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MicroRNA-145 regulates oncolytic herpes simplex virus-1 for selective killing of human non-small cell… Li, Jhy-Ming; Kao, Kuo-Chin; Li, Li-Fu; Yang, Tsung-Ming; Wu, Chean-Ping; Horng, Yan-Ming; Jia, William W; Yang, Cheng-Ta Jul 22, 2013

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RESEARCH Open AccessMicroRNA-145 regulates oncolytic herpes simplexvirus-1 for selective killing of human non-smallcell lung cancer cellsJhy-Ming Li1,2, Kuo-Chin Kao1, Li-Fu Li1, Tsung-Ming Yang3, Chean-Ping Wu4, Yan-Ming Horng4,William WG Jia5 and Cheng-Ta Yang1,6*AbstractBackground: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, andnovel treatment modalities to improve the prognosis of patients with advanced disease are highly desirable.Oncolytic virotherapy is a promising approach for the treatment of advanced NSCLC. MicroRNAs (miRNAs) may bea factor in the regulation of tumor-specific viral replication. The purpose of this study was to investigate whethermiRNA-145 regulated oncolytic herpes simplex virus-1 (HSV-1) can selectively kill NSCLC cells with reduced collateraldamage to normal cells.Methods: We incorporated 4 copies of miRNA-145 target sequences into the 3′-untranslated region of an HSV-1essential viral gene, ICP27, to create AP27i145 amplicon viruses and tested their target specificity and toxicity onnormal cells and lung cancer cells in vitro.Results: miRNA-145 expression in normal cells was higher than that in NSCLC cells. AP27i145 replication wasinversely correlated with the expression of miRNA-145 in infected cells. This oncolytic HSV-1 selectively reduced cellproliferation and prevented the colony formation of NSCLC cells. The combination of radiotherapy and AP27i145infection was significantly more potent in killing cancer cells than each therapy alone.Conclusions: miRNA-145-regulated oncolytic HSV-1 is a promising agent for the treatment of NSCLC.Keywords: Oncolytic virus, MicroRNA, Lung cancer, RadiotherapyBackgroundNSCLC is the leading cause of cancer-related mortalityworldwide [1]. Traditional treatment modalities includesurgical resection, chemotherapy, radiotherapy, and tar-geted therapy. Patients in the early stages of the diseaseare better suited for surgical treatment. In advanced dis-ease, NSCLC cells are often resistant both to chemother-apeutic agents owing to alterations in certain signalpathways [2,3] and to radiotherapy via anti-apoptoticgene overexpression [4]. Targeted therapy with receptor-tyrosine kinase inhibitors has improved progression-freeand overall survival in patients with advanced NSCLC,particularly those harboring activating mutations. How-ever, despite initial responses and long remissions, theinevitable development of secondary resistance leads totreatment failure [5]. Recently, the use of oncolyticviruses has been identified as a novel potential strategyfor cancer treatment owing to its capacity to destroytumor cells both in vitro and in vivo with minimal collat-eral damage to normal cells [6]. In recent decades, anumber of oncolytic virus vectors have been developedwith mutations in genes associated with virulence orviral DNA synthesis to confine viral replication to cancercells and avoid causing disease [7,8]. One approach toengineering replication selectivity is the deletion of viralgenes, which causes inefficient viral replication in nor-mal cells but expansion in tumor cells. This approachwas first described with herpes simplex virus type-1(HSV-1) with thymidine kinase-negative modification,* Correspondence: yang1946@cgmh.org.tw1Department of Thoracic Medicine, Chang Gung Memorial Hospital,5 Fu-Hsing Street, Kweishan, 333, Taoyuan, Taiwan6Department of Respiratory Therapy, Chang Gung University, Taoyuan,TaiwanFull list of author information is available at the end of the article© 2013 Li et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Li et al. Virology Journal 2013, 10:241http://www.virologyj.com/content/10/1/241which attenuates the neurovirulence of HSV to treat hu-man gliomas [9]. HSV-1 is a common human