| Background&Objective:Gastric cancer (GC) is the fourth most common cancer and the second most common cause of cancer-related death worldwide. Chemothearpy is a mainstay of GC treatment and therefore understanding the mechanisms of GC chemoresistance may lead to improved clinical outcomes for GC patients. The WD40repeat-containing62(WDR62) is a recently identified centrosome-associated gene, which plays important roles in DNA replication and cell cycle progression. WDR62protein mediates cellular signalling, transcription, mitotic and apoptotic functions. Notably, endogenous WDR62strongly accumulates at the spindle poles of dividing cells but not in the nucleus during mitosis, suggesting a critical role of WDR62in tumor cell proliferation. However, the involvement of WDR62in human malignancies remains unknown. Based on the important physiological functions of WDR62, we hypothesized that elevated WDR62expression facilitates the development of human GC and significantly influences GC chemosensitivity and therapeutic outcomes. Therefore, the present study aimed to determine the level of WDR62expression in GC tissues as well as evaluate the association between WDR62and the clinicopathological features and prognosis of human GC. Furthermore, the involvement of WDR62in GC cell proliferation, invasion, migration and cell cycle distribution was investigated using in vitro and in vivo models.Methods:Matched cancer and adjacent non-tumorous stomach tissues were randomly collected from372GC patients receiving surgical resection at Xiangya Hospital. Each tumor sample was subjected to haematoxylin&eosin and immunochemistry staining. Diagnoses of gastric adenocarcinomas were confirmed by two independent experienced pathologists. Quantitative real-time PCR and Western blotting were used to check the expression of WDR62. Clinicopathological features were also recoded to analyze the relationship between WDR62, clinicopathological variables and prognosis in GC patients.Following surgical resection, all patients received chemotherapy of5-fluorouracil (5-FU) combined with cisplatin (CF). Clinical follow-up data were obtained from all372cases of GC patients. The follow-up period was defined as the interval between the date of operation and that of the patient’s death or the last follow-up. Time to progression was measured from the initiation of chemotherapy to the progressive disease. Apart from tissues, the cell lines of AGS, HGC-27, MGC-803, BGC-823, SGC7901, SGC7901/VCR, SGC7901/DDP, SGC7901/ADM and293T were cultured in Dulbecco’s modified Eagle medium (DMEM). Quantitative real-time PCR and Western blotting were used to check the expression level of WDR62. To maintain the multi-drug-resistant (MDR) phenotype, vincristine (1μg/mL) and adriamycin (1μg/mL) were added to the culture medium for SGC7901/VCR and SGC7901/ADM cells. Lentiviral short-hairpin RNA (LV-shRNA) interference plasmids (pLKO.1-shRNA) were constructed and transfected to BGC-823and SGC7901/VCR cells. Expression of WDR62, cyclin B, CDK1and caspase3were confirmed by qRT-PCR and Western blotting. Several signal molecules were also checked.We then conducted chemosensitivity detection, wound healing and invasion assays, cell viability assay, colony formation assay, cell cycle analysis, Hoechst assay and Annexin V staining to elucidate roles of WDR62in GC cells apoptosis, proliferation and chemosensitivity in vitro. Data of LV-shRNA-WDR62treated group was compared to LV-shRNA-Control treated group.To study in vivo, the BGC-823GC cells and multidrug-resistant GC cells of SGC7901/VCR were respectively infected with LV-shRNA-Control and LV-shRNA-WDR62lentivirus and injected subcutaneously into BALB/c mice. The nude mice were checked and tumors sizes were measured every3days from the eighth day after injection. On the31st day after inoculation, all mice were sacrificed and the tumor masses were weighed. The tumor tissues were verified by hematoxylin&eosin staining. In addition, IHC staining against WDR62and Ki67were performed on serial sections of each tumor.Results:The relative expressions levels of human WDR62mRNA examined by real-time qRT-PCR, calculated by2-△Ct and normalized to the internal control of GAPDH mRNA revealed that the average expression level of WDR62mRNA in GC tissues was higher than that in adjacent non-tumorous tissues (NTs)(0.0065±0.0016versus0.0027±0.0009, p <0.001). Poorly differentiated GC tumors exhibited higher WDR62mRNA expression levels than well differentiated tumors (0.0058±0.0019versus0.0022±0.00059, p<0.001). These data implicate that the increased WDR62mRNA expression observed in GC is correlated with poor tumor differentiation. The intensity of WDR62staining was associated with poor GC tumor differentiation.80.1%(298/372) of the tumor samples exhibited positive WDR62staining, while only23.9%(89/372) of the NTs displayed positive WDR62staining. The mean IS value in GC tissues was also significantly higher than that in NTs (8.3±0.8versus1.7±0.7, p<0.01). Furthermore, GC patients with higher WDR62expression (IS≥2) have poorer prognoses than those with lower WDR62expression (IS<2)(mean survival time:22.0±2.2months versus43.0±5.6months, p=0.003). A Cox regression analysis indicated that poor tumor differentiation (hazard ratio [HR]=2.383, p=0.009), lymph node metastasis (HR=1.205; p=0.012) and high WDR62expression (HR=2.365, p=0.006) were found to be independent prognostic factors for the survival time of GC patients. In GC cells, WDR62mRNA and protein levels were significantly higher in poor differentiation cell lines BGC-823and four multidrug resistance (MDR) cell lines (SGC7901/VCR, SGC7901/CDDP, SGC7901/ADM, and SGC7901), as compared with cell lines lacking drug resistance. Heightened WDR62mRNA and protein levels in MDR cell lines implicate WDR62involvement in the molecular mechanisms underlying cell differentiation and chemoresistance in GC. WDR62enhanced GC cell growth but not cell metastasis, knockdown of WDR62induced apoptosis of GC cells by G2/M cell-cycle arrest by reducing the expression of Cyclin B and CDK1protein, but enhanced the expression of Caspase3. WDR62down-regulation partly reversed the resistance of GC cells toward chemotherapeutic drugs by inhibiting Akt and p38-MAPK signal transduction pathways. Conclusions:WDR62expression was significantly increased in GC tissues and cell lines and was associated with poor differentiation and prognosis of GC. WDR62expression was elevated in GC multidrug resistant cells. Supressing WDR62significantly decreased cell proliferation and induced G2/M phase arrest of GC cells. Consistently, WDR62knockdown inhibited gastric carcinogenesis in nude mice. Regulation of Akt/p38-MAPK/MDR1expression and activation by WDR62contributed to the chemoresistance of GC cells. WDR62overexpresses in GC and the suppression of WDR62inhibits GC cell growth by inducing G2/M cell-cycle arrest. Above all, WDR62may be a novel prognostic marker and a potential chemotherapy target for GC. |
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