scholarly journals Upregulation of MiR-340-5p Reverses Cisplatin Sensitivity by Inhibiting the Expression of CDK6 in HepG2 Cells

2021 ◽  
Vol 69 (2) ◽  
pp. 57-66
Author(s):  
Sha Tian ◽  
Zhuo Liu ◽  
Qing Zhou ◽  
Ruoxia Wu ◽  
Xiaodi Huang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Liver Cancer ◽  
Hepg2 Cells ◽  
Luciferase Reporter ◽  
Cycle Arrest ◽  
Cisplatin Sensitivity ◽  
New Ideas ◽  
Lower Drug ◽  
Dual Luciferase Reporter Assay

Cisplatin (CDDP) has been successfully used in chemotherapy for liver cancer. However, the development of CDDP resistance in HepG2 cells usually leads to relapse and a worsening prognosis. MiR-340-5p has attracted much attention because of its ability to affect cell resistance. This project is intended to explore the role of miR-340-5p and CDK6 in CDDP-R HepG2 cells and provide new ideas for the treatment of liver cancer. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-340-5p and CDK6. We constructed a CDDP-resistant model of HepG2 cells to examine the effect of miR-340-5p on the drug sensitivity of HepG2 cells. CDDP-R HepG2 cells were transfected with miR-340-5p overexpression plasmid and CDK6 silencing plasmid. QRT-PCR was used to detect the expression of miR-340-5p and CDK6. A western blot was performed to determine the expression of CDK6, CyclinD1, and CyclinD2. CCK-8, flow cytometry, TUNEL and Clonogenic assays were also carried out to detect CDDP-R HepG2 cells. There is a targeting relationship between miR-340-5p and CDK6. The drug resistance of CDDP-R HepG2 cells was significantly higher than that of CDDP-S HepG2 cells. CDDP-R HepG2 cells transfected with both miR-340-5p overexpressing plasmid and CDK6 silencing plasmid showed a lower proliferation ability, cell cycle arrest in the G0/G1 phase, and lower drug resistance compared with single CDDP-R HepG2 cells. Overexpression of miR-340-5p aggravated CDDP-R HepG2 cells' apoptosis and inhibited cell viability. Overexpression of miR-340-5p could reverse the sensitivity of HepG2 cells to CDDP by inhibiting the expression of CDK6 in HepG2 cells.

scholarly journals Possible Role of IRS-4 in the Origin of Multifocal Hepatocellular Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2560
Author(s):  
Luis G. Guijarro ◽  
Patricia Sanmartin-Salinas ◽  
Eva Pérez-Cuevas ◽  
M. Val Toledo-Lobo ◽  
Jorge Monserrat ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Cancer ◽  
Hepg2 Cells ◽  
Cellular Polarity ◽  
Ki 67 ◽  
Tissue Samples ◽  
Vitro Analysis ◽  
Tumoral Cells

New evidence suggests that insulin receptor substrate 4 (IRS-4) may play an important role in the promotion of tumoral growth. In this investigation, we have evaluated the role of IRS-4 in a pilot study performed on patients with liver cancer. We used immunohistochemistry to examine IRS-4 expression in biopsies of tumoral tissue from a cohort of 31 patient suffering of hepatocellular carcinoma (HCC). We simultaneously analyzed the expression of the cancer biomarkers PCNA, Ki-67, and pH3 in the same tissue samples. The in vitro analysis was conducted by studying the behavior of HepG2 cells following IRS-4 overexpression/silencing. IRS-4 was expressed mainly in the nuclei of tumoral cells from HCC patients. In contrast, in healthy cells involved in portal triads, canaliculi, and parenchymal tissue, IRS-4 was observed in the cytosol and the membrane. Nuclear IRS-4 in the tumoral region was found in 69.9 ± 3.2%, whereas in the surrounding healthy hepatocytes, nuclear IRS-4 was rarely observed. The percentage of tumoral cells that exhibited nuclear PCNA and Ki-67 were 52.1 ± 7%, 6.1 ± 1.1% and 1.3 ± 0.2%, respectively. Furthermore, we observed a significant positive linear correlation between nuclear IRS-4 and PCNA (r = 0.989; p < 0.001). However, when we correlated the nuclear expression of IRS-4 and Ki-67, we observed a significant positive curvilinear correlation (r = 0.758; p < 0.010). This allowed us to define two populations, (IRS-4 + Ki-67 ≤ 69%) and (IRS-4 + Ki-67 > 70%). The population with lower levels of IRS-4 and Ki-67 had a higher risk of suffering from multifocal liver cancer (OR = 16.66; CI = 1.68–164.8 (95%); p < 0.05). Immunoblot analyses showed that IRS-4 in normal human liver biopsies was lower than in HepG2, Huh7, and Chang cells. Treatment of HepG2 with IGF-1 and EGF induced IRS-4 translocation to the nucleus. Regulation of IRS-4 levels via HepG2 transfection experiments revealed the protein’s role in proliferation, cell migration, and cell-collagen adhesion. Nuclear IRS-4 is increased in the tumoral region of HCC. IRS-4 and Ki-67 levels are significantly correlated with the presence of multifocal HCC. Moreover, upregulation of IRS-4 in HepG2 cells induced proliferation by a β-catenin/Rb/cyclin D mechanism, whereas downregulation of IRS-4 caused a loss in cellular polarity and in its adherence to collagen as well as a gain in migratory and invasive capacities, probably via an integrin α2 and focal adhesion cascade (FAK) mechanism.

