Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT06427278 |
| Other study ID # |
RHDIRB2020110301/2022 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
August 1, 2019 |
| Est. completion date |
January 1, 2023 |
Study information
| Verified date |
May 2024 |
| Source |
Ain Shams University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Elucidate the role of lncRNA CCDC144NL-AS1, hsa-miR-143-3p, and HMGA2 protein as non-invasive
epigenetic molecular biomarkers in liquid biopsy of CRC Egyptian patients, individually or as
an interaction arm and in comparison, to the conventional protein TMs. In addition, the
investigators investigated the potential role of lncRNA CCDC144NL-AS1 as a mediator for
development and/or progression of the cancer phenotype as well as CRC metastasis and its
relation to both hsa-miR-143-3p and HMGA2, clinically and in silico.
Description:
1. Introduction 1.1. Background Colorectal cancer (CRC) is one of the malicious
malignancies worldwide, accounting for nearly 8 % of all annual deaths [1]. It is
considered Egypt's 7th most prevalent cancer, representing about 3.47 % of male tumors
and 3 % of female tumors, respectively [2]. By 2030, there will be an estimated 60 %
increase in incidence and mortality for CRC globally [3]. Early-stage CRC is usually
asymptomatic, but when symptoms do manifest, timely detection is essential because any
delay in the diagnosis may increase mortality rates [4].
1.2. Problem Surgical resection could cure 90 % of CRCs in the early stages.
Nevertheless, the majority of patients frequently have poor prognosis since they are
detected at an advanced stage [5]. Although colonoscopy tissue biopsy is commonly used
for CRC diagnosis, yet, it is an invasive high-risk test, not convenient to be
implemented in routine medical examination for longitudinal monitoring or prognosis and
is considered partially representative of inter-metastatic or tumoral genetic
heterogeneity [6]. The classical circulating tumor biomarkers (TMs) as carbohydrate
antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) [7] are used as follow-up or
prognosis markers, despite of their limited sensitivity and specificity [8]. Therefore,
there is an urgent need for more efficient prognostic molecular biomarkers which could
be "epi/genetic molecular marker(s)" that have 2 benefits, first, harbor potential
therapeutic target(s) and second, augment classical circulating TMs, in order to improve
CRC precision. Liquid biopsy has emerged as a minimally invasive diagnostic tool to
analyze tumoral genetic and epigenetic molecular markers released into the circulation.
Liquid biopsy captures, better, tumor genetic heterogeneity, reflecting the dynamic
picture of tumor molecular landscape with lower processing time and lower cost than
tissue biopsy [9]. Long non-coding RNAs (lncRNAs) are RNA molecules with >200
nucleotides length, that regulate gene expression at the transcriptional,
post-transcriptional, and translational levels, but cannot code for proteins synthesis
[10-12]. Multiple cancer types exhibit deregulations of lncRNAs, which are involved in
all cancer hallmarks, including cancer genesis, progression, and metastasis [13,14].
Emerging studies revealed several lncRNAs are implicated in CRC tumorigenesis,
metastasis, and progression [15,16]. Beyond their potential for diagnosis, lncRNAs would
also serve as possible therapeutic targets [17]. Coiled-lncRNA Coil Domain Containing
144 N-Terminal-Like antisense 1 (CCDC144NL-AS1) located on the 17p11.2 human chromosome,
is a novel oncogenic lncRNA recently reported to be involved in carcinogenesis [18].
