Autophagy Regulation Gene DRAM1 Promotes Proliferation And Metastasis Of OSCC

Objective:To screen the autophagy regulatory genes with the largest multiple related to the prognostic of Oral Squamous Cell Carcinoma(OSCC),and to investigate the effects of autophagy regulatory genes on the proliferation and metastasis of OSCC.Methods:1)The Cancer Genome Atlas(TCGA)database were used to collect the m RNA expression data and clinical data of OSCC patients.Screen the differential expression signal pathways between 330 OSCC samples and32 normal tissue samples by GSVA.The effect of autophagy level on lymph node metastasis and poor prognosis in 260 OSCC patients was investigated by differential analysis.2)Differential analysis was conducted to explore the expression differences of all autophagy related regulatory genes in OSCC tissues and normal tissues.The autophagy risk assessment model was constructed by Lasso regression analysis to screen out autophagy regulatory genes related to OSCC prognosis.The factors with the largest multiple of difference were used as research subjects to explore the changes in their expression levels and their relationship with patients’ age,gender,TNM grade,pathological grade and survival rate in TCGA database.3)The changes of DRAM1 expression level in 30 OSCC tissues and12 paracancer tissues and its relationship with 30 OSCC patients’ age,gender,TNM grade,pathological grade and survival rate were investigated by immunohistochemistry.4)The level of autophagy in OSCC cells was detected by real-time Fluorescent Quantitative PCR(q RT-PCR)and Western Blot(WB).5)OSCC cell lines with stable DRAM1 knockdown were constructed,and the effect of DRAM1 knockdown on autophagy level of OSCC cells was verified by WB,and the effect of DRAM1 expression on proliferation,migration and invasion ability of OSCC cells was explored by in vitro functional experiment.Results:1)By GSVA and difference analysis,autophagy regulation score was significantly higher in OSCC tissues than in normal tissues,and was positively correlated with lymph node metastasis and poor prognosis.2)Compared with normal tissues,6 autophagy regulation genes were down-regulated and 40 genes were up-regulated in OSCC tissues;Six prognostic autophagy regulation genes were screened by constructing autophagy risk assessment model,among which DRAM1 had the largest multiple of difference,and its expression was significantly positively correlated with pathological grade and lymph node metastasis,while negatively correlated with five-year survival rate.3)Immunohistochemical analysis showed that DRAM1 was highly expressed in OSCC tissues compared with paracancer tissues,and its expression was significantly positively correlated with pathological grade and lymph node metastasis,and negatively correlated with five-year survival rate.4)The expression of autophagy related factors(P62,BECLIN1,LC3B)was up-regulated in OSCC cell lines(CAL27 and SCC4)compared with normal oral keratinocytes(HOK).5)DRAM1 was down-regulated in OSCC cell lines(CAL27 and SCC4),autophagy related factor P62 was up-regulated,and BECLIN1 and LC3B were significantly down-regulated.At the same time,CCK8 showed that DRAM1 knockdown resulted in down-regulation of cell proliferation.Scratch test and Transwell test showed that DRAM1 inhibited cell migration and invasion.Conclusion:1)Autophagy was significantly increased in OSCC,which was positively correlated with the poor prognosis of OSCC.2)DRAM1 is an autophagy regulatory gene associated with the prognosis of OSCC,and is highly expressed in OSCC tissues and cells,leading to poor prognosis of OSCC patients by promoting the proliferation and metastasis of OSCC.