scholarly journals Echinacoside exerts anti-tumor activity via the miR-503-3p/TGF-β1/Smad aixs in liver cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wen Li ◽  
Jing Zhou ◽  
Yajie Zhang ◽  
Jing Zhang ◽  
Xue Li ◽  
...  
Keyword(s):  
Liver Cancer ◽  
Cancer Cells ◽  
Hepg2 Cells ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Expression Levels ◽  
Liver Cancer Cells ◽  
Anti Tumor Activity ◽  
Tgf Β1

Abstract Background Echinacoside (ECH) is the main active ingredient of Cistanches Herba, which is known to have therapeutic effects on metastatic tumors. However, the effects of ECH on liver cancer are still unclear. This study was to investigate the effects of ECH on the aggression of liver cancer cells. Methods Two types of liver cancer cells Huh7 and HepG2 were treated with different doses of ECH at different times and gradients. MTT and colony formation assays were used to determine the effects of ECH on the viability of Huh7 and HepG2 cells. Transwell assays and flow cytometry assays were used to detect the effects of ECH treatment on the invasion, migration, apoptosis and cell cycle of Huh7 and HepG2 cells. Western blot analysis was used to detect the effects of ECH on the expression levels of TGF-β1, smad3, smad7, apoptosis-related proteins (Caspase-3, Caspase-8), and Cyto C in liver cancer cells. The relationship between miR-503-3p and TGF-β1 was detected using bioinformatics analysis and Luciferase reporter assay. Results The results showed that ECH inhibited the proliferation, invasion and migration of Huh7 and HepG2 cells in a dose- and time-dependent manner. Moreover, we found that ECH caused Huh7 and HepG2 cell apoptosis by blocking cells in S phase. Furthermore, the expression of miR-503-3p was found to be reduced in liver tumor tissues, but ECH treatment increased the expression of miR-503-3p in Huh7 and HepG2 cells. In addition, we found that TGF-β1 was identified as a potential target of miR-503-3p. ECH promoted the activation of the TGF-β1/Smad signaling pathway and increased the expression levels of Bax/Bcl-2. Moreover, ECH could trigger the release of mitochondrial Cyto C, and cause the reaction Caspases grade. Conclusions This study demonstrates that ECH exerts anti-tumor activity via the miR-503-3p/TGF-β1/Smad aixs in liver cancer, and provides a safe and effective anti-tumor agent for liver cancer.

scholarly journals miR-34a Inhibits Cell Proliferation by Targeting SATB2 in Hepatocellular Carcinoma

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Gang Wu ◽  
Zhixi Li ◽  
Youyu Wang ◽  
Xueming Ju ◽  
Rui Huang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Hepg2 Cells ◽  
Hepatoma Cell ◽  
Luciferase Reporter ◽  
Hepatoma Cell Line ◽  
Novel Therapy ◽  
The Third ◽  
Direct Target