LncRNA CCDC144NL-AS1 was found to be upregulated in various cancers including gastric
cancer (GC) [18], hepatocellular carcinoma (HCC) [19], non-small cell lung cancer
(NSCLC) [20], ovarian cancer (OC) [21], osteosarcoma [22], and CRC [23]. However, its
clinical role as biomarker for CRC needs to be elucidated. MicroRNAs (miRNAs or miRs)
are single-stranded, small RNA molecules that are 18-25 nucleotides length [24]. They
have a regulatory function in a variety of physiological processes, involving cell
differentiation, growth, apoptosis, immunological response, hematopoiesis, and
proliferation [25]. Several studies have shown miRs have crucial role in both initiation
and progression of CRC, besides their potential as molecular biomarkers and possible
therapeutic hits [26-28]. Hsa-miR-143 located on the human chromosome 5q32 [29] is a
tumor-suppressor miR reported to be down-regulated via miR-mediated post-transcriptional
gene silencing in several human cancers including prostate cancer [30], cervical cancer
[31], OC [32], and B-cell lymphoma [33]. The 3' arm of the miR precursor product,
hsa-miR-143-3p is down regulated in CRC and if it would contribute to CRC initiation
[34] will be studied currently clinically. LncRNA-miR interaction plays an essential
role during various cancer development [35,36]. CCDC144NL-AS1 was reported to act as
competing endogenous RNA (ceRNA) for hsa-miR-143-3p during GC progression, via competing
with the common binding regions of miRs, in order to sequester them and therefore, alter
the expression of miRs downstream target genes or proteins [18]. Being approved
experimentally by Fan et al. [37] in GC lncRNA CCDC144NL-AS1 upregulation in GC tissues
and sponging hsa-miR-143-3p followed by upregulated expression of its direct endogenous
target protein. Similarly, the clinical correlation between lncRNA CCDC144NL-AS1 and
hsa-miR-143-3p in CRC patients' peripheral blood samples, that haven't been estimated
previously, could further clarify our understanding of CRC molecular pathogenesis and
proof the cancer findings documented in silico. High Mobility Group AT-hook 2 (HMGA2)
gene encoded by 5 exons and an open reading frame of 330 base pair, is found at human
chromosome band 12q13-15 [38]. The adult normal cell's HMGA2 protein concentration is
minimal or absent under normal physiological conditions, but it is highly expressed
during embryogenesis [38] and carcinogenesis [39,40]. Patients with CRC who have HMGA2
overexpression are experiencing worse prognosis [41]. HMGA2 takes part in almost every
stage of biological activity of CRC, including cell division, proliferation, apoptosis,
senescence, tumor invasion, epithelial-tomesenchymal transition (EMT), DNA repairing
mechanism, and stem cell ability of self-renewal [42]. HMGA2 in osteosarcoma, was
reported to be positively modulated by CCDC144NL-AS1 [22].
1.3. Aim The oncogenic lncRNA CCDC144NL-AS1 if being involved in CRC pathogenesis, by
acting as a ceRNA/sponging the tumor suppressor hsamiR-143-3p, and further, upregulating
the expression of its endogenous target HMGA2 as an interaction arm will be highlighted
in the current study.
1.4. Objectives Elucidate the role of lncRNA CCDC144NL-AS1, hsa-miR-143-3p, and HMGA2
protein as non-invasive epigenetic molecular biomarkers in liquid biopsy of CRC Egyptian
patients, individually or as an interaction arm and in comparison, to the conventional
protein TMs. In addition, we investigated the potential role of lncRNA CCDC144NL-AS1 as
a mediator for development and/or progression of the cancer phenotype as well as CRC
metastasis and its relation to both hsa-miR-143-3p and HMGA2, clinically and in silico.
2. Subjects 2.1. Sample size and power of the study Based on the previous study by Zhang et
al. in 2019 lncRNA CCDC144NL-AS1 was normally distributed with a standard deviation
(3.2) and large effect size (0.85) [43]. If the true differences between the CRC group
and the control group means are 1 and 3.3, respectively, the study group sizes are 34
patients and 34 control subjects. Total sample size was 68 cases, which was increased by
25 % for expected losses, and total sample was finally 90 subjects, 60 CRC subjects and
30 control (2:1). This is to be able to reject the null hypothesis, that the population
means of the experimental groups are equal with probability (power) of 0.8. The Type I
error probability associated with this test of this null hypothesis is (0.05). Sample
size estimation was performed by G power* sample size online calculator
http://www.gpower.hhu.de/en.html, depending on two-sided confidence level 95 %.
