Part Ⅰ:Molecular Mechanism Of RASSF6 In Promoting Esophageal Squamous Cell Carcinoma Cell Proliferation,Migration And Invasion Part Ⅱ:The Function And Molecular Mechanism Of LncRNA-581 In Esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the common malignant tumors in China.The 2018 China Cancer report showed that the incidence and mortality of esophageal cancer ranked the sixth and fourth in China.There are two pathological types of esophageal cancer,esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma,the former is the main type in China.In spite of the development of diagnosis and treatment technology,the pathogenesis of ESCC is concealed,its progress is rapid,and the five-year survival rate is only 10-30%.Therefore,it is of great significance to study the mechanism of ESCC and to screen the molecular markers and effective drug targets of esophageal cancer.Ras-associated domain family protein 6(RASSF6)is one member of the Ras associated domain protein family.In colon cancer,breast cancer and melanoma,the expression of RASSF6 was significantly reduced due to epigenetic modification.RASSF6 could affecte the growth and apoptosis of tumor by regulating FAK,Hippo and other signaling pathways.However,our transcriptomics sequencing data in the early stage showed that the expression of RASSF6 in ESCC was increased abnormally,which was different from that in other cancers.Therefore,we study the function and mechanism of RASSF6 in the development of ESCC.In this study,we first tested the expression of RASSF6 in 43 pairs of ESCC tissue.The results showed that the mRNA and protein expression levels of RASSF6 in ESCC were significantly higher than that of adjacent tissues.The subsequent immunohistochemical staining of 84 esophageal squamous cell carcinoma tissues showed that the expression of RASSF6 in ESCC was increased,and its abnormal expression was significantly correlated with the adverse prognosis of patients with ESCC.Then we conducted CoBRA and found no methylation in the promoter region of RASSF6 in ESCC tissues.The vitro functional experiments showed that overexpression of RASSF6 promoted the proliferation,colony formation and migration and invasion of ESCC cells,while knockdown of RASSF6 inhibited the proliferation,colony formation,invasion and migration of ESCC cells,and inducing the Gl/S phase arrest.Animal experiments showed that the abilities of subcutaneous tumor formation and lung metastasis of ESCC were significantly attenuated after knockdown of RASSF6.We found that RASSF6 bound to the tumor suppressor gene TRIM16 by immunoprecipitation combined with mass spectrometry.Further studies have shown that RASSF6 could promote the ubiquitination process of TRIM16,thereby affecting the stability of TRIM16 protein.Mechanism studies have found that RASSF6 could affect the expression of Cyclin D1 and Snail by regulating TRIM 16,and promoted the proliferation and migration and invasion of ESCC cells.At the same time,TRIM 16 partially restored the effect of RASSF6 on the malignant phenotype of ESCC.In summary,this study found that RASSF6 was abnormally highly expressed in ESCC for the first time.Furthermore,RASSF6 could promote the development of ESCC by combining TRIM 16.All of these provide a new idea for the diagnosis and treatment of ESCC.Esophageal cancer is one of the common malignant tumors in China.The 2018 China Cancer report showed that the incidence and mortality of esophageal cancer were respectively rank sixth and fourth in China.There are two pathological types of esophageal cancer:esophageal squamous cell carcinoma and esophageal adenocarcinoma.Most patients with esophageal cancer in China are esophageal squamous cell carcinoma.Despite the development of diagnosis and treatment technology,the pathogenesis of esophageal squamous cell carcinoma is concealed and the progress is rapid,the five-year survival rate is still maintained at the lower level of the scale.Therefore,it is of great clinical significance to study the mechanism of the occurrence and development and to screen the molecular markers and effective drug target of esophageal cancer.Long-chain non-encoded RNA(Long non-coding RNA,LncRNA)is a class of non-encoding RNA transcribed by RNA polymerase Ⅱ,longer than 200nt in length,and with little protein encoding ability.The kind of it is abundant,the sondary structure of it is complicated,and the expression is organizationally specific.Recent studies have shown that LncRNA can regulate gene expression at multiple levels through its own folding or binding DNA,RNA or protein,and affect the proliferation,metastasis,metabolism and apoptosis of tumor cells.In the early work,91 cases of esophageal squamous cell carcinoma and paired adjacent tissues were sequenced by transcription group.Comparing the two groups of data,it was found that the expression of LncRNA-581 in esophageal squamous cell carcinoma was upregulated,and there was no report about it.The NONCODE database shows that LncRNA-581 has four transcription copies and only expresses in placental tissue.The UCSC database shows that the expression of LncRNA-581 in esophageal cancer is increased without protein coding ability.The qPCR of 85 patients with esophageal carcinoma and adjacent tissues isolated from the transcription group sequencing samples showed that LncRNA-581 was highly expressed in esophageal squamous cell carcinoma.The results of RACE and Northern Blot showed that LncRNA-581 in esophageal squamous cell carcinoma was 668nt in length.LncRNA-581 was located in the cytoplasm.Cell function experiments showed that LncRNA-581 promoted the proliferation,cloning,and invasion and migration of esophageal squamous cell carcinoma.It was found that the tumor forming ability of esophageal squamous cell tumor was weakened after stably knocking down LncRNA-581.Preliminary mechanism studies have found that LncRNA-581 can combine and influence the expression of HNP-1,which in turn affected the polarization process of M1 macrophages.The above studies showed that LncRNA-581 was upregulated in esophageal squamous cell carcinoma.It could combine and influence the expression of HNP-1,affecting the polarization of monocytes to macrophages,and then promoted the malignant process of esophageal squamous cell carcinoma.We will further explore the effects of LncRNA-581 on the immune escape of esophageal squamous cell carcinoma.