Abstract:

Background and purpose: Hepatocellular carcinoma (HCC) is still one of the most prevalent malignancies in China and throughout the world, while most of these patients are in intermediate or advanced stage when diagnosed. The resistance of advanced HCC to chemotherapy or interventional therapy is one of the crucial factors significantly limiting the survival and prognosis of patients. However, the molecular mechanisms involved have not been fully clarified. We previously found that the long non-coding RNA LINC00601 was highly expressed in oxaliplatin resistant HCC cells. This study aimed to explore the role and specific mechanism of LINC00601 in regulating the sensitivity of HCC cells to oxaliplatin chemotherapy, to provide a novel target to identify proper candidates for oxaliplatin chemotherapy, and the theoretical basis for the treatment of patients with advanced HCC. Methods: Human HCC cell line MHCC-97H and Hep-3B were induced to establish oxaliplatin resistant cell lines 97H-OXR and 3B-OXR by long-term low-dose oxaliplatin treatment in vitro. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to detect the expression of LINC00601 in cell lines. Cell counting kit-8 (CCK-8) assay was performed to detect the effect of LINC00601 gene silencing or overexpression on the sensitivity of HCC cells to oxaliplatin treatment. Flow cytometry and Western blot experiments were conducted to detect the alteration of cell apoptosis. Finally, Western blot and immunofluorescence staining were applied to explore the nuclear translocation of growth arrest and DNA damage-inducible protein 45α (GADD45A), and RNA pull-down together with RNA immunoprecipitation (RIP) experiments were carried out to validate the combination of LINC00601 and GADD45A. Results: Compared with the control cells, LINC00601 expression level was significantly increased in oxaliplatin resistant cells. Both induced-oxaliplatin-resistant cells and LINC00601-overexpressed MHCC-97H cells were observed to be less sensitive to oxaliplatin administration. On the contrary, the down-regulation of LINC00601 expression in oxaliplatin resistant cells significantly increased the sensitivity to oxaliplatin treatment. Meanwhile, upregulated expression of LINC00601 inhibited the apoptosis level of HCC cell lines after oxaliplatin treatment, which was consistent with the sensitivity levels of different cell lines to oxaliplatin as above. In addition, the overexpression of LINC00601 reduced the nuclear translocation of GADD45A and the expression of GADD45A in the nucleus. Furthermore, the combination of LINC00601 and GADD45A was observed. Conclusion: LINC00601 might regulate the apoptotic progress of cells after oxaliplatin chemotherapy by interfering with the nuclear translocation of GADD45A, which eventually lead to the resistance to oxaliplatin.

Key words: Hepatocellular carcinoma, Long non-coding RNA, Oxaliplatin, Chemotherapy resistance