| Hepatocellular carcinoma (HCC) is one of the most common malignant cancers, which is of poor prognosis. Therefore, to explore the pathogenesis of HCC is a major topic of clinical research. Cripto-1 (CR-1) plays a significant role in embryonic development, tumor development and metastasis. CR-1 is overexpressed in human breast, colon, and lung cancers. Furthermore, CR-1 expression level has a positive correlation with tumor infiltration depth, lymph node metastasis and recurrence, and a negative correlation with prognosis. CR-1 antisense oligonucleotides and neutralizing antibodies against CR-1 were able to significantly inhibit in vitro and in vivo growth of human breast, colon, ovarian, testicular carcinoma cells and leukaemia cells. Until now, there is never any evidence that has shown a relationship between CR-1 expression and carcinogenesis of HCC. Therefore, this study will identify the expression of CR-1 in hepatocellular carcinoma patients and cell lines, and explore the role of CR-1 in the pathogenesis of human HCC using a model that CR-1 specific expressed in transgenic mice liver. Furthermore, the implication of the role of CR-1 in HCC might provide new insight into understanding the molecular mechanism involved in HCC carcinogenesis and progression, and may find new targets for early diagnosis and treatment. In this study, we performed the following researches to elucidate the characteristics and regulatory mechanisms of CR-1.METHODS1. Detection the expression of CR-1 in human hepatocellular carcinoma tissues and liver cancer cell lines.The expressions of CR-1 in human hepatocellular carcinoma tissue specimens and hepatoma cell lines (HepG2, Huh-7, SK-Hep-1, LO2 and PLC) were detected by quantitative real time PCR. real-time reverse polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry respectively.2. Using RCLG transgenic mice to explain the role of CR-1 in the development of liver cancer.RCLG/Alb-Cre double transgenic mice were obtained by mating RCLG transgenic mice with Alb-Cre transgenic mice, and then mediated by Cre to recombinate in transgenic mouse liver and achieved CR-1 transgenic overexpression. At different time points (3 weeks,3 months,6 months and 8 months), samples of liver tissue were collected for histopathology, other related testing and analysis in order to identify the role of CR-1 and related mechanisms in the development of liver cancer:①RT-PCR detection the expression of CR-1 in RCLG/Alb-Cre double transgenic mouse liver.②General observation the liver of overexpression CR-1 transgenic mouse.③To identified the pathologie changes in RCLG/Alb-Cre double transgenic mouse liver by HE staining sections.(ie, normal tissue→hyperplasia→atypical hyperplasia→carcinoma in situ, etc.).④Labeled with BrdU or EdU for cell proliferation test to evaluate liver cell proliferation of the double transgenic mice:Mice were killed 2 hours after (hour, h) intraperitoneal injection of BrdU (100 mg/kg body wt) or 4h after intraperitoneal injection EdU (100 mg/kg body wt).⑤Detected correlated genes expression levels by quantitative PCR (such as TGF-β1, Notch1, IL-6, IL-1 etc.) to further confirm the above pathological changes.⑥Based on the pathological changes of the organization to determine the appropriate point time for collecting double transgenic mouse liver tissue, further extracting total RNA, and then using high-throughput methods (such as gene chip) to screen the transgenic mice with liver differentially expressed genes after overexpression of CR-1, to study the closely genes that related in the development of liver cancer and signaling pathways, etc.;⑦Process "⑥" screened out the specific genes and signaling pathways, which may play an important role in the development of liver cancer, and then conventional molecular biology techniques and methods were used to further confirm the specific role and molecular mechanism.RESULTS1. The CR-1 in human hepatocellular carcinoma tissues and liver cancer cell lines.1.1 To detect expression of CR-1 in human hepatocellular carcinoma tissues by RT-PCRThe expression levels of Cripto-1 mRNA were examined in 21HCC tissue samples,18 adjacent samples and 1 normal liver tissue specimens by semiquantitative RT-PCR analysis. Cripto-1 was higher expressed in 15 of 21(71.4%) carcinomatous tissues compared with 5 of 18 (27.8%) in adjacent cancer tissues (P=0.01).1.2 Quantitive real-time PCR was used to detect the expression of CR-1 in HCC tissuesThe results showed that the expression levels of CR-1 were consistent with the expression levels of mRNA detected by semiquantitative RT-PCR.1.3 Comfired CR-1 expression in hepatocellular carcinoma issues by Western blotWestern blot showed the expression levels of Cripto-1 protein were consistent with the expression levels of mRNA.1.4 Immunohistochemical further examining the expression of CR-1 in liver tissue specimensCripto-1 staining was mostly observed in the nucleus and cytoplasm of carcinoma cells. No specific Cripto-1 staining was observed in normal adjacent heptic epithelial cells and stroma cells in surrounding tissues.1.5 Detection of CR-1 in liver cancer cell lines by RT-PCR. The expression of CR-1 gene expression in the cell linesderived hepatoma (HepG2, Huh-7, SK-Hep -land the PLC) showed that CR-1 was mainly expressed short transcripts of 1.7kb. The LO2 cell line is expressed both thel.7Kb and 2.0kb transcripts.In summary, CR-1 is overexpressed in human HCC issues and hepatoma-derived cell lines.2. Using RCLG transgenic mice to explore the role of CR-1 in HCC development2.1 Conditional expression of human CR-1 in RCLG transgenic mice mediated by Cre/lox P systemBy in vivo bioluminescence imaging, Luc expression activation was detected in liver of RCLG/Alb-Cre double transgenic mice. 2.2 Pathological changes in the liver of RCLG/Alb-Cre double transgenic miceHE staining for histopathological analysis showed that there is no significantly pathological change in the liver of RCLG/Alb-Cre double transgenic mice at the age of 3,6 and 8 months, but BrdU or EdU cell proliferation assay indicates that the hepatocytes in double transgenic mice demonstrate the obvious proliferation;2.3 Detection of the expression levels of the downstream of CR-1 by real-time PCRThe expression levels of TGF-β, Notchl, IL-6 and IL-1 genes in the liver of 3-week-old RCLG/Alb-Cre double transgenic mice have no significant changes, but increase 2 to 3 times in the liver of 6-month-old RCLG/Alb-Cre double transgenic mice;2.4 Microarray screening the differentially expressed genes in liver of RCLG/Alb-Cre double transgenic miceTo detect the key genes differentially expressed in 6 months of transgenic mice and littermate control, total RNA were extracted and cDNA microarray screening were used to screening the differentially expressed genes in liver of RCLG/Alb-Cre double transgenic mice.Conclusions1. CR-1 expression in HCC is upregulated and CR-1 in HCC cell lines and tissues mainly expressed 1.7kb short transcripts; 2. The expression of CR-1 and Luc in liver of RCLG/Alb-Cre double transgenic mice is successfully activated;3. There is no significantly pathological change in the liver of RCLG/Alb-Cre double transgenic mice at the age of 3,6 and 8 months, but BrdU or EdU cell proliferation assay indicates that the hepatocytes in double transgenic mice demonstrate the obvious proliferation;4. The expression levels of TGF-P, Notchl, IL-6 and IL-1 genes in the liver of 3-week-old RCLG/Alb-Cre double transgenic mice have no significant changes, but increase 2 to 3 times in the liver of 6-month-old RCLG/Alb-Cre double transgenic mice;... |
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