virus thatcan infect most mammalian cells. However, gene dele-tion might reduce the killing capability of HSV mutantsin cancer in vivo. Another engineering approach is toplace an immediate-early (IE) gene essential for HSV-1replication under the control of a cell-specific promoteror enhancer [10]. However, our previous study [11]revealed that HSV-1 infection might up-regulate theactivities of various cellular promoters and telomerase inboth tumor and nontumor cells. The viral IE gene prod-uct, infected cell protein 0 (ICP0), responds by de-regulating cellular promoter activity and activatingtelomerase. Such nonspecific up-regulation of promoteractivities can result in a loss of specific control of geneexpression and might cause nonselective toxicity of theHSV-1 mutant in normal human cells and tissues.Micro-RNAs (miRNAs) are non-coding small RNAsbound to the 3′-untranslated region (3′-UTR) oftargeted messenger RNAs and affect either messengerRNA cleavage or translational inhibition of genes at thepost-transcriptional level [12,13]. Recent reports havesuggested that miRNAs play critical roles in the tumori-genesis and progression of various human cancers[14-16]. miRNA-145 is down-regulated in several malig-nancies including lung cancer [14,16,17], colon cancer[18], ovarian cancer [19], and prostate cancer [20,21],and has been identified as a tumor-suppressive miRNA.Therefore, tumor-specific targeting of oncolytic HSV-1may be achieved at the translational level by incorporat-ing multiple copies of miRNA-145 target sequences intothe 3′-UTR of an essential IE gene such as infection cellprotein 27 (ICP27) or infection cell protein 4 (ICP4). Leeet al. [22] have used an amplicon system to show thatmiR-143 and miRNA-145 inhibit the expression of theICP4 gene at the translational level by targeting the cor-responding 3′-UTR in a dose-dependent manner andthus selectively enable HSV-1 mutant replication inprostate cancer cells. In principle, this system shouldalso permit unimpeded translation of the ICP27 gene inlung cancer cells and subsequent oncolysis but protectnormal cells owing to degradation of the amplicon tran-script by miRNA-145. In the present study, we investi-gated the expression of miRNA-145 in normal cells andNSCLC cells and tested miRNA145-regulated ICP27oncolytic HSV-1 for its capacity to kill NSCLC cells. Wealso studied the therapeutic potential of concurrentviroradiotherapy in NSCLC cells.ResultsDifferential expression of miRNA-145 expressed in variouscell linesmiRNA-145 is reportedly down-regulated in lung cancertissues [23,24]. To investigate the level of miRNA-145expression in normal and lung cancer cell lines, weextracted total RNA with TRIzol® and measured themiRNA-145 expression level using quantitative reversetranscription polymerase chain reaction (RT-PCR).miRNA-145 is highly expressed in normal cells, includ-ing human umbilical vein endothelial cells (HUVECs)and cells obtained from pneumonia/heart failure associ-ated pleural effusions (PL1 and PL2), but it is signifi-cantly down-regulated in human NSCLC cells A549,H460, H838, and H1975 (Figure 1). The miRNA-145expression levels in HUVECs, PL2, A549, H460, H838,and H1975 were 0.376, 0.763, 0.0308, 0.01278, 0.0328,and 0.0392, respectively, relative to PL1 cells. These dataindicate that miRNA-145 expression is a biomarker fordifferentiating normal cells and NSCLC cells.Expression levels of ICP27 in various cell lines afterinfection by AP27i145Because miRNA-145 expression in NSCLC cells is lowerthan that in normal cells, we constructed an miRNA-145 target sequence to regulate ICP27 expression andpromoted viral replication in NSCLC cells. To investi-gate the expression levels of ICP27 mRNA and protein,the normal and lung cancer cell lines were infected withAP27i145 at MOI of 0.1. For assaying the mRNA expres-sion of ICP27, the total RNA of virus-infected cells wasextracted with illustra RNAspin Mini Kit (GE HealthcareLife sciences; 25-0500-70) and the mRNA expressionlevel of ICP27 was measured using quantitative reversemiRNA-145 expression level ( 