thrombospondin 1 (THBS1) and Fork head box
O1 (FOXO1).

Introduction
Diseases linked to glucose and lipid metabolism disorders, including obesity, diabetes and
(MetS), have become a global health problem in last few years (Sliwinska et al., 2017).
These metabolic disorders are associated with growing incidence of unhealthy lifestyle characteri
zed by restricted physical activity and excessive caloric consumption. There are several roots for
both obesity and T2D, of which lifestyle and genetics are the most significant ones and medical
community is working hard to figure out the genetic factors and mutations that lead to a risk of
(MetS). It has become a global epidemic health issue occurring due to increasing rate of T2D and
obesity (Lam & LeRoith, 2012). Both T2D and obesity are associated with insulin resistance
(Algoblan et al., 2014). It is a recognized fact that being overweight or obese progresses the
likelihood of developing T2D; its progression and particularly bad glycemic control give rise to
various life-threatening problems. Statistics from the International Diabetes Federation reveal
that the number of diabetic patients globally reached 370 million in 2011, and by 2030 this
number will be almost 550 million (Mashitisho & Mashitisho, 2016). Obesity and diabetes are
major global health issues and considered to be strongly linked (Felber & Golay, 2002). Impaired
healing is a severe complication of diabetes that can lead to reduce physical activity and, in some
situations to permanent injuries and limb amputation (Siqueira et al., 2009).
There are genetic markers responsible for the onset and progression of metabolic diseases.
MicroRNAs (miRNAs) have lately appeared as important mediators of metabolic processes,
playing important roles in the maintenance/alteration of physiological mechanisms, including
insulin signaling, lipid metabolism, energy equilibrium and metabolic homeostasis (Iacomino &
Siani, 2017). They are small (18–25 nucleotides in length), noncoding RNAs that control cellular
activity by suppressing gene expression primarily at post-transcription level (Ambros, 2004).
Moreover, alterations in miRNA expression are closely related to human diseases (Kim et al.,
2012). MiRNAs present in body fluids including blood have demonstrated their potential as
novel biomarkers of diabetes mellitus and cardiovascular disorders (Fiedler et al., 2011).
Latest studies prove that miRNAs play considerable role in T2D. Dysregulation of microRNA
expression leads to significant changes in the expression of their target genes, so in our study we
will measure the expression of miR-182 and its target genes (THBS1 and FOXO1) in obese
diabetic individuals, as it plays important role in metabolic processes, promoting glucose
metabolism and also because, little attention has been given to its relationship with obesity and
diabetes.
THBS1 is a real adipokine, strongly expressed in obese individuals that are insulin-resistant; also
prominently associated with inflammation of adipose tissue, inhibition of angiogenesis, and
progression of fibrosis (Varma et al., 2007). Insulin resistance is the major driving factor and a
key feature that leads to T2D, and the forkhead box O family of transcription factors are
recognized targets of insulin, thus providing a connection between transcription and metabolic
control by insulin, acting as potential targets to control genetics of T2D. Methodical studies infer
that miR-182 regulates the use of muscle glucose by targeting FoxO1 and PDK4 that adjust fuel
preference through PDHC (Zhang et al., 2016). FOXO1 plays a significant part in both types
of adipocytes that are being increasingly recognized as main regulator of the energy homeostasis
of the whole body and as a primary therapeutic target for metabolic syndrome. (Dansen &
Kalkhoven, 2015).

Statement of problem
Over 415 million people around the world are estimated to have diabetes, and obesity is thought
to be the primary risk factor accounting for 80-85 percent of the danger of developing T2D
leading to ' diabesity ' connotation. Type 2 Diabetes formerly known as adult-onset Diabetes is
now progressively seen in kids, teenagers and younger adults as a result of increasing rates of
Obesity, poor nutrition and sedentary lifestyle. Both obesity and T2D are multifactorial maladies
that are affected by various genetic and environmental factors. In spite of so much research going
on, there is still continuing increase in prevalence of both, which demands that extensive efforts
should be made to identify the disease-affecting genes for a better understanding of disease
pathogenesis, improved clinical treatment objectives and disease prediction. Recently, extensive
research on the link between regulatory microRNAs (miRNAs) and metabolic diseases has been
conducted and miRNAs have been shown to regulate insulin production, insulin secretion, and
insulin action contributing to maintenance of metabolic homeostasis. Several researches have
indicated that miR-182-5p is linked with a number of ailments, including cancer, heart disease,
and leukemia. However association with diabetes or obesity has rarely been reported. So in this
research expression of miR-182 and its target genes would be analyzed in obese diabetic
individuals of Pakistan, as it can be a therapeutic diagnostic tool for the pathogenesis and
development of metabolic syndrome.

