Animals ; Humans ; MicroRNAs ; genetics ; Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Neoplasm ; genetics ; RNA, Small Interfering ; genetics

Alternative splicing of pre-mRNA is an important mechanism for regulating gene function at the post-transcription level and for producing proteomic diversity in higher eukaryotes. The alternative splicing is regulated by the interaction between diverse cis-acting elements and trans-acting factors. Alternative splicing events of oncogenes, tumor suppressor genes and metastasis suppressor genes are associated with the initiation and development of human neoplasms. The protein isoforms sourced from alternative splicing take part in regulating the gene transcription, cell cycle, apoptosis of cells, and playing a role in tumor growth. It is possible for molecular therapy to target directly isoforms of protein produced by alternative splicing or to interfere with the process of alternative splicing.
Alternative Splicing ; genetics ; Humans ; Neoplasms ; genetics ; RNA Precursors ; metabolism ; RNA, Neoplasm ; analysis ; Transcription, Genetic

OBJECTIVETo detect tumor cells in the peripheral blood of patients with hepatocellular carcinoma (HCC) by using the mRNA of the MAGE-1 and MAGE-3 genes as specific tumor markers.

METHODSPeripheral blood was obtained from 25 HCC patients and 20 healthy volunteers. The mRNA of the MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR. The MAGE-1 and MAGE-3 transcripts in the tumor tissues of these HCC patients were also detected by RT-PCR.

RESULTSOf the 25 HCC patients, MAGE-1 and MAGE-3 mRNA were positive in 44% (11/25) and 36% (9/25) of PBMCs respectively, and in 68% (17/25) and 56% (14/25) of HCC tissues respectively. In the PBMCs of the 25 HCC patients, 16 (64%) samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMCs from the patients whose tumors did not express the MAGE genes, nor in the PBMCs from the 20 healthy donors. The positive rate of MAGE mRNA in the PBMCs was closely correlated with the TNM stages and the diameter of tumors, but there was no correlation between the positive rate of MAGE mRNA in PBMCs and tumor differentiation degree or serum alpha-FP level. Of 9 HCC patients whose serum alpha-FP was normal or slightly elevated (< 50 ng/ml), 6 were MAGE-1 and/or MAGE-3 mRNA positive in their PBMCs.

CONCLUSIONMAGE-1 and MAGE-3 mRNA could be specifically detected with high percentage in the PBMCs of HCC patients by our method. They can be used as specific tumor markers for the detection of the circulating HCC cells, and the detection results may be helpful to evaluate the prognosis of HCC patients.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; Humans ; Leukocytes, Mononuclear ; Liver Neoplasms ; genetics ; Melanoma-Specific Antigens ; Neoplasm Proteins ; RNA, Messenger ; genetics

Objective To investigate the effects of microRNA-18a (miR-18a) on migration and invasion of hepatocellular carcinoma (HCC) cells, and its possible mechanism associated with Dicer l.Methods HepG2 and HepG2.2.15 cells were transfected with miR-18a inhibitor using Lipofectamine. Cell invasion was evaluated by transwell invasion assay, and cell migration was detected by transwell migration and wound-healing assays. Moreover, luciferase reporter assay was used to identify whether Dicer expression was regulated by miR-18a. Real-time RT-PCR and western blot were performed to analyze Dicer 1 expression. In addition, a functional restoration assay was performed to investigate whether miR-18a promotes HCC cell migration and invasion by directly targeting Dicer 1.Results miR-18a inhibitor can suppress the migration and invasion of HCC cells. Furthermore, suppression of Dicer l expression by small interfering RNA essentially abolished the inhibition of cell migration and invasion induced by miR-18a inhibitor, restorating these activities to levels similar to the parental HCC cells. Interestingly, suppression of miR-18a in HCC cells resulted in enhanced expression of Dicer l. In addition, the results of a luciferase assay demonstrated targeted regulation of Dicer l by miR-18a.Conclusion Our findings suggest that miR-18a promotes migration and invasion of HCC cells by inhibiting Dicer l expression.
Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Movement ; DEAD-box RNA Helicases ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; MicroRNAs ; genetics ; metabolism ; Neoplasm Invasiveness ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Neoplasm ; genetics ; metabolism ; Ribonuclease III ; genetics ; metabolism

OBJECTIVEThe aim of this review was to assess RNA interference (RNAi) and its possibility as a potential and powerful tool to develop highly specific double-stranded RNA (dsRNA) or small interfering RNA (siRNA) based gene-silencing therapeutics.

DATA SOURCESThe data used in this review were obtained from the current RNAi-related research reports.

STUDY SELECTIONdsRNA-mediated RNAi has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides siRNAs trigger gene-specific silencing in mammalian cells has further expanded the utility of RNAi in to the mammalian system.

DATA EXTRACTIONThe currently published papers reporting the discovery and mechanism of RNAi phenomena and application of RNAi on gene function in mammalian cells were included.

DATA SYNTHESISSince the recent development of RNAi technology in the mammalian system, investigators have used RNAi to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous siRNA or siRNA expressing vector. The general and sequence-specific inhibitory effects of RNAi that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality.

