The Invasive Suppression And Treatment Effect Of Bit1 Targetedsmall Molecule RNA In Esophageal Squamous Cell Carcinoma(cells)

BackgroundEsophageal squamous cell carcinoma(ESCC) is one of the most common malignancies in China, especially in Henan province. The tumorigenesis of ESCC is a multistage and progressive process. The mechanism imbalanced between restraining and promoting apoptosis may play very important role in tumor genesis and growth. Bit1(Bcl-2 inhibitor of transcription1) is a protein that was found in 2004, the function of normal physiological is still not clear. One study showed that Bit1 protein was localized in the mitochondrial intermembrane space in the physiological condition, when it was released by mitochondria in response to apoptotic stimuli into the cytoplasm or the Bit1 protein was expressed in the cytoplasm, it can combine AES(amino-terminal enhancer of split), induce cell de-adhesion and promote cell apoptosis. But the latest researches suggest that Bit1 is located in the golgiosome and has an antiapoptotic function. Recently, there are few conflicted reports about the relationship between the Bit1 and tumor. What is more, the studies related Bit1 and esophageal squamous cell carcinoma(ESCC) have never been appeared.To explore the relationship between Bit1 and generation of ESCC, especially between Bit1 and the invasion and metastasis of ESCC, we used RNAi, Western blot, transwell assays and established a model of transplanted tumor on nude mouse to detect the impaction of down-regulated Bit1 expression in the genesis and metasis of ESCC in vivo and vitro. Then we can take a novel target and may have a new opportunity to the early diagnosis and the treatment of ESCC. Methods1. We detected Bit1 protein expression in six ESCC cell lines with various differentiation grades through western blot method.2. We used transwell method to check the impact on the invasion of ESCC through down-regulated Bit1 protein expression by RNAi technology.3. We investigated the effect on the tumor genesis of ESCC via establishing a transplated tumor modle through down-regulated Bit1 protein expression by RNAi technology continuously.4. SPSS 17.0 statistical software package was used for statistical treatment. For the measurement data that were presented as mean±SD. Differences among the groups were determined by one-way ANOVA, and then multiple comparisons between groups were tested by LSD t-test. For the count data, rank sum test was used in the comparisons between groups. The level of significant difference was α=0.05. Results1. The expression of Bit1 protein in different ESCC cell linesThe results showed that poorly differentiated ESCC cell lines expressed significantly(p<0.05) higher Bit1 in protein level compared with the differentiated well.2. Down-regulation of Bit1 expression in ESCC cells affects tumour cell invasion in vitroTo explore whether Bit1 is involved in migration and invasion of ESCC cells, the transfected EC9706 cell lines were used for wound healing and transwell assays as described below. We observed the inhibition efficiency of RNA interference vecter targeting to Bit1 gene was reached 62% at 72 h and the great effects of migration and invasion after Bit1-silencing, cells were significantly decreased migration and invasion to the bottom surface of the insert compared with conduct none(P<0.05).3. Down-regulation of Bit1 expression in ESCC cells affects tumour occurrence and development in vivoThe results showed that the tumor prevalence was 100% after injected transfected or untreated EC9706 cells to the animals. On day 5, there was a significant difference(p<0.05) in tumour volumes between animals injected with transfected p Super-negative vector cells and those with p Super Bit1-si RNA cells. From day 9 to 21, 10μg p Super-negative xenografts group were significantly larger than those of 10μg p Super Bit1-si RNA group conducted by treatment, but there never was a difference between dealt with 5μg p Silencer Bit1-si RNA and 5μg p Super-negative group. Conclusions1. The expression of Bit1 protein in ESCC cell lines might have a difference due to the different differention grades.2. Down-regulation of Bit1 expression can inhibit the biological behaviour of human ESCC cells in invasion ability in vitro.3. Down-regulation of Bit1 expression can inhibit tumor growth in nude mice in vivo.