Objective:
The main aim of this study is to measure the expression of miR-182 and its target genes; THBS1
and FOXO1 in blood samples of obese diabetic patients that may be useful for identifying novel
diagnostic and therapeutic approaches to treat metabolic Syndrome. It will also provide an onset
about how diabetes and obesity are increasing and what are the major risk factors in Pakistani
population. The study will focus on the metabolic complications related to diabetes and obesity
as well.

Research Methodology.

Study subjects:
Total hundred diabetic individuals and hundred control samples will be enlisted in this study.
Upon consent, a questionnaire survey will collect data concerning patient’s history, biography
and any other data required for work purpose. A total of hundred blood samples will be collected
from diabetic patients with different classes of BMI and with/without metabolic complications as
well as from hundred healthy controls.

Blood Samples Processing:

The blood samples will be processed according to a standardized operating procedure to collect
plasma for miRNAs analysis.

RNA Extraction

Using standard protocol, RNA will be extracted. It will be stored at temperature 80°C. For the
extraction of RNA, TRIzol® method will be used. After extraction, it will be quantified by Nano
drop Spectrometer

cDNA synthesis:
cDNA will be synthesized from extracted RNAs using cDNA synthesis kit. Reverse transcription
will be carried out by using a specific amount of RNA from each sample.

RT-PCR:
Primers will be designed using primer software for target genes. Quantification of target genes
will be done by RT-PCR. Quantitative estimation of amplified genes will be done using SYBER
Green dye. The reaction will be carried out using the following components: qPCR system,
specific sense and anti-sense primers, SYBER Green dye and CDNA. Reaction conditions will be
set as required.

Target Genes Identification and Bioinformatic Analysis

Statistical Analysis:
A statistical analysis will be performed to analyze significant relation of miR-182 with its target
genes.

Expected result:

MiRNAs are key players in complex physiological and metabolic processes, including glucose
and lipid metabolism. For amelioration of cardiometabolic disease, several miRNAs are being
targeted. This approach is becoming very promising strategy for the treatment of metabolic
disorders. We are studying the role miRNAs because they are emerging as key regulators of
metabolism. It is expected that miRNA-182 expression will significantly vary in obese diabetic
individuals. Moreover, the expression of three different genes will provide us evidence as they
are being regulated by miRNA-182.

Bibliography:
Sliwinska, A., Kasinska, M. A., & Drzewoski, J. (2017). MicroRNAs and metabolic disorders –
where are we heading? Archives of Medical Science, 4, 885-896. doi:10.5114/aoms.2017.65229

Lam, D., & LeRoith, D. (2012). The worldwide diabetes epidemic. Current Opinion In
Endocrinology & Diabetes And Obesity, 19(2), 93-96. doi: 10.1097/med.0b013e328350583a

Algoblan, A., Alalfi, M., & Khan, M. (2014). Mechanism linking diabetes mellitus and
obesity. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 587.
doi:10.2147/dmso.s67400

Liu, Y., Dong, J., & Ren, B. (2018). MicroRNA-182-5p contributes to the protective effects of
thrombospondin 1 against lipotoxicity in INS-1 cells. Experimental And Therapeutic Medicine.
doi: 10.3892/etm.2018.6883

Mashitisho, M., & Mashitisho, B. (2016). Early insulin therapy in patients with type 2 diabetes
mellitus. Journal Of Endocrinology, Metabolism And Diabetes Of South Africa, 21(1), 13-15.
doi: 10.1080/16089677.2016.1160539

