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

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 ◽

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

Cancer Cell International ◽

10.1186/s12935-020-01601-4 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Yang Gu ◽

Shulan Zhang

Keyword(s):

Ovarian Cancer ◽

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.

Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer

10.21203/rs.2.23093/v1 ◽

2020 ◽

Author(s):

Yang Gu ◽

Shulan Zhang

Keyword(s):

Ovarian Cancer ◽

Molecular Mechanisms ◽

Target Genes ◽

Luciferase Reporter ◽

Remarkable Change ◽

Differentially Expressed Mirnas ◽

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.

Microtranscriptome of contrasting sugarcane cultivars in response to aluminum stress

10.1101/645267 ◽

2019 ◽

Author(s):

Renan Gonçalves Silva ◽

Thiago Mateus-Rosa ◽

Suzelei de Castro França ◽

Pratibha Kottapalli ◽

Kameswara Rao Kottapalli ◽

...

Keyword(s):

Target Genes ◽

Abiotic Factors ◽

Nutrient Deficiency ◽

Aluminum Stress ◽

Factors Affecting ◽

Aluminum Ions ◽

AbstractAlthough metallic elements are required for plant growth, aluminum ions (Al+3) can be considered one of the major abiotic factors affecting productivity. In plants, the presence of Al+3 can result in inhibition of root growth triggering water and nutrient deficiency. Plants under stress conditions undergo gene expression changes in specific genes or post-transcriptional gene regulators as miRNAs that can led to resistance. In this study, we investigated the miRNAs involved in the sugarcane response to aluminum stress. Four miRNA libraries were generated using sugarcane roots of two contrasting (tolerant and sensitive) sugarcane cultivars growing under aluminum stress to identify the miRNAs involved in the sugarcane response. Here we present the first miRNAs sequencing of sugarcane response under aluminum stress. The contrast of the cultivars seen in the field was reflected in the micro transcriptome with opposing expression profile. We selected 394 differentially expressed miRNAs, in both cultivars, 22% were common between cultivars. Real time quantitative polymerase chain reaction was used to validate the differentially expressed miRNAs through high-throughput sequencing in sugarcane roots. Target genes prediction was also analyzed. Our results indicated miRNAs that modulated specific target genes involved in roots development and plant aluminum stress response. Those genes can be the answer to tolerance in sugarcane and used in breeding programs to develop tolerant cultivars.

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.

Long non-coding RNA CCDC183-AS1 acts AS a miR-589-5p sponge to promote the progression of hepatocellular carcinoma through regulating SKP1 expression

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01861-6 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

He Zhu ◽

Hongwei Zhang ◽

Youliang Pei ◽

Zhibin Liao ◽

Furong Liu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Cell Lines ◽

Human Cancer ◽

Luciferase Reporter ◽

Migration And Invasion ◽

High Morbidity ◽

Regulatory Relationship

Abstract Background Hepatocellular carcinoma (HCC) is a common type of malignant human cancer with high morbidity and poor prognosis, causing numerous deaths per year worldwide. Growing evidence has been demonstrated that long non-coding RNAs (lncRNAs) are closely associated with hepatocarcinogenesis and metastasis. However, the roles, functions, and working mechanisms of most lncRNAs in HCC remain poorly defined. Methods Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of CCDC183-AS1 in HCC tissues and cell lines. Cell proliferation, migration and invasion ability were evaluated by CCK-8 and transwell assay, respectively. Animal experiments were used to explore the role of CCDC183-AS1 and miR-589-5p in vivo. Bioinformatic analysis, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the regulatory relationship between CCDC183-AS1, miR-589-5p and SKP1. Results Significantly upregulated expression of CCDC183-AS1 was observed in both HCC tissues and cell lines. HCC patients with higher expression of CCDC183-AS1 had a poorer overall survival rate. Functionally, overexpression of CCDC183-AS1 markedly promoted HCC cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo, whereas the downregulation of CCDC183-AS1 exerted opposite effects. MiR-589-5p inhibitor counteracted the proliferation, migration and invasion inhibitory effects induced by CCDC183-AS1 silencing. Mechanistically, CCDC183-AS1 acted as a ceRNA through sponging miR-589-5p to offset its inhibitory effect on the target gene SKP1, then promoted the tumorigenesis of HCC. Conclusions CCDC183-AS1 functions as an oncogene to promote HCC progression through the CCDC183-AS1/miR-589-5p/SKP1 axis. Our study provided a novel potential therapeutic target for HCC patients.

Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows

Journal of Dairy Research ◽

10.1017/s0022029919000785 ◽

2019 ◽

Vol 86 (4) ◽

pp. 425-431 ◽

Cited By ~ 3

Author(s):

Zhi Chen ◽

Jingpeng Zhou ◽

Xiaolong Wang ◽

Yang Zhang ◽

Xubin Lu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Differential Expression ◽

Target Genes ◽

Interleukin 1 ◽

Luciferase Reporter ◽

Expression Of Genes ◽

Chinese Holstein Cows ◽

Reporter Assays

AbstractWe established a mastitis model using exogenous infection of the mammary gland of Chinese Holstein cows with Staphylococcus aureus and extracted total RNA from S. aureus-infected and healthy mammary quarters. Differential expression of genes due to mastitis was evaluated using Affymetrix technology and results revealed a total of 1230 differentially expressed mRNAs. A subset of affected genes was verified via Q-PCR and pathway analysis. In addition, Solexa high-throughput sequencing technology was used to analyze profiles of miRNA in infected and healthy quarters. These analyses revealed a total of 52 differentially expressed miRNAs. A subset of those results was verified via Q-PCR. Bioinformatics techniques were used to predict and analyze the correlations among differentially expressed miRNA and mRNA. Results revealed a total of 329 pairs of negatively associated miRNA/mRNA, with 31 upregulated pairs of mRNA and 298 downregulated pairs of mRNA. Differential expression of miR-15a and interleukin-1 receptor-associated kinase-like 2 (IRAK2), were evaluated by western blot and luciferase reporter assays. We conclude that miR-15a and miR-15a target genes (IRAK2) constitute potential miRNA–mRNA regulatory pairs for use as biomarkers to predict a mastitis response.

Progression-Mediated Changes in Mitochondrial Morphology Promotes Adaptation to Hypoxic Peritoneal Conditions in Serous Ovarian Cancer

Frontiers in Oncology ◽

10.3389/fonc.2020.600113 ◽

2021 ◽

Vol 10 ◽

Author(s):

Joseph P. Grieco ◽

Mitchell E. Allen ◽

Justin B. Perry ◽

Yao Wang ◽

Yipei Song ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Model ◽

Gynecological Cancer ◽

Metastatic Potential ◽

Adaptation To Hypoxia ◽

Mitochondrial Morphology ◽

Serous Ovarian Cancer ◽

Tumor Initiating Cells ◽

Mitochondrial Dynamic ◽

Functional Changes

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases.

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.

The Expression of microRNA in Adult Rat Heart with Isoproterenol-Induced Cardiac Hypertrophy

Cells ◽

10.3390/cells9051173 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1173 ◽

Cited By ~ 1

Author(s):

Mailin Gan ◽

Shunhua Zhang ◽

Yuan Fan ◽

Ya Tan ◽

Zhixian Guo ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Signaling Pathway ◽

Natriuretic Peptide ◽

Target Genes ◽

Pathological Condition ◽

Wnt Signaling Pathway ◽

Mitogen Activated Protein Kinase ◽

Luciferase Reporter

Cardiac hypertrophy is a common pathological condition and an independent risk factor that triggers cardiovascular morbidity. As an important epigenetic regulator, miRNA is widely involved in many biological processes. In this study, miRNAs expressed in rat hearts that underwent isoprenaline-induced cardiac hypertrophy were identified using high-throughput sequencing, and functional verification of typical miRNAs was performed using rat primary cardiomyocytes. A total of 623 miRNAs were identified, of which 33 were specifically expressed in cardiac hypertrophy rats. The enriched pathways of target genes of differentially expressed miRNAs included the FoxO signaling pathway, dopaminergic synapse, Wnt signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, and Hippo signaling pathway. Subsequently, miR-144 was the most differentially expressed miRNA and was subsequently selected for in vitro validation. Inhibition of miR-144 expression in primary myocardial cells caused up-regulation of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The dual luciferase reporter system showed that ANP may be a target gene of miR-144. Long non-coding RNA myocardial infarction associated transcript (LncMIAT) is closely related to heart disease, and here, we were the first to discover that LncMIAT may act as an miR-144 sponge in isoproterenol-induced cardiac hypertrophy. Taken together, these results enriched the understanding of miRNA in regulating cardiac hypertrophy and provided a reference for preventing and treating cardiac hypertrophy.