Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer

Luciferase Reporter ◽

Remarkable Change ◽

Control Plasmid ◽

Target Binding

Abstract Background: The molecular mechanisms of ovarian cancer (OC) remain unclear. We sought to comprehensively identify miRNAs that are aberrantly expressed in OC. Methods: Differentially expressed miRNAs were screened from six pairs of primary and metastatic OC tissues; their possible functions were then analyzed and target genes were predicted by bioinformatics. Then gene expression profiling results were established by reverse transcription quantitative polymerase chain reaction and western blot. Target binding between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Results: Fifteen miRNAs and 10 target mRNAs were differentially expressed in primary and metastatic OC tissues. ITGB3, TGFβ2 and TNC correlated to miRNA function in metastatic OC. Compared with primary OC, RNA levels of hsa-miR-141-3p, hsa-miR-7-5p and hsa-miR-187-5p in metastatic OC tissues were potently decreased ( p < 0.05). However, a statistically prominent difference in hsa-miR-584-5p level between the two groups ( p > 0.05) was not observed. Comparing to primary OC, TGFβ2 and TNC were markedly increased ( p < 0.05). Luciferase activity was remarkably decreased after co-transfection of a wild-type TGFβ2 3’-UTR plasmid and miR-7-5p compared with a control plasmid, but no remarkable change after co-transfection of mutant TGFβ2 3’-UTR and miR-7-5p was demonstrated. Conclusions: Fifteen miRNAs and 10 mRNAs were differentially expressed in metastatic OC tissues compared with primary OC tissues, which suggested that they may participate in invasive and metastatic processes. Hsa-miR-141-3p, hsa-miR-187-5p and hsa-miR-7-5p expression was prominently lower in metastatic OC than in primary OC, while TGFβ2 and TNC expression was markedly higher in metastatic OC tissues. Hsa-miR-7-5p may bind to the TGFβ2 3’-UTR to inhibit its expression.

High-Throughput Sequencing Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer with experimental validations

10.21203/rs.2.23093/v3 ◽

2020 ◽

Author(s):

Yang Gu ◽

Shulan Zhang

Keyword(s):

Ovarian Cancer ◽

High Throughput Sequencing ◽

Human Cancer ◽

Gynecological Cancer ◽

Metastatic Potential ◽

Luciferase Reporter ◽

Downstream Target ◽

Abstract Background: Ovarian cancer (OC) is a common gynecological cancer and characterized by high metastatic potential. MicroRNAs (miRNAs, miRs) have the promise to be harnessed as prognostic and therapeutic biomarkers for OC. Herein, we sought to identify differentially expressed miRNAs and mRNAs in metastatic OC, and to validate them with functional experiments. Methods: Differentially expressed miRNAs and miRNAs were screened from six pairs of primary OC tissues and metastatic tissues using an miRStar™ Human Cancer Focus miRNA & Target mRNA PCR Array. Then, gene expression profiling results were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The binding affinity between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Expression of miR-7-5p and TGFβ2 was manipulated to assess their roles in malignant phenotypes of highly metastatic HO-8910PM cells. Results: MiRNA profiling and sequencing identified 12 miRNAs and 10 mRNAs that were differentially expressed in metastatic tissues. Gene ontology and Pathway analyses determined that 3 differentially expressed mRNAs (ITGB3, TGFβ2 and TNC) were related to OC metastasis. The results of RT-qPCR confirmed that the decrease of miR-7-5p was most significant in OC metastasis, while TGFβ2 was up-regulated in OC metastasis. Moreover, miR-7-5p targeted and negatively regulated TGFβ2. MiR-7-5p overexpression accelerated HO-8910PM cell viability and invasion, and TGFβ2 overexpression reversed the results. Meanwhile, simultaneous miR-7-5p and TGFβ2 overexpression rescued the cell activities. Conclusions: This study characterizes differentially expressed miRNAs and mRNAs in metastatic OC, where miR-7-5p and its downstream target were most closely associated with metastatic OC. Overexpression of miR-7-5p targets and inhibits TGFβ2 expression, thereby inhibiting the growth and metastasis of OC.