Hepatocellular carcinoma (HCC) is the most common type of malignancy of the liver and has been reported as the third most frequent cause of cancer associated death worldwide. Accumulating evidence showed that the expression of miR-34a was abnormal in HCC patients; however, the role of miR-34a in HCC is not clear. In this study, we have observed low expression of the miR-34a in both HCC tissues and hepatoma cell line as compared to normal control. Further to investigate the role of miR-34a in HCC development, HepG2 cells were transfected with miR-34a mimic. Following transfection, miR-34a expression was significantly increased, which further repressed proliferation of HepG2 cells. Bioinformatics, Luciferase Reporter, RT-qPCR, and western blotting assays indicated that special AT-rich sequence-binding protein-2 (SATB2) is a direct target of miR-34a in HCC cells. There was a negative correlation between the expression levels of SATB2 and miR-34a. Investigation into the molecular mechanism indicated that miR-34a regulated cell proliferation through inhibiting SATB2. Therefore, the results of the present study may improve understanding regarding the role of miR-34a in regulating cell proliferation and contribute to the development of novel therapy of HCC.

scholarly journals Cyperus articulatus L. (Cyperaceae) Rhizome Essential Oil Causes Cell Cycle Arrest in the G2/M Phase and Cell Death in HepG2 Cells and Inhibits the Development of Tumors in a Xenograft Model

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2687
Author(s):  
Mateus L. Nogueira ◽  
Emilly J. S. P. de Lima ◽  
Asenate A. X. Adrião ◽  
Sheila S. Fontes ◽  
Valdenizia R. Silva ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Gas Chromatography ◽  
Cell Death ◽  
Liver Cancer ◽  
Essential Oil ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Hepg2 Cells ◽  
Cycle Arrest

Cyperus articulatus L. (Cyperaceae), popularly known in Brazil as “priprioca” or “piriprioca”, is a tropical and subtropical plant used in popular medical practices to treat many diseases, including cancer. In this study, C. articulatus rhizome essential oil (EO), collected from the Brazilian Amazon rainforest, was addressed in relation to its chemical composition, induction of cell death in vitro and inhibition of tumor development in vivo, using human hepatocellular carcinoma HepG2 cells as a cell model. EO was obtained by hydrodistillation using a Clevenger-type apparatus and characterized qualitatively and quantitatively by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detection (GC-FID), respectively. The cytotoxic activity of EO was examined against five cancer cell lines (HepG2, HCT116, MCF-7, HL-60 and B16-F10) and one non-cancerous one (MRC-5) using the Alamar blue assay. Cell cycle distribution and cell death were investigated using flow cytometry in HepG2 cells treated with EO after 24, 48 and 72 h of incubation. The cells were also stained with May–Grunwald–Giemsa to analyze the morphological changes. The anti-liver-cancer activity of EO in vivo was evaluated in C.B-17 severe combined immunodeficient (SCID) mice with HepG2 cell xenografts. The main representative substances of this EO sample were muskatone (11.6%), cyclocolorenone (10.3%), α-pinene (8.26%), pogostol (6.36%), α-copaene (4.83%) and caryophyllene oxide (4.82%). EO showed IC50 values for cancer cell lines ranging from 28.5 µg/mL for HepG2 to >50 µg/mL for HCT116, and an IC50 value for non-cancerous of 46.0 µg/mL (MRC-5), showing selectivity indices below 2-fold for all cancer cells tested. HepG2 cells treated with EO showed cell cycle arrest at G2/M along with internucleosomal DNA fragmentation. The morphological alterations included cell shrinkage and chromatin condensation. Treatment with EO also increased the percentage of apoptotic-like cells. The in vivo tumor mass inhibition rates of EO were 46.5–50.0%. The results obtained indicate the anti-liver-cancer potential of C. articulatus rhizome EO.

scholarly journals Activation of the miR-371/372/373 miRNA Cluster Enhances Oncogenicity and Drug Resistance in Oral Carcinoma Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9442
Author(s):  
Shu-Chun Lin ◽  
Hsiao-Li Wu ◽  
Li-Yin Yeh ◽  
Cheng-Chieh Yang ◽  
Shou-Yen Kao ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcriptional Activation ◽  
Mirna Cluster ◽  
Human Stem Cells ◽  
Oncogenic Potential ◽  
Synergistic Activation ◽  
Cisplatin Sensitivity ◽  
The Individual ◽  
Cell Subclones

