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LINC01419-mediated epigenetic silencing of ZIC1 promotes metastasis in hepatocellular carcinoma through the PI3K/Akt signaling pathway

Abstract

Hepatocellular carcinoma (HCC) is a rapidly growing tumor characterized by a high potential for vascular invasion and metastasis. The purpose of our study is to explore the regulation mechanism of long noncoding RNA (lncRNA) LINC01419 on cell-cycle distribution and metastasis in hepatocellular carcinoma (HCC) by regulating zinc finger of the cerebellum (ZIC1) through PI3K/Akt signaling pathway. Bioinformatics analysis and dual-luciferase reporter assay were used to analyze LINC01419 and related genes in HCC, and their expression in HCC tissues and adjacent normal tissues were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. Then, HCC cell lines were subjected to the construction of LINC01419/ZIC1 overexpression/knockdown cells utilizing lentiviral vectors. RIP and ChIP assays were applied to identify the LINC01419-binding protein. BSP and MSP assays were used to determine gene methylation. According to the results, LINC01419 was highly expressed in HCC tissues and cells, while ZIC1 was poorly expressed. LINC01419 targeted and downregulated ZIC1 expression. Furthermore, LINC01419 increased the methylation of ZIC1 promoter and repressed ZIC1 expression. PI3K/Akt signaling pathway was activated by LINC01419 overexpression and ZIC1 knockdown, under which conditions, the HCC cell self-renewal and proliferation were promoted while cell apoptosis was attenuated, accompanied by accelerated formation and metastasis of xenografted tumors in mice. In conclusion, LINC01419 enhances the methylation of ZIC1 promoter, inhibits ZIC1 expression, and activates the PI3K/Akt signaling pathway, thereby enhancing the malignant phenotypes of HCC cells in vitro as well as tumor formation and metastasis in vivo.

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Fig. 1: Construction of lentiviral vector using a pLVX-IRES-Neo plasmid.
Fig. 2: LncRNA LINC01419 participates in HCC progression.
Fig. 3: LncRNA LINC01419 is highly expressed in HCC tissues and SMMC7721 cells and participates in HCC development.
Fig. 4: ZIC1 gene, downregulated in SMMC7721 cells, is involved in the regulation of HCC development through the PI3K/Akt signaling pathway.
Fig. 5: LncRNA LINC01419 epigenetically silences ZIC1 by methylation in the promoter region of ZIC1 gene.
Fig. 6: LncRNA LINC01419 modulates the biological function of SMMC7721 cells by downregulating ZIC1 gene.
Fig. 7: LncRNA LINC01419 stimulates the activation of the PI3K/Akt signaling pathway by inhibiting the expression of ZIC1 gene.
Fig. 8: LncRNA LINC01419 promotes HCC tumor growth and metastasis in vivo by reducing the expression of ZIC1.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

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Acknowledgements

We acknowledge and appreciate our colleagues for their valuable efforts and comments on this paper.

Funding

The study was supported by Science & Technology Department of Sichuan Province Applied Basic Research Program (No. 2020YJ0462).

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YH and KC conceived and designed research. RL performed experiments and analyzed data. YL prepared figures. HY and JG drafted paper. YH and KC edited and revised paper. All authors have approved the final version of paper.

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Correspondence to Hongji Yang or Jun Gong.

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The authors declare no competing interests.

Ethical approval

The study was approved by the Ethics Committee of Sichuan Provincial People’s Hospital, Henan Provincial People’s Hospital. Signed informed consents of all study procedures including the collection of tumor and adjacent normal tissues were obtained from all patients participating in this study. Animal experiments were preapproved by the Institutional Review Board of Sichuan Provincial People’s Hospital, Henan Provincial People’s Hospital. All efforts were made to minimize the number and pain of animals used in the experiments.

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Hou, Y., Chen, K., Liao, R. et al. LINC01419-mediated epigenetic silencing of ZIC1 promotes metastasis in hepatocellular carcinoma through the PI3K/Akt signaling pathway. Lab Invest (2021). https://doi.org/10.1038/s41374-021-00539-z

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