Recombinant MS2 Phage Virus-like Particles Containing IncRNA MEG3 Target EGFR-positive Hepatocellular Carcinoma

Heptocellular carcinoma (HCC) is one of the most common cancers with a high mortality rate in the worldwide. The traditional therapy methods, such as surgery, radiotherapy and chemotherapy, only have good effect on early stage of cancers, and a relative poor effect on late stage of cancers. Since the pathogenesis of HCC is complicated and may be related to many different signaling molecules, recently molecular targeted therapy is getting more and more attention. Long non-coding RNAs (lncRNAs) are broadly defined as endogenous cellular non-coding RNA molecules that are longer than 200 nucleotides, which used to be "noise" of genome transcription. However, with further research on lncRNAs, it is reported that lncRNAs can perform multiple functions as signals, decoys, guides, and scaffolds and be closely related with cancers. Maternally expressed gene 3 (MEG3), the product of which functions as a non-coding RNA, is normally or highly expressed in many normal human organs or tissues. Whereas, its expression is lower or absent in some kinds of tumors and in several different human carcinoma cell lines, including HCC, indicating it may act as a novel tumor suppressor. However, the deficiencies such as limited transduction efficiency, cytotoxicity, and integration-induced tumorigenesis, remain a concern that significantly limits the effect and applications of MEG3 therapy.In this study, we constructed and expressed MS2 virus-like particles (VLPs) contained lncRNA MEG3 RNA fragments, which could not only protect RNA from degradation of RNase but also didn’t affect the normal function of MEG3 RNA. In the meanwhile, we conjugated the GE11 polypeptide to the surface of MS2 VLPs, thus rendering them target EGFR positive cells and solving the problem of lncRNAs delivery. These targeted VLPs provide a new delivery strategy for therapy against EGFR-positive HCC.First of all, we explored the mechanism of GE11-mediated entry of VLPs. The experiment in vitro showed that a dose-dependent change in MEG3 RNA level was documented in four different cell lines, and the expression level was stable for more than 72 h. The targeted delivery was mainly dependent on clathrin-mediated endocytosis and caveolin-meidated endocytosis and it didn’t activate the EGFR tyrosine kinase, which didn’t cause the activation of downstream signaling pathways. Then, we discussed the therapy effect of lncRNA MEG3 encapsulted in VLPs after entering cells. The experiments in vitro and in vivo verified that after the entry of VLPs containing lncRNA MEG3 RNA, the recombinant VLPs could significantly inhibit cell proliferation, promote cell apoptosis, make the cell cycle arrest at G0/G1 phase, and effectively improve the tumor cell population dependence, thereby inhibiting tumor metastasis. Overexpression of MEG3 increased the level of p53 protein expression in the HepG2, while the expression level of MDM2 protein was decreased, and the mRNA expression of GDF15 gene in the downstream of p53 signaling pathway was also increased. In addition, because the cell proliferation of p53-deficient Hep3B cells was also inhibited, we hypothesized that the tumor suppressor function of MEG3 was achieved through the MDM2-p53-GDF15 signaling pathway and non-p53 dependent signaling pathways.In conclusion, in vitro and in vivo results show that the new lncRNA delivery vehicle based on MS2 VLPs crosslinked with GE11 peptides possesses the advantages of simple preparation, good stability and high cellular uptake rate, no influence on the RNA function and no toxic and side effects. However, the immunogenicity of protein macromolecules cannot be ignored. We will explore new methods to modify the surface molecular of MS2 VLPs, thus making them more suitable for therapy in vivo.This study provides new ideas and technical means on lncRNA based cancer therapy and also can be applied to other cancers or diseases, and thus playing a greater role in the future.