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated deaths worldwide. Family members in miR-371/372/373 miRNA cluster, which is localized at human chromosome 19q13.4, are co-expressed in both human stem cells and malignancies. The individual miRNA in this cluster are also involved in modulating the pathogenesis of malignancies as either oncogenes or suppressors. The 19q13 region is frequently gained in head and neck cancers. High expression of miR-372 and miR-373 are survival predictors for OSCC. However, the role of the miR-371/372/373 cluster in oral carcinogenesis remains to be fully investigated. We use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system to establish OSCC cell subclones that had the miR-371/372/373 cluster deleted. In addition, further subclones were established that had the promoter of this cluster deleted. Concordant silencing in SAS cells of miR-371/372/373 decreased oncogenic potential, increased cisplatin sensitivity, activated p53, and upregulated the expression of Bad and DKK1. We also employed the CRISPR/dCas9 synergistic activation mediator system, which allowed robust transcriptional activation of the whole miR-371/372/373 cistron. Upregulation of endogenous miR-371/372/372 expression increased both oncogenicity and drug resistance. These were accompanied by a slight activation of AKT, β-catenin, and Src. This study identifies the oncogenic role of the miR-371/372/373 cluster in OSCC. Using CRISPR based strategy can be a powerful paradigm that will provide mechanistic insights into miRNA cluster functionality, which will also likely help the development of targeting options for malignancies.

scholarly journals Knockdown of LincRNA PADNA Promotes Bupivacaine-Induced Neurotoxicity by miR-194/FBXW7 Axis

2020 ◽  
Author(s):  
Fan Yuning ◽  
Chen Liang ◽  
Wang Tenghuan ◽  
Nan Zhenhua ◽  
Shengkai Gong
Keyword(s):  
Functional Analysis ◽  
Western Blot ◽  
Cell Viability ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Drg Neurons ◽  
Dual Luciferase Reporter Assay

Abstract The aim of the study was to explore the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Mouse DRG neurons were cultured in vitro and treated with bupivacaine to establish the neurotoxicity model. Caspase3 activity, cell viability, tunel assay were analyzed to assess the role of lincRNA PADNA. Dual-luciferase reporter assay was used to determine the binding target of lincRNA PANDA. The expression of lincRNA PADNA was significantly increased with the increasing concentration of bupivacaine. Functional analysis revealed that knockdown of lincRNA PADNA accelerated the caspase3 activity and inhibited the cell viability. Western blot showed that knockdown of lincRNA PADNA promoted the occurrence of cleaved-caspase3. We also proved that lincRNA PADNA may bind with miR-194. Overexpression of miR-194 could rescued the function of lincRNA PADNA, suggesting that lincRNA PADNA may sponge miR-194. In addition, we provided new evidences that lincRNA PADNA/miR-194/FBXW7 axis play an important role in the neurotoxicity process. We performed comprehensive experiments to verify the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Our study provided new evidences and clues for prevention of neurotoxicity.

scholarly journals MiR-375 gene therapy attenuates drug resistance of hepatocellular carcinoma cells by inhibiting cell autophagy

2020 ◽  
Author(s):  
Dan Wang ◽  
Jingbo Yang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Drug Resistance ◽  
Western Blotting ◽  
Regulatory Mechanism ◽  
Negative Control ◽  
Carcinoma Cells ◽  
Luciferase Reporter ◽  
Sorafenib Treatment ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Abstract Objective To probe into the regulatory mechanism of miR-375 in hepatocellular carcinoma (HCC) cells under sorafenib treatment. Methods Western blotting and qRT-PCR were applied to measure the expressions of miR-375 and SIRT5 in parental HCC cells (HepG2 and Huh7) and sorafenib-resistant HCC cells (HepG2/so and Huh7/so). HepG2/so cells were accordingly transfected with miR-375 mimic, miR-375 inhibitor, sh-SIRT5, pcDNA3.1-SIRT5 or negative control. Western blotting measured the expressions of p62, LC3I and LC3II in HCC cells. CCK-8 and flow cytometry assessed the survivability and apoptosis of HCC cells, respectively. Bioinformatics techniques and dual-luciferase reporter assay predicted and verified the targeting relationship between miR-375 and SIRT5. Results MiR-375 was under-expressed and SIRT5 was over-expressed in HCC cells. Autophagy inhibitor impaired the survival of HepG2/so cells transfected with miR-375 inhibitor. Autophagy activator enhanced the drug resistance of HepG2/so cells transfected with miR-375 mimic. MiR-375 suppressed the drug resistance of HepG2/so cells by inhibiting autophagy. SIRT5 enhanced the drug resistance of HepG2/so cells by promoting autophagy and it could be targeted by miR-375. Conclusion MiR-375 suppresses autophagy to attenuate the drug resistance of HCC cells by regulating SIRT5. The findings of this study may provide new therapeutic targets for treating hepatocellular carcinoma.