High-Throughput Sequencing Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer with Experimental Validations

10.21203/rs.2.23093/v4 ◽

2020 ◽

Author(s):

Yang Gu ◽

Shulan Zhang

Keyword(s):

Ovarian Cancer ◽

High Throughput Sequencing ◽

Human Cancer ◽

Gynecological Cancer ◽

Metastatic Potential ◽

Luciferase Reporter ◽

Downstream Target ◽

Abstract Background: Ovarian cancer (OC) is a common gynecological cancer and characterized by high metastatic potential. MicroRNAs (miRNAs, miRs) have the promise to be harnessed as prognostic and therapeutic biomarkers for OC. Herein, we sought to identify differentially expressed miRNAs and mRNAs in metastatic OC, and to validate them with functional experiments.Methods: Differentially expressed miRNAs and miRNAs were screened from six pairs of primary OC tissues and metastatic tissues using an miRStar™ Human Cancer Focus miRNA & Target mRNA PCR Array. Then, gene expression profiling results were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The binding affinity between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Expression of miR-7-5p and TGFβ2 was manipulated to assess their roles in malignant phenotypes of highly metastatic HO-8910PM cells.Results: MiRNA profiling and sequencing identified 12 miRNAs and 10 mRNAs that were differentially expressed in metastatic tissues. Gene ontology and Pathway analyses determined that 3 differentially expressed mRNAs (ITGB3, TGFβ2 and TNC) were related to OC metastasis. The results of RT-qPCR confirmed that the decrease of miR-7-5p was most significant in OC metastasis, while TGFβ2 was up-regulated in OC metastasis. Moreover, miR-7-5p targeted and negatively regulated TGFβ2. MiR-7-5p overexpression accelerated HO-8910PM cell viability and invasion, and TGFβ2 overexpression reversed the results. Meanwhile, simultaneous miR-7-5p and TGFβ2 overexpression rescued the cell activities.Conclusions: This study characterizes differentially expressed miRNAs and mRNAs in metastatic OC, where miR-7-5p and its downstream target were most closely associated with metastatic OC. Overexpression of miR-7-5p targets and inhibits TGFβ2 expression, thereby inhibiting the growth and metastasis of OC.

Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer

10.21203/rs.2.23093/v2 ◽

2020 ◽

Author(s):

Yang Gu ◽

Shulan Zhang

Keyword(s):

Cell Cycle ◽

Ovarian Cancer ◽

Molecular Mechanisms ◽

Cell Function ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Dual Luciferase Reporter Assay

Abstract Background: The molecular mechanisms of ovarian cancer (OC) remain unclear. We sought to comprehensively identify microRNAs (miRNAs) that are aberrantly expressed in metastatic OC. Methods: Differentially expressed miRNAs were screened from six pairs of primary and metastatic OC tissues; their possible functions were analyzed and target genes were predicted by bioinformatics. Then gene expression profiling results were established by reverse transcription quantitative polymerase chain reaction and western blot. Targeting relationship between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. CCK-8, Transwell assay, scratch test and flow cytometry were used for cell function detection after miR-7-5p overexpression. Results: Twelve miRNAs and 10 target mRNAs were differentially expressed in primary and metastatic OC tissues. ITGB3, TGFβ2 and TNC correlated to miRNA function in metastatic OC. Among all 7 miRNAs, expression of hsa-miR-141-3p, hsa-miR-7-5p, hsa-miR-187-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p in metastatic OC tissues was obviously lower than that in primary OC tissues ( p < 0.05). Moreover, there was a significant correlation between hsa-miR-7-5p and TGFβ2 in OC tissues. Dual-luciferase reporter assay confirmed that hsa-miR-7-5p negatively targeted TGFβ2. After miR-7-5p overexpression, the OC cell viability and invasion were reduced, the cell cycle was blocked ( p < 0.05). Conclusions: Hsa-miR-141-3p, hsa-miR-187-5p, hsa-miR-7-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p expression was prominently lower in metastatic OC than in primary OC, while TGFβ2 expression was markedly increased in metastatic OC tissues. Hsa-miR-7-5p bound to TGFβ2 3’-UTR to inhibit its expression. Hsa-miR-7-5p targeted TGFβ2 to inhibit cell proliferation, invasion and cell cycle entry.

