Ignite Creation Date:
2024-06-16 @ 11:48 AM
Last Modification Date:
2024-10-26 @ 3:30 PM
Study NCT ID:
NCT06427720
Status:
COMPLETED
Last Update Posted:
2024-05-24
First Post:
2024-05-20
Brief Title:
LINC00511miR-185-3p Axis and miR-301a-3p Markers for Breast Cancer Diagnosis
Sponsor:
Ain Shams University
Organization:
Ain Shams University
Study Overview
Official Title:
Competitive Endogenous Role of the LINC00511miR-185-3p Axis and miR-301a-3p From Liquid Biopsy as Molecular Markers for Breast Cancer Diagnosis
Status:
COMPLETED
Status Verified Date:
2024-05
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Breast cancer is a leading cause of death among women globally with significant incidence in Egypt It is characterized by aggressive behavior and resistance to treatment necessitating early detection methods Research has revealed that many cancer-related mutations are in non-coding DNA including microRNAs miRNAs and long non-coding RNAs LncRNAs miRNAs like miR-185-3p and miR-301a and LncRNAs such as LINC00511 are potential biomarkers for cancer diagnosis due to their roles in gene regulation and stability in circulation This study aims to explore the diagnostic utility of these biomarkers in breast cancer
Detailed Description:
1 Introduction 11 Background Breast cancer is one of the leading causes of death for women globally according to the world health organization WHO the number of cancer cases expected in 2025 will be 193 million cases In Egypt cancer is an increasing problem and especially breast cancer 1
Breast cancer is the most common female malignant tumor and a major threat to womens health and its development is considerably aggressive local invasion early metastases and multidrug resistance to chemotherapy 2 3 Therefore developments of minimally-invasive markers for early detection of breast cancer are of great interest
The cancer genome Atlas TCGA revealed that many of the mutations and copy number changes found in cancer do not overlap with protein coding genes 4 but are frequently located in non-coding DNA-including non-coding RNA genes 5 MicroRNAs miRNAs were among the first non-coding RNAs to be investigated cancer and their roles as therapeutic targets or biomarkers in cancer 6
MiRNAs are short single-stranded RNA sequences usually 19-23 nucleotides that control gene expression in a variety of physiological and developmental process thus having a critical role in posttranscriptional regulation of gene expression in a broad range of biological systems 7 8 MiRNAs mediate the repression of target mRNA by base pairing to complementary sequence in the 3-UTR causing transcript destabilization translational repression or both 9 It has been reported that miRNAs also modulate gene expression by binding to other regions including protein-coding exons 10 and can even induce gene expression in mammalian cells 11
Cell-free circulating miRNAs usually exist bound to ribonucleoprotien complexes or high-density lipoprotein or they are released from cells in lipid vesicles micro-vesicles exosomes or apoptotic bodies 12-15Thus circulating miRNAs may reflect homeostatic response of the organism as well as signs of disease progression Owing to their stability and resistance to endogenous RNAse activity these miRNAs have been proposed as diagnostic and prognostic biomarkers for diseases including cancer 16
Among these miRNAs MiR-185-3p has been identified as a tumor suppressor gene in various types of cancer The expression of miR-185-3p was decreased in breast cancer cells MDA-MB-231 and MCF-7 Moreover miR-185-3p was over-expressed in the LINC00511 silenced transfection while decreased in the enhancedLINC00511 plasmid transfection 17
MicroRNA-301a is another oncomiR that has been related to tumor progression in several types of cancer For example in prostate cancer gastric cancer as well as pancreatic cancer 18-21 However its role in breast cancer is not fully investigated
