Ignite Creation Date:
2024-06-16 @ 11:49 AM
Last Modification Date:
2024-10-26 @ 3:30 PM
Study NCT ID:
NCT06432413
Status:
COMPLETED
Last Update Posted:
2024-05-29
First Post:
2024-05-22
Brief Title:
Emerging Role of NOTCH-related Long Non-coding RNAs as Biomarkers in Liquid Biopsy From Colorectal Cancer Patients
Sponsor:
Ain Shams University
Organization:
Ain Shams University
Study Overview
Official Title:
Emerging Role of NOTCH-related Long Non-coding RNAs as Biomarkers in Liquid Biopsy From Colorectal Cancer Patients
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:
This work aims to Investigate the role of circulating notch associated lncRNAs SNHG3 and LUNAR1 as possible non invasive prognostic biomarkers for colorectal cancer CRC monitoring via measuring the gene expression level of lncRNAs SNHG3 and LUNAR1 in serum of CRC patients compared with control subjects Also to investigate the correlation between SNHG3 and LUNAR1 expression levels and CRC clinicopathological features and their relevance for CRC patients clinico-pathological features outcomes assessment
Detailed Description:
1 INTRODUCTION Background Colorectal cancer CRC has been identified as a major public health concern given its high incidence and mortality rates 1 In 2020 CRC was the third-most prevalent malignancy after breast and lung cancer accounting for 10 of new cases and ranking second in terms of mortality with 94 of deaths 2 Unfortunately by year 2030 more than 22 million new cases and 11 million fatalities from CRC are globally anticipated 3
Problem Despite significant improvements in CRC surgical or radiological interventional treatment approaches with neo-adjuvant therapies patients prognosis remains bleak 45 Metastases and post-surgical tumor recurrence are prevalent particularly in more advanced cancer cases 6 which may account for the increased number of cases and fatalities prediction being against the national efforts for achieving Egypt Vision 2030 implementing SDGs
Problem statement Conventionally used CRC prognostic markers for monitoring treatment outcome and pointing to cancer recurrence are carbohydrate antigen 199 CA199 andor carcinoembryonic antigen CEA that are not that sensitive 7 per patients subclassification or stratification related to CRC pathological characteristics for more efficient and earlier prognosis Thence a growing demand for sensitive and precise bio-molecular markers better correlating with CRC prognostic markers andor CRC clinical outcome 8 that is if successful would constitute the bases for Better Health SDG3 and less cancer recurrence and therefore less mortality
Liquid biopsies are used for cancer diagnosis or prognosis like breast cancer leukemia liver cancer or CRC via measurement of tumor-derived bio-molecular markers including circulating ncRNAs including long non-coding RNAs lncRNAs microRNA exosomal ncRNAs oncogenes or tumor suppressor genes and their mutations tumor-related-cytokines and down-stream target proteins 9-22
After extensive literature search and mining to mind-the-research-gaps for better Cancer Epigenetics Study a Step-toward ncRNA-Precision we have chosen in the current work two notch-related lncRNAs to study in relation-to-CRC prognosis
Notch-signaling pathway is an ubiquitous cascade within species to control a broad variety of biological events including cell division proliferation and cell death 2324 Recent investigations have revealed the fundamental role of Notch-cascade in CRC evolution 25 Intestinal epithelial cells homeostatic self-renewal and tumor-promoting transformation can both be managed by Notch signaling 26 Stimulation of Notch-cascade can be epigenetically triggered by dysregulated non-protein coding RNAs ncRNAs expressions 27 a hot area of research nowadays to figure out the impact of Notch-related ncRNAs on CRC risk andor progression
The significant utility of tumor-expressed Notch-associated lncRNAs as prognostic malignancy indicators proves how they are connected to carcinogenesis or metastasis and therefore reflect the outcomes in different cancer types including CRC 28-31
Small Nucleolar RNA Host Gene3 SNHG3 is 4950bp lncRNA situated on chromosome 1p353 32 In breast cancer upregulated SNHG3 triggers Notch system activation as a result of its competitive binding to human homo sapiens hsa micro-RNA miR hsa-miR-154-3p exacerbating proliferation and metastasis of cancer cells 33 Moreover SNHG3 positively regulates Notch1 expression in ovarian cancer through hsa-miR-139-5p suppression accelerating tumor cells proliferation and migration 34 SNHG3 exerted a carcinogenic role in prostate cancer osteosarcoma glioma gastric cancer laryngeal cancer bladder cancer and CRC 32 Huang et al reported SNHG3 elevated expression in CRC cells and tissues stimulating cancer progression through sponging hsa-miR-182-5p 35 Therefore SNHG3 is considered as a malignancy enhancer that regulates Notch system in various cancer types