High-throughput sequencing identification of differentially expressed microRNAs in metastatic ovarian cancer with experimental validations

Cancer Cell International ◽

10.1186/s12935-020-01601-4 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Yang Gu ◽

Shulan Zhang

Keyword(s):

Ovarian Cancer ◽

High Throughput Sequencing ◽

Human Cancer ◽

Gynecological Cancer ◽

Metastatic Potential ◽

Luciferase Reporter ◽

Downstream Target ◽

Abstract Background Ovarian cancer (OC) is a common gynecological cancer and characterized by high metastatic potential. MicroRNAs (miRNAs, miRs) have the promise to be harnessed as prognostic and therapeutic biomarkers for OC. Herein, we sought to identify differentially expressed miRNAs and mRNAs in metastatic OC, and to validate them with functional experiments. Methods Differentially expressed miRNAs and mRNAs were screened from six pairs of primary OC tissues and metastatic tissues using a miRStar™ Human Cancer Focus miRNA and Target mRNA PCR Array. Then, gene expression profiling results were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The binding affinity between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Expression of miR-7-5p and TGFβ2 was manipulated to assess their roles in malignant phenotypes of highly metastatic HO-8910PM cells. Results MiRNA profiling and sequencing identified 12 miRNAs and 10 mRNAs that were differentially expressed in metastatic tissues. Gene ontology and Pathway analyses determined that 3 differentially expressed mRNAs (ITGB3, TGFβ2 and TNC) were related to OC metastasis. The results of RT-qPCR confirmed that the decrease of miR-7-5p was most significant in OC metastasis, while TGFβ2 was up-regulated in OC metastasis. Moreover, miR-7-5p targeted and negatively regulated TGFβ2. MiR-7-5p overexpression accelerated HO-8910PM cell viability and invasion, and TGFβ2 overexpression reversed the results. Meanwhile, simultaneous miR-7-5p and TGFβ2 overexpression rescued the cell activities. Conclusions This study characterizes differentially expressed miRNAs and mRNAs in metastatic OC, where miR-7-5p and its downstream target were most closely associated with metastatic OC. Overexpression of miR-7-5p targets and inhibits TGFβ2 expression, thereby inhibiting the growth and metastasis of OC.

Characterization of microRNAs during Embryonic Skeletal Muscle Development in the Shan Ma Duck

Animals ◽

10.3390/ani10081417 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1417

Author(s):

Chuan Li ◽

Ting Xiong ◽

Mingfang Zhou ◽

Lei Wan ◽

Suwang Xi ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Target Genes ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Luciferase Reporter ◽

Meat Production ◽

Skeletal Muscle Development ◽

Poultry skeletal muscle provides high quality protein for humans. Study of the genetic mechanisms during duck skeletal muscle development contribute to future duck breeding and meat production. In the current study, three breast muscle samples from Shan Ma ducks at embryonic day 13 (E13) and E19 were collected, respectively. We detected microRNA (miRNA) expression using high throughput sequencing following bioinformatic analysis. qRT-PCR validated the reliability of sequencing results. We also identified target prediction results using the luciferase reporter assay. A total of 812 known miRNAs and 279 novel miRNAs were detected in six samples; as a result, 61 up-regulated and 48 down-regulated differentially expressed miRNAs were identified between E13 and E19 (|log2 fold change| ≥ 1 and p ≤ 0.05). Enrichment analysis showed that target genes of the differentially expressed miRNAs were enriched on many muscle development-related gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially mitogen-activated protein kinase (MAPK) signaling pathways. An interaction network was constructed using the target genes of the differentially expressed miRNAs. These results complement the current duck miRNA database and offer several miRNA candidates for future studies of skeletal muscle development in the duck.

Integrated analysis of sex-biased mRNA and miRNA expression profiles in the gonad of the discus fish (Symphysodon aequifasciatus)

10.1101/492264 ◽

2018 ◽

Cited By ~ 1

Author(s):

