Ignite Creation Date:
2024-06-16 @ 11:48 AM
Last Modification Date:
2024-10-26 @ 3:30 PM
Study NCT ID:
NCT06427278
Status:
COMPLETED
Last Update Posted:
2024-05-23
First Post:
2024-05-19
Brief Title:
Implication of Long Non-coding RNA CCDC144NL-AS1Micro RNA-143-3p Axis Expression Level as Novel Signature in Colorectal Cancer
Sponsor:
Ain Shams University
Organization:
Ain Shams University
Study Overview
Official Title:
CCDC144NL-AS1Hsa-miR-143-3pHMGA2 Interaction In-silico and Clinically Implicated in CRC Progression Correlated to Tumor Stage and Size in Case-controlled Study Step Toward ncRNA Precision
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:
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 andor 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
Detailed Description:
1 Introduction 11 Background Colorectal cancer CRC is one of the malicious malignancies worldwide accounting for nearly 8 of all annual deaths 1 It is considered Egypts 7th most prevalent cancer representing about 347 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
12 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 epigenetic molecular markers that have 2 benefits first harbor potential therapeutic targets 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 1314 Emerging studies revealed several lncRNAs are implicated in CRC tumorigenesis metastasis and progression 1516 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 17p112 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 3536 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 havent 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 cells HMGA2 protein concentration is minimal or absent under normal physiological conditions but it is highly expressed during embryogenesis 38 and carcinogenesis 3940 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
13 Aim The oncogenic lncRNA CCDC144NL-AS1 if being involved in CRC pathogenesis by acting as a ceRNAsponging 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
14 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 andor 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 21 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 32 and large effect size 085 43 If the true differences between the CRC group and the control group means are 1 and 33 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 21 This is to be able to reject the null hypothesis that the population means of the experimental groups are equal with probability power of 08 The Type I error probability associated with this test of this null hypothesis is 005 Sample size estimation was performed by G power sample size online calculator httpwwwgpowerhhudeenhtml depending on two-sided confidence level 95
22 Study design Case-controlled retrospective mono-center study 23 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 IC form This study was conducted in accordance to the Declaration of Helsinki Guidelines approved in 2013 44 24 Study participants 241 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
2411 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
2412 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
242 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 11 1515 Control subjects were recruited randomly during routine check-up examinations for themselves or during blood donation
243 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 DM and hypertension HTN were recorded to determine the non communicable diseases statusimpact 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 III 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 31 In silico databases search and analysis accessed on October 2021 and revised July 2023 311 Identification of the investigated ncRNAs by bioinformatics The HUGO Gene Nomenclature Committee HGNC httpswww genenamesorg supported by National Human Genome Research Institute grant National Center for Biotechnology Information NCBI httpswwwncbinlmnihgov NCBI Genome Data Viewer GDV 46 USAgov and Ensembl Databases httpswwwensemblorgindex
html for human ncRNAs genes characterization Ensembl release 109 CCDC144NL-AS1 and MIR-143 are shown in Table 1
312 LncRNA disease v20 expression The LncRNA and Disease Database version 20 47 httpwwwrnanutnetlncrnadiseaseindexphphome to explore lncRNA CCDC144NL-AS1 expression in different cancer types retrieved from validated experimental results in publications or predicted httpwww rnanutnetlncrnadiseaseindexphphomedetailLDA0044869
313 Expression via ENCORI pan-cancer analysis platform 48 httpsrnasysucomencoripanCancerphp of miR lncRNA or genes across 32 types of Cancers The expression box-plot values of genes from RNA-seq data were scaled with log2FPKM 001 while the ones from miRNA-seq data were scaled with log2RPM 001
314 Association via miREnvironment database 4950 httpwwwcuilabcnmirenfragment-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
315 Interaction via the lncRNASNP2-human 51 The lncRNA CCDC144NL-AS1 or HMGA2 as non-conserved targets of miRNAhsa-miR-143 httpwwwnoncodeorggene_trans_searchphp search_typekeywordkeywordCCDC144NL-AS1sbtSearch
3151 Binding targets interaction Binding targets for hsa-miR-143-3p predicted via database RNAhybrid 22 httpsmybiosoftwarecomrna 22-v2-microrna-target-detectionhtml 52 httpbibiservcebitecuni -bielefelddernahybrid 53 and RNA22 v2 microRNA target detection httpscmjeffersonedurna22Interactive 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
