| [Background]Renal cell carcinoma (RCC) is one of the most common urinary malignant tumors. Renal cell carcinoma is resistant to chemotherapy and radiotherapy, with only small subsets of patients responding to immunotherapy. Although targeted therapy showed some prospects, however, there are individual differences. As a result, five-year survival rate is less than 10% for advanced and metastatic RCC patients. The fundamental reason for all above is that the mechanism of renal carcinogenesis and RCC progression remains unclear. Count of study has demonstrated that NF-κB signaling pathway is continuously activated in RCC tissus. In our previous study, we found Follistatin-like protein 1(FSTL1) may be related with RCC metastasis using microarray assay, meanwhile, the transcription level of FSTL1 had a tendency to decrease from normal renal tissue to primary cancer tissue till metastasis tissue. FSTL1 is a new found inflammation related molecule produced by non-immune cells. In this project, we try to explore the roles of FSTL1 in renal carcinogenesis and progression based on the population-based study, and further reveal the possible mechanisms at molecular level, which would be helpful to understand renal carcinogenesis and provide scientific evidence for new therapeutic targets and prognostic biomarkers.[Material and Method]1. Real-time PCR and Immunohistrochemistry were utilized to exam the expression pattern of FSTL1 in three RCC cell lines (786-0, ACHN, and NRCC, which was established in our laboratory from a Chinese Han RCC patient) and 95 RCC clinical samples, respectively. The association between FSTL1 expression level and RCC tumor stage was analyzed. Kaplan-Meier survival curve was employed to evaluate the prognostic value of FSTL1 protein expression in RCC patients.2. FSTLl related tagger SNPs were selected through searching international HapMap project and running Haploview software. In case-control study, Taqman probe RT-PCR was used to identify the genotypes of six SNP candidates (rs 11708686, rs2673704, rs1259293, rs1402372, rs1105219, rs1259339) in 417 RCC patients and 855 healthy controls. Online analysis tool (http://ihg.gsf.de) was used to exam Hardy-Weinberg equilibrium (HWE) in controls. Logistic regression model was used to explore the correlation between SNPs variation and RCC susceptibility. Kaplan-Meier analysis was used to explore the association of SNP genotype and RCC patients’ prognosis.3. Construction of FSTLleukaryotic expression vector and shRNA vectors which were specific on silencing FSTL1 gene. Transfection of these plasmids into RCC cell lines (786-0, ACHN, NRCC) and over-expression or silencing effect was detected by RT-PCR. Soft agar assay, transwell invasion assay, flow cytometry assay and RT-PCR were used to explore the effect of FSTL1 on the maliganancy phenotype of RCC cells.4. Affymetix Human U133 Plus 2.0 gene chip was used to identify gene expression change in FSTL1 gene silencing NRCC cells. Then the differently expressed genes were subjected to perform pathway enrichment analysis and functional prediction by DIVID and GSEA software, The significant genes and pathways involved were verified by RT-PCR, western blot and reporter assay.5. The crosstalk in FSTL1 downstream pathways was checked in mimic imflammation and hypoxic microenvironment of tumor cell growth in vitro using RT-PCR and western blot. The candidate molecules potentially mediated the interaction were identified using immunoprecipitation and mass spectrometry.[Result]1. Compared with human embryonic kidney epithelial cells 293T, the expression of FSTL1 was down-regulated in RCC cell lines (ACHN, MRCC and NRCC) (p<0.05), while up-regulated in RCC cell line 786-O (p<0.05). The expression of FSTL1 protein in tumor tissues decreased compared with paired adjacent normal tissues (p<0.001). A significant negative correlation was found between FSTL1 expression in tumor tissue and histological stages (p=0.013, r=-0.264). The average postsurgical survival time for RCC patients who had positive FSTL1 expression in tumor tissues was 115.84 months, and the average postsurgical survival time was 97.22 months for the RCC patients who had negative FSTL1 expression (Log-rank test, p=0.138).2. All six SNPs were conformed to HWE in healthy controls (p>0.05). Taking RCC patients as cases, compared with rs1 259293 TT genotype, CC genotype was significantly associated with increased risk of RCC (OR=2.004,95%CI=1.190-3.375, p=0.009). When taking ccRCC patients as cases, the major histological type of RCC, similar result was obtained (OR=2.014, 95%CI=1.171-3.463, p=0.011), suggesting