Long Noncoding RNA KCNQ1OT1 Induces Resistance of HCC Cells To Cisplatin Through Regulating The miR-26a/CCND2 Molecular Axis

Qrt Pcr ◽

Gene Assay ◽

Proliferation And Invasion ◽

Hcc Cells

Abstract Objective: To explore the molecular mechanism by which LncRNA KCNQ1OT1 regulated the miR-26a/CCND2 molecular axis to participate in the resistance of Hepatocellular carcinoma(HCC) cells to cisplatin.Methods: Cancer tissue and corresponding para-carcinoma tissue specimens were collected from 25 HCC patients with complete data admitted from January 2018 to December 2018 at The Transplantation Center of the Third Xiangya Hospital. Then, the expression levels of KCNQ1OT1, miR-26a and CCND2 in HCCtissues and cell lines were detected through qRT-PCR. Meanwhile, the sensitivity of HCC cells to cisplatin was examined through Transwell and Annexin V-FITC/PI double staining flow cytometry. Further, the targeted relationships among KCNQ1OT1, miR-26a and CCND were verified through dual-luciferase reporter gene assay, and the regulatory relationships were detected through Western blotting and qRT-PCR.Results: KCNQ1OT1 was highly expressed in HCC tissues and cisplatin-resistant cell lines; meanwhile, over-expression of KCNQ1OT1 promoted the resistance of Huh7/CDDP cells to cisplatin. Dual-luciferase reporter gene assay verified that, KCNQ1OT1 targeted miR-26a and down-regulated its expression level. miR-26a suppressed Huh7/CDDP cell proliferation and invasion, while promoting their apoptosis, thus down-regulating the promoting effect of KCNQ1OT1 on the cisplatin resistance of HCC cells. miR-26a negatively regulated CCND2 expression, while KCNQ1OT1 down-regulated the suppression of miR-26a on CCND2 to promote Huh7/CDDP cell proliferation and invasion and to suppress apoptosis, thereby up-regulating the resistance of HCCcells to cisplatin. Conclusions: LncRNA KCNQ1OT1 regulates the miR-26a/CCND2 molecular axis to induce the resistance of HCC cells to cisplatin.

Hsa_circ_0060927 Is a Novel Tumor Biomarker by Sponging miR-195-5p in the Malignant Transformation of OLK to OSCC

Frontiers in Oncology ◽

10.3389/fonc.2021.747086 ◽

2022 ◽

Vol 11 ◽

Author(s):

Siming Xu ◽

Yuhan Song ◽

Yanxiong Shao ◽

Haiwen Zhou

Keyword(s):

Cell Proliferation ◽

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Qrt Pcr ◽

Cerna Network ◽

Gene Assay ◽

And Migration

ObjectiveTo investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).MethodsWe performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.ResultsThe results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.ConclusionHsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis.

MiR-486 protects against acute myocardial infarction via regulation of PTEN

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v20i9.10 ◽

2021 ◽

Vol 20 (9) ◽

pp. 1845-1853

Author(s):

Qinfeng Han ◽

Zhong Xu ◽

Xiaolei Zhang ◽

Kun Yang ◽

Zhifei Sun ◽

...

Keyword(s):

Myocardial Infarction ◽

Akt Signaling ◽

Reporter Gene Assay ◽

Expression Level ◽

Luciferase Reporter ◽

Akt Signaling Pathway ◽

Qrt Pcr ◽

Purpose: To investigate the effect of miR-486 on rats with acute myocardial infarction (AMI), and its mechanism of action.Methods: A rat model of AMI was established. They were randomly divided into 4 groups, namely, sham, model, agomiR-486 and antagomiR-486 groups, respectively. Rats in these different groups were treated with agomiR-21 (5 μL, 40 nmol/mL), antagomiR-21 (5 μL, 40 nmol/mL) or agomiR-NC (5 μL, 40 nmol/mL), respectively. Then, key miRNAs were sorted out using gene-chip assay and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Luciferase reporter gene assay was conducted to determine the interaction between miR-486 and gene of PTEN. After intraperitoneal injection of agomiR-486 and antagomiR-486, hemodynamics was measured to determine the effect of miR-486 on myocardial function of the rats. The effect of miR-486 expression level on the expression of myocardial enzymes in serum, the morphology of myocardial tissues, and the apoptosis of myocardial tissues in rats, were investigated. Additionally, the effect of miR-486 expression level on PTEN/AKT signaling pathway in the rats was determined by Western blotting.Results: The results of gene-chip and qRT-PCR assays revealed that there were 8 differentially expressed genes in rat myocardial tissues in the model group when compared with the sham group. MiR-486 improved the cardiac function of rats and the morphology of myocardial tissues, but reduced AMI-induced apoptosis of myocardial cells and the expression of myocardial enzymes (markers of myocardial injury) in a dose-dependent manner (p < 0.05). The results of luciferase reporter gene assay showed that PTEN was a direct target of miR-486. In rat models of AMI, a raised expression of miR-486 remarkably suppressed the protein expression level of PTEN and up-regulated that of p-AKT/AKT (p < 0.05).Conclusion: MiR-486 protects against AMI in rats probably by targeting PTEN and activating the AKT signaling pathway. The results of the current study may provide new insights for the treatment of AMI.