Leukemia-associated nc-insulin-like growth factor1 receptor IGF1R-Activator RNA1 LUNAR1 serves as a downstream target of Notch-signaling in the same time LUNAR1 acts through Notch-signaling stimulation LUNAR1 is a transcript of 491 nucleotides nt gene on chromosome 15q263 with four exons and poly A tail 36 LUNAR1 was identified to be elevated in CRC tissues triggered by Notch1 stimulation accelerating CRC progression via retaining IGF1R expression 37 being a positive regulator of cell division Aim of the study Per few research publications on both notch-related lncRNAs SNHG3 and LUNAR1 in CRC prognosis or CRC risk assessment as well as patients stratification based on clinico-pathological characteristics therefore assessment of the clinical utility of SNHG3 and LUNAR1 lncRNAs fold change expressions in CRC patients peripheral blood liquid biopsy is alarming as step toward implementing ncRNA precision Study objectives to assess first the expression level and pattern of Notch-associated lncRNAs SNHG3 and LUNAR1 in peripheral blood liquid biopsy polled from treatment-naïve Egyptian CRC patients cohort compared to age-matched and sex-matched apparently healthy volunteer subjects as controls Second to evaluate lncRNAs SNHG3 and LUNAR1 expression usefulness as sensitive non-invasive prognostic bio-molecular markers for CRC monitoring Third to investigate the correlation between SNHG3 and LUNAR1 expression with CRC clinic-pathological features Finally to explore the relevance of the investigated lncRNAs for CRC patients clinical features outcomes assessment All these objectives to be confirmed or ruled out by in silico analysis and bioinformatics databases search SUBJECTS 21 Sample Size and Power of the Study In accordance with prior reference studies 3638 the sample size was estimated utilizing sample size online calculator httpsriskcalcorgsamplesize for comparison of the area under the curve AUC with a null hypothesis value done January 2021 through using two-sided significance level of 005 and the power 1-beta of 095 as the two-sided confidence level of 95 Groups will be 45 samples for CRC patients and 17 controls for SNHG3 and 48 CRC patients vs 16 controls for LUNAR1
22 Study Design Case-controlled retrospective observational study The study was carried out from June 2021 to October 2022
23 Institutional Review Board IRB Statement This study was ethically approved by the review board Research Ethical Committee REC of Faculty of Pharmacy Ain Shams University REC ID 15 2021 that was approved by the Faculty of Medicine Ain Shams University Hospitals Ain Shams University REC This research investigation was conducted out in compliance with Declaration of Helsinki principles World Medical Association Declaration of Helsinki Ethical principles for medical research involving human subjects 2013 Every volunteering participant in the study whether apparently healthy controls or CRC patients were fully aware of the studys objective and signed a written comprehensive informed consent IC form ethically-approved were considered in
24 Study Participants 241 Patients group 70 CRC Egyptian patients admitted to the Oncology Center of the Faculty of Medicine Ain Shams University Hospitals ASUH or the Oncological Surgery Department of Dar-El-Shafa Hospital before surgical treatment or CRC neo-adjuvant treatment if they were eligible and agreed to participate signed the IC were recruited in the research Male-to-female was 3040 with an age-range minimum-maximum 24 -79 years
242 Apparently healthy control group 26 randomly-chosen age-matched and sex-matched healthy subjects served as the control group Male-to-female was 917 and age range minimum-maximum 35 -78 years Control group subjects were chosen from those who came to visit hospital workers in the hospital or from blood donors at the ASUH Blood Donation Unit None of the control group did take any medications or have any diseases upon questioner and blood samples was taken for CBC
