Decreased expression of miR-23b is associated with poor survival of endometrial cancer patients

doi:10.21203/rs.3.rs-1422217/v1

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Decreased expression of miR-23b is associated with poor survival of endometrial cancer patients

https://doi.org/10.21203/rs.3.rs-1422217/v1

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Endometrial cancer (EC) is one of the most common types of cancer of the female reproductive system. EC is classified into two types (EC1 and EC2). MiRNAs are single-stranded RNA molecules that regulate gene expression posttranscriptionally. They have aberrant expression profiles in cancer, including EC. This study aimed to assess the level of expression of a panel of 16 miRNAs in both types of EC and healthy endometrium (HE). A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). RNA was isolated from collected material and the expression of selected miRNAs was determined using the real-time qPCR. We found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a were downregulated in EC in comparison to HE. Moreover, the expression of miR-34a-5p and miR-146-5p was higher in EC1 compared to EC2. Analysis of The Cancer Genome Atlas (TCGA) database confirmed decreased levels of miR-23b, miR-125b-5p, and miR-199a-3p in EC. Decreased miR-23b expression was associated with worse survival of EC patients.

miRNA

endometrial cancer

biomarkers

miR-23b

TCGA

Endometrial cancer (EC) arises from the epithelial lining of the uterus (endometrium) and is the most common cancer of the female reproductive system in the USA ¹. It is estimated that 65 950 new cases will be diagnosed in 2022, representing 3.5% of all new cancer cases in the USA, and the disease will be fatal to 12 550 patients, which is equivalent to 2.1% of all cancer deaths ². The incidence and mortality are steadily increasing in the population which is associated with many factors, including the growing prevalence of obesity - one of the major risk factors for the development of the EC ³. The 5-year relative survival is estimated at 81.1% ⁴. EC can be classified based on two different classification systems. The traditional classification proposed in 1981 by Bokhman ⁵, divides EC into two types, where type 1 of EC (EC1) is defined as an estrogen-dependent tumor associated with endometrial hyperplasia, and type 2 of EC (EC2) as an estrogen-independent tumor which is associated with endometrial atrophy. Another classification of EC divides tumors based on the histopathological characteristics, where EC can be categorized into endometrioid carcinoma, serous carcinoma, clear-cell adenocarcinoma, carcinosarcoma, and other types ⁶.

MiRNAs (microRNAs) are endogenous, small single-stranded non-coding RNA molecules that regulate gene expression at the posttranscriptional level ⁷. They play a significant role in a broad range of biological processes such as cellular proliferation, differentiation, and apoptosis ^8–11. The biogenesis of miRNAs involves multiple steps that occur at each step of the synthesis of the functional molecule ^8,10. The expression of miRNAs is controlled by numerous transcription factors, two of which, p53 and c-Myc, appear to play a key role in this process ⁹. The dysregulation of miRNAs may result in the initiation of carcinogenesis, which can affect all hallmarks of cancer as defined by Hanahan and Weinberg ¹², including replicative immortality, proliferative signaling, immune evasion, deactivation of growth suppressors, or inducing angiogenesis ^9,13. Notably, miRNAs can act as both: oncogenes or tumor suppressors, as dictated by their target genes ¹⁴. Over the past decade, a body of evidence of dysregulation of miRNA expression in a variety of cancer types has been described. The focus on this subject by numerous research groups, including our, resulted in reports on the role of miRNAs as cancer biomarkers, as well as markers of cancer invasiveness and metastasis ^11,15−18.

This study aimed to further characterize the pathogenesis of EC by determining the level of miRNAs typically involved in cancer development. The study was conducted on microdissected tissue samples of both types of EC: EC1 and EC2 and the healthy endometrial tissue.

In this study, we enrolled 30 primary EC patients, previously untreated, and 15 control patients with healthy endometrium (HE) operated due to other gynecological pathologies (leiomyoma). The endometrium of these patients was histopathologically confirmed to be normal. To ensure that material obtained for miRNAs expression analysis contained only EC or HE tissue, we used Laser Capture Microdissection (LCM) to precisely dissect only specific fragments of tissue. From each FFPE sample of EC, only tumor tissue was dissected using LCM. In HE samples, only glandular endometrial tissue was dissected (Fig. 1). From the collected material we have determined the level of: miR-21-3p, miR-23b, miR-34a-5p, miR-96-5p, miR-125b-5p, miR-134-5p, miR-146-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-199a-3p, miR-200b-3p, miR-211-3p, miR-221-3p, miR-410-3p, and miR-451a.