Long non-coding RNA LncRNAs are those longer than 200 nucleotides and many of them can also act as primary transcripts to produce short RNAs Generally LncRNAs have been implicated in gene-regulatory roles such as chromatin dosage compensation imprinting epigenetic regulation cell cycle control nuclear and cytoplasmic trafficking and cell differentiation 22 Furthermore pre-mRNA interaction where the primary transcript is interacting with LncRNAs and ends up with a different functional spliced sequence or is degraded into endogenous small interfering RNAs miRNA sponges modulation of protein activity or localization and facilitation of riboprotein complex formation Not in the least LncRNAs can stand as precursors for smaller fragments like miRNAs or piRNAs The main function of ciRNAs consists in miRNAs capturing through complementary interactions functioning like miRNAs sponge 23 LncRNAs have already been implicated in human disease including cancer 24
Long intergenic noncoding RNA 00511 LINC00511 is an oncogene that influences tumor size metastasis and poor prognosis LINC00511 binds histone methyltransferase EZH2 and specifies the histone modification pattern on p57 25 It also acts as an oncogene in squamous cell carcinoma and pancreatic ductal adenocarcinoma 26 In particular little is known whether LncRNAs can serve as biomarkers for cancer prognosis or diagnosis 27 12 Problem The role of these biomarkers are not investigated yet in breast cancer early diagnosis
13 Hypothesis Tumor Suppressor or Oncogenic Biomarkers and LncRNA used in early diagnosis of breast cancer
2 Aim of the Work
The ultimate aim of the work is to provide a clearer picture of the functions of miR-185-3p miR-301a in association with LINC00511in the early diagnosis of breast tumors with measuring both sensitivity and specificity
3 Previous Studies Findings in half page No previous clinical studies were made on the aspect of the diagnostic utilities of the studied biomarkers
4 Problem Statement
The role of these biomarkers are not investigated yet in breast cancer early diagnosis in Egyptian patients
5 Research Significance
Primary Outcome
To identify Peripheral blood circulating Tumor Suppressor or Oncogenic Small Biomarkers breast cancer and to compare with normal control expression
Secondary Outcome
To calculate sensitivity and specificity of miR-185-3p miR-301a and LINC00511 as compared to the protein-based markers CEA and CA15-3 as diagnostic markers
6 Research Objectives
1 Analyze the expression level of two miRNA miR-185-3p miR-301a and their predicted interacted lncRNA LINC00511 as minimal noninvasive molecular markers for diagnosing the primary breast cancer
2 Compare miR-185-3p miR-301a and LINC00511 with the protein based markers CEA and CA15-3 as diagnostic markers regarding to sensitivity and specificity
3 Study the relation between miR-185-3p miR-301a and LINC00511and the clinicopathological characters of breast cancer
4 Research Methodology
This study will first be approved by the Ethical Committee of Faculty of Pharmacy Ain Shams University and by the Ethical Committee of National Cancer Institute New Cairo Moreover it will be performed in adherence to the Declaration of Helsinki Guidelines where two groups will be included and all subjects will give their written consent to be archived
Group 1 the control group A total of 50 healthy volunteers who are age matched healthy female volunteers not suffering from any disease or taking any medication
Group 2 Malignant Breast Tumor 50 women with non-metastatic primary breast cancer Patients recently diagnosed who havent yet received any chemotherapy or radiotherapy attending the National Cancer Institute New Cairo
Full history will be taken for all cases and control The characteristics of the breast cancer patients with regards to age menopausal status histopathological type tumor size lymph node metastasis and tumor grade as well as estrogen receptor ER and progesterone receptor PR will be collected for data analysis
Exclusion criteria
Patients with
Blood disorder diseases -Any cancer other than breast cancer
Liver Cirrhosis Uterine Urinary bladder diseases
Methods
Routine Work
Blood Sampling
5 ml blood will be collected into plain vaccutainer tubes for serum preparation Serum samples will be stored at -80 ºC until biochemical assessment at the Biochemistry Department Research Lab Faculty of Pharmacy Ain Shams University
Assay of miR-185-3p miR-301a and LINC00511by Quantitative PCR
