The Mechanism Of LncRNA SNHG1 Regulating The Development Of Esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the most common digestive system malignancies in humans,and its incidence ranks eighth in the incidence of malignant tumors worldwide.Esophageal cancer also has a high incidence in China.According to the pathological type of esophageal cancer,it can be divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).Among them,about 90%of patients with esophageal cancer in China are ESCC.Because the early onset symptoms of esophageal cancer are not obvious,and there is a lack of early screening methods for esophageal cancer,most patients are already in the advanced stage at the time of treatment,and the patient's therapeutic efficacy and prognosis are poor.At present,the pathogenesis of esophageal cancer is still not very clear.Therefore,in-depth study of the pathogenesis of esophageal cancer and the search for molecular markers for early diagnosis and potential targets for clinical treatment are very effective in improving the quality of life and improving the prognosis of patients with esophageal cancer.Significance.Long non-coding RNA(LncRNA)is a type of RNA that is longer than 200 nucleotides but does not encode a protein.It can regulate gene expression at multiple levels including transcription,post-transcription,and apparent abnormality.In recent years,with the deepening of LncRNA research,it is found that LncRNA expression is abnormal in tumors,which can affect the biological behaviors such as tumor cell proliferation,apoptosis,migration and invasion,and is closely related to the occurrence and development of tumors.Molecular markers and judgment of patient prognosis.Studies have shown that LncRNA SNHG1 is highly expressed in a variety of tumors,but studies on whether SNHG1 regulates the development of ESCC and its mechanism of action are rare.Micro RNA(miRNA)is a type of single-stranded small-molecule RNA with a length of 18 to 25 nucleotides.It can bind to specific mRNAs or regulate the protein translation process of specific mRNAs to regulate gene expression.miR-204 is localized to human chromosome 9 and is abnormally expressed in a variety of tumors,including breast cancer,gastric cancer,non-small cell lung cancer,and esophageal cancer,and is involved in tumorigenesis and development.Recent studies have shown that there is still a close interaction between LncRNA and miRNA,both of which are involved in the occurrence and development of diseases.According to the prediction of the biological information website,it was found that there were binding sites between SNHG1 and miR-204 gene sequence,and miR-204 may be a target gene of SNHG1.Whether SNHG1 regulates miR-204 expression and affects the biological behavior of ESCC cells is unknown.Bioinformatics software predictions also show that homeobox gene 8(HOXC8)is a target gene of miR-204.Studies have shown that HOXC8 is involved in the proliferation,growth,and migration of non-small cell lung cancer and other tumor cells,which is closely related to tumorigenesis and development.It has been reported that the median survival time of patients with high expression of HOXC8 is significantly lower than that of patients with low expression of HOXC8.HOXC8 expression is an independent factor affecting the prognosis of ESCC patients and can be used as a prognostic marker of ESCC.However,the expression level of HOXC8 in ESCC tissues and cells and its effect on the biological behavior of ESCC cells are also unknown.Therefore,in order to clarify the role of SNHG1 in ESCC and the possible molecular mechanisms,the following three parts of the study were conducted:Part I:First,the expression of SNHG1 in ESCC tissues was detected,and its expression and clinical pathological characteristics of patients were analyzed;Detect the expression levels of SNHG1 in ESCC cell lines EC9706,KYSE450,KYSE150 and Eca109,and observe the effects of down-regulating SNHG1 on the cell cycle,apoptosis,proliferation,migration and invasion of EC9706 and KYSE150 cells;Part ?:Using miR-204/HOXC8 axis as the entry point,explore the molecular mechanism of SNHG1 affecting EC9706 and KYSE150 cell cycle,apoptosis,proliferation,migration and invasion;Part III:Establish a nude mouse model of esophageal squamous cell carcinoma xenografts,and explore the effect of SNHG1 on tumor growth of esophageal squamous cell carcinoma xenografts in nude mice.Part ? Expression of SNHG1 in ESCC and its effect on cell biological behaviorBackground:LncRNA is a kind of noncoding RNA with a length of more than 200 nucleotides.It plays an important role in the occurrence and development of malignant tumors and participates in the malignant biological behavior of tumor cells.The study shows that the abnormal expression of lncRNA in tumor is closely related to the clinicopathological characteristics and prognosis of patients,and can be used as a molecular