Ignite Creation Date:
2024-10-26 @ 3:36 PM
Last Modification Date:
2024-10-26 @ 3:36 PM
Study NCT ID:
NCT06531902
Status:
COMPLETED
Last Update Posted:
None
First Post:
2024-05-16
Brief Title:
LncRNA Nicotinamide Nucleotide Transhydrogenase-Antisense RNA1 NNT-AS1 in CRC
Sponsor:
None
Organization:
None
Study Overview
Official Title:
LncRNA NNT-AS1Hsa-miR-485-5pHSP90 Axis In-silico and Clinical Prospect correlated-to Histologic Grades-based CRC Stratification A Step Toward ncRNA Precision
Status:
COMPLETED
Status Verified Date:
2024-07
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
No
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The current study will compare the blood expression level of lncRNA NNT-AS1 and hsa-miR-485-5p as well as heat shock protein 90 HSP90 serum levels in both healthy and CRC Egyptian patients cohort peripheral blood samples Second examine if lncRNA NNT-AS1hsa-miR-485 5pHSP90 axis or individually investigated would be used as a non-invasive molecular precision biomarker in liquid biopsy for better diagnostic utility for CRC patients Also based on CRC patients group stratification by histologic grades 1-3 Finally investigate the probable linkcorrelation between lncRNA NNT-AS1hsa-miR-485-5pHSP90 as an axis or individually in CRC Egyptian patients cohort in relation to demographic data or clinicopathological characteristics All findings will be confirmed or ruled out by in silico means as well
Detailed Description:
Colorectal cancer CRC is a heterogeneous malignancy that affects the colon and rectum It is the third most prevalent and lethal malignancy with roughly 19 million new cases and nearly 94 of cancer-related deaths in 2020 worldwide The CRC survival rate mostly relies on the diseases stage at the time of diagnosis Thus early detection of CRC provides a higher 5-year survival probability of 80-90 compared to just 14 in the late stage with distant metastases Colonoscopy is the gold standard method for CRC diagnosis Alongside its great accuracy it is an invasive technique with considerable mortality risks such as intestinal perforation and bleeding limiting its usage for screening Fecal occult blood test carbohydrate antigen 19-9 CA19-9 and carcinoembryonic antigen CEA are the currently available CRC early diagnosis screening tumor markers TMs tests Although CEA and CA19-9 tests are simple non-invasive affordable and safe their diagnostic accuracy is limited Screening and early diagnosis for CRC have been identified as the most significant factor for lowering mortality Long non-coding RNA lncRNA which are more than 200 nucleotides non-coding RNAs ncRNAs are either over-expressed or down-regulated in numerous malignancies and act as potential molecular mediators for various tumors progression Overexpressed lncRNAs act as oncogenes enhancing tumor cell proliferation apoptosis invasion metastasis and autophagy Several lncRNAs have been linked to the incidence metastasis and therapeutic resistance of CRC Recent research suggested that the lncRNA Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 NNT-AS1 which is localized in the 5p12 human chromosome is involved in gene expression regulation and plays important roles in a variety of cancers such as breast cancer cervical cancer hepatocellular carcinoma and osteosarcoma however insufficient research was performed about lncRNA NNT-AS1 role in CRC MicroRNAs miRNAs are small ncRNAs with 22 nucleotides length that undertake a variety of biological functions such as cell proliferation apoptosis and angiogenesis The mature miRNA species also known as the miRNA-5p guide strand and - 3p species passenger miRNA can originate from both the 5 and 3 arms of the precursor duplex The arm that is destined to be loaded into the RNA-induced silencing complex is the guide strand Passenger miRNAs were assumed to be entirely destroyed however deep sequencing investigations have revealed that certain minor miRNAs retain and in fact play a meaningful role in gene regulation Numerous studies have found a substantial association between miRs dysregulation and tumor metastasis Homo sapiens hsa-miR-485-5p is located on the human chromosome 14q32 and has been identified as an anticancertumor suppressor gene in several human malignancies through targeting and regulating the expression of downstream cell proliferation and invasion genes The regulatory function of hsa-miR-485-5p in CRC is still undetermined