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Research Article
Bhudev Das
Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), Amity University Campus, Sector-125, Noida, 201313, Uttar Pradesh, India
Corresponding Author
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MicroRNAs as cancer biomarkers in serum, plasma and other body fluids are often used but analysis of miRNA in urine is limited. We investigated the expression of selected miRNAs in the paired urine, serum, cervical scrape and tumor tissue specimens from the women with cervical precancer and cancer with a view to identify if urine miRNAs could be used as reliable non-invasive biomarkers for an early diagnosis and prognosis of cervical cancer. Expression of three oncomiRs (miR-21, miR-199a, and miR-155-5p) and three tumor suppressors (miR-34a, miR-145, and miR-218) in cervical pre-cancer, cancer and normal controls including cervical cancer cell lines were analyzed using qRT-PCR. The expression of miRNAs was correlated with various clinicopathological parameters, including HPV infection and survival outcome. We observed a significant overexpression of the oncomiRs and the downregulation of tumor suppressor miRNAs. A combination of miR-145-5p, miR-218-5p, and miR-34a-5p in urine yielded 100% sensitivity and 92.8% specificity in distinguishing precancer and cancer patients from healthy controls. The expression of miR-34a-5p and miR-218-5p were found to be independent prognostic factors for overall survival of cervical cancer patients. We conclude that the evaluation of specific miRNA expression in non-invasive urine samples may serve as reliable biomarker for early detection and prognosis of cervical cancer.
Keywords
MicroRNAs, serum, plasma and other body fluids, HPV infection, survival outcome
Figure 1
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Figure 4
The full text of this article is available to read as a PDF.
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFiguresTables.pdf
Figure S1: Differential expression of six miRNAs in urine, serum, cervical scrape, and tissue biopsies derived from healthy controls, pre-cancer and cervical cancer patients. The miRNA expression level of miR-21-5p, miR-155-5p, miR-199a-5p, miR-145-5p, miR218 -5p, and miR-34a-5p in (a) urine, (b) serum, (c) cervical scrape, and (d) tissue biopsies. Urine and serum samples were taken from pre-cancer and cervical cancer patients and compared to samples from healthy controls. In case of cervical scrape, the samples were derived from pre-cancer patients compared to samples from healthy volunteers, while in case of tissue biopsies, the samples were derived from cancer patient compared to samples from adjacent non-malignant tissues. **p≤ 0.01, ***p ≤ 0.001. Figure S2: Correlation of expression pattern of miRNAs in cervical tissue biopsies and paired urine using Pearson’s correlation coefficient. Pearson’s correlation scatter plots for the correlation between the fold-change expression levels of (a) miR-21-5p, (b) miR-155-5p, (c) miR-199a-5p, (d) miR-145-5p, (e) miR-34a-5p, and (f) miR-218-5p in tissue biopsies and paired urine of cervical cancer patients. Figure S3: Expression levels of miR-21-5p, miR-155-5p, miR-199a-5p, miR-145-5p, miR-218-5p, and miR-34a-5p in HPV16-positive and HPV16-negative urine, serum, and cervical scrape samples of cervical pre-cancer (a-c) and urine, serum, and tissue biopsies of cervical cancer patients (d-f). Figure S4: (a-f) Differential expression of six miRNAs in pre-cancer, cancer and normal urine, and the ROC analyses of six miRNA detection. Box plot of miRNA expression in urine (upper: miR‐21-5p, miR‐155-5p and miR‐199a-5p; lower: miR‐145-5p, miR‐218-5p and miR‐34a-5p is normalized to U6. The lines inside the boxes indicate the medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers indicate the interval between and outside the 10th and 90th percentiles. A statistically significant difference is determined by Kruskal–Wallis test with Dunn's multiple comparison post‐hoc test, normal urine. Figure S5: The target gene prediction and function analyses. The Venn diagrams represent the overlapping target genes of (a) miR-21-5p, (b) miR-155-5p, (c) miR-199a-5p, (d) miR-34a-5p, (e) miR-145-5p, and (f) miR-218-5p predicted by miRDB, Target Scan and DIANA microT‐CDS online analysis tools. Figure S6A: Top 10 Gene ontology enrichment in (a) Biological, (b) Molecular, and (c) Cellular processes. Figure S6B: Top 10 pathway enrichment analysis, including (a) KEGG and (b) REACTOME pathways. Table S1: The expression of miRNAs in HPV positive and HPV negative cervical cancer cell line Table S2: The expression of miRNAs according to HPV infection status in urine, serum, cervical scrape, and tissue biopsies derived from cervical pre-cancer and cancer patients Table S3: The correlation of the expression of urine, serum and cervical scrape miRNAs with the clinicopathological parameters of cervical pre-cancer patients Table S4: The correlation of the expression of urine and serum miRNAs with the clinicopathological parameters of cervical cancer patients Table S5: Primers for amplification of human papilloma virus gene sequence Table S6: MiRNA primer sequences used for Real-time PCR
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Research Article
Bhudev Das
Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), Amity University Campus, Sector-125, Noida, 201313, Uttar Pradesh, India
