| Part 1 The expression and clinical significance of lncRNA DGCR5 in esophageal squamous cell carcinoma tissuesObjective:To investigate the expression of long non-coding RNA(lncRNA)DiGeorge syndrome critical region gene 5(DGCR5)in esophageal squamous cell carcinoma(ESCC)tissues,and to analyze its relationship with clinicopathological features and prognosis of ESCC patients.Methods:1.The expression of DGCR5 in ESCC data set from TCGA database was analyzed by bioinformatics method.2.Seventy pairs of ESCC tissues and para-cancerous tissues resected at the Fourth Hospital of Hebei Medical University from August 2016 to March 2017 were collected for this study.The expression of DGCR5 in ESCC tissues was detected by RT-qPCR.3.According to the expression level of DGCR5 in ESCC tissues,Logrank Test was used to analyze the relationships between the expression of DGCR5 in ESCC tissue and clinicopathological parameters.4.Kaplan-Meier analysis was used to investigate the correlation between the expression of DGCR5 and the survival of ESCC patients.5.Univariate and multivariate Cox regression model were used to evaluate the effect of DGCR5 on the prognosis of ESCC patients.Results:1.TCGA database analysis showed that the expression of DGCR5 in ESCC tissues was significantly increased than that in normal esophageal tissues(P<0.01).2.High expression of DGCR5 was observed in 70 case(58.57%)ofESCC tissues,low expression of DGCR5 was 41.43%.The expression of DGCR5 in ESCC tissues was significantly higher than that in precancerous tissues(P<0.01),which was consistent with the results of TCGA database analysis.3.The correlation between the expression of DGCR5 in 70 patients with ESCC and clinical pathological was analyzed.The expression level of DGCR5 was significantly correlated with TNM stage,invasion range and lymph node metastasis in ESCC patients(all P<0.01),but not related to sex and age(P>0.05).4.Kaplan-Meier analysis showed that high expression of DGCR5 was significantly correlated with poor prognosis of ESCC patients(P<0.01).5.Univariate analysis was found that the high expression of DGCR5 was an important factor for poor prognosis of ESCC patients regardless of TNM stage and lymph node metastasis(P<0.01).In addition,Multivariate Cox regression analysis showed that the expression of DGCR5 was an independent risk factor for prognosis of ESCC patients(P<0.01).Summary:1.DGCR5 was highly expressed in ESCC tissues,which is closely related to TNM stage,invasion range,lymph node metastasis and poor prognosis.2.The expression of DGCR5 was an independent risk factor for the prognosis of ESCC patients and may be an important biomarker for early diagnosis and prognosis evaluation of ESCC.Part 2 The function of lncRNA DGCR5 in esophageal squamous cell carcinoma cellsObjective:To investigate the effects of DGCR5 on the proliferation,invasion,migration and apoptosis of ESCC cells.Methods:1.RT-qPCR was used to detect the DGCR5 expression in ESCC cell lines(TE1,Kyse170,Yes-2,Kyse150 and Eca9706)and cells with higher expression of DGCR5 were selected for subsequent functional experiments.2.ESCC cells knocked down or overexpressed DGCR5 gene were successfully constructed.RT-qPCR was detected the transfection efficiency.3.CCK-8 assay was used to detect the cell proliferation of ESCC cells with DGCR5 low-expression or over-expression.4.Wound healing and transwell assays were used to detect the cell migration of ESCC cells with DGCR5 low-expression or over-expression.5.Matrigel assays was used to detect the cell invasion of ESCC cells with DGCR5 low-expression or over-expression.6.Flow cytometry assay was used to detect cell apoptosis of ESCC cells with DGCR5 low-expression or over-expression.7.Western-blot was performed to examine the expression of related apoptosis proteins of ESCC cells with DGCR5 low-expression or overexpression.Results:1.DGCR5 was highly expressed in ESCC cells(TE1,Kyse170,Yes-2,Kyse150 and Eca9706).TE1 and Kyse170 were selected for further study.2.CCK-8 assay showed that knocking down DGCR5 gene expression could inhibit the proliferation of TE1 and Kyse 170 cells(P<0.05),while overexpressing DGCR5 gene could promote the