243 Inclusion and Exclusion Criteria Patients came to the Oncology Center of ASUH or the Oncological Surgery Department of Dar-El-Shafa Hospital who experienced a range of colonic symptoms such as constipation abdominal discomfort rectal bleeding and abrupt weight loss were included in the study when diagnosis of CRC was clinically confirmed by abdominal radiography colonoscopy and histopathology
Exclusion criteria patients who received chemotherapy radiotherapy or undergone surgery Patients with other types of cancer were also excluded Individuals with missing data were not included
244 Patients Pathological and Clinical Data For each CRC participant colonoscopy outcomes abdominal radiographic imaging pathological evaluations were used to define CRC staging Tumor lymph-node metastasis TNM staging criteria was based on the American Joint Committee on Cancer AJCC 39 criterion
CRC study participants family history hypertension HTN or diabetes mellitus DM were recorded as non-communicable diseases NCD to correlate them to notch-related lncRNAs studied
Inflammatory conditions such as ulcerative colitis tumor size tumor location if rectal colonic or rectosigmoid being mucinous tumor or not tumor invasion depth lymph-node metastasis LNM presence of vascular invasion or not tumor differentiation status adenocarcinoma moderately differentiated adenocarcinoma or poorly differentiated adenocarcinoma as well as the presence of the sub histologic features signet ring cell or not were collected from eligible volunteering CRC patients files
3 Methods 31 In Silico Analysis 311 Notch-related lncRNA Bioinformatics from various databases via accessed January 2021 and revised July 2023 Gene Set Enrichment Analysis GSEA 40 with ClusterProfiler utilizing the Kyoto Encyclopedia of Genes and Genomes KEGG 4142 from Genome net httpswwwgenomejp was used to analyze the functional enrichment of genes diseases networks drugs and pathways related to Notch-signaling Ensemble database search 43 httpswwwensemblorgindexhtml for the potential probable Notch-related lncRNAs SNHG3 and LUNAR1 genes National Center for Biotechnology Information NCBI httpswwwncbinlmnihgov search for characterization of the investigated hsa lncRNAs SNHG3 gene and transcripts 2 and hsa lncRNAs LUNAR1 gene and transcript 1 HUGO Gene Nomenclature Committee HGNC 44 httpswwwgenenamesorg hsa lncRNAs genes SNHG3 and LUNAR1
312 LncRNADisease v30 Expression LncRNA and Disease Database version 30 45 to explore the Notch-related lncRNAs expression CRC retrieved from validated experimental results in publications or predicted httpwwwrnanutnetlncrnadiseaseindexphphome 313 Expression via ENCORI Project Pan-Cancer Analysis Platform 46 httpsrnasysucomencoripanGeneDiffExpphp of 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 Differential expression Analysis for SNHG3 LUNAR1 and its transcript IGF1R expression levels in CRC tumor samples vs control samples
314 Functional Enrichment Analysis and Targeted Pathways KEGG Targeted Pathways and STRING Protein-Protein Interaction PPI Networks version 115 httpsstring-dborg 4748 Accessed on July 2023
LncRNAWiki 20 LncRNA - LncRNAWiki - CNCB-NGDChttpsngdccncbaccnlncrnawiki Accessed August 30th 2023 32 Blood Samples From CRC patients and healthy volunteers five milliliters of peripheral venous blood fluid biopsy were collected and stored in clot-activating polymer gel vacutainers Greiner Bio-One GmbH Australia The samples were centrifuged in vacutainers at a speed of 4000 rpm for ten minutes at room temperature 25C The obtained serum was aliquoted and stored at -80C in RNAse-free Eppendorf tubes
321 Total RNA Extraction Using the miRNeasy Mini kit Cat No 217004 Qiagen Hilden Germany total RNA was isolated from sera in accordance with the protocols instructions Aliquots of the extracted RNA were kept at -80C after being dissolved in 30 μl of RNase-free water
322 Quantitation of purified RNA Using a NanoDrop-1000 spectrophotometer Thermo Scientific Wilmington DE USA the isolated RNAs purity and concentration are evaluated RNAs quantity was determined in the sample utilizing absorbance at 260 nm A260 1 44 ngμl In addition the purity of RNA was evaluated utilizing A260280 nm ratios The acceptable 260280 ratio ranges from 18 to 21 whereas the 260230 ratio is more than 17
323 Reverse Transcription Complementary DNA cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor Cat No 4374966 Applied Biosystems ThermoFisher Scientific USA according to the manufacturers regulations In a 20 µl reaction volume reverse transcription was carried out at 25 C for 10 minutes 37 C for 120 minutes and then underwent heat inactivation for 5 minutes at 85 C The produced cDNA was maintained at -80C till further investigations