LncRNAs-miRs interaction would control the incidence andor progression of various cancer types as well as being considered as potential target for future treatment invention LncRNA NNT-AS1 was found to promote proliferation migration and invasion of cholangiocarcinoma via sponging hsa-miR-485 This sponging process elevated β-catenin and B-cell chronic lymphocytic leukemia CLLlymphoma 9 BCL9 in response LncRNA NNT-AS1s relation to hsa-miR-485-5p in CRC remains to be determined clinically In most cells under non-stress settings heat shock protein 90 HSP90 are highly conserved molecular chaperones that make up 1-2 of all cellular proteins Important functions of HSP90 proteins include protein folding and degradation Physiologically the molecular chaperone HSP90AB1 interactions with other co-chaperones play a crucial part for folding of the newly generated proteins or in stabilizing and refolding denatured proteins following stress but pathologically is involved in tumorigenesis For instance within osteosarcoma hsa-miR-485-5p binds to the 3
-untranslated region UTR of HSP90 messenger RNA mRNA to reduce HSP90 protein production hence exerting a tumor suppressor effect Therefore research is warranted to determine the clinical significance of the lncRNA NNT-AS1hsa-miR-485-5pHSP90 axis in CRC patients
The current study will compare the blood expression level of lncRNA NNT-AS1 and hsa-miR-485-5p as well as HSP90 serum levels in both healthy and CRC Egyptian patients cohort peripheral blood samples Second examine if lncRNA NNT-AS1hsa-miR-485-5pHSP90 axis or individually investigated would be used as non-invasive molecular precision biomarker in liquid biopsy for better diagnostic utility for CRC patients Also based on CRC patients group stratification by histologic grades 1-3 Finally investigate the probable linkcorrelation between lncRNA NNT-AS1hsa-miR-485-5pHSP90 as an axis or individually in CRC Egyptian patients cohort in relation to demographic data or clinicopathological characteristics All findings will be confirmed or ruled out by in silico means as well
31 Patient group A total of 60 CRC patients were enrolled in the study CRC patients were treatment-naïve Egyptian patients cohort admitted to the Clinical Oncology Clinic National Cancer Institute NCI Cairo University Cairo Egypt Male-to-female 11 3030 and their age range 24-76 years
32 Control group 28 age-matched and sex-matched apparently healthy volunteers randomly selected from subjects enlisted during normal check-up examination or during blood donation The control group participants were not taking any medication or suffering from any disease Controls age range is 40-60 years and 1315 male-to-female For the patients group n 60 a full history was recorded If a patient met the inclusion criteria and after giving their approval for participation in the study and signing the informed consent peripheral blood samples were taken at the diagnosis time If a patient met the exclusion criteria blood samples were not taken Patient inclusion criteria were those who visited the Colonoscopy Unit for colorectal examination and had a variety of colonic symptoms including CRC noticeable symptoms constipation abdominal pain rectal bleeding and sudden weight loss CRC diagnosis was clinically confirmed by colonoscopy abdominal radio-imaging and histopathological examination Patients exclusion criteria included individuals receiving chemotherapy radiation or undergone surgery patients with blood disorders or any cancer other than CRC Individuals with inadequate data or missing histopathological diagnoses as well as those with distant metastases were excluded from the study
321 Patients Pathological and Clinical Data The clinical assessment of CRC patients tumor was done at the Pathology Unit NCI Cairo Cairo University In addition to a patients colorectal surgery history the complete family history of cancer disease was recorded for all CRC participants At the NCI CRC staging was determined by the colonoscopic results abdominal radiography pathological analyses and clinical decisions relying on these results and the American Joint Committee on Cancer AJCC criteria 2010 Patients are categorized into three stages stage III local cancer stage III lymph node LN localized involvement N1-x and stage IV distant metastasis M1 LN involvement with enlargement incidence as either no N0 or present N1 was noted from patient files