biological marker for tumor diagnosis and prognosis.SNHG1 is a potential biomarker in the diagnosis and treatment of colorectal cancer and non-small cell lung cancer.At present,the expression of SNHG1 in esophageal squamous cell carcinoma is relatively rare.Aim:To explore the expression of lncRNA SNHG1 in esophageal squamous cell carcinoma and its effect on the biological behavior of esophageal squamous cell carcinoma.Methods:The cancer tissues and corresponding paracancerous tissues of 53 ESCC patients were collected.The expression of SNHG1 in ESCC cancer tissues and adjacent tissues were detected by qRT-PCR.According to the mean value of SNHG1 expression in ESCC patients,ESCC patients were divided into SNHG1 high expression patients and low expression patients.Chi-square test was used to analyze the correlation between SNHG1 expression and age,gender,TNM stage,tumor location and prognostic of ESCC patients.To compare the expression levels of SNHG1 in cancer tissues of ESCC patients with different age,gender,TNM stage and tumor location.Normal esophageal epithelial cells Het-1A and ESCC cell lines EC9706,KYSE450,KYSE150,Eca109 were cultured in vitro,and the expression level of SNHG1 was detected by qRT-PCR.EC9706 and KYSE150 cells were divided into blank group,si-NC group and si-SNHG1 group.Cells in si-NC group and si-SNHG1 group were transfected with si-NC and si-SNHG1 respectively,and the blank group was not treated.The expression of SNHG1 in transfected cells was detected by qRT-PCR.The cell cycle changes and apoptosis rates of EC9706 and KYSE150 were detected by flow cytometry,CCK-8 method was used to detected cell proliferation.Trans well was used to detect cell migration and invasion.Results:(1)The results of qRT-PCR showed that the expression level of SNHG1 in ESCC tissues was significantly higher than that in adjacent tissues(P<0.05).(2)The expression of SNHG1 was closely related to the TNM stage and prognosis of ESCC patients(P<0.05),and was not related to the age,sex and tumor location of ESCC patients(P>0.05).(3)The higher the TNM stage of ESCC patients,the higher the expression level of SNHG1 in cancer tissues.There was no significant difference in the expression of SNHG1 in ESCC patients with different age,gender and tumor location(P>0.05).(4)Compared with Het-1 A cells,the expression levels of SNHG1 in ESCC cell lines EC9706,KYSE450,KYSE150 and Eca109 were significantly increased(P<0.05).Compared with ESCC cell lines,the expression levels of SNHG1 in EC9706 and KYSE150 cells were relatively high.Therefore,EC9706 and KYSE150 cells were selected as the next experimental study subjects.(5)Compared with the si-NC group,the expression level of SNHG1 in EC9706 and KYSE150 cells of si-SNHG1 group were significantly decreased(P<0.01),the percentage of cells in G0-G1 phase were significantly increased(P<0.01),and the S phase were significantly decreased(P<0.01).P<0.01),the apoptotie rate were significantly increased(P<0.001),the cell OD value was significantly reduced(P<0.001),and the number of cell migration and invasion were significantly decreased(P<0.01).There was no significant difference between the blank group and the si-NC group(P>0.05).Conclusion:SNHG1 was highly expressed in ESCC patients' cancer tissues,and its high expression was closely related to TNM staging and prognosis.SNHG1 was highly expressed in ESCC cell lines.Down-regulating SNHG1 expression could block EC9706 and KYSE150 cell cycle progression,promote apoptosis,and inhibit cell proliferation,migration and invasion.Part ? Study on the molecular mechanism of the effect of SNHG1 on the biological behavior of ESCC cellsBackground:Both lncRNA and miRNA,as important gene expression regulators,play an important role in the occurrence and development of tumors.There is also a close interaction between them.The interaction between them is involved in the occurrence and development of many diseases.It was found that lncrna can affect the occurrence and development of tumor by miRNA.Through the prediction of bioinformatics website,it was found that there was a binding site between snhgl and miR-204 gene sequence,and miR-204 might be the target gene of SNHG1.Bioinformatics software also predicted that homeobox gene 8(HOXC8)is the target gene of miR-204.At first,homeobox gene(HOX gene)was found in the process of studying Drosophila embryo development.At present,39 HOX genes have been identified.All members of HOX gene family encode their corresponding transcription factors and play a regulatory role in the expression of a series of genes.HOX gene encodes proteins that form a transcription factor regulatory network.In the normal life activities of the body,it participates in the communication process of cells.When the protein encoded by HOX gene changes,it can lead to the production of tumor.The expression level of HOXC8 in ESCC tissues and cells and its effect on the biological