Effects Of Minichromosome Maintenance Protein7(MCM7) Knockdown On Biological Functions Of Hepatocellular Carcinoma Cells

With more than 850,000 new cases emerged each year,liver cancer is one of the major diseases affecting human health.Among all primary liver cancers,hepatocellular carcinoma(HCC)is the most common portion.Therefore,understanding the mechanisms of HCC development is crucial to discover the novel bio-markers and potentially therapeutic targets.We have been working on exploring the functional mechanisms of HCC cancer stem cell,and developing new strategies to suppress HCC-tumor stem cells or tumor metastasis.In previous work,we found that MCM7,an important member of the minichromosome maintenance(MCM)protein family,might play a key role in the metastasis and HCC-tumor stem cells.This family,which currently has ten members,is an indispensable component of DNA pre-replication complex and takes part in the precise initiation of DNA replication and the repair of DNA damage.Our previous results also showed that MCM7 was highly expressed in clinical tumor tissue of 80 HCC patients and its expression was positively correlated with clinical pathology grade,tumor size,AFP level,but survival time was in reverse.In order to clarify its roles in the development of HCC,in this study,we first constructed stable cells with low MCM7 expression by knocking down MCM7 with lentiviral transfection of MCM7 interference vector.Then we studied the effects of MCM7 knockdown on HCC cell’s biological function and explored the underlying mechanism of MCM7.This research is divided into three parts:Part Ⅰ:Establishment and identification of stable cell lines with low MCM7expression.Methods:Small interfering RNA(siRNA)which can silence the expression of MCM7 gene was predicted by the website(www.sigma.com).Then according to the principle of constructing the shRNA vector,we designed the sense and antisense oligonucleotide sequences of the MCM7-shRNA gene.After the designed interference fragment was added to the pSicoR vector,MCM7 knockdown in four HCC cell lines,Huh-7.5.1,PLC/PRF/5,Hep3B,HCCLM3,was conducted by using lentiviral vector transduction system.Cells expressing GFP~+fluorescence were selected through flow sorting.Western blot was performed to verify whether the MCM7 expression was reduced in the constructed cell lines.Results:After screened by flow sorting,each of the HCC cell lines showed green fluorescence strongly.These evidences proved that the constructed pSicoR-shMCM7interference plasmid was expressed in these cells.In addition,Western blot analysis showed that MCM7 protein levels were down-regulated in all cell lines,which also proved that the construction of stable HCC cell lines was successful.Part Ⅱ:Effect of knockdown of MCM7 on the biological function of HCC cellsMethods:Through drawing the growth curve and detecting the colony formation,we explored the effects of down-regulation of MCM7 on HCC cells proliferation.We also explored effects of MCM7 knockdown on the proliferation of HCC cells in a three-dimensional model,which imitating the microenvironment of tumor.Using cell migration assays and flow cytometry,we also explored the role of MCM7 in tumor cell migration and apoptosis.To observe effects of MCM7 knockdown on the tumorigenic ability of HCC cells,Huh7.5.1 and HCCLM3 cells with/without MCM7 knockdown were slected to inject into the xenograft mice model.Then,the mices were observed twice a week.After 40 days,the mice were sacrificed amd the subcutaneous tumos were harvested and analyzed.Results:After analyzing the growth curve,we found that the cell proliferation of the MCM7 knockdown group was depressed significantly.The number of colony formation in the MCM7 knockdown group was less than that in the control group.These results suggested that MCM7 knockdown inhibits the ability of HCC proliferation,including in a 3D culture model.In addition,in the experiments of cell scratch and cell apoptosis,MCM7 knockdown inhibited cell migration ability and promoted apoptosis.Further,xenograft tumor model was established to confirm the biological role of MCM7 in HCC in vivo.We found that the tumor formation rates and tumor sizes in the MCM7 knockdown group were significantly smaller than that in the control group.This indicated that knockdown of MCM7 might inhibit the tumorigenic ability of HCC cells in vivo.Part Ⅲ:Prediction of MCM7 target genes.Methods:HCC cells with MCM7 knockdown were detected by the gene chip.The differently expressed genes were screened through P value and Fold change value.The standard is up-or down-regulated fold change value≥1.5 and P≤0.05.Real-time PCR was the preliminary work checking changed miRNA.Further the analyses of ceRNA among the obtained differential genes were used to explore the underlying mechanism.Results:Fifty pairs of ceRNA were obtained by screening.Further GO and KEGG analyses were performed among obtained ceRNA relationship pairs.GO analysis showed that MCM7 knockdown could negatively regulate NF-κB transcription factor activity,MAP kinase activity,typical Wnt signaling pathway,ERK1 and ERK2 cascade,which have important roles in the progression of cancer.The results of KEGG analysis showed that the changes in signaling pathways with MCM7 knockdown mainly contained cell metabolism,apoptosis,and stem cell function.Furthermore,we confirmed that MCM7 knockdown could upregulate miRNA-194 expression and downregulate miRNA-629 expression.Based on the previous work of the laboratory,our study investigated the functional changes of HCC cells with low MCM7 expression.These results indicated that MCM7knockdown not only inhibited cell proliferation,cell migration and promoted cell apoptosis in vitro,but also inhibited the tumorigenic ability of HCC cell in vivo.These results provided a certain experimental basis and theoretical support in making MCM7becoming a potential new drug target.