Circ_0000005 Facilitates Proliferation, Apoptosis, Migration and Invasion of Acute Myeloid Leukemia Cells via Modulating miR-139-5p/Tspan3 Axis

10.21203/rs.3.rs-57994/v1 ◽

2020 ◽

Author(s):

Juan Tong ◽

Huilan Liu ◽

Changcheng Zheng ◽

Xiaoyu Zhu ◽

Xiang Wan ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Regulatory Function ◽

Qrt Pcr ◽

Abstract Background: Accumulating circular RNAs (circRNAs) are reported to be abnormally expressed in diverse cancers, hematologic malignancies included. This study aimed to investigate the biological function and underlying mechanisms of circ_0000005 in acute myeloid leukemia (AML).Materials and methods: Bone marrow samples were enrolled from AML patients with normal samples as controls. Circ_0000005, miR-139-5p and tetraspanin 3 (Tspan3) were detected by qRT-PCR and Western blot, respectively. AML cell lines (KG1 and HL60) were used as cell models. CCK-8, Transwell and flow cytometry assays were adopted to study the biological functions of circ_0000005 on AML cells in vitro. The interrelation between circ_0000005 and miR-139-5p was detected by bioinformatics, qRT-PCR, luciferase reporter gene assay, RNA pull-down assay, and RNA-binding protein immunoprecipitation (RIP) assays. Ultimately, Western blot, qRT-PCR, luciferase reporter gene assay were adopted to corroborate the interrelation between miR-139-5p and its target Tspan3. Results: Circ_0000005 was demonstrably elevated in both AML clinical samples and cell lines. Circ_0000005 overexpression promoted the viability, migration and invasion of AML cells, and repressed the apoptosis of AML cells, while silencing circ_0000005 showed opposite biological effects. Circ_0000005 interacted with miR-139-5p and repressed its expression, and Tspan3 was proved to be negatively regulated by miR-139-5p. Circ_0000005 could promote the expression of Tspan3 via repressing miR-139-5p, and the oncogenic functions of circ_0000005 were dependent on its regulatory function on miR-139-5p/Tspan3 axis.Conclusion: Circ_0000005 facilitates the malignant phenotypes of AML cells via miR-139-5p/Tspan3 axis. Circ_0000005 may serve as a potential therapeutic target in AML.

Screening and validation of differentially expressed microRNAs and target genes in hypertensive mice induced by cytomegalovirus infection

Bioscience Reports ◽

10.1042/bsr20202387 ◽

2020 ◽

Vol 40 (12) ◽

Cited By ~ 1

Author(s):

YunZhong Shi ◽

DongMei Xi ◽

XiaoNi Zhang ◽

Zhen Huang ◽

Na Tang ◽

...

Keyword(s):

Target Genes ◽

Reporter Gene Assay ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Mcmv Infection ◽

Qrt Pcr ◽

Gene Assay ◽

Differentially Expressed Mirnas

Abstract Introduction: Multiple studies have suggested an association between cytomegalovirus (CMV) infection and essential hypertension (EH). MicroRNAs (miRNAs) play a critical role in the development of EH by regulating the expression of specific target genes. However, little is known about the role of miRNAs in CMV-induced EH. In the present study, we compared the miRNA expression profiles of samples from normal and murine cytomegalovirus (MCMV)-infected C57BL/6 mice using high-throughput sequencing analysis. Methods: We collected the thoracic aorta, heart tissues, and peripheral blood from 20 normal mice and 20 MCMV-infected mice. We identified differentially expressed miRNAs in the peripheral blood samples and predicted their target genes using bioinformatics tools. We then experimentally validated them using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the target genes with double luciferase reporter gene assay. Results: We found 118 differentially expressed miRNAs, among which 9 miRNAs were identified as potential MCMV infection-induced hypertension regulators. We then validated the expression of two candidate miRNAs, mmu-miR-1929-3p and mcmv-miR-m01-4-5p, using qRT-PCR. Furthermore, the dual-luciferase reporter gene assay revealed that the 3′-untranslated region (UTR) of endothelin A receptor (Ednra) messenger RNA (mRNA) contained a binding site for mmu-miR-1929-3p. Collectively, our data suggest that MCMV infection can raise the blood pressure and reduce mmu-miR-1929-3p expression in C57BL/6 mice. Moreover, we found that mmu-miR-1929-3p targets the 3′-UTR of the Ednra mRNA. Conclusion: This novel regulatory axis could aid the development of new approaches for the clinical prevention and control of EH.

