Study On The Function Of Long Non-coding RNA LINC00680 In The Proliferation And Invasion Of Esophageal Squamous Cell Carcinoma And Its Correlation With The Prognosis And Micrometastasis

Background As we know,esophageal cancer is one of the most common malignant tumors in the world.According to the reported studies,among the malignant tumors,the incidence rate of esophageal cancer ranks the 8th and the mortality ranks the 6th.Incidence rate of esopahgeal cancer of male is significantly higher than that of female.There are about 250000 newly diagnosed cases of esophageal cancer in China every year,accounting for more than half of the new cases in the world.Esophageal squamous cell carcinoma(ESCC)is the most common pathological type in China and even in East Asia.The pathological types and common sites of esophageal squamous cell carcinoma and esophageal adenocarcinoma are different,and their risk factors,biological behavior,common sites and treatment sensitivity are also different.Therefore,relevant studies on esophageal squamous cell carcinoma are currently in full progress,and it is expected to find the unique molecular biological mechanism of the occurrence and development of Asian esophageal squamous cell carcinoma and its biological behavior.Esophageal squamous cell carcinoma also associated with poor prognosis.Lymphatic metastasis at early stage of esophageal squamous cell carcinoma may be one of the most important reasons leading to poor prognosis.In recent years,it has been reported that with the rapid development of sequencing technology,researchers have found that the coding sequence of proteins accounts for a very small proportion,and only less than 1-2% of the whole human genome is involved in proteome coding.For a long time,the function of non-coding RNAs was neglected because they were not directly involved in the proteome coding.Since it was found that non-coding RNAs are involved in the P53 pathway and also participate in the biological regulation process,more and more studies have focused on non-coding RNAs.Long non-coding RNA(lnc RNA)is a special type of RNA molecule.It refers to RNA molecules with a length of over 200 nucleotides in the transcript,which do not directly participate in the coding of proteins,or only participate in the coding of short polypeptides.Compared with RNA related to protein coding,the research on lnc RNA has just started,and the research on gene expression of lnc RNA and its role in tumorigenesis and development will continue to be deepened.It is reasonable to believe that lnc RNA may be another star molecule after the coding RNA.In the field of esophageal cancer research,according to literature reports,there have also been some studies on the involvement of lnc RNA in various processes of tumor occurrence and development.For example,some researchers have identified that lnc RNA regulates the biological process of esophageal cancer by regulating the methylation level of downstream target genes.Some researchers have proved that certain lnc RNA could be used in early screening and diagnosis of esophageal cancer as a potential biomarker.Some researchers have proved that certain lnc RNA was associated with the proliferation,invasion and adhesion of tumor cells.Some researchers have found that high expression of lnc RNA related to esophageal squamous cell carcinoma can promote the invasion and metastasis of esophageal cancer cells and induce drug resistance in tumor cells.These results show that during the occurrence and development of esophageal cancer,the expression level of some lnc RNAs changes characteristically.These lnc RNAs regulate the expression of downstream target genes by regulating some important signal transduction pathways,thus affecting the occurrence and development of esophageal cancer.Nowadays,the study of cancer pathogenesis with sequencing technology is a hot topic in the field of esophageal cancer research.In our previous work,we completed second generation sequencing of esophageal squamous carcinoma tissues and its corresponding normal tissue adjacent to carcinoma.The expression levels of lnc RNAs were compared.The screening of esophageal squamous carcinoma samples found significant changes have taken place in the expression levels of lnc RNAs,suggesting that the lnc RNAs with different expression levels may play an important role in occurrence or development of esophageal cancer.So we proposed using molecular biology and cell biology experiment,culturing esophageal squamous cancer cells by in vitro system,establishing animal model of esophageal squamous carcinoma,collecting medical records of esophageal cancer patients,to explore the function of differentially expressed lnc RNA in the proliferation and invasion of ESCC,and to study its relationship with the prognosis.In the study,we expect to further reveal the mechanisms of occurrence and development in esophageal squamous cell carcinoma.We expect to provide effective and specific biomarkers for the diagnosis,treatment or prognosis of esophageal squamous cell carcinoma,and provide new directions for its clinical diagnosis and treatment if it is possible.Methods In this study,42 pairs of ESCC and matched adjacent non-tumor tissue samples from ESCC cases were used,and the non-tumor tissue samples were taken from the adjacent normal tissues of the corresponding patients with autologous cancer.All patients included in the study got histopathology,clinical diagnosis and underwent surgical treatment in our hospital.All patients received thoracoscopic combined laparoscopic esophagectomy and left cervical anastomosis,followed by thoracic and abdominal 2-field lymph node dissection or cervical,thoracic and abdominal three-field lymph node dissection.Patients did not receive any antitumor therapy before surgery.Clinical information was collected from medical records.Survival data were