Ignite Creation Date:
2024-05-06 @ 8:17 PM
Last Modification Date:
2024-10-26 @ 3:24 PM
Study NCT ID:
NCT06320418
Status:
ACTIVE_NOT_RECRUITING
Last Update Posted:
2024-03-20
First Post:
2024-02-22
Brief Title:
The Epigenetic Regulatory Role of P-element Induced Wimpy Testis Piwi Interacting RNA-823 piR-823 in Ovarian Cancer Progression
Sponsor:
Ain Shams University
Organization:
Ain Shams University
Study Overview
Official Title:
The Epigenetic Regulatory Role of P-element Induced Wimpy Testis Piwi Interacting RNA-
Status:
ACTIVE_NOT_RECRUITING
Status Verified Date:
2024-03
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:
Ovarian cancer OC has one of the highest mortality rates for female malignant tumors attributed to advanced cancer stages upon diagnosis as well as a high recurrence rate Piwi-interacting RNA-823 piR-823 is a single-stranded non-protein coding RNA ncRNA star molecule in epigenetics research Extensive cellular regulatory functions and aberrant expression of piR-823 have been implicated in carcinogenesis Therefore the findings of piwi-ncRNA dysregulated-expression in OC Egyptian female patients cohort could be employed as a potential novel mechanism for OC precision a step toward ncRNA-precision
Detailed Description:
1 Introduction Ovarian cancer OC represents one of the most aggressive female tumors worldwide and is the second most prevalent malignant tumor after breast cancer In Egypt OC represents the fourth most common female cancer Current treatments become ineffective after several cycles of management due to the high recurrence rate which is about 80-85 Thus it is urgent to explore novel targets for OC treatment 1 OC represents a spectrum of diseases not a single disease entity Piwi-interacting RNA piRNA is a single-stranded non-coding RNA ncRNA with a nucleotide sequence of 23-35 that was discovered in mammalian germ cells It has become a star molecule in non-coding RNA research due to its extensive cellular regulatory functions 3 In recent years many studies have found that piRNA is involved in tumor occurrence and development in many types of cancer such as gastric cancer liver cancer lung cancer breast cancer and other cancers 4 piR-52207 and piR-33733 participate in OC oncogenesis through involvement in numerous cell signaling pathways at the post-transcriptional level 5 Nevertheless the clinical significance and biological mechanisms of piRNAs in the progression of OC remain largely unknown piR-823 was one of the first piRNAs recognized to be linked to malignancy and was identified in white blood cells cancer cell lines and blood plasma piR-823 exhibits a pan-cancer expression pattern in various types of cancer such as stomach colon breast esophageal liver prostate and renal cancer 6 7 More recently piRNA-823 has been documented to be involved in tumorigenesis by regulating de novo DNA methylation and angiogenesis in multiple myeloma 3 However their functional mechanisms as well as their epigenetic regulatory role in OC have not been investigated Thus investigating the regulatory role played by piR-823 could provide a way to explore the application of ncRNAs as a prognostic mechanism in OC constituting a potential target for drug development RESEARCH OBJECTIVES Investigate the expression level of piR-823 in OC tissue samples Demonstrate the correlation between piR-823 and the progression of OC Correlate piR-823 and OC clinicopathological characteristics
2 Subjects 21 Sample size and power of the study Sample size calculation is based on the previous study by Jie Yin et al 2017 Sample size estimation performed by G power sample size online calculator httpwwwgpowerhhudeenhtml depending on two-sided confidence level 95 The sample size to be able to reject the null hypothesis that the population means of the studied groups are equal with probability power 095 and type I error probability associated with this test of this null hypothesis is 005
22 Study design Case-controlled retrospective observational study mono-center 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 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 24 Study participant 241 Patients group A total of 39 female their age range 13- 75 years patients diagnosed with primary malignant ovarian tumor were enrolled in the study OC patients were treatment-naïve Egyptian patients cohort admitted to the Gynecology and Obstetrics Department or the Oncology Dept Ain Shams University Hospitals Cairo Egypt Ovarian cancer tissue samples were collected during surgical resection and confirmed by postoperative pathological examinations