behavior of ESCC cells,as well as whether mir-204 can affect the biological behavior of ESCC cells by regulating the expression of HOXC8,are unknown..Aim:Taking the miR-204/HOXC8 axis as the starting point,the molecular mechanism of SNHGl's effect on the cell cycle,apoptosis,migration and invasion of ESCC cell lines EC9706 and kyse150 was studied.Methods:qRT-PCR was used to detect miR-204 and HOXC8 mRNA expression in ESCC tissues and adjacent tissues.The expression levels of miR-204 and HOXC8 mRNA in normal esophageal epithelial cells Het-1A and ESCC cell lines EC9706,KYSE450,KYSE150 and Eca109 were detected by qRT-PCR.Starbase bioinformatics software predicted that there were binding sites between SNHG1 and miR-204 sequence,and that there were binding sites between 3'UTR and miR-204 sequence of HOXC8.Dual luciferase reporter gene and RNA immunoprecipitation(RIP)experiments demonstrated the targeted regulation of SNHG1 and miR-204 in EC9706 and KYSE150 cells,as well as miR-204 and HOXC8.EC9706 and KYSE150 cells were divided into pcDNA group(transfected with empty vector),SNHG1 group(transfected with SNHG1 overexpression vector),si-NC group(transfected with nonsense negative sequence)and si-SNHG1 group(transfected SNHG1 small interfering RNA),qRT-PCR method was used to detect the effect of up-regulating or down-regulating SNHG1 on miR-204 expression in cells.EC9706 and KYSE150 cells were divided into miR-204 group(transfected with miR-204 mimics),miR-NC group(transfected with mimics control sequence),si-SNHG1+anti-miR-204 group(co-transfected with SNHG1)Small interfering RNA and miR-204 inhibitor)and si-SNHG1+anti-miR-NC group(co-transfected with SNHG1 small interfering RNA and inhibitor negative control sequence).Then cell cycle changes and apoptosis rate of EC9706 and KYSE150 cells were detected by flow cytometry in each group,cell proliferation was detected by CCK-8 method and migration and invasion were detected by Transwell.EC9706 and KYSE150 cells were divided into miR-204 group(transfected with miR-204 mimics),miR-NC group(transfected with mimics control sequence),and anti-miR-204 group(transfected with miR-204 inhibitor)and ani-miR-NC group(transfection inhibitor negative control sequence),Western Blot was used to detect the effect of up-regulating or down-regulating miR-204 on HOXC8 protein expression in cells.EC9706 and KYSE150 cells were divided into miR-204+pcDNA group(co-transfected with miR-204 mimics and empty vector)and miR-204+HOXC8 group(co-transfected with miR-204 mimics and HOXC8 over-vector vector),Western Blot was used to detect the expression of HOXC8 protein in each group of cells,Flow cytometry was used to detect cell cycle changes and apoptotic rates in each group,CCK-8 method was used to detect cell proliferation,and Transwell was used to detect cell migration and invasion in each group.Results:(1)Compared with Het-1 A cells,the expression levels of miR-204 in ESCC cell lines EC9706,KYSE450,KYSE150 and Eca109 were significantly decreased(P<0.05),while HOXC8 mRNA expression level was significantly increased(P<0.05)..Compared with adjacent tissues,the expression level of miR-204 in ESCC tissues was significantly decreased(P<0.05),and the expression level of HOXC8 mRNA was significantly increased(P<0.05).(2)The dual luciferase reporter gene results showed that the luciferase activity of the miR-204 group with the plasmid SNHG1-WT was significantly lower than that of the miR-NC group with the plasmid SNHG1-WT(P<0.001);co-transfection with the plasmid SNHG1-MUT,The luciferase activity of the miR-204 group was not significantly different from that of the miR-NC group(P>0.05).The results of RNA immunoprecipitation(RIP)showed that the amount of SNHG1 captured by the miR-204 group was significantly higher than that of the miR-NC group(P<0.001).(3)Compared with the pcDNA group,the expression levels of miR-204 in EC9706 and KYSE15 cells in the SNHG1 group were significantly decreased(P<0.001);compared with the si-NC group,the expression levels of miR-204 in the EC9706 and KYSE150 cells in the si-SNHG1 group were increased(P<0.001).(4)Compared with the miR-NC group,the expression levels of miR-204 in the EC9706 and KYSE150 cells in the miR-204 group were significantly increased(P<0.01),and the percentage of cells in the G0-G1 phase were significantly increased(P<0.01),the apoptotic rates were significantly increased(P<0.001),the cell OD value were significantly reduced(P<0.001),and the number of cell migration and invasion were significantly reduced(P<0.01).Compared with the si-SNHG1+anti-miR-NC group,the expression levels of miR-204 were significantly decreased of the EC9706 and KYSE150 cells in the si-SNHG1+anti-miR-204 group(P<0.01),and the percentage of cells in the G0-G1 phase were significantly decreased(P<0.01),S phase increased significantly(P<0.01),apoptosis rates were decreased significantly(P<0.001),and the number of cell migration and invasion were increased significantly(P<0.01),the cell OD value were significantly increased(P<0.001).