| Background:Hepatoblastoma(HB)is a special type of childhood liver cancer that plays an important role in children's tumors,and its incidence is increasing worldwide,especially in North America and Europe.HB is mainly derived from human immature liver precursor cells.According to histological classification,this tumor can be classified into epithelial-derived or mixed epithelial/mesenchymal tissue.The treatment of hepatoblastoma is adjuvant chemotherapy,radiotherapy,transplantation,and the like.Although some progress has been made in the treatment of this type of tumor,the effect is still not obvious.With the development of biotechnology,the role of gene therapy in anti-cancer has received more and more attention.Long Non-coding RNA(lncRNA)plays an important role in tumor research,affecting various aspects of tumor biological activity,such as transcription,epigenetics and regulation of mRNA expression.They can form pathways by interacting with miRNAs or mRNAs in human cancers.For example,lncRNA-MUF interacts with ANXA2 and miR-34a,making mesenchymal stem cells unable to induce hepatocarcinogenesis;LncRNA HNF1A-AS1 mediated miR-34a/SIRT1/p53 axis promotes colon cancer metastasis progression.In this paper,lncRNA OIP5-AS1 was selected for research.Several studies have shown that LncRNA OIP5-AS1 has a regulatory role in certain diseases that modulate biological activity.For example,it can control mitosis and the like by inhibiting GAK expression to regulate proliferation and apoptosis of multiple myeloma cells.In human cancer,microRNAs are some very important biomarkers.They can be tumor promoters or tumor suppressors for various human cancers.Studies have shown that miRNA can exert the anticancer effect of miRNA by targeting the seed region of downstream mRNA by inhibiting the translation of mRNA.For example,has-miR-495 targeting FAM83D acts as a tumor suppressor in colorectal cancer(CRC).MiR-186-5p is a miRNA whose specific biological behavior has not been reported in hepatoblastoma This article discusses its anti-tumor effect in hepatoblastoma.Zinc finger E-box binding to homolog 1(ZEB1)is a transcription factor that has been shown to be rich in carcinogenic properties.ZEB1 has previously been reported to be highly expressed in many types of human cancers,such as colon cancer,glioblastoma,and prostate cancer It has also been found to be involved in ceRNA patterns of hepatocellular carcinoma(HCC),thyroid cancer(TC),colorectal cancer(CCAR)and the like.These previous reports have helped us to further our research.Through the above reports and introductions,we understand the importance of lncRNA,miRNA and mRNA.The CeRNA pattern is a biological axis composed of three genes.For example,lncRNA CCAT1 acts as a ceRNA to regulate the miR-181a-5p/HOXA1 axis in myeloma;NSCLC,medium MALAT1-miR-124-STAT3 pathway.In this study,we explored a ceRNA pathway in HB to study the function of the LncRNA OIP5-AS1/miRNA-186-5p/ZEB 1(mRNA)axisAfter reviewing the data obtained from the previous clinical and tissue microarrays,the relationship between the expression of LncRNA OIP5-AS1 in patients with clinical hepatoblastoma and the TNM staging,hepatocarcinoma cell proliferation,metastasis and epithelial-mesenchymal transition EMT were explored.To explore the relationship between LncRNA OIP5-AS1 and miRNA-186-5p and epithelial-mesenchymal transition key molecule EMT-tfs,and this will further elucidate the effect of ceRNA pattern on hepatocarcinoma in tumor cell invasion-metastasis The mechanism provides a certain new theoretical basis for the diagnosis of HCC metastasis,and provides strategy for clinical individualized treatmentPurpose:Based on the clinical and tissue microarray data of hepatoblastoma,this experiment was performed by knocking down IncRNA OIP5-AS1 to explore whether it can pass the miR-186-5p/ZEB1 signal axis,and then proliferate in hepatoblastoma cells in ceRNA mode.Invasion