MiRNA and LncRNAs will be extracted from serum using Qiagen miRNeasy Mini Kit according to the manufactures protocol and their concentration and purities will be detected by nanodrop spectrophotometer
Enriched RNA will be reverse transcribed with miroRNA or LncRNAs reverse transcription kit according to the manufactures protocol
Expression of miRNA and LncRNAs will be quantified by qRT-PCR using human MicroRNA and LncRNAs assay kits and their specific primers
The expression levels of the investigated miRNA will be evaluated using the Ct method 28 The cycle threshold Ct value is the number of qPCR cycles required for the fluorescent signal to cross a specific threshold Ct will be calculated by subtracting the Ct values of RNU6-2 and GAPDH from those of investigated miRNAs and LncRNAs respectively The Ct will be calculated by subtracting the Ct of the control samples from the Ct of the cancer samples
Examination of Hormone Receptors and Protein-based tumor Markers Both estrogen receptor progesterone receptor and human epidermal growth factor receptor 2 HER-2neu in addition to CEA and CA153 tumor markers using ELISA kits will be obtained from National Cancer Institute New Cairo
SAMPLE SIZE ESTIMATION The aim of this study is to investigate the Role of some circulating MiRNAs from independent control and breast cancer cases with 1 control per experimental subject Based on previous studyHu et al 2012 the oncogenic small biomarkers expression was normally distributed with standard deviation 29 and large effect size 109 If the true differences in the breast cancer group and control group means is 225 we will need to study 25 experimental subjects and 25 control subjects to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with probability power 08 The Type I error probability associated with this test of this null hypothesis is 005
As regards the tumor suppressor expression means was normally distributed with large effect size 14 If the true differences in the breast cancer group and control group means is 14 we will need to study 15 experimental subjects and 15 control subjects to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with probability power 08 The Type I error probability associated with this test of this null hypothesis is 005
Total sample size will be 80 cases this number will be increased by 25 for expected losses and total sample is 100 subjects 50 subjects per group
Sample size estimation was performed by G power sample size calculator
Statistical Analysis
SPSS 210 software package SPSSChicagoIL will be used to perform the statistical analyses T-test or Mann-Whitneys U test will be used to compare the difference of continuous variables in two groups as appropriate Pearsons Chi-square analysis or Fishers exact test will be employed to compare the difference of categorical variables Receiver operating characteristic ROC curve and area under the ROC curve AUC will be plotted to determine the best cutoff point that discriminates between cancer and non-cancer groups the sensitivities and specificities will be calculated for the miRNAs and their diagnostic efficacy For all analyses a two-tailed P value of 005 or less will be considered as statistically significant
5 Research Time PlanFrame i Time plan Qyear Actions Q1 Preparation of the official documents for sample collection data collection and ethical approval and calculate sample size for control and patients Q2 Sample collection from patients with selected criteria and separation of serum in aliquots Q3 Continue sample collections and separation of serum in aliquots Extraction of RNA from the samples Q4 Collection of control samples and Extraction of RNA Q1 cDNA synthesis for all samples qPCR Q2 Statistical analysis of the data and starting to write a paper collection of related articles for thesis writing Q3 Publication of the paper and finalizing the thesis for revision
ii Paper title The Diagnostic Utility of Tumor Suppressor or Oncogenic Small Biomarkers in Association with LncRNA in Breast Cancer
Authors
Marwa M Mohamed Dr Eman Fouad Sanad Dr Reham Ali Abbas El-Shimy Nadia M Hamdy
Corresponding Submitting Author Prof Nadia M Hamdy First Author Marwa M Mohamed Contributing AuthorS Dr Eman Fouad Sanad Dr Reham Ali Abbas El-Shimy
