Potential Target For Gene Therapy Research Articles

Alterations of Axis Inhibition Protein 1 (AXIN1) in Hepatitis B Virus-Related Hepatocellular Carcinoma and Overexpression of AXIN1 Induces Apoptosis in Hepatocellular Cancer Cells

Axis inhibition protein 1 (AXIN1) is a negative regulator of Wnt/beta-catenin signaling via regulating the level of beta-catenin. However, the role of AXIN1 in the tumorigenesis and progression of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is less clear. PCR sequence analysis, immunohistochemistry, and Western blot were performed on 22 HBV-related HCC samples and corresponding nontumor liver tissues to detect variants in AXIN1 gene and the expression level of AXIN1. Human hepatoma cell lines SNU475 and SNU423 were transfected with pCDNA3.1-AXIN1-myc or AXIN1 G425S-myc mutant. The growth curve and apoptosis rate of cell lines, phosphorylation of beta-catenin, and cell cycle regulatory proteins depending on beta-catenin transcriptional activity were detected. We identified four mutations of AXIN1 in 22 primary HBV-related HCCs and demonstrated a lower expression of AXIN1 in HBV-related HCC tissues than that in paired adjacent nontumor tissues. Overexpression of AXIN1 wild-type but not AXIN1 mutant inhibited the growth of HCC cell lines, accelerated their apoptosis, and negatively regulated beta-catenin-dependent transcriptional activity. Our study revealed that alterations of AXIN1 were involved in HBV-related HCC. Overexpression of AXIN1 but not AXIN1 mutant negatively regulated beta-catenin-dependent transcriptional activity and downregulated the level of cell cycle regulatory proteins, suggesting that AXIN1 may be a potential target for gene therapy of primary HCC.

Prognostic value of centromere protein-A expression in patients with epithelial ovarian cancer

Altered expression of centromere protein-A (CENP-A) is observed in various types of human cancers. However, the clinical significance and pathological role of CENP-A in epithelial ovarian cancer (EOC) remains unclear. The main objective of this investigation was to clarify the relationships between CENP-A expression and the clinicopathological features of patients with EOC. Real-time quantitative PCR and Western blot were performed to examine CENP-A expression in 20 pairs of fresh-frozen EOC tissues and corresponding noncancerous tissues. Using immunohistochemistry, we performed a retrospective study of the CENP-A expression levels on 120 archival EOC paraffin-embedded samples. Prognostic outcomes correlated with CENP-A were examined using Kaplan-Meier analysis and Cox proportional hazards model. Our results showed that the expression levels of CENP-A mRNA and protein in EOC tissues were both significantly higher than those in noncancerous tissues. By immunohistochemistry, the data revealed that high CENP-A expression was significantly correlated with pathological grade (P = 0.02) and International Federation of Gynecology and Obstetrics stage (P = 0.006). Consistent with these results, we found that high expression of CENP-A was significantly correlated with poor survival in EOC patients (P < 0.001). Furthermore, Cox regression analyses showed that CENP-A expression was an independent predictor of overall survival. Our data suggest that CENP-A could play an important role in EOC and might serve as a valuable prognostic marker and potential target for gene therapy in the treatment of EOC.

Low expression of LOC285194 is associated with poor prognosis in colorectal cancer

BackgroundThe long non-coding RNAs (lncRNAs) study has gradually become one of the hot topics in the field of RNA biology. One lncRNA which has attracted attention is LOC285194, a lncRNA demonstrated the potential tumor-suppressor role in osteosarcoma. The aim of this study was to examine the expression of LOC285194 in colorectal cancer (CRC) patients and to investigate the relationship between this lncRNA levels and existing clinicopathologic parameters and patient survival.MethodsThe expression of LOC285194 was detected by quantitative real-time polymerase chain reaction in pairs of tumorous and adjacent normal tissues of 81 colorectal cancer patients with a follow-up of 5 years, as well as in three colorectal cancer cell lines and normal intestinal mucous cell line. Then, we analyzed the potential relationship between this lncRNA levels in tumor tissues and existing clinicopathological features of CRC, and clinical outcome.ResultsThe relative expression levels of LOC285194 was significantly lower in tumor tissues (p < 0.001) and colorectal cancer cell lines compared with adjacent normal tissues and normal intestinal mucous cell line. In addition, low expression of LOC285194 was correlated with larger tumor size (p = 0.015), higher tumor stage (p = 0.034), and more distant metastasis (p = 0.046). Kaplan-Meier analysis indicated that patients with low LOC285194 expression had a poor disease free survival (p = 0.010). Moreover, multivariate analysis showed that decreased expression of LOC285194 was an independent predictor of disease-specific survival.ConclusionOur data indicate that LOC285194 might be a novel prognostic indicator in colorectal cancer and may be a potential target for diagnosis and gene therapy.