MiR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a are downregulated in EC

The analysis of 16 miRNAs revealed that the levels of expression of certain miRNAs were significantly downregulated in EC compared to HE. miR-23b was downregulated 4.54 times, (Fig. 2a, p<0.0001), miR-125b-5p was downregulated 7.15 times (Fig. 2b, p=0.0005), miR-199a-3p was downregulated 11.11 times (Fig. 2c, p<0.0001), miR-221-3p was downregulated 4.54 times, (Fig. 2d, p=0.0029), and miR-451a was downregulated 17.24 times (Fig. 2e, p<0.0001). In contrast, there were no statistically significant differences between the expression of miR-21-3p, miR-34a-5p, miR-96-5p, miR-134-5p, miR-146-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-200b-3p, miR-211-3p, and miR-410-3p in EC compared to HE (Fig. 3a-k, p>0.05).

MiR-34a-5p and miR-146-5p are upregulated in EC1 compared to EC2

Next, we analyzed the differences in miRNAs expression profiles between EC1 and EC2. Out of 30 EC samples used in this study, 18 samples were EC1 type and 12 samples were EC2. The expression of two miRNAs was upregulated in EC1 compared to EC2. These were miR-34a-5p (Fig. 4a, upregulated 5.43 times, p=0.031) and miR-146-5p (Fig. 4b, upregulated 3.50 times, p=0.0479). There was no differences in expression of miR-21-3p, miR-23b, miR-96-5p, miR-125b-5p, miR-134-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-199a-3p, miR-200b-3p, miR-211-3p, miR-221-3p, miR-410-3p, and miR-451a between EC1 and EC2 specimens (Fig. 5a-n, p>0.05).

MiR-23b, miR-125b-5p miR-199a-3p, miR-221-3p are downregulated in TCGA cohort

To confirm our findings, we analyzed the expression of investigated miRNAs in The Cancer Genome Atlas (TCGA) on the cohort of 418 EC tissues and 32 HE controls using OncomiR database ¹⁹. The levels of miR-23b (Fig. 6a, downregulated 1.87 times, p<0.0001), miR-125b-5p (Fig. 6b, downregulated 2.34 times, p<0.0001) miR-199a-3p (Fig. 6c, downregulated 1.26 times, p=0.0067), and miR-221-3p (Fig. 6d, downregulated 1.45 times, p=0.0110) were significantly downregulated in EC compared to HE. There were no differences in expression levels of miR-451a in EC compared to HE (Fig. 6e, p>0.05).

Decreased miR-23b expression is associated with worse survival

Further, we checked whether the expression of downregulated miRNAs in the EC tissue correlated with the survival of patients. We analyzed the patients' survival and miRNAs level in TCGA EC cohort using OncoLnc ²⁰. We found that decreased expression of miR-23b was associated with worse survival (Fig. 7a, p=0.0203). The decreased expressions of miR-125-5p, miR-199a-3p, miR-221-3p, or miR-451a were not correlated with the survival of EC patients (Fig. 7b-e, p>0.05).

Aberrant expression of miRNAs has been observed in many types of cancers, including EC ^11,21−23. Our recent systematic review revealed that miRNAs are crucial regulators of EC progression ¹⁵. By analyzing 115 articles, we identified 106 dysregulated miRNAs involved in the modulation of the EC invasiveness and metastasis. They regulate not only EC cells invasion and migration but also influence metastasis and tumor growth. Moreover, the expression of several miRNAs was correlated with clinical parameters of EC patients ¹⁵. In this study, we analyzed the expression of 16 miRNAs with a well-established role in tumor tissues of EC1 and EC2 as well as in healthy endometrium ¹¹. We identified five miRNAs, miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a, that are downregulated in EC compared to HE.

