An Artificial Interfering LncRNAi Competitively Consumes Onco-miRNAs To Exert Antitumor Efficacy In Hepatocellular Carcinoma

【Background & Objectives】Recently, bioinformatics analysis showed that mi RNAs regulate a significant number of human genes, and play a key role in various biological processes. Compared with normal tissue, miRNA expression profile in tumor tissue changes, indicating that tumor development closely relates to mi RNAs. MiRNAs, as oncogenes or oncosuppressor gene, regulate the expression and function of their related target genes to play a significant role in cancer malignant progression. MiRNAs highly expressed in tumor cells, as oncogenic miRNAs, inhibit translation of tumor suppressor genes. Therefore, the therapy approach targeted these OncomiRs is invaluable.At present, there are numerous tumor therapy strategies targeted miRNAs, including miRNAs inhibitors and antisense sequence that block the expression of mi RNAs. However, most of these current treatments target single miRNA or a family of mi RNAs. The regulation mechanism of miRNAs is extremely complex, for which a mi RNAs may regulate multiple target genes, meanwhile a target gene may be regulated by multiple miRNAs. Obviously, the effect of the treatment aimed to single miRNA on tumors is limited. So, we designed an artificial interfering lncRNA(lncRNAi) which contained complementary sequences of multiple Oncomi Rs seed sequences. The function of that is to compete with OncomiRs’ target genes to bind to OncomiRs, which protects OncomiRs’ target genes by consuming OncomiRs. If so, only when the copy number of LncRNAi was significantly higher than OncomiRs, could it show better protective effect. Consequently, in previous study, we have constructed an oncolytic adenovirus vector that could replicate in HCC. In this study, we made use of this adenovirus to express LncRNAi. The LncRNAi expression mediated by cancer-selectively oncolytic adenovirus competitively consumes Oncomi Rs to achieve the targeted intervention against cancer cells.【Method】1. Construction and identification of adenoviral vector: The adenovirus AdSVPE1a-lncR, which expressed LncRNAi, was recombined by using the previously generated Survivin promoter-regulated oncolytic adenovirus AdSurp. Encoding LncRNAi, the sequence contained six copies of complementary sequences of 12 Oncomi Rs’ seed sequences. Meanwhile, AdSVPeGFP-lncR, which acted as the positive-control adenovirus carrying LncRNAi, was constructed by instead of E1 a gene with enhanced green fluorescent protein(eGFP). The negative-control adenorvirus Ad5-EGFP was constructed in previous study.2. Cell culture: The human HCC cell lines(HepG2, Hep3 B, SMMC-7721, MHCC97 H, MHCC97 L, Huh-7 and PLC/PRF/5) and the human liver cell lines(L02 and WRL-68) were obtained from the Cell Bank of Cellular and Biochemical Institute(Shanghai Institute for Biological Science, Chinese Academy of Science), and cultured according to the methods provided by the supplier.3. Examination of gene expression: The cell lines used herein were infected with different intensity of experimental adenovirus and control adenovirus. After the total protein was extracted from the cells, the expression of the protein was examined by Western Blot.4. Adenovirus specific proliferative activity: The aforementioned cell lines were infected with AdSVPE1a-lncR or AdSVPeGFP-lncR at an MOI of 1 pfu/cell. After 24 h, 48 h and 72 h, the cells were collected to detect the viral titers with the Tissue Culture Infectious Dose 50(TCID50) method.5. Adenovirus-mediated LncRNAi gene expression: The aforementioned cell lines were infected with AdSVPE1a-lncR or AdSVPeGFP-lncR at an MOI of 1 pfu/cell. The expression of LncRNAi was measured by RT-PCR and qRT-PCR.6. Cell proliferation assay: HCC cell lines and normal liver cell lines were infected with AdSVPE1a-lncR, AdSVPeGFP-lncR and Ad5-EGFP were taken as controls. Cell viability was tested by MTT assay.7. Migration and invasion assays: The aforementioned cell lines were infected with AdSVPE1a-lncR, AdSVPeGFP-lncR and Ad5-EGFP were taken as controls. Cell migration and invasion were detected by Transwell chamber assay.8. Cell cycle and apoptosis: The aforementioned cell lines were infected with AdSVPE1a-lncR or AdSVPeGFP-lncR or Ad5-EGFP, and the parental cells were taken as blank control. Cell cycle and apoptosis were evaluated by flow cytometry.9. Gene expression profiles analysis: Huh-7 was infected with AdSVPE1 alncR or Ad5-eGFP at an MOI of 1 pfu/cell, taking the parental cells as blank control. After 48 h, the total mRNA was extracted from the cells, and analysis of gene expression profiles was examined by gene expression microarray.10. Animal experiment: The HCC xenograft models in nude mice were established, and treated with experimental adenovirus. The effect on tumor growth was observed and recorded.