2 –Figure 1 Expression levels of microRNA (miRNA)-145 in normalcells and non-small cell lung cancer (NSCLC) cells. Expressionlevels of miRNA-145 in various cell lines were determined usingquantitative reverse transcription polymerase chain reaction assay.Expression levels of miRNA-145 were normalized to an internalcontrol (miRNA-93) to obtain ΔCT values, and then all ΔCT valueswere compared with cells obtained from pneumonia/heart failureassociated pleural effusions (PL1) to give -ΔΔCT. HUVECs, humanumbilical vein endothelial cells.Li et al. Virology Journal 2013, 10:241 Page 2 of 9http://www.virologyj.com/content/10/1/241transcription polymerase chain reaction (RT-PCR). Theresult showed that ICP27 mRNA was highly expressedin human NSCLC cells A549, H460, H838, and H1975than that in HUVECs, PL1 and PL2 (Figure 2a). TheICP27 mRNA expression levels in HUVECs, PL2, A549,H460, H838, and H1975 were 2.025, 2.84, 39.921, 57.19,33.376, and 25.904 folds relative to that in PL1 cells,respectively. For the assessment of the ICP27 proteinexpression, the total proteins of virus-infected cellswere extracted with protein extraction reagent andthen measured by Western blotting using anti-ICP27specific antibody. As shown in Figure 2, the proteinexpression levels of ICP27 were compatible with themRNA expression levels of ICP27 in all tested cells(Figure 2b). These data indicated that the cell infectedby AP27i145 could express ICP27, and the expressionof viral protein ICP27 was much higher in malignantcells than in non-malignant cells.Comparisons of cytolytic effects between 5dl1.2and AP27i145 HSV-1The HSV-1 amplicon AP27i145 was generated by carry-ing the ICP27 gene under a cytomegalovirus (CMV)promoter with 4 copies of miRNA-145 complementarytarget sequences in the 3′-UTR. The amplicon plasmidalso contained a viral origin of replication and packagingsignal, which helped replicate and package the ampliconvirus with a replication-deficient recombinant ICP27-helper virus, 5dl1.2, in host cells. The 5dl1.2 helper viruslacks the ICP27 gene and cannot replicate by itself [25].Both cancer cells and normal cells were infected with5dl1.2 or AP27i145 at a multiplicity of infection (MOI)of 0.001 to 0.1. The numbers of viable cells werecounted 5 days after the treatments.As shown in Figure 3, no significant difference wasfound in survival ratios (the ratio of viable cells in thevirus-treated group to those in the mock-infected group)of in AP27i145- and 5dl1.2-treated normal cell lines atvarious doses (71.2328% ± 3.6243 vs. 72.6027% ± 3.6243,74.2574% ± 7.8586 vs. 75.2475% ± 7.9079, and 70.8333 ±8.6736 vs. 63.8888 ± 1.3888 for HUVECs, PL1, and PL2,respectively; p > 0.05 in all comparisons). In A549, H460,H838, and H1975 NSCLC cells, the survival ratios of theAP27i145-infection groups were significantly lower thanthose of respective 5dl1.2-infected groups at an MOIof 0.1 on post-infection day 5 (19.8260 ± 1.3386% vs.84.3478 ± 1.7391%, 17.7083 ± 4.3351% vs. 64.5833 ±4.1666%, 28.1578 ± 4.7441% vs. 70.0000 ± 4.6557%, and7.6923 ± 2.2205% vs. 62.8205 ± 3.3919%, respectively; p <0.05 in all comparisons). The cytotoxicity of AP27i145 wassignificantly stronger than that of 5dl1.2 at an MOIof 0.01 in A549 and H460 cells (69.5652 ± 2.3006% vs.93.9130 ± 3.0122% and 79.1666 ± 4.1666% vs. 97.9166 ±2.0833%, respectively; p < 0.05 in both comparisons; seeFigure 3). This result indicated that AP27i145 causedcytotoxicity more efficiently in NSCLC cells than innormal cells.Correlation of miRNA-145 expression and AP27i145replication in cellsBecause miRNA-145 expression was down-regulated(see Figure 1) and the AP27i145 HSV-1 was more cyto-toxic in NSCLC cells (see Figure 3) than in normal cells,we further investigated whether the replication ofAP27i145 correlated with miRNA-145 expression incells. All tested cells were treated with AP27i145 at anMOI of 0.1. The media were collected 5 days after treat-ment. Using the plaque assay, we examined the virus-induced cytopathic effect on cells to determine the virustiters. The correlation of AP27i145 replication andmiRNA-145 