MiR-23b plays contrary roles in different types of cancer but is a tumor suppressor miRNA in EC ^24,25. MiR-23b targets metastasis-associated in colon cancer-1 (MACC1) inhibiting EC cells proliferation, invasion, and migration. Moreover, miR-23b suppresses EC metastasis in vivo in a murine model ²⁵. The expression of miR-23b was downregulated in EC-derived cell lines compared to the normal fallopian epithelial cells It is also downregulated in FFPE EC tissues in the miRNA-profiling study ²⁶. Moreover, the expression of miR-23b was lower in grade 3 EC1 compared to grade 1 tumors ²⁷. We found that miR-23b is downregulated in EC tissues compared to HE regardless of the EC type. Additionally, analysis of the TCGA cohort revealed that decreased expression of miR-23b was correlated with the poor survival of EC patients.

MiR-125b-5p expression was downregulated in EC tissue in our study which was confirmed in the TCGA cohort. In EC, miR-125b-5p acts as a tumor suppressor miRNA and inhibits invasion of EC cells by directly targeting protooncogene ERBB2 ²⁸. However, the expression of miR-125b-5p was not correlated with patients' survival, even though the decreased level of miR-125b-5p was found to be associated with higher histological grade and myometrial invasion ²⁹.

MiR-199a-3p is another miRNA that was downregulated in EC compared to HE ^30,31. Also, the increased level of miR-199a-3p was associated with better progression-free survival and overall survival of EC patients ³², however, it was not the case for TCGA cohort patients.

Moreover, we found that miR-221-3p and miR-451a were downregulated in EC tissue compared to HE regardless of EC type. Notably, miR-221-3p was confirmed to be downregulated in the TCGA cohort of EC patients. Of note, miR-221 was identified as an oncomiRNA in other types of cancer, including breast cancer and liver cancer ³³. Its overexpression promotes tumor cell migration, invasiveness, and proliferation ³³. Our results suggest that the role of miR-221 is not critical for tumor growth in EC since its expression is decreased in this pathology. This is contrary to published data on other cancers, where miR-221 and miR-451a both act as tumor suppressor miRNAs, including ovarian cancer and cervical cancer ³⁴.

Further, we found that the expression of the majority of analyzed miRNAs was very similar in both types of EC. Only miR-34a-5p and miR-146-5p levels differed between both types, namely the expression of miR-34a-5p and miR-146-5p was upregulated in EC1 compared to EC2. There are discordant data concerning the level of expression of miR-34a-5p in EC compared to HE ^35,36. Nonetheless, miR-34a-5p was identified to act as tumor suppressor miRNA in EC. It targets Notch1, L1CAM, MMSET and thus inhibits EC cells migration, invasion, and EMT in vitro as well as tumor growth in vivo ^36–38. There are no studies regarding the role or the level of expression of miR-146-5p in EC. However, the expression of miR-146-5p was increased by estrogen in the plasma of rats with prostate cancer ³⁹, which may suggest that upregulation of miR-146-5p may be related to estrogens in EC1.

As miRNAs reveal different expression patterns in healthy and cancerous tissues, they have great potential to be diagnostically and prognostically valuable biomarkers as well as potential therapeutic targets ⁴⁰. So far, a variety of miRNAs with different expression patterns in normal and malignant endometrial tissue have been identified ^41,42. MiRNAs expression can be determined in FFPE tissues and this evaluation could be performed in addition to the standard histopathological examination ⁴³. Moreover, using laser capture microdissection (LCM) it is possible to precisely dissect only tumor tissue without contamination of non-malignant cells surrounding tumor ^44,45.^41,42. In this study, we dissected only neoplastic tissues or glandular healthy endometrium, so the analyses were not disturbed by adjacent tissues. The main limitation of our study is a low number of specimens of EC tissue and a lack of complete clinical data of included patients. For this reason, we were unable to correlate the expression of analyzed miRNAs with e.g. survival of our patients. Therefore, further studies are required to assess the clinical relevance of studied miRNAs in EC, especially the role of miR-23b as a prognostic biomarker.

