Abstract
Hepatocellular carcinoma (HCC) is one of the most malignant tumors because of high operative mortality, recurrence rate and lack of survival benefit, thus entailing the need to develop new therapy. microRNAs (miRNAs) play pivotal regulatory roles in cancers and tumor suppressor miRNAs can modulate gene regulatory networks to inhibit tumor cell proliferation, invasion and metastasis, hence holding great promise as a therapeutics strategy for HCC. Additionaly, long non-coding RNAs (lncRNAs) can manipulate a variety of cellular processes and may offer a therapeutic opportunity. However, delivery into miRNA or lncRNA into cancer cells remain to be a roadblock to their applications in cancer therapy. In this regard, baculovirus has emerged as a promising gene delivery vector with growing applications. Therefore, the overriding objective of this study was to combine baculovirus-mediated gene delivery for long-term non-coding RNA regulation for HCC therapy. To combat HCC, long-term expression of the anti-cancer gene specifically in HCC cells is desired. Since miR-122 downregulation and miR-151 overexpression are implicated in HCC development and metastasis, we first aimed to develop a baculovirus-mediated, miRNA-based gene therapy approach to inhibiting HCC tumorigenesis and metastasis. To this end, we constructed 3 baculovirus vectors exploiting the Sleeping Beauty system (SB) for sustained transgene expression: Bac-T2-s151 expressed miR-151-specific sponges, Bac-T2-m122 harbored a cluster comprising 8 copies of pre-miR-122 while Bac-T2-Dual accommodated both cassettes. By co-transducing Mahlavu cells with another SB transposase-expressing Bac-SB, Bac-T2-s151 significantly attenuated miR-151 levels while Bac-T2-m122 offset the miR-122 deficiency in Mahlavu cells for 6 weeks. Moreover, Bac-T2-Dual simultaneously suppressed miR-151 and elevated miR-122 levels for 6 weeks. All 3 vectors modulated the expression of genes associated with metastastis, arrested cell cycle, impeded cell growth and alleviated the migration/invasion. Intratumoral injection of the baculovirus into the mice bearing Mahlavu tumor effectively repressed the tumor growth and enhanced the survival rate. Immunohistochemistry showed fewer Ki-67+ cells and more cleaved caspase-3 cells with baculovirus-mediated miR-122 overexpression and miR-151 suppression. These data implicated the potentials of enhancing miR-122 and suppressing miR-151 levels in HCC to suppress tumor development and metastasis. Next, we demonstrated that several lncRNAs (e.g. LincRNA-p21、MEG3 and PTENP1) are significantly down-regulated in Mahlavu cells. Since PTENP1 lncRNA can regulate PI3K/AKT pathway and is associated with tumorigenesis, we attempted to restore the intracellular PTENP1 lncRNA and constructed SB-based hybrid baculovirus vectors for sustained lncRNA expression. Bac-SB/W expressed the SB100X transposase with a WPRE sequence at the 3’ end of mRNA, while Bac-T2-PTENP1-3’ expressed the 3’-untranslated region of PTENP1 lncRNA. By co-transducing Mahlavu cells with Bac-SB/W and Bac-T2-PTENP1-3’ (SB/PTENP1-3’ group), the PTENP1 levels were significantly elevated for 5 weeks. SB/PTENP1-3’ suppressed the expression or phosphorylation of proteins associated with the PI3K/AKT pathway (e.g. p-PDK1, p-AKT, p-mTOR, Bcl-2 and HIF-1) and activated GSK3 and p53 proteins. Furthermore, SB/PTENP1-3’ suppressed the proliferation of Mahlavu cells, induced cell apoptosis and autophagy which was enhanced the cell death. Intratumoral co-injection of Bac-SB/W and Bac-T2-PTENP1-3’ into immunocompetent mice bearing Mahlavu tumors effectively mitigated the tumor growth, suppressed intratumoral cell growth, elicited cell apoptosis, triggered autophagy and inhibited angiogenesis. These data collectively demonstrated that the SB-based PTENP1 lncRNA-expressing baculovirus was able to suppress HCC. In summary, this study demonstrates the potential of the SB-based hybrid baculovirus vectors for persistent modulation of noncoding RNA, either miRNA o lncRNA, for HCC therapy. Keywords: baculovirus, hepatocellular carcinoma, miR-122, miR-151, PTENP1, cancer therapy