324 Expression Measurement of lncRNAs Using Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction qRT-PCR QRT-PCR was carried out in a 20 µl reaction using the TaqMan Gene Expression Master Mix Cat No 4370048 Applied Biosystems ThermoFisher Scientific USA In order to determine the expression levels of lncRNAs SNHG3 and LUNAR1 TaqMan gene expression assays for human SNHG3 Hs05055352_s1 Cat No 4448892 ThermoFisher Scientific USA and human LUNAR1 Hs03829521_s1 Cat No 4426961 ThermoFisher Scientific USA were used and the TaqMan gene expression assay for human glyceraldehyde 3-phosphate dehydrogenase GAPDH Cat No 4326317E ThermoFisher Scientific USA was used as an endogenous standard to normalize the values The reaction was conducted using the StepOne qRT-PCR technique Applied Biosystems CA USA The thermal cycling strategy was as follows a stage of initial uracil-N-glycosylase UNG incubation at 50C for 2 minutes followed by activation step lasting 10 minutes at 95 C proceeded by 40 cycles of denaturation for 15 sec at 95 C annealing and extending for 1 minute at 60 C
Per the manufacturer the assay limit of detection LoD can detect cDNA template from 1pg to 100 ng in nuclease free water
The cycle threshold Ct technique as a fold change 2-ΔΔCt was used to calculate and normalize the lncRNAs expression levels utilizing GAPDH as the housekeeping gene ΔCt was obtained by subtracting the Ct values of GAPDH from either SNHG3 or LUNAR1 Ct values 49 where ΔΔCt ΔCtCRC samples - ΔCthealthy control samples 325 CEA and CA19-9 determination by electrochemiluminescence immunoassay One portion of the obtained sera was utilized for the purpose of determining CEA and CA19-9 tumor markers Electrochemiluminescence immunoassay utilizing Cobas e 602 developed by Roche Diagnostics GmbH Germany was used to determine the serum concentrations of CEA Cat No 11731629 322 and CA199 Cat No 11776193500 according to the manufacturers protocol
326 Routine biochemical testing results record from patients files Liver function tests aspartate aminotransferase AST and Alanine aminotransferase ALT as well as serum creatinine and serum urea levels hemoglobin Hgb platelet count lymphocytes count and prothrombin time PT were all gathered from patients files
327 Ratios and Indices In meters participants heights and in kilograms their weights were recorded to calculate Body mass index BMI in kgm2 where overweight subjects have BMI of 25-299 kgm2 30 kgm2 or over are obese while 185-249 kgm2 is indicative of normal weight httpswwwnhlbinihgovhealtheducationallose_wtBMIbmicalchtm The immune response-related inflammation indicator biomarker able to predict the accompanying inflammatory disorder severity is the platelet-to-lymphocytes ratio PLR 1050
33 Statistical Analysis With the aid of GraphPad Prism version 901 GraphPad Software San Diego CA USA SPSS 230 statistical package for social studies software IBM Armonk NY MedCalc Statistic Software version 191 MedCalc Software by Ostend Belgium and Microsoft Office Excel 2019 statistical analysis was carried out
Chi-square test χ2 was utilized to assess the associations between participants characteristics and the groups The Shapiro-Wilk normality test as well as Kolmogorov-Smirnov test were applied to determine the pattern of the normal distribution for the both groups and subgroups of data Data was expressed as mean SD when it passed the normality test Students t test was utilized to determine any significant differences between two normally distributed groups Additionally one-way ANOVA F followed by post hoc Tukeys multiple comparison test was used when required to determine significant variations between multiple groups While the data that didnt pass the normality test was expressed as median interquartile range IQR 25th percentile-75th percentile The Mann-Whitney U test was used to pinpoint the significant changes between two sets of participants Moreover the Kruskal-Wallis H test and consequently Dunns multiple comparison test were used when needed to determine whether there were statistically significant differences between different groups
The receiver operating characteristic ROC curve as well as AUC were implemented to evaluate the abilities of serum lncRNAs SNHG3 and LUNAR1 for discrimination between groups and with groups for subgrouping identification The best cut-off sensitivities SNs specificities SPs as well as negative and positive predictive values NPVs and PPVs respectively were all determined using the ROC curve with AUC estimated range from 0 to 1