2.2. Study design Case-controlled retrospective mono-center study. 2.3. Institutional
Review Board (IRB) statement The Research Ethics Committee of the Faculty of Pharmacy,
Ain Shams University, granted ethical permission to conduct the study, 2022. All
participants (controls or patients) were fully cognizant of the purpose of the study and
signed a written, ethically-approved, informed consent (I⋅C) form. This study was
conducted in accordance to the Declaration of Helsinki Guidelines approved in 2013 [44].
2.4. Study participants 2.4.1. Patients group A total of 60 primary CRC treatment-naïve
Egyptian patients admitted to the Dar Al Shefa Hospital, Cairo, Egypt, were enrolled in
the study.
2.4.1.1. Patients' inclusion criteria. Patients visiting the Colonoscopy Unit for
colorectal examination, suffering from variable colonic symptoms, including the CRC
alarming symptoms, constipation, abdominal pain, rectal bleeding, and sudden weight
loss. CRC diagnosis was clinically confirmed by colonoscopy, abdominal radio-imaging,
and histopathologically.
2.4.1.2. Patients' exclusion criteria. Patients suffering from inflammatory disorders,
hematological disorders, any cancer other than CRC, or those receiving chemotherapy,
radiation, or have undergone surgery, patients with hematological disorders, or any
cancer other than CRC. Individuals with inadequate data or missing histopathological
diagnosis, as well as those with distant metastases at the time of diagnosis were
excluded from the study.
2.4.2. Control group 30 age-matched and sex-matched apparently healthy volunteers, not
receiving any medications or suffering from any disease, age range 30-60 years, were
included as controls, male-to-female 1:1 (15/15). Control subjects were recruited
randomly during routine check-up examinations for themselves or during blood donation.
2.4.3. Patients demographic, clinical, and pathological data The patients' demographic
data including age, gender, smoking status, and the patient full history, retrieved from
the hospital medical records. In addition to patients' colorectal surgery history, the
complete family history of cancer, as well as history of diabetes mellitus (D.M) and
hypertension (HTN) were recorded to determine the non communicable diseases
status/impact. From patients' files, the following chemistry lab results were recorded,
for statistical correlations, CEA, CA19-9 and routine biochemical testing of liver
function profiling of alanine aminotransaminase (ALT), aspartate aminotransaminase
(AST), and serum albumin, kidney function tests including serum creatinine and serum
urea. Hemoglobin (Hgb) as well as prothrombin time (PT), platelet count, lymphocytes
count, lactate dehydrogenase (LDH), and C-reactive protein (CRP) were all measured in
blood at the Clinical Biochemistry Lab, Dar Al Shefa Hospital, Cairo, Egypt. Tumor site,
tumor size, tumor type if mucinous or not, LN metastasis (LNM), tumor invasion or
vascular invasion, tumor differentiation, tumor grade, tumor-node-metastasis (TNM)
staging, inflammation status as inflammatory bowel disease (IBD), and CRC locations if
colonic or rectal, as well as if transverse, sigmoid, rectosigmoid, and rectal, were
collected from the patients' files at the Statistics Unit at Dar Al Shefa Hospital,
Cairo.
Pathological records according to the American Joint Committee (AJCC) on Cancer 2010
criteria [45] were collected as well. Patients were categorized into three stages from
II to IV, Stage I/II is the local cancer with no LN involvement (N0), Stage III where LN
involvement (N1-x) is there, and Stage IV with distant metastasis (M1). CRC staging was
determined by the colonoscopy results, abdominal radiography, pathological findings, and
clinical evaluation, where the early stage T2 indicates invasion of the muscularis
propria by the tumor, T3 indicates the tumor penetration to the subserosa and the
muscularis propria, while late tumor stage T4 indicates that it has penetrated the
rectum or several colon layers.