In this study, we found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a were downregulated in endometrial cancer compared to healthy endometrium. Additionally, the expression of miR-34a-5p and miR-146-5p were higher in EC1 than in EC2. Decreased miR-23b expression is associated with worse survival of EC patients. There is a need for further studies assessing the potential clinical use of described miRNAs as biomarkers.

Patients tissue

A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). The FFPE samples have been obtained from the Department of Pathology, Medical Center of Postgraduate Education, Warsaw, Poland. Patients data are presented in Table 1. The study was conducted following the Declaration of Helsinki and was approved by the Bioethical Committee Medical University of Warsaw (AKBE/78/2021, 17 May 2021). The patient’s consent was waived due to the performed anonymization and retrospective character of the study.

Hematoxylin and Eosin Staining

Resected tumors were formalin-fixed and paraffin-embedded according to the standard protocol in the tissue processor. Thereafter the samples were cut on microtome and hematoxylin and eosin-stained for the pathologist examination.

All samples were cut with a microtome to 10 µm slices and were mounted on glass slides with a drop of UltraPure DNAse/RNAse-free water. Then, samples were incubated in a fume hood at 56 °C for one hour to increase slices’ adherence. Mounted slices were hematoxylin and eosin-stained according to the standard protocol in a set of stains, alcohol solutions, and xylene. Slides were immediately subjected to LCM.

Laser capture microdissection

Stained and dehydrated sections of EC or HE were subjected to LCM-aided dissection of regions containing only neoplastic tissues or glandular healthy endometrium. Approximately 10 mm2 of each region was marked to dissect with LCM system (PALM Robo, Zeiss, Germany). These regions were selected by a board-certified pathologist. Each LCM was preceded by optimization of LCP energy and spot distance to provide a full dissection of marked areas. LCM was performed under the following conditions: LCP energy—80–90, LCP spot distance—25 μm, magnification—5 ×, tissue collected in 20 μl of Digestion Buffer (Norgen Biotek) in 500 μl sterile PCR-tube cap. Caps were sealed back with tubes, centrifuged briefly, and placed on wet ice until further steps ^44-47.

RNA isolation and RT-qPCR

Norgen Biotek FFPE RNA/DNA Purification Plus Kit was used for RNA isolation according to the manufacturer guidelines. RNA was eluted with 30 µl ultrapure, molecular-grade water, and stored at − 80 °C until the next steps. The purity and quantity of isolated RNA were assessed by the absorbance measurements at wavelengths of 260/280 nm using the NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). Samples with 260/280 ratios between 1.8 and 2.1 were used for further analysis. RNA was then subjected to reverse transcription using Mir-X™ miRNA FirstStrand Synthesis (Takara, Clontech) followed by real-time qPCR using SYBR® qRT-PCR (Thermo Fisher Scientific). Primers sequences used in the study are presented in Table 2. U6 (Takara, Clontech) was used as an endogenous control for the analysis of microRNA expression. The 2^-ΔCt method was used to calculate relative expression using the mean Ct values of target genes and the control.

Bioinformatical analysis

Analysis of TCGA database of Uterine Corpus Endometrial Carcinoma (EC) cohort was performed using OncomiR database ¹⁹. Analysis of the association of the miRNAs expression and EC patients' survival from the TCGA cohort (n=533) was performed using OncoLnc ²⁰. Patients were divided based on miRNA level low: high 75:25.

Data processing and analysis

Data were collected and processed with Excel 2016 (Microsoft, USA). Statistical analyses were conducted with GraphPad Prism 8.4.3 (GraphPad Software Inc.) using the Mann-Whitney and log-rank tests. Data distribution was tested using the Shapiro-Wilk test. All values in Fig. 2, Fig. 3, Fig. 4, Fig. 5 are represented as median and 95% CI. Data in Fig 6. is presented as a violin plot with the median indicated as dashed line and quartiles indicated as solid lines. Data in Fig. 7 is presented as a Kaplan-Meier plot. A P-value of < 0.05 was considered statistically significant.

Acknowledgements

This research was funded by the National Centre of Science, grant number 2021/41/N/NZ5/02772 (K.K.). The APC was funded by the Medical University of Warsaw.