6 References List
1 D A Ragab M Sharkas S Marshall and J ReenBreast cancer detection using deep convolutional neural networks and support vector machines PeerJ vol 7 p e6201 2019
2 A E Cyr and J AMargenthalerMolecular profiling of breast cancer Surgical Oncology Clinics of North America vol 23 no 3 Pp 451-462 2014
3 W J GradisharTreatment of metastatic breast cancer in JNCCN Journal of the National Comprehensive Cancer Network 2014 vol 12 no 5 SUPPL pp 759-761
4 H L Martin L Smith and D C Tomlinson Multidrug-resistant breast cancer Current perspectives Breast Cancer Targets and Therapy vol 6 no 0 Pp 1-13 2014
5 S Nik-Zainal et al Landscape of somatic mutations in 560 breast cancer whole-genome sequences Nature vol 534 no 7605 pp 47-54 2016
6 M T Maurano et al Systematicc localization of common disease-associated variation in regulatory DNA Science 80- vol 337 no 6099 pp 1190-1195 2012
7 M V Iorio and C M Croce MicroRNA dysregulation in cancer Diagnostics monitoring and therapeutics A comprehensive review EMBO Molecular Medicine vol 4 no 3 Pp 143-159 2012
8 D Baek J Villen C Shin F D Camargo S P Gygi and D P Bartel The impact of microRNAs on protein output Nature vol 7209 pp 64-71 2008
9 L P Lim et al Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs Nature vol 433 no 7027 pp 769-773 2005
10 W Filipowicz S N Bhattacharyya and N Sonenberg Mechanisms of post-transcriptional regulation by microRNAs Are the Answers in sight Nature Reviews Genetics vol 9 no 2 Pp 102-114 2008
11 J J Forman A Legesse-Miller and H A CollerA search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence Proc Natl Acad Sci vol 105 no 39 pp 14879-14884 2008
12 S Vasudevan Y Tong and J A Steitz Switching from repression to activation MicroRNAs can up-regulate translation Science 80- vol 318 no 5858 pp 1931-1934 2007
13 K C Vickers B T Palmisano B M Shoucri R D Shamburek and A T Remaley MicroRNAs are transported in plasma and delivered to recipient cells by high-denisty lipoproteins Nat Cell Biol vol 13 no 4 pp 423-435 2011
14 H Valadi K Ekstrom A Bossios M Sjostrand J J Lee and J O Lotvall Exosome-mediated transfer of mRNA and microRNAs is a novel mechanism of genatic exchange between cells Nat Cell Biol vol 9 no 6 pp 654-9 2007
15 J L Lindenberg et al Funcional delivery of viral miRNAs via exosomes Proc Natl Acad Sci vol 107 no 14 pp 6328-6333 2010
16 A Turchinovich L Weiz and B Burwinkel Extracellular miRNAs The mystery of their origin and function Trends Biochem Sci vol 37 no 11 pp 460-465 2012
17 L J Chin and F J Slack A truth serum for cancer---- microRNAs have major potential as cancer biomarkers Cell Res vol 18 no 10 pp 983-984 2008
18 Lu et al Journal of Experimental Clinical Cancer Research 2018
19 Cui L et al Expression of MicroRNA-301a and its Functional Roles in Malignant Melanoma Cellular physiology and biochemistry international journal of experimental cellular physiology biochemistry and pharmacology 40 230-244 2016
20 Shi Y K Zang Q L Li G X Huang Y Wang S Z Increased expression of microRNA-301a in nonsmall-cell lung cancer and its clinical significance Journal of cancer research and therapeutics 12 693-698 2016
21 Chen Z et al miR-301a promotes pancreatic cancer cell proliferation by directly inhibiting Bim expression Journal of cellular biochemistry 113 3229-3235 2012
22 Ma X et al Modulation of tumorigenesis by the pro-inflammatory microRNA miR-301a in mouse models of lung cancer and colorectal cancer Cell discovery 1 15005 2015
23 O Wapinski and H Y Chang Long noncoding RNAs and human disease Trends in Cell Biology vol 21 no6 pp 354-361 2011
24 D Gulei N Mehterov S M Nabavi A G Atanasov and I Berindan-Neagoe Targeting ncRNAs by plant secondary metabolites The ncRNAs game in balance towards malignancy inhibition Biotechnology advances vol 36 no 6 Pp 1779-1799 2018
25 T Derrien R Guigo and R Johnson The long non-coding rnas A new player in the dark matter Frontiers in Genetics vol 2 no Jan 2012
26 Ding J Yang C Yang S LINC00511 interacts with miR-765 and modulate tongue squamous cell carcinoma progression by targeting LAMC2 J Oral Pathol Med 20184746876
