DNA Polymerases as targets for gene therapy of hepatocellular carcinoma.
BACKGROUND
Hepatocyte carcinoma (HCC) is one of the most common malignancies worldwide. Despite many achievements in diagnosis and treatment, HCC mortality remains high due to the malignant nature of the disease. Novel approaches, especially for targeted therapy, are being extensively explored. Gene therapy is ideal for such purpose for its specific expression of exogenous genes in HCC cells driven by tissue-specific promoter. However strategies based on correction of mutations or altered expressions of genes responsible for the development/progression of HCC have limitations because these aberrant molecules are not presented in all cancerous cells. In the current work, we adopted a novel strategy by targeting the DNA replication step which is essential for proliferation of every cancer cell.


METHODS
A recombinant adenovirus with alpha fetoprotein (AFP) promoter-controlled expressions of artificial microRNAs targeting DNA polymerases α, δ, ε and recombinant active Caspase 3, namely Ad/AFP-Casp-AFP-amiR, was constructed.


RESULTS
The artificial microRNAs could efficiently inhibit the expression of the target polymerases in AFP-positive HCC cells at both RNA and protein levels, and HCC cells treated with the recombinant virus Ad/AFP-Casp-AFP-amiR exhibited significant G0/1 phase arrest. The proliferation of HCC cells were significantly inhibited by Ad/AFP-Casp-AFP-amiR with increased apoptosis. On the contrary, the recombinant adenovirus Ad/AFP-Casp-AFP-amiR did not inhibit the expression of DNA polymerases α, δ or ε in AFP-negative human normal liver cell HL7702, and showed no effect on the cell cycle progression, proliferation or apoptosis.


CONCLUSIONS
Inhibition of DNA polymerases α, δ and ε by AFP promoter-driven artificial microRNAs may lead to effective growth arrest of AFP-positive HCC cells, which may represent a novel strategy for gene therapy by targeting the genes that are essential for the growth/proliferation of cancer cells, avoiding the limitations set by any of the individually altered gene.
Institute of Occupational Diseases, Zhejiang Academy of Medical Sciences, 182 Tianmushan Road, Hangzhou, 310013, Zhejiang, P.R. China. liuh_1985@163.com.
Hao
H
Liu
Department of Surgical Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou, 310016, Zhejiang, P.R. China. ellequn@126.com.
Qun
Q
Wei
Institute of Occupational Diseases, Zhejiang Academy of Medical Sciences, 182 Tianmushan Road, Hangzhou, 310013, Zhejiang, P.R. China. 845834078@qq.com.|School of Laboratory Medicine and Life Science, Wenzhou Medical University, Chashan Higher Educational Park, Wenzhou, 325035, Zhejiang, P.R. China. 845834078@qq.com.
Jia
J
Wang
Institute of Occupational Diseases, Zhejiang Academy of Medical Sciences, 182 Tianmushan Road, Hangzhou, 310013, Zhejiang, P.R. China. hxm349@yahoo.com.cn.
Xiaoming
X
Huang
Clinical Research Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, P.R. China. lichunchun.1@163.com.
Chunchun
C
Li
Clinical Research Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, P.R. China. qlzheng@hotmail.com.
Qiaoli
Q
Zheng
Clinical Research Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, P.R. China. caoj@zju.edu.cn.
Jiang
J
Cao
Institute of Occupational Diseases, Zhejiang Academy of Medical Sciences, 182 Tianmushan Road, Hangzhou, 310013, Zhejiang, P.R. China. zhenyujia@yahoo.com.
Zhenyu
Z
Jia
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors / Q000235:genetics*; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000235:genetics*; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D058990:Molecular Targeted Therapy; D011401:Promoter Regions, Genetic; D023041:Xenograft Model Antitumor Assays
D035683:MicroRNAs; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III