expression was normalized at log 10, andthe data were analyzed using SPSS software. Figure 4shows the strong negative correlation between theNormal cell lines Cancer cell linesICP27expressionlevel(2–CT)020406080HUVECs PL1 PL2 A549 H460 H838 H1975HUVECs PL1 PL2 A549 H460 H838 H1975ICP27actin(a)(b)Figure 2 Expression levels of ICP27 in normal cells andnon-small cell lung cancer cells after infection with AP27i145.The mRNA and protein expression levels of ICP27 in AP27i145-infectedcells was determined using quantitative reverse transcriptionpolymerase chain reaction assay and Western blot assays. (a) Theexpression of ICP27 mRNA in AP27i145-infected cells. The mRNAexpression level of ICP27 was normalized to an internal control (actin)to obtain ΔCT values, and then all ΔCT values were compared withPL1 cells to give -ΔΔCT. (b) The expression of ICP27 protein inAP27i145-infected cells. The protein expressions of ICP27 in normalcells and non-small cell lung cancer cells after infection withAP27i145 were examined by Western blotting with antibodiesspecifically against ICP27 and β-actin.Li et al. Virology Journal 2013, 10:241 Page 3 of 9http://www.virologyj.com/content/10/1/2410204060801001200 0.001 0.01 0.1 0.001 0.01 0.10.001 0.01 0.1 0.001 0.01 0.10.001 0.01 0.10.001 0.01 0.10.001 0.01 0.15dl1.2A P27i145HUVECsMOISurvivalRatio%02040608010012005dl1.2A P27i145MOISurvivalRatio%5dl1.2A P27i145PL1MOISurvivalRatio%5dl1.2A P27i145*SurvivalRatio%MOI**H460Sur vi valRat io%A549MOI**MOI*H838PL2MOIH1975SurvivalRatio%0 002040608010012002040608010012002040608010012000204060801001205dl1.2A P27i1455dl1.2A P27i1450020406080100120SurvivalRatio%05dl1.2A P27i145Figure 3 (See legend on next page.)Li et al. Virology Journal 2013, 10:241 Page 4 of 9http://www.virologyj.com/content/10/1/241replication of AP27i145 HSV-1 and miRNA-145 expres-sion in AP27i145-infected cells (r = -0.842). The dataindicated that the AP27i145 HSV-1 replicated highly inNSCLC cells with low miRNA-145 expression.Inhibition of NSCLC cell growth in vitro by oncolyticAP27i145 HSV-1Furthermore, we elucidated the capability of AP27i145to inhibit NSCLC cell growth in vitro by assessing col-ony formation. We seeded A549, H460, H838, andH1975 (5 × 105 cells) in 10-cm dishes and infected themwith viruses at an MOI of 0.1. Twenty-four hours later,we seeded 1 × 103 infected cells in 6-well plate and cul-tured them for 12 days. After staining the resultant col-onies with methylene blue, we checked the existingcolonies in the well. In contrast to the 5dl1.2-infectedgroups, the AP27i145-infected groups displayed no col-ony growth (Figure 5). The results revealed the inhibi-tory capability of AP27i145 on the growth of NSCLCcells in vitro.Effects of concurrent viroradiotherapy on NSCLC cellsWe investigated the cytotoxicity of the combined effectsof radiation and AP27i145 in NSCLC cells. A549, H460,H838, and H1975 cells were treated with 5dl1.2 orAP27i145 at an MOI of 0.01. Because the ratios of viablecell numbers in the AP27i145-infection group to those inthe mock-infected group (survival ratio) were not less than50% (78.4211 ± 4.3091%, 69.5652 ± 2.3006%, 79.1666 ±4.1666%, and 98.71 ± 3.39%, respectively; see Figure 3), adistinctly additive efficacy was detected. Culture me-dium alone was used for mock infection. These cellswere subsequently treated with radiation at 0, 2, 4, or8 Gy 72 h after infection. The cells were then collectedand counted on day 5. In A549 and H838 lung cancercells, the survival ratios in the AP27i145-treatedgroups at 2 and 4 Gy or 2 and 8 Gy were significantlylower than those in the 5dl1.2-treated groups (2-wayanalysis of variance [ANOVA], p < 0.05 in 4 compari-sons); however, the survival ratios in the AP27i145-treated groups were not significantly different thanthose in the 5dl1.2-treated groups at 8 or 4 Gy ra-diation. In H460 and H1975 lung cancer cells, the survivalratios in AP27i145-treated groups were significantly lowerthan those in the 5dl1.2-treated groups from 2 to 8 Gy(See figure on previous page.)Figure 3 Cytotoxicity of viral infection in