Circ_0072088 mediates hepatic fibrosis renders epithelial to mesenchymal transition through miR-330-3p/CDK1 axis in AML12 hepatocytes

doi:10.21203/rs.3.rs-1730326/v2

Download PDF

Research Article

Circ_0072088 mediates hepatic fibrosis renders epithelial to mesenchymal transition through miR-330-3p/CDK1 axis in AML12 hepatocytes

https://doi.org/10.21203/rs.3.rs-1730326/v2

This work is licensed under a CC BY 4.0 License

Version 2

posted

You are reading this latest preprint version

Hepatic fibrosis was one of the causes to hepatocellular cancer (HCC), owing to limited therapeutic options. And hepatocytes can induce liver fibrogenesis via epithelial-mesenchymal transition (EMT). Multiple circRNAs exert critical role in the occurrence of liver fibrosis via working as microRNA (miRNA) sponges. Circ_0072088 is upregulated in hepatocellular cancer (HCC) and promote proliferation, migration of HCC cells. However, the potential research mechanism for circ_0072088 action against the fibrotic progression remain still unknown. The treatment of AML12 hepatocytes with TGF-β1 contributed to dramatically increased in the levels of circ_0072088. Additionally, in terms of mechanism, circ_0072088 was authenticated to function as miR-330-3p sponge, not only noticeable increase EMT, but also induced fibrosis in AML12 hepatocytes. MiR-330-3p could weaken the roles of circ_0072088 acceleration. Moreover, we proved that CDK1, a miR-330-3p target, was time-dependently augmented in the TGF-β1 treated AML12 hepatocytes, and its overexpression weakens the miR-330-3p regulatory effects.Our findings provided a unique role of the circ_0072088 mediates hepatic fibrosis renders epithelial-mesenchymal transition at least partially through miR-330-3p/CDK1 axis, thus suggesting its tremendous therapeutic potential in the treatment of liver diseases.

circ_0072088

miR-330-3p

CDK1

hepatic fibrosis

EMT

Aberrant sedimentation of extracellular matrix (ECM) can induce hepatic fibrogenesis processes, which results in injure to the normal hepatic parenchyma[1, 2]. Recent studies have shown that delayed therapy of hepatic fibrosis can bring about the development of cirrhosis, which eventually result in liver failure, especially hepatocellular cancer (HCC)[3, 4]. Although the principal source of extracellular matrix (ECM) are conventionally considered from tissue fibroblasts, EMT are also regard as a main mechanism for the aberrant sedimentation of ECM in hepatic, renal and pulmonary fibrosis[5–7]. Additionally, growing evidence indicates that activated AML12 hepatocytes are deemed to the principal, but not the only, origin of ECM-generating via the EMT in liver fibrosis[8]. The EMT is a dynamic biological process, and undergo morphological alterations from epithelial to fibroblast-like in the fibrosis process[9, 10]. Unfortunately, in spite of excellent advances in clinical execute, there is no specific medicine for hepatic fibrosis. Hence, the exploitation of effective drugs for its supervisison is crucial.

Increasing studies have found that non-coding RNAs have attracted reinforced consideration by reason of its impact in development of mammalian[11, 12]. A newfound form of non-coding RNAs is discovered, and the RNAs are covalently closed continuous loops, is named circular RNAs (circRNAs)[13, 14]. MicroRNAs (miRNAs), the short non-coding RNAs, can restrain translation process through binding to the 3′-untranslated region (UTR) of target mRNAs[15]. Based on emerging evidence, circRNAs can give play to their biological functions and participate in the regulation of various human physiological processes through acting as “miRNA sponges”[16, 17]. Furthermore, it has been reported that dysregulation of a number of circRNAs and miRNAs is implicated in pathological progression of many human diseases, including hepatic fibrosis[18, 19]. For example, downregulation of circCREBBP in carbon tetrachloride (CCl4)-triggered hepatic stellate cells (HSCs) accelerated collagen deposition and overexpression of pro-fibrosis genes through act as miR-1291 sponge and binding to the 3′-UTR of LEFTY2 protein, manifesting that circCREBBP promote the occurrence and development of hepatic fibrosis via miR-1291/LEFTY2 axis[20].

