| Objective: The current researches of basic oncology are mainly focusing on the molecular mechanism of metastasis,while the exploration of early detection and more effective treatment strategies are still concerned equally.Eukaryotic translation initiation factors 3H(EIF3H)is a kind of protein that plays important roles in the process of protein translation initiation in eukaryotic cells,and is one of the important subunits of EIFs family.In recent years,many studies have reported that the amplification and overexpression of EIF3H in various malignant tumors,such as gastric cancer,colon cancer,liver cancer,breast cancer,etc.,which can affect abilities of the growth,proliferation,apoptosis,invasion,drug resistance and other phenotypes.However,the role of gene EIF3H in the occurrence and development of ovarian cancer and the related molecular mechanism are still unclear.In the course of this study,the expression of EIF3H in epithelial ovarian cancer was mainly analyzed,and the relationship between EIF3 h and apoptosis,proliferation and migration of ovarian cancer was studied,and its related mechanism was further studied.Methods: This study is divided into three parts:The first part: Using immunohistochemical method,129 epithelial ovarian cancer tissues and 19 normal ovarian tissues were selected as the research objects,and the expression of EIF3H was analyzed,and the relationship between the expression of EIF3H and the prognosis of ovarian cancer was analyzed.The second part: 1.The mRNA level of EIF3H in several ovarian cancer cell lines(SK-OV-3,Caov3,HO-8910,OVCAR3)was detected by qRT-PCR,and the cell lines with higher expression level were determined,and they were taken as the follow-up research objects;2.Three different types of interference vectors were designed to infect the corresponding target cells with lentivirus,and the expression of EIF3H in different groups of ovarian cancer cells was detected by qRT-PCR,and the si RNA-EIF3H with the best interference effect was selected as the post-infection condition.Furthermore,qRT-PCR and Western blot were used to detect the expression of EIF3H gene and protein in ovarian cancer cells infected with lentivirus.3.SKOV3 and HO-8910 cell lines were selected and divided into control group(sh Ctrl)and experimental group(sh EIF3H)and transfected with different lentivirus vectors,respectively.The apoptosis ability of the two groups was detected and compared by HCS cell proliferation and FACS cell apoptosis experiment(single staining method).4.Scratch test and Transwell test were used to detect and compare the cell migration and transfer ability of the two groups.5.In vivo experiments further verified the effect of EIF3H on the proliferation of ovarian cancer cells.The third part: 1.The potential downstream gene regulated by EIF3H gene was inferred by chip technology and biological analysis,and the expression of mRNA and protein was further verified by qRT-PCR and Western blot respectively,and finally SPINK7 was selected as the downstream gene;2.CO-IP experiment was used to verify the interaction between EIF3H and SPINK7 protein;3.Quantitative qRT-PCR was used to detect the mRNA level of SPINK7 in various ovarian cancer cell lines(SKOV3,Caov3,HO-8910,OVCAR3),and the cell lines with higher expression level were determined,and they were taken as the follow-up research objects;Three different types of interfering vectors were designed to infect the corresponding target cells with lentivirus,and the expression of interfering RNA was analyzed by qRT-PCR method,focusing on its SPINK7 expression in different groups of ovarian cancer cells,and the si RNA with the best interference effect was determined,which was regarded as the infection condition in the later experiment.4.Recovery experiment: Divided into four groups: blank control group NC(EIF3H+sh SPINK7),EIF3H up-regulation group(EIF3H+NC-sh SPINK7),SPINK7down-regulation group(NC-EIF3H+sh SPINK7),EIF3H up-regulation and SPINK7down-regulation group(EIF3H+sh SPINK7).The lentiviral vectors up-regulating EIF3H and/or down-regulating SPINK7 were infected into HO-8910 cells,and the expression changes of corresponding mRNA and protein of EIF3H and SPINK7 were compared by qRT-PCR and Western blot,further verifying the