Screening And Fuctional Study Of Metastasis-related Genes Based On Gene Expression Profiling Assay In Esophageal Squamous Cell Carcinoma

【Background】Esophageal squamous cell carcinoma (ESCC) is one of the most common digestivemalignant cancers in China. Although the advancements in surgery, chemotherapy,radiotherapy and molecular target therapy, the prognosis of ESCC patients is still poorbecause most patients are presented with local invasion, lymph node and distant metastasisdue to the highly aggressive characteristics. The mechanism of ESCC metastasis is stillunclear, resulting in lack of ideal therapeutic method. Therefore, further studying thepotential mechanisms involved in ESCC metastasis is critical for improving the prognosisof patients. Several single molecules play significant roles in the metastasis of ESCC.However, the complex process of ESCC metastasis is involved in multiple genes andsignal ways. Thus, overall understanding of ESCC metastasis-related genes is significantfor patient diagnosis and therapy, which can be carried out by the gene expressionprofiling assay for the screening on ESCC metastasis-related genes. 【Aims】To screen on the ESCC metastasis-related genes, and to investigate the roles and theunderlying mechanisms of SDR9C7, one of the most differential genes, in the ESCCmetastasis process, with the aims of better understanding the potential mechanisms ofESCC metastasis and providing a new theoretical target for treatment of ESCC metastasis.【Methods】1. The gene expression profiling assay was performed to identify the differences in geneexpression profiles between primary ESCC tumors that were with LN metastases (N+)and those without LN metastases（N-）.2. Immunohistochemical staining was performed to evaluate the expression of the shortchain dehydrogenase/reductase family9C, member7(SDR9C7) in ESCC tissues, twotranscriptions of which were observed7times more frequently in N+compared with N-tumors.3. In vitro repeated transwell assays were performed to screen on the ESCC sub-cell lineswith highly invasive ability (EC9706-P and EC109-P), and low invasiveability(EC9706-N and EC109-N). Transwell assays were performed to analyze thedifference of invasive ability between the two pairs of sub-cell lines. Then western blotwas used to detect the SDR9C7expression in the4cell lines.4. The Lentivirus-mediated siRNA target SDR9C7was constructed, and transfected intothe highly invasive cell lines with high SDR9C7expression. After the interferentialeffects have been confirmed by western blot analyses, MTT, plate cloning formationand nude mouse subcutaneous tumorigenesis assays were performed to detect effects ofSDR9C7gene silencing on the growth of ESCC cells. Then transwell and metastaticability in nude mouse induced through caudal vein injection assays were uses toevaluate the effects of SDR9C7gene silencing on migration, invasion and metastasis ofESCC cell lines.5. Western blot analyses were performed to detect the effects of SDR9C7inhibiting on the expression of metastasis-related proteins including MMP11, E-cadherin and V-EGF, andimmunohistochemical staining was used to analyze the correlation of SDR9C7andMMP11expression in ESCC tissues.【Results】1. The results of gene expression profiling assays showed that a total of26transcriptswere increasingly expressed in N+tumors to23different genes, and a total of32transcripts with decreased expression in N+tumors correspond to30different genes.Two transcripts of the SDR9C7gene were present7times more frequently in N+tumorscompared with N-tumors, indicating that SDR9C7might be a significant prognosticsignature for ESCC metastasis.2. The expression of SDR9C7was further detected in104ESCC tissues byimmunohistochemical staining, and presented positive staining in the cytoplasm ofESCC tissues. Positive expression of SDR9C7was significantly correlated withlymphatic invasion and LN metastasis (P＜0.001). However, SDR9C7expression hadno significant correlation with age, sex, differentiation and T stage. The total survivalrate of all patients with ESCC during the observation period was41.3%. The meanfollow-up time was31.8months with a median value of24months. The survival ratein patients with positive SDR9C7expression was29.9%(20/67), which wassignificantly lower that the survival rate in patients with negative SDR9C7expression62.2%（23/37）. The Kaplan–Meier postoperative survival analyses showed that thefollowing factors significantly correlated with postoperative survival: differentiation(P=0.005), LN metastasis (P<0.001), lymphatic invasion (P<0.001), T stage (P<0.001)and SDR9C7expression (P=0.001). Multivariate regression analyses revealed thatdifferentiation (P=0.02), lymphatic invasion (P=0.03) and T stage (P=0.001) wereindependent prognostic factors, however, SDR9C7expression was not an independentprognostic factor (P=0.31).3. Transwell assays showed that the levels of migration and invasion of the highly invasive cell lines EC109-P and EC9706-P were significantly stronger than the matchednon-invasive cell lines EC109-N and EC9706-N. The western blot analyses showed thatSDR9C7expression was markedly higher in EC109-P and EC9706-P cells comparedwith the matched non-invasive cell lines, indicating that SDR9C7might be associatedwith the invasive phenotype of the ESCC cells.4. Western blot analyses confirmed that SDR9C7protein was significantlydown-regulated by lentivirus-mediated SDR9C7siRNA transfection in both EC109-Pand EC9706-P cells. MTT, plate cloning formation and nude mouse subcutaneoustumorigenesis assays showed that there was no significant difference in the growth ratebetween SDR9C7knockdown cells compared with the controls both in vitro and invivo (P>0.05). The results of the transwell assays showed that the migration andinvasion of SDR9C7siRNA-transfected EC109-P and EC9706-P cells were notablyreduced compared with untreated cells or cells transfected with a control siRNA.Consistent with the in vitro results, the animal experiments showed that liver and lungmetastases were apparently recognized in mice injected with Con-EC9706-P cells, butfew metastases were observed in mice injected with SDR9C7-siRNA transfected cells.Histological analyses revealed that the number and the size of metastatic nodules inthe lungs and livers of mice were significantly smaller in the controls (P<0.01). Thus,down regulated SDR9C7inhibited the metastasis of ESCC in vivo and in vivo.5. Western blot analyses showed that inhibiting SDR9C7expression can markedlyrepress the expression of MMP11, but no obvious alteration observed on VEGF orE-cadherin expression. The results of immunohistochemical staining showed asignificant correlation was found between SDR9C7and MMP11expression in ESCCtissues (r=0.56, p＜0.001；Spearman’s rank test).【Conclusions】1. A total of53genes were found to be significantly different expressed in ESCC with orwithout metastasis, indicating that multiple genes play important roles in themetastasis of ESCC. Two transcripts of the SDR9C7gene were present7times more frequently in N+tumors compared with N-tumors;2. SDR9C7expression in tumors significantly correlated with the metastasis and poorprognosis in patients with ESCC;3. The ESCC metastatic cell models were successfully established, providing a platformfor study of ESCC metastasis research;4. Inhibiting SDR9C7expression could significantly repress the invasion and metastasisof ESCC cells.5. The effects of SDR9C7on the metastasis of ESCC might be partly through regulatingof MMP11expression.