proliferation of TE1 and Kyse170 cells(P<0.05).3.Wound healing and transwell assay showed that knocking down DGCR5 gene expression could inhibit the migration of TE1 and Kyse170 cells(P<0.01),while overexpressing DGCR5 gene could promote the migration of TE1 and Kyse 170 cells(P<0.01).4.Matrigel assays showed that knockdown of DGCR5 gene expression could inhibit the invasion of TE1 and Kyse170 cells(P<0.01),while overexpression of DGCR5 gene could promote the invasion of TE1 and Kyse 170 cells(P<0.01).5.Flow cytometry assays showed that knockdown of DGCR5 gene expression could promote the apoptosis of TE1 and Kyse170 cells(P<0.05),while overexpression of DGCR5 gene expression could inhibit the apoptosis of TE1 and Kyse170 cells(P<0.05).6.Western-blot analysis showed that the expression of apoptosis-related protein Bax was increased,while Bcl-2 and Mcl-1 protein was decreased in TE1 and Kyse170 cells after knocking down DGCR5 expression(P<0.05).Overexpression of DGCR5 decreased the expression of apoptosis-related protein Bax,while Bcl-2 and Mcl-1 protein was increased in TE1 and Kyse170 cells(P<0.01).Summary:1.DGCR5 was highly expressed in ESCC cells.2.knockdown of DGCR5 could inhibit proliferation,migration and invasion of ESCC cells,while enhance apoptosis.3.Overexpression of DGCR5 could promote proliferation,migration and invasion of ESCC cells,while inhibit apoptosis.Part 3 The regulation mechanism of lncRNA DGCR5 in esophageal squamous cell carcinoma cellsObjective:To explore the possible mechanism of DGCR5 in ESCC cells.Methods:1.Using the TCGA database to select the genes interact with DGCR5 and pick out the genes with Pearson value>0.5.Then classified and analyzed the role and function of these genes with KEGG pathway analysis.2.FISH and RNA cytoplasmic and nuclear purification was separately used to detect the subcellular localization of DGCR5 in ESCC cells.3.StarBase database was used to predict RNA binding proteins that might interact with DGCR5.Serine/arginine-rich splicing factor 1(SRSF1)was selected for study,and the interaction between SRSF1 protein and DGCR5 was reversely verified by RNA-binding protein immunoprecipitation(RIP)4.RT-qPCR assay was used to detect the expression level of SRSF1mRNA in TE1 and Kyse170 cells after knockdown or overexpression of DGCR5.Western-blot assay was used to detect the expression of SRSF1 protein in TE1 and Kyse170 cells after knockdown or overexpression of DGCR5.5.Flow cytometry showed that DGCR5 could influence the apoptosis of ESCC cells by regulating the expression of Mcl-1.6.RT-qPCR assay was used to detect the expression of Mcl-1L and Mcl1S in TE1 and Kyse170 cells with knockdown or overexpression of SRSF1.7.Western-blot assay was used to detect the expression of Mcl-1 protein in TE1 and Kyse170 cells with knockdown or overexpression of SRSF1.8.RT-qPCR assay was used to detect the expression of Mcl-1L and Mcl1S in TE1 and Kyse170 cells with knockdown DGCR5 and overexpression of SRSF1 together.9.Western-blot assay was used to detect the expression of Mcl-1 protein in TE1 and Kyse 170 cells with knockdown DGCR5 and overexpression of SRSF1 together.Results:1.The genes related to DGCR5 in ESCC were screened from TCGA database.There were 178 genes with Pearson value>0.5,including 73 genes with positive expression and 105 genes with negative expression.KEGG pathway analysis showed that DGCR5 was correlated with apoptosis signaling pathway.GESA analysis showed that there was a negative correlation between DGCR5 expression level and apoptosis signaling pathway.2.DGCR5 was mainly located in the nucleus of TE1 and Kyse170 cells.3.StarBase database analysis showed that DGCR5 could combine with EIF4A3,FMRP,FUS,LIN28A,SRSF1 and UPF1 proteins.On the basis of literature reports and previous work,SRSF1 protein was selected for subsequent experimental research.RIP assay showed that DGCR5 could interact with SRSF1 protein.4.RT-qPCR assay showed that there were no correlations between the expression of DGCR5 and SRSF 1 in ESCC cells(P>0.05).Western-blot assay showed that knockdown of DGCR5 could inhibit the expression of SRSF1 protein in the nucleus of TE1 and Kyse170 cells,while