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 07 Dec, 2020
No community comments so far
Editorial decision: Major revision
On 28 Dec, 2020
Reviews received
Received 24 Dec, 2020
Reviewers agreed
On 18 Dec, 2020
Reviewers agreed
On 17 Dec, 2020
Reviewers invited
Invitations sent on 15 Dec, 2020
Editor assigned
On 15 Dec, 2020
Editor invited
On 03 Dec, 2020
Submission checks complete
On 02 Dec, 2020
First submitted
On 25 Nov, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Obstetrics & Gynecology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
MicroRNAs as cancer biomarkers in serum, plasma and other body fluids are often used but analysis of miRNA in urine is limited. We investigated the expression of selected miRNAs in the paired urine, serum, cervical scrape and tumor tissue specimens from the women with cervical precancer and cancer with a view to identify if urine miRNAs could be used as reliable non-invasive biomarkers for an early diagnosis and prognosis of cervical cancer. Expression of three oncomiRs (miR-21, miR-199a, and miR-155-5p) and three tumor suppressors (miR-34a, miR-145, and miR-218) in cervical pre-cancer, cancer and normal controls including cervical cancer cell lines were analyzed using qRT-PCR. The expression of miRNAs was correlated with various clinicopathological parameters, including HPV infection and survival outcome. We observed a significant overexpression of the oncomiRs and the downregulation of tumor suppressor miRNAs. A combination of miR-145-5p, miR-218-5p, and miR-34a-5p in urine yielded 100% sensitivity and 92.8% specificity in distinguishing precancer and cancer patients from healthy controls. The expression of miR-34a-5p and miR-218-5p were found to be independent prognostic factors for overall survival of cervical cancer patients. We conclude that the evaluation of specific miRNA expression in non-invasive urine samples may serve as reliable biomarker for early detection and prognosis of cervical cancer.
Figure 1
Figure 2
Figure 3
Figure 4
The full text of this article is available to read as a PDF.
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFiguresTables.pdf
Figure S1: Differential expression of six miRNAs in urine, serum, cervical scrape, and tissue biopsies derived from healthy controls, pre-cancer and cervical cancer patients. The miRNA expression level of miR-21-5p, miR-155-5p, miR-199a-5p, miR-145-5p, miR218 -5p, and miR-34a-5p in (a) urine, (b) serum, (c) cervical scrape, and (d) tissue biopsies. Urine and serum samples were taken from pre-cancer and cervical cancer patients and compared to samples from healthy controls. In case of cervical scrape, the samples were derived from pre-cancer patients compared to samples from healthy volunteers, while in case of tissue biopsies, the samples were derived from cancer patient compared to samples from adjacent non-malignant tissues. **p≤ 0.01, ***p ≤ 0.001. Figure S2: Correlation of expression pattern of miRNAs in cervical tissue biopsies and paired urine using Pearson’s correlation coefficient. Pearson’s correlation scatter plots for the correlation between the fold-change expression levels of (a) miR-21-5p, (b) miR-155-5p, (c) miR-199a-5p, (d) miR-145-5p, (e) miR-34a-5p, and (f) miR-218-5p in tissue biopsies and paired urine of cervical cancer patients. Figure S3: Expression levels of miR-21-5p, miR-155-5p, miR-199a-5p, miR-145-5p, miR-218-5p, and miR-34a-5p in HPV16-positive and HPV16-negative urine, serum, and cervical scrape samples of cervical pre-cancer (a-c) and urine, serum, and tissue biopsies of cervical cancer patients (d-f). Figure S4: (a-f) Differential expression of six miRNAs in pre-cancer, cancer and normal urine, and the ROC analyses of six miRNA detection. Box plot of miRNA expression in urine (upper: miR‐21-5p, miR‐155-5p and miR‐199a-5p; lower: miR‐145-5p, miR‐218-5p and miR‐34a-5p is normalized to U6. The lines inside the boxes indicate the medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers indicate the interval between and outside the 10th and 90th percentiles. A statistically significant difference is determined by Kruskal–Wallis test with Dunn's multiple comparison post‐hoc test, normal urine. Figure S5: The target gene prediction and function analyses. The Venn diagrams represent the overlapping target genes of (a) miR-21-5p, (b) miR-155-5p, (c) miR-199a-5p, (d) miR-34a-5p, (e) miR-145-5p, and (f) miR-218-5p predicted by miRDB, Target Scan and DIANA microT‐CDS online analysis tools. Figure S6A: Top 10 Gene ontology enrichment in (a) Biological, (b) Molecular, and (c) Cellular processes. Figure S6B: Top 10 pathway enrichment analysis, including (a) KEGG and (b) REACTOME pathways. Table S1: The expression of miRNAs in HPV positive and HPV negative cervical cancer cell line Table S2: The expression of miRNAs according to HPV infection status in urine, serum, cervical scrape, and tissue biopsies derived from cervical pre-cancer and cancer patients Table S3: The correlation of the expression of urine, serum and cervical scrape miRNAs with the clinicopathological parameters of cervical pre-cancer patients Table S4: The correlation of the expression of urine and serum miRNAs with the clinicopathological parameters of cervical cancer patients Table S5: Primers for amplification of human papilloma virus gene sequence Table S6: MiRNA primer sequences used for Real-time PCR
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