normal and NSCLC cells. Cells were treated with varying doses of AP27i145 or 5dl1.2 herpessimplex virus-1 (HSV-1). Culture medium alone was used for mock infection. Triplet cultures were performed for each treatment, and viable cellswere counted on day 5. The results are expressed as a percentage of the mock-infected cells (survival ratio). Data are expressed as means ±standard error (SE). MOI, multiplicity of infection.Regression line“r= -0.842”Log (virus titer) H460H1975A549H838HUVECsPL1PL27.06.56.05.55.04.54.01.21.00.00.20.40.60.8miRNA - 145 expression level (2 –       CT)Figure 4 Correlation of miRNA-145 expression and virusreplication in normal and NSCLC cells. Cells were treated withAP27i145 at an MOI of 0.1. The media were collected 5 days aftertreatment, and viral titer was determined using 7B cells. Thecorrelation of AP27i145 replication and miRNA-145 expression wasnormalized at log 10 and calculated using SPSS software.H460Mock infectionMOI of 0.15dl1.2AP27i145H1975MOI of 0.15dl1.2AP27i145A5495dl1.2AP27i145H838MOI of 0.1MOI of 0.15dl1.2AP27i145Mock infection Mock infectionMock infection(a) (b)(c) (d)Figure 5 Effects of AP27i145 infection on cell growth in vitro.HSV-1 infection was performed at an MOI of 0.1. Twenty-four hoursafter infection, cells were seeded onto 6-well culture plates at aconcentration of 103 cells per well and were cultured for additional12 days. The colonies were stained with methylene blue forphotography. The experiments were performed in triplicate, andrepresentative data are shown. (a) H460, (b) H1975, (c) A549, and(d) H838 cells.Li et al. Virology Journal 2013, 10:241 Page 5 of 9http://www.virologyj.com/content/10/1/241radiation (2-way ANOVA, p < 0.05 in 6 comparisons;Figure 6).DiscussionThis study was proposed to determine whether we couldselectively direct the oncolytic activity of a mutant herpesvirus to kill NSCLC cells. miRNA-145 is reportedly down-regulated in various lung cancer tissues [26], which sug-gests that the 3′-UTR of miRNA-145 might regulate theexpression of viral replication genes for selective targetingof tumor cells but not normal tissues. We analyzed the ex-pression of miRNA-145 in normal and NSCLC cells usingreal-time quantitative RT-PCR. As expected, the expres-sion of miRNA-145 in NSCLC cells was much lower thanthat in normal cells (see Figure 1). Based on this lowermiRNA-145 expression pattern in cancer cells, a new classof oncolytic HSV-1 was developed and designated asAP27i145. AP27i145 is an amplicon virus carrying theICP27 gene, an essential gene for HSV replication underthe control of a CMV promoter.Four copies of miRNA-145 complementary target se-quences in the 3′-UTR of ICP27 gene may result in re-stricted replication of virus owing to the high miRNA-145 expression in normal cells. A significant differencewas found in survival ratios between AP27i145- and 5dl1.2-infected NSCLC cells at MOIs of 0.01 and 0.1 (see Figure 3).Accordingly, the cytotoxicity of AP27i145 at an MOI of0.01 in H460 and A549 was significantly more efficient thanthat in H838 and H1975 (see Figure 3), which is consistentwith the lower expression of miRNA-145 in A549 andH460 cells (see Figure 1). These data suggest that the infec-tion of AP27i145 and the lower expression of miRNA-145lead to considerably stronger oncolysis in NSCLC cells.Furthermore, the relatively higher expression level ofmiRNA-145 in normal cells significantly decreased thecytotoxicity of AP27i145, which suggests that the 4 copiesof the 3′-UTR miRNA binding site sufficiently and selec-tively inhibit oncolytic HSV-1 replication in normal cells.We studied the correlation between the replication ofAP27i145 and miRNA-145 expression in AP27i145-infectedcells. A strong negative correlation was found betweenthe replication of AP27i145 and miRNA-145 expressionin AP27i145-infected cells, confirming that the expressionlevel of miRNA-145 may affect AP27i145 HSV-1 replica-tion in cells. Furthermore, we demonstrated thatAP27i145 selectively inhibits colony formation in NSCLCcells. The results of the present study are compatiblewith