Of note, circ_0072088 is up-regulated in hepatocellular carcinoma (HCC). Overexpression of circ_0072088 is capable of expediting the migration, proliferation, and inhibit the apoptosis of human HCC cell lines (HepG2, SNU-398) to accelerate progression of HCC by sponging miR-375 to activate JAK2/STAT3 signaling[21]. However, the exact role of circ_0072088 in the developmental process of hepatic fibrosis remain largely unclear.

In the research paper, we research intended to further dissect the effects and undering mechanism with circ_0072088 in liver fibrosis and EMT, which mediate ECM generating in AML12 hepatocytes, so as to provide more theoretical foundation for find feasible biomarkers and potential targets in hepatic fibrosis.

Hepatocytes culture

AML12 hepatocytes, an immortalized mouse hepatocytes cell line, were routinely cultured in a 1:1 mixture of Dulbecco's Modifified Eagle's Medium (DMEM)/Ham's F-12 medium containing pre-mixed ITS (5 µg/ml; insulin, transferrin and sodium selenite), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. AML12 hepatocytes were cultured at 37 ℃ under humidified atmosphere with 5% CO₂, and were gived with 2 ng/ml of TGF-β1 (R&D System, USA) to construct a liver fibrosis cell model.

Cell transfections

Circ_0072088 and CDK1 cDNA were synthesized and cloned into pcDNA3.1 plasmid to construct overpression vector. When the AML12 hepatocytes cultured 70% confluence, pcDNA3.1-circ_0072088 (circ_0072088), pcDNA3.1 CDK1 (CDK1) si-circ_0072088, antimiRNA oligonucleotide (AMO)-330-3p, miRNA-330-3p mimics or negative controls (NC) were transfected into AML12 hepatocytes through Lipofectamine 2000 reagent (Invitrogen, USA). All the overpression vector were provided by Aibosi company (Shanghai, China). Following culturing for 6 h, the culture medium was replaced with complete medium for 48 h at 37 ℃.

Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR)

Total RNA from AML12 hepatocytes were harvested using 1 ml TRIzol reagent (Invitrogen, USA). The harvested RNA (1 µg) was implemented to reverse transcribed into cDNA using the Reverse Transcription kit (Toyobo, Japan) with random primer in each sample. After that, a SYBR Green PCR Master Mix kit (Toyobo, Japan) was used to confirm the circ_0072088, miR-330-3p levels through quantitative polymerase chain reaction. MiR-330-3p were normalized using small nuclear U6, while circ_0072088 was normalized to the gene GAPDH. The relative expression levels of the circRNA, miRNA or mRNA were quantified by Ct (2-ΔΔCT) method. The primer sequences were as follows:

circ_0072088: Forward, 5′-AATGGTCTGCAGTCCTGTGT-3′ and Reverse, 5′-TGCCTGTAACTCCTCTTCAGT-3′;

miR-330-3p: Forward, 5′-CGGCAAAGCACACGGCCTG-3′ and Reverse, 5′-TGCGTGTCGTGGAGTCGGC-3′;

GAPDH: Forward, 5′-ACTCCACTCACGGCAAATTC-3′ and Reverse, 5′-TCTCCATGGTGGTGAAGACA-3′;

U6: Forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and Reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′;

