| Objective(1) To explore the expressing of STAT3 in human colorectal carcinoma and study the impact on angiogenesis.(2) To observe the impact of STAT3 gene silencing on HT-29 cell growth and cell cycle distribution.(3) To observe the effect of STAT3 signal on tumor growth of human colorectal carcinoma in nude mice.(4) To investigate the effect of STAT3 signaling pathway on tumor angiogenesis of human colorectal carcinoma in nude mice, and probe the primary mechanism.Methods(1) STAT3 and CD34 expressing in the clinical colorectal carcinomas were studied by the way of immunohistochemistry. MVD were counted according to CD34 expression in tumor tissues. The correlation between the expression of STAT3 and MVD was statistically analyzed.(2) Three different siRNAs of STAT3 were designed and synthesized. After being screened, the most effective siRNA was found. According to this sequence, the short hairpin DNA of STAT3 was constructed. The shRNA duplex was ligated into the recombinant vector pRNAT-U6.2/Lenti. The recombinant vector was conformed by the restriction map and DNA sequencing. The correct recombinant lentiviral vectors were packaged in 293T cells. Viruses in the supernatant were collected and the titer was measured. HT-29 cells transfected with viruses were selected by G418 and transfected cells were gained and harvested. Real-time PCR and Western blot were used to detect the interference effects. Cell growth was assessed by MTT assay and cell cycle distribution was detected by flow cytometry.(3) HT-29, HT-29-GFP and HT-29-shSTAT3 cells were respectively injected subcutaneously into the back of the nude mice of each group. During the tumor growth, the appearance and size of each tumor were examined.(4) On 30th day all mice were killed. Tumor tissues were removed for histopatho- logical and CD34 immunohistochemical analysis and taken to extract total RNA for angiogenesis superarray assay.Results(1) The positive rate of STAT3 expressing in colorectal carcinomas was 63.6%, which was significantly higher than that in the normal group(P<0.01). MVD in colorectal carcinoma tissus was 47.55±12.15, which was significantly higher than that in the normal group(P<0.01). In colorectal carcinoma, the positive rate of STAT3 expression and MVD were closely related to malignancy degree,Duke's stage and lymph node metastasis (P<0.05), and MVD was closely correlated to the expression of STAT3(r=0.788).(2) The restriction map and DNA sequencing demonstrated that the recombinant lentiviral vector of RNA interference of STAT3 gene was constructed successfully. Virus particles were packaged in 293T cells, and the title of viruses was 2×107 TU/ml. In HT-29 cells transfected with viruses effectively, the expressing level of STAT3 was down-regulated as Real-time PCR and Western blot analyses demonasrated. In transfected group, MTT assay showed the growth of HT-29 cells was suppressed and FCM assay indicated the proportion of cells at G0/G1 and S phase was 68.73±2.88% and 22.93±1.10% respectively, which was great different from that of the control group (P<0.01).(3) A tumor was formed in every nude mouse. On day 15 after inoculating, tumor volums of the HT-29-shSTAT3 group were less than those of HT-29 group and HT-29-GFP group (P<0.05). On day 30 after inoculating, the difference was more obvious (P<0.01), the inhibitory rate of tumor growth was 57.46%.(4) MVD in the HT-29-shSTAT3 group was less than that in the other two groups (P<0.01).(5) The result of angiogenesis superarray indicated that there were 15 down- regulated genes and 7 up-regulated genes in tumor tissues of the HT-29-shSTAT3 mice group.Conclusions(1) STAT3 expressing in colorectal carcinoma is significantly higher. It may play an important role in the development and metastasis of colorectal carcinoma. It is suggested that angiogenesis is closely correlated to STAT3 signaling pathway in colorectal carcinoma.(2) STAT3 singnaling pathway plays an important role in HT-29 cells growth. STAT3 targeting shRNA can silence the STAT3 gene remarkably, induce HT-29 cell growth inhibition and block cell cycle at G0/G1 phase.(3) In nude mice, STAT3 singnal is important to the growth of human colorectal carcinoma.(4) In human colorectal carcinoma, STAT3 signal can promote angiogenesis.(5) In human colorectal carcinoma, STAT3 singnal may participate in regulating angiogenesis through modulating expression of VEGF-A, MMP-2, ECGF1, EPHB4, IGF1, NRP1, NRP2, STAB1, TNF,VEGFR-1 and BAI1.
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