| Research Background:Cervical cancer is the third most common cancer among women worldwide and the fourth leading cause of cancer death,accounting for 9%of new cancers and 8%of total cancer deaths.More than 85%of cases of cervical cancer morbidity and mortality occur in developing countries.(Global Cancer Statistics)As the largest developing country in China,its morbidity and mortality are far above average.A large body of literature indicates that long-chain non-coding RNA(LncRNA)plays a key role in tumorigenesis and development;NEAT1 is a LncRNA located in the nucleus involved in the progression of many cancers;however,the biological function of LncRNA NEAT1 in cervical cancer Not fully studied.At the same time,PI3K/AKT/mTOR signaling pathway plays a classical pathway to promote cell proliferation and migration,and regulates the role of various LncRNAs in order to participate in the development of tumors.Research purposes:To explore the expression level of LncRNA NEAT 1 in human cervical cancer tissues or cells,the relationship between LncRNA NEAT1 and clinicopathological features and overall survival rate of cervical cancer patients;on the basis of the above,study the effect of LncRNA NEAT 1 on proliferation and migration of cervical cancer cells,and Explore the specific molecular mechanisms and conduct targeted interventions to provide relevant targets for the treatment of cervical cancer.Research method:(1)Collect 62 cervical cancer patients with cancer tissue and adjacent tissues(requires the distance of adjacent tissues from the cancer center>5cm)and collect their clinical data from September 2012 to September 2017.The patient did not receive radiotherapy,chemotherapy and other related treatments before surgery.At the same time,the clinical data of 62 patients with cervical cancer were collected.The database was established and statistically inductive analysis was performed.The expression level of LncRNA NEAT1 in cervical cancer specimens was detected by qRT-PCR.Comparison of adjacent tissues;(2)Cell culture of four kinds of cervical cancer cells(HeLa,ME-180,SiHa,CaSki),normal cervical epithelial cells(H8),and their expression levels of LncRNA NEAT1 were detected by qRT-PCR.SPSS software version 19.0(IBM Corp.,Armonk,NY,USA)for statistical analysis,repeated 3 measurements,data expressed as x±s;(3)evaluation of 62 cervical cancer patients LncRNA NEAT1 expression levels and clinical pathology parameters Correlation between pathological type,FIGO grade,tumor size,degree of differentiation,lymphatic metastasis,etc.,and Kaplan-Meier method was used to analyze the correlation between LncRNA NEAT 1 expression level and overall survival of patients.(4)Transfecting the plasmid of si-NEAT1 into CaSki and HeLa cells,causing low expression of LncRNA NEAT1 in CaSki and HeLa cells,transfection of pcDNA-NEAT1 into SiHa cells,high expression of LncRNA NEAT1 in SiHa cells,and transfection by qRT-PCR.Efficiency,MTT assay,colony formation assay to determine cell proliferation ability;transwell assay to detect the migration ability of the above cells;and compared with the blank control cells;(5)Western blot detection of low expression of LncRNA NEAT1 and overexpression of LncRNA NEAT1 cervical cancer Activation of the PI3K/AKT/mTOR signaling pathway.(6)Using the activators and inhibitors of PI3K/AKT/mTOR signaling pathway IGF-1 and LY294002 to treat LncRNA NEAT1 and overexpressing LncRNA NEAT1 cervical cancer cells,using MTT assay and colony formation assay to determine cell proliferation ability;Transwell assay for detection The ability of the cells to migrate;and compared with the cells of the blank control group.Research resu t:(1)NEAT1 is highly expressed in human cervical cancer tissues and cervical cancer cell lines.High expression of NEAT1 predicts poor prognosis in patients with cervical cancer(P=0.005,?2=7.735);(2)cellular activity of CaSki cells with low expression of NEAT1 From 0.592±0.257 to 0.360±0.091,the number of cell colonies decreased from 136.667±13.503 to 71.667±7.506(t=-18.76,P=0.003),and the migration ability decreased from 100.333±9.866 to 58.333±5.859(t=-8.08,P=0.015);the activity of HeLa cells with low expression of NEAT 1 decreased from 0.516±0.208 to 0.328±0.069(t=2.98,P=0.041),and the number of cell colonies decreased from 128.667±13.317 to 65.667±7.024(t=-5.54,P=0.031),the migration capacity decreased from 123.667±12.097 to 67.667±7.095(t=-6.03,P=0.026);the cell viability of SiHa cells with high expression of NEAT1 increased from 0.314±0.013 to 0.728±0.146(t=12.16,P=0.017),the number of cell colonies increased from 84.667±12.014 to 150.667±18.037(t=7.27,P=0.018);the migration ability increased from 127.333±16.042 to 231.333±31.786(t=4.92,PP=0.039).In vivo experiments confirmed that the growth of tumors with low expression of NEAT 1 cell line was significantly slower than that of tumor cells with high expression of LncRNA NEAT1 cell line.(3)In CaSki and HeLa cells with low expression of LncRNA NEAT1,PI3K/AKT/mTOR signal is weakened,and in SiHa with high expression of LncRNA NEAT1,PI3K/AKT/mTOR signal is enhanced,and the quantitative analysis results have statistical significance;With low expression of NEAT1 in CaSki and HeLa cells,the inhibitor of PI3K/AKT/mTOR signaling pathway,IGF-1,can enhance cell proliferation and migration.In SiHa cells with high expression of LncRNA NEAT1,LY294002,an inhibitor of PI3K/AKT/mTOR signaling pathway,can inhibit cell proliferation,migration ability.Research conclusion:1.Up-regulation of LncRNA NEAT 1 expression in human cervical cancer tissues and various cervical cancer cell lines;2.The level of LncRNA NEAT 1 expression is related to the clinicopathological features and overall survival rate of patients with cervical cancer;3.Overexpression or knockdown of LncRNA NEAT1 affects cervical cancer cell proliferation and migration;4.LncRNA NEAT1 is involved in the activation of the PI3K/AKT/mTOR signaling pathway in cervical cancer;5.PI3K/AKT/mTOR pathway participates in LncRNA NEAT1 to regulate cell proliferation and migration6.LncRNA NEAT1 may play an oncogene role in the development of cervical cancer.LncRNA NEAT1 may be a new therapeutic target for cervical cancer and is closely related to the PI3K/AKT/mTOR signaling pathway. |
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