HMGA2 Contributes Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma And Is Regulated By MiR-204-5p And MiR-137

Objective:Invasion and metastasis are the main reasons influencing the prognosis of patients with esophageal squamous cell carcinoma(ESCC).Therefore,it is of great importance to make a further exploring the molecular mechanism for improving the outcome of patients with ESCC.High mobility group protein A2(HMGA2),a small non-histone chromatin protein,could regulate other gene transcription by altering chromatin architecture.Previous studies have demonstrated that HMGA2 was overexpressed in many malignant neoplasms,and closely related to the tumorigenesis,development and poor outcome.However,the expression and regulation mechanism of HMGA2 in ESCC have not been elucidated.MicroRNAs(mi RNAs)are a set of endogenous,noncoding RNAs that bind to partially complementary sequences of target mRNAs resulting in posttranscriptional regulation of gene expression.miR-204-5p and miR-137 have been demonstrated to be lower expressed in many human malignances,and closely associated with tumor cell differentiation,metastasis and apoptosis.However,the role of miR-204-5p,miR-137 in ESCC is still unknown.Therefore,the objective of this research is to investigate the expression levels of HMGA2,miR-204-5p and miR-137,and analyze the associations with clinicopathological factors as well as the prognosis of ESCC.Besides,we explored the role of HMGA2,miR-204-5p and miR-137 on the proliferation and metastasis of ESCC cells by vitro experiments,and further clarify the mechanism of mi R-204-5p and miR-137 in the regulating of the expression of HMGA2 of ESCC cells to provide theoretical evidence for elucidating the molecular mechanism of ESCC cells metastasis and to find new targets for tumor gene therapy in the future.Methods:1.We adopted the fluorescent quantitative RT-PCR(qRT-PCR)and Western blot techniques to detect the expression level of HMGA2 mRNA and protein in 52 fresh ESCC tissues and corresponding adjacent non-tumor tissues.Furthermore,immunohistochemical(IHC)was used to detect the expression of HMGA2 protein in paraffin-embedded ESCC tissue from 178 patients.The relationship between HMGA2 expression and clinicopathological parameters as well as the prognosis of ESCC patients were further analyzed.2.RNA interference was used to down-regulate the expression level of HMGA2 in cell lines.MTT assay was performed to detect the role of HMGA2 on cell proliferation.Plate clone formation assay was performed to observe the effect of HMGA2 on the ability of cell clone.The effect of HMGA2 on the ability of invasion and metastasis was quantified by scratch and transwell assay.After the expression of HMGA2 was interfered,Western blot and Immunofluorescence assay were used to observe the changes of EMT related markers.Finally,the relationship between HMGA2 expression and EMT related markers in ESCC tissues was detected by IHC assay.3.The expression of miR-204-5p and mi R-137 in 52 fresh ESCC tissues and corresponding adjacent non-tumor tissues was detected by q RT-PCR assay.The relationship between these two micro RNAs expression and clinicopathological parameters were further analyzed.After miR-204-5p and miR-137 expression were up-regulated by transfecting with miR-204-5p mimic and miR-137 mimic,the methods mentioned above were used to further detect the changes of proliferation,invasion and migration of ESCC cells.Western blot assays were used to detect the changes of expression of HMGA2 protein.Finally,the associations between miR-204-5p,miR-137 expression and HMGA2 mRNA expression were also analyzed in ESCC tissues.4.To identify the targeting regulatory role of miR-204-5p and miR-137 on HMGA2 gene,Target Scan and miRanda assays were used to analyze that HMGA2 might be one of the target genes of mi R-204-5p or miR-137.Dual luciferase reporter assay was performed to identify the two miRNAs directly regulated HMGA2 expression.After cotransfection of miR-204-5p mimic or miR-137 mimic and HMGA2 plasmid into ESCC cells.Scratch and transwell assays were used to analyze the effects on the ability of invasion and metastasis.Results:1.The qRT-PCR and Western blot assays showed that the expression of HMGA2 mRNA and protein were significantly up-regulated in tumor tissues when compared with adjacent non-tumor tissues(52.8% vs.27.5%,P=0.004).Clinicopathological analysis indicated that high HMGA2 expression level was associated with tumor size,depth of tumor invasion,lymph nodes metastasis and TNM stage(P<0.05).The 5-year overall survival rate of patients with high expression of HMGA2 was significantly lower than that of patients with low expression of HMGA2(24.9% vs 42.3%,P=0.009).The Multivariate survival analysis indicated that HMGA2 expression can be an independent prognosis biomarker in ESCC patients(HR 1.484,95% CI 1.012-2.175,P=0.040).2.HMGA2 was highly expressed in the Kyse-30 and Ec-109 ESCC cell lines,siRNA technique could significantly down-regulate the expression level of HMGA2 in ESCC cell lines.MTT and colony formation assays showed that down-regulation of HMGA2 expression could significantly decrease the ability of ESCC cell proliferation and colony formation.In the scratch and transwell assays,down-regulation of HMGA2 expression could obviously reduce the ability of ESCC cell invasion and metastasis.3.In the western blot assay,the expression of E-cadherin,one epithelial marker,was significantly increased after interfering with the expression of HMGA2 in Kyse-30 and Ec-109 cell lines.The expression of interstitial markers,N-cadherin,Vimentin and Snail,were obviously decreased.The immunofluorescence a ssays showed that the expression of E-cadherin was up-regulated and the expression of N-cadherin was down-regulated after down-regulating the HMGA2 expression.Meanwhile,?-catenin was transformed from cytoplasm and nucleus to the cell membrane in cancer cells.The ?-catenin binding to cell membrane obviously increased.4.The qRT-PCR assay showed that the expression of miR-204-5p and miR-137 were significantly down-regulated in tumor tissues when compared with adjacent non-tumor tissues(P<0.01).The expression level of miR-204-5p was significantly related to depth of tumor invasion and lymph nodes metastasis.The expression level of miR-137 was significantly associated with tumor size and depth of tumor invasion(P<0.05).After up-regulating the expression of miR-204-5p or miR-137 in Kyse-30 and Ec-109 cells,the abilities of proliferation,invasion and metastasis of cancer cells were obviously inhibited,and the expression levels of HMGA2 protein were significantly decreased.Correlation analysis showed tha t the expression of miR-204-5p or miR-137 in ESCC tissues were significantly and negatively correlated with HMGA2 m RNA expression level(r2=0.609 and 0.682,all P <0.001).5.Bioinformatics software analysis Target Scan and miRnada indicated that HMGA2 might be one of the target genes of miR-204-5p or miR-137.Dual luciferase reporter gene assay indicated that HMGA2 might be the direct target of these two microRNAs.The inhibitory effect of miR-204-5p or miR-137 on the invasion and metastasis of tumor cells could be partially weakened by HMGA2 plasmid expression.Conclusions:1.HMGA2 was overexpressed in ESCC and significantly related to tumor cells proliferation,invasion and metastasis.High expression of HMGA2 was related to poor prognosis,and may be regarded as one target gene in the treatment for ESCC.2.HMGA2 participated in the progress of ESCC cells proliferation,colony formation,invasion and metastasis,which was one malignant phenotype of ESCC.HMGA2 could regulate the EMT procession thro?gh regulating the ?-catenin translocation to promote the tumor cells metastasis.3.Both miR-204-5p and miR-137 were lower expressed in ESCC and had certain relationship with tumor proliferation,invasion and metastasis.4.HMGA2 was a direct target gene of mi R-204-5p and mi R-137.miR-204-5p or miR-137 could inhibit the invasion and metastasis of cancer cells by down-regulating the expression of HMGA2.