Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation.

Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focus on U1 snRNA genes, we investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation.

Sex-lethal regulates back-splicing and generation of the sex-differentially expressed circular RNAs.

Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a SXL mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through PAR-CLIP. After splicing assay of mini-genes with mutations in the SXL-binding sites, we revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas SXL-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.

Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila.

The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.

[Dendritic cell vaccine modified by murine mAFP gene enhances immunoprotective effect on liver carcinogenesis and tumor development in mice].

OBJECTIVE: To construct a dendritic cell vaccine transduced by murine alpha-fetoprotein (mAFP) gene, and evaluate its immunoprotective effect on C57BL/6J mice during the induction of hepatocellular carcinoma by diethylnitrosamines, carbon tetrachloride and ethanol. METHODS: Dendritic cells (DCs) were induced and augmented by murine IL-4 and GM-CSF, and transfected by recombinant adenovirus engineered with mAFP gene. Major MHC class I and II, B7.1 (CD80), B7.2 (CD86), CD18a, and CD54 molecules on DC were analyzed by FACS. 80 C57BL/6J male mice were randomly divided into 4 groups (20 mice per group): Simple DC inoculated group, pAdBM5-mAFP-DC inoculated group, pAdBM5-mAFP plasmid inoculated group, and PBS control group. They were immunized once with 5 x 10(5) DCs (0.1 ml)/mouse administered s. c. in the left flank or 100 mg pAdBMS-mAFP plasmid/mouse administered i. m. in the left tibialis anterior muscle. Inoculation was conducted once a week for 4 weeks after 3 times consecutive immunization initially. At the same time of immunization, DEN/CCl4/ethanol were given to induce hepatocellular carcinoma. Tumor incidence was assessed after 20 weeks. RESULTS: A transgenic DC vaccine was successfully constructed and the mAFP transgenic DCs expressed high level molecules of major MHC class I and II , B7.1, B7.2, CD18a, and CD54. After the 20-week induction, the incidence of primary hepatocellular carcinoma (PLC) was 70.0% in simple DC inoculated group, 25.0% in pAdBMS-mAFP-DC inoculated group, 65.0% in pAdBM5-mAFP plasmid inoculated group, and 75.0% in PBS control group. There was a significant difference between group B and other groups (P < 0.05). CONCLUSION: mAFP transgenic DC tumor vaccine inoculation may induce strong immunoprotection against liver carcinogenesis and tumor development and reduce PLC incidence induced by DEN/CCl4/ethanol.

[Effects of hMIP-1beta gene modification on in vivo tumorigenicity and vaccine efficacy of tumor cells].

UNLABELLED: OBJECTIVE To explore the effects of human macrophage inflammatory protein-1 beta (hMIP-1beta) modification on the in vivo tumorigenicity and vaccine efficacy of tumor cells. METHODS: Murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus carring the hMIP-1beta gene (AdhMIP-1beta). The efficacy of gene transfection was tested by X-gal staining. The hMIP-1beta level in the supernatant of hMIP-1beta gene-modified CT26 cells was assayed by ELISA, and the chemotactic activity for CD4+ T cells, CD8+ T cells, NK cells and immature dendritic cells (imDCs) were assayed by a transwell chamber. The changes of growth characteristics and in vivo tumorigenicity of hMIP-1beta gene-modified CT26 cells were also assessed. BALB/c mice were immunized with hMIP-1beta gene-modified CT26 tumor vaccine and the antitumor effect was evaluated. RESULTS: hMIP-1beta gene could be transfected into CT26 cells by AdhMIP-1beta with an efficiency over 95%. The level of hMIP-1beta in the culture supernatant of hMIP-1beta gene-modified CT26 cells was 980 pg/ml and the supernatant displayed ramarkable chemotactic activity to CD4+ T cells, CD8+ T cells, NK cells and imDCs compared with LacZ gene-modified CT26 cells and control. When the hMIP-1beta gene-modifited CT26 cells were subcutaneously inoculated in BALB/c mice, the tumorigencity was delayed and suppressed, and overt necrosis and lymphocyte infiltration were observed in the tumor tissue, but not in those inoculated with LacZ gene-modified CT26 cells or parental CT26 cells. The mice immunized with hMIP-1beta gene-modified CT26 tumor vaccine could induce tumor specific CTL activity and nonspecific NK activity, and exhibited resistance to later challenge with wild-type CT26 cells. CONCLUSION: hMIP-1beta gene-modified CT26 cells exhibit decreased tumorigenicity, and hMIP-1beta gene-modified tumor vaccine may induce a powerful specific and nonspecific antitumor response. The data suggested that hMIP-1beta gene-modified tumor vaccine may play a potent role in prevention of metastasis and recurrence of malignant tumors.