【Results】1. Oncolytic adenovirus specifically replicated in HCC cell lines: The Survivin promoter was used in the oncolytic adenovirus AdSVPE1a-lncR to regulate the expression of E1 a, which achieved the selective expression of LncRNAi in tumor cells. Western Blot analysis showed that the expression of Survivin in various HCC cell lines were different, whereas it was negative in normal liver cell lines. The E1 a expression levels were determined by the expression level of Survivin in HCC cells. So the AdSVPE1a-lncR replication rate was high in some Survivin highly expressed HCC cells, up to 68,465.66 times(Huh-7). However, the adenovirus replication was weak in L02 or WRL-68.2. Oncolytic adenovirus-mediated LncRNAi expression in HCC cell lines: qRT-PCR showed that the expression level of LncRNAi in HCC cells was higher than that in normal liver cells.3. Oncolytic adenovirus-mediated LncRNAi expression decreased HCC cell viability: The oncolytic adenovirus exhibited the strongest cytotoxic activity in Hep3 B and Huh-7. The cell viability of Hep3 B decreased to 50% or less at an MOI of 0.5 pfu/cell, and to 10% at an MOI of 2 pfu/cell. Meanwhile, the cell viability of Huh-7 decreased to 50% or less at an MOI of 1 pfu /cell, and to 10% at an MOI of 100 pfu/cell. This antitumor effect in other HCC cell lines was evident, but not in normal liver cell lines.4. Oncolytic adenovirus-mediated LncRNAi expression inhibited HCC cell migration and invasion: Transwell assay indicated that the AdSVPE1a-lncR and AdSVPeGFP-lncR had an effect on HCC cell migration and invasion, compared with the blank control group. And the effect of AdSVPE1a-lnc R was significantly stronger than AdSVPeGFP-lncR. Both of them had no inhibition on normal liver cell lines.5. Oncolytic adenovirus-mediated LncRNAi expression stimulated cell cycle distribution changes: Experimental and control viruses had no effect on the cell cycle in normal liver cells. But in HCC cell lines, the AdSVPE1a-lncR stimulated cell cycle distribution to change. However, the changes in various cells were different. In PLC/PRF/5, Hep3 B and Huh-7, the cell cycle arrested in G0/G1 phase. At the same time, the adenorvirus in HepG2 and MHCC97 H induced S phase arrest.6. Oncolytic adenovirus-mediated LncRNAi expression induced cell apoptosis: After infection with the AdSVPE1a-lncR, the percentages of apoptotic cells were all increased in HCC cell lines, especially in Hep3 B, HepG2, PLC/PRF/5 and Huh-7. Whereas, it had no statistical difference in BEL-7402, WRL-68 and L02. Postive-control adenovirus AdSVPe GFP-lncR showed no effect on cell apoptosis.7. Oncolytic adenovirus-mediated LncRNAi expression had an effect on gene expression profiles: Huh-7 and HepG2 were infected with AdSVPE1a-lncR, which lead to the changes in gene expression profiles. The results suggested that 708 genes were upregulated, most of which were tumor suppressor genes(PTEN, p27kip1, TIMP3, RECK), and 628 genes were downregulated, most of which were oncogenes(p38/MAPK, Survivin, CDK4, c-myc).8. Oncolytic adenovirus-mediated LncRNAi expression suppressed the xenograft tumor growth: The HCC xenografts in nude mice were established. Compared with the blank control group, the tumor growth of experimental group was significantly inhibited after treatment; even the size of tumor was smaller than that before treatment at 42 ed day. The AdSVPeGFP-lncR also showed that the antitumor effect on tumor growth after 28 days, but the tumor continued to grow. The blank control group did not exhibit a repression effect.【Conclusion】In our previous study, we constructed and identified the vector which used the Survivin promoter to regulate the oncolytic adenovirus replication. The oncolytic adenorvius in this study was created based on the vector mentioned above, which could express the LncRNAi. The LncRNAi contained complementary sequences of 12 OncomiRs’ seed sequences. These OncomiRs could contribute to the development of liver cancer through a variety of mechanisms, including miR-21, by cancer-selectively oncolytic adenovirus, which competitively consumes Oncomi Rs to widely block signal pathways, exerts a better antitumor efficacy in HCC. The therapy strategy overcomes the limit of single-miRNA antitumor efficacy and the defect that cancer cells can easily regain viability through the bypass pathways. This adenorvirus regulated by Survivin promoter just replicates in Survivin-positive HCC cells, and releases progeny viruses to kill more tumor cells without affecting normal cells. Simultaneously, the oncolytic adenovirus-mediated LncRNAi expression is restricted to have an influence on these OncomiRs, which increases the safety and efficiency of the virus vector. In this study, the antitumor strategy, aimed to consume the miRNAs highly expressed in HCC, is not only applied to the treatment of HCC, but also may provide a reference for treatment of other tumors.