rs1259293 CC genotype was a risk factor for RCC and ccRCC. In this study, rs11708686, rs1105219, rsl259339, rs1402372 and rs2673704 was not significantly related to the risk of RCC or ccRCC. Survival analysis of 309 RCC patients showed that RCC patients with rs1259293 CC genotype tended to have poor prognosis compared with those with rs1259293 TT/CT genotypes (p=0.022).3. As compared with control (shsiscramble), cell colony formation ability, mobility and invasion capabilities were significantly increased in stable silencing FSTL1 NRCC cells (shFSTLl-2). E-cadherin expression level was significantly increase while N-cadherin expression level was significantly decreased in NRCC shFSTLl-1/2 cells. Similar phenomenon was detected in ACHN and 786-0 cells. After silencing FSTL1 in NRCC cells, cells in G0/G1 stage were decreased while cells in S and G2/M stages were increased, CD99, N-cadherin and EpCAM expression were increased, while CD24 expression were decreased. All together, over expression of FSTL1 could inhibit RCC cells growth, migration and invasion, while silencing FSTL1 could enhance cell growth, migration and invasion abilities, promote cell cycle from S stage into G stage, decrease cell adhesion ability, and initiate EMT process.4. Gene expression analysis showed, after silencing FSTL1 expression, a total of 105 differently expressed genes were found in NRCC shFSTL1-1/2 cells, including 57 up regulated genes and 48 down regulated genes. Taking false discovery rate less than 25% as threshold, GSEA analysis showed 12 functional gene subsets were down regulated and 23 functional gene subsets were up regulated after silencing FSTL1 expression in NRCC cells, including TNFa, NF-κB and HIF related functional gene subsets were significantly enriched and up regulated (p<0.05). After silencing FSTL1 gene in ACHN,786-O and NRCC cells, the transcriptional levels of IL-6 and HIF1α were significantly increased (p<0.05). HIF1α transcriptional level was decreased with over expression of FSTL1 gene expression in ACHN and 786-0 cells caused (p<0.05). No significant change was found in the transcription level of IL-6. The expression of IκBα was lower in NRCC shFSTLl-1/2 cells than in NRCC shsiscramble cells with the treatment of TNFa for 30 min by Western blot, which suggested that silencing FSTL1 expression could increase NF-κB signal pathway activity. In ACHN and 786-0 cells, NF-κB transcriptional activity was decreased after silencing FSTL1 by NF-κB reporter assay, however, NF-κB transcriptional activity was increased with overexpression of FSTL1. Thus, FSTL1 may play as a tumor suppressor by inhibiting NF-κB and HIF signaling pathway activities.5. DFX and TNFa were used to treat RCC cells and mimic hypoxia and inflammational tumor microenvironment. IL-6 expression was higher in 786-0 and ACHN cells under treatment with DFX. PGKl.the target gene of HIF signaling pathway, its expression was higher in ACHN cells with treatment of TNFa. Downregulation of IκBα was detected in ACHN, MRCC and NRCC cells with treatment of DFX by western blot, suggesting NF-κB signaling pathway activity up regulated. Upregulation of HIF2α was deteceted in 786-0, ACHN and MRCC cells with treatment of TNFa, suggesting HIF signal pathway activity up regulated. Furthermore, in 786-0, MRCC and NRCC cells with treatment of DFX or TNFa alone or both, p-Akt and p-GSK expression were both increased significantly. Altogether, these results indicated that there was a crosstalk between HIF and NF-κB signaling pathway, and FSTL1 was the upstream gene of these two signaling pathways. The interaction of NF-kB and HIF signaling pathways may enhance PI3K pathway activity. Utilizing Immunoprecipitation and Mass Spectrometry method, TUBB, ACTN4, CCT3, SPTAN1, IQGAP1, LMNA, PDIA3, YWHAQ and RPLPO were identified as potential mediators in the interaction process.[Conclusion]FSTL1 expression in tumor tissues of RCC patients was down regulated, compared with paired adjacent normal tissues, and it was negatively correlated with pathological stages. It is first to report that rs 1259293 was significantly associated with RCC risk. Genotype CC of rsl259293 is the risk factor for renal carcinognesis and has the relationship with poor prognosis. Silencing FSTL1 in RCC cells would increase cell growth and proliferation abilities, enhance cell migration and invasion capability, improve cell cycles from G1 stage to S stage and initiate EMT process. In RCC cells, FSTL1 may play as a tumor suppressor by inhibiting the activities of HIF and NF-κB signaling pathway. |
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