| Malignant neoplasm is a serious disease threatening human health. As aresult of the high complexity and diversity of tumor development andprogression, it is very difficult to understand the development mechanism andfind the therapeutic methods for malignancies. Tumor immunotherapy hasbeen proven to be the fourth effective anti-tumor strategy following surgery,radiotherapy, chemotherapy for malignancies. In recent years, B7-H3, as anew member of B7immunoregulatory superfamily, overexpressed in multipletumor types, is significantly correlated with biological characateristics oftumor and considered to be a new tumor maker and potential theraputic target.Objective:Our study aims at examing the expression of B7-H3in human esophagealsquamous cell carcinoma (ESCC), corresponding adjacent tissues andesophageal squamous atypical hyperplasia tissues and determinng itscorrelations with clinicopathological data and overall survival in paitients withESCC.In addtion, We analyzed the expression of B7-H3in esophagealcarcinoma cells lines of TE-1, TE-13, Eca-109. RNA interference was used toinvestigate the effect of B7-H3on the biological behavior of esophagealcarcinoma cell lines such as proliferation, invasion in vitro.Methods:1The expression of B7-H3and the intensity of tumor infiltrating CD8+Tlymphocytes in pathologic specimens of82ESCC paitients, correspondingadjacent tissues and esophageal squamous atypical hyperplasia tissues wereevaluated by immunohistochemical assay.2RT-PCR (Reverse Transcription-Polymerase Chain Reaction) wasperformed to evaluate the expression of B7-H3in esophageal carcinoma cells lines of TE-1, TE-13, Eca-109.3RT-PCR, Western Blot were performed to evaluate the effect of mRNAand protein expression of B7-H3in Eca-109cells after siRNA transfection.4The proliferation activity of esophageal carcinoma cells after B7-H3siRNA interference was detected by MTT assay.5The cell motility and migration ability of esophageal carcinoma cellsafter B7-H3siRNA interference was evaluated on wound scrape assay.6The invasion ability of esophageal carcinoma cells after B7-H3siRNAinterference was revealed in vitro assay on Matrigel filters.Results:1Immunohistochemical assay showed that B7-H3positive staningmainly localized in the cytoplasm and cell membrane of tumor cells. In thesetumor specimens, expression of B7-H3was found in98.8%(81/82) ofsamples, while it were almost negative in adjacent tissue and atypicalhyperplasia tissue. The abnormal expression of B7-H3was correlated with age,patient’s tumor grade and TNM stage (P<0.05). In these tumor specimens,36samples is CD8+T lymphocyte slight infiltration and46is severe. Meanwhile,the expression of B7-H3in cancer tissues negatively correlated with theintensity of tumor infiltrating CD8+T lymphocytes (P=0.003) andpostoperative prognosis (P<0.05).2we performed RT-PCR to test whether esophageal carcinoma cellsexpress the costimulatory molecule B7-H3in vitro. All tested culturedesophageal carcinoma cell lines constitutively expressed B7-H3mRNA undernormal conditions (TE-10.382±0.008, TE-130.399±0.008, Eca-1090.428±0.012. The expression of B7-H3in Eca-109cells was slightly higher thanits expression level in TE-1, TE-13cells, while there was no statisticallysignificant diffrerence.3To determine the efficiency of RNA interference we first analyzed thelevels of B7-H3gene expression in the B7-H3siRNA (0.1285±0.0017),control siRNA (0.5324±0.0072), and untransfected (0.5403±0.0129) groups.There was markedly decreased gene expression after transfection of B7-H3 siRNA for48hours compared with transfection in the control siRNA anduntransfected groups (P<0.01). However, there was no significant differencebetween the control siRNA and untransfected groups (P>0.05).4The Western blotting assay shows that the protein expression wasdown-regulated after transfection of B7-H3siRNA (0.4214±0.0048) for48hours compared with transfection in the control siRNA (0.5006±0.0129) anduntransfected (0.4921±0.0148) groups (P<0.05). However, there was nosignificant distinguish between the control siRNA and untransfected groups(P>0.05). This indicates that the siRNA down-regulated B7-H3gene wasspecific and efficient.5The MTT assay was used to study the effect of B7-H3siRNAinterference on Eca-109cell proliferation. Untranfected, B7-H3or controlsiRNA transfected Eca-109cells were plated at8,000cells per well in a96-well plate for24,48or72hours. There was no statistical significance incellular proliferation among B7-H3siRNA, control siRNA, and untransfectedgroups.6To determine whether B7-H3acts as a tumor migration regulator weused the wound scrape assay to evaluate cell motility. siRNA inhibition ofB7-H3significantly decreased Eca-109cell migration in the wound scrapemodel. Time course analysis of wound closure showed that a monolayer wasreestablished within a significantly shorter period in the untranfected group orcontrol siRNA transfected group than in the B7-H3siRNA transfected group(P<0.05). Yet, there was no significant difference between the control siRNAand untransfected groups (P>0.05).7After down-regulating, the gene expression of B7-H3by siRNA in vitroassay on Matrigel filters revealed that the number of invaded Eca-109cellswas decreased up to50%. There was no significant difference between thecontrol siRNA and untransfected groups (P>0.05).Conclusion:1B7-H3is overexpressed in ESCC tissues; B7-H3expression issignificantly correlated with tumor progression and prognosis and may become a promising indicator of monitoring esophageal carcinoma progress.2B7-H3expression in ESCC negatively correlated with the intensity oftumor infiltrating CD8+T lymphocytes, may be involved in tumor immuneescape.3B7-H3siRNA interference significantly decreased Eca-109cellmigration and invasion abilities, suggesting B7-H3may regulate tumorinvasiveness and metastasis in a novel way. It may serve as a potentialtherapeutic target for tumor immunotherapy. |
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