overexpression of DGCR5 gene could promote the expression of SRSF1 protein(P<0.01).However,knockdown or overexpression of DGCR5 had no effect on the expression level of SRSF1 protein in the cytoplasm of TE1 and Kyse170 cells(P>0.05).5.Flow cytometry showed that knocking down DGCR5 could promote the apoptosis of TE1 and Kyse170 cells,and overexpressing Mcl-1 can inhibit the apoptosis of TE1 and Kyse170 cells,while knocking down DGCR5 and overexpressing Mcl-1 could partially restore the ability of inhibiting the apoptosis of TE1 and Kyse170 cells(P<0.01).6.RT-qPCR assay showed that knockdown of SRSF1 inhibited the rate of Mcl-1L/Mcl-1S in TE1 and Kyse170 cells(P<0.01),while overexpression of SRSF1 promoted the rate of Mcl-1L/Mcl-1S in(P<0.01).7.Western-blot assay showed that knocking down SRSF1 inhibited the expression of Mcl-1 protein in TE1 and Kyse170 cells(P<0.01),while overexpression of SRSF1 promoted the expression of Mcl-1 protein(P<0.01).8.RT-qPCR assay showed that knocking down of DGCR5 and overexpressing of SRSF1 together could partially restore the expression rate of Mcl-1L/Mcl-1S ratio in TE1 and Kyse170 cells(P<0.01).9.Western-blot assay showed that knocking down of DGCR5 and overexpressing of SRSF 1 together could partially restore the expression level of Mcl-1 protein in TE1 and Kyse170 cells(P<0.01).Summary:1.DGCR5 was located in the nucleus of ESCC cells,and could bind to RNA binding protein SRSF 1.2.SRSF1 could regulate the alternative splicing of Mcl-1 in ESCC cells,and promote the increase of Mcl-1L subtype,which leading to the high expression of Mcl-1 protein.3.DGCR5 could regulate Mcl-1 alternative splicing through binding to SRSF1 protein,which affecting the anti-apoptosis of ESCC cells.Part 4 The study on the mechanism of lncRNA DGCR5 in esophageal squamous cell carcinoma in vivoObjective:To verify the mechanism of DGCR5 in esophageal squamous cell carcinoma in vivo.Methods:1.Kyse170 cells were transfected with siRNA-DGCR5(si-DGCR5)and negative control(si-NC)plasmids,respectively.Then transfected Kyse170 cells were subcutaneously injected into the right flank of 4-week-old female BALB/c nude mice to establish xenograft tumor model.2.The growth of transplanted tumor in each group was observed,and the tumor size and weight in each group were measured.3.RT-qPCR assay detected the expression levels of DGCR5,SRSF1 and Mcl-1L/Mcl-1S mRNA in the two groups.4.Immunohistochemical staining assay was used to detect the expression level of SRSF1 and Mcl-1 protein in transplanted tumor tissues of each group.Results:1.Tumor growth curve showed that the growth rate of transplanted tumor in si-DGCR5 group was significantly lower than that in si-NC group(P<0.05).The experimental results showed that compared with the si-NC group,the size and weight of transplanted tumors were significantly suppressed in si-DGCR5 group(P<0.05).2.Compared with si-NC group,RT-qPCR results showed that the expression level of DGCR5 and the ratio of Mcl-1L/Mcl-1S mRNA in siDGCR5 group of transplanted tumor tissues of nude mice were lower(P<0.05),but the expression level of SRSF1 mRNA had no obvious difference(P>0.05).3.Immunohistochemical staining assay showed that compared with the control group,the protein levels of SRSF1 and Mcl-1 in transplanted tumor tissues of nude mice decreased significantly(P<0.05).Summary:DGCR5 could promote the growth of xenografts of esophageal cancer cells in nude mice by regulating the expression of SRSF1 and Mcl-1 protein,which revealed that DGCR5/SRSF1/Mcl-1 axis might play a carcinogenic role in vivo.Conclusions:1.DGCR5 was up-regulated in ESCC tissues,and its high expression was related to advanced TNM stage,deeper tumor invasion degree,more lymph node metastasis and poor prognosis of ESCC patients.2.DGCR5 could promote the proliferation,invasion and migration of ESCC cells,and inhibit its apoptosis.3.DGCR5 could directly bind to SRSF1 protein and enhance its stability,which further promoted the alternative splicing event of Mcl-1,and then led to the increase of Mcl-1L subtype,thereby playing its role in promoting malignant biological behavior in ESCC. |
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