| BACKGROUND:In recent years, with the improvement of living standard and alteration of food habits, the morbidity and mortality of CRC has increased year by year in China. Conventional treatments of colorectal cancer include surgical ablation, chemotherapy and radiotherapy. Besides good short-term effect, these treatments also have had many defects such as significant toxicity and high recurrence rate, especially for patients with metastasis. With the rapid development of molecular biology, gene therapy, which has manifested good prospect for the therapy of CRC, is an important part of tumor biotherapy. Therefore, novel targeted approaches based on the mechanism of colorectal cancer development, will develop a promising path for the treatment of this disease.Notch signaling pathway is an evolutionarily highly conserved mechanism for cell-cell communication, which comprises Notch receptor, Notch ligand(DSL protein) and the transcription factor CSL (a DNA binding protein). The mammalian family of Notch receptors consists of 4 members (Notch1-4); Jaggedl is one main ligand of the Notch receptors. Notch signaling controls cell differentiation, proliferation and apoptosis, and plays an important role in embryogenesis and cell fate determination. There are the reports showing that Notch signaling is involved in the development of some cancers. The deregulation of Notch receptors and ligands and the aberrant activation of Notch signaling are found in a series of malignant tumors. Until now, the role of Notch signaling in CRC and its underlying mechanism are still unknown. The relationship between Notch signaling and colorectal cancer is a new field of researching, and it may also be a new target of gene therapy for colorectal cancer. OBJECTIVES:1. To investigate the expression of Notch1 receptor and Jaggedl ligand protein in CRC, and to explore its relation with clinical pathological characteristic. 2. To determine the expression of Notch1 protein and mRNA in CRC cell lines, and to screen a Notch1 over-expression target cell line to further research.3. To explore the role of Notch signaling expression in proliferation and apoptosis of CRC, and its possible mechanism.MATERIALS AND METHODS:1. Materials:①Clinical specimens:77 CRC,32 colorectal adenoma,31 tissues adjacent to CRC, and 31 tissues far from CRC as normal control;②CRC cell lines: COLO205, HT29, LoVo, SW480 and SW1116.2. Notch signaling pathway associated protein expression in CRC tissues and cell lines was examined by immunohistochemistry/immunocytochemistry PV-9000 staining method and Western blotting analysis.3. Notch signaling pathway associated gene mRNA expression in CRC cell lines was analyzed by means of reverse transcriptase-PCR.4. Notch signaling in CRC cells was regulated by RNA interference and specific inhibitor DAPT.Notch1-RNAi subgroup:①no RNAi group(NR, with Normal saline)②negative control group(NC, with negative control viral supernatant);③RNAi group(R, with viral supernatant).DAPT subgroup:DAPT concentration at 6.25μM,12.5μM,25μM and 50μM for MTT assay, and DMSO as control. Subsequently, grouping according to MTT results:①25μM DAPT treatment group;②50μM DAPT treatment group;③negative control group(DMSO).5. Cell proliferation was assessed by 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide MTT assays.6. Cell cycle was analyzed by Flow cytometry propidium iodide(PI) staining.7. Cell apoptosis was assessed by FCM Annexinⅴ/PI dual-staining and TUNEL staining.RESULTS:1. The expression of Notch1 and Jagged1 in colorectal cancer tissues:(1)The expression of Notch1 was detected in normal colorectum tissues, tissues adjacent to CRC, the colorectal adenoma tissues and the CRC tissues, the positive rate of Notch1 expression, which was increasing tendency, was 22.6%,19.4%, 78.1% and 89.6%, respectively. The expression of Notch1 was significantly higher in the cases with CRC and with colorectal adenoma than that in normal colorectum and in tissues adjacent to CRC (P=0.000), but no significant differences between the cases with CRC and colorectal adenoma, and normal colorectum tissues and tissues adjacent to CRC (P>0.05)(2)The expression of Jagged1 was detected in normal colorectum tissues, tissues adjacent to CRC, the colorectal adenoma tissues and the CRC tissues. The positive rate of Notch1 expression, which was also increasing tendency, was 12.9%,12.9%,81.2% and 85.7%, respectively. The expression of Jagged1 was significantly higher in the CRC and colorectal adenoma than that in normal colorectum and tissues adjacent to CRC (P=0.000), but no significant differences between the CRC and colorectal adenoma, normal colorectum and tissues adjacent to CRC (P>0.05). The expression of Jagged1 was significantly lower in moderately differentiated and well-differentiated CRC (83.05%) than that in poorly differentiated CRC (94.44%, P=0.001). The lower differentiation degree is, the higher expression of Jagged1 is, and negative correlation was showed between the two aspects(rs=-0.355, P=0.002) The expression of Jagged1 was significantly lower in Dukes stage A+B cases (76.32%) than that in Dukes stage C+D cases (94.87%, P=0.029). The expression levels of Jagged1 were getting higher from Dukes A to Dukes D stage, and positive correlation was showed between the two aspects (rs=0.385, P=0.001). The expression levels of Jagged1 were higher in lymph node metastasis of CRC (94.44%) than those in non-lymph node metastasis of CRC (78.05%, P=0.031), positive correlation was also showed between the two aspects (rs=0.246,P=0.03).