3. Methods 3.1. In silico database(s) search and analysis (accessed on October 2021 and
revised July 2023) 3.1.1. Identification of the investigated ncRNAs by bioinformatics
The HUGO Gene Nomenclature Committee (HGNC) https://www. genenames.org/ supported by
National Human Genome Research Institute grant, National Center for Biotechnology
Information (NCBI) https://www.ncbi.nlm.nih.gov/, NCBI Genome Data Viewer (GDV) [46]
(USA.gov) and Ensembl Databases https://www.ensembl.org/index.
html for human ncRNAs genes characterization (Ensembl release 109) CCDC144NL-AS1 and MIR-143
are shown in Table 1.
3.1.2. LncRNA disease v2.0 expression The LncRNA and Disease Database (version 2.0) [47]
http://www.rnanut.net/lncrnadisease/index.php/home to explore lncRNA CCDC144NL-AS1 expression
in different cancer types retrieved from validated experimental results in publications or
predicted http://www. rnanut.net/lncrnadisease/index.php/home/detail/LDA0044869.
3.1.3. Expression via ENCORI pan-cancer analysis platform [48]
https://rnasysu.com/encori/panCancer.php of miR, lncRNA or genes across 32 types of Cancers.
The expression box-plot values of genes from RNA-seq data were scaled with log2(FPKM +0.01),
while the ones from miRNA-seq data were scaled with log2(RPM + 0.01).
3.1.4. Association via miREnvironment database [49,50]
http://www.cuilab.cn/miren#fragment-1of curated and collected experimentally supported miRNA
and various environmental factors (394 factor) interplay and their associated phenotypes
(June-28, 2011, the original miREnvironment Database was released, Last update: Sep-9, 2012)
for analysis for has-miR-143-3p as prediction of association between environmental factors
and human disease.
3.1.5. Interaction via the lncRNASNP2-human [51] The lncRNA CCDC144NL-AS1 or HMGA2 as
non-conserved targets of miRNA:hsa-miR-143 http://www.noncode.org/gene_trans_search.php
search_type=keyword&keyword=CCDC144NL-AS1&sbt=Search.
3.1.5.1. Binding targets interaction. Binding targets for hsa-miR-143-3p predicted via
database RNAhybrid 2.2 https://mybiosoftware.com/rna 22-v2-microrna-target-detection.html
[52], http://bibiserv.cebitec.uni -bielefeld.de/rnahybrid [53] and RNA22 v2 microRNA target
detection https://cm.jefferson.edu/rna22/Interactive/ by Jefferson Computational Medicine
Center (CMC) [54] to predict hsa-miR-143-3p interaction with either lncRNA CCDC144NL-AS1 or
HMGA2 as target, and database RNA22 v2 microRNA target detection.
3.1.6. Functional enrichment analysis, targeted pathways and heatmaps 3.1.6.1. KEGG targeted
pathways, clusters/heatmap using DIANA. Reverse Search [55]
https://dianalab.e-ce.uth.gr/html/universe/index. php?r=mirpath to identify in KEGG
pathways-involved miRs using Mirpath, using the DIANA-TarBase v7.0 Heatmap with cluster
dendrogram with statistically significant results in red by a posteriori analysis method
after an enrichment analysis is performed, p value threshold set at 0.05 and MicroT threshold
set at 0.8 (Accessed on July 25th 2023). Functional enrichment analysis using STRING version
11.5 https://string-db.org/ [56]. Finally, using the Genome Browser (UCSC) [57] Dec. 2013
initial release; June 2022 patch release 14, selected top genes Targets of HMGA2 interactions
and pathways from curated databases and textmining https://genome.ucsc.edu/cgi-bin/hgGateway
(Accessed on July 25th, 2023). 3.2. Blood samples Five milliliters of peripheral venous blood
were withdrawn from controls and CRC patients, under strict sterile conditions, following
standard international biosecurity safety procedures, into polymer gel clot activator
vacutainers (Greiner Bio-One GmbH, Australia). At room temperature (25 ◦C), completely
coagulated samples were centrifuged at 4000 rpm for 10 min. Sera obtained were aliquoted into
three DNase/ RNase free Eppendorf tubes and stored at -80 ◦C for molecular analysis.