242 Control group A total of 17 normal ovarian tissue samples were collected from patients with benign uterine diseases uterine such as myoma who underwent uterine and ovarian resection Controls age range is 45-62 years For the patients group n 39 a full history was recorded Patient enrolled into the study If met the inclusion criteria and after giving their approval for participation in the study and signed the informed consent Inclusion criteria This study included newly diagnosed untreated cases of histopathologically confirmed OC patients Exclusion criteria Individuals receiving chemotherapy radiation and patients with history of other cancer other than OC Additionally individuals with inadequate data or missing histopathological diagnoses
243 Patients demographic data and pathological data Patients were either in the reproductive premenopausal or postmenopausal phase The patients demographic data including age menopausal status ascites and the patient full history retrieved from the patients medical records collected from Ain Shams Hospitals in addition to the original pathology reports of the following data tumor site tumor size tumor type lymph node metastasis distant metastasis tumor stage and tumor grade were obtained from the Department of Pathology Ain Shams Hospitals OC histological staging made in accordance to the International Federation of Gynecology and Obstetrics FIGO criteria
3 Methods
31 in silico analysis Databases mining for targeting pathways and functional enrichment analysis in addition to Binding interaction predications 32 Tissue samples Ovarian fresh tissue samples were collected from the controls and OC patients underwent surgical resection at the Department of Obstetrics and Gynecology Ain shams university All resected tissue samples were stored at -20C after adding RNA Stabilization Reagent Qiagen Hilden Germany 33 Total RNA extraction and quantification Using the miRNeasy Mini kit Cat No217004 Qiagen Hilden Germany according to the manufacturers protocol from tissue samples Total RNA extraction was done The isolated RNA was eluted in 40 μL of RNase-free water Then the isolated RNA quantified using NanoDrop 1000 spectrophotometer Thermo Scientific Wilmington DE USA the concentration and purity of all RNA samples were determined was stored at -80 C in aliquots
34 cDNA synthesis and measurement of piR-823 expression using qRT-PCR Total RNA will be reverse transcribed by miSCRIPT Kit Qiagen Hilden Germany Real-time PCR analysis subsequently performed using a miRcury LNA SYBR Green PCR Kit Qiagen 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 piR823 using the miRCURY LNA miRNA endogenous control 35 cDNA synthesis and measurement of mRNA expression using qRT-PCR RevertAid First Strand cDNA Synthesis Kit Thermo Scientific Rockford IL USA was used for cDNA synthesis according the manufacture instructions qRT-PCR made using Power SYBR Green PCR Master Mix Thermo Scientific Rockford IL USA GAPDH primer was used as endogenous control The RNA relative expression was computed and normalized as fold change using the cycle threshold Ct method 2--ΔΔCt
36 Statistical analysis
Data will be collected excel tabulated and tested for normality by Kolmogorov-Smirnov test
Between-group differences in age variability will be compared by the χ2 test and by analysis of variance for age variability Multivariate logistic regression with adjustments for age will be used to show the association between selected piR-823 expression and OC risk
Normally distributed variables will be expressed as mean SEM and analyzed using two samples independent t-test Median interquartile range will be used to express nonparametric data and subsequently analyzed using Mann Whitney U test
Pearsons Chi-square analysis or Fishers exact test employed to compare the difference of categorical variables
For all analyses a two-tailed P value of 005 or less considered statistically significant
2 Subjects 21 Sample size and power of the study Sample size calculation is based on the previous study by Jie Yin et al 2017 Sample size estimation performed by G power sample size online calculator httpwwwgpowerhhudeenhtml depending on two-sided confidence level 95 The sample size to be able to reject the null hypothesis that the population means of the studied groups are equal with probability power 095 and type I error probability associated with this test of this null hypothesis is 005