determined by outpatient and telephone follow-up.We used total RNA extraction reagent TRIzol reagent to isolate and extract total RNA from specimens.The quality control of RNA samples was done.Second generation sequencing was performed.By establishing of c DNA library and bioinformatics analysis and processing of lnc RNA sequencing data,differential expression of lnc RNA was screened.We used q RT-PCR to detect the expression levels of lnc RNAs.Nucleoplasmic separation assay was used to verify the main positions of differentially expressed lnc RNAs.Small interfering RNA(si RNAs)and a negative control(NC)without specific targets were selected to transfect ESCC cell lines.The proliferation of the transfected cell lines was observed.Transwell assay was used to detect the ability of migration and invasion of transfected cells.Transfected cells with NC or si RNA were subcutaneously injected into nude mice.At the end of the observation period,tumor tissue was taken out and weighed.Differently expressed lnc RNA and potential prognostic indicators were combined for analysis.Relevant clinicopathological data were collected,including: gender,age,smoking status,family history,tumor location,tumor length,degree of differentiation,T staging,lymph node metastasis,TNM staging.We investigated whether differentially expressed lnc RNAs have different expressions in different clinical and pathological conditions.Kaplan-Meier survival analysis was used to study whether each clinicopathological feature had a significant influence on the postoperative survival rate.We used log-rank method to test and draw the survival curves.Univariate and multivariate analysis were conducted with COX proportional risk model,and independent risk factors affecting survival were obtained.Micrometastasis of regional lymph node was detected with combined application of Ber-Ep4 and CD44v6 monoclonal antibodies.For differentially expressed lnc RNA and local lymph node micrometastasis in each patient,sperman level correlation analysis was used to explore the correlation between differentially expressed lnc RNA and positive lymph node micrometastasis.Results RNA quality test showed that each sample met the requirements.The RNA integrity of each sample was good.All samples were controlled by RNA quality requirements.Two pairs of esophageal squamous cell carcinoma tissues and their corresponding adjacent normal tissue samples were sequenced by second generation sequencing technology,and differentially expressed lnc RNAs were screened out.We analyzed the expression levels of differential lnc RNAs.181 differentially expressed lnc RNAs were screened out.86 lnc RNAs were relatively highly expressed in ESCC tissues,while 95 lnc RNAs were relatively low expressed in ESCC tissues.Biological information analysis showed that LINC00680 was one of the lnc RNAs with the most up-regulated expression in ESCC tissues.Therefore,LINC00680 was selected as the target lnc RNA in this study.Gene Expression Profiling Interactive Analysis(GEPIA) and database retrieval of CCLE(Cancer Cell Line Encyclopedia)were used.Results showed that LINC00680 was also highly expressed in a variety of cancer cells.UCSC Genome Browser was used to annotate the target lnc RNA gene.LINC00680 was significantly overexpressed in 90.5%(38/42)of cancer tissues,and the expression of LINC00680 in tumor tissues was significantly different from that in adjacent normal tissues,with a median upregulation multiple of 2.09(P<0.001).We performed q RT-PCR analysis to evaluate the expression of LINC00680 in ESCC cell lines.We found that LINC00680 expression was higher in KYSE140 and KYSE510 cell lines than in normal human esophageal epithelial cell line(HEEC).The results of nucleoplasmic separation showed that most LINC00680 expression was concentrated in the nucleus rather than the cytoplasm.After we transfected cell lines with si RNA(si RNA-1 and si RNA-2),the expression levels of LINC00680 were effectively inhibited.Cell proliferation experiments showed that LINC00680 was significantly inhibited by si RNA knockout in KYSE140 and KYSE510 cell lines.The transwell results showed that after we transfected cells with si RNA(si RNA-1 and si RNA-2),abilities of migration and invasion of were significantly reduced.In animal experiment,the results showed that after LINC00680 gene knockout,the growth of xenograft was inhibited in vivo.We found that LINC00680 expression level was correlated with age,smoking status,tumor length,lymph node metastasis and TNM stage of the patients(P<0.05).The overall survival rate of patients with higher LINC00680 expression level was significantly lower.Univariate analysis showed that in addition to the expression of LINC00680,N stage and TNM stage were also important prognostic factors for overall survival of ESCC patients.However,gender,age,smoking status,family history,tumor location,tumor size,degree of differentiation and T stage were not significantly correlated with survival rate.Multivariate analysis showed that LINC00680 expression level was the only independent predictive factor of overall survival rate(HR:3.192,95%ci :1.197-8.510,P=0.020).Detection of micrometastasis of regional lymph node showed that co-positive expression rate of Ber-Ep4 and CD44v6 was 26.2%(11/42).Spearman correlation analysis showed that the micrometastasis and LINC00680 expression level were positively correlated(r=0.487,P=0.001).Conclusions In conclusion,for the first time,our study identified LINC00680 as a novel potential oncogene in ESCC which may plays an important role in cell proliferation,migration and invasion.LINC00680 should be a potential important prognostic factor in ESCC.However,the underlying mechanisms involved in LINC00680 associated cell migration and invasion in ESCC remains unclear.Thus,further researches are needed to study the detailed mechanisms of LINC00680 in ESCC.