Targeting SNHG3/miR-186-5p reverses the increased m6A level caused by platinum treatment through regulating METTL3 in esophageal cancer

Cancer Cell International ◽

10.1186/s12935-021-01747-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Mingxin Zhang ◽

Minghua Bai ◽

Li Wang ◽

Ning Lu ◽

Jia Wang ◽

...

Keyword(s):

Esophageal Cancer ◽

Caspase 3 ◽

Regulatory Mechanism ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Qrt Pcr ◽

Platinum Based Chemotherapy ◽

Abstract Background Platinum-based chemotherapy is a mainstay for treating esophageal cancer patients. In this manuscript, we have provided clues for influence of platinum on overall m6A level and further investigated the potential regulatory mechanism. Methods qRT-PCR was used to measure SNHG3 and miR-186-5p expression; ELISA and western blot were used to measure the expression of METTL3. CCK8 was used to measure the cell proliferation rate. Caspase 3/7 activity was used to measure the apoptosis rate. Dual luciferase reporter gene assay and RNA pull down assay were used to investigate the potential crosstalk between miR-186-5p and SNHG3; and miR-186-5p and METTL3. Results m6A level was increased when treated with platinum (CDDP, CPB and L-OHP). Besides, SNHG3 expression was induced and miR-186-5p expression was suppressed by platinum. Furthermore, SNHG3 could promote the m6A level, however miR-186-5p inhibited the m6A level through targeting METTL3. SNHG3 interacts with miR-186-5p to negatively regulate the expression of miR-186-5p; and miR-186-5p might bind to the 3′UTR of METTL3 to regulate its expression. Conclusion Platinum can increase the overall m6A level of esophageal cancer. SNHG3/miR-186-5p, induced by platinum, was involved in regulating m6A level by targeting METTL3. Our manuscript has provided clues that regulating m6A level might be a novel way to enhance the platinum efficacy.

miR-138-5p inhibits the metastasis of prostate cancer by targeting FOXC1

10.21203/rs.3.rs-16546/v1 ◽

2020 ◽

Author(s):

Dapeng Zhang ◽

Xiaodong Liu ◽

Qingwei Zhang ◽

Xin Chen

Keyword(s):

Cell Lines ◽

Poor Prognosis ◽

Bioinformatics Analysis ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Over Expression ◽

Qrt Pcr ◽

Abstract Background: This study aimed to uncover the regulatory effect of miR-138-5p on the metastasis of PCa cells, and further explore the potential regulatory mechanisms via regulating FOXC1. Methods: 60 pairs tumor specimens from PCa patients were collected to determine the expression level of miR-138-5p by qRT-PCR. Subsequently, over-expression of miR-138-5p were established to explore the proliferation and metastasis of miR-138-5p in PCa cell lines was analyzed by CCK-8, Tranwell assay and Wounding healing assay, respectively. Bioinformatics analysis and luciferase reporter gene assay were performed to search for the target genes of miR-138-5p, and FOXC1 was selected. Finally, the biological role of miR-138-5p and FOXC1 in the progression of PCa was clarified by a series of rescue experiments. Results: The results of qRT-PCR revealed that miR-138-5p was lowly expressed in PCa tissues and cell lines. Besdies, these PCa patients with low-miR-138-5p had a higher Gleason score, lymph node metastasis, bone metastasis and poor prognosis of PCa, compared with the patients with high-miR-138-5p. Over-expression of miR-138-5p inhibited the viability, migratory and invasive capacities of PC-3 and DU-145 cells. Bioinformatics analysis and luciferase reporter gene assay suggested that FOXC1 was predicted to be the target of miR-138-5p. Moreover, FOXC1 level was negatively correlated to that of miR-138-5p in PCa tissues. Importantly, FOXC1 could reverse miR-138-5p mimic induced-inhibition of PCa malignant progression. Conclusions: Downregulated miR-138-5p was closely associated with Gleason score, distant metastasis and poor prognosis of PCa patients. In addition, miR-138-5p alleviated the malignant progression of PCa by targeting and downregulating FOXC1.

miR-138-5p inhibits the metastasis of prostate cancer by targeting FOXC1

10.21203/rs.3.rs-16546/v2 ◽

2020 ◽

Author(s):