Author Contributions

K.K. acquired funding, planed and conducted the experiments, analyzed the results, and wrote the manuscript. T.M.G. conceived the experiments, analyzed the results, prepared figures, supervised the study, and wrote the manuscript. A.K. collected and analyzed clinical data of the patients. A.M. wrote the manuscript. J.W. and J.O. collected and provided HE and EC samples and performed histopathological analysis of the samples. A.G. participated in the experiments. P.K.W. acquired funding and supervised the study. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data that support the findings of this study are available from T.M.G. or K.K. upon reasonable request. Data from TCGA are publicly available.

Competing Interests Statement

The authors declare no conflict of interest.

Makker, V.et al. Endometrial cancer. Nature Reviews Disease Primers 7, 88, doi:10.1038/s41572-021-00324-8 (2021).
Siegel, R. L., Miller, K. D., Fuchs, H. E. & Jemal, A. Cancer statistics, 2022. CA: a cancer journal for clinicians 72, 7-33, doi:10.3322/caac.21708 (2022).
Calle, E. E., Rodriguez, C., Walker-Thurmond, K. & Thun, M. J. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. The New England journal of medicine 348, 1625-1638, doi:10.1056/NEJMoa021423 (2003).
National Cancer Institute Surveillance, Epidemiology, and End results program. Cancer Stat facts: Uterine Cancer.2021. Available at: https://seer.cancer.gov/statfacts/html/corp.html. Accessed August 1, 2021. .
Bokhman, J. V. Two pathogenetic types of endometrial carcinoma. Gynecologic oncology 15, 10-17, doi:10.1016/0090-8258(83)90111-7 (1983).
Masood, M. & Singh, N. Endometrial carcinoma: changes to classification (WHO 2020). Diagnostic Histopathology 27, 493-499, doi:https://doi.org/10.1016/j.mpdhp.2021.09.003 (2021).
Mohr, A. M. & Mott, J. L. Overview of microRNA biology. Semin Liver Dis 35, 3-11, doi:10.1055/s-0034-1397344 (2015).
Ali Syeda, Z., Langden, S. S. S., Munkhzul, C., Lee, M. & Song, S. J. Regulatory Mechanism of MicroRNA Expression in Cancer. International journal of molecular sciences 21, doi:10.3390/ijms21051723 (2020).
Peng, Y. & Croce, C. M. The role of MicroRNAs in human cancer. Signal transduction and targeted therapy 1, 15004, doi:10.1038/sigtrans.2015.4 (2016).
Gebert, L. F. R. & MacRae, I. J. Regulation of microRNA function in animals. Nature Reviews Molecular Cell Biology 20, 21-37, doi:10.1038/s41580-018-0045-7 (2019).
Grzywa, T. M., Klicka, K. & Włodarski, P. K. Regulators at Every Step-How microRNAs Drive Tumor Cell Invasiveness and Metastasis. Cancers (Basel) 12, doi:10.3390/cancers12123709 (2020).
Hanahan, D. Hallmarks of Cancer: New Dimensions. Cancer Discov 12, 31-46, doi:10.1158/2159-8290.Cd-21-1059 (2022).
Ruan, K., Fang, X. & Ouyang, G. MicroRNAs: novel regulators in the hallmarks of human cancer. Cancer letters 285, 116-126, doi:10.1016/j.canlet.2009.04.031 (2009).
Zhang, B., Pan, X., Cobb, G. P. & Anderson, T. A. microRNAs as oncogenes and tumor suppressors. Developmental biology 302, 1-12, doi:10.1016/j.ydbio.2006.08.028 (2007).