3.2.1. Total RNA extraction Using the miRNeasy Mini kit (Cat. No.217004; Qiagen, Hilden,
Germany) according to the manufacturer's protocol, from serum samples, RNA extraction was
done. The isolated RNA was eluted in 40 μL of RNase-free water.
3.2.2. Quantitation of purified RNA including miRNAs Using a NanoDrop® 1000 spectrophotometer
(Thermo Scientific, Wilmington, DE, USA) the concentration and purity of all RNA samples were
determined. Absorbance at 260 nm was used to measure the conc. of RNA in the sample, whereas
260/280 and 260/230 nm ratios were used to evaluate RNA purity. After quantification, the
isolated and eluted RNA was stored at
-80 ◦C in aliquots. 3.2.3. Reverse transcription and measurement of ncRNAs expression
3.2.3.1. cDNA synthesis and measurement of lncRNA CCDC144NL-AS1 expression using qRT-PCR.
Total RNA was reverse transcribed into cDNA using the Xpert cDNA Synthesis Kit (Cat. No.
GK80.0100; Grisp Research Solutions, Rua Alfredo Allen, Portugal) containing Xpert Reverse
Transcriptase (RNase H-), RNA-dependent DNA polymerase appropriate for cDNA synthesis from
long RNA templates, following the manufacturer's protocol. The synthesized cDNA was then
stored at - 20 ◦C till qRT-PCR. The expression of lncRNA CCDC144NL-AS1 was measured using the
Xpert Fast SYBR (Cat. No. GE20.0100; Grisp Research Solutions, Rua Alfredo Allen, Portugal)
in accordance with the manufacturer protocol. The primer RT2 lncRNA qPCR Assay for Human
CCDC144NL-AS1 (Hs04941765 m1, Cat. No. 4426961) was used to assess the level of expression of
the lncRNA CCDC144NL-AS1. The human GAPDH primer (LPH31725A-200, Cat. No. 330701) was used as
endogenous control to normalize the expression of lncRNA CCDC144NLAS1.
3.2.3.2. cDNA synthesis and measurement of hsa-miR-143-3p expression using qRT-PCR. The
miRCURY LNA RT Kit was used for cDNA synthesis (Cat. No.339340, Qiagen, Hilden, Germany) as
proposed by the manufacturer's instructions. The resulting cDNA was kept at - 20 ◦C until
quantification. qRT-PCR was used to measure the expression of the hsamiR- 143-3p using the
miRCURY LNA miRNA PCR Assay (Cat. No. 339306, Qiagen, Hilden, Germany). The primer SNORD38B
(hsa) miRCURY LNA miRNA PCR Assay (YP00203901, Cat. No. 339306) was used as endogenous
control to normalize the expression of hsa-miR-143-3p. The reaction was carried out using the
PCRmax Eco™48 qRT-PCR system (PCRmax, Staffordshire, USA).
Primers sequences are listed in Table 2. All these primers were designed by Qiagen
https://www.qiagen.com/workflow-configurator/ workflows
intcmp=CM_QF_WFC_1121_OTHERS_QB_nav_products, except for lncRNA CCDC144NL-AS1 that was
obtained from Thermofisher https://www.thermofisher.com/taqman-gene-expression/pr
oduct/Hs04941765_m1 (Accessed on October 2021). The RNA relative expression was computed and
normalized as fold change using the cycle threshold (Ct) method (2- ΔΔCt) with GAPDH or
SNORD38B (hsa) as the internal control for lncRNA CCDC144NL-AS1 and hsa-miR-143-3p,
respectively. ΔCt was determined by subtracting the Ct values of GAPDH and SNORD38B (hsa)
from those of the lncRNA CCDC144NL-AS1 and hsa-miR-143-3p, respectively; where ΔΔCt = ΔCt
cancer samples - ΔCt control samples [58].