Investigation of the relationship between chromobox homolog 8 and nucleus pulposus cells degeneration in rat intervertebral disc

Here, we aimed to investigate the expression of chromobox homolog 8 (CBX8) in nucleus pulposus (NP) cells from rat intervertebral disc (IVD) and its function in DNA damage and repair. NP cells were isolated from healthy rat IVD for immunohistochemistry staining. Small interfering RNA (siRNA) of CBX8 was applied for gene silencing, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine mRNA levels of CBX8, type II collagen, and proteoglycans. Cell proliferation and cell cycle were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming assay, and flow cytometry. Hydrogen peroxide (H2O2) was added to simulate DNA oxidative damage, and expression of CBX8 was examined using RT-PCR and Western blot. After five passages, mRNA levels of type II collagen and proteoglycans decreased but that of CBX8 increased. When CBX8 was silenced by siRNA, the expressions of CBX8, type II collagen and proteoglycans declined, and the cell growth was inhibited. Besides, cell cycle was slowed down as most cells were arrest in G0/G1 phase. Furthermore, CBX8 expression went up responding to DNA oxidative damage caused by H2O2. The data indicated that CBX8 plays important roles in cell proliferation and DNA damage. Cell proliferation and cell cycle were stimulated by CBX8, which may be associated with INK4A-ARF pathway. Moreover, CBX8 plays a role in DNA damage which made it a potential gene therapy target for treatment of disc degeneration.

Knockdown of Phospholipase C-Epsilon by Short-Hairpin RNA-Mediated Gene Silencing Induces Apoptosis in Human Bladder Cancer Cell Lines

Transitional cell carcinoma of bladder (TCCB) is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. To study the pathogenesis of TCCB, we investigated roles of Phospholipase C (PLC)ɛ, an effector of Ras and Rap small GTPases. RNA interference was used to knockdown PLCɛ expression in human bladder cancer cell lines (BIU-87 and T24). The expression levels of PLCɛ mRNA and protein were detected by reverse transcriptase-polymerase chain reaction and Western blot, respectively. Flow cytometry (FCM) was used to detect distribution of cell cycle. Cellular apoptosis was reflected by transmission electron microscopy and the expression of bcl-2 and bax. We found that PLCɛ could be efficiently knocked down by shRNA. FCM assay showed that the pGenesil-PLCɛ-transfected cells were arrested at the G0/G1 phase. Silence of PLCɛ might induce apoptosis via modulation of bcl-2 and bax. In conclusion, our results suggest that PLCɛ plays an important role in the pathogenesis of human bladder cancer cells. PLCɛ may be used as a potential target of gene therapy for bladder cancer in future.

Co-overexpression of cortactin and CRKII increases migration and invasive potential in oral squamous cell carcinoma

Cortactin stimulates cell migration, invasion, and experimental metastasis. Overexpression of cortactin has been reported in several human cancers. CRK was originally identified as an oncogene product of v-CRK in a CT10 chicken retrovirus system. Overexpression of CRKII has been reported in several human cancers. CRKII regulates cell migration, morphogenesis, invasion, phagocytosis, and survival; however, the underlying mechanisms are not well understood. We evaluated the possibility of the combination of cortactin and CRKII as an appropriate molecular target for cancer gene therapy. The expression of cortactin and CRKII in 70 primary oral squamous cell carcinomas and 10 normal oral mucosal specimens was determined immunohistochemically, and the correlation of cortactin and CRKII co-overexpression with clinicopathological factors was evaluated. Co-overexpression of cortactin and CRKII was detected in 31 of 70 oral squamous cell carcinomas, the frequency being significantly greater than in normal oral mucosa. In addition, cortactin and CRKII co-overexpression was more frequent in higher-grade cancers according to the T classification, N classification, and invasive pattern. RNAi-mediated co-suppression of cortactin and CRKII expression reduced the migration and invasion potential of an oral squamous cell carcinoma cell line, OSC20. Downregulation of cortactin and CRKII expression also reduced the expression of vimentin, fibronectin, and N-cadherin. These results indicate that the co-overexpression of cortactin and CRKII may be tightly associated with an aggressive phenotype of oral squamous cell carcinoma. Therefore, we propose that the combination of cortactin and CRKII could be a potential molecular target of gene therapy by RNAi-targeting in oral squamous cell carcinoma.

