Mechanism Of SPRY4-IT1 On The Metastasis Of Esophageal Squamous And Its Roles As A Marker For Lymphadenectomy In Three Fields

Esophageal cancer(EC)is one of the deadliest malignant tumors worldwide.According to the 2018 Global Cancer Statistics,the incidence of esophageal cancer is seven among all malignant tumors and the mortality is six.It is different from European and American,esophageal squamous cell carcinoma is one of the most common cancer in China.Due to diagnostic difficulties,most patients with esophageal squamous cell carcinoma have a high rate of metastasis and recurrence,and the 5-year OS rate is about20%.Inhibition of esophageal squamous cell carcinoma is very important in the treatment and it has become the main focus of current research.Non-coding transcription is an important part of the human transcriptome,and long non-codingRNA is a subtype of non-codingRNA with a length of more than 200 nucleotides.Recently,evidence has shown that lncRNA plays an important regulatory role in the development of malignant tumors,for example,lncRNA 91 H increases the invasive phenotype of breast cancer cells,and lncRNA XIST promotes the growth,migration and invasion of bladder cancer cells.However,there are few reports on the role of lncRNA in the progression of esophageal squamous cell carcinoma.The underlying mechanism which lncRNAs play a role in ESCC has not been fully elucidated.LncRNA SPRY4-IT1 is a novel lncRNA that has been found to be abnormally expressed in some malignant tumors and has the function of an oncogene.However,its role in ESCC has not been reported.In this study,we found that lncRNA SPRY4-IT1 was abnormally expressed in ESCC tissues.Furthermore,we explored the molecular mechanism of lncRNA SPRY4-IT1 in the occurrence and development of ESCC.Part one The expression and function of lncRNA SPRY4-IT1 in esophageal squamous cell carcinoma.Objective:1.To investigate the expression of lncRNA SPRY4-IT1 in esophageal squamous cell carcinoma and paracancerous normal tissues.2.To investigate the expression level of lncRNA SPRY4-IT1 in esophageal squamous cell carcinoma and its relationship with the prognosis of patients.Methods: We got 92 patients with thoracic segment esophageal squamous cell carcinoma tissue and paired normal tissue adjacent to carcinoma.All patients underwent R0 esophagectomy in the Department of Thoracic Surgery of the Fourth Hospital affiliated to the Hebei Medical University and Hebei General Hospital between Jun 2011 and Dec 2015.Primary cancer tissues and paired adjacent noncancerous tissues(≥2cm from the edge of carcinoma)were snap-frozen in liquid nitrogen immediately after resection and then stored at-80°C forRNA extraction.No patient received chemotherapy,radiotherapy,immunotherapy and traditional Chinese medicine prior to specimen collection.To analyze the expression of SPRY4-IT1,PCR assay was performed on cancer tissues and paracancerous tissues.Results:1.Expression of SPRY4-IT1 in esophageal carcinoma and paracancerous tissuesThe expression of SPRY4-IT1 gene in ESCC tissue is higher than the corresponding normal tissue adjacent to cancer.92 pairs of tissues were detected by RT-PCR technology.The results showed that the expression in cancer tissues was significantly higher than that in normal tissues adjacent to cancer,and the expression was up-regulated by more than 2 times in 86.9%cases.According to the relative median of SPRY4-IT1 expression,92 ESCC patients were divided into two groups.The high expression of SPRY4-IT1 is related to tumor differentiation(P=0.026),T classification(P=0.010),lymph node metastasis(P=0.007)and clinical stage(P<0.005).However,there was no significant correlation with the patient’s age,gender,smoking status,drinking or tumor location(P> 0.05).These observations indicate that the increase of SPRY4-IT1 expression may have a certain correlation with the progress and development of ESCC.2.High expression level of SPRY4-IT1 is associated with poor prognosis of ESCC patients.The results of this study show that the expression level of SPRY4-IT1,lymph node metastasis,and clinical stage are significantly related to the overall survival rate of ESCC patients.The expression level of SPRY4-IT1,lymph node metastasis,and TNM staging are independent prognostic factors that affect the overall survival of ESCC patients.These results indicate that the expression level of SPRY4-IT1 in ESCC patients