3.2.4. Quantification of HMGA2 protein concentration by ELISA HMGA2 protein concentration was
measured in serum samples by commercially available ELISA kits from Bioassay Technology
Laboratory (Cat.No. E7513Hu, Jiaxing, China) according to the manufacturer's instructions.
The reaction is based on pre-coating the ELISA plate with Human HMGA2 antibody and HMGA2
present in added samples binds to antibodies coating the wells. Biotinylated human HMGA2
antibody was added to bind HMGA2 in samples. A second detector antibody was then added to
bind the biotinylated HMGA2 antibody. A substrate solution was added that reacts with the
enzyme-antibody-target complex to produce a measurable signal measured at 450 nm.
3.2.5. Indices and ratios 3.2.5.1. Body mass index (BMI kg/m2) was calculated in kg/m2 for
each participant using the website https://www.nhlbi.nih.gov/health /educational/lose
wt/BMI/bmicalc.htm. Normal weight individuals have BMI of 18.5-24.9 kg/m2, overweight BMI as
25-29.9 kg/m2, and 30 kg/m2 or more for obesity. 3.2.5.2. Platelets-to-lymphocytes ratio
(PLR) was calculated by dividing the patient platelet count (x103 cell/μL) by the lymphocyte
count (x103 cell/μL). PLR is an inflammation indicator and immune response-related predictor
that has a stronger link with inflammatory diseases severity than the
neutrophils-to-lymphocytes ratio (NLR) [59] or either individual cells alone.
3.3. Statistical analysis Data were collected and excel tabulated in Microsoft Excel 2019.
Statistical package for social studies software SPSS 26.0 (IBM, Armonk, NY)
(https://www.ibm.com/products/spss-statistics), and GraphPad Prism® version 8.01 (GraphPad
Software, San Diego, USA) (htt ps://www.graphpad.com/scientific-software/prism/) were
utilized for figures, while MedCalc Statistical Software version 19.2.6 of (MedCalc Software
by Ostend, Belgium) (https://www.medcalc.org) was used for the receiver operating
characteristic (ROC) curve analysis. Data were tested for normality using Shapiro-Wilk
normality test for both groups and subgroups data. Since the patients' data were not normally
distributed, data were expressed as median (interquartile range: IQR (25th percentile-75th
percentile). Mann-Whitney (U) or Kruskal-Wallis (H) were conducted to compare between any two
or more independent groups, respectively.
The ROC curve was used to find the best cutoff, sensitivities (SNs), specificities (SPs),
negative predictive values (NPVs), and positive predictive values (PPVs), with an area under
the curve (AUC) calculated ranged from 0 to 1. In medical testing, negative likelihood ratios
(LRs) are used to understand the diagnostic or prognostic test utility. Essentially, the LR
indicates the likelihood that a patient has a condition or disease. The likelihood that they
have the disease or condition increases with the ratio. A low ratio, on the other hand,
indicates that they most likely do not. Therefore, a physician can use these ratios to either
rule in or rule out an illness. The ratio, which expresses how likely it is for someone to
have the disease or condition, supports the SNs and SPs identified by the ROC curve. An
alternative definition of the LR is SN and SP, where negative LR = (100 - SN)/SP. Spearman's
correlation coefficient r was used to evaluate the correlation between various variables.
Additionally, the expression levels of the lncRNA CCDC144NL-AS1, hsamiR- 143-3p, and HMGA2
protein were set to Spearman correlation r, and the link among two variables-one continuous
and one dichotomous- was assessed using point-biserial correlation. Significance level was
set if the two-tailed statistical analysis test p-value is <0.05.