Effect of GINS2 on Proliferation and Apoptosis in Leukemic Cell Line

Although previous researches have demonstrated that GINS2 express abundantly and abnormally in many malignant solid tumors, such as breast cancer, melanoma and hepatic carcinoma. However, the role and precise molecular mechanism in acute promyelocytic leukemia (APL) are rarely reported. In this current study, we investigated the possible effect and particular mechanism of GINS2 in occurrence and development of APL. We synthesized interference plasmid targeted GINS2 successfully in vitro and also constructed recombinant adenovirus vector carrying GINS2 gene in order to down-regulate or up-regulate GINS2 expression from two aspects of positive and negative in APL. After siRNA were transfected into HL60 cells, both GINS2 expression level of mRNA and protein in interfering group were down-regulated when compared with control groups. Together, MTT and flow cytometry technology showed that cell growth was significantly inhibited. Moreover, the expression lever of Bax was distinctly increased whereas Bcl2 was dramatically decreased in transfected group. Further experiments revealed that down-regulation of GINS2 expression inhibited DNA replication and had a G2/M phase block in HL60 cells. What's more, ATM, CHK2, and P53 gene could involve in the pathogenic signaling pathways of HL60 cells when GINS2 gene was down-regulated. On the contrary, after HL60 cells were infected by recombinant adenovirus vector which contained GINS2 gene, we observed that over-expression of GINS2 could promote HL-60 cell proliferation. What's more, GINS2 might implicate a potential target for leukemia gene therapy.

Understanding velocardiofacial syndrome

Improved recognition of velocardiofacial syndrome (VCFS) has led to increasing awareness of VCFS by otolaryngologists. Understanding the developmental biologic processes affected in VCFS patients will help improve treatment and outcomes. Advanced application of molecular labeling techniques has better outlined the role of T-Box transcription factor 1 (TBX1) as the primary genetic anomaly leading to VCFS. TBX1 plays multiple roles during branchial, cardiac, and craniofacial development and increased understanding of how these systems are affected by TBX1 mutations will improve patient outcomes. Furthermore, additional modifiers of TBX1 expression have been identified that may explain the variability of VCFS phenotypes. The phenotypic spectrum of VCFS may include cardiac anomalies, velopharyngeal insufficiency, aberrant calcium metabolism, and immune dysfunction. Recent interest has focused on the cognitive and neuropsychiatric manifestations of VCFS. Improved understanding of the biology of VCFS associated mutations has the potential to improve therapeutic outcomes. This article will discuss recent developmental biologic understanding of the role of TBX1 and genetic modifiers generating the phenotypic variability seen in VCFS patients. Special attention is given to advances in the realms of immunodeficiency, hypocalcemia, cardiac and arterial patterning anomalies, velopharyngeal insufficiency, as well as cognitive and psychiatric problems. Enhanced understanding of the multiple systems affected by TBX1 mutations will result in improved patient outcomes and improved family education. Future research will lead to improved detection of potential targets for gene therapy and change the way physicians counsel families and treat patients.

Suppression of IGHG1 gene expression by siRNA leads to growth inhibition and apoptosis induction in human prostate cancer cell

