MicroRNA-145-5p/SPATS2 Axis Exacerbates Tumor Growth,Migration And Invasion In Hepatocellular Carcinoma

BackgroundPrimary liver cancer is one of the most common solid tumors in the world.Hepatocellular carcinoma(HCC)is the most common pathological type in primary liver cancer,accounting for about 75-80%of liver cancer.HCC has high infiltration.Therefore,in order to improve the survival rate and quality of life of patients with liver cancer,exploring new types of liver cancer early biomarkers and effective drug treatment targets is of great significance for improving the clinical prognosis and significance of liver cancer in order to early diagnose and prognosis liver cancer,treatment strategy.Spermatogenesis associated serine rich 2(SPATS2),also known as SCR59,p59SCR,SPATA10,Nbla00526.The gene encoding this protein is located on chromosome 12,at 12q13.12,and contains 16 exons.Some scientists found that SPATS2 is significantly down-regulated in human oral epithelial cells after Bisphenol A treatment.Subsequently,the upper limit analysis of gene expression was performed on 97 frozen tissues from surgically resected squamous cell carcinoma(SCC)and adenocarcinoma(AD),and SPATS2 was found as a new complementary biomarker to distinguish SCC from AD.Recently,some researchers have demonstrated that SPATS2 is involved in the growth of colorectal cancer cells mediated by lncRNA SNHG5.In addition,there is a study show that SPATS2 is related to the progression of prostate cancer.However,the function of SPATS2 in HCC has not been comprehensively studied.In this study,we firstly test SPATS2 expression could be an independent prognostic factor of overall survival in HCC.Then analyzed the The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)databases and found that showed that the expression of SPATS2 was abnormally elevated and closely related to the malignant phenotype.And further applied bioinformatics technology to predict and verify the related microRNAs,and their interactions with SPATS2,and their impact on the development of liver cancer.In the era of precise tumor treatment,we are exploring a potential new,effective target for HCC treatment.In conclusion,our results find that SPATS2 is upregulated in HCC tissues and cell lines.Knockdown of SPATS2 dampens HCC development and metastasis by regulating cell cycle and cell apoptosis.Furthermore,SPATS2 is a direct target of miR-145-5p and miR-145-5p exerts its tumor suppressive function via negatively regulating SPATS2 expression.Taken together,our results provide a profound insight of SPATS2/MiR-145-5p axis in understanding HCC development and progression,which might be used as a promising therapeutic target of HCC.The first part:the expression and biological function of SPATS2 in HCCObjective:Confirm the expression of SPATS2 in HCC tissues in the occurrence and development of HCCMethod:1.Western blot was used to detect the expression of SPATS2 in next eight pairs of HCC tissues and adjacent tissues.2.Immunohistochemical detection of SPATS2 protein expression in liver cancer tissues3.TCGA and GEO public databases and its follow-up data of HCC microarray data were used to analyze the relationship between the mRNA expression of SPATS2 in tissues and clinical phenotype of HCC.4.Explore the relationship between SPATS2 expression and the clinical phenotype of liver cancer and patient prognosis in the ZZU liver cancer cohort,and further analyze the relationship between SPATS2 mRNA and clinical characteristics and prognosis of liver cancer patients using the TCGA database and GEO database.Result:1.SPATS2 protein is highly expressed in liver cancer tissues,and the worse the phenotype,the higher the expression level;2.Analysis by bioinformatics technology show that SPATS2 mRNA is highly expressed in liver cancer tissue,and the worse the prognosis and clinical phenotype,the higher the expression level.The second part:Expression of SPATS2 in HCC cell lines and its functionObjective:To study the expression of SPATS2 in liver cancer cell lines and its effect on tumor cell functionMethod:1.Expression of SPATS2 in basic molecular biology experiments in HCC cell lines;2.Establishing SPAT2 silencing hepatocellular carcinoma cell lines via lentivirus transfection,and detecting the biological functions of SPATS2 on the proliferation,migration,invasion and tumorigenicity of HCC cells;3.Application of bioinformatics to analyze the possible biological functions of SPATS2 in HCC cells;4.Using SPATS2 silenced HCC cell lines to explore the effects of SPAT2 on apoptosis and cell cycle function of hepatocellular carcinoma