those of Lee et al. [22], who reported miRNA-145-dependent replication of the CMV-ICP4-145T virus in aprostate cancer cell line.Survival Ratio%Mock Infection Mock Infection MOI of 0.01Mock Infection Mock Infection(a) A549 (b) H460(c) H838 (d) H1975MOI of 0.01* *** ***0204060801001200G Y 2G Y 4G Y 8G Y5dl1.2A P27i145MOI of 0.01***MOI of 0.01**0204060801001200G Y 2G Y 4G Y 8G YSurvival Ratio%0204060801001200G Y 2G Y 4G Y 8G Y0204060801001200G Y 2G Y 4G Y 8G Y5dl1.2A P27i145020406080100120Survival Ratio%Survival Ratio%Survival Ratio%Survival Ratio%0204060801001200G Y 2G Y 4G Y 8G Y 0G Y 2G Y 4G Y 8G Y5dl1.2A P27i1450204060801001200G Y 2G Y 4G Y 8G YSurvival Ratio%020406080100120Survival Ratio%0G Y 2G Y 4G Y 8G Y5dl1.2A P27i145Figure 6 Cytotoxicity of combined radiation and AP27i145 HSV-1 treatment. (a) A549, (b)) H460, (c) H838, and (d) H1975 lung cancer cellswere treated with AP27i145 or 5dl1.2 HSV-1 at an MOI of 0.01. Culture medium alone was used for mock infection. Cells were subsequentlytreated with radiation at 0, 2, 4, or 8 Gy 48 h after infection. On day 3 after irradiation, 3 cultures for each combination treatment were countedfor viable cells. The results are expressed as a percentage of the mock-infected and mock-irradiated cells (survival ratio). Data are expressedas means ± SE.Li et al. Virology Journal 2013, 10:241 Page 6 of 9http://www.virologyj.com/content/10/1/241Oncolytic HSV can lyse infected tumor cells andspread viral progeny for further infection and killing ofneighboring cancer cells. Some investigators have sug-gested that combining oncolytic HSV-1 treatment withionizing radiation could increase viral replication toinduce tumor cell death [27-29]. Ionizing radiation canaugment the expression of the HSV-1 late gene ICP34.5to expand viral replication. Our data indicate that theeffects of combined treatment modalities includingAP27i145 HSV-1 infection and radiation inhibit cellgrowth with significantly more potency than that ofmonotherapy. The cytotoxicity of combined AP27i145HSV-1 infection and irradiation increases in a dose-dependent manner for the treatment modalities. However,the augmentation of the treatment effect via combinedmodalities varied by cell type.Josson et al. [30] have reported that irradiation at 6 Gydecreases prostate cancer cell expression of miRNAs.Conversely, irradiation might activate p38 mitogen-activated protein kinase and up-regulate dynamin 2,thereby enhancing the activity of the CMV promoter[31]. The genetic design of AP27i145 may thus be morespecific to the rise in virus production by irradiation.In conclusion, this study demonstrates that regulatingICP27 expression with miRNA-145 can control HSV-1to kill NSCLC cells selectively in vitro. The combinationof this virotherapy with irradiation significantly en-hanced the cytotoxicity of AP27i145 in NSCLC cells.miRNA-145-regulated oncolytic HSV-1 is a promisingagent for the treatment of NSCLC.MethodsPlasmid constructsFour copies of miRNA-145 complementary sequences(miRNA-145T) were constructed as described previously[22]. The ICP27 gene (~1.8 kb) and the miRNA-145Tfragments, excised by XhoI and XbaI digestion, werethen cloned into the pcDNA3.0-neo vector (Invitrogen,Carlsbad, CA, USA), which contained the virus ori, viralpackaging signal, and human CMV promoter to generateCMV-ICP27-145T plasmids. The ICP27 gene, including4 copies of miRNA-145 complementary target sequencesin the 3′-UTR, was controlled by the CMV promoterfor expression.Virus recombinationThe replication-deficient ICP27 helper virus (5dl1.2) andAP27i145 HSV-1 were packaged, propagated, and titeredin 7B cells (ICP4- and ICP27-transformed African greenmonkey kidney cells). Monolayer 7B cells were transfectedwith 24 μg amplicon plasmid DNA (pCMV-ICP27-145T)using Lipofectamine 2000 (Invitrogen) according tomanufacturer instructions. Twenty-four hours after trans-fection, cells were superinfected with 5dl1.2 virus at MOIof 1, the pCMV-ICP27-145T plasmid was thus packagedinto the capsid of AP27i145 exclusively, and the virus wascollected 3 days after