To investigate the immunoglobulin G (IgG) expression in prostate cancer cell lines and explore the effects of IGHG1 gene knockdown on PC3 cell growth and apoptosis. Flow cytometry, qPCR and western blot were used to demonstrate IgG expression in prostate cancer cell lines. PC3 cells were transfected with designed siRNA, the expression of IgG was determined by qPCR and western blot, the proliferation and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt (MTS) and flow cytometry. The percentages of IgG in LNCaP cell membrane and cytoplasm were 2.96 and 89.22 % by flow cytometer, those of PC3 cell were 86.73 and 90.99 % respectively. The average level of IgG1 mRNA expression in PC3 cell line was significantly higher than that in LNCaP cell line (3.08 ± 0.15 vs 1.00 ± 0.37, P = 0.001). The protein level of IgG expression of PC3 cell line was 1.92 ± 0.15, compared with LNCaP cell line (1.05 ± 0.86). The expression of IgG1 mRNA and protein level in transfected PC3 cells decreased, with significant statistical differences from the blank control group (P < 0.01). The PC3 cell growth inhibition rates were 31.3 and 43.3 % in 48 and 72 h respectively. The rate of apoptotic PC3 cells were 5.29 ± 0.41 % in experimental group higher than that in control group (1.49 ± 0.29 %) (P < 0.01). IgG was identified in prostate cancer cells, and the siRNA targeted silencing of IGHG1 can inhibit cell viability and promote apoptosis, which might therefore act as a potential target in prostate cancer gene therapy.

Novel Possible Pharmaceutical Research Tools: Stem Cells, Gene Delivery and their Combination

Both stem cell research and gene delivery are very promising fields of today's biomedical research. In the present review we first attempt to summarize the state of the art in stem cell research. We describe the major categories of stem cells based on cell sources: embryonic, fetal, postnatal and induced pluripotent stem cells. We then present new data on stem cell cultures of dental pulp origin as examples of the progress of postnatal stem cell research. Afterwards, we briefly summarize the most promising achievements in the field of gene delivery. As an example of such advances, we describe novel in vitro and in vivo gene delivery studies to demonstrate that salivary glands are highly potential targets for gene therapy: they can be used to produce therapeutic peptides delivered either into the oral cavity or into the systemic circulation. Finally, we describe and compare studies combining the use of stem cells and gene delivery. We conclude that stem cell therapy and gene delivery alone are both very exciting research areas, and they may act in synergy when used in combination.

Novel Possible Pharmaceutical Research Tools: Stem Cells, Gene Delivery and their Combination

Both stem cell research and gene delivery are very promising fields of today’s biomedical research. In the present review we first attempt to summarize the state of the art in stem cell research. We describe the major categories of stem cells based on cell sources: embryonic, fetal, postnatal and induced pluripotent stem cells. We then present new data on stem cell cultures of dental pulp origin as examples of the progress of postnatal stem cell research. Afterwards, we briefly summarize the most promising achievements in the field of gene delivery. As an example of such advances, we describe novel in vitro and in vivo gene delivery studies to demonstrate that salivary glands are highly potential targets for gene therapy: they can be used to produce therapeutic peptides delivered either into the oral cavity or into the systemic circulation. Finally, we describe and compare studies combining the use of stem cells and gene delivery. We conclude that stem cell therapy and gene delivery alone are both very exciting research areas, and they may act in synergy when used in combination.

Growth inhibition and apoptosis of human B-cell lymphoma in vitro and in vivo by Bcl-2 short hairpin RNA

Bcl-2 is overexpressed in various types of human tumors, including Burkitt's lymphoma, and it is involved in tumorigenesis and chemoresistance, therefore, it is regarded as a potential target of gene therapy. In this study, RNA interference using short hairpin RNA (shRNA)-mediated RNA interference was introduced into Burkitt's lymphoma Raji cells to validate its effects on Bcl-2 expression and cell proliferation invitro and invivo. We constructed two types of Bcl-2 shRNA plasmid (pGenesil-1-Bcl-2-1 and pGenesil-1-Bcl-2-2) and negative control shRNA plasmid (pGenesil-1-NC) and stably transfected them into Raji cells. The expression levels of Bcl-2 mRNA and protein were assayed by RT-PCR, flow cytometry and western blotting. Cell proliferation was determined by cell count assay. The antitumor activities and apoptosis of the two types of Bcl-2 shRNA plasmid were evaluated in BALB/c nude mice bearing Burkitt's lymphoma inoculated with Raji cells. The results showed that the expression levels of Bcl-2 mRNA and protein decreased, compared with either the pGenecil-1-NC or the untransfected cell group (P<0.05). The cell proliferation assay showed that Bcl-2 shRNA significantly inhibited the growth of Raji cells (P<0.01). Furthermore, the tumor growth of the Bcl-2 shRNA cell group was dramatically lower and smaller than that of the negative control or untransfected cell group (P<0.01). Bcl-2 protein expression in the untransfected and the pGenesil-1-NC group were markedly higher than that of the pGenesil-1-Bcl-2-1 and the pGenesil-1-Bcl-2 group by immunohistochemistry (both P<0.01) and the results using transmission electron microscopy showed that Bcl-2 shRNA significantly induced Raji cell apoptosis. Additionally, the inhibition effect of pGenesil-1-Bcl-2-1 was better than that of pGenesil-1-Bcl-2-2. It has been suggested that vector-based Bcl-2 shRNA could effectively reduce the expression of Bcl-2 and induce apoptosis and growth inhibition of Burkitt's lymphoma Raji cells. Vector-based Bcl-2 shRNA could be a potential gene therapeutic strategy against human Burkitt's lymphoma.

