The Sttudy Of Long Non-coding RNA HOXA11-AS In Promoting The Liver Metastasis Of Colorectal Cancer

Until recently,Colorectal Cancer（CRC）is still a most common malignant in China,with an increasing morbidity in recent years.The survival of patients with CRC is closely related to the occurrence of metastasis,especially to the liver.Therefore,in order to improve the survival of CRC patients,earlier diagnosis and treatment of CRC patients with liver metastases is very important.Long non-coding RNAs（lncRNAs）have been proved to play key roles in gene regulation and thus affect various aspects of cellular homeostasis,including proliferation,survival,migration or genomic stability.HOMEOBOX A11 antisense RNA（HOXA11-AS）is a newly identified lncRNA.Its expression is related to glioma,uterine cervix carcinoma,epithelial ovarian cancer,and lung adenocarcinoma.In this study,HOXA11-AS expression was up-regulated in the tissues of CRC patients with liver metastasis.The knockdown and overexpression of HOXA11-AS inhibited and promoted the migration and invasion of colon cancer cells,respectively.Moreover,we confirmed that HOXA11-AS functions as a ceRNA to regulate PADI2 expression by sponging miR-125a-5p.The HOXAll-AS/miR-125a-5p/PADI2 regulatory network may represent a novel therapeutic target for liver metastasis of CRC.Part ⅠGenome-wide analysis of long noncoding RNA（lncRNA）expression in colorectal cancer tissues from patients with liver metastasisBackground:Until recently,Colorectal Cancer（CRC）is still a most common malignant in China,with an increasing morbidity in recent years.The survival of patients with CRC is most closely related to the occurrence of metastasis;and liver is the most frequent target organ of metastasis.According to the existing data,long noncoding RNAs（lncRNAs）may play a crucial role in the process of liver metastasis of CRC.In this study,we performed a genome-wide analysis of lncRNA expression to identify novel targets for the further study of liver metastasis in CRC,Material and Methods:Primary cancer samples of CRC patients with liver metastasis（Exp G）or without metastasis（Ctrl G）are obtained in the First Affiliated Hospital,College of Medicine,Zhejiang University.All the samples were analyzed using human 8x60K lncRNA/mRNA v3.0 microarrays chips（Arraystar）to find differentially expressed lncRNAs and mRNAs.The differentially expressed lncRNAs and mRNAs were identified through fold-change filtering.Gene ontology（GO）and pathway analyses were employed by using standard enrichment computational methods.Several mRNAs with high normalized intensity and high fold changes were selected to be further verified by quantitative reverse transcription-polymerase chain reaction（qRT-PCR）.The verified mRNAs and the differentially expressed lncRNAs in microarray analysis are integrated in a coding-noncoding gene co-expression network（CNC）to explain their relationship.Thereafter,several lncRNAs with high normalized intensity and high fold changes were selected to be further verified by qRT-PCR.Futhermore,Competing endogenous RNAs（CeRNA）analysis are employed to predict the potential signaling pathways of several further selected verified lncRNAs.Results:Be compared with the Ctrl G,2636 differentially expressed lncRNAs（1600 IncRNAs are up-regulated and 1036 IncRNAs are down-regulated over two-folds）,as well as 1584 differentially expressed mRNAs（548 are up-regulated and 1036 are down-regulated over two-folds）are found in Exp G.GO and pathway analysis of the up-regulated or down-regulated mRNAs are processed independently,and different results are obtained.In the section of biological process,cellular component and molecular function,single-organism process,membrane part and transporter activity are found as the most important component in the up-regulated mRNAs,respectively;While cellular process,membrane,and binding are found as the most important component in the down-regulated mRNAs,respectively.27 signaling pathways in up-regulated mRNAs and 51 signaling pathways in down-regulated mRNAs were targeted in the pathway analysis.Ten mRNAs with high normalized intensity and high fold changes were selected to be further verified by qRT-PCR,and mRNA NQO2（NM₀00904）were proved to be consistent with the microarray analysis.Thereafter,NQ02 and the differentially expressed IncRNAs were integrated in the CNC,and then 769 IncRNAs were found to have potential relationship with NQO2.Among ten selected IncRNAs,which with high normalized intensity and high fold changes,seven of them were further verified to be consistent with the microarray analysis by qRT-PCR.Among the above 7 verified IncRNAs,4 IncRNAs（NR₀46711、NR₀36537、NR-03658O 和 NR₀02795）were chosen to under CeRNA analysis.Conclusions:Aberrantly expressed IncRNAs were expected to play a crucial role in the liver metastatic process of CRC.Our present finding provided potential novel targets for the future exploring of liver metastasis of CRC.Part ⅡThe lncRNA HOXA11-AS functions as a competing endogenous RNA to regulate PADI2 expression by sponging miR-125a-5p in liver metastasis of colorectal cancerBackground:Colorectal cancer is one of the most common malignancies.Metastasis is the primary factor affecting the prognosis of colorectal cancer,and the liver is the most common target organ of metastatic colorectal cancer.According to existing studies,long noncoding RNAs（IncRNAs）may play a crucial role in colorectal liver metastasis.In the first part of this study,we use microarray assay and qRT-PCR verification method to find several potential lncRNAs associated with liver metastasis of colorectal cancer.The subsequent CeRNA analysis showed that HOXA11-AS could potentially be used as a competitive endogenous RNA,adsorption of miR-125a-5p to regulate the expression of PADI2,so as to promote colorectal cancer with liver metastasis.Material and Methods:Samples in this part were collected from patients admitted to the Department of Colorectal Surgery,First Affiliated Hospital,Zhejiang University,Hangzhou,China,between April 2016 and September 2016.In total,30 primary CRC samples were obtained from 15 patients with CRC and liver metastasis（Exp G）and 15 patients with CRC without metastasis（Ctrl G）.The differentially expression of HOXA11-AS,miR-125a-5p and PADI2 were verified by qRT-PCR.Two colon cancer cells were selected,according to the HOXA11-AS expression level,to undergo the subsequent studies.We employed construct generation and transient transfection,qRT-PCR,Western Blot,Dual-luciferase reporter gene assays,migration and invasion assay to verify the function of HOXA11-AS,miR-125a-5p and PADI2 in the process of liver metastasis of CRC.Results:The expression of HOXA11-AS and PAD 12 in Exp G were significantly increased,while the expression of miR-125a-5p was significantly decreased by qRT-PCR.The expression of HOXA11-AS were detected in six colon cancer cell lines,and it was higher in more aggressive cell lines（Co1o205,HCT116,Lovo and SW620）than the less aggressive cell lines（Caco-2 and SW480）.The highest HOXA11-AS expression cell（SW620）and the lowest HOXA11-AS expression cell（SW480）were selected for the subsequent studies.Overexpression or knockdown studies of HOXA11-AS or PADI2,as well as gain-/loss-of-function studies of miR-125a-5p,revealed a positive correlation between HOXA11-AS and PADI2 and a negative correlation with miR-125a-5p,confirming their involvement in the regulation of liver metastasis in CRC cell lines.Using dual-luciferase reporter gene assays,we identified HOXA11-AS and PAD 12 as endogenous sponges for miR-125a-5p.Conclusions:This study supports a role for HOXA11-AS in the promotion of liver metastasis by functioning as a ceRNA sponge for miR-125a-5p to promote PADI2 expression.Our findings indicate a novel HOXA11-AS/miR-125a-5p/PAD12 regulatory network in CRC liver metastasis.Our findings also show that HOXA11-AS is an important molecular marker in predicting liver metastasis and a potential target for CRC therapy.