yuanshuai Fu ◽

Zhe Xu ◽

Zaizhong Chen ◽

Bin Wen ◽

Jianzhong Gao

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

High Efficiency ◽

Expression Profiles ◽

Gonad Development ◽

Cdna Libraries ◽

Integrated Analysis ◽

Illumina Hiseq ◽

The discus fish (Symphysodon aequifasciatus) is an ornamental fish that is well-known around the world. Phenotype investigation indicated that there are no significant differences in appearance between males and females of the discus fish. To better understand the sexual development mechanisms and obtain a high efficiency sex identification method in the artificial reproduction process of the discus fish, we constructed six cDNA libraries from three adult testes and three adult ovaries, and perform RNA-sequencing for identifying sex-biased candidate genes, microRNA (miRNA), and metabolic pathway using the Illumina Hiseq 4000. A total of 50,082 non-redundant genes (unigenes) were identified, of which 18,570 unigenes were significantly overexpressed in testes, and 11,182 unigenes were significantly overexpressed in ovaries, and 8 differentially expressed unigenes were validated by quantitative Real-Time PCR (qPCR). A total of 551 miRNAs were identified, of which 47 miRNAs were differentially expressed between testes and ovaries, and 7 differentially expressed miRNAs and one non-differential miRNA were validated by qPCR. Twenty-four of these differentially expressed miRNAs and their 15 predicted target genes constituted 41 important miRNA-mRNA interaction pairs, which may be important candidates for sex-related miRNAs and sex-related genes in the discus fish. Some of vital sex-related metabolic pathways were also identified that may play key roles in regulating gonad development of the discus fish. These results can provide important insights to better understand molecular mechanisms for sexual dimorphism in gonads development.

Identification and validation of key miRNAs and miRNA–mRNA regulatory network associated with uterine involution in postpartum Kazakh sheep

Archives Animal Breeding ◽

10.5194/aab-64-119-2021 ◽

2021 ◽

Vol 64 (1) ◽

pp. 119-129

Author(s):

Heng Yang ◽

Lin Fu ◽

Qifeng Luo ◽

Licai Li ◽

Fangling Zheng ◽

...

Keyword(s):

Mirna Target ◽

Molecular Mechanisms ◽

Target Genes ◽

Gene Prediction ◽

Biological Effects ◽

Illumina Hiseq ◽

Go Annotation ◽

Uterine Involution ◽

Abstract. MicroRNAs (miRNAs) are widely expressed in different mammalian tissues and exert their biological effects through corresponding target genes. miRNA target genes can be rapidly and efficiently identified and screened by combining bioinformatics prediction and experimental validation. To investigate the possible molecular regulatory mechanisms involving miRNAs during uterine involution in postpartum ewes, we used Illumina HiSeq sequencing technology to screen for the number and characteristics of miRNAs in faster uterine involution and normal uterine involution group. A total of 118 differentially expressed miRNAs, including 33 known miRNAs and 85 new miRNAs, were identified in the hypothalamic library, whereas 54 miRNAs, including 5 known miRNAs and 49 new miRNAs, were identified in the uterine library. Screening with four types of gene prediction software revealed 73 target genes associated with uterine involution, and subsequently, GO annotation and KEGG pathway analysis were performed. The results showed that, in the hypothalamic–uterine axis, uterine involution in postpartum ewes might primarily involve two miRNA-target gene pairs, namely, miRNA-200a–PTEN and miRNA-133–FGFR1, which can participate in GnRH signal transduction in the upstream hypothalamus and in the remodeling process at the downstream uterus, through the PI3K–AKT signaling pathway to influence the recovery of the morphology and functions of the uterus during the postpartum period in sheep. Therefore, identification of differentially expressed miRNAs in this study fills a gap in the research related to miRNAs in uterine involution in postpartum ewes and provides an important reference point for a comprehensive understanding of the molecular mechanisms underlying the regulation of postpartum uterine involution in female livestock.

Circulating miRNA Profiling in Plasma Samples of Ovarian Cancer Patients

International Journal of Molecular Sciences ◽

10.3390/ijms20184533 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4533 ◽

Cited By ~ 8

Author(s):

András Penyige ◽

Éva Márton ◽

Beáta Soltész ◽

Melinda Szilágyi-Bónizs ◽

Róbert Póka ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Patients ◽

Target Genes ◽

Enrichment Analysis ◽

Cancer Development ◽

Plasma Samples ◽

Circulating Mirna ◽

Potential Biomarkers

Ovarian cancer is one of the most common cancer types in women characterized by a high mortality rate due to lack of early diagnosis. Circulating miRNAs besides being important regulators of cancer development could be potential biomarkers to aid diagnosis. We performed the circulating miRNA expression analysis in plasma samples obtained from ovarian cancer patients stratified into FIGO I, FIGO III, and FIGO IV stages and from healthy females using the NanoString quantitative assay. Forty-five miRNAs were differentially expressed, out of these 17 miRNAs showed significantly different expression between controls and patients, 28 were expressed only in patients, among them 19 were expressed only in FIGO I patients. Differentially expressed miRNAs were ranked by the network-based analysis to assess their importance. Target genes of the differentially expressed miRNAs were identified then functional annotation of the target genes by the GO and KEGG-based enrichment analysis was carried out. A general and an ovary-specific protein–protein interaction network was constructed from target genes. Results of our network and the functional enrichment analysis suggest that besides HSP90AA1, MYC, SP1, BRCA1, RB1, CFTR, STAT3, E2F1, ERBB2, EZH2, and MET genes, additional genes which are enriched in cell cycle regulation, FOXO, TP53, PI-3AKT, AMPK, TGFβ, ERBB signaling pathways and in the regulation of gene expression, proliferation, cellular response to hypoxia, and negative regulation of the apoptotic process, the GO terms have central importance in ovarian cancer development. The aberrantly expressed miRNAs might be considered as potential biomarkers for the diagnosis of ovarian cancer after validation of these results in a larger cohort of ovarian cancer patients.