can be used as one of the prognostic factors for patients with esophageal cancer.Conclusions:1.In this study,the cancer tissues and adjacent tissues of ESCC patients were detected and analyzed,and it was found that SPRY4-IT1 was abnormally highly expressed in patients with esophageal cancer,which suggests that SPRY4-IT1 may become one of the biomarkers for early detection of esophageal cancer.2.SPRY4-IT1 is up-regulated in esophageal cancer tissues and ultimately affects the prognosis of patients,indicating that SPRY4-IT1 may be involved in the pathogenesis of esophageal cancer and play the role of oncogenes.Part two Experiment on the Regulation of ABCA1 by SPRY4-IT1Objective: To clarify the mechanism of lncRNA SPRY4-IT1 on the occurrence and metastasis of esophageal squamous cell carcinoma;further explore whether lncRNA SPRY4-IT1 regulates ABCA1,leading to changes in intracellular cholesterol content and cell membrane fluidity,and ultimately affect the lymph node metastasis of esophageal squamous cell carcinoma.Methods: The human esophageal cancer cell lines TE1,TE13,and Eca109 were all retained and passaged by the Cancer Institute of the Fourth Hospital of Hebei Medical University;bioinformatics methods,q RT-PCR and wetsern blotting were used to predict the downstream target genes of SPRY4-IT1.The expression level of ABCA1 in clinical samples of esophageal cancer was detected,and the ABCA1 overexpression plasmid and SPRY4-IT1 mimics were co-transfected into TE-1 cells.CCK-8 test,flow cytometry and western blotting were used to detect their influences on cell proliferation and protein expression.Results: The expression of SPRY4-IT1 in esophageal cancer tissue was up-regulated by q RT-PCR detection,and the expression of ABCA1 was also increased;the expression of ABCA1 and SPRY4-IT1 in esophageal cancer tissue was roughly synergistically correlated.Dual luciferase method,q RT-PCR,flow cytometry and western blotting found that SPRY4-IT1 binds to the 3’-UTR site of ABCA1.After transfection of SPRY4-IT1 mimics,the mRNA and protein expression levels of ABCA1 increased significantly;after transfection of SPRY4-IT1 inhibitor,the mRNA and protein expression levels of ABCA1 decreased significantly.Conclusions: SPRY4-IT1 binds to the 3’-UTR of ABCA1 gene,and there is a signal pathway regulation relationship.SPRY4-IT1 promotes the proliferation of esophageal cancer cells and inhibits cell apoptosis by enhancing the expression of ABCA1.Part three Study on SPRY4-IT1 as an evaluation index for three-field lymph node dissectionObjective:1.To study the correlation between the expression of lncRNA SPRY4-IT1 in esophageal squamous cell carcinoma and survival.2.To study the correlation between lncRNA SPRY4-IT1 and ESCC lymph node metastasis and poor prognosis,and to guide the most suitable strategy for lymph node dissection of ESCC.Methods: The Real-time PCR method was used to detect 92 cases of esophageal cancer tissues and corresponding normal tissues adjacent to cancer in the thoracic surgery department of the Fourth Affiliated Hospital of Hebei Medical University and Hebei General Hospital from June 2011 to December2015.The relationship was analyzed between SPRY4-IT1 expression level and lymph node metastasis,pathological characteristics and patient prognosis.Results: Statistical analysis of clinical data found that the high expression of SPRY4-IT1 was related to tumor differentiation,T classification,lymph node metastasis and pathological stage,but not to the patient’s age,gender,smoking status,drinking,tumor location,and lymph node recurrence.Further analysis of the relationship between the expression level of SPRY4-IT1 and lymph node metastasis found that the increase in its expression level is related to the increased risk of cervical and upper mediastinal lymph node metastasis.According to the follow-up results,there is no significant correlation between the risk of recurrence of cervical and upper mediastinal lymph nodes and the expression level of SPRY4-IT1.Conclusions: Our data supports the hypothesis that the high expression of SPRY4-IT1 is associated with a high risk of lymph node metastasis,and it has potential application value in guiding the three-field lymph node dissection in patients with thoracic esophageal squamous cell carcinoma.In the future,a prospective randomized controlled trial with a large sample of patients is needed to confirm this conclusion.