[Expression and significance of EGF mRNA and EGFR mRNA in hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Many evidences demonstrated that the epidermal growth factor receptor (EGFR) subfamily played an important role in they initiation and progression of various cancers. But it is not clear whether there is a relationship between EGFR and hepatocellular carcinoma (HCC). The aims of the present study were to explore the expression and significance of the epidermal growth factor (EGF) mRNA and EGFR mRNA in human HCC tissues. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was employed to determine the expression of EGF mRNA and EGFR mRNA in 60 HCC tissues and their adjacent liver tissues. RESULTS: The positive rate of EGF mRNA was significantly lower in the HCC tissue (60%, 36/60) than in the adjacent liver tissue (80%, 48/60) (P< 0.05). The positive rate of EGFR mRNA in the HCC tissue (60%, 36/60) was markedly higher than in the adjacent liver tissue (41.67%, 25/60) (P< 0.05). The expression of EGFR mRNA in the HCC tissue was significantly correlated with the clinical stage, the portal vein tumor thrombus, the presence of extrahepatic metastasis, and the recurrence of tumor, the number of tumor, but not correlated with the diameter of tumor, the level of serum alpha-fetoprotein (AFP), the differentiation of tumor and the liver cirrhosis in the adjacent tissue. The detectable rate of EGF mRNA was correlated with the diameter of tumor but not correlated with the clinical stage, the portal vein tumor thrombus, the presence of extrahepatic metastasis, the recurrence of tumor, the number of tumor, the level of serum AFP, the differentiation of tumor and the liver cirrhosis in the adjacent tissue. CONCLUSIONS: EGF may not be involved in the initiation and progression of HCC, whereas, EGFR relates to the initiation and progression and the recurrence of HCC. EGFR can be considered as a marker for predicting the metastasis and recurrence of HCC.

Changed clinical aspects of primary liver cancer in China during the past 30 years.

BACKGROUND: Primary liver cancer (PLC) is one of the most frequently seen tumors in China. Thirty years ago, patients with PLC were often detected at relatively late stage, with a palpable mass or marked clinical symptoms and poor prognosis. In the past 30 years, the diagnosis and treatment of PLC have been greatly improved with better prognosis. METHODS: In order to study the changes of PLC during the 30 years, the clinical data of 3250 patients with PLC from 10 medical institutions of China were collected, analyzed, and compared with those of 3254 PLC patients before the 30 years. RESULTS: In the 3250 patients aged 1-80 years, with an average age of 49.1 years, the male to female ratio (2.3:1) was lower than that before the 30 years. 73.5% of the 3250 patients sought medical advice within 3 months after the onset of the disease in contrast to 63.8% before the 30 years. Compared with those patients before the 30 years the symptoms and signs were alleviated generally. The HBsAg positive rate was 81.0%, but the HCV-Ab positive rate was 13.2%. The AFP level in 75% of patients was elevated, but in the remaining 25% was normal. 1912 patients (58.8%) were confirmed pathologically. Among them 1755 patients (91.8%) had hepatocellular carcinoma. The overall resection rate was 46.3%. Those who had early, middle, late stage carcinoma accounted for 29.9%, 51.5%, and 18.6% respectively in contrast to 0.4%, 47.0%, and 52.6% reported before the 30 years. The 1-, 3-, 5-year survival rates of the patients were 66.1%, 39.7%, and 32.5% respectively, whereas 93.5%, 70.1%, and 59.1% for the early stage patients, and 65.3%, 30.5%, and 23.5% for the middle stage patients. The half and 1-year survival rates of the late stage patients were 52.5%, and 14.7%, respectively. CONCLUSION: Comparison with the clinical data before and after the 30 years show that PLC can be diagnosed early. More PLC patients tend to undergo resection while receiving a better conservative treatment, which ensures a prognosis.

[Comprehensive therapy for primary liver cancer: a report of 607 cases].

BACKGROUND & OBJECTIVE: Progress has been made in the field of early detection and early treatment of primary liver cancer (PLC), but many PLC patients remain unresectable, because their tumors are advanced or coexist with liver cirrhosis. Even if the tumor can be resected, the recurrent rate is more than 60%. This study aimed to investigate the efficacy of comprehensive therapy of PLC to improve the outcome. METHODS: A retrospective analysis of 607 patients with PLC received comprehensive treatment in Affiliated Tumor Hospital, Guangxi Medical University from 1985 to 2001. Among them, 423 cases were treated with various modes of hepatectomy: 134 with irregular hepatectomy, 95 with local radical resection, 123 with regular liver lobectomy or liver segment resection, 54 with semi-hepatectomy or more, 17 with hepatectomy combined with section of other organ. The other 184 nonresectable cases were treated with various combinations of therapy, such as ligation of hepatic artery, microwave coagulation, inter tumor injection of ethanol, cryosurgery, radio-frequency (RF), and intraperitoneal chemotherapy. RESULTS: 69.7%(423/607) of the whole group received liver resection, the overall mortality rate within one month after operation was 1.2%(5/423), and the 3-, 5-, 10-year survival rates were 42.7%(218/511), 37.5%(123/328), and 26.5%(26/98), respectively. For the resection group,the 3-, 5-, 10-year survival rates were 57.2%(203/355), 51.3%(118/230), and 35.3%(24/68), respectively. For the nonresectable group, the 3-, 5-, 10-year survival rates were 9.6%(15/156), 5.1%(5/98), and 6.7%(2/30), respectively. CONCLUSION: Surgery-predominant comprehensive therapy is effective modality for resectable PLC. Postoperative individualized comprehensive treatment can prevent tumor recurrence and improve postoperative effect.

