Effects And Its Molecular Mechanism Of TROP2 On Malignant Biological Behaviors Of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC)is the most common malignant tumor in the oral and maxillofacial cancer.Its occurrence is polygenic and multistage,but the exact mechanism is still unclear.Early cervical metastasis,local recurrence and distant metastasis are the leading causes of death.At present,the main treatment methods are surgical treatment,chemotherapy and radiotherapy.Although the technology has improved,the fiveyear survival rate of tumor patients is still low.Oral surgery often cause facial damage,so the patient's quality of life decreased significantly.Therefore,it is urgent to find new tumor treatment methods,tumor markers for molecular diagnosis and prognosis prediction.Some studies have found that TROP2 is generally highly expressed in tumor cells of gastric cancer,cervical cancer,esophageal cancer and can promote abnormal cell proliferation,metastasis and invasion.However,the role of TROP2 in OSCC invasion and metastasis remains unclear.Our study will discuss the specific role of TROP2 in OSCC in vitro and in vivo experiments.The molecular mechanism of PI3K/Akt signaling pathway is further explored.Objectives(1)To observe TROP2 expression in OSCC tissues and precancerous tissues,and analyze the relationship between TROP2 expression and clinicopathological factors and prognosis.(2)To observe the malignant biological behavior influence of TROP2 in vivo and in vitro for OSCC cell proliferation,apoptosis,migration and invasion.(3)To investigative the molecular mechanisms of TROP2 in malignant biological behaviors of OSCC.Methods 1.The expression of TROP2 in OSCC tissues was detected by qRT-PCR and immunohistochemistry staining and analysis the correlation with clinicopathological factorsThe expression of TROP2 mRNA was detected by qRT-PCR in 31 fresh postoperative frozen tissues of OSCC,precancerous lesions and adjacent tissues.Immunohistochemical methods were used to detect the expression of TROP2 in OSCC tissues,precancerous lesions tissues and paracancerous tissues.The correlation between TROP2 expression and the clinical staging and prognosis of OSCC was investigated by combining the clinicopathological data.GSEA v2.2.2 software(http://www.broadinstitute.org/gsea)was used to analysis the biological pathways,and a composed network of TROP2 predicted protein interactions was builded.2.TROP2 overexpression and knockdown plasmid was constructed and identifiedTROP2 overexpressed plasmid and knockdown plasmid were synthesized.The constructed plasmids were transformed and the plasmids were extracted.Part of the colony was sent to sequence to confirm whether the TROP2 sequence was correctly cloned into the vector.3.Effects of TROP2 on proliferation,migration,invasion,apoptosis and cell cycle of oral squamous cell carcinoma cells were observed in vitro and in vivoThe influence of TROP2 in vivo and in vitro for OSCC cell proliferation,apoptosis,migration and invasion malignant biological behaviors was observed.(1)The expression of TROP2 was detected by Western Blot,qRT-PCR and FACS in OSCC cells,then the low expression and high expression cell lines of TROP2 were selected.(2)Fluorescence microscopy,qRT-PCR and Western Blot were used to detect the transfection efficiency of oral squamous cell carcinoma cell lines with high TROP2 expression after TROP2 knockdown and overexpression of TROP2 in oral squamous cell carcinoma cell lines with low TROP2 expression.(3)The effects of shRNA-TROP2 and OE-TROP2 on the proliferation of OSCC cells were observed by CCK 8 experiments.(4)The effects of shRNA-TROP2 and OE-TROP2 on the migration and invasion of OSCC cells were observed by scratch experiment,transwell chamber migration experiment and transwell chamber invasion experiment.(5)The effects of shRNA-TROP2 and OE-TROP2 on apoptosis and cell cycle of OSCC cells were investigated by FACS.(6)The effects of shRNA-TROP2 were observed on subcutaneous xenograft growth of OSCC cells in vivo.4.The possible molecular mechanism of TROP2 on the malignant biological behaviors of OSCC malignancy was investigatedThe effects of shRNA-TROP2 and OE-TROP2 on PI3K/Akt pathway of protein expression were dected by Western Blot,including Akt,PTEN,p-Akt,PI3K-P85 and PDK1.Results 1.TROP2 expression in oral tissues and its relationship with prognosis of oral squamous cell carcinomaTROP2 mRNA expression in OSCC was higher than that in oral precancerous tissues(P=0.008)and adjacent normal tissues(P<0.001).The positive expression rate of TROP2 in OSCC tissues was significantly higher than that in paracancerous tissues and precancerous tissues,and the differences were statistically significant(P<0.05).TROP2 expression was associated with tumor differentiation,lymph node metastasis,TNM stage,nerve infiltration and vascular infiltration.High TROP2 expression was associated with poor overall survival and disease-free survival.Multivariate analysis showed that TROP2 was an independent prognostic factor of OSCC.The results of bioinformatic analysis suggested that TROP2 was related to CCN1 through ATF2 and acted with PDK1.PIK3R1/PTEN/AKT1 taked PDK1 as the center and interacted with each other.2.Effects of TROP2 on proliferation,migration,invasion,apoptosis and cell cycle of oral squamous cell carcinoma cellsCell experiments showed that high TROP2 expression promoted cell proliferation,migration,invasion and inhibited apoptosis.The proportion of S phase cells was increased obviously.Knockdown TROP2 inhibited cell proliferation,migration,invasion and promoted apoptosis.The proportion of S phase cells was decreased obviously.In vivo,subcutaneous xenograft tumor growth was significantly inhibited in nude mice after TROP2 knockdown.3.Effects of TROP2 on protein expression in the PI3K/Akt signaling pathwayshRNA-TROP2 inhibited the expression of p-Akt,PI3K-P85 and PDK1 proteins in the PI3K/Akt signaling pathway,while PTEN expression was enhanced and Akt expression remained unchanged.OE-TROP2 activated the expression of p-Akt,PI3K-P85 and PDK1 in the PI3K/Akt signaling pathway,while the expression of PTEN was decreased and the expression of Akt remained unchanged.Conclusions1.TROP2 was highly expressed in OSCC tissues and associated with tumor differentiation,lymph node metastasis,TNM stage,nerve infiltration and vascular invasion.TROP2 was an independent prognostic factor of OSCC.2.TROP2 promoted the proliferation,migration and invasion of OSCC cells,inhibited apoptosis and induced S phase arrest.TROP2 interference inhibited tumor growth in nude mice.3.TROP2 may effect malignant biological behaviors of OSCC cells including proliferation,invasion,metastasis,apoptosis through the PI3K/Akt signaling pathway,thus promoting the growth and metastasis of OSCC.