Abstract
In spite of the development of new therapies to cancers, hepatocellular carcinoma (HCC) is still 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 and strong therapy. Small non-coding RNAs consisting of 21-25 nucleotides called microRNAs (miRNAs) induce mRNA degradation or suppress mRNA translation by binding to the 3’-untranslated region of target mRNAs. Deregulation of miRNAs facilitates cancer development through the upregulation of oncogenes and the silencing of tumor suppressor genes, which plays 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. For gene suppression, a novel RNA-guided CRISPR interference (CRISPRi) technology was recently well-developed, which only requires co-expression of dead Cas9 (dCas9) and single guide RNA (sgRNA) that target specific genomic sequences without permanently altering genomic DNA. We found that the miR-221&222 are overexpressed in Mahlavu cells compared with normal liver sample, and the tumor suppressor gene, PTEN, is relatively low expressed in hepatocarcinoma cells, especially Mahlavu cells. We intend to develop a baculovirus-mediated, CRISPRi-based gene regulation approach to specificly inhibit miR-221/222 expression. We constructed 2 baculovirus vectors exploiting the Sleeping Beauty system (SB) for sustained transgene expression: Bac-dCas9K-gRNAabc expressed dCas9 protein and three sgRNAs that target upstream of miR-221/222 cluster, Bac-dCas9K-gRNAØ expressed dCas9 protein and sgRNA that doesn’t have targeting site. By co-transducing Mahlavu cells with another SB transposase-expressing Bac-SB/W, we found that Bac-dCas9K-gRNAabc couldn’t stably attenuate miR-221/222 levels. To solve this problem, we design another 2 baculovirus which contain 2 and 3 nucleus localization signals (NLS), and we also optimize the sgRNA scaffold sequence to enhance the affinity between dCas9 protein and sgRNA. As the result, we still couldn’t stably inhibit the highly expressed oncomiR.