regulatory effect of EIF3H on SPINK7.5.Group as above,transfect the corresponding regulatory vectors of the target gene into ovarian cancer cells respectively,and detect and compare the changes of apoptosis,cloning and migration ability of different groups of cells by FACS method,cloning experiment,scratch experiment and Transwell migration experiment respectively;6.Combined with the mechanism of regulating classical pathway proteins and inflammatory factors verified by SPINK7 in the literature,WB method was used to detect the regulatory function of EIF3H on inflammatory factors and classical pathway proteins in ovarian cancer cells.7.Divided into four groups: blank control group(sh Ctrl),blank control +P53 inhibitor group(sh Ctrl+P53 inhibitor),knock-down EIF3H group(sh EIF3H)and knock-down EIF3H+P53 inhibitor group(sh EIF3H+P53 inhibitor).Lentiviral vector infected cell HO-8910 to verify the effect of P53 inhibitor on the expression of classical pathway protein regulated by target gene EIF3H.Results: Part Ⅰ: 1.Cmpared with normal ovarian tissues,EIF3H protein was significantly higher in ovarian cancer tissues(P<0.01),and the difference was statistically significant.The expression of EIF3H protein in ovarian cancer tissue is related to age,pathological type,clinical stage and prognosis(P<0.05).Part Ⅱ: 1.The mRNA expression level of EIF3H in ovarian cancer cells with statistical significance(P<0.05).2.After the ovarian cancer cell line was infected with lentivirus of si RNA,the results of q PCR and WesternBlot showed that of EIF3H in sh EIF3H group were lower than those in sh Ctrl group(P<0.05).3.The experimental results of cell proliferation and apoptosis show that compared with the control group,the ability of cell proliferation is significantly reduced(P<0.05),while the rate of apoptosis is obviously increased in the sh EIF3H group(P<0.05)accompanied by the reduction of EIF3 h expression.4.The results of cell cycle experiment suggested that when EIF3 h expression was knocked down in ovarian cells,the ratio of cells in G2 phase in sh EIF3H group was also significantly increased compared with the control group(P<0.05).5.Wound-healing assay and Transwell assay showed that the cell migration ability of sh EIF3H group was much lower than that of control group after 24 h scratch(P<0.05).6.The results of in vivo experiments indicated that the proliferation ability of tumor cells in nude mice was significantly decreased after EIF3H was knocked out,and the difference was statistically significant(P<0.05).Part Ⅲ:1.The mRNA and protein expression of downstream genes were screened out by PCR and WB validation biogenic analysis,and the potential downstream gene SPINK7 was verified and identified by CO-IP experiment.2.The results of qRT-PCR indicated that the expression of SPINK7 in HO-8910 and SK-OV-3 was relatively higher(P<0.05),but there was no significant difference in A2780 cells,compared with IOSE80 cells.After infecting HO-8910 cells with the interfering RNA of SPINK7 gene by lentivirus,the expression of SPINK7 in different groups of ovarian cancer cells was significantly downregulated.3.The results of qRT-PCR on mRNA level show that,after infection:Compare to NC(EIF3H+sh SPINK7)group,in EIF3H+NC-sh SPINK7 group,EIF3H mRNA level had no significant change,SPINK7 mRNA level had no significant change.Compare to NC(EIF3H+sh SPINK7)group,in NC-EIF3H+sh SPINK7 group,EIF3H mRNA level increased obviously(P<0.05),SPINK7 mRNA level decreased obviously(P<0.001).Compare to EIF3H+NC-sh SPINK7 group,in EIF3H+sh SPINK7 group,EIF3H mRNA level had no significant change,SPINK7 mRNA level decreased obviously(P<0.001).Compare to NC-EIF3H+sh SPINK7 group,in EIF3H+sh SPINK7 group,EIF3HmRNA level had no significant change,SPINK7 mRNA level increased obviously(P<0.001).4.The results of Western Blot show that,after infection :Compare to NC(EIF3H+sh SPINK7)group,in NC-EIF3H+sh SPINK7 group EIF3H protein level decreased obviously,SPINK7 protein level decreased obviously.Compare to NC(EIF3H+sh SPINK7)group,in EIF3H+NC-sh SPINK7 group EIF3H protein level increased obviously,SPINK7 protein level had no significant change.Compare to NCEIF3H+sh SPINK7 group,in sh SPINK7+EIF3H group EIF3H protein level increased obviously,SPINK7 protein level