(5)The double luciferase reporter gene results showed that the luciferase activity of the miR-204 group was significantly lower than that of the miR-NC group(P<0.001);the co-transfection with the plasmid HOXC8-MUT was co-transfected with the plasmid HOXC8-WT.The luciferase activity of the miR-204 group was not significantly different from that of the miR-NC group(P>0.05).The results of RNA immunoprecipitation(RIP)showed that the expression of HOXC8 in the miR-204 group was significantly higher than that in the miR-NC group(P<0.001).(6)Compared with the miR-NC group,HOXC8 protein expression levels were significantly decreased in the miR-204 group EC9706 and KYSE15 cells(P<0.001);compared with the anti-miR-NC group,the anti-miR-204 group EC9706 and KYSE150 were compared with the anti-miR-NC group.The expression level of HOXC8 protein in cells was significantly increased(P<0.001).(7)Compared with miR-204+pcDNA group,HOXC8 protein expression level was significantly increased in EC9706 and KYSE150 cells in miR-204+HOXC8 group(P<0.01),and the percentage of G0-G1 phase cells was significantly decreased(P<0.01),S phase.Significantly elevated(P<0.01),the apoptotic rate was significantly decreased(P<0.001),the cell OD value were significantly increased(P<0.001)and the number of cell migration and invasion was significantly increased(P<0.01).Conclusion:miR-204 was low expressed in ESCC tissues and cell lines,and HOXC8 mRNA was highly expressed.In EC9706 and KYSE15 cells,SNHG1 targeted negative regulation of miR-204 expression,and miR-204 targeted negative regulation of HOXC8 expression.Down-regulating SNHG1 could up-regulate the expression of miR-204 and then down-regulate the expression of HOXC8 protein,which could block the progression of ESCC cell cycle,promote cell apoptosis,and inhibit cell proliferation,migration and invasion.Part ? Effect of Silencing SNHG1 on Transplanted Tumor of Esophageal Squamous Cell Carcinoma in Nude MiceBackground:The occurrence and development of esophageal squamous,cell carcinoma is mainly due to the abnormal expression of genes.With the rapid progress of human gene technology,through the study of the change of gene molecular level and the relationship between different genes,to explore the relationship between a certain gene change and the occurrence and development of tumor,directly through the regulation of abnormal expression of genes can achieve the purpose of tumor treatment.Short hairpin interfering RNA(shRNA)technology is a new gene therapy technology.It silences the abnormal expression of oncogene at the gene molecular level,and then it can effectively and specifically inhibit the growth of tumor.Since shRNA technology was found,it has been widely used in the field of tumor gene therapy.Aim:By constructing the recombinant Lentivirus Expression Vector sh-SNHG1,further study in nude mice was carried out in order to show whether snhgl can affect the growth of esophageal squamous cell carcinoma subcutaneously transplanted tumor in nude mice.Methods:Establish a subcutaneous xenograft model of esophageal squamous cell carcinoma in nude mice.Nude mice were divided into Empty group:EC9706 cells;sh-NC group:EC9706 cells transfected with sh-NC;sh-SNHG1 group:EC9706 cells transfected with sh-SNHG1.Observing the growth of subcutaneous xenografts in nude mice,measuring the volume of tumors by measuring the length and short diameter of the transplanted tumors,and plotting the growth curve of the transplanted tumors.After 5 weeks,the mice were sacrificed by cervical dislocation,the tumor was removed and weighed.The expression of SOHG1 and miR-204 in transplanted tumor tissues were detected by qRT-PCR.The expression of HOXC8 protein in transplanted tumor tissues was detected by Western blot and immunohistochemistry.Results:(1)Compared with the Empty group and the sh-NC group,the subcutaneous xenografts of the sh-SNHG1 group grew slowly,and the volume and.weight of the tumors decreased significantly(P<0.05).(2)Compared with the Empty group and the sh-NC group,the expression level of SNHG1 in subcutaneous xenografts tissue of nude mice in the sh-SNHG1 group was significantly reduced(P<0.05).The expression level of miR-204 was significantly increased(P<0.05),and the expression level of HOXC8 protein was significantly reduced(P<0.05).Conclusion:Silencing of SNHG1 could inhibit the growth of esophageal squamous cell carcinoma in nude mice by up-regulating miR-204 expression and then inhibiting the expression of HOXC8 protein.General conclusion:(1)LncRNA SNHG1 is highly expressed in ESCC and ESCC cell lines,which belongs to the oncogenic gene;(2)LncRNA SNHG1 promotes the expression of HOXC8 protein by targeting miR-204,promotes the cell cycle process,proliferation,migration and invasion of ESCC cells,and inhibits cell apoptosis.