Western blot

In brief, protein extraction was acquired from AML12 hepatocytes using RIPA lysis buffer (Solarbio, China) supplemented with 1% protease inhibitors on ice. BCA reagent (Beyotime, China) was applied to quantified concentration of protein. For the western blot analysis, SDS-PAGE (10% gels) was used to separate each protein sample. The separated protein was transferred form the gels onto nitrocellulose (NC) membranes, and then the membranes were blocked with 5% non-fat milk in the PBS for 2 h. Later on, the blocked NC membranes were incubated with the specific primary antibodies of α-SMA (Cell Signaling Technology, USA), E-cadherin (Cell Signaling Technology, USA), vimentin (Cell Signaling Technology, USA), CDK1 (Abcam, USA), collagen-1 (Abcam, USA), collagen-4 (Abcam, USA) and GAPDH (Zhongshanjinqiao, China) in PBS at 4 ℃ overnight. Following by incubation used the secondary antibody (LI-COR, USA) for 1 h at room temperature. The results were visualized by Odyssey v3.0 software Imaging system (LI-COR Biosciences, USA), and quantified thoughtout the measured band intensity (area×OD).

Luciferase reporter assays

The binding sequence of miR-330-3p with CDK1 and circ_0072088 was forecasted using the CircInteractome website (https://circinteractome.nia.nih.gov/) and TargetScan (http://www.targetscan.org/vert_80/). The luciferase reporter vectors for mutated (MUT) or wild-type (WT) 3'-UTR regions of CDK1 and circ_0072088 were constructed using psi-CHECK2 vectors (Promega, USA). The WT and MUT luciferase reporter vector (100 mg) were co-transfected with miR-330-3p mimic or mimic NC into the HEK 293T cells through Lipofectamine 2000 reagent (Invitrogen, USA). Fluorescence enzyme activity were analyzed using the dual-luciferase reporter gene assay system (Promega, USA).

RNA Pull-Down Assay

Firstly, The streptavidin magnetic beads (Thermo Fisher Scientifific) was applied to incubate biotinylated circ_0072088 or miR-330-3p probe for 2 h. Thereafter, the magnetic beads were added into AML12 hepatocytes at 4 ℃ overnight. Finally, RT-qPCR assay was accomplished to pull down and analysis the interaction circ_0072088 and miR-330-3p.

Cell viability assay

Cell viability was assessed by Cell Counting Kit-8 assay (CCK8; Dojindo, Japan) as previously described[22]. Briefly, AML12 hepatocytes (70% confluence) were seeded onto 96-well plates by a density of 1x10⁴ cells/well, and the indicated transfection before TGF-β1 treatment for 48 h. Subsequently, 10% CCK8 working solution was added to the per group, whereafter incubation at 37 ℃ for 1h. The optical density (OD) was gauged at 450 nm wavelength.

Statistical analysis

All data were presented as the mean ± standard error of the mean (SME). Each experiment was repeated at least five times independently. Comparisons between the differences among multiple groups were analyzed using one-way analysis of variance (ANOVA). P < 0.05 was considered to indicate a statistically significant. GraphPad Prism version 5.0 software was used to statistical analysis (GraphPad Software, USA).

Circ_0072088 knockdown inhibited EMT and fibrosis in AML12 hepatocytes

In order to explore biological effect of circ_0072088 in AML12 hepatocytes treatment of TGF-β1, we first investigate the levels of circ_0072088 expression in AML12 pretreated with or without TGF-β1 by RT-qPCR. We fould that circ_0072088 was time-dependently increased in AML12 hepatocytes treated with TGF-β1 compared with that in control group (Figure 1A). What’s more, RT-qPCR assay was accomplished to verify high transfection efficiency of si-circ_0072088 in AML12 hepatocytes (Figure 1B). Western blot assay showed that circ_0072088 deficiency can augment E-cadherin, and reduce α-SMA and vimentin proteins in AML12 hepatocytes (Figure 1C-F). Next, the levels of collagen-1 and collagen-4 protein (EMT markers) expression were performed using western blot assay. We manifested that collagen-1 and collagen-4 proteins were decreased by knockdown of circ_0072088 (Figure 1G-I), suggesting that circ_0072088 knockdown inhibited ECM accumulation. Meanwhile, under-exprssion of circ_0072088 aggrandized cell viability in AML12 hepatocytes using CCK-8 assay (Figure 1J). Overall, circ_0072088 knockdown might inhibit liver fibrosis and EMT development.