(3)There was positive correlation between the expressions of Notch1 and Jagged1 protein in CRC(rs=0.305, P=0.007).2. The expression of Notch1 protein and mRNA in CRC cell lines:The expression of Notch1 protein, which was no difference, could be detected in all five CRC cell lines by immunocytochemistry staining (P>0.05); The expression of Notch1mRNA and its protein was also observed by RT-polymerase chain reaction and western blotting to a different degree. The mRNA and protein levels of Notch1 were relative high in COLO205, HT29, SW480 and SW1116 cell lines while relative low in LoVo cells. Therefore, HT29 cell line, which had Notch1 over-expression, was selected for further research.3. Identification and preparation of the retro virus-mediated expression system containing double strands DNA for RNA interference on human Notch1 gene(pSiRNA-Notch1):(1)The pSiRNA-Notch1 recombinant retroviral vector had been constructed correctly by PCR, restriction endonucleases and gene sequencing.(2) The titer assayed on NIH3T3 cells was up to 234×104CFU/ml in pSiRNA-Notch1 group, and 185×104CFU/ml in negative control group.4. The effect of pSiRNA-Notch1 on HT29 cell line:(1) The effect on expression of Notch1mRNA:pSiRNA-Notch1 interfering HT29 cell at 72h, the expression of Notch1mRNA in R group was significant lower than that in NC group and NR group(P<0.05),but no significant difference between NC group and NR group(P>0.05);(2) The effect of pSiRNA-Notch1 on cell morphology:Under inverted microscope, HT29 cells, in which the expression levels of Notch1 were down-regulation, were showed dramatic morphologic changes such as unclear cell outline, cell edge coarseness;(3) The effect on cell growth:OD490nm value by MTT had no significant difference among R group, NC group and NR group at 24h(P>0.05). But the data was significantly lower in R group than that in NC group and in NR group at 48h, 72h and 96h (P=0.000), inhibitory rate was significantly higher (P=0.000).No differences were found at each time point between NC group and NR group (P＞0.05)(4) The effect on cell cycle:The percentage of G1 of HT29 cells by FCM was significant higher in R group than that in NC group and NR group at 48h (P<0.01) but no significant difference between the latter two groups (P>0.05)(5) The effect on cell apoptosis:Compared to NC group and NR group, the number of HT29 cell apoptosis was significantly increased in R group at 48h(P< 0.05), but no difference was found between the latter two groups (P>0.05)(6) The effect on expression of P21 and PUMA mRNA:The expression levels of p21 and PUMA mRNA were significant higher in R group, than those in NC group and NR group at 72h (P<0.01), but no significant difference was found between the latter two groups(P>0.05)5. Effect of DAPT on HT29 cell line:(1) The effect of DAPT on cell morphology:In inverted microscope, HT29 cells, in which Notch signaling was inhibited by DAPT at 48h, were showed dramatic morphologic changes, characterized by cell dispersing, rounding and extended processes.(2) The effect of DAPT on cell growth:HT29 cells growth was significant slower at 24h with 50μM DAPT, at 72h with 25μM DAPT and at 96h with 12.5μM DAPT than that in negative control group(P<0.01,0.05). This was time-dependent and concentration-dependent. But no significant differences were found at each time point between 6.25μM DAPT group and negative control group (P>0.05).25μM DAPT and 50μM DAPT were selected in sequent study according to the MTT results;(3) The effect of DAPT on cell cycle:Comparing with control, the percentage of G1 of HT29 cells was significantly higher in 25μM DAPT group and 50μM DAPT group in a dosage dependent manner at 48h (P<0.01);(4) The effect of DAPT on cell apoptosis:Compared with control group, the number of HT29 cell apoptosis was significantly increased in 25μM DAPT group and 50μM DAPT group at 48h in a dose-dependent manner(P=0.000)CONCLUTIONS:1. The expression levels of Notch1 and Jagged1 have been gradually increasing in the stage of canceration. It suggests that expression levels of Notch1 and Jagged1 are relevant to carcinomatous change of CRC. The expression of Notch1 gene in many kinds of CRC cell lines is observed to a different degree, which further confirmed the relationship between Notch signaling and CRC.2. The expression of Jagged1 in CRC is negatively correlated with differentiation degree, and positively correlated with Duke's stage as well as lymph node metastasis of CRC. It shows the relation between expression levels of Jagged1 and malignant degree as well as prognosis.3. Notch1 siRNA and DAPT could suppress CRC cell proliferation and promote cell apoptosis. DAPT could suppress the survival of CRC cells, block cell cycle at G0-G1, and promote cell apoptosis in both concentration-and time-dependent manner.4.Down-regulation of Notch signaling could inhibit HT29 cell growth, lead to cell cycle blocking and cell apoptosis by up-regulation of P21 and PUMA, which are regulated by Notch signaling.
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