316 Functional enrichment analysis targeted pathways and heatmaps 3161 KEGG targeted pathways clustersheatmap using DIANA Reverse Search 55 httpsdianalabe-ceuthgrhtmluniverseindex phprmirpath to identify in KEGG pathways-involved miRs using Mirpath using the DIANA-TarBase v70 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 005 and MicroT threshold set at 08 Accessed on July 25th 2023 Functional enrichment analysis using STRING version 115 httpsstring-dborg 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 httpsgenomeucsceducgi-binhgGateway Accessed on July 25th 2023 32 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
321 Total RNA extraction Using the miRNeasy Mini kit Cat No217004 Qiagen Hilden Germany according to the manufacturers protocol from serum samples RNA extraction was done The isolated RNA was eluted in 40 μL of RNase-free water
322 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 260280 and 260230 nm ratios were used to evaluate RNA purity After quantification the isolated and eluted RNA was stored at
-80 C in aliquots 323 Reverse transcription and measurement of ncRNAs expression 3231 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 GK800100 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 manufacturers 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 GE200100 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
3232 cDNA synthesis and measurement of hsa-miR-143-3p expression using qRT-PCR The miRCURY LNA RT Kit was used for cDNA synthesis Cat No339340 Qiagen Hilden Germany as proposed by the manufacturers 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 Eco48 qRT-PCR system PCRmax Staffordshire USA
Primers sequences are listed in Table 2 All these primers were designed by Qiagen httpswwwqiagencomworkflow-configurator workflows intcmpCM_QF_WFC_1121_OTHERS_QB_nav_products except for lncRNA CCDC144NL-AS1 that was obtained from Thermofisher httpswwwthermofishercomtaqman-gene-expressionpr oductHs04941765_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
324 Quantification of HMGA2 protein concentration by ELISA HMGA2 protein concentration was measured in serum samples by commercially available ELISA kits from Bioassay Technology Laboratory CatNo E7513Hu Jiaxing China according to the manufacturers 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
325 Indices and ratios 3251 Body mass index BMI kgm2 was calculated in kgm2 for each participant using the website httpswwwnhlbinihgovhealth educationallose wtBMIbmicalchtm Normal weight individuals have BMI of 185-249 kgm2 overweight BMI as 25-299 kgm2 and 30 kgm2 or more for obesity 3252 Platelets-to-lymphocytes ratio PLR was calculated by dividing the patient platelet count x103 cellμL by the lymphocyte count x103 cellμL 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
33 Statistical analysis Data were collected and excel tabulated in Microsoft Excel 2019 Statistical package for social studies software SPSS 260 IBM Armonk NY httpswwwibmcomproductsspss-statistics and GraphPad Prism version 801 GraphPad Software San Diego USA htt pswwwgraphpadcomscientific-softwareprism were utilized for figures while MedCalc Statistical Software version 1926 of MedCalc Software by Ostend Belgium httpswwwmedcalcorg 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 - SNSP Spearmans 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 005
12 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 epigenetic molecular markers that have 2 benefits first harbor potential therapeutic targets 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 1314 Emerging studies revealed several lncRNAs are implicated in CRC tumorigenesis metastasis and progression 1516 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 17p112 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 3536 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 havent 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 cells HMGA2 protein concentration is minimal or absent under normal physiological conditions but it is highly expressed during embryogenesis 38 and carcinogenesis 3940 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
13 Aim The oncogenic lncRNA CCDC144NL-AS1 if being involved in CRC pathogenesis by acting as a ceRNAsponging 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
14 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 andor 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 21 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 32 and large effect size 085 43 If the true differences between the CRC group and the control group means are 1 and 33 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 21 This is to be able to reject the null hypothesis that the population means of the experimental groups are equal with probability power of 08 The Type I error probability associated with this test of this null hypothesis is 005 Sample size estimation was performed by G power sample size online calculator httpwwwgpowerhhudeenhtml depending on two-sided confidence level 95
22 Study design Case-controlled retrospective mono-center study 23 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 IC form This study was conducted in accordance to the Declaration of Helsinki Guidelines approved in 2013 44 24 Study participants 241 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
2411 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
2412 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
242 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 11 1515 Control subjects were recruited randomly during routine check-up examinations for themselves or during blood donation