[Changes of clinical aspect of primary liver cancer in China during the past 30 years--control study for 3,250 cases with primary liver cancer].

OBJECTIVE: To study the changes of the clinical aspects of primary liver cancer (PLC) during the past 30 years. METHODS: The clinical data of 3,250 patients with PLC, from 10 regions of China were collected, analyzed, and compared with the clinical data of 3254 PLC cases 30 years before. RESULTS: The 3,250 patients were aged 1- 80, with an average age of 49.1 years, younger than those 30 years before (43.7 years). The male to female ratio was 2.3:1, lower than that 30 years before (7.7:1). 73.5% of them sought medical advice within 3 months after the onset in comparison of 63.8% 30 years before. Compared with those 30 years before the symptoms and signs were alleviated in general. The HBsAg positive rate was 81.0%, the HCV-Ag positive rate was 13.2%, and the alpha-fetoprotein positive rate was 75%. 1912 cases underwent pathological examination of which 91.8% were diagnosed as with hepatocellular carcinoma. The overall resection rate was 46,3%. Those of early, median, and late stages accounted for 29.9%, 51.5%, and 18.6% respectively in comparison with the rates of 0.4%, 47.0%, and 52.6% 30 years before. The one-year survival rate, three-year survival rate, and five-year survival rate were 66.1%, 39.7%, and 32.5% respectively for the whole group, 93.5%, 70.1%, and 59.1% for the early stage patients, and 65.3%, 30.5%, and 23.5% respectively for the median stage patients. The half-year survival rate and one-year survival rate of the late stage patients were 52.5% and 14.7% respectively. Compared with the data 30 years before a lower percentages of the patients died of hepatic coma, hemorrhage of upper digestive tract and hemorrhage due to rupture of tumor, and a higher percentage of then died of asthenia universalis and other causes. CONCLUSION: In comparison with the situation 30 years ago, PLC can be diagnosed earlier. More patients undergo resection. The prognosis of PLC has been improved greatly.

[Surgery-predominant comprehensive therapy for 134 patients with small hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Although surgical resection is the primary choice modality in treatment of small hepatocellular carcinoma(HCC), the 5-year recurrent rate after resection was as high as 35.4%-43.5%. This study was designed to investigate the efficacy of surgery-predominant comprehensive therapy for small HCC in reducing the recurrent rate and improving the outcome. METHODS: A total of 134 cases of small HCC (< or = 5 cm in diameter) received surgery-predominant comprehensive treatment in The Affiliated Tumor Hospital, Guangxi Medical University from 1985 to 2001. The median age of the patients was 45 years old (range,18-70 years). Of 134 cases, 121 were treated with hepatectomy: 16 with irregular hepatectomy, 83 with local radical resection, 12 with regular liver lobe resection or liver segment resection, 2 with left semi-hepatectomy, and 8 with hepatectomy and gallbladder resection. In the other 13 cases of nonresectable small HCC, they received multimodality treatments by various combinations of hepatic artery ligation and anticancer agents by hepatic artery infusion, microwave coagulation, ethanol injection into tumor, cryosurgery,radio-frequency (RF), and hepatic artery chemoembolization therapy. RESULTS: Of 134 HCC patients, 90.3% received liver resection and no operative death occurred. For the surgery group, the 1-, 3-, 5-, and 10-year survival rates were 89.3%, 74.4%, 64.6%, and 43.8%, respectively; the 1-, 3-, and 5-year recurrent rates were 11.9%, 23.8%, and 32.1%, respectively. For the total group,the 1-, 3-,5-, and 10-year survival rates were 88.8%, 72.2%, 63.4%, and 41.7%, respectively; the 1-, 3-, and 5-year recurrent rates were 15.9%, 29.1%, and 36.6%, respectively. CONCLUSIONS: Surgical resection remains primary choice modality in treatment of small HCC; postoperative comprehensive treatment is important for preventing tumor recurrence and improving the long-term results.