The CRC patients family history smoking status the noncommunicable diseases status as diabetes mellitus DM and hypertension HTN were recorded Tumor site tumor size mucinous or not lymph node metastasis LNM tumor invasion or vascular invasion tumor differentiation tumor grade tumor-node-metastasis TNM staging inflammation status as inflammatory bowel disease IBD CRC location if colonic or rectal as well as if transverse sigmoid rectosigmoid and rectal CRC clinical symptoms were assessed by the NCI Biochemical Analysis Unit or the Statistics Unit collected from patients files Hemoglobin Hgb prothrombin time PT erythrocyte sedimentation rate ESR platelet count lymphocyte count lactate dehydrogenase LDH C-reactive protein CRP were all recorded from patient files done at the NCI Cairo Egypt Central Clinical Biochemistry Lab 42 Blood samples Five milliliters of peripheral venous blood liquid biopsy were collected from the antecubital vein from controls and CRC patients into clot activator polymer gel vacutainers Vacutainers of completely coagulated samples were centrifuged at 4000 rpm for 10 min at room temperature 25 C The collected serum was aliquoted and kept at - 80 C in three DNase RNase free Eppendorf tubes until analysis
43 Total RNA extraction Total RNA was extracted from serum samples according to the procedure guidelines The extracted RNA was dissolved in 40 µL RNase free water and stored in aliquots at - 80 C
44 Quantitation of purified RNA including miRNAs The extracted RNAs purity and concentration are assessed using a spectrophotometer The quantity of RNA in the sample was assessed using absorbance at 260 nm A260 1 44 ngµL and RNA purity was assessed using absorbance at 260280 nm ratios
45 Reverse transcription and expression measurement of ncRNAs using quantitative real-time reverse transcription polymerase chain reaction qRT-PCR 451 lncRNA NNT-AS1 The real-time RT2 First Strand Kit was used for complementary DNA cDNA synthesis as directed by the manufacturers instructions The obtained cDNA was stored at - 20 C After reverse transcription qRT-PCR was utilized to measure the expression of the lncRNA NNT-AS1 to evaluate the expression level of the lncRNA NNT-AS1 the primer RT2 lncRNA qPCR Assay for Human NNT-AS1 LPH15988A-200 Cat No 330701 was used and data were normalized using RT2 lncRNA qPCR Assay for Human GAPDH LPH31725A-200 Cat No 330701 as endogenous control 452 hsa-miR-485-5p cDNA synthesis was performed as directed by the procedure guidelines The obtained cDNA was stored at - 20 C After reverse transcription qRT-PCR was utilized to measure the expression of the hsa-miR-485-5p To detect the expression level of the hsa-miR-485-5p the primer hsa-hsa-miR-485-5p was used and data were normalized miRNA PCR Assay YP00203901 Cat No 339306 as endogenous control The ncRNA expression level as fold change was calculated and normalized by using the cycle threshold Ct approach as fold change 2- ΔΔCt with GAPDH or SNORD38B hsa as the housekeeping genes for lncRNA NNT-AS1 and hsa-miR-485-5p respectively ΔCt was calculated by deducting the Ct values of GAPDH and SNORD38B hsa from the ones of the lncRNA NNT-AS1 and hsa-miR-485-5p under study respectively
ΔΔCt ΔCt cancer samples - ΔCt control samples where ΔCtCt target - Ct reference 453 HSP90 protein quantification by Enzyme-linked immunosorbent assay ELISA The HSP90 was quantified by ELISA using the commercially available kit according to the manufacturers instructions The Human HSP90 alpha solid-phase sandwich ELISA measures the amount of target bound between a matching antibody pair In the wells of the supplied microplate a target-specific antibody has been pre-coated In these wells samples standards or controls bind to the immobilized capture antibody The sandwich is constructed by adding the second detector antibody followed by the addition of a substrate solution that interacts with the enzyme-antibody-target combination to give a quantifiable signal This signals strength is proportional to the concentration of the target in the original material
454 CEA and CA19-9 measurement by electrochemiluminescence immunoassay CA19-9 and CEA serum concentrations were measured using an electrochemiluminescence immunoassay using Cobas e 602 RocheDiagnostics North America
4541 Routine biochemical testing Liver function tests alanine aminotransferase ALT aspartate aminotransferase AST and serum albumin Kidney function indicators serum creatinine and urea were determined for all participants
