Abstract:

Methods The GES-1 cells from normal gastric mucosa，gastric cancer MGC-803 cells，MKN-45 cells，and HGC-27 cells were collected. The hsa_ circ_ 0009735 small interfering RNA and negative control were transfected into the MGC-803 cells， and the gastric cancer MGC-803 cells were divided into si-hsa_ circ_ 0009735 group and negative control group；the control plasmid and hsa_circ_0009735 over-expression plasmid were transfected into the HGC-27 cells， and the gastric cancer HGC-27 cells were divided into OE-hsa_circ_0009735 group and control plasmid group， and the MGC-803 cells transfected with autophagic plasmid pcDNA-eGFP-LC3 were divided into blank group and different concentrations （0.25， 0.50， 1.00 and 2.00 mg·L^－1） of rapamycin groups.The expression levels of hsa _ circ_ 0009735 in the gastric cancer tissue and cells were detected by real-time fluorescence quantitative PCR （RT-qPCR）method；laser confocal microscope was used to observe the morphology of the MGC-803 cells and the number of autophagosomes in the MGC-803 cells in various groups； Transwell chamber experiment was used to detect the number of migration cells in various groups； flow cytometry was used to detect the percentages of the cells at different cell cycles in various groups； Western blotting method was used to detect the expression levels of microtuble-associated protein light chain 3Ⅱ（LC3 Ⅱ），microtuble-associated protein light chain 3Ⅰ（LC3 Ⅰ），E-cadherin， Cyclin D1， Vimentin，and N-cadherin proteins in the cells in various groups. Results The PCR products were sequenced and showed cyclization sites，which was matched with the sequences of circ RNA hsa_circ_0009735. Compared with the sequences of adjacent tissue， the expression level of hsa_ circ_ 0009735 in gastric cancer tissue was increased （P<0.05）； compared with GES-1 cells， the expression level of hsa_ circ_ 0009735 in the MGC-803 cells was increased （P<0.05）.There was no significant change of the morphology of the MGC-803 cells among 0.25 and 0.50 mg·L^－1 rapamycin groups.Compared with blank group，the number of granules in the MGC-803 cells in 1.00 mg·L^－1 rapamycin group was increased and the boundary was unclear； in 2.00 mg·L^－1 rapamycin group， more MGC-803 cells died. The laser confocal microscope observation results showed that the number of autophagosomes in the MGC-803 cells in 1.00 mg·L^－1rapamycin group had no significant difference compared with 2.00 mg·L^－1 rapamycin group （P>0.05）.Compared with blank group， the ratio of LC3 Ⅱ/LC3 Ⅰ in the MGC-803 cells in different concentrations of rapamycin groups were increased （P<0.05）， the expression levels of hsa_circ_0009735 were decreased （P<0.01）. Compared with negative control group， the expression level of hsa_circ_0009735 in the MGC-803 cells in si hsa_ circ_ 0009735 group was decreased（P<0.01）， the ratio of LC3 Ⅱ/LC3 Ⅰ was increased （P<0.01），the number of migration cells was decreased （P<0.01）， the percentage of cells at G₁ phase was increased （P<0.05）， the percentage of cells at S phase was decreased （P<0.01）， the percentage of cells at G₂ phase was decreased （P<0.05）， and the expression levels of Cyclin D1， Vimentin，and N-cadherin proteins in the cells were decreased （P<0.05）； compared with control plasmid group， the expression level of hsa _ circ -0009735 in the HGC-27 cells in OE-hsa_ circ_0009735 group was increased significantly （P<0.01）， the ratio of LC3 Ⅱ/LC3 Ⅰ was decreased （P<0.01）， the number of migration cells was decreased （P<0.01）， the percentage of the cells at G₁ phase was decreased （P<0.01）， the percentage of the cells at S phase was increased （P<0.05）， the percentage of cells at G₂ phase was increased （P<0.05）， the expression level of E-cadherin protein in the cells was decreased （P<0.05）， and the expression levels of Cyclin D1， Vimentin，and N-cadherin proteins in the cells were increased （P<0.05）. Conclusion High expression of hsa_circ_0009735 in the GC cells may promote the EMT process and affect the migration and the cell cycle， and inhibit the autophagy. Objective To investigate the expressions of circular RNA hsa_circ_0009735 in the gastric cancer tissue and cells， and to explore its effects on the epithelial mesenchymal transformation（EMT）， migration， cell cycle，and autophagy of the gastric cancer cells.

Key words: Hsa_circ_0009735, Stomach neoplasms, Epithelial mesenchymal transformation, Metastasis, Cell cycle, Autophagy