Hsa_circ₀005379 Affects The Malignant Biological Behavior Of Oral Squamous Cell Carcinomas Through The GFR Signaling Pathway

Purpose: Oral squamous cell carcinoma is a common malignancy in oral and maxillofacial surgery.In recent years,oral squamous cell carcinoma incidence showed younger trend,which is life-threatening and seriously affects the quality of patients’ life.Study the possible mechanism of the oral cancer development will provide an important basis on the early diagnosis and treatment.Circular RNA is a non-coding internal RNA that has recently been discovered.The specific structure and biological function makes it-important in many tumors.Circ RNA is becoming an important biomarker for clinical diagnosis.By investigating the effect of hsa_circ₀005379 on the malignant biological behavior,we will explore the potential therapeutic targets in the oral squamous cell carcinoma development.Methods: Eight pairs of oral and squamous cell carcinoma specimens from patients were selected.Differential circular RNAs were screened by high-throughput sequencing.The expression of hsa_circ₀005379 in 37 oral and precancerous tissues was determined by q RT-PCR.The expression of hsa_circ₀005379 in oral squamous cell carcinoma cell lines was determined by q RT-PCR.The lentiviral expression plasmid or si RNA is transfected into cell lines,which resulting in high expression of hsa_circ₀005379 or or knock down.Cell proliferation,migration,invasion and apoptosis were measured by CCK-8 assay,scratch assay,co-culture experiment,Transwell assay,Hoechst staining,Annexin VFITC/PI flow cytometry experiment,and Western blot.The nude mice were tested by HE staining and immunohistochemistry.Results: High-throughput sequencing showed that the expression of hsa_circ₀005379 was significantly downregualated in the specimens from 8 pairs of oral squamous cell carcinomas.The results of q RT-PCR showed that the expression of hsa_circ₀005379 was significantly lower in the 37 pairs of cancer tissues than in the adjacent normal tissues（P<0.05）.Clinical data analysis showed that the expression of hsa_circ₀005379 was negatively correlated with OSCC differentiation and tumor size.The q RT-PCR results showed that the expression level of hsa_circ₀005379 in the four oral squamous cell carcinoma cell lines SCC9,SCC15,SCC25,and CAL27 was significantly lower than that in HOK cells（P<0.001）.Transfection of the HBLV lentivirus high expression plasmid showed that the expression of hsa_circ₀005379in oral squamous cell carcinoma SCC25 and CAL27 were increased by 4 and 6 fold,respectively.The difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.The results of si RNA interference experiments showed that the expression of hsa_circ₀005379 in SCC25 and CAL27 were decreased by approximately 75% and85%,respectively.The difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.The results of CCK-8 proliferation assay showed that the proliferation rate of HBLV-hsa_circ₀005379 cells was lower in the SCC25 and CAL27 cell lines than in the HBLV-MOCK group.The difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.EDU proliferation experiments showed that the proliferation rate of HBLV-hsa_circ₀005379 cells was lower than that of HBLV-MOCK.The difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.The scratch assay showed that the migration speed of HBLV-hsa_circ₀005379 overexpression group was significantly lower than that of the HBLV-MOCK group.The difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.Transwell experiments showed that the number of cells migrated in the HBLV-hsa_circ₀005379 group was significantly lower than that in the HBLV-MOCK group,and the difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.Transwell with matrigel experiment showed that the number of migrating cells in the HBLV-hsa_circ₀005379 group was significantly lower than that in the HBLV-MOCK group,which was statistically significant（PSCC25<0.001,PCAL27<0.001）.The results of Hoechst staining showed that the number of apoptotic cells in the HBLV-hsa_circ₀005379 group was significantly higher than that in the HBLV-MOCK group,which the difference was statistically significant（PSCC25<0.001,PCAL27<0.001）.Western blot results showed that compared with the HBLV-MOCK group,the HBLV-hsa_circ₀005379 group showed significant changes in proliferative,invasive,apoptotic,and EMT-related protein expression levels.Knock down or overexpression of hsa_circ₀005379 will cause a significant change in the expression of EGFR.Increasing or inhibiting the phosphorylation of EGFR does not affect the expression of hsa_circ₀005379.The results of subcutaneous tumorigenesis in nude mice showed that overexpression of hsa_circ₀005379 inhibited the growth of OSCC in nude mice.HE staining showed that the differentiation of tumor cells in HBLV-hsa_circ₀005379 group was significantly higher than that of HBLV-MOCK group.Immunohistochemical staining results showed that changes of hsa_circ₀005379 expression will lead to the change in EMT and other related indicators.Annexin V-FITC/PI double labeling results showed that the early apoptotic rates in the HBLV-MOCK group were 0.31% and 0.43%,respectively,and the early apoptotic rates in the HBLV-hsa_circ₀005379 group were 1.12% and 0.91%,respectively.The early apoptotic rates in the HBLV-MOCK + Cetuximab group were35.77% and 15.22%,respectively.The early apoptotic rates in the HBLV-hsa_circ₀005379 + Cetuximab group were38.35% and 17.88%,respectively.Conclusion: hsa_circ₀005379 affects the malignant biological behavior of oral squamous cell carcinoma through the EGFR signaling pathway...