455 Ratios and indices Body mass index BMI calculation in kgm2 was done for all the participants where normal weight 185-249 kgm2 overweight 25-299 kgm2 and obesity BMI of 30 kgm2 or greater morbid obesity Platelet-to-Lymphocytes ratio PLR is an immune response-related indicator and systematic inflammation biomarker which is superior to the neutrophil-to-lymphocytes ratio for correlation with inflammatory disease severity
-untranslated region UTR of HSP90 messenger RNA mRNA to reduce HSP90 protein production hence exerting a tumor suppressor effect Therefore research is warranted to determine the clinical significance of the lncRNA NNT-AS1hsa-miR-485-5pHSP90 axis in CRC patients
The current study will compare the blood expression level of lncRNA NNT-AS1 and hsa-miR-485-5p as well as HSP90 serum levels in both healthy and CRC Egyptian patients cohort peripheral blood samples Second examine if lncRNA NNT-AS1hsa-miR-485-5pHSP90 axis or individually investigated would be used as non-invasive molecular precision biomarker in liquid biopsy for better diagnostic utility for CRC patients Also based on CRC patients group stratification by histologic grades 1-3 Finally investigate the probable linkcorrelation between lncRNA NNT-AS1hsa-miR-485-5pHSP90 as an axis or individually in CRC Egyptian patients cohort in relation to demographic data or clinicopathological characteristics All findings will be confirmed or ruled out by in silico means as well
31 Patient group A total of 60 CRC patients were enrolled in the study CRC patients were treatment-naïve Egyptian patients cohort admitted to the Clinical Oncology Clinic National Cancer Institute NCI Cairo University Cairo Egypt Male-to-female 11 3030 and their age range 24-76 years
32 Control group 28 age-matched and sex-matched apparently healthy volunteers randomly selected from subjects enlisted during normal check-up examination or during blood donation The control group participants were not taking any medication or suffering from any disease Controls age range is 40-60 years and 1315 male-to-female For the patients group n 60 a full history was recorded If a patient met the inclusion criteria and after giving their approval for participation in the study and signing the informed consent peripheral blood samples were taken at the diagnosis time If a patient met the exclusion criteria blood samples were not taken Patient inclusion criteria were those who visited the Colonoscopy Unit for colorectal examination and had a variety of colonic symptoms including CRC noticeable symptoms constipation abdominal pain rectal bleeding and sudden weight loss CRC diagnosis was clinically confirmed by colonoscopy abdominal radio-imaging and histopathological examination Patients exclusion criteria included individuals receiving chemotherapy radiation or undergone surgery patients with blood disorders or any cancer other than CRC Individuals with inadequate data or missing histopathological diagnoses as well as those with distant metastases were excluded from the study
321 Patients Pathological and Clinical Data The clinical assessment of CRC patients tumor was done at the Pathology Unit NCI Cairo Cairo University In addition to a patients colorectal surgery history the complete family history of cancer disease was recorded for all CRC participants At the NCI CRC staging was determined by the colonoscopic results abdominal radiography pathological analyses and clinical decisions relying on these results and the American Joint Committee on Cancer AJCC criteria 2010 Patients are categorized into three stages stage III local cancer stage III lymph node LN localized involvement N1-x and stage IV distant metastasis M1 LN involvement with enlargement incidence as either no N0 or present N1 was noted from patient files The CRC patients family history smoking status the noncommunicable diseases status as diabetes mellitus DM and hypertension HTN were recorded Tumor site tumor size mucinous or not lymph node metastasis LNM tumor invasion or vascular invasion tumor differentiation tumor grade tumor-node-metastasis TNM staging inflammation status as inflammatory bowel disease IBD CRC location if colonic or rectal as well as if transverse sigmoid rectosigmoid and rectal CRC clinical symptoms were assessed by the NCI Biochemical Analysis Unit or the Statistics Unit collected