F-box Protein FBXO31 Is Down-regulated In Gastric Cancer And Negatively Regulated By MiR-17 And MiR-20a

Background:Gastric cancer (GC) is a kind of common malignant tumor and ranks the second in terms of cancer-related deaths. Asia, especially China, is a high incidence rate of GC area. Since it is difficult to be diagnosed in the early stage of this disease, most of the patients have reached the advanced or metastatic stage once be diagnosed. Conventional treatments with surgery, chemotherapies or radiation therapy play a minor role in improving the patients’survival rate. Thus, it is crucial to explore the exact molecular mechanism of gastric cancer development and find the new diagnostic or prognostic markers and therapeutic targets to improve the survival rate of gastric cancer patientsUbiquitin-dependent proteasome pathway plays a key role in degradation of proteins. Ubiquitination requires three types of enzyme:ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes(E2), and ubiquitin ligases (E3). FBXO31 takes part in the formation of skpl-cullin-F-box(SCF) ubiquitin E3 ligase complex and works in an important role in the substrate recognition during this pathway.FBXO31 is involved in cell cycle regulation, DNA damage response, signal transduction and neuronal development processes by recognizing and binding to the target proteins, such as CyclinD1, Cdt1, MKK6, Par6c, etc. and making them ubiquitination and degradation. In terms of tumorigenesis, the expression of FBXO31 was significantly reduced in breast cancer and hepatocellular carcinoma. Therefore it was initially considered to be a tumor suppressor gene. However, the opposite results were obtained in the esophageal squamous cell carcinoma. The high expression of FBXO31 implied a poor prognosis. Therefore, the role of FBX031 is different in various tumors.In this study we first detected the expression of FBXO31 in gastric carcinoma and corresponding adjacent tissues, analyzed whether the FBXO31 expression level was associated with the clinicopathological variables and postoperative survival of GC patients. We then detected the effect of FBXO31 on cell proliferation and cell cycle progression in gastric cancer cells, explored the molecular mechanism of the biological effects caused by FBXO31. Finally we explored the molecular mechanism of FBXO31 expressing disorder in gastric cancer in terms of miRNA mediated post-transcriptional regulation.Objective:We explore the role of FBXO31 in gastric tumorigenesis and the regulation mechanism of FBXO31, in order to find a new diagnostic target.Methods:1. FBX031 mRNA and protein expression in gastric cancer tissues and the corresponding non-cancerous normal mucosa tissues were determined by qRT-PCR、Western blot and IHC and we explored whether the FBXO31 expression level was associated with the clinicopathological variables and patients’survia.I2. We transfected FBXO31 siRNA or vectors expressing wild-type FBXO31 into gastric cancer cells.,Then we detected the cell proliferation and cell-cycle distribution By colony formation assay and flow cytometric analysis. We also used Western blot to detect thechange of cell cycle-related proteins.3. We detected the effect of FBXO31 on tumor formation ability in nude mice xenograft model.4. Databases, such as pictar and TargetScan, were used to predict the potential miRNA targeting FBX031. After transfecting the potential miRNA into the cell lines, we detected whether FBXO31 was regulated by the candidate miRNAs using qRT-PCR, Western blot and dual luciferase reporter assays.5. We used qRT-PCR to detect the candidate miRNAs expression in gastric cancer tissues and the corresponding non-cancerous normal mucosa tissues and analysed the relationship between FBXO31 expression and candidate miRNAs expression.Results:1. In 53 paired GC tissues, we found that the FBXO31 mRNA level was significantly lower in 38 (71.7%) GC tissues compared with the matched non-cancerous normal tissues. In 52 paired samples, the FBXO31 protein level was significantly lower in 37 (71.2%) GC tissues compared to the corresponding non-cancerous normal tissues. The FBXO31 expression was associated with the clinicopathological variables. The lower expression usually determined the poorer prognosis.2. High expression of FBXO31 in gastric cancer cells significantly inhibited colony formation capacity and made cell cycle arrest in G1 phase, while FBXO31 siRNA could increase the colony formation capacity and promote cell cycle progression. Further studies showed that FBXO31 could significantly inhibit the expression of CyclinDl.3. High expression of FBXO31 impaired the tumor formation ability in vivo.4. miR-20a and miR-17 directly bind to the 3’untranslated region (UTR) of FBXO31 and inhibit FBXO31 expression in human gastric cancer cells.5. The expression of miR-20a and miR-17 is noticeable higher in GC tissues compared with the matched non-cancerous normal tissues. FBXO31 expression was negatively associated with mir-17 and 20a expression in patient tissues.Conclusion:Our findings indicated that FBXO31 expression was dramatically decreased in human gastric cancer tissue and significantly correlated with patient low survival. High expression of FBXO31 in gastric cancer cells significantly inhibited colony formation capacity, made cell cycle arrest in G1 phase and impaired the tumor formation ability in vivo. Therefore FBX031 was considered to be a tumor suppressor gene in GC. miR-20a and miR-17 directly binded to the 3’untranslated region (UTR) of FBXO31 and inhibited FBXO31 expression in human gastric cancer cells. miR-20a and miR-17 overexpression contributed to the decreased expression of FBXO31 in gastric cancer partly. Therefore, effective therapy targeting the miR-20a (miR-17)-FBXO31-CyclinDl pathway may help control gastric cancer.