The Effect And Mechanism Of Sestrin2 On Oral Squamous Cell Carcinoma Through MTOR And MAPK Pathway Regulation

Objective: The purpose of this study is to investigate the biological effects of Sestrin2(Sesn2)on the proliferation,migration,invasion,and apoptosis of oral squamous cell carcinoma(OSCC)cell lines while preliminarily exploring the mechanisms based on the PI3K/AKT/mTOR and MAPK pathways.Methods: Firstly,RNA-seq-related data to Sesn2 in normal tissues and oral squamous cell carcinoma projects were collected from The Cancer Genome Atlas(TCGA),differential analysis was performed on paired and unpaired samples.Meanwhile,immunohistochemical staining images were obtained from The Human Protein Atlas(HPA)database to evaluate the expression and potential role of Sesn2 in OSCC.Then,small interfering RNA(si RNA)and lentivirus transfection techniques were used to knockdown and overexpress the Sesn2 gene,respectively.Subsequently,the effects of Sesn2 on cell proliferation,toxicity,and viability were measured by CCK-8 and Brdu assay in the CAL27 and TSCCA cell lines.The effects of Sesn2 on the cell cycle distribution of CAL27 and TSCCA cells were detected through a flow cytometry.The Trans-well and cell scratch assays were used to reflect the effects of Sesn2 on the in vitro migration and invasion abilities of CAL27 and TSCCA cell lines.Hoechst33342 staining and Caspase 3 enzyme activity detection were used to evaluate the effects of Sesn2 on the in vitro apoptosis of both cell lines by observing changes in nuclear morphology and enzyme activity.Finally,the expression levels of key molecular proteins in the PI3K/AKT/mTOR and MAPK pathways were detected by Western Blot.Results:(1)According to the analysis of RNA-seq data,the expression of Sesn2 in OSCC was 10.6% higher than that in normal tissues in the analysis of unpaired samples(P<0.001).In the difference analysis of paired samples,the expression of Sesn2 in OSCC was 12.5% higher than that in normal tissues(P<0.01).At the same time,the results of immunohistochemical staining images obtained from HPA database showed that the expression level of Sesn2 was higher in head and neck squamous cell carcinoma.(2)According to the results of CCK-8 and Brdu detection,the knockdown of Sesn2 inhibited the proliferation of CAL27 and TSCCA cells in vitro,which caused that the proportion of G0/G1 in the cycle decreased while that of G2/M phase increased without significant change in S phase.At the same time,Trans-well assay and Cell Scratch assay showed that the inhibition of Sesn2 expression significantly reduced the migration and invasion ability of both cell lines.In addition,the apoptotic bodies and Caspase 3enzyme activity of CAL27 and TSCCA cells increased with the increase of apoptosis.However,the biological behaviors of CAL27 cells after overexpression of Sesn2 were on the contrary.(3)The protein expression levels of PI3 K,P-AKT,P-ERK,P-P38,mTOR,MMP-2,MMP9 and MMP-7 decreased while the expression of Caspase 3 protein increased after Sesn2 being knockdown.However,it is the opposite while there was no significant change in BAX/BCL-2 after overexpression and knockdown of Sesn2.Conclusion:Sesn2 may play a stimulative role in the development and progression of oral squamous cell carcinoma through PI3K/AKT/mTOR and MAPK pathways.