from patients files Hemoglobin Hgb prothrombin time PT erythrocyte sedimentation rate ESR platelet count lymphocyte count lactate dehydrogenase LDH C-reactive protein CRP were all recorded from patient files done at the NCI Cairo Egypt Central Clinical Biochemistry Lab 42 Blood samples Five milliliters of peripheral venous blood liquid biopsy were collected from the antecubital vein from controls and CRC patients into clot activator polymer gel vacutainers Vacutainers of completely coagulated samples were centrifuged at 4000 rpm for 10 min at room temperature 25 C The collected serum was aliquoted and kept at - 80 C in three DNase RNase free Eppendorf tubes until analysis
43 Total RNA extraction Total RNA was extracted from serum samples according to the procedure guidelines The extracted RNA was dissolved in 40 µL RNase free water and stored in aliquots at - 80 C
44 Quantitation of purified RNA including miRNAs The extracted RNAs purity and concentration are assessed using a spectrophotometer The quantity of RNA in the sample was assessed using absorbance at 260 nm A260 1 44 ngµL and RNA purity was assessed using absorbance at 260280 nm ratios
45 Reverse transcription and expression measurement of ncRNAs using quantitative real-time reverse transcription polymerase chain reaction qRT-PCR 451 lncRNA NNT-AS1 The real-time RT2 First Strand Kit was used for complementary DNA cDNA synthesis as directed by the manufacturers instructions The obtained cDNA was stored at - 20 C After reverse transcription qRT-PCR was utilized to measure the expression of the lncRNA NNT-AS1 to evaluate the expression level of the lncRNA NNT-AS1 the primer RT2 lncRNA qPCR Assay for Human NNT-AS1 LPH15988A-200 Cat No 330701 was used and data were normalized using RT2 lncRNA qPCR Assay for Human GAPDH LPH31725A-200 Cat No 330701 as endogenous control 452 hsa-miR-485-5p cDNA synthesis was performed as directed by the procedure guidelines The obtained cDNA was stored at - 20 C After reverse transcription qRT-PCR was utilized to measure the expression of the hsa-miR-485-5p To detect the expression level of the hsa-miR-485-5p the primer hsa-hsa-miR-485-5p was used and data were normalized miRNA PCR Assay YP00203901 Cat No 339306 as endogenous control The ncRNA expression level as fold change was calculated and normalized by using the cycle threshold Ct approach as fold change 2- ΔΔCt with GAPDH or SNORD38B hsa as the housekeeping genes for lncRNA NNT-AS1 and hsa-miR-485-5p respectively ΔCt was calculated by deducting the Ct values of GAPDH and SNORD38B hsa from the ones of the lncRNA NNT-AS1 and hsa-miR-485-5p under study respectively
ΔΔCt ΔCt cancer samples - ΔCt control samples where ΔCtCt target - Ct reference 453 HSP90 protein quantification by Enzyme-linked immunosorbent assay ELISA The HSP90 was quantified by ELISA using the commercially available kit according to the manufacturers instructions The Human HSP90 alpha solid-phase sandwich ELISA measures the amount of target bound between a matching antibody pair In the wells of the supplied microplate a target-specific antibody has been pre-coated In these wells samples standards or controls bind to the immobilized capture antibody The sandwich is constructed by adding the second detector antibody followed by the addition of a substrate solution that interacts with the enzyme-antibody-target combination to give a quantifiable signal This signals strength is proportional to the concentration of the target in the original material
454 CEA and CA19-9 measurement by electrochemiluminescence immunoassay CA19-9 and CEA serum concentrations were measured using an electrochemiluminescence immunoassay using Cobas e 602 RocheDiagnostics North America
4541 Routine biochemical testing Liver function tests alanine aminotransferase ALT aspartate aminotransferase AST and serum albumin Kidney function indicators serum creatinine and urea were determined for all participants
455 Ratios and indices Body mass index BMI calculation in kgm2 was done for all the participants where normal weight 185-249 kgm2 overweight 25-299 kgm2 and obesity BMI of 30 kgm2 or greater morbid obesity Platelet-to-Lymphocytes ratio PLR is an immune response-related indicator and systematic inflammation biomarker which is superior to the neutrophil-to-lymphocytes ratio for correlation with inflammatory disease severity
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None