Decreased Expression of MicroRNA-375 in Nonsmall Cell Lung Cancer and its Clinical Significance

Emerging evidence has shown the association of aberrant microRNA-375 (miR-375) expression with tumourigenesis in many types of human malignancy. This prospective study characterized the contribution of miR-375 to the initiation and progression of nonsmall cell lung cancer (NSCLC). The real-time reverse transcription-polymerase chain reaction was used to examine miR-375 levels prospectively in 96 pairs of samples of NSCLC tissue and adjacent noncancerous tissue (> 2 cm from cancer tissue). The relationship between miR-375 levels and clinico pathological features was also explored. MiR-375 was downregulated in 89% (85/96) of NSCLC samples compared with matched noncancerous tissue samples. Decreased miR-375 correlated significantly with advanced disease stage and lymphatic metastasis. Univariate and multivariate Cox proportional hazard regression analyses revealed that underexpression of miR-375 was an unfavourable prognostic factor for overall survival in NSCLC. This study suggested that miR-375 is a novel prognostic indicator in NSCLC and might be a potential target for diagnosis and gene therapy.

Silencing phospholipid scramblase 1 expression by RNA interference in colorectal cancer and metastatic liver cancer

Phospholipid scramblase 1 (PLSCR1) not only participates in the transbilayer movement of phospholipids, but also plays a role in the pathogenesis and progression of cancers. The present study aimed to evaluate the effect of silencing PLSCR1 expression by RNA interference in colorectal cancer (CRC) and metastatic liver cancer. The expression of PLSCR1 in CRC and metastatic liver cancer samples was assessed by immunohistochemistry. The cultured cells with the highest expression were selected for subsequent experiments. We designed three siRNA oligonucleotide segments targeted at PLSCR1. Successful transfection was confirmed. The biological behavior of the cells in proliferation, adhesion, migration and invasion was determined. PLSCR1 protein expression increased significantly in the majority of CRC and metastatic liver cancer samples compared with normal samples. Lovo cells had the highest expression of PLSCR1. The siRNA-390 oligonucleotide segment had the best silencing effect. After transfection, Lovo cell proliferation was significantly inhibited compared with the controls in the MTT assay. Laminin and fibronectin adhesion assays showed Lovo cell adhesion was also significantly inhibited. In the migration assay, the number of migrating cells in the PLSCR1 siRNA-390 group was 50+/-12, significantly lower than the number in the siRNA-N group (115+/-28) and in the control group (118+/-31). In an invasion test, the number of invading cells in the PLSCR1 siRNA-390 group was 60+/-18, significantly lower than that in the siRNA-N group (97+/-26) and the control group (103+/-24). PLSCR1 is overexpressed in CRC and metastatic liver cancer. Silencing of PLSCR1 by siRNA inhibits the proliferation, adhesion, migration and invasion of Lovo cells, which suggests that PLSCR1 contributes to the tumorigenesis and tumor progression of CRC. PLSCR1 may be a potential gene therapy target for CRC and associated metastatic liver cancer.

Decreased Expression of the ARID1A Gene Is Associated with Poor Prognosis in Primary Gastric Cancer