increased obviously.Compare to EIF3H+NCsh SPINK7 group,in sh SPINK7+EIF3H group EIF3H protein level decreased obviously,SPINK7 protein level decreased obviously.5.Compared with NC(EIF3H+sh SPINK7)group,the apoptosis of HO-8910 cells in EIF3H+ NC-Shspink7 group was significantly decreased(P<0.001),and the number of cell clones was significantly increased(P<0.001).Cell mobility was significantly increased at 48h(P<0.001);The apoptosis of NC-EIF3H+sh SPINK7 group was significantly increased(P<0.001),and the number of cell clones was significantly decreased(P<0.001).Cell mobility at 48 h was significantly decreased(P<0.05).Compared with EIF3H+NC-sh SPINK7 group,the apoptosis of cells in EIF3H+sh SPINK7 group was significantly increased(P<0.001),the number of cell clones was significantly decreased(P<0.001),and the mobility of cells at 48 h was significantly decreased(P<0.001).Compared with NC-EIF3H+sh SPINK7 group,cell apoptosis was significantly decreased in EIF3H+sh SPINK7 group(P<0.001),the number of cell clones was significantly increased in EIF3H+sh SPINK7 group(P<0.001),and cell mobility was significantly increased at 48h(P<0.001).6.The results of Western Blot on classical apoptosis proteins manifested that the expressions of Bax,Caspase-1,P53 and Vimentin were up-regulated,while there was no significant change in the expression of CCNB1,N-Cadherin and p-P53 in sh EIF3H group,compared with sh Ctrl group after lentivirus infection.The results of WB on detection of inflammatory factors showed that,compared with the sh Ctrl group,the expressions of TLR4,HMGB1,CD133 were downregulated after lentvirus infection,while the proteins MCP-1,SOX2,OCT4 were not detected,compared with the sh Ctrl group.7.P53 inhibitor experiment: WB results indicated that after lectin infection,compared with NC group,the expression of Bcl-2was up-regulated,EIF3H and Bax down-regulated,whie the expressions of FAS,MDM2 and p-P53 were not significantly changed in sh EIF3H group.Compared with NC group,the expressions of Bax,Bcl-2 and FAS were down-regulated,EIF3H not significantly changed,while the expressions of protein MDM FAS2 and p-P53 were up-regulated in sh EIF3H+P53 inhibitor group.Compared with sh EIF3H group,the expressions of Bax,Bcl-2 and FAS were down-regulated,while the expressions of proteins EIF3H,MDM2and p-P53 were up-regulated in sh EIF3H+P53 inhibitors group.8.The results of FACS apoptosis assay suggested that compared with sh Ctrl group,apoptosis in sh EIF3H group was significantly increased(P<0.001),while apoptosis in sh Ctrl+P53 inhibitor group was significantly decreased(P<0.01).Compared with sh EIF3H group,apoptosis was significantly decreased in sh EIF3H+P53 inhibitor group(P<0.001).Compared with sh Ctrl+P53 inhibitor group,apoptosis was significantly increased in sh EIF3H+P53inhibitor group(P<0.001).The results of cell cycle experiment indicated that,compared with sh Ctrl group,there was no change in S phase in sh EIF3H group,but significantly increased in G2 phase(P<0.05).No change in S phase in sh Ctrl+P53 inhibitor group,and no change in G2 phase.Compared with sh EIF3H group,sh EIF3H+P53 inhibitor group significantly decreased in S phase(P<0.05)and G2 phase(P<0.001).Compared with sh Ctrl+P53 inhibitor group,cells in sh EIF3H+P53 inhibitor group were significantly increased in S phase(P<0.05),and significantly decreased in G2 phase(P<0.001).Conclusions: EIF3H protein is significantly higher expressed in epithelial ovarian cancer tissues,and the expression in ovarian cancer tissues is significantly related to the pathological registration of patients and the prognosis of patients.Knocking down the EIF3H gene in human ovarian cancer cell line can significantly inhibit the proliferation and migration of ovarian cancer cells and promote apoptosis.EIF3H may be involved in the occurrence and development of ovarian cancer by regulating the downstream gene SPINK7,and change the expression of apoptosis pathway proteins and inflammatory factors.This mechanism can be influenced by P53 inhibitors,which can further change the ability of apoptosis and cell cycle.Briefly,EIF3H gene may become a new direction of target gene detection and treatment of ovarian cancer. |
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