CONCLUSIONSIt is certain that the ability of RNAi in mammals to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that RNAi might one day be used to treat human diseases.

Animals ; Antigens, Neoplasm ; Gene Silencing ; Genes, abl ; Genetic Therapy ; Humans ; Neoplasm Proteins ; genetics ; RNA Interference

OBJECTIVETo explore the possibility of MAGE-4 gene encoding protein used as a target for immunotherapy in HCC patients.

METHODSThe expression of MAGE-4 gene in tumor tissues and tumor adjacent non-HCC liver tissues was examined by the RT-PCR method. The relationship between positive expression rate of MAGE-4 gene and other clinical and lab data including AFP, AFU, anti-HCV, HBsAg, AFP mRNA, and the diameter of the tumors in HCC patients was also determined.

RESULTSThe positive expression rate of MAGE-4 gene was significantly higher in the tumor than in tumor surrounding tissues (38.7% vs 0%, P<0.01), while the positive expression rate of MAGE-4 gene had no relationship with the clinical and lab data (P>0.05).

CONCLUSIONSThe high frequency of MAGE-4 gene expression in HCC suggests the possibility of MAGE-4 gene encoding protein as a target for immunotherapy in HCC patients, but the expression has no relationship with the tumor metastasis and the recurrence of HCC.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; pathology ; Neoplasm Proteins ; genetics ; RNA, Neoplasm ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo silence annexin II gene expression by using small interference RNA (siRNA) in prostate cancer cell line PC3.

METHODSFor in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin II gene. The other pair was negative control. The 8 nucleotides at the 3' end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin II protein and its mRNA. 3H thymidine was used to measure DNA synthesis.

RESULTSThe siRNA sequence specific to annexin II gene was capable of inhibiting the expression of annexin II protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.

CONCLUSIONSThe protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin II might be involved in DNA synthesis.

Annexin A2 ; genetics ; Cell Line, Tumor ; DNA Replication ; DNA, Neoplasm ; genetics ; Humans ; Male ; Promoter Regions, Genetic ; genetics ; Prostatic Neoplasms ; genetics ; RNA Interference ; RNA, Neoplasm ; genetics ; RNA, Small Interfering ; genetics ; Transcription, Genetic

A trans-splicing ribozyme which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful agent for tumor-targeted gene therapy. In this study, we evaluated in vivo function of the hTERT-targeting trans-splicing ribozymes by employing the molecular analysis of expression level of genes affected by the ribozyme delivery into peritoneal carcinomatosis mice model. To this effect, we constructed adenoviral vector encoding the specific ribozyme. Noticeably, more than four-fold reduction in the level of hTERT RNA was observed in tumor nodules by the systemic infection of the ribozyme-encoding virus. Such hTERT RNA knockdown in vivo induced changes in the global gene expression profile, including the suppression of specific genes associated with anti-apoptosis including bcl2, and genes for angiogenesis and metastasis. In addition, specific trans-splicing reaction with the targeted hTERT RNA took place in the tumors established as peritoneal carcinomatosis in mice by systemic delivery of the ribozyme. In conclusion, this study demonstrates that an hTERT-specific RNA replacement approach using trans-splicing ribozyme represents a potential modality to treat cancer.
Animals ; Cell Line ; Gene Expression/*physiology ; Genetic Vectors ; Humans ; Mice ; Neoplasm Metastasis ; Neoplasms/genetics/pathology ; RNA, Catalytic/genetics/*metabolism ; RNA, Messenger/genetics/metabolism ; RNA, Neoplasm/genetics/metabolism ; Telomerase/antagonists & inhibitors/genetics/*metabolism ; Trans-Splicing/*genetics

OBJECTIVETo explore the association of FMNL2 expression with the metastatic potential of colorectal cancer cells.

METHODSFMNL2 mRNA and protein expressions in 6 human colorectal cancer cell lines were detected by real-time RT-PCR and immunohistochemical method, respectively, and analyzed for their correlations to the in vitro invasiveness of the cell lines evaluated by Boyden assay. In SW620 and SW480/M5 cell lines, the expression of FMNL2 was repressed by FMNL2 short hairpin RNA (shRNA), and the changes in the invasiveness of the cells were observed.

RESULTSFMNL2 was highly expressed in SW480/M5, LoVo and SW620 cells derived from metastatic colorectal cancers in comparison with that in LS174T, SW480 and HT29 cells, which were derived from primary colorectal cancers. In vitro analysis of the cell invasiveness demonstrated that SW480/M5, LoVo and SW620 cells had higher invasiveness than LS174T, SW480 and HT29 in vitro. In SW480/M5 and SW620 cells, transfection with FMNL2 shRNA resulted in significantly lowered cell invasiveness.

CONCLUSIONFMNL2 may play an important role in the invasion and metastasis of colorectal cancer.

Colorectal Neoplasms ; metabolism ; pathology ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Proteins ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Tumor Cells, Cultured

Apoptosis ; Cell Proliferation ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; RNA Interference ; Securin