Felber, J., & Golay, A. (2002). Pathways from obesity to diabetes. International Journal Of
Obesity, 26(S2), S39-S45. doi: 10.1038/sj.ijo.0802126

Siqueira, M., Li, J., Chehab, L., Desta, T., Chino, T., & Krothpali, N. et al. (2009). Impaired
wound healing in mouse models of diabetes is mediated by TNF-α dysregulation and associated
with enhanced activation of forkhead box O1 (FOXO1). Diabetologia, 53(2), 378-388. doi:
10.1007/s00125-009-1529-y

Iacomino, G., & Siani, A. (2017). Role of microRNAs in obesity and obesity-related
diseases. Genes & Nutrition, 12(1). doi:10.1186/s12263-017-0577-z

Ambros, V. (2004). The functions of animal microRNAs. Nature, 431(7006), 350-355. doi:
10.1038/nature02871

Kim, K., Park, S., Jung, S., Kim, E., Jogeswar, G., & Ajita, J. et al. (2012). miR-182 is a
negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through
targeting FoxO1. Journal Of Bone And Mineral Research, 27(8), 1669-1679. doi:
10.1002/jbmr.1604
Fiedler, J., Jazbutyte, V., Kirchmaier, B., Gupta, S., Lorenzen, J., & Hartmann, D. et al. (2011).
MicroRNA-24 Regulates Vascularity After Myocardial Infarction. Circulation, 124(6), 720-730.
doi: 10.1161/circulationaha.111.039008

Varma, V., Yao-Borengasser, A., Bodles, A., Rasouli, N., Phanavanh, B., & Nolen, G. et al.
(2007). Thrombospondin-1 Is an Adipokine Associated With Obesity, Adipose Inflammation,
and Insulin Resistance. Diabetes, 57(2), 432-439. doi: 10.2337/db07-0840

Matsuo, Y., Tanaka, M., Yamakage, H., Sasaki, Y., Muranaka, K., Hata, H.,…Satoh-Asahara, N.
(2015). Thrombospondin 1 as a novel biological marker of obesity and metabolic
syndrome. Metabolism, 64(11), 1490-1499. doi:10.1016/j.metabol.2015.07.016

Zhang, D., Li, Y., Yao, X., Wang, H., Zhao, L., & Jiang, H. et al. (2019). miR-182 Regulates
Metabolic Homeostasis by Modulating Glucose Utilization in Muscle.

Dansen, T. B., & Kalkhoven, E. (2015). Targeting FOXO1 as an option to treat obesity? Cell
Cycle, 14(16), 2558-2558. doi:10.1080/15384101.2015.1060779

Tentative Time Table:

Phase- I (0-11/2 months) Sampling and Literature Review
Phase-II (2-3 months) RNA Extraction + cDNA synthesis
Phase-III (3-4 months) Real time PCR (qPCR)
Phase-IV (1 month) Statistical Analysis and Thesis Writing

No. of Months 1 2 3 4 5 6 7 8 9 10 11 12
Sampling & Literature Review
RNA Extraction & cDNA Synthesis
Real time PCR (qPCR)
Statistical Analysis
Thesis Writing
PART II

Name of Student: _____________________ Signature______________ Dated__________

Recommendation by the Research Supervisor

Name _________________________________ Signature _____________ Date___________

Signed by Supervisory Committee

S. # Name of Committee Member Designation Signature and Date

1
2
3
4

Approved by Departmental Advisory Committee

Certified that the synopsis has been seen by members of DAC and considered it suitable for
putting up to BASAR.

Secretary
Departmental Advisory Committee

Name: _______________________ ___________Signature:__________________________

Date:________________________

Chairman/HoD ____________________________________

Signature: ______________________________

Date ________________
PART III

Dean, Faculty of Sciences

________________ Approved for placement before BASAR.

________________ Not Approved on the basis of following reasons

Signature ______________________________ Date ________________

Secretary BASAR

________________ Approved from BASAR.

________________ Not Approved on the basis of the following reasons

Signature ______________________________ Date ________________

Dean Faculty of Sciences

Remarks:

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

Signature:_____________________________ Date______________________