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Modulatory Role of Vaginal-Isolated Lactococcus lactis on the Expression of miR-21, miR-200b, and TLR-4 in CAOV-4 Cells and In Silico Revalidation

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Abstract

Ovarian cancer (OC) is a leading cause of death among women worldwide. Various evidences suggest that oncomiRs and Toll-like receptor 4 (TLR-4) signaling pathways appear to be key players in the initiation and progression of OC. It seems there exists a continuous intercommunication between cancer cells and normal microbiota of the vagina. The biological impacts of vaginal isolated lactococcus lactis on CAOV-4 cells were investigated using several molecular biology experiments, including flow cytometry, DAPI staining, DNA ladder, and scratch assay. The expression of microRNAs (miRNAs/miRs) 21, 200b, and TLR-4 in the CAOV-4 cells was also evaluated by the real-time RT-PCR assay. Furthermore, an integrative in silico analysis was conducted using normalized web-available microarray data (GSE14407) to revalidate the experimental findings and identify potential biomarkers in ovarian cancer. Protein-protein interactions (PPIs) network was studied by means of the STRING database using Cytoscape v3.6.1. The miRNA target genes were identified using the dbDEMC v2.0, miRTarBase, and miRDB databases. Our data demonstrated that L. lactis probiotic candidate downregulates TLR-4, miR-21, and miR-200b expression levels, which correlates with induction of apoptosis as confirmed by DAPI staining, DNA ladder assay, annexin V/PI staining, and inhibition of migration validated by scratch assay. By in silico analysis, several targets (miR-17-5p-BCL2, miR-21-5p-MKNK2, miR-129-5p-CDK6) were identified, while BCL2, CCNB1, and VEGFA were found as the hub proteins in the miRNA-target and PPI networks. Further, downregulation of the TLR-4, miR-21, and miR-200b was partially validated by the in silico analysis. Based on our findings, the vaginal isolated probiotic strain presents great potential to control the ovarian cancer which may provide beneficial impact on the clinical management of ovarian cancer.

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Funding

The Research Center for Pharmaceutical Nanotechnology (RCPN) at the Biomedicine Institute, Tabriz University of Medical Sciences, provided financial support.

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Author notes

  1. Yalda Rahbar Saadat, Mohammad Mostafa Pourseif and Sepideh Zununi Vahed contributed equally to this work and should be considered as co-first authors.

Authors and Affiliations

  1. Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran

    Yalda Rahbar Saadat, Mohammad Mostafa Pourseif, Abolfazl Barzegari, Yadollah Omidi & Jaleh Barar

  2. Kidney Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

    Sepideh Zununi Vahed

  3. Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

    Yadollah Omidi & Jaleh Barar

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  1. Yalda Rahbar Saadat
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  2. Mohammad Mostafa Pourseif
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  4. Abolfazl Barzegari
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Contributions

Y.O., A.B., and J.B. designed the research. Y.R.S. and S.Z.V. performed experiments. A.B. performed the bacterial characterization and western blot assay. M.M.P. performed in silico analysis. YO reviewed the in silico data. Y.R.S. and S.Z.V. analyzed the data. All the authors discussed the results. Y.R.S. and M.M.P. drafted the manuscript. J.B. critically revised the manuscript for important intellectual content. All authors read and gave final approval of the submission of the last version of the paper.

Corresponding authors

Correspondence to Abolfazl Barzegari or Jaleh Barar.

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The present study was approved by the Ethics and Human Rights Committee of Tabriz University of Medical Sciences, Tabriz, Iran (Ethical code: IR.TBZMED.REC.1397.1051) and written informed consent was obtained after enough explanation about the purpose of the study.

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Rahbar Saadat, Y., Pourseif, M.M., Zununi Vahed, S. et al. Modulatory Role of Vaginal-Isolated Lactococcus lactis on the Expression of miR-21, miR-200b, and TLR-4 in CAOV-4 Cells and In Silico Revalidation. Probiotics & Antimicro. Prot. 12, 1083–1096 (2020). https://doi.org/10.1007/s12602-019-09596-9

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