BackgroundThe ARID1A gene encodes adenine-thymine (AT)-rich interactive domain-containing protein 1A, which participates in chromatin remodeling. ARID1A has been showed to function as a tumor suppressor in various cancer types. In the current study, we investigated the expression and prognosis value of ARID1A in primary gastric cancer. Meanwhile, the biological role of ARID1A was further investigated using cell model in vitro.Methodology/Principal FindingsTo investigate the role of ARID1A gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were used to examine the ARID1A expression in paired cancerous and noncancerous tissues. Results revealed decreased ARID1A mRNA (P = 0.0029) and protein (P = 0.0015) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues, and in gastric cancer cell lines. To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Data revealed that the loss of ARID1A expression was significantly correlated with T stage (P = 0.001) and grade (P = 0.006). Consistent with these results, we found that loss of ARID1A expression was significantly correlated with poor survival in gastric cancer patients (P = 0.003). Cox regression analyses showed that ARID1A expression was an independent predictor of overall survival (P = 0.029). Furthermore, the functions of ARID1A in the proliferation and colony formation of gastric cell lines were analyzed by transfecting cells with full-length ARID1A expression vector or siRNA targeting ARID1A. Restoring ARID1A expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing ARID1A expression in gastric epithelial cell line significantly enhanced cell growth rate.Conclusions/SignificanceOur data suggest that ARID1A may play an important role in gastric cancer and may serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.

Inhibitory Effect of p53 Upregulated Modulator of Apoptosis Targeting siRNA on Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis in Rats

Objectives: The effect of p53 upregulated modulator of apoptosis (PUMA) in hypoxia/reoxygenation-induced cardiomyocyte injuries in rats was investigated. Methods: PUMA-targeting (si-PUMA) and scramble siRNAs were designed and transfected into primarily rat cardiomyocytes in vitro. Results: RT-PCR and Western blot analysis showed that 50 nmol/l of si-PUMA can specifically inhibit PUMA expression. MTT assay and lactate dehydrogenase activity detection showed that the cell survival rate in the si-PUMA group was enhanced and that the lactate dehydrogenase enzymatic activity dramatically decreased compared with the control group (p < 0.01). Spectrophotometry, as well as annexin V and propidium iodide staining, combined with flow cytometry, revealed that caspase-3 activity in the si-PUMA group was downregulated and the apoptotic rate was decreased (p < 0.01). RT-PCR also showed that Bax expression was downregulated and Bcl-2 expression was upregulated in the si-PUMA group, compared with the control group (p < 0.05). si-PUMA protects cardiomyocytes from apoptosis. Conclusion: PUMA mediates hypoxia/reoxygenation-induced cardiomyocyte apoptosis, which can be a potential target of gene therapy for ischemia/reperfusion cardiomyocyte injuries.

Inhibition of autocrine IGF-II on effect of human HepG2 cell proliferation and angiogenesis factor expression

Abnormal expression of insulin-like growth factor II (IGF-II) is associated with the hepatocyte malignant transformation and hepatocellular carcinoma (HCC) progress. In this study, specific IGF-II miRNA plasmids were constructed and transfected to HepG2 cells to knockdown IGF-II expression for observing effects on the cell proliferation, survival, apoptosis, angiogenesis, and anchorage-independent colony formation. IGF-II mRNA was evaluated by quantitative real-time polymerase chain reaction, and the level of IGF-II or vascular endothelial growth factor (VEGF) was quantitatively analyzed by ELISA. Our data shown that downregulation of IGF-II expression resulted in the viability alteration, proliferation inhibition, and apoptosis occurrence of HepG2 cells. The level of VEGF expression in the supernatant of HepG2 cells in the IGF-II-miRNA-transfected group was significantly decreasing (P < 0.01) than those in the untransfected group or the miRNA-neg-transfected group, with the susceptibility to anoikis and decreasing of anchorage-independent colony formation of HepG2 cells. Thus, we conclude that IGF-II is a potential molecular target for HCC gene therapy.

Abstract 3917: Regulatory role of ROBO-1, a novel tumor suppressor on Androgen receptor and Wnt signaling during castration-resistant prostate cancer development: A novel molecular target for gene therapy