Negative likelihood ratios LRs are employed in medical testing to evaluate the marker discriminative efficiency The ratio which denotes the likelihood that a person has the disease or condition confirms the SNs and SPs identified by the ROC curve SN and SP provide a different meaning for the LR with negative LR equal to 100-SNSP
Multiple regression models were used to assess the influence of the participants demographic and patients clinicopathological data as independent factors on the expression levels of the lncRNAs SNHG3 and LUNAR1 that we considered as the dependent variables Correlation between numerous variables was assessed using Spearmans correlation coefficient r Statistical analysis tests are set significant when the two-tailed p-value is 005
Problem Despite significant improvements in CRC surgical or radiological interventional treatment approaches with neo-adjuvant therapies patients prognosis remains bleak 45 Metastases and post-surgical tumor recurrence are prevalent particularly in more advanced cancer cases 6 which may account for the increased number of cases and fatalities prediction being against the national efforts for achieving Egypt Vision 2030 implementing SDGs
Problem statement Conventionally used CRC prognostic markers for monitoring treatment outcome and pointing to cancer recurrence are carbohydrate antigen 199 CA199 andor carcinoembryonic antigen CEA that are not that sensitive 7 per patients subclassification or stratification related to CRC pathological characteristics for more efficient and earlier prognosis Thence a growing demand for sensitive and precise bio-molecular markers better correlating with CRC prognostic markers andor CRC clinical outcome 8 that is if successful would constitute the bases for Better Health SDG3 and less cancer recurrence and therefore less mortality
Liquid biopsies are used for cancer diagnosis or prognosis like breast cancer leukemia liver cancer or CRC via measurement of tumor-derived bio-molecular markers including circulating ncRNAs including long non-coding RNAs lncRNAs microRNA exosomal ncRNAs oncogenes or tumor suppressor genes and their mutations tumor-related-cytokines and down-stream target proteins 9-22
After extensive literature search and mining to mind-the-research-gaps for better Cancer Epigenetics Study a Step-toward ncRNA-Precision we have chosen in the current work two notch-related lncRNAs to study in relation-to-CRC prognosis
Notch-signaling pathway is an ubiquitous cascade within species to control a broad variety of biological events including cell division proliferation and cell death 2324 Recent investigations have revealed the fundamental role of Notch-cascade in CRC evolution 25 Intestinal epithelial cells homeostatic self-renewal and tumor-promoting transformation can both be managed by Notch signaling 26 Stimulation of Notch-cascade can be epigenetically triggered by dysregulated non-protein coding RNAs ncRNAs expressions 27 a hot area of research nowadays to figure out the impact of Notch-related ncRNAs on CRC risk andor progression
The significant utility of tumor-expressed Notch-associated lncRNAs as prognostic malignancy indicators proves how they are connected to carcinogenesis or metastasis and therefore reflect the outcomes in different cancer types including CRC 28-31
Small Nucleolar RNA Host Gene3 SNHG3 is 4950bp lncRNA situated on chromosome 1p353 32 In breast cancer upregulated SNHG3 triggers Notch system activation as a result of its competitive binding to human homo sapiens hsa micro-RNA miR hsa-miR-154-3p exacerbating proliferation and metastasis of cancer cells 33 Moreover SNHG3 positively regulates Notch1 expression in ovarian cancer through hsa-miR-139-5p suppression accelerating tumor cells proliferation and migration 34 SNHG3 exerted a carcinogenic role in prostate cancer osteosarcoma glioma gastric cancer laryngeal cancer bladder cancer and CRC 32 Huang et al reported SNHG3 elevated expression in CRC cells and tissues stimulating cancer progression through sponging hsa-miR-182-5p 35 Therefore SNHG3 is considered as a malignancy enhancer that regulates Notch system in various cancer types