27 Zhao X Liu Y Li Z Zheng S Wang Z Li W Bi Z Li L Jiang Y Luo Y Lin Q Fu Z Rufu C Linc00511 acts as a competing endogenous RNA to regulate VEGFA expression through sponging hsa-miR-29b-3p in pancreatic ductal adenocarcinoma J Cell Mol Med 201822655-67
28 S H Kim et al Correlation of ultrasound finfings with histology tumor grade and biological markers in breast cancer ActaOncol Madar vol 47 no 8 pp 1531-1538 2008
29 M H Zweig and G Campbell Receiver-operating characteristic ROC plots A fundamental evaluation tool in clinical medicine Clinical Chemistry vol 39 no 4 Pp 561-577 1993
30Hu Z B J Dong L E Wang H X Ma J B Liu Y Zhao J H Tang X Chen J C Dai Q Y Wei C Y Zhang H B Shen 2012 Serum microRNA profiling and breast cancer risk the use of miR-484191 as endogenous controls Carcinogenesis 33 828-834
Breast cancer is the most common female malignant tumor and a major threat to womens health and its development is considerably aggressive local invasion early metastases and multidrug resistance to chemotherapy 2 3 Therefore developments of minimally-invasive markers for early detection of breast cancer are of great interest
The cancer genome Atlas TCGA revealed that many of the mutations and copy number changes found in cancer do not overlap with protein coding genes 4 but are frequently located in non-coding DNA-including non-coding RNA genes 5 MicroRNAs miRNAs were among the first non-coding RNAs to be investigated cancer and their roles as therapeutic targets or biomarkers in cancer 6
MiRNAs are short single-stranded RNA sequences usually 19-23 nucleotides that control gene expression in a variety of physiological and developmental process thus having a critical role in posttranscriptional regulation of gene expression in a broad range of biological systems 7 8 MiRNAs mediate the repression of target mRNA by base pairing to complementary sequence in the 3-UTR causing transcript destabilization translational repression or both 9 It has been reported that miRNAs also modulate gene expression by binding to other regions including protein-coding exons 10 and can even induce gene expression in mammalian cells 11
Cell-free circulating miRNAs usually exist bound to ribonucleoprotien complexes or high-density lipoprotein or they are released from cells in lipid vesicles micro-vesicles exosomes or apoptotic bodies 12-15Thus circulating miRNAs may reflect homeostatic response of the organism as well as signs of disease progression Owing to their stability and resistance to endogenous RNAse activity these miRNAs have been proposed as diagnostic and prognostic biomarkers for diseases including cancer 16
Among these miRNAs MiR-185-3p has been identified as a tumor suppressor gene in various types of cancer The expression of miR-185-3p was decreased in breast cancer cells MDA-MB-231 and MCF-7 Moreover miR-185-3p was over-expressed in the LINC00511 silenced transfection while decreased in the enhancedLINC00511 plasmid transfection 17
MicroRNA-301a is another oncomiR that has been related to tumor progression in several types of cancer For example in prostate cancer gastric cancer as well as pancreatic cancer 18-21 However its role in breast cancer is not fully investigated
Long non-coding RNA LncRNAs are those longer than 200 nucleotides and many of them can also act as primary transcripts to produce short RNAs Generally LncRNAs have been implicated in gene-regulatory roles such as chromatin dosage compensation imprinting epigenetic regulation cell cycle control nuclear and cytoplasmic trafficking and cell differentiation 22 Furthermore pre-mRNA interaction where the primary transcript is interacting with LncRNAs and ends up with a different functional spliced sequence or is degraded into endogenous small interfering RNAs miRNA sponges modulation of protein activity or localization and facilitation of riboprotein complex formation Not in the least LncRNAs can stand as precursors for smaller fragments like miRNAs or piRNAs The main function of ciRNAs consists in miRNAs capturing through complementary interactions functioning like miRNAs sponge 23 LncRNAs have already been implicated in human disease including cancer 24
