**5. Application prospect of targeting CLU gene**

#### **5.1. Antisense oligonucleotide (ASO) therapy**

ASO is a useful technique to inhibit specific-targeted CLU genes, with a small synthetic natural nucleic acid analogue, that can complementary to CLU mRNA that induce degradation or inhibit translation into protein [52]. It is considered to be a potent inhibitor of sCLU expression in vitro, in vivo, and in human clinical trials, with no apparent effect on the expression of nCLU. Custirsen (OGX-011, 5′-CAGCAGCA GAGTCTTCATCAT-3′, 50 nM) is a novel 2′-methoxy-ethyl-modified phosphorothioate ASO, which is a 21-nucleoside complement to target the translation initiation site of CLU gene exon II mRNA translation initiation site with one CpG motif [53]. Hence, OGX-011 plays the role of chemosensitization by influencing the anti-apoptotic protein sCLU instead of the proapoptotic protein nCLU. Xiu et al. have reviewed the current state of research on clusterin, to predict future research directions and to analyze the potential of the clinical application of custirsen in HCC [54]. However, the median overall survival of HCC cases were ms 23.4 mo in the group of the OGX-011 combining anti-cancer drugs (docetaxel/prednisone) and ms 22.2 mo in the other group of cases with docetaxel/prednisone alone. No significant difference was found between two groups. Some potential key factors might contribute to its results, and still want to do more clinical trials to be ongoing [55].

#### **5.2. Reversal MDR by specific shRNA-targeted sCLU**

Resistance of tumor cells to chemotherapy continues to be a major clinical obstacle to extend the survival rate of patients with HCC. Recently, one of the major strategies for liver cancer is surgical resection with adjuvant anti-HCC drug chemotherapy [10]. However, the HCC patients always tend to acquire MDR during tumor progression. MDR-related P-glycoprotein (P-gp), encoded by MDR1 gene is positively linked closely with chronic liver diseases, because of its drug efflux via ATP-binding cassette (ABC) family transporters, which can decrease intracellular concentration of anti-HCC drugs. Targeting sCLU to sensitize cancer cells to chemotherapy has become an attractive new strategy for cancer treatment. Previous studies found sCLU expressed in line with P-gp via immunohistochemical staining. The sCLU expression was analyzed in human hepatoma cells and chemoresistant counter-part HepG2/ADM cells. After transfection of shRNA-1 (5′-GTAAGTACGTC AATAAGGA-3′) into HepG2/ADM cells, the inhibition of CLU expression was 73.68% at mRNA level with obvious enhancement in chemosensitivity, and increasing apoptosis induced by doxorubicin [56]. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-gp. Moreover, silencing CLU led to downregulation of β-catenin, suggesting that downregulation of CLU might be a key point to reverse MDR of HepG2/ADM cells [57].