Klicka, K., Grzywa, T. M., Klinke, A., Mielniczuk, A. & Włodarski, P. K. The Role of miRNAs in the Regulation of Endometrial Cancer Invasiveness and Metastasis-A Systematic Review. Cancers (Basel) 13, doi:10.3390/cancers13143393 (2021).
Donkers, H., Bekkers, R. & Galaal, K. Diagnostic value of microRNA panel in endometrial cancer: A systematic review. Oncotarget 11, 2010-2023, doi:10.18632/oncotarget.27601 (2020).
Banno, K.et al. MicroRNAs in endometrial cancer. International journal of clinical oncology 18, 186-192, doi:10.1007/s10147-013-0526-9 (2013).
Kontomanolis, E. N. & Koukourakis, M. I. MicroRNA: The Potential Regulator of Endometrial Carcinogenesis. Microrna 4, 18-25, doi:10.2174/2211536604666150710094418 (2015).
Sarver, A. L., Sarver, A. E., Yuan, C. & Subramanian, S. OMCD: OncomiR Cancer Database. BMC Cancer 18, 1223, doi:10.1186/s12885-018-5085-z (2018).
Anaya, J. OncoLnc: linking TCGA survival data to mRNAs, miRNAs, and lncRNAs. PeerJ Computer Science 2, e67 (2016).
Zaheer, U.et al. Expression profile of MicroRNA: An Emerging Hallmark of Cancer. Curr Pharm Des 25, 642-653, doi:10.2174/1386207322666190325122821 (2019).
Lu, J.et al. MicroRNA expression profiles classify human cancers. Nature 435, 834-838, doi:10.1038/nature03702 (2005).
Petrovic, N., Ergün, S. & Isenovic, E. R. Levels of MicroRNA Heterogeneity in Cancer Biology. Mol Diagn Ther 21, 511-523, doi:10.1007/s40291-017-0285-9 (2017).
Guo, Y. X.et al. The Role of miR-23b in Cancer and Autoimmune Disease. J Oncol 2021, 6473038, doi:10.1155/2021/6473038 (2021).
Chen, S.et al. The role of metastasis-associated in colon cancer 1 (MACC1) in endometrial carcinoma tumorigenesis and progression. Mol Carcinog 56, 1361-1371, doi:10.1002/mc.22599 (2017).
Jayaraman, M.et al. Identification of novel diagnostic and prognostic miRNA signatures in endometrial cancer. Genes Cancer 8, 566-576, doi:10.18632/genesandcancer.144 (2017).
Kalinkova, L.et al. Discriminating miRNA Profiles between Endometrioid Well- and Poorly-Differentiated Tumours and Endometrioid and Serous Subtypes of Endometrial Cancers. International journal of molecular sciences 21, doi:10.3390/ijms21176071 (2020).
Shang, C., Lu, Y. M. & Meng, L. R. MicroRNA-125b down-regulation mediates endometrial cancer invasion by targeting ERBB2. Med Sci Monit 18, Br149-155, doi:10.12659/msm.882617 (2012).
Buchynska, L. G., Borykun, T. V., Iurchenko, N. P., Nespryadko, S. V. & Nesina, I. P. Expression of microRNA in tumor cells of endmetrioid carcinoma of endometrium. Exp Oncol 42, 289-294, doi:10.32471/exp-oncology.2312-8852.vol-42-no-4.15522 (2020).
Wu, D., Huang, H.-j., He, C.-n. & Wang, K.-y. MicroRNA-199a-3p Regulates Endometrial Cancer Cell Proliferation by Targeting Mammalian Target of Rapamycin (mTOR). International Journal of Gynecologic Cancer 23, 1191-1197, doi:10.1097/IGC.0b013e31829ea779 (2013).
Cohn, D. E.et al. Comprehensive miRNA profiling of surgically staged endometrial cancer. American Journal of Obstetrics and Gynecology 202, 656.e651-656.e658, doi:10.1016/j.ajog.2010.02.051 (2010).
