Study On The Effect Of NSG1 On The Biological Behavior Of Esophageal Squamous Cell Carcinoma And Its Mechanism

Background and ObjectivesAs a highly invasive tumor,esophageal squamous cell carcinoma(ESCC)development often leads to lymph node metastasis,showing high malignancy.It is the sixth leading cause of cancer death worldwide and does a great harm to human health.At present,the overall therapeutic effect of most treatments for ESCC are still poor.Therefore,to further explore the mechanism of the occurrence and development of ESCC and identify novel molecular targets related to ESCC for improving the survival rate and prognosis of ESCC patients is essential.Our study found that there are a large amount of autoantibodies against neuron specific gene family member 1(NSG1)in serum of ESCC patients.NSG1 is a 21 k Da endosomal protein specifically expressed by neuronal cells and is highly enriched in neurons.Currently,the study on NSG1 mainly focuses on its role on neural development and regulation,while little attention has been paid to the correlation between NSG1 and malignancies.The results of immunohistochemistry staining showed that NSG1 protein was highly expressed in the cancer tissues of ESCC patients,prompt that the aberrant expression of NSG1 in cancer tissues might lead to a large amount of release of autoantibodies against NSG1 in the serum of ESCC patients,whether aberrant expression of NSG1 is involved in the occurrence,development process of ESCC is worthy of being discussed,therefore,our study transfected si RNA which was targeted NSG1 into ESCC cells with relatively high expression of NSG1 and found that the knockdown of NSG1 can obviously inhibit cell viability,migration and invasion of the ESCC cells and decreased the phosphorylation level of ERK protein which is the key effector molecule of MEK/ERK signaling pathway that closely related to epithelial mesenchymal transition.On the basis of this study,we plans to(1)constructed lentivirus expression system for NSG1 in ESCC cells,and detect the influence of NSG1 overexpression on the cell viability,migration and invasion of ESCC cells(2)western blot was used to detect the the phosphorylation level of MEK,ERK of MEK/ERK signaling pathway and molecular markers of EMT in ESCC cells.To explore whether NSG1 affects the EMT of ESCC cells via the MEK/ERK signaling pathway,and participates in the occurrence and development of ESCC.Methods1.The expression of NSG1 protein in 20 pairs of ESCC cancer tissues and matched paracancerous tissues were detected by immunohistochemistry staining.2.The expression of NSG1 m RNA and protein of five ESCC cell lines including TE-1,KYSE-150,KYSE-410,KYSE-30 and ECA-109 were detected by RT-q PCR and western blot,respectively,and the cell lines with relatively low and high expression of NSG1 were applied for the subsequent experiments.3.The expression of NSG1 in ESCC cells with relatively poor NSG1 expression was enhanced by the constructed lentivirus expression system for NSG1,while the expression of NSG1 in ESCC cells with relatively high NSG1 expression was knocked down by the si RNA for NSG1.The m RNA and protein expression level of NSG1 in transfected cells were determined by RT-q PCR and western blot.4.CCK-8 assay was used to detect the influence of NSG1 overexpression/knockdown on the viability of ESCC cells5.The effect of NSG1 overexpression/knockdown on migration of ESCC cells was detected by transwell assay and the wound healing assay.6.The effect of NSG1 overexpression/knockdown on invasion of ESCC cells was detected by transwell assay.7.The effect of NSG1 overexpression/knockdown on ESCC cell cycle distribution was evaluated by the flow cytometry assay.8.Western blot was used to detect the the expression level of p-MEK,p-ERK of MEK/ERK signaling pathway and molecular markers of EMT in ESCC cells.Results1.The positive rate of NSG1 protein expression in ESCC cancer tissues was 90%(18/20),which was significantly higher than 5%(1/20)in matched paracancerous tissues(P<0.01).2.The results of RT-q PCR and western blot showed that NSG1 was relatively highly expressed in TE-1 cells,while relatively poor expressed in KYSE-150,KYSE-30,KYSE-410 and ECA-109 cells,and the expression level of NSG1 in KYSE-150 was the lowest.3.The results of RT-q PCR and western blot showed that the expression of NSG1 m RNA and protein were significantly enhanced in KYSE-150 cells while transfected the constructed lentivirus expression system for NSG1.si RNA-NSG1 was transfected into TE-1 cells to reduced the the expression of NSG1 m RNA and protein.4.Compared with negative control group(NC),the vilitialy,invasion and migration of KYSE-150 cells with overexpression of NSG1 were significantly enhanced,the cell percentage in G0/G1 phase were significantly reduced(P<0.001)followed by the cell percentage in S phase was significantly increased(P<0.001),On the other hand,compared with NC group,the viability,invasion and migration were significantly inhibited,the cell percentage in G0/G1 phase were significantly increased(P<0.01)followed by the cell percentage in S phase was significantly reduced(P<0.01).5.Compared with NC group,the expression of p-MEK and p-ERK in MEK/ERK signaling pathway were significantly upregulated,the expression of EMT-related transcription factors snail,slug as well as mesenchymal marker vimentin were significantly upregulated,and the expression of epithelial marker E-cadherin was significantly downregulated in KYSE-150 cells with overexpression of NSG1.On theother hand,the expression of p-MEK and p-ERK in MEK/ERK signaling pathway were significantly downregulated,the expression of EMT-related transcription factors snail,slug as well as mesenchymal marker vimentin were significantly downregulated,and the expression of epithelial marker E-cadherin was significantly upregulated in TE-1 cells with knockdown of NSG1.Conclusion1.NSG1 is highly expressed in the cancer tissues of ESCC patients and aberrant expression of NSG1 can enhance the viability,migration,and invasion of ESCC cells and promote the transformation of G1/S phase,suggesting that the aberrant expression of NSG1 is closely related to the malignant biological behavior of ESCC cells,prompting that NSG1 may play a potential carcinogenic role in the occurrence and development of ESCC disease.2.The expression of NSG1 may promote the EMT of ESCC cells by activating the MEK/ERK signaling pathway,and then participate in the occurrence and development process of ESCC.