superinfection. The AP27i145viruses were then amplified and propagated by infectingmore 7B cells. The virus titer was determined with aplaque-forming assay in 7B cells.Cell linesThe cell lines HUVECs, A549 (ATCC CCL-185), H460(ATCC HTB-177), H838 (ATCC CRL-5844), and H1975(ATCC CRL-5908) were purchased from American TypeCulture Collection (ATCC; Manassas, VA, USA). Thepleural effusion cell lines PL1 and PL2 were culturedfrom clinical specimens of 2 consecutive patientswithout malignancy. Cytological studies found no can-cer cells in these specimens. This study was approvedby the Chang Gung Medical Foundation review board,and written consent was obtained from both patients.All cells were cultured in M199, Dulbecco’s modifiedEagle medium, or Roswell Park Memorial Institutemedium complete medium containing 10% fetal bo-vine serum.Real-time quantification of miRNA-145 using stem-loopRT-PCRFor miRNA-145 quantification, the pulsed reverse tran-scription (RT) reaction described by Chen et al. [32,33]was performed to convert all miRNAs into correspond-ing complementary DNAs in a single RT reaction.Briefly, 10 μL reaction mixture containing miRNA-145-specific stem-loop RT primers (5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGGGATTC-3′ , final 2 mM each), internal control-specific stem-loop RT primers (miRNA-93; 5′-CTCAACGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTACCTGC-3′, final2 mM each), 500 mM deoxyribonucleotide triphosphate,0.5 μL Superscript III (Invitrogen), and 1 μg total RNAwere used for the RT reaction. The pulsed RT reactionwas performed as follows: 16°C for 30 min, followed by 50cycles at 20°C for 30 s, 42°C for 30 s, and 50°C for 1 s. RTproducts were diluted 20-fold before being used formiRNA quantitative PCR. Then, 1 μL diluted RT productwas used as a template for a 10-μL PCR. Briefly, 1X SYBRMaster Mix (Applied Biosystems, Foster City, CA), 200nM miRNA-145-specific forward primer (5′-CGGCGGGTCCAGTTTTCCCAGG-3′), internal control-specific for-ward primer (miRNA-93; 5′-CGGCGGCAAAGTGCTGTTCGTG-3′), and 200 nM universal reverse primer (5′-CTGGTGTCGTGGAGTCGGCAATTC-3′) were usedfor each PCR. The conditions for quantitative PCR were95°C for 10 min, followed by 40 cycles at 95°C for 15 sand at 63°C for 32 s. All quantity PCR reactions wereperformed on an ABI Prism 7500 Fast Real-Time PCRsystem (Foster City, CA).Li et al. Virology Journal 2013, 10:241 Page 7 of 9http://www.virologyj.com/content/10/1/241Western blotCells (5 × 105/well) were plated in 10-cm culture platesand incubated for 24 h at 37°C. After the incubation, thecells were infected with AP27i145 at MOI of 0.1 for 1 hand then all media were changed to fresh medium. Afterthree days virus infection, the remaining cells in the dishwere collected via trypsinization. The total cell lysatesfrom virus-infected cells were extracted with lysis buffer(M-PER Mammalian Protein Extraction Reagent; 78501;Thermo Fisher Scientific Inc, Rockford, IL;) and theanalysis of Western bolt were performed using mouseanti-HSV-1/2 ICP27 monoclonal antibody (sc-69806;Santa Cruz Biotechnology, Santa Cruz, CA) and goatanti-actin polyclonal antibody (sc-1616; Santa CruzBiotechnology, Santa Cruz, CA) Horseradish peroxid-ase conjugated goat anti-mouse, or donkey anti-goatantibody was used as the secondary antibody (SantaCruz Biotechnology). Chemiluminescence detectionwas carried out by using ECL Plus™ (GE Healthcare,Piscataway, NJ) and executed according to the manu-facturer’s instructions.Cytotoxicity assayCells (1 × 105/well) were plated in 6-well culture platesand incubated for 24 h at 37°C before infection. Theywere then mock infected or infected with either 5dl1.2or AP27i145 at various doses (MOIs of 0.001, 0.01, and0.1). After infection for 1 h, all media were changed tofresh media. On day 5 after infection, the remaining cellsin the wells were collected via trypsinization andsuspended in phosphate-buffered saline (PBS). An equalvolume of 0.4% trypan blue (Sigma-Aldrich, St. Louis,MO, USA) was added to the cell suspension. Viable cellswere subsequently determined