胰RNA对瘤细胞作用的研究——胰RNA对离体瘤细胞的杀伤作用及其有效组分的初步探讨

在体外培养瘤细胞的实验中,胰RNA提取物不仅对艾氏腹水癌细胞而且对其他瘤细胞(S_(180)、P_(388)、H_(22))也具有杀伤作用。在胰RNA提取物中,对瘤细胞具有杀伤作用的活性物质不是外源性凝集素样多糖类或能透过半透膜的低分子化合物,而是具有一定二级结构的低分子RNA。

胰RNA对瘤细胞作用的研究——抑制动物体内实体瘤及提高细胞免疫的机能

^(125)Ⅰ-胰RNA能参入体外培养的瘤细咆中抑制DNA合成因而导致瘤细胞死亡。在以上实验基础上,我们又在动物体内注射胰RNA,观察对三种(S_(180)、EC、P_(388))带瘤小鼠的影响,其结果:(1)抑瘤率为68.3％。(2)连续观察90天,给药组与对照组比较,两组的平均存活日数差异非常显著(P<0.01),生命延长率为372％。(3)T.B淋巴细胞百分率及病理组织学变化,给药组与对照组比较都有明显差异。

Down-regulation of STAT3 expression by vector-based small interfering RNA inhibits pancreatic cancer growth

AIM:To evaluate the effect of RNA interference (RNAi) mediated silence of signal transduction and activation of transcription (STAT)3 on the growth of human pancreatic cancer cells both in vitro and in vivo.METHODS:STAT3 specific shRNA was used to silence the expression of STAT3 in pancreatic cancer cell line SW1990.The anti-growth effects of RNAi against STAT3 were studied in vitro and in experimental cancer xenografts in nude mice.The potential pathways involved in STAT3 signaling were detected using reverse transcription polymerase chain reaction and western blotting.RESULTS:The expression of the STAT3 was inhibited using RNAi in SW1990 cells.RNAi against STAT3 inhibited cell proliferation,induced cell apoptosis and significantly reduced the levels of CyclinD1 and Bcl-xL when compared with parental and control vector-transfected cells.In vivo experiments showed that RNAi against STAT3 inhibited the tumorigenicity of SW1990 cells and significantly suppressed tumor growth when it was directly injected into tumors.CONCLUSION:STAT3 signaling pathway plays an important role in the progression of pancreatic cancer,and silence of STAT3 gene using RNAi technique may be a novel therapeutic option for treatment of pancreatic cancer.

Expression and alteration of insulin-like growth factor II-messenger RNA in hepatoma tissues and peripheral blood of patients with hepatocellular carcinoma

AIM: To investigate the clinical values of serum free insulin-like growth factor Ⅱ (IGF-Ⅱ) levels and IGF-Ⅱ mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood for diagnosis of HCC and monitoring of extrahepatic metastasis.METHODS: Total RNAs were extracted from HCC tissues or peripheral blood mononuclear cells from patients with HCC, liver diseases devoid of cancer, non-hepatic tumors,and healthy controls, respectively. IGF-Ⅱ cDNAs were synthesized through random primers and reversetranscriptase, amplified by polymerase chain reaction (PCR), and confirmed by DNA sequencing analysis. Serum free IGF-Ⅱ levels in patients with different liver diseases were analyzed by an enzyme-linked immunosorbent assay.RESULTS: The amplified fragments of IGF-Ⅱ mRNA by RT-PCR were identical to originally designed ones with a size of 170 bp and confirmed by sequencing analysis.The dilution experiments revealed that the lowest sensitivity of our system was 2 ng/L of total RNA. The positive frequencies of IGF-Ⅱ mRNA were 100% in HCC tissues,53.3% in para-cancerous tissues, and 0% in non-cancerous tissues, respectively. The serum free IGF-Ⅱ levels were significantly higher in HCC than those in chronic hepatitis or liver cirrhosis. The positive frequency of circulating IGF-Ⅱ mRNA was 34.2% in HCC, no amplified fragment was found in other liver diseases, extrahepatic tumors,and normal controls, respectively. The circulating IGF-Ⅱ mRNA correlated with the stage of HCC, and its positive rate was 100% in HCC with extrahepatic metastasis and 35.5% in HCC with AFP-negative. No significant correlation was found between tumor sizes and circulating IGF-Ⅱ mRNA fragment.CONCLUSION: The abnormal expressions of free IGF-Ⅱ and IGF-Ⅱ mRNA are useful tumor markers for HCC diagnosis, differentiation of extrahepatic metastasis and monitoring postoperative recurrence.