Leukemia-associated nc-insulin-like growth factor1 receptor IGF1R-Activator RNA1 LUNAR1 serves as a downstream target of Notch-signaling in the same time LUNAR1 acts through Notch-signaling stimulation LUNAR1 is a transcript of 491 nucleotides nt gene on chromosome 15q263 with four exons and poly A tail 36 LUNAR1 was identified to be elevated in CRC tissues triggered by Notch1 stimulation accelerating CRC progression via retaining IGF1R expression 37 being a positive regulator of cell division Aim of the study Per few research publications on both notch-related lncRNAs SNHG3 and LUNAR1 in CRC prognosis or CRC risk assessment as well as patients stratification based on clinico-pathological characteristics therefore assessment of the clinical utility of SNHG3 and LUNAR1 lncRNAs fold change expressions in CRC patients peripheral blood liquid biopsy is alarming as step toward implementing ncRNA precision Study objectives to assess first the expression level and pattern of Notch-associated lncRNAs SNHG3 and LUNAR1 in peripheral blood liquid biopsy polled from treatment-naïve Egyptian CRC patients cohort compared to age-matched and sex-matched apparently healthy volunteer subjects as controls Second to evaluate lncRNAs SNHG3 and LUNAR1 expression usefulness as sensitive non-invasive prognostic bio-molecular markers for CRC monitoring Third to investigate the correlation between SNHG3 and LUNAR1 expression with CRC clinic-pathological features Finally to explore the relevance of the investigated lncRNAs for CRC patients clinical features outcomes assessment All these objectives to be confirmed or ruled out by in silico analysis and bioinformatics databases search SUBJECTS 21 Sample Size and Power of the Study In accordance with prior reference studies 3638 the sample size was estimated utilizing sample size online calculator httpsriskcalcorgsamplesize for comparison of the area under the curve AUC with a null hypothesis value done January 2021 through using two-sided significance level of 005 and the power 1-beta of 095 as the two-sided confidence level of 95 Groups will be 45 samples for CRC patients and 17 controls for SNHG3 and 48 CRC patients vs 16 controls for LUNAR1
22 Study Design Case-controlled retrospective observational study The study was carried out from June 2021 to October 2022
23 Institutional Review Board IRB Statement This study was ethically approved by the review board Research Ethical Committee REC of Faculty of Pharmacy Ain Shams University REC ID 15 2021 that was approved by the Faculty of Medicine Ain Shams University Hospitals Ain Shams University REC This research investigation was conducted out in compliance with Declaration of Helsinki principles World Medical Association Declaration of Helsinki Ethical principles for medical research involving human subjects 2013 Every volunteering participant in the study whether apparently healthy controls or CRC patients were fully aware of the studys objective and signed a written comprehensive informed consent IC form ethically-approved were considered in
24 Study Participants 241 Patients group 70 CRC Egyptian patients admitted to the Oncology Center of the Faculty of Medicine Ain Shams University Hospitals ASUH or the Oncological Surgery Department of Dar-El-Shafa Hospital before surgical treatment or CRC neo-adjuvant treatment if they were eligible and agreed to participate signed the IC were recruited in the research Male-to-female was 3040 with an age-range minimum-maximum 24 -79 years
242 Apparently healthy control group 26 randomly-chosen age-matched and sex-matched healthy subjects served as the control group Male-to-female was 917 and age range minimum-maximum 35 -78 years Control group subjects were chosen from those who came to visit hospital workers in the hospital or from blood donors at the ASUH Blood Donation Unit None of the control group did take any medications or have any diseases upon questioner and blood samples was taken for CBC
243 Inclusion and Exclusion Criteria Patients came to the Oncology Center of ASUH or the Oncological Surgery Department of Dar-El-Shafa Hospital who experienced a range of colonic symptoms such as constipation abdominal discomfort rectal bleeding and abrupt weight loss were included in the study when diagnosis of CRC was clinically confirmed by abdominal radiography colonoscopy and histopathology
Exclusion criteria patients who received chemotherapy radiotherapy or undergone surgery Patients with other types of cancer were also excluded Individuals with missing data were not included
244 Patients Pathological and Clinical Data For each CRC participant colonoscopy outcomes abdominal radiographic imaging pathological evaluations were used to define CRC staging Tumor lymph-node metastasis TNM staging criteria was based on the American Joint Committee on Cancer AJCC 39 criterion
CRC study participants family history hypertension HTN or diabetes mellitus DM were recorded as non-communicable diseases NCD to correlate them to notch-related lncRNAs studied