Joint RNA-Seq and miRNA Profiling Analyses to Reveal Molecular Mechanisms in Regulating Thickness of Pod Canopy in Brassica napus

Genes ◽

10.3390/genes10080591 ◽

2019 ◽

Vol 10 (8) ◽

pp. 591 ◽

Cited By ~ 1

Author(s):

Chen ◽

Huo ◽

Yang ◽

Jian ◽

Qu ◽

...

Keyword(s):

Brassica Napus ◽

Molecular Mechanisms ◽

Target Genes ◽

Oilseed Crop ◽

Flower Bud ◽

Mirna Profiling ◽

Yield Formation ◽

Architecture Component

Oilseed rape (Brassica napus) is the second largest oilseed crop worldwide. As an architecture component of B. napus, thickness of pod canopy (TPC) plays an important role in yield formation, especially under high-density cultivation conditions. However, the mechanisms underlying the regulation of TPC remain unclear. RNA and microRNA (miRNA) profiling of two groups of B. napus lines with significantly different TPC at the bolting with a tiny bud stage revealed differential expressions of numerous genes involved in nitrogen-related pathways. Expression of several nitrogen-related response genes, including ASP5, ASP2, ASN3, ATCYSC1, PAL2, APT2, CRTISO, and COX15, was dramatically changed in the thick TPC lines compared to those in the thin TPC lines. Differentially expressed miRNAs also included many involved in nitrogen-related pathways. Expression of most target genes was negatively associated with corresponding miRNAs, such as miR159, miR6029, and miR827. In addition, 12 (including miR319, miR845, and miR158) differentially expressed miRNAs between two plant tissues sampled (stem apex and flower bud) were identified, implying that they might have roles in determining overall plant architecture. These results suggest that nitrogen signaling may play a pivotal role in regulating TPC in B. napus.

A systemic study of indoxacarb resistance in Spodoptera litura revealed complex expression profiles and regulatory mechanism

Scientific Reports ◽

10.1038/s41598-019-51234-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Li Shi ◽

Yao Shi ◽

Ya Zhang ◽

Xiaolan Liao

Keyword(s):

Spodoptera Litura ◽

Molecular Mechanisms ◽

Target Genes ◽

Expression Profiles ◽

Resistance Mechanisms ◽

Glutathione S Transferase ◽

Important Pest ◽

Resistant Strains

Abstract The tobacco cutworm, Spodoptera litura, is an important pest of crop and vegetable plants worldwide, and its resistance to insecticides have quickly developed. However, the resistance mechanisms of this pest are still unclear. In this study, the change in mRNA and miRNA profiles in the susceptible, indoxacarb-resistant and field indoxacarb-resistant strains of S. litura were characterized. Nine hundred and ten co-up-regulated and 737 co-down-regulated genes were identified in the resistant strains. Further analysis showed that 126 co-differentially expressed genes (co-DEGs) (cytochrome P450, carboxy/cholinesterase, glutathione S-transferase, ATP-binding cassette transporter, UDP-glucuronosyl transferase, aminopeptidase N, sialin, serine protease and cuticle protein) may play important roles in indoxacarb resistance in S. litura. In addition, a total of 91 known and 52 novel miRNAs were identified, and 10 miRNAs were co-differentially expressed in the resistant strains of S. litura. Furthermore, 10 co-differentially expressed miRNAs (co-DEmiRNAs) had predicted co-DEGs according to the expected miRNA-mRNA negative regulation pattern and 37 indoxacarb resistance-related co-DEGs were predicted to be the target genes. These results not only broadened our understanding of molecular mechanisms of insecticide resistance by revealing complicated profiles, but also provide important clues for further study on the mechanisms of miRNAs involved in indoxacarb resistance in S. litura.