Circ_0072088 directly targeted miR-330-3p

CircInteractome dataset was implemented to predicte the targets binding to circ_0072088. We found that circ_0072088 could firsthand bind with miR-330-3p sponges (Figure 2A). Next, RNA pull-down and dual-luciferase reporter assays were applied to identify the conclusion. Luciferase reporter assay indicated that miR-330-3p obviously restrained luciferase activity of circ_0072088-WT. Nevertheless, the luciferase activity of circ_0072088-MUT was unchanged in HEK 293T cells (Figure 2B). Moreover, RNA pull-down assay proved higher improvement of circ_0072088 and miR-330-3p in Ago2 group than those in IgG group (Figure 2C, D). Meanwhile, we also found that the expression of miR-330-3p was time-dependently reduced in TGF-β1 induced AML12 hepatocytes (Figure 2E). At last, we studied the influence of circ_0072088 on miR-330-3p level. The RT-qPCR results demonstrated that the expression level of miR-330-3p was augmented by knockdown of circ_0072088 in AML12 hepatocytes (Figure 2F). These data indicated that circ_0072088 could directly bind with miR-330-3p sponges and might play critical roles in liver fibrosis.

Circ_{_}0072088 gived play to its role by targeting miR-330-3p

To evaluate whether circ_0072088 influenced EMT process by targeting miR-330-3p, AML12 hepatocytes was transfected with pcDNA3.1-circ_0072088, miR-330-3p mimic and NC. We found that e-cadherin protein was markedly decreased, and α-SMA and vimentin proteins were observably increased in AML12 hepatocytes with transfect circ_0072088 using western blot assay. Moreover, miR-330-3p upregulation weakened circ_0072088 effect (Figure 3A-D). Meanwhile, the expression levels of collagen-1 and collagen-4 (ECM markers) were decreased in AML12 hepatocytes co-transfected with miR-330-3p mimic and pcDNA3.1-circ_0072088. These research results indicated that the effect of circ_0072088 induce ECM accumulation was suppressed through upregulation of miR-330-3p (Figure 3E-G). Cell viability was significantly decreased by circ_0072088 treatment, whereas miR-330-3p mimic significantly reversed this effect compared with NC group (Figure 3H). Collectively, circ_0072088 regulated EMT and hepatic fibrosis by targeting miR-330-3p.

MiR-330-3p bind CDK1 3′-UTR

To obtain further explore the undering mechanism, the TargetScan database was applied to comfirm gene targets. We found that a binding site for miR-330-3p was ensured on the CDK1 3′-UTR (Figure 4A). Meanwhile, miR-330-3p obviously restrained luciferase activity of CDK1-WT. Nevertheless, the luciferase activity of CDK1-MUT was not affected using luciferase reporter assay (Figure 4B). Based on the bioinformatics analysis and luciferase reporter assay, we evalated the role of miR-330-3p on CDK1. Firstly, we obtained that CDK1 protein was time-dependently augment in the TGF-β1 treated AML12 hepatocytes (Figure 4C). Besides, miR-330-3p could reduce the expression level of CDK1 at protein (Figure 4D). We also found that miR-330-3p knockdown by AMO‑330-3p increased CDK1 protein level compared with control and miR-NC cells (Figure 4E). Therefore, it was concluded that miR-330-3p directly targeted CDK1 3′-UTR.

Knockdown of circ_0072088 indirectly restrain CDK1 expressionby miR-330-3p

To explored whether miR-330-3p regulate EMT process through CDK1 changes, pcDNA-CDK1, miR-330-3p mimic were transfected to AML12 hepatocytes. We found that e-cadherin protein was markedly increased, and α-SMA and vimentin proteins were observably decreased in AML12 hepatocytes with transfect miR-330-3p using western blot assay. Moreover, CDK1 upregulation weakened miR-330-3p effect (Figure 5A-D). Meanwhile, miR-330-3p overexpression inhibited collagen-1 and collagen-4 at protein level, which was restored by CDK1 (Figure 5E-G). Taken together, miR-330-3p regulated EMT and fibrosis in AML12 hepatocytes, which could be reversed by targeting CDK1.