Inflammatory conditions such as ulcerative colitis tumor size tumor location if rectal colonic or rectosigmoid being mucinous tumor or not tumor invasion depth lymph-node metastasis LNM presence of vascular invasion or not tumor differentiation status adenocarcinoma moderately differentiated adenocarcinoma or poorly differentiated adenocarcinoma as well as the presence of the sub histologic features signet ring cell or not were collected from eligible volunteering CRC patients files
3 Methods 31 In Silico Analysis 311 Notch-related lncRNA Bioinformatics from various databases via accessed January 2021 and revised July 2023 Gene Set Enrichment Analysis GSEA 40 with ClusterProfiler utilizing the Kyoto Encyclopedia of Genes and Genomes KEGG 4142 from Genome net httpswwwgenomejp was used to analyze the functional enrichment of genes diseases networks drugs and pathways related to Notch-signaling Ensemble database search 43 httpswwwensemblorgindexhtml for the potential probable Notch-related lncRNAs SNHG3 and LUNAR1 genes National Center for Biotechnology Information NCBI httpswwwncbinlmnihgov search for characterization of the investigated hsa lncRNAs SNHG3 gene and transcripts 2 and hsa lncRNAs LUNAR1 gene and transcript 1 HUGO Gene Nomenclature Committee HGNC 44 httpswwwgenenamesorg hsa lncRNAs genes SNHG3 and LUNAR1
312 LncRNADisease v30 Expression LncRNA and Disease Database version 30 45 to explore the Notch-related lncRNAs expression CRC retrieved from validated experimental results in publications or predicted httpwwwrnanutnetlncrnadiseaseindexphphome 313 Expression via ENCORI Project Pan-Cancer Analysis Platform 46 httpsrnasysucomencoripanGeneDiffExpphp of 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 Differential expression Analysis for SNHG3 LUNAR1 and its transcript IGF1R expression levels in CRC tumor samples vs control samples
314 Functional Enrichment Analysis and Targeted Pathways KEGG Targeted Pathways and STRING Protein-Protein Interaction PPI Networks version 115 httpsstring-dborg 4748 Accessed on July 2023
LncRNAWiki 20 LncRNA - LncRNAWiki - CNCB-NGDChttpsngdccncbaccnlncrnawiki Accessed August 30th 2023 32 Blood Samples From CRC patients and healthy volunteers five milliliters of peripheral venous blood fluid biopsy were collected and stored in clot-activating polymer gel vacutainers Greiner Bio-One GmbH Australia The samples were centrifuged in vacutainers at a speed of 4000 rpm for ten minutes at room temperature 25C The obtained serum was aliquoted and stored at -80C in RNAse-free Eppendorf tubes
321 Total RNA Extraction Using the miRNeasy Mini kit Cat No 217004 Qiagen Hilden Germany total RNA was isolated from sera in accordance with the protocols instructions Aliquots of the extracted RNA were kept at -80C after being dissolved in 30 μl of RNase-free water
322 Quantitation of purified RNA Using a NanoDrop-1000 spectrophotometer Thermo Scientific Wilmington DE USA the isolated RNAs purity and concentration are evaluated RNAs quantity was determined in the sample utilizing absorbance at 260 nm A260 1 44 ngμl In addition the purity of RNA was evaluated utilizing A260280 nm ratios The acceptable 260280 ratio ranges from 18 to 21 whereas the 260230 ratio is more than 17
323 Reverse Transcription Complementary DNA cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor Cat No 4374966 Applied Biosystems ThermoFisher Scientific USA according to the manufacturers regulations In a 20 µl reaction volume reverse transcription was carried out at 25 C for 10 minutes 37 C for 120 minutes and then underwent heat inactivation for 5 minutes at 85 C The produced cDNA was maintained at -80C till further investigations
324 Expression Measurement of lncRNAs Using Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction qRT-PCR QRT-PCR was carried out in a 20 µl reaction using the TaqMan Gene Expression Master Mix Cat No 4370048 Applied Biosystems ThermoFisher Scientific USA In order to determine the expression levels of lncRNAs SNHG3 and LUNAR1 TaqMan gene expression assays for human SNHG3 Hs05055352_s1 Cat No 4448892 ThermoFisher Scientific USA and human LUNAR1 Hs03829521_s1 Cat No 4426961 ThermoFisher Scientific USA were used and the TaqMan gene expression assay for human glyceraldehyde 3-phosphate dehydrogenase GAPDH Cat No 4326317E ThermoFisher Scientific USA was used as an endogenous standard to normalize the values The reaction was conducted using the StepOne qRT-PCR technique Applied Biosystems CA USA The thermal cycling strategy was as follows a stage of initial uracil-N-glycosylase UNG incubation at 50C for 2 minutes followed by activation step lasting 10 minutes at 95 C proceeded by 40 cycles of denaturation for 15 sec at 95 C annealing and extending for 1 minute at 60 C