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective： To construct a lentiviral expression vector for RNA interference （RNAi） of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods： Three pairs of human VIM gene short hairpin RNA（shRNA） sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides （dsOligo） were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction （PCR） and DNA sequencing analysis. Real-time PCR and Westemblotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 calls were validated by real-time PCR and Western-blotting. Results： An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2× 10^9TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion： An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.

Advanced pancreatic ductal adenocarcinoma-Complexities of treatment and emerging therapeutic options

Pancreatic ductal adenocarcinoma is a devastating disease with a poor prognosis regardless of stage. To date the mainstay of therapy for advanced disease has been chemotherapy with little incremental im-provements in outcome. Despite extensive research investigating new treatment options the current practices continue to utilise fluorouracil or gemcitabine containing combinations. The need for novel the-rapeutic approaches is mandated by the ongoing poor survival rates associated with this disease. One such approach may include manipulation of ribosome biogenesis and the nucleolar stress response, which has recently been applied to haematological malignancies such as lymphoma and prostate cancer with promising results. This review will focus on the current therapeutic options for pancreatic ductal adenocarcinoma and the complexities associated with developing novel treatments, with a particular emphasis on the role of the nucleolus as a treatment strategy.

Development of cystathionine gamma-lyase-specific microRNAs

The gasotransmitter role of H2S in mammalian has been extensively studied, and cystathionine gammalyase （CSE） is the major H2S-producing enzyme in vascular system. Dysregulation of CSE/H2S system was found in various pathophysiological conditions. MicroRNA （miRNA） and short interfering RNA （siRNA） are known to inhibit gene expression by mRNA degradation and/or translation repression. The regulation of CSE expression by miRNA and siRNA has been reported recently, but the off-target effect of miRNA and the lower knockdown efficiency of siRNA have shadowed the application of these approaches. In the present study, we designed CSE-specific miRNAs based on the rules of miRNA-mRNA complementary and human CSE cDNA sequence. The CSE-specific miRNAs significantly inhibited CSE expression and H2S production, increased reactive oxygen species generation, and induced proliferation of human aorta smooth muscle cells （HASMCs）. The designed CSE-specific miRNAs specifically targeted on CSE gene as evidenced by the direct inhibition of luciferase activity from reporter gene containing human CSE 3t-UTR sequence. The expression of other genes, such as estrogen receptor a, heme oxygenase 1, specificity protein 1, and 3-mercaptopyruvate sulfurtransferase, was not affected by the CSE-specific miRNAs.Compared with CSE-siRNAs, CSE-specific miRNAs dis- played significantly higher efficacy in suppressing CSE expression and H2S production, miR-143, a highly expressed miRNA in vascular system, down-regulated the expressions of CSE as well as other genes, such as insulin-like growth factor binding protein 5 and kruppel-like factor 4. miR-143 suppressed H2S production but had no effect on HASMC proliferation. In conclusion, CSE-specific miRNAs designed in our study provide a highly effective research tool for regulating CSE expression and H2S production. These CSE-specific miRNAs have potential as being novel therapeutic agents for treating vascular disorders related to abnormal oxidative stress and SMC growth.

Stable expression of antisense hTR inhibits in vitro pancreatic cancer cell growth

OBJECTIVE: To clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex. METHODS: A 593 bp full length hTR cDNA was subcloned into a mammalian expression vector pcDNA3.1(-) in the antisense orientation to construct an antisense hTR expression plasmid. These were introduced into panc1 cells, a human pancreatic carcinoma cell line, by lipofectin and G418-resistant stable transformants were expanded. Resulting stable clones were screened for the presence of the hTR insert by PCR with T7 and BGH reverse primers located on the flanks of the multiclonal site of the pcDNA3.1 vector. Cell growth rate, hTR expression, telomerase activity and anchorage-independent growth properties were analyzed. RESULTS: Significant downregulation of endogenous hTR was evident in the antisense-hTR transformed cells and telomerase activity was markedly decreased compared to control cells in standard TRAP assays. Furthermore, cell proliferation and the anchorage-independent growth ability in antisense-hTR expressing cells were significantly decreased compared with control parental cells. However, no crisis or senescence phenomena were observed. CONCLUSIONS: These data indicate that hTR may be a critical component of human telomerase activity and suggest that downregulation of the RNA component of human telomerase is a possible target for anticancer strategies.