Long interspersed nuclear element ORF-1 protein promotes proliferation and resistance to chemotherapy in hepatocellular carcinoma

Abstract

To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1 (LINE-1), ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma (HCC) cells. MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells. Cell proliferation inhibition and the IC50 were calculated by the Origin 8.0 software. Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes, including p53, p27, p15, Bcl-2, mdr, and p-gp. To corroborate the proliferation and anchor-independent growth results, the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF-1p on the apoptosis regulation. LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs (cisplatin and epirubicin) in HepG2 cells. The IC50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L. Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin. The IC50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L. Interestingly, down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel. The IC50 decreased from 35.90 nmol/L to 7.36 nmol/L. However, overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells. Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression. As a protein arrested in the nucleus, LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors. Indeed, LINE-1 ORF-1p promoted HepG2 cell proliferation, anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15, p21, p53, and Bcl-2 genes. LINE-1 ORF-1p promotes HepG2 cell proliferation and plays an important role in the resistance of chemotherapeutic drugs. By establishing novel roles and defining the mechanisms of LINE-1 ORF-1p in HCC chemotherapeutic drug resistance and cell proliferation regulation, this study indicates that LINE-1 ORF-1p is a potential target for overcoming HCC chemotherapeutic resistance.