Long intergenic noncoding RNA 00511 LINC00511 is an oncogene that influences tumor size metastasis and poor prognosis LINC00511 binds histone methyltransferase EZH2 and specifies the histone modification pattern on p57 25 It also acts as an oncogene in squamous cell carcinoma and pancreatic ductal adenocarcinoma 26 In particular little is known whether LncRNAs can serve as biomarkers for cancer prognosis or diagnosis 27 12 Problem The role of these biomarkers are not investigated yet in breast cancer early diagnosis
13 Hypothesis Tumor Suppressor or Oncogenic Biomarkers and LncRNA used in early diagnosis of breast cancer
2 Aim of the Work
The ultimate aim of the work is to provide a clearer picture of the functions of miR-185-3p miR-301a in association with LINC00511in the early diagnosis of breast tumors with measuring both sensitivity and specificity
3 Previous Studies Findings in half page No previous clinical studies were made on the aspect of the diagnostic utilities of the studied biomarkers
4 Problem Statement
The role of these biomarkers are not investigated yet in breast cancer early diagnosis in Egyptian patients
5 Research Significance
Primary Outcome
To identify Peripheral blood circulating Tumor Suppressor or Oncogenic Small Biomarkers breast cancer and to compare with normal control expression
Secondary Outcome
To calculate sensitivity and specificity of miR-185-3p miR-301a and LINC00511 as compared to the protein-based markers CEA and CA15-3 as diagnostic markers
6 Research Objectives
1 Analyze the expression level of two miRNA miR-185-3p miR-301a and their predicted interacted lncRNA LINC00511 as minimal noninvasive molecular markers for diagnosing the primary breast cancer
2 Compare miR-185-3p miR-301a and LINC00511 with the protein based markers CEA and CA15-3 as diagnostic markers regarding to sensitivity and specificity
3 Study the relation between miR-185-3p miR-301a and LINC00511and the clinicopathological characters of breast cancer
4 Research Methodology
This study will first be approved by the Ethical Committee of Faculty of Pharmacy Ain Shams University and by the Ethical Committee of National Cancer Institute New Cairo Moreover it will be performed in adherence to the Declaration of Helsinki Guidelines where two groups will be included and all subjects will give their written consent to be archived
Group 1 the control group A total of 50 healthy volunteers who are age matched healthy female volunteers not suffering from any disease or taking any medication
Group 2 Malignant Breast Tumor 50 women with non-metastatic primary breast cancer Patients recently diagnosed who havent yet received any chemotherapy or radiotherapy attending the National Cancer Institute New Cairo
Full history will be taken for all cases and control The characteristics of the breast cancer patients with regards to age menopausal status histopathological type tumor size lymph node metastasis and tumor grade as well as estrogen receptor ER and progesterone receptor PR will be collected for data analysis
Exclusion criteria
Patients with
Blood disorder diseases -Any cancer other than breast cancer
Liver Cirrhosis Uterine Urinary bladder diseases
Methods
Routine Work
Blood Sampling
5 ml blood will be collected into plain vaccutainer tubes for serum preparation Serum samples will be stored at -80 ºC until biochemical assessment at the Biochemistry Department Research Lab Faculty of Pharmacy Ain Shams University
Assay of miR-185-3p miR-301a and LINC00511by Quantitative PCR
MiRNA and LncRNAs will be extracted from serum using Qiagen miRNeasy Mini Kit according to the manufactures protocol and their concentration and purities will be detected by nanodrop spectrophotometer
Enriched RNA will be reverse transcribed with miroRNA or LncRNAs reverse transcription kit according to the manufactures protocol
Expression of miRNA and LncRNAs will be quantified by qRT-PCR using human MicroRNA and LncRNAs assay kits and their specific primers
The expression levels of the investigated miRNA will be evaluated using the Ct method 28 The cycle threshold Ct value is the number of qPCR cycles required for the fluorescent signal to cross a specific threshold Ct will be calculated by subtracting the Ct values of RNU6-2 and GAPDH from those of investigated miRNAs and LncRNAs respectively The Ct will be calculated by subtracting the Ct of the control samples from the Ct of the cancer samples