cells.Result:1.Cellular immunofluorescence method was used to verify the localization of SPATS2 in cells.SPATS2 was mainly expressed in the cytoplasm;2.Western blot test also found that SPAT2 expression in liver cancer was significantly higher than that in normal liver cells.After applying shRNA transfection technology to interfere with the expression level of SPATS2 protein in liver cancer cells,compared with the control group,SPATS2 silencing significantly inhibited the cell proliferation and colony formation of SMMC-7721 and HepG2 cells,and also significantly reduced the invasion of Migration capabilities.In addition,knockdown of SPATS2 significantly inhibited the expression of migration-related proteins(such as CXCR4,MMP-2,MMP-7,and MMP-9),which is consistent with reduced invasive ability of HCC cells.3.Applying the comprehensive results of bioinformatics analysis,the signal pathway associated with SPAT2 in liver cancer may be related to the cell cycle.4.SPATS2 markedly promoted HCC cell proliferation and invasion through downregulating the expression of p21 and p27,while upregulating the expression of EZH2,survinvin,c-myc and cyclin D1.The third part:Effect of SPATS2 on proliferation and metastasis of HCC in vivoObjective:Explore the effects of SPATS2 on the proliferation and metastasis of liver cancer in vivoMethod:1.Establish subcutaneously implanted HCC model in nude mice by using SPATS2 silenced SMMC-7721 cells,and explore the effect of SPATS2 on HCC proliferation;2.Establish a nude mouse orthotopic HCC model using SPATS2 silenced SMMC-7721 cells and explore the effect of SPATS2 on HCC.Result:1.HCC xenograft model showed that SPATS2 knockdown markedly dampened HCC tumor development,with reduced tumor volume and tumor weight.Furthermore,SPATS2 and Ki-67 IHC staining results confirmed that knockdown of SPATS2 drastically decreased the expression of SPATS2 and Ki-67.The fourth part:Screening and identification of miR-145-5p targeting SPATS2 gene and its effect on the biological behavior of HCC cellsObjective:Explore the microRNAs that may be involved in regulating the expression of SPATS2,their interactions with SPATS2,and their effects on the development of HCC.Method:1.Screening microRNAs regulating SPAT2 expression using bioinformatics methods and luciferase reporter assay.2.Real-time PCR and Western blot verify the miR-145-5p targeted regulation of SPATS2 expression.3.Replenishment experiments prove that SPATS2 mediates the regulation of miR-145-5p on HCC cell proliferation.4.Replenishment experiments explore the function of SPATS2 in miR-145-5p apoptosis and cell cycle.Result:1.Both bioinformatics analysis and Pearson correlation analysis have verified that miR-145-5p has a regulatory effect on SPATS2 expression;mRNA levels and protein levels have found that miR-145-5p has a negative regulatory effect on SPATS2 expression.2.miR-145-5p overexpression could significantly decrease SPATS2 expression while miR-145-5p inhibitor could enhance SPATS2 expression both at mRNA and protein levels.3.miR-145-5p/SPATS2 regulatory axis has an effect on the apoptosis and cell cycle processes of HCC cells.The full text of the conclusions1.The expression of SPATS2 was significantly increased in HCC,and was closely related with metastasis,recurrence and clinical prognosis of patients.SPATS2 played a role of cancer-promoting gene in the occurrence and development of HCC.2.The expression of SPATS2 was significantly upregulated in four HCC cell lines.We performed loss-of-function studies using shRNAs targeting SPATS2 and the RNA silence efficiency was determined by western blot.SPATS2 knockdown notably inhibited cell proliferation,colony formation and DNA synthesis.3.HCC xenograft model showed that SPATS2 knockdown markedly dampened HCC tumor development,with reduced tumor volume and tumor weight.4.miR-145-5p overexpression could significantly decrease SPATS2 expression while miR-145-5p inhibitor could enhance SPATS2 expression both at mRNA and protein levels.Functionally,miR-145-5p overexpression remarkably inhibited the proliferation and invasion of HCC cells.However,overexpression SPATS2 together with miR-145-5p partially reversed the inhibitory effect of miR-145-5p overexpression.These results demonstrate that miR-145-5p might inhibit the proliferation and invasion of HCC by regulating SPATS2.5.SPATS2 is a direct target of miR-145-5p and miR-145-5p exerts its tumor suppressive function via negatively regulating SPATS2 expression.SPATS2/miR145-5p axis in understanding HCC development and progression,which might be used as a promising therapeutic target of HCC.