Dapeng Zhang ◽

Xiaodong Liu ◽

Qingwei Zhang ◽

Xin Chen

Keyword(s):

Cell Lines ◽

Bioinformatics Analysis ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Over Expression ◽

Qrt Pcr ◽

Gene Assay ◽

Proliferation And Metastasis

Abstract Background: This study aimed to uncover the effect of miR-138-5p on the proliferation and metastasis of PCa cell lines, and further explore the potential regulatory mechanisms via regulating FOXC1.Methods: 60 pairs tumor tissues and corresponding paracancerous tissues from PCa patients were collected to determine the expression level of miR-138-5p by qRT-PCR. Subsequently, over-expression of miR-138-5p were established to explore the proliferation and metastasis of miR-138-5p in PCa cell lines was analyzed by CCK-8, Tranwell assay and Wounding healing assay, respectively. Bioinformatics analysis and luciferase reporter gene assay were performed to search for the target genes of miR-138-5p, and FOXC1 was selected. Finally, the biological role of miR-138-5p and FOXC1 in the progression of PCa was clarified by a series of rescue experiments. Results: The results of qRT-PCR revealed that miR-138-5p was lowly expressed in PCa tissues and cell lines. Besdies, these PCa patients with low-miR-138-5p had a higher Gleason score, lymph node metastasis and poor prognosis of PCa, compared with the patients with high-miR-138-5p. Over-expression of miR-138-5p inhibited the viability, migratory and invasive capacities of PC-3 and DU-145 cells. Bioinformatics analysis and luciferase reporter gene assay suggested that FOXC1 was predicted to be the target of miR-138-5p. Moreover, FOXC1 level was negatively correlated to that of miR-138-5p in PCa tissues. Importantly, FOXC1 could reverse miR-138-5p mimic induced-inhibition of PCa malignant progression.Conclusions: Downregulated miR-138-5p was closely associated with Gleason score, distant metastasis and poor prognosis of PCa patients. In addition, miR-138-5p alleviated the malignant progression of PCa by targeting and downregulating FOXC1.

[34] Luciferase reporter gene assay in mammalian cells

Methods in Enzymology - Recombinant DNA Part G ◽

10.1016/0076-6879(92)16036-j ◽

1992 ◽

pp. 386-397 ◽

Cited By ~ 26

Author(s):

Allan R. Brasier ◽

David Ron

Keyword(s):

Reporter Gene ◽

Mammalian Cells ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Upregulation of miR-324-5p Inhibits Proliferation and Invasion of Colorectal Cancer Cells by Targeting ELAVL1

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504018x15166183598572 ◽

2019 ◽

Vol 27 (5) ◽

pp. 515-524 ◽

Cited By ~ 15

Author(s):

Chijiang Gu ◽

Mingyuan Zhang ◽

Weiliang Sun ◽

Changzheng Dong

Keyword(s):

Colorectal Cancer ◽

Luciferase Reporter ◽

Gene Assay ◽

Colorectal Cancer Cells ◽

Colorectal Cell ◽

Sw480 Cells ◽

Direct Target ◽

Proliferation And Invasion

Colorectal cancer (CRC) is a common clinical cancer that remains incurable in most cases. miRNAs are reported to play a part in the development of various tumors. In the present study, we found that miR-324-5p was downregulated in CRC cells, while ELAV (embryonic lethal, abnormal vision, Drosophila)-like protein 1 (ELAVL1) showed a higher expression. miR-324-5p transfection significantly inhibited the proliferation as well as invasion in both SW620 and SW480 cells. miR-324-5p mimic transfection markedly decreased the expression of ELAVL1. Luciferase reporter gene assay confirmed that ELAVL1 is a direct target of miR-324-5p. Furthermore, cancer invasion factors uPA, uPAR, and MMP-9 were found to drop significantly in miR-324-5p-transfected groups. To conclude, our findings indicate that miR-324-5p may play a suppressive role in colorectal cell viability and invasion, at least in part, through directly targeting ELAVL1. Therefore, miR-234-5p might function as a promising candidate for CRC treatment and deserves deeper research.

Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D1 Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay

Molecules ◽

10.3390/molecules23102585 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2585 ◽

Cited By ~ 7

Author(s):

Lehao Wu ◽

Weiyue Zhang ◽

Xin Qiu ◽

Chaoran Wang ◽

Yanfang Liu ◽

...

Keyword(s):

Dopamine Receptors ◽

Reporter Gene ◽

Reporter Gene Assay ◽

D1 Receptor ◽

Assay System ◽

Luciferase Reporter ◽

Antagonistic Effects ◽

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.