via direct microscopiccounting with trypan blue exclusion. All counts werecarried out on 3 samples.Colony-forming assayCells (5 × 105/well) were plated onto 10-cm culturedishes and incubated for 24 h at 37°C. The cells werethen mock infected or infected with either 5dl1.2 orAP27i145 at an MOI of 0.1. After 1 h of infection, allmedia were changed to fresh media and subsequentlycultured for 24 h. All cells (1 × 103/well) were thenseeded into 6-well culture plates and cultured for 12days. After removal of the media, the wells were rinsedtwice with PBS. Glutaraldehyde (1.25%) in PBS wasadded to each well, and the plates were incubated for 30min at room temperature to allow for cell fixation. After2 rinses with distilled water, 0.05% methylene blue solu-tion was added to each well, and plates were incubatedfor 30 min at room temperature to facilitate staining ofthe colonies. After 2 rinses with distilled water, theplates were dried and photographed.IrradiationCells (1 × 105/well) were plated onto 6-well cultureplates and incubated for 24 h at 37°C before infection.Subsequently, they were either mock infected or infectedwith 5dl1.2 or AP27i145 at an MOI of 0.01. Forty-eighthours after infection, the cells were irradiated (0, 2, 4, or8 Gy in a single fraction) with a 6-MeV electron beamgenerated by a linear accelerator (Clinac 21EX; Varian,Palo Alto, CA, USA) at a dose rate of 300 cGy min-1.On day 3 after irradiation, 3 cultures for each combin-ation treatment were counted for viable cells.Statistical analysesResults were expressed as means ± standard error. Statis-tical comparisons were made with a 2-sided t-test. Two-way ANOVA was used to analyze the data acquiredthrough radiation dosing in both treatment groups. Thefirst factor was the treatment (AP27i145 vs. 5dl1.2 groups),and the second factor was radiation dose (0 ~ 8 Gy). A pvalue less than 0.05 was accepted as significant.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsLJM contributed to data acquisition and analysis and drafted the manuscript;KKC contributed to data acquisition and revision of the manuscript; LLFprovided technical assistance; YTM worked on aspects of the study relatingto the cohort of patients with lung pleural effusion; WCP was involved indata acquisition and analysis; HYM was involved in data acquisition andanalysis; JWWG provided technical assistance; YCT contributed to the studydesign, data analysis, and revision of the manuscript. All authors have readand approved the final manuscript.AcknowledgementThis study was supported by a Chang Gung Research Project grant(CMRPG3b0091) to C.-T. Yang.Author details1Department of Thoracic Medicine, Chang Gung Memorial Hospital,5 Fu-Hsing Street, Kweishan, 333, Taoyuan, Taiwan. 2Graduate Institute ofAnimal Science, College of Agriculture, National Chiayi University, Chiayi,Taiwan. 3Division of Pulmonary and Critical Care Medicine, Chang GungMemorial Hospital, Chiayi, Taiwan. 4Department of Animal Science, NationalChiayi University, Chiayi, Taiwan. 5Departments of Surgery, University ofBritish Columbia, Vancouver, BC, Canada. 6Department of RespiratoryTherapy, Chang Gung University, Taoyuan, Taiwan.Received: 11 March 2013 Accepted: 6 June 2013Published: 22 July 2013References1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancerstatistics. CA Cancer J Clin 2001, 61:69–90.2. Shanker M, Willcutts D, Roth JA, Ramesh R: Drug resistance in lung cancer.Lung Cancer: Targets and Ther 2010, 1:23–36.3. Monzo M, Rosell R, Taron M: Drug resistance in non-small cell lungcancer. Lung Cancer 2001, 34(Suppl 2):S91–S94.4. 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Cancer Gene Ther 2010, 17:120–130.doi:10.1186/1743-422X-10-241Cite this article as: Li et al.: MicroRNA-145 regulates oncolytic herpessimplex virus-1 for selective killing of human non-small cell lung cancercells. Virology Journal 2013 10:241.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitLi et al. Virology Journal 2013, 10:241 Page 9 of 9http://www.virologyj.com/content/10/1/241

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