Per the manufacturer the assay limit of detection LoD can detect cDNA template from 1pg to 100 ng in nuclease free water
The cycle threshold Ct technique as a fold change 2-ΔΔCt was used to calculate and normalize the lncRNAs expression levels utilizing GAPDH as the housekeeping gene ΔCt was obtained by subtracting the Ct values of GAPDH from either SNHG3 or LUNAR1 Ct values 49 where ΔΔCt ΔCtCRC samples - ΔCthealthy control samples 325 CEA and CA19-9 determination by electrochemiluminescence immunoassay One portion of the obtained sera was utilized for the purpose of determining CEA and CA19-9 tumor markers Electrochemiluminescence immunoassay utilizing Cobas e 602 developed by Roche Diagnostics GmbH Germany was used to determine the serum concentrations of CEA Cat No 11731629 322 and CA199 Cat No 11776193500 according to the manufacturers protocol
326 Routine biochemical testing results record from patients files Liver function tests aspartate aminotransferase AST and Alanine aminotransferase ALT as well as serum creatinine and serum urea levels hemoglobin Hgb platelet count lymphocytes count and prothrombin time PT were all gathered from patients files
327 Ratios and Indices In meters participants heights and in kilograms their weights were recorded to calculate Body mass index BMI in kgm2 where overweight subjects have BMI of 25-299 kgm2 30 kgm2 or over are obese while 185-249 kgm2 is indicative of normal weight httpswwwnhlbinihgovhealtheducationallose_wtBMIbmicalchtm The immune response-related inflammation indicator biomarker able to predict the accompanying inflammatory disorder severity is the platelet-to-lymphocytes ratio PLR 1050
33 Statistical Analysis With the aid of GraphPad Prism version 901 GraphPad Software San Diego CA USA SPSS 230 statistical package for social studies software IBM Armonk NY MedCalc Statistic Software version 191 MedCalc Software by Ostend Belgium and Microsoft Office Excel 2019 statistical analysis was carried out
Chi-square test χ2 was utilized to assess the associations between participants characteristics and the groups The Shapiro-Wilk normality test as well as Kolmogorov-Smirnov test were applied to determine the pattern of the normal distribution for the both groups and subgroups of data Data was expressed as mean SD when it passed the normality test Students t test was utilized to determine any significant differences between two normally distributed groups Additionally one-way ANOVA F followed by post hoc Tukeys multiple comparison test was used when required to determine significant variations between multiple groups While the data that didnt pass the normality test was expressed as median interquartile range IQR 25th percentile-75th percentile The Mann-Whitney U test was used to pinpoint the significant changes between two sets of participants Moreover the Kruskal-Wallis H test and consequently Dunns multiple comparison test were used when needed to determine whether there were statistically significant differences between different groups
The receiver operating characteristic ROC curve as well as AUC were implemented to evaluate the abilities of serum lncRNAs SNHG3 and LUNAR1 for discrimination between groups and with groups for subgrouping identification The best cut-off sensitivities SNs specificities SPs as well as negative and positive predictive values NPVs and PPVs respectively were all determined using the ROC curve with AUC estimated range from 0 to 1
Negative likelihood ratios LRs are employed in medical testing to evaluate the marker discriminative efficiency The ratio which denotes the likelihood that a person has the disease or condition confirms the SNs and SPs identified by the ROC curve SN and SP provide a different meaning for the LR with negative LR equal to 100-SNSP
Multiple regression models were used to assess the influence of the participants demographic and patients clinicopathological data as independent factors on the expression levels of the lncRNAs SNHG3 and LUNAR1 that we considered as the dependent variables Correlation between numerous variables was assessed using Spearmans correlation coefficient r Statistical analysis tests are set significant when the two-tailed 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