243 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 DM and hypertension HTN were recorded to determine the non communicable diseases statusimpact 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 III 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 31 In silico databases search and analysis accessed on October 2021 and revised July 2023 311 Identification of the investigated ncRNAs by bioinformatics The HUGO Gene Nomenclature Committee HGNC httpswww genenamesorg supported by National Human Genome Research Institute grant National Center for Biotechnology Information NCBI httpswwwncbinlmnihgov NCBI Genome Data Viewer GDV 46 USAgov and Ensembl Databases httpswwwensemblorgindex
html for human ncRNAs genes characterization Ensembl release 109 CCDC144NL-AS1 and MIR-143 are shown in Table 1
312 LncRNA disease v20 expression The LncRNA and Disease Database version 20 47 httpwwwrnanutnetlncrnadiseaseindexphphome to explore lncRNA CCDC144NL-AS1 expression in different cancer types retrieved from validated experimental results in publications or predicted httpwww rnanutnetlncrnadiseaseindexphphomedetailLDA0044869
313 Expression via ENCORI pan-cancer analysis platform 48 httpsrnasysucomencoripanCancerphp of miR lncRNA or genes across 32 types of Cancers The expression box-plot values of genes from RNA-seq data were scaled with log2FPKM 001 while the ones from miRNA-seq data were scaled with log2RPM 001
314 Association via miREnvironment database 4950 httpwwwcuilabcnmirenfragment-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
315 Interaction via the lncRNASNP2-human 51 The lncRNA CCDC144NL-AS1 or HMGA2 as non-conserved targets of miRNAhsa-miR-143 httpwwwnoncodeorggene_trans_searchphp search_typekeywordkeywordCCDC144NL-AS1sbtSearch
3151 Binding targets interaction Binding targets for hsa-miR-143-3p predicted via database RNAhybrid 22 httpsmybiosoftwarecomrna 22-v2-microrna-target-detectionhtml 52 httpbibiservcebitecuni -bielefelddernahybrid 53 and RNA22 v2 microRNA target detection httpscmjeffersonedurna22Interactive 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
316 Functional enrichment analysis targeted pathways and heatmaps 3161 KEGG targeted pathways clustersheatmap using DIANA Reverse Search 55 httpsdianalabe-ceuthgrhtmluniverseindex phprmirpath to identify in KEGG pathways-involved miRs using Mirpath using the DIANA-TarBase v70 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 005 and MicroT threshold set at 08 Accessed on July 25th 2023 Functional enrichment analysis using STRING version 115 httpsstring-dborg 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 httpsgenomeucsceducgi-binhgGateway Accessed on July 25th 2023 32 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
321 Total RNA extraction Using the miRNeasy Mini kit Cat No217004 Qiagen Hilden Germany according to the manufacturers protocol from serum samples RNA extraction was done The isolated RNA was eluted in 40 μL of RNase-free water
322 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 260280 and 260230 nm ratios were used to evaluate RNA purity After quantification the isolated and eluted RNA was stored at
-80 C in aliquots 323 Reverse transcription and measurement of ncRNAs expression 3231 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 GK800100 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 manufacturers 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 GE200100 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
3232 cDNA synthesis and measurement of hsa-miR-143-3p expression using qRT-PCR The miRCURY LNA RT Kit was used for cDNA synthesis Cat No339340 Qiagen Hilden Germany as proposed by the manufacturers 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 Eco48 qRT-PCR system PCRmax Staffordshire USA
Primers sequences are listed in Table 2 All these primers were designed by Qiagen httpswwwqiagencomworkflow-configurator workflows intcmpCM_QF_WFC_1121_OTHERS_QB_nav_products except for lncRNA CCDC144NL-AS1 that was obtained from Thermofisher httpswwwthermofishercomtaqman-gene-expressionpr oductHs04941765_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
324 Quantification of HMGA2 protein concentration by ELISA HMGA2 protein concentration was measured in serum samples by commercially available ELISA kits from Bioassay Technology Laboratory CatNo E7513Hu Jiaxing China according to the manufacturers 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
325 Indices and ratios 3251 Body mass index BMI kgm2 was calculated in kgm2 for each participant using the website httpswwwnhlbinihgovhealth educationallose wtBMIbmicalchtm Normal weight individuals have BMI of 185-249 kgm2 overweight BMI as 25-299 kgm2 and 30 kgm2 or more for obesity 3252 Platelets-to-lymphocytes ratio PLR was calculated by dividing the patient platelet count x103 cellμL by the lymphocyte count x103 cellμL 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
33 Statistical analysis Data were collected and excel tabulated in Microsoft Excel 2019 Statistical package for social studies software SPSS 260 IBM Armonk NY httpswwwibmcomproductsspss-statistics and GraphPad Prism version 801 GraphPad Software San Diego USA htt pswwwgraphpadcomscientific-softwareprism were utilized for figures while MedCalc Statistical Software version 1926 of MedCalc Software by Ostend Belgium httpswwwmedcalcorg 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 - SNSP Spearmans 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 005
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