The Tumor Formation And Mechanism Analysis Of Human Cervical Cancer Cells Stably Transfected With LRAT And RDH12 Genes In Nude Mice

BACKGROUND AND PURPOSE:Cervical cancer is one of the most common cancers in females.It has been demonstrated that HPV infection alone is insufficient while the abnormal expression of related genes is critical in development of cervical cancer.Our studies have screened the differential expressed genes between cervical cancer tissues and matched adjacent non-tumor tissues by RNA-Seq technology.We discovered that the expression of the retinol metabolism related genes(LRAT and RDH12)were lower in cervical cancer tissue.In our experiments in vitro,we found that overexpression of LRAT?RDH12 helps inhibiting the cell proliferation,transferation and invation.In the cervical cancer cells with overexpression of LRAT/RDH12,the expression of proteins involved in MAPK/NF-kB signal pathway changed accordingly.The expression of LRAT/RDH12 increased,the expression of P38 MAPK/NF-kB decreased.In this study,we are trying to carry on experiments in vivo,verify the relation between LRAT/RDH12 gene and the MAPK/NF-kB signal pathway.By establishing the nude mouse model,this project aims to analyze the tumor growth curve in nude mice injected with Hela cell of LRAT/RDH12 overexpression.In order to clarify the effect and mechanism of retinol metabolism related genes LRAT/RDH12 in the progress of cervical cancer,the study will also draw the tumor tissues from nude mice to make immunohistochemistry analysis and western blot experiments.In this way,this study can provide important theoretical supports and molecular targets for cervical cancer.METHODS:1.Growing the stably transfected cell:using recombinant plasmid to culture two kinds of Hela cell lines stabled transfected by LRAT and RDH12 gene.2.Establishing the nude-mouse transplanted tumor model:using original Hela cell in control group to explore the best concentration of Hela cell to form tumor tissues.We make different concentration of Hela cell suspension:5×106/mL?1×107/mL?5×107/mL?1×108/mL.3.Observing the tumor growth condition.Recording the volume size of tumor tissues and drawing the tumor growth curve.4.Drawing the tumor tissues from nude mice to make immunohistochemistry analysis and western blot experiments.Verifying the relation between LRAT/RDH12 gene and expression of P38,P50 and P65 in the MAPK/NF-kB signal pathway.RESULTS:Culturing and acquiring the Hela cell lines stabled transfected by LRAT and RDH12 gene.By qPCR technique,we verify that the overexpression of LRAT gene in stably transfected Hela cell is over 20 thousand times than blank control group,while the overexpression of RDH12 gene is over 30 thousand times.Putting two kinds of Hela cell above into western blot experiment,we get the expected results:in the two kinds Hela cells stabled transfected by LRAT and RDH12 gene,the expression of P38,P50 and P65 in the MAPK/NF-kB signal pathway are all decreasing.In establishing nude-mouse transplanted tumor model,We get the 5×107/mL as the best concentration of Hela cell suspension to form tumor tissues.The nude-mouse transplanted tumor model is built successfully.By observing and recording the tumor growth condition,we discovered the nude mice in LRAT and RDH12 group has slower tumor growth rate than control group of original Hela cell.The inhibiting effect of LRAT gene is stronger than RDH12 gene.Drawing the tumor tissue of 3 weeks from nude mice in control group and two experimental group,carrying on immunohistochemistry analysis and western blot experiments in three groups.The results show the overexpression of LRAT/RDH12 gene can reduce the expression of P38,P50 and P65.In this way,the activity of MAPK/NF-kB signal pathway can be suppressed.CONCLUSION:The overexpression of LRAT/RDH12 gene can effectively inhibit the development of cervical cancer.This phenomenon is related with suppressing the protein in MAPK/NF-kB signal pathway.LRAT and RDH12 gene have the potential to be the new targets for cervical cancer treatment in the future.