Examination of Hormone Receptors and Protein-based tumor Markers Both estrogen receptor progesterone receptor and human epidermal growth factor receptor 2 HER-2neu in addition to CEA and CA153 tumor markers using ELISA kits will be obtained from National Cancer Institute New Cairo
SAMPLE SIZE ESTIMATION The aim of this study is to investigate the Role of some circulating MiRNAs from independent control and breast cancer cases with 1 control per experimental subject Based on previous studyHu et al 2012 the oncogenic small biomarkers expression was normally distributed with standard deviation 29 and large effect size 109 If the true differences in the breast cancer group and control group means is 225 we will need to study 25 experimental subjects and 25 control subjects to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with probability power 08 The Type I error probability associated with this test of this null hypothesis is 005
As regards the tumor suppressor expression means was normally distributed with large effect size 14 If the true differences in the breast cancer group and control group means is 14 we will need to study 15 experimental subjects and 15 control subjects to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with probability power 08 The Type I error probability associated with this test of this null hypothesis is 005
Total sample size will be 80 cases this number will be increased by 25 for expected losses and total sample is 100 subjects 50 subjects per group
Sample size estimation was performed by G power sample size calculator
Statistical Analysis
SPSS 210 software package SPSSChicagoIL will be used to perform the statistical analyses T-test or Mann-Whitneys U test will be used to compare the difference of continuous variables in two groups as appropriate Pearsons Chi-square analysis or Fishers exact test will be employed to compare the difference of categorical variables Receiver operating characteristic ROC curve and area under the ROC curve AUC will be plotted to determine the best cutoff point that discriminates between cancer and non-cancer groups the sensitivities and specificities will be calculated for the miRNAs and their diagnostic efficacy For all analyses a two-tailed P value of 005 or less will be considered as statistically significant
5 Research Time PlanFrame i Time plan Qyear Actions Q1 Preparation of the official documents for sample collection data collection and ethical approval and calculate sample size for control and patients Q2 Sample collection from patients with selected criteria and separation of serum in aliquots Q3 Continue sample collections and separation of serum in aliquots Extraction of RNA from the samples Q4 Collection of control samples and Extraction of RNA Q1 cDNA synthesis for all samples qPCR Q2 Statistical analysis of the data and starting to write a paper collection of related articles for thesis writing Q3 Publication of the paper and finalizing the thesis for revision
ii Paper title The Diagnostic Utility of Tumor Suppressor or Oncogenic Small Biomarkers in Association with LncRNA in Breast Cancer
Authors
Marwa M Mohamed Dr Eman Fouad Sanad Dr Reham Ali Abbas El-Shimy Nadia M Hamdy
Corresponding Submitting Author Prof Nadia M Hamdy First Author Marwa M Mohamed Contributing AuthorS Dr Eman Fouad Sanad Dr Reham Ali Abbas El-Shimy
6 References List
1 D A Ragab M Sharkas S Marshall and J ReenBreast cancer detection using deep convolutional neural networks and support vector machines PeerJ vol 7 p e6201 2019
2 A E Cyr and J AMargenthalerMolecular profiling of breast cancer Surgical Oncology Clinics of North America vol 23 no 3 Pp 451-462 2014
3 W J GradisharTreatment of metastatic breast cancer in JNCCN Journal of the National Comprehensive Cancer Network 2014 vol 12 no 5 SUPPL pp 759-761
4 H L Martin L Smith and D C Tomlinson Multidrug-resistant breast cancer Current perspectives Breast Cancer Targets and Therapy vol 6 no 0 Pp 1-13 2014
5 S Nik-Zainal et al Landscape of somatic mutations in 560 breast cancer whole-genome sequences Nature vol 534 no 7605 pp 47-54 2016
