Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.

YB-1 stabilizes HIV-1 genomic RNA and enhances viral production

HIV-1 utilizes cellular factors for efficient replication. The viral RNA is different from cellular mRNAs in many aspects, and is prone to attacks by cellular RNA quality control systems. To establish effective infection, the virus has evolved multiple mechanisms to protect its RNA. Here, we show that expression of the Y-box binding protein 1 (YB-1) enhanced the production of HIV-1. Downregulation of endogenous YB-1 in producer cells decreased viral production. YB-1 increased viral protein expression by stabilizing HIV-1 RNAs. The stem loop 2 in the HIV-1 RNA packaging signal was mapped to be the YB-1-responsive element. Taken together, these results indicate that YB-1 stabilizes HIV-1 genomic RNA and thereby enhances HIV-1 gene expression and viral production.

Construction of recombinant adenovirus vector expressing shRNAs targeting COX-2, AKT1 and PIK3R1 gene and its inhibition effect on proliferation of human gastric adenocarcinoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector that expresses small hairpin RNAs (shRNA) against COX-2, AKT1 and PIK3R1 gene and to evaluate its potential for suppressing the cell proliferation of human gastric adenocarcinoma SGC701 cell in vitro and in vivo, which will enable the development of a gene therapy protocol for the treatment of human gastric adenocarcinoma.</p><p><b>METHODS</b>Three strips of shRNA targeting AKT1, COX-2 and PIK3R1, was subcloned into adenovirus expression vector. After verification, it was amplified and titered. The recombinant adenovirus expression vector was infected into human gastric adenocarcinoma SGC7901 cells in vitro and the infected cells were injected in nude mice. The mRNA and protein expression levels of AKT1, COX-2 and PIK3R1 were determined by real-time PCR and Western blot respectively. Cell proliferation in vitro was determined by methyl thiazolyltetrazolium (MTT) assay and flow cytometry, tumor growth in vivo was measured by volume of tumor in nude mice.</p><p><b>RESULTS</b>Restriction digestion and sequencing analysis showed that the rAd5-C-A-P adenovirus expression vector was constructed successfully. It significantly inhibited the expression of AKT1, COX-2 and PIK3R1, and cell growth was inhibited over 70% as indicated by MTT assay and accompanied with G0/G1 phase arrest. Cell growth on matrigel matrix showed that the rAd5-C-A-P transfected cells were detached from the matrix or grew in a scattered clustering pattern, indicating poor cell growth activities in 2-D matrigel. Tumor growth in nude mice in the C + A + P group was inhibited (P<0.01).</p><p><b>CONCLUSION</b>shRNA targeting COX-2, AKT1 and PIK3R1 down regulated significantly the expression of the three genes in a sequence-specific manner, exerted proliferation inhibition effect on SGC7901 cells in vitro and in vivo.</p>

Recombinant adenovirus-mediated RNA silencing of tissue factor expression in human islet: an in vitro study / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a replication-incompetent recombinant adenovirus mediating short hairpin RNA (shRNA)-induced tissue factor gene silencing in the islet.</p><p><b>METHODS</b>Four pairs of complementary oligonucleotides were designed and synthesized to create double-stranded oligonucleotides (ds oligo). The ds oligos were cloned into Pentr/U6 vector to construct the shuttle plasmid pENTR/U6-shRNA, which was transduced into human islets via liposome after sequence verification. The plasmid with the best silencing effect was identified by real-time RT-PCR, followed by homologous recombination with the adenovirus backbone plasmid. The functional clone was transfected into 293A cells to amplify the adenovirus, whose silencing effect against TF expression was tested using real-time RT-PCR and Western blotting.</p><p><b>RESULTS</b>The pENTR/U6-shRNA shuttle plasmid was constructed and verified by sequencing. The recombinant adenovirus-mediated shRNA against TF was constructed, and real-time RT-PCR and Western blotting demonstrated that the strongest silencing effect of the adenovirus against TF occurred on the 4th day following islet transfection.</p><p><b>CONCLUSION</b>Replication-incompetent recombinant adenovirus-mediated shRNA against TF has been successfully constructed, which has good silencing effect against TF expression in human islet in vitro.</p>

An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.</p><p><b>METHODS</b>The double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.</p><p><b>RESULT AND CONCLUSION</b>DNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.</p>

Short hairpin RNA-mediated survivin gene silencing inhibits invasion and metastasis of human colon carcinoma cell line SW480 in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of short hairpin RNA (shRNA) targeting survivin on adhesion and invasion of human colon carcinoma cell line SW480 in vitro.</p><p><b>METHODS</b>According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to prepare the hairpin construct as the DNA templates for the target shRNA. The shRNA templates were cloned into shRNA expression vector pRNAT-U6.1/Neo, and the resulted vector pRNAT-U6.1/Neo-survivin was transfected into SW480 cells using Lipofectamine 2000. Western blotting was performed to evaluate survivin gene silencing induced by shRNA transfection at the protein level, and the biological behaviors of the SW480 cells were investigated by cell-matrix adhesion, invasion and gelatin-zymography assays.</p><p><b>RESULTS</b>Western blotting revealed significantly lowered survivin protein expression in transfected SW480 cells, and survivin gene silencing induced by shRNA significantly suppressed the metastatic potential of SW480 cells in association with suppressed MMPs activity.</p><p><b>CONCLUSIONS</b>Survivin may play an important role in modulating human colorectal carcinoma cell invasion and metastasis, and survivin gene silencing can inhibit human colorectal cancer cell invasion and the production of MMP-2 and MMP-9. Survivin may affect invasion and metastasis of human colorectal carcinoma cells via regulating the production of MMPs.</p>

A Small Cryptic Plasmid pZMO1 of Zymomonas mobilis ATCC10988

The nucleotide sequence of pZMO1, a small cryptic plasmid of Zymomonas mobilis ATCC10988 was determined. Analysis of 1,680 bp of sequence revealed 69% identity with Shigella sonnei plasmid, pKYM and 61% identity with Nostoc sp. ss DNA replicating plasmid. Analysis of a deduced amino acid sequence of an orf of pZMO1 revealed 75% identity and 90% similarity with the repA gene of Synechocystis sp. plasmid pCA2.4. The upstream region of the repA gene of pZMO1 possesses six directed repeat sequences and two inverted repeat sequences at downstream of the IR consensus sequence of nick region of rolling circle replication (RCR) plasmid. A typical terminator hairpin structure was found at the downstream region of repA gene. Degradation of single-stranded plasmid DNA by S1 nuclease was detected by Southern hybridization. It suggests that pZMO1 replicates by a rolling circle mechanism in Z. mobilis ATCC10988 cells.