22 Study design Case-controlled retrospective observational study mono-center 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 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 24 Study participant 241 Patients group A total of 39 female their age range 13- 75 years patients diagnosed with primary malignant ovarian tumor were enrolled in the study OC patients were treatment-naïve Egyptian patients cohort admitted to the Gynecology and Obstetrics Department or the Oncology Dept Ain Shams University Hospitals Cairo Egypt Ovarian cancer tissue samples were collected during surgical resection and confirmed by postoperative pathological examinations
242 Control group A total of 17 normal ovarian tissue samples were collected from patients with benign uterine diseases uterine such as myoma who underwent uterine and ovarian resection Controls age range is 45-62 years For the patients group n 39 a full history was recorded Patient enrolled into the study If met the inclusion criteria and after giving their approval for participation in the study and signed the informed consent Inclusion criteria This study included newly diagnosed untreated cases of histopathologically confirmed OC patients Exclusion criteria Individuals receiving chemotherapy radiation and patients with history of other cancer other than OC Additionally individuals with inadequate data or missing histopathological diagnoses
243 Patients demographic data and pathological data Patients were either in the reproductive premenopausal or postmenopausal phase The patients demographic data including age menopausal status ascites and the patient full history retrieved from the patients medical records collected from Ain Shams Hospitals in addition to the original pathology reports of the following data tumor site tumor size tumor type lymph node metastasis distant metastasis tumor stage and tumor grade were obtained from the Department of Pathology Ain Shams Hospitals OC histological staging made in accordance to the International Federation of Gynecology and Obstetrics FIGO criteria
3 Methods
31 in silico analysis Databases mining for targeting pathways and functional enrichment analysis in addition to Binding interaction predications 32 Tissue samples Ovarian fresh tissue samples were collected from the controls and OC patients underwent surgical resection at the Department of Obstetrics and Gynecology Ain shams university All resected tissue samples were stored at -20C after adding RNA Stabilization Reagent Qiagen Hilden Germany 33 Total RNA extraction and quantification Using the miRNeasy Mini kit Cat No217004 Qiagen Hilden Germany according to the manufacturers protocol from tissue samples Total RNA extraction was done The isolated RNA was eluted in 40 μL of RNase-free water Then the isolated RNA quantified using NanoDrop 1000 spectrophotometer Thermo Scientific Wilmington DE USA the concentration and purity of all RNA samples were determined was stored at -80 C in aliquots
34 cDNA synthesis and measurement of piR-823 expression using qRT-PCR Total RNA will be reverse transcribed by miSCRIPT Kit Qiagen Hilden Germany Real-time PCR analysis subsequently performed using a miRcury LNA SYBR Green PCR Kit Qiagen 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 piR823 using the miRCURY LNA miRNA endogenous control 35 cDNA synthesis and measurement of mRNA expression using qRT-PCR RevertAid First Strand cDNA Synthesis Kit Thermo Scientific Rockford IL USA was used for cDNA synthesis according the manufacture instructions qRT-PCR made using Power SYBR Green PCR Master Mix Thermo Scientific Rockford IL USA GAPDH primer was used as endogenous control The RNA relative expression was computed and normalized as fold change using the cycle threshold Ct method 2--ΔΔCt
36 Statistical analysis
Data will be collected excel tabulated and tested for normality by Kolmogorov-Smirnov test
Between-group differences in age variability will be compared by the χ2 test and by analysis of variance for age variability Multivariate logistic regression with adjustments for age will be used to show the association between selected piR-823 expression and OC risk
Normally distributed variables will be expressed as mean SEM and analyzed using two samples independent t-test Median interquartile range will be used to express nonparametric data and subsequently analyzed using Mann Whitney U test
Pearsons Chi-square analysis or Fishers exact test employed to compare the difference of categorical variables
For all analyses a two-tailed P value of 005 or less considered statistically significant
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