We further elucidated the regulate mechanism of circ_0072088 regulate CDK1 expression in hepatic fibrosis. AML12 was transfected with si-circ_0072088, AMO-330-3p, and AMO-NC. Circ_0072088 silence reduced the expression level of CDK1 at protein, while knockdown of miR-330-3p could restore the effect (Figure 5H). Collectively, our data suggested that circ_0072088 deficiency could restrain CDK1 expression acting as miR-330-3p sponges.

Extensive studies indicate that epithelial-mesenchymal transition (EMT) can reduce the development of hepatic fibrosis in AML12 hepatocytes. However, the potential research mechanisms that circRNA induce hepatic fibrosis via EMT needs to be further elucidated.

Under physiological conditions, Epithelial to mesenchymal transition (EMT) occupy a significant position in embryogenesis, cancer, fibrosis progression and tissue remodeling and all that[23–25]. Recent data have pointed out that EMT has been deemed to serves a substantial role in hepatic fibrosis progression for a long time[5]. Because our aim is to study the relationship between fibrosis and EMT, we focused to type II EMT. In the present work, we found that inhibit the expression of circ_0072088 can restrain TGF-β1 induce the expression of EMT-related proteins and liver fibrosis process.

CircRNAs have become a hot spot as novel non-coding RNAs. Unlike linear RNA, circRNAs have been found that are closed continuous loop structures, and relatively stable in the cytoplasm[26]. Signifificantly, circRNAs regulate various physiological processes. For example, circular RNA circ_0090231 promotes atherosclerosis via enhancing endothelial cell pyroptosis, and circ_NOTCH3 promote tumorigenesis of hepatocellular carcinoma by miR-875-5p/ZNF146 axis[27, 28]. However, the underlying mechanism largely remain to be elucidated in circRNAs regulate EMT to hepatic fibrosis. Increasing evidence has suggested that circRNAs could give play to their biological functions and participate in the regulation of various human physiological processes through acting as “miRNA sponges”. To examine the molecular mechanism that circ_0072088 regulate EMT via sponging miRNAs in hepatic fibrosis, we searched the target miRNAs that bind to circ_0072088 using CircInteractome dataset. We had demonstrated that miR-330-3p might be sponged by circ_0072088 using RNA pull-down and luciferase reporter assay. Moreover, down-expression of circ_0072088 significantly attenuated the EMT progression to play anti-fibrosis role by binging to miR-330-3p sponges in AML12 hepatocytes, proved that circ_0072088 regulate EMT in hepatic fibrosis progression via sponging miR-330-3p in hepatocytes. Overall, the current work was focused on research the substantial role of circ_0072088 in the liver fibrosis processes as well as the underlying mechanism.

MiRNAs are crucial organismal adjustment factors which come into play biological function and take part in a mass of pathophysiological processes by complementary nucleotide sequence-specific binding to the 3′-UTR of the downstream mRNA to restrain translation process of target gene[29]. Several studies have provided evidence that various miRNAs adjust the process of hepatic fibrosis, including miR-98-5p and miR-34a[30, 31], but no direct evidence demonstrated that miR-330-3p regulate the process of hepatic fibrosis.

Recent evidence have demonstrated that CDK1 may be associated with the cancer by enriching the EMT process[32, 33]. Moreover, targetScan website prediction show that CDK1 can bind to the 3′-UTR of miR-330-3p. Meanwhile, we found that CDK1 was upregulated in AML12 hepatocytes treatment of TGF-β1. In addition, upregulate of circ_0072088 memorably increased the expression of CDK1 proposing that circ_0072088 could function as miR-330-3p sponge to adjust and control CDK1 expression. The investigation of this article has provided neoteric testimony into the role of circRNA-miRNA-mRNA on illness progression after the development of hepatic fibrosis.