6 M T Maurano et al Systematicc localization of common disease-associated variation in regulatory DNA Science 80- vol 337 no 6099 pp 1190-1195 2012
7 M V Iorio and C M Croce MicroRNA dysregulation in cancer Diagnostics monitoring and therapeutics A comprehensive review EMBO Molecular Medicine vol 4 no 3 Pp 143-159 2012
8 D Baek J Villen C Shin F D Camargo S P Gygi and D P Bartel The impact of microRNAs on protein output Nature vol 7209 pp 64-71 2008
9 L P Lim et al Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs Nature vol 433 no 7027 pp 769-773 2005
10 W Filipowicz S N Bhattacharyya and N Sonenberg Mechanisms of post-transcriptional regulation by microRNAs Are the Answers in sight Nature Reviews Genetics vol 9 no 2 Pp 102-114 2008
11 J J Forman A Legesse-Miller and H A CollerA search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence Proc Natl Acad Sci vol 105 no 39 pp 14879-14884 2008
12 S Vasudevan Y Tong and J A Steitz Switching from repression to activation MicroRNAs can up-regulate translation Science 80- vol 318 no 5858 pp 1931-1934 2007
13 K C Vickers B T Palmisano B M Shoucri R D Shamburek and A T Remaley MicroRNAs are transported in plasma and delivered to recipient cells by high-denisty lipoproteins Nat Cell Biol vol 13 no 4 pp 423-435 2011
14 H Valadi K Ekstrom A Bossios M Sjostrand J J Lee and J O Lotvall Exosome-mediated transfer of mRNA and microRNAs is a novel mechanism of genatic exchange between cells Nat Cell Biol vol 9 no 6 pp 654-9 2007
15 J L Lindenberg et al Funcional delivery of viral miRNAs via exosomes Proc Natl Acad Sci vol 107 no 14 pp 6328-6333 2010
16 A Turchinovich L Weiz and B Burwinkel Extracellular miRNAs The mystery of their origin and function Trends Biochem Sci vol 37 no 11 pp 460-465 2012
17 L J Chin and F J Slack A truth serum for cancer---- microRNAs have major potential as cancer biomarkers Cell Res vol 18 no 10 pp 983-984 2008
18 Lu et al Journal of Experimental Clinical Cancer Research 2018
19 Cui L et al Expression of MicroRNA-301a and its Functional Roles in Malignant Melanoma Cellular physiology and biochemistry international journal of experimental cellular physiology biochemistry and pharmacology 40 230-244 2016
20 Shi Y K Zang Q L Li G X Huang Y Wang S Z Increased expression of microRNA-301a in nonsmall-cell lung cancer and its clinical significance Journal of cancer research and therapeutics 12 693-698 2016
21 Chen Z et al miR-301a promotes pancreatic cancer cell proliferation by directly inhibiting Bim expression Journal of cellular biochemistry 113 3229-3235 2012
22 Ma X et al Modulation of tumorigenesis by the pro-inflammatory microRNA miR-301a in mouse models of lung cancer and colorectal cancer Cell discovery 1 15005 2015
23 O Wapinski and H Y Chang Long noncoding RNAs and human disease Trends in Cell Biology vol 21 no6 pp 354-361 2011
24 D Gulei N Mehterov S M Nabavi A G Atanasov and I Berindan-Neagoe Targeting ncRNAs by plant secondary metabolites The ncRNAs game in balance towards malignancy inhibition Biotechnology advances vol 36 no 6 Pp 1779-1799 2018
25 T Derrien R Guigo and R Johnson The long non-coding rnas A new player in the dark matter Frontiers in Genetics vol 2 no Jan 2012
26 Ding J Yang C Yang S LINC00511 interacts with miR-765 and modulate tongue squamous cell carcinoma progression by targeting LAMC2 J Oral Pathol Med 20184746876
27 Zhao X Liu Y Li Z Zheng S Wang Z Li W Bi Z Li L Jiang Y Luo Y Lin Q Fu Z Rufu C Linc00511 acts as a competing endogenous RNA to regulate VEGFA expression through sponging hsa-miR-29b-3p in pancreatic ductal adenocarcinoma J Cell Mol Med 201822655-67
28 S H Kim et al Correlation of ultrasound finfings with histology tumor grade and biological markers in breast cancer ActaOncol Madar vol 47 no 8 pp 1531-1538 2008
29 M H Zweig and G Campbell Receiver-operating characteristic ROC plots A fundamental evaluation tool in clinical medicine Clinical Chemistry vol 39 no 4 Pp 561-577 1993
30Hu Z B J Dong L E Wang H X Ma J B Liu Y Zhao J H Tang X Chen J C Dai Q Y Wei C Y Zhang H B Shen 2012 Serum microRNA profiling and breast cancer risk the use of miR-484191 as endogenous controls Carcinogenesis 33 828-834
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None