Cohn, D. E.et al. Comprehensive miRNA profiling of surgically staged endometrial cancer. Am J Obstet Gynecol 202, 656.e651-658, doi:10.1016/j.ajog.2010.02.051 (2010).
Song, J.et al. Potential Value of miR-221/222 as Diagnostic, Prognostic, and Therapeutic Biomarkers for Diseases. Frontiers in Immunology 8, doi:10.3389/fimmu.2017.00056 (2017).
Bai, H. & Wu, S. miR-451: A Novel Biomarker and Potential Therapeutic Target for Cancer. Onco Targets Ther 12, 11069-11082, doi:10.2147/OTT.S230963 (2019).
Notaro, S.et al. Evaluating L1CAM expression in human endometrial cancer using qRT-PCR. Oncotarget 7, 40221-40232, doi:10.18632/oncotarget.9574 (2016).
Dong, P., Xiong, Y., Yue, J., Hanley, S. J. B. & Watari, H. miR-34a, miR-424 and miR-513 inhibit MMSET expression to repress endometrial cancer cell invasion and sphere formation. Oncotarget 9, 23253-23263, doi:10.18632/oncotarget.25298 (2018).
Wang, Z.et al. MicroRNA-34a inhibits cells proliferation and invasion by downregulating Notch1 in endometrial cancer. Oncotarget 8, 111258-111270, doi:10.18632/oncotarget.22770 (2017).
Schirmer, U.et al. Role of miR-34a as a suppressor of L1CAM in endometrial carcinoma. Oncotarget 5, 462-472, doi:10.18632/oncotarget.1552 (2014).
Nakamura, N., Davis, K., Yan, J., Sloper, D. T. & Chen, T. Increased estrogen levels altered microRNA expression in prostate and plasma of rats dosed with sex hormones. Andrology 8, 1360-1374, doi:10.1111/andr.12780 (2020).
He, B.et al. miRNA-based biomarkers, therapies, and resistance in Cancer. International journal of biological sciences 16, 2628-2647, doi:10.7150/ijbs.47203 (2020).
Coll-de la Rubia, E.et al. Prognostic Biomarkers in Endometrial Cancer: A Systematic Review and Meta-Analysis. Journal of clinical medicine 9, doi:10.3390/jcm9061900 (2020).
Hutt, S.et al. The role of biomarkers in endometrial cancer and hyperplasia: a literature review. Acta oncologica (Stockholm, Sweden) 58, 342-352, doi:10.1080/0284186x.2018.1540886 (2019).
Klopfleisch, R., Weiss, A. T. & Gruber, A. D. Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues. Histol Histopathol 26, 797-810, doi:10.14670/hh-26.797 (2011).
Grzywa, T. M.et al. Higher Mutation Burden in High Proliferation Compartments of Heterogeneous Melanoma Tumors. Int J Mol Sci 22, doi:10.3390/ijms22083886 (2021).
Grzywa, T. M.et al. miR-410-3p is induced by vemurafenib via ER stress and contributes to resistance to BRAF inhibitor in melanoma. PLoS One 15, e0234707, doi:10.1371/journal.pone.0234707 (2020).
Pełka, K.et al. miR-96-5p, miR-134-5p, miR-181b-5p and miR-200b-3p heterogenous expression in sites of prostate cancer versus benign prostate hyperplasia-archival samples study. Histochem Cell Biol 155, 423-433, doi:10.1007/s00418-020-01941-2 (2021).
Grzywa, T. M.et al. Lineage-dependent role of miR-410-3p as oncomiR in gonadotroph and corticotroph pituitary adenomas or tumor suppressor miR in somatotroph adenomas via MAPK, PTEN/AKT, and STAT3 signaling pathways. Endocrine 65, 646-655, doi:10.1007/s12020-019-01960-7 (2019).