scholarly journals LncRNA LINC01303 Promotes the Progression of Oral Squamous Cell Carcinomas via the miR-429/ZEB1/EMT Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Bo Sun ◽  
Xianyu Zheng ◽  
Weilong Ye ◽  
Pengcheng Zhao ◽  
Guowu Ma
Keyword(s):  
Squamous Cell ◽  
Luciferase Reporter ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Cytoplasmic Rna ◽  
Transwell Invasion Assay ◽  
Dual Luciferase Reporter Assay

Objectives. The aim of this research was to uncover the biological role and mechanisms of LINC01303 in oral squamous cell carcinoma (OSCC). Materials and Methods. Real-time quantitative PCR (qRT-PCR) was used to determine LINC01303 expression in OSCC tissues. Subcellular distribution of LINC01303 was examined by nuclear/cytoplasmic RNA fractionation and FISH experiments. The role of LINC01303 in the growth of TSCCA and SCC-25 was examined by CCK-8 assay, colony formation, transwell invasion assay in vitro, and xenograft tumor experiment in vivo. Dual-luciferase reporter assay was used to verify the interaction between LINC01303 and miR-429. RNA pull‐down assay was used to discover miR-429‐interacted protein, which was further examined by qRT-PCR, western blot, and rescue experiments. Results. LINC01303 expression was higher in OSCC tissues compared with adjacent nontumor tissues. LINC01303 was found to be localized in the cytoplasm of OSCC cells. Knockdown of LINC01303 inhibited OSCC cell proliferation and invasion, whereas increasing the expression of LINC01303 showed the opposite effects. Furthermore, LINC01303 served as a miR-429 “sponge” and positively regulated ZEB1 expression. Moreover, LINC01303 promoted OSCC through miR-429/ZEB1 axis both in vivo and in vitro. Conclusions. LINC01303 plays an oncogenic role in OSCC and is a promising biomarker for OSCC patients.

scholarly journals GATA1-induced upregulation of LINC01503 promotes carboplatin resistance in ovarian carcinoma by upregulating PD-L1 via sponging miR-766-5p

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yao Li ◽  
Yan Zhai ◽  
Yuxuan Chen
Keyword(s):  
Ovarian Carcinoma ◽  
Inhibitory Concentration ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Invasive Ability ◽  
Protein Levels ◽  
Reverse Transcription Quantitative Pcr ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Abstract Background Ovarian Carcinoma (OCa) is a high-mortality malignancy derived from female reproductive system. Increasing evidence has identified long non-coding RNAs (lncRNAs) as important regulators in OCa chemoresistance. In this study, we intended to explore the role of LINC01503 in OCa resistance to carboplatin (CBP). Methods Gene expression was measured by reverse transcription-quantitative PCR (RT-qPCR) in OCa cells. Western blot was adopted to detect protein levels of GATA1, PD-L1, E-cadherin, N-cadherin, Vimentin, Bcl-2, Bax, cleaved caspase-3. To assess the effects of LINC01503 on the resistance of OCa cells to CBP, Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and flow cytometry experiments were performed to evaluate half-maximal inhibitory concentration (IC50), cell viability, migrative and invasive ability, as well as cell apoptosis. Dual-luciferase reporter assay was employed to assess the associations between the genes. Results LINC01503 was upregulated in CBP-resistant OCa cells. LINC01503 knockdown reduced CBP resistance in OCa cells. Besides, GATA-binding protein 1 (GATA1) activated LINC01503 transcription in CBP-resistant OCa cells. MiR-766-5p was lowly expressed in CBP-resistant cells and confirmed as a target for LINC01503. In addition, miR-766-5p overexpression increased CBP sensitivity in OCa cells. PD-L1 was verified as the target of miR-766-5p. Besides, LINC01503 upregulated PD-L1 level by regulating miR-766-5p. Furthermore, rescue experiments showed that PD-L1 overexpression abrogated the inhibited impacts of blocking LINC01503 on CBP resistance in OCa cells. Conclusion GATA1-induced LINC01503 expedited CBP resistance in OCa cells via the miR-766-5p/PD-L1 axis, providing a new target for improving the efficacy of OCa chemotherapy. Graphical Abstract

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