To sum up, the expression of circ_0072088 was signifificantly increased in the TGF-β1 treated AML12 hepatocytes. Furthermore, downregulation of circ_0072088 markedly decreased the EMT related proteins and upregulated miR-330-3p levels in the TGF-β1 induced AML12 hepatocytes. Meanwhlie, Knockdown of circ_0072088 indirectly restrain CDK1 expression by miR-330-3p. Our data supported the notion that circ_0072088 knockdown can amoliorate hepatic fibrosis by targeting the miR-330-3p/CDK1 axis to improve EMT, and may be a potential preventive agent for the anti-fibrotic therapy.

Conflict of interest

The authors have no conficts of interest to declare that are relevant to the content of this article.

Ethical approval

This work does not involve animals or human subjects.

Consent for publication

Not applicable.

Funding

This work was supported by Young Talents Foundation of Harbin Medical University Cancer Hospital (No. JJQN2021-10).

Author Contributions

MD, LZ participated in study design. ML, YX, YJ, SL, XT and DZ performed the experiments. ML, YX and YJ analyzed the data. MD and LZ contributed to the writing of the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgement

Not applicable.

X. Yu, N. Elfimova, M. Muller, D. Bachurski, U. Koitzsch, U. Drebber, E. Mahabir, H.P. Hansen, S.L. Friedman, S. Klein, H.P. Dienes, M. Hoesel, R. Buttner, J. Trebicka, V. Kondylis, I. Mannaerts, M. Odenthal, Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion, Cellular and molecular gastroenterology and hepatology, (2022).
K. Baghaei, S. Mazhari, S. Tokhanbigli, G. Parsamanesh, H. Alavifard, D. Schaafsma, S. Ghavami, Therapeutic potential of targeting regulatory mechanisms of hepatic stellate cell activation in liver fibrosis, Drug discovery today, 27 (2022) 1044–1061.
Ji, Q. Zhao, Y. Qin, H. Tong, Q. Wang, M. Yu, C. Mao, T. Lu, J. Qiu, C. Jiang, Germacrone improves liver fibrosis by regulating the PI3K/AKT/mTOR signalling pathway, Cell biology international, 45 (2021) 1866–1875.
Y. Yang, Z. Min, Effect of long noncoding RNA AK021443 on promoting hepatic fibrosis in vitro, Molecular medicine reports, 23 (2021).
D. Kong, Z. Zhang, L. Chen, W. Huang, F. Zhang, L. Wang, Y. Wang, P. Cao, S. Zheng, Curcumin blunts epithelial-mesenchymal transition of hepatocytes to alleviate hepatic fibrosis through regulating oxidative stress and autophagy, Redox biology, 36 (2020) 101600.
H. Zhang, J. Xing, L. Zhao, Lysine-specific demethylase 1 induced epithelial-mesenchymal transition and promoted renal fibrosis through Jagged-1/Notch signaling pathway, Human & experimental toxicology, 40 (2021) S203-S214.
L. Ding, Y. Li, Y. Yang, S. Song, H. Qi, J. Wang, Z. Wang, J. Zhao, W. Zhang, L. Zhao, D. Zhao, X. Li, Z. Wang, Wenfei Buqi Tongluo Formula Against Bleomycin-Induced Pulmonary Fibrosis by Inhibiting TGF-beta/Smad3 Pathway, Frontiers in pharmacology, 12 (2021) 762998.
J.H. Park, J. Yoon, Schizandrin inhibits fibrosis and epithelial-mesenchymal transition in transforming growth factor-beta1-stimulated AML12 cells, International immunopharmacology, 25 (2015) 276–284.