Table 1

The histopathological and clinical data of patients.

ID	Age	Type of EC/ HE	Grading	TNM
101	53	Endometrioid EC (EC1)	G2	pT3b N0
102	66	Endometrioid EC (EC1)	G1	pT1b N0
103	60	Endometrioid EC (EC1)	G3	pT1a N1
104	46	Endometrioid EC (EC1)	G1	pT1a N0
105	60	Endometrioid EC (EC1)	G1	pT2 N0
106	70	Endometrioid EC (EC1)	G1	pT1a N0
107	63	Endometrioid EC (EC1)	G2	pT2 N0
108	68	Endometrioid EC (EC1)	G1	pT1b Nx
109	64	Endometrioid EC (EC1)	G1	pT3b N0
110	79	Endometrioid EC (EC1)	G2	pT1b N0
111	57	Endometrioid EC (EC1)	G1	pT1a N0
112	65	Endometrioid EC (EC1)	G1	pT3b N0
113	64	Endometrioid EC (EC1)	G1	pT1a N0
114	52	Endometrioid EC (EC1)	G2	pT2 N0
115	62	Endometrioid EC (EC1)	G2	pT3a N0
116	68	Endometrioid EC (EC1)	G1	pT1b N1
117	71	Endometrioid EC (EC1)	G2	pT1a N0
118	61	Endometrioid EC (EC1)	G1	pT1a N0
119	64	Serous EC (EC2)	High grade	pT3a N0
120	84	Serous EC (EC2)	n/a	pT1a N0
121	80	Serous EC (EC2)	High grade	pT3b N1a
122	79	Serous EC (EC2) + Endometrioid EC (EC1)	High grade + G1	pT3c N0 + pT1a N0
123	68	Serous EC (EC2)	High grade	pT3b N0
124	64	Serous EC (EC2)	High grade	pT1b N1
125	78	Serous EC (EC2)	High grade	pT1b Nx
126	73	Serous EC (EC2)	n/a	pT3b N1
127	84	Serous EC (EC2)	High grade	pT1a N0
128	69	Serous EC (EC2)	High grade	pT2 N1a
129	78	Serous EC (EC2)	High grade	pT3b N2
130	66	Serous EC (EC2)	High grade	pT3b N2
131	23	HE	n/a	n/a
132	46	HE	n/a	n/a
133	44	HE	n/a	n/a
134	41	HE	n/a	n/a
135	49	HE	n/a	n/a
136	50	HE	n/a	n/a
137	48	HE	n/a	n/a
138	55	HE	n/a	n/a
139	37	HE	n/a	n/a
140	51	HE	n/a	n/a
141	46	HE	n/a	n/a
142	52	HE	n/a	n/a
143	51	HE	n/a	n/a
144	47	HE	n/a	n/a
145	50	HE	n/a	n/a

Table 2

List of primers sequences

miRNA	Sequence
hsa-miR-21-3p	5’-CAACACCAGTCGATGGGCTGT-3’
hsa-miR-23b	5’-ATCACATTGCCAGGGATTACC-3’
hsa-miR-34a-5p	5’-TGGCAGTGTCTTAGCTGGTTGT-3’
hsa-miR-96-5p	5’-TTTGGCACTAGCACATTTTTGCT-3’
hsa-miR-125b-5p	5’-TCCCTGAGACCCTAACTTGTGA-3’
hsa-miR-134-5p	5’-TGTGACTGGTTGACCAGAGGGG-5’
hsa-miR-146-5p	5’-TGAGAACTGAATTCCATGGGTT-5’
hsa-miR-150-5p	5’-TCTCCCAACCCTTGTACCAGTG-3’
hsa-miR-181b-5p	5’-AACATTCATTGCTGTCGGTGGGT-3’
hsa-miR-182-5p	5’-TTTGGCAATGGTAGAACTCACACT-3’
hsa-miR-199a-3p	5’-ACAGTAGTCTGCACATTGGTTA-3’
hsa-miR-200b-3p	5’-TAATACTGCCTGGTAATGATGA-3’
hsa-miR-211-3p	5’-GCAGGGACAGCAAAGGGGTGC-3’
hsa-miR-221-3p	5’-AGCTACATTGTCTGCTGGGTTTC-3’
hsa-miR-410-3p	5’-AATATAACACAGATGGCCTGT-3’
hsa-miR-451a	5’-AAACCGTTACCATTACTGAGTT-3’

No competing interests reported.

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Decreased expression of miR-23b is associated with poor survival of endometrial cancer patients

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Abstract

Figures

Introduction

Results

MiR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a are downregulated in EC

MiR-34a-5p and miR-146-5p are upregulated in EC1 compared to EC2

MiR-23b, miR-125b-5p miR-199a-3p, miR-221-3p are downregulated in TCGA cohort

Decreased miR-23b expression is associated with worse survival

Discussion

Conclusions

Materials And Methods

Patients tissue

Hematoxylin and Eosin Staining

Laser capture microdissection

RNA isolation and RT-qPCR

Bioinformatical analysis

Data processing and analysis

Declarations

Acknowledgements

Author Contributions

Data Availability Statement

Competing Interests Statement

References

Tables

Additional Declarations

Status:

Version 1