Expression Of LncRNA TUSC7 In Serum Of Patients With Esophageal Squamous Cell Carcinoma And Its Effect On Tumor Cell Invasion And Metastasis

The esophageal cancer(EC)is a kind of malignant tumor with high incidence in the digestive tract,and has the higher metastasis and invasion ability.According to the histological types,the esophageal squamous cell carcinoma(ESCC)and the esophageal adenoeareinoma(EAC)have both had higher incidence in China,with the majority of the countries being ESCC.The early symptoms of esophageal squamous cell carcinoma are atypical and most patients have advanced metastasis at the time of initial diagnosis,resulting in a 5-year survival rate of less than 10% in patients with esophageal squamous cell carcinoma.Therefore,it is of great significance to explore and screen new effective biomarkers for early or prognostic metastasis of esophageal squamous cell carcinoma.Long non-coding RNAs(LncRNAs)are nucleotide molecules with a length of more than 200 bp,encoding little or no proteins.With the large-scale application of high-throughput sequencing technology and LncRNA chip detection,a large number of LncRNAs have been found in tumor tissues and body fluids,playing a key role in the occurrence and development of tumors as oncogenes or tumor suppressor genes.In recent years,more and more studies have shown that LncRNAs are involved in the invasion and metastasis of a variety of malignant tumors,including lung cancer,bladder cancer,gastric cancer,colorectal cancer and esophageal squamous cell cancer.Therefore,the exploration of LncRNAs related to esophageal squamous cell carcinoma has become one of the important topics in this field.LncRNA TUSC7 is also known as limbic system-associated membrane protein antiRNA3(LSAMP Antisense RNA3;LOC285194;Gene ID: 285194),2105 bp long,composed of four exons,located on chromosome 3Q13.31,belonging to anti-lncrNA,Pasic et al.first proposed it as the main tumor suppressor Gene for the treatment of malignant tumor osteosarcoma in 2010.In recent years,many clinical studies have shown that TUSC7 is low-expressed in a variety of malignant tumors and has the effect of tumorsuppressor gene,which is closely related to clinicopathological characteristics and prognosis.However,whether TUSC7 is differentially expressed in the serum of patients with esophageal squamous cell carcinoma and what biological function it plays in the process of esophageal squamous cell carcinoma metastasis have not been reported.LncRNA TUSC7 is also known as limbic system-associated membrane protein antiRNA3(LSAMP Antisense RNA3;LOC285194;Gene ID: 285194),2105 bp,consisting of four exons,located at chromosome 3q13.31.Pasic and others first proposed it as a major tumor suppressor gene for the treatment of malignant tumor osteosarcoma in 2010.In recent years,many clinical studies have shown that TUSC7 is low-expressed in a variety of malignant tumors and has the effect of tumor suppressor gene,which is closely related to clinicopathological characteristics and prognosis.However,whether TUSC7 is differentially expressed in the serum of patients with esophageal squamous cell carcinoma and what biological function it plays in the process of esophageal squamous cell carcinoma metastasis have not been reported.In this study,Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression level of LncRNA TUSC7 in serum of 60 patients with esophageal squamous cell carcinoma and 60 healthy subjects and analyze its relationship with clinicopathological characteristics.Meanwhile,the expression level of TUSC7 in four esophageal squamous cell lines(KYSE-30,EC-9706,KYSE-150,ECa-109)was detected by using normal esophageal epithelial cells as the control,and a highly expressed strain of esophageal squamous cell line KYSE-30 was selected as the object of subsequent in vitro experiments.Further,by overexpressing or inhibiting TUSC7 in vitro,the effects of TUSC7 on the proliferation,invasion and migration of ESCC cells were detected by MTT assay,cell trace assay and Transwell cell invasion assay,and the effects of EMT markers(MMP-9,E-cadherin,N-cadherin and Vimentin)were detected by Western blot.It provides a theoretical basis for further study of the mechanism of TUSC7 in ESCC invasion and metastasis.The main results are as follows:1.The expression level of LncRNA TUSC7 in serum of ESCC patients was lower than that of healthy control group(P<0.05).Based on the cut-off point of TUSC7 medianexpression level,60 patients were divided into TUSC7 high expression group(n=30)and TUSC7 low expression group(n=30).TUSC7 low expression group was significantly correlated with TNM stage and more lymph node metastasis and local infiltration(P<0.01).2.Among the four types of human esophageal squamous cell carcinoma cells,TUSC7 expression was down-regulated in 3 strains(ECa-109,EC-9706,KYSE-30)and normal esophageal epithelial cells(P<0.05),but there was no significant difference in KYSE150(P>0.05).3.qRT-PCR detection of KYSE-30 cells after overexpression or inhibition of TUSC7 showed that the relative expression of TUSC7 was significantly increased in the plenti-TUSC7 transfection group.The relative expression of TUSC7 was significantly decreased in the Si-TUSC7 transfection group.4.MTT assay showed that the proliferation capacity of KYSE-30 cells transfected with Plenti-TUSC7 was significantly inhibited at 24 h and 48h(P<0.01).The proliferation of KYSE-30 cells in the Si-TUSC7 transfection group was significantly enhanced at 24 h and 48h(P<0.05).5.Cell scratch and transwell cell invasion experiments showed that the migration and invasion ability of KYSE-30 cells in the transfected Plenti-TUSC7 group was significantly lower than that in the plenti-NC group(P<0.05).The migration and invasion of KYSE-30 cells in the Si-TUSC7 transfection group were significantly higher than those in the Si-NC group(P<0.05).6.Westen blot showed that overexpression of TUSC7 significantly increased E-cadherin expression in KYSE-30 cells compared with plenti-NC control group(P<0.01),while decreased expression of MMP-9,N-cadherin and Vimentin in KYSE-30 cells(P<0.01).Compared with the control group of Si-NC group,E-cadherin expression was significantly down-regulated in the Si-TUSC7 group(P<0.01),while MMP-9,N-cadherin and Vimentin protein expression were significantly up-regulated in the Si-TUSC7 group(P<0.05).In summary,TUSC7 was down-regulated in ESCC serum and cell lines,significantly associated with TNM stage and more lymph node metastasis and local infiltration,andpromoted tumor cell migration and invasion through EMT.