G. Shu, A. Yusuf, C. Dai, H. Sun, X. Deng, Piperine inhibits AML-12 hepatocyte EMT and LX-2 HSC activation and alleviates mouse liver fibrosis provoked by CCl4: roles in the activation of the Nrf2 cascade and subsequent suppression of the TGF-beta1/Smad axis, Food & function, 12 (2021) 11686–11703.
T. Weng, D. Yan, D. Shi, M. Zhu, Y. Liu, Z. Wu, T. Tang, L. Zhu, H. Zhang, H. Yao, L. Li, The MSP-RON pathway regulates liver fibrosis through transforming growth factor beta-dependent epithelial-mesenchymal transition, Liver international: official journal of the International Association for the Study of the Liver, 41 (2021) 1956–1968.
P. de la Cruz-Ojeda, R. Flores-Campos, E. Navarro-Villaran, J. Muntane, The Role of Non-Coding RNAs in Autophagy During Carcinogenesis, Frontiers in cell and developmental biology, 10 (2022) 799392.
C. Guay, C. Jacovetti, M.B. Bayazit, F. Brozzi, A. Rodriguez-Trejo, K. Wu, R. Regazzi, Roles of Noncoding RNAs in Islet Biology, Comprehensive Physiology, 10 (2020) 893–932.
Y. Liang, N. Liu, L. Yang, J. Tang, Y. Wang, M. Mei, A Brief Review of circRNA Biogenesis, Detection, and Function, Current genomics, 22 (2021) 485–495.
Y.L. Wu, H.F. Li, H.H. Chen, H. Lin, Emergent Roles of Circular RNAs in Metabolism and Metabolic Disorders, International journal of molecular sciences, 23 (2022).
W. Sun, Y. Li, D. Ma, Y. Liu, Q. Xu, D. Cheng, G. Li, C. Ni, ALKBH5 promotes lung fibroblast activation and silica-induced pulmonary fibrosis through miR-320a-3p and FOXM1, Cellular & molecular biology letters, 27 (2022) 26.
W. Zhou, Y.X. Chen, B. Ke, J.K. He, N. Zhu, A.F. Zhang, X.D. Fang, W.P. Tu, circPlekha7 suppresses renal fibrosis via targeting miR-493-3p/KLF4, Epigenomics, 14 (2022) 199–217.
Y. Zhu, F. Zha, B. Tang, T.T. Ji, X.Y. Li, L. Feng, S.J. Bai, Exosomal hsa_circ_0125310 promotes cell proliferation and fibrosis in diabetic nephropathy via sponging miR-422a and targeting the IGF1R/p38 axis, Journal of cellular and molecular medicine, 26 (2022) 151–162.
L. Ma, J. Wei, Y. Zeng, J. Liu, E. Xiao, Y. Kang, Y. Kang, Mesenchymal stem cell-originated exosomal circDIDO1 suppresses hepatic stellate cell activation by miR-141-3p/PTEN/AKT pathway in human liver fibrosis, Drug delivery, 29 (2022) 440–453.
S. Zhu, X. Chen, J.N. Wang, J.J. Xu, A. Wang, J.J. Li, S. Wu, Y.Y. Wu, X.F. Li, C. Huang, J. Li, Circular RNA circUbe2k promotes hepatic fibrosis via sponging miR-149-5p/TGF-beta2 axis, FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 35 (2021) e21622.
Y.R. Yang, S. Hu, F.T. Bu, H. Li, C. Huang, X.M. Meng, L. Zhang, X.W. Lv, J. Li, Circular RNA CREBBP Suppresses Hepatic Fibrosis Via Targeting the hsa-miR-1291/LEFTY2 Axis, Frontiers in pharmacology, 12 (2021) 741151.
L. Li, C. Xiao, K. He, G. Xiang, Circ_0072088 promotes progression of hepatocellular carcinoma by activating JAK2/STAT3 signaling pathway via miR-375, IUBMB life, 73 (2021) 1153–1165.
R. Mu, S. Ye, R. Lin, Y. Li, X. Guo, L. An, Effects of Peroxiredoxin 6 and Its Mutants on the Isoproterenol Induced Myocardial Injury in H9C2 Cells and Rats, Oxidative medicine and cellular longevity, 2022 (2022) 2576310.