Abstract Introduction and Objectives: Prostatic epithelial cells while progressing from premalignant stages to malignant stages (hormone-sensitive and castration-resistance phenotypes) undergo a lot of phenotypical changes which include increased clonal expansion; and modulation in cytoskeleton and cell adhesion characteristics. During organogenesis, the Roundabout-1 (ROBO-1) regulates cell proliferation, migration and adhesion. We for the first time (1) established the mechanismαbased role of ROBO1 tumor suppressor gene in the development of castrationαresistant CaP (CRPC) and (2) determined its significance as potential target for gene therapy. Methods: Immunoblot analysis, real time PCR and immunohistochemistry was used to analyze human prostatic tissues and cell lines for ROBO1. ROBOα1 was (i) overαexpressed by transfecting ROBO1αexpressing plasmid (ii) knocked down by employing vectorαbased shRNA. ROBO1αsuppressed and ROBO1αoverexpressing cells were tested for proliferation, clonogenicity, sensitivity towards chemotherapeutic agents, androgen receptor (AR)α binding to target genes, AR and Tcfαtranscriptional activities by employing 3[H]thymidine uptake, softαagar, luciferase reporter and ChIP assays. Results: We provide evidence that mRNA and protein levels of ROBO1 are lost in (i) CRPC cells, ii) prostatic tissues of NKx3.1/Pten mouse model and (ii) human prostatic tissues during the progression of disease from early stage (low) to late stage (highαGleason score). We show that Loss of ROBO-1 was observed to increase (i) AR transcriptional activity, (ii)Tcf-transcriptional activity (marker of Wnt signaling) (iii) accumulation of β-catenin and (iv) Akt activity and (v) proliferation of CRPC cells. Importantly, CRPC cells (which survived therapy) exhibited high potential to proliferate after repopulation when ROBO-1 was silenced. We provide compelling evidence that under ablation conditions ROBO1 is lost in some malignant CaP cells which results in a subtype that is castration-resistant. We show that ROBO-1 is inactivated due to epigenetic changes in CRPC cells under castration-conditions. ROBO-1-deficient CRPC cells were more chemoresistant and exhibited increased AR-activity than ROBO-1-expressing counterparts. ROBO1αexpressing CRPC cells exhibited reduced tumorigenicity as compared to ROBO-1-silenced CRPC cells. Finally, reactivation of ROBO-1 by gene therapy resulted in decreased AR/Wnt signaling and sensitization of CRPC cells to chemotherapy. Conclusions: We suggest that inactivation of ROBO1 during androgen ablation treatment could be one of the possible mechanisms involved in the recurrence of disease in CaP patients. Therefore ROBO-1 offers a potential to be exploited as a molecular target for gene therapy to treat CRPC condition in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3917. doi:1538-7445.AM2012-3917

Increased Expression of MicroRNA-221 in Gastric Cancer and Its Clinical Significance

MicroRNA-221 (miR-221), which is located on chromosome X, is an oncogenic microRNA that is involved in several human malignancies. This study aimed to characterize the role of miR-221 in the initiation and malignant progression of gastric cancer. The level of miR-221 expression in 92 pairs of primary gastric tumour tissue and adjacent nontumour tissue was quantified by real-time reverse transcription-polymerase chain reaction. The relationship between the miR-221 level and clinicopathological features was explored. MiR-221 was up-regulated in 88% (81/92) of gastric cancer tissue samples compared with their paired adjacent nontumour tissue samples. High expression of miR-221 showed a significant correlation with advanced tumour-node-metastasis stage, local invasion and lymphatic metastasis. Univariate and multivariate Cox proportional hazard regression analyses revealed that miR-221 overexpression was an unfavourable prognostic factor for overall survival in patients with gastric cancer. These findings suggest that miR-221 is a novel prognostic indicator in gastric cancer and may be a potential target for diagnosis and gene therapy.

In vitro Study of Nucleostemin Gene as a Potential Therapeutic Target for Human Lung Carcinoma

ObjectiveNucleostemin (NS) is a GTP-conjugated protein located in the nucleoli of stem cells and some cancer cells, and maintains cell self-renewal. We aimed to evaluate NS as a potential target for lung carcinoma gene therapy by investigating NS gene expression and its effect on A549 cell proliferation. MethodsNS mRNA and protein expression in A549, HepG2, SMMC-7721, HeLa, and U251 cells was analyzed by RT-PCR and western blotting following transfection of NS siRNAs and negative control siRNA (NC). The effect on cell proliferation was also analyzed by MTT assays. ResultsNS mRNA and protein were both expressed in A549 cells and four other tumor cell lines; the relative expression levels were similar in all five cell lines. The three pairs of NS siRNA, either transfected alone or cotransfected into A549 cells, could effectively inhibit the expression of NS mRNA and protein. Moreover, the interference ratio showed an obvious concentration-dependent relationship. NS siRNA treatment resulted in significant inhibition of A549 cell proliferation by 35.7%. ConclusionNS gene was not only highly expressed but also played an important role in A549 cell proliferation. Thus, targeting of NS may be a promising novel strategy for the treatment of lung carcinoma.