The Effect Of Interfering Gene TRIP13 On Proliferation Of HCT116 Colorectal Cancer Cells

Background:Epidemiology,both the morbidity and mortality of colorectal cancer(colorectal cancer,CRC)are human concerns.In our country,the incidence of CRC in all tumors is ranking third to fifth.In western countries,the incidence of CRC in all tumors is ranking second to third.Otherwise,CRC is the fourth leading cause of cancer-related death in the world.Many patients were diagnosed advanced stage CRC when they first came to the hospital.After surgery,radiotherapy,chemotherapy and other treatments,the rate of the occurrence of drug resistance,progression,recurrence is high.Gene diagnosis and treatment provide a new direction for the prevention and treatment of CRC.The discovery of genes associated with CRC and its pathogenic mechanisms are the prerequisite.Thyroid hormone receptor factor 13(TRIP13)gene is located in chromosome 5 short arm 1 zone 5 band.The TRIP13 gene is a protein-coding gene and is also identified as an oncogene.The overexpression of TRIP13 gene can lead to a variety of human cancers.TRIP13 gene has not been confirmed to be related to CRC,and the relationship between TRIP13 gene and CRC remains to be further studied.Objectives:In this study,we surpose to examine the expression of TRIP13 gene in colorectal cancer HCT116 cells firstly.Then infect HCT116 cells with lentivirus containing the RNA interference sequence of the TRIP13 gene.Finally a series of cell function tests(Including cell cycle detection,apoptosis detection,clonal formation detection,MTT detection)were performed on HCT116 cells after knockdown of TRIP13 gene.To investigate the effect of TRIP13 on the proliferation of HCT116 cells and toexplore the role of TRIP13 in the proliferation of HCT116 cells,and to provide preliminary experimental evidence for further diagnosis and treatment of colorectal cancer.Methods:(1)The expression of the target gene in the target cell was detected by Real-time Quantitative Polymerase Detection System(qPCR),and The HCT116 cells were infected with lentivirus containing the RNA interference sequence of the TRIP13 gene.The efficiency of TRIP13 gene knockdown was detected by qPCR in mRNA level.If the TRIP13 gene knockdown efficiency <50%,we continue lentivirus infection.If the TRIP13 gene knockdown efficiency> 50%,we began with the next experiment of cell function testing.(2)In vitro functional part of the experiment,using the method of Propidium(PI)staining and flow cytometry(FCM)to detect cell cycle,in order to investigate the effect of TRIP13 gene on HCT116 cells growth cycle.Apoptosis was detected by Annexin V-APC single-dose flow cytometry to explore the association between TRIP13 gene and the HCT116 cells apoptosis.The Giemsa staining clone formation assay was used to explore the tumorigenic ability of the HCT116 cells after lentivirus infection.The MTT assay was used to verify the purpose effects of TRIP13 gene on HCT116 cells proliferation.(3)Statistical analysis was performed by using Spss 21.0 software.Results:(1)The expression of TRIP13 gene in HCT116 cells is high.The efficiency of Lentivirus infection in the purpose cells had been achieved more than 80%,and the state of the cells was normal.After the Lentivirus infection,the expression of TRIP13 gene in HCT116 cells was inhibited by mRNA(p <0.05),and the knockdown efficiency was 85.5%.(2)Cell cycle test results: Compared with the control group(shCtrl group),the cells in the S phase of the experimental group(shTRIP13 group)were decreased(P<0.05),and the cells in the G1 phase increased(P <0.05).There was no significant change in the G2 / M phase cells.(3)Apoptosis test results: Compared with the shCtrl group,shTRIP13 group increased the number of apoptotic(P <0.05).(4)MTT test results: compared with shCtrl group,shTRIP13 group cell proliferation slowed down.Conclusions:Interference with the target gene TRIP13 on the colorectal cancer cell HCT116 inhibit its proliferation.TRIP13 gene may be a potential target for the therapy of CRC.