L. Shao, Y. Zhu, B. Liao, G. Wang, L. Huang, L. Yu, D. Bai, Effects of Curcumin-mediated photodynamic therapy on autophagy and Epithelial-mesenchymal transition of lung cancer cells, Photodiagnosis and photodynamic therapy, (2022) 102849.
S. Ha, K.W. Chung, J. Lee, H.Y. Chung, H.R. Moon, Renal tubular PAR2 promotes interstitial fibrosis by increasing inflammatory responses and EMT process, Archives of pharmacal research, 45 (2022) 159–173.
R. Mullins, A. Pal, T.F. Barrett, M.E. Heft Neal, S.V. Puram, Epithelial-mesenchymal plasticity in tumor immune evasion, Cancer research, (2022).
L. Gao, T. Xia, M. Qin, X. Xue, L. Jiang, X. Zhu, CircPTK2 Suppresses the Progression of Gastric Cancer by Targeting the MiR-196a-3p/AATK Axis, Frontiers in oncology, 11 (2021) 706415.
Y. Ge, W. Liu, W. Yin, X. Wang, J. Wang, X. Zhu, S. Xu, Circular RNA circ_0090231 promotes atherosclerosis in vitro by enhancing NLR family pyrin domain containing 3-mediated pyroptosis of endothelial cells, Bioengineered, 12 (2021) 10837–10848.
L. Bao, M. Wang, Q. Fan, Hsa_circ_NOTCH3 regulates ZNF146 through sponge adsorption of miR-875-5p to promote tumorigenesis of hepatocellular carcinoma, Journal of gastrointestinal oncology, 12 (2021) 2388–2402.
J. Gu, H. Xu, Y. Chen, N. Li, X. Hou, MiR-223 as a Regulator and Therapeutic Target in Liver Diseases, Frontiers in immunology, 13 (2022) 860661.
Y. Ma, X. Yuan, M. Han, Y. Xu, K. Han, P. Liang, S. Liu, J. Chen, H. Xing, miR-98-5p as a novel biomarker suppress liver fibrosis by targeting TGFbeta receptor 1, Hepatology international, (2022).
J. Zhang, H. Wang, L. Yao, P. Zhao, X. Wu, MiR-34a promotes fibrosis of hepatic stellate cells via the TGF-beta pathway, Annals of translational medicine, 9 (2021) 1520.
B. Zhang, M. Zhang, Q. Li, Y. Yang, Z. Shang, J. Luo, TPX2 mediates prostate cancer epithelial-mesenchymal transition through CDK1 regulated phosphorylation of ERK/GSK3beta/SNAIL pathway, Biochemical and biophysical research communications, 546 (2021) 1–6.
C.X. Wu, X.Q. Wang, S.H. Chok, K. Man, S.H.Y. Tsang, A.C.Y. Chan, K.W. Ma, W. Xia, T.T. Cheung, Blocking CDK1/PDK1/beta-Catenin signaling by CDK1 inhibitor RO3306 increased the efficacy of sorafenib treatment by targeting cancer stem cells in a preclinical model of hepatocellular carcinoma, Theranostics, 8 (2018) 3737–3750.

No competing interests reported.

Download PDF

Version 2

posted

You are reading this latest preprint version

Circ_0072088 mediates hepatic fibrosis renders epithelial to mesenchymal transition through miR-330-3p/CDK1 axis in AML12 hepatocytes

Status:

Version 2

Abstract

Figures

Introduction

Materials And Methods

Hepatocytes culture

Cell transfections

Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR)

Western blot

Luciferase reporter assays

RNA Pull-Down Assay

Cell viability assay

Statistical analysis

Results

Discussion

Conclusion

Declarations

Funding

Author Contributions

Acknowledgement

References

Additional Declarations

Supplementary Files

Status:

Version 2