and metastasis and epithelial-mesenchymal transition processes(EMT)are regulated.Materials and methods:1.Real-time quantitative PCR was used to detect tissue samples from 80 patients with hepatoblastoma(40 cases cancer tissue matched with 40 cases of adjacent normal tissues),and q-PCR was used to explore human normal liver cell lines L-02,QSG-7701,and hepatoblastoma cells.The expression of lncRNA OIP5-AS1 in SMMC-7721,HepG2,HuH-6 cells.The expression levels were correlated with TNM staging of tumors,hepatoblastoma proliferation,metastasis,and epithelial-mesenchymal transition EMT.2.Construction and transfection of the knockdown plasmid of lncRNA OIP5-AS1,knocking down OIP5-AS1 in HepG-2 and HuH-6 cell lines,and detecting the expression of OIP5-AS1 in cells by q-PCR,using scratch test The clone formation assay and the Transwell chamber assay were used to detect cell metastasis,invasion and proliferation,and the expression of EMT-related proteins was detected by Western Blot.3.Construct a stable cell line of HuH-6 knockout IncRNA OIP5-AS1 and find the downstream target mircoRNA miRNA-185a-5p by miRNA sequencing technology.4.Using the nucleoplasm separation experiment to explore the specific expression position of IncRNA OIP5-AS1 in HepG2 and HuH-6 cells,and to explore OIP5-AS1 by real-time quantitative PCR verification,dual luciferase reporter experiment and base pairing relationship prediction analysis.The ability to bind to the downstream target miRNA-186-5p and the regulatory relationship between the two were verified by rescue experiments.5.Using several commonly used miRNA databases,we searched for the target gene ZEB1 of miRNA-186-5p and explored the binding ability of miRNA-186-5p to the downstream target gene ZEB1 using the same method as in the fourth step.The regulatory relationship between the two is verified by a rescue experiment.The regulatory relationship between lncRNA OIP5-AS1 and ZEB1 was verified by real-time quantitative PCR.6.Using the inhibitor of miRNA-186-5p and the knock-out plasmid of si-ZEB1,LncRNA OIP5-AS1/miRNA-186 was verified in the constructed HuH-6 stable cell line knocking out lncRNA OIP5-AS1-5p/ZEB 1(mRNA)Axis function7.Finally,the expression of EMT-related protein in each step of the fifth step was detected by Western Blot to verify that lncRNA OIP5-AS1 can pass the miR-186-5p/ZEB 1 signal axis,and then in hepatoblastoma cells.In the ceRNA model,tumor cell proliferation,migration and invasion-metastasis(EMT)are regulatedResults:1.LncRNA OIP5-AS1 is highly expressed in hepatoblastoma tissues and cell lines and has a significant correlation with various clinical indicators of clinical patients The results of Kaplan-Meier survival curve indicate that the low expression group,in contrast,the high expression group has a shorter survival rate,and the high expression of lncRNA OIP5-AS1 suggests a poor prognosis in patients with hepatoblastoma2.After knocking out lncRNA OIP5-AS1 in the hepatoblastoma cell lines HepG2 and HuH-6,cell proliferation,metastasis and epithelial-to-glycan transfer(EMT)processes were significantly inhibited3.In hepatoblastoma,lncRNA OIP5-AS1 and miR-186-5p can be combined by base pairing,and the same miR-186-5p can also be paired with ZEB1 by base pairing Binding is performed to form a ceRNA regulation mode4.LncRNA OIP5-AS1/miRNA-186-5p/ZEB 1(mRNA)signal axis may be present in hepatoblastoma,regulated by lncRNA OIP5-AS1,miR-186-5p and ZEB1 expression,and further in HB cells with the ceRNA model,tumor cell proliferation,migration and invasion-metastasis(EMT)are regulatedConclusions:1.In hepatoblastoma,the high expression status of lncRNA OIP5-AS1 suggests a poor prognosis in patients with hepatoblastoma2.LncRNA OIP5-AS1 can regulate tumor cell proliferation,migration and invasion-metastasis(EMT)in hepatoblastoma cells via the miR-186-5p/ZEB1 signal axis... |
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