Expression Of TIM-1 In Cervical Cancer Tissue And Its Effect On Invasion And Metastasis Of Cervical Cancer Cells

Objective: It has been reported that TIM-1 is involved in the biological behavior of a variety of malignant tumors,however,the expression of TIM-1in cervical cancer cells and its effect on the progression and metastasis of cervical cancer remain unclear.The purpose of this study was to explore the correlation between TIM-1 expression and the occurrence and development of cervical cancer,and to explore the mechanism of TIM-1 involvement in the invasion and metastasis of cervical cancer,so as to find a new target for the diagnosis and treatment of cervical cancer.Methods:1.Using immunohistochemical method to detect 80 cases of human cervical cancer group,30 cases of cervical intraepithelial neoplasia and 10 cases of normal cervical tissue in TIM-1 protein expression,and combining with clinical and pathological characteristics analysis of TIM-1 expression with age,pathological type,differentiation degree,FIGO stage,tumor diameter,infiltrating depth,whether involving vaginal stump,with and without lymph node metastasis,in presence of nerve invasion,correlation parameters such as presence of vascular invasion.2.The expression of TIM-1 in three cervical cancer cell lines with different HPV phenotypes,He La(HPV-18 positive),Si Ha(HPV-16 positive)and C-33A(HPV-negative)were detected by q RT-PCR for m RNA and Western blotting for protein.3.Overexpression of TIM-1 lentivirus vector and negative lentivirus empty vector were transfected into He La and Si Ha cells with relatively low expression of TIM-1,and the transfected cells were screened by purinomycin.Fluorescence microscopy and flow cytometry were used to detect the transfection efficiency.The expression of TIM-1 was verified by q RT-PCR and Western blotting.4.The effect of overexpression of TIM-1 on proliferation of cervical cancer cell lines was observed in vitro by CCK-8 experiment and plate clone formation experiment.5.The effect of TIM-1 on the cell cycle of cervical cancer was detected by PI single staining flow cytometry.The effect of TIM-1 on apoptosis of cervical cancer cells was detected by Annexin V-APC/PI double staining flow cytometry.6.The effect of TIM-1 on tumor-forming ability of cervical cancer cells was observed in vivo by subcutaneous tumor-forming experiment in nude mice.7.The effect of TIM-1 on the migration ability of cervical cancer cells was observed in vitro by scratch healing test and Transwell migration test.Transwell invasion assay was used to observe the effect of TIM-1 on the invasion ability of cervical cancer cells.8.Western blotting was used to detect the effects of overexpression of TIM-1on cyclin D1,p53,Bcl-2 and Bax,which were related to cell cycle apoptosis.9.Western blotting was used to detect the effects of TIM-1 overexpression on metastasis related molecules E-cadherin,N-cadherin,Vimentin,Snail1,MMP-2 and VEGF.10.Western blotting was used to detect the effects of TIM-1 overexpression on PI3 K,AKT,p AKT and m TOR proteins related to the PI3K/AKT/m TOR pathway.Results:1.Immunohistochemical results showed that the expression of TIM-1 protein in cervical cancer tissues(81.25%,65/80)was significantly higher than that in cervical intraepithelial neoplasia tissues(40%,12/30)and normal cervical tissues(0%,0/10),with statistical significance(P = 0.000).TIM-1 expression was negatively correlated with vaginal stump involvement(r =-0.303,P =0.006),and had no significant difference with age,pathological type,degree of differentiation,FIGO stage,tumor diameter,invasion depth,lymph node metastasis,nerve invasion,and vascular invasion(P > 0.05)2.The results of q RT-PCR and Western blotting showed that TIM-1 was expressed in three cervical cancer cell lines(C-33 A,He La and Si Ha),and the expression was relatively low in He La and Si Ha cells.3.After transfection with the expression of TIM-1 lentivirus and screening with purinomycin,both fluorescence microscopy and flow cytometry analysis showed that the transfection efficiency of the stable transfection strains of He La and Si Ha cells reached more than 90%.q RT-PCR and Western blotting results showed that the expression level of TIM-1 m RNA and protein in the TIM-1group of He La and Si Ha cells was significantly higher than that in the MOCK group and NC group(P < 0.001),and there was no significant difference between the MOCK group and the NC group(P > 0.05).4.CCK-8 experiment showed that the absorbance of the TIM-1 group was significantly higher than that of the MOCK group and the NC group from the fourth day of He La cell inoculation,and the data was statistically significant(P < 0.01),and there was no significant difference between the MOCK group and the NC group(P > 0.05).From the second day after inoculation,the absorbance of Si Ha cells in TIM-1 group was significantly higher than that in MOCK group and NC group,and the data was statistically significant(P <0.001),while there was no significant difference between the MOCK group and the NC group(P > 0.05).The results of clone formation experiments showed that the clone formation ability of He La and Si Ha cells in TIM-1 group was significantly higher than that in MOCK group and NC group(P < 0.001),while there was no significant difference between the MOCK group and the NC group(P > 0.05).5.Flow cytometry cell cycle detection results showed that the number of cells in S phase of cell cycle in the TIM-1 group was significantly increased after overexpression of TIM-1 in He La cells compared with the MOCK group and the NC group(P < 0.001),the number of cells in the G2 phase of the cell cycle was significantly increased(P < 0.001),the number of cells in the G1 phase of the cell cycle was significantly reduced(P < 0.001),while there was no significant difference between the MOCK and NC groups(P > 0.05).After the overexpression of TIM-1 in Si Ha cells,the number of cells in the S phase of cell cycle in the TIM-1 group was significantly increased compared with that in the MOCK group and the NC group(P < 0.001),the number of cells in the G1 phase of the cell cycle was significantly reduced(P < 0.001),while there was no significant difference between the MOCK and NC groups(P >0.05).Flow cytometry assay showed that the apoptosis rate of TIM-1 group in He La cells and Si Ha cells was lower than that in MOCK group and NC group,and it was statistically significant(P < 0.001),while there was no significant difference between the MOCK and NC groups(P > 0.05).6.Subcutaneous tumor-formation experiments in nude mice showed that tumor growth rate,tumor fluorophore,tumor size and tumor weight of Si Ha cell in TIM-1 group were significantly higher than those in MOCK group and NC group(P < 0.05),while there was no significant difference between the MOCK group and the NC group(P > 0.05).7.Compared with MOCK and NC groups,TIM-1 of He La and Si Ha cells showed relatively narrower scratch spacing at 24 h and 48 h after scratch(P <0.001),while there was no significant difference between the MOCK and NC groups(P > 0.05).Tranwell cell migration assay showed that,compared with MOCK and NC groups,the number of cells per high power field(×200)penetrated the membrane in TIM-1 group of He La and Si Ha cells was significantly increased,and the difference was statistically significant(P <0.001),while there was no statistical significance between the MOCK and NC groups(P > 0.05).Transwell cell invasion assay showed that,compared with MOCK group and NC group,the number of cells per high power field(×200)penetrating membrane in TIM-1 group of He La and Si Ha cells was significantly increased,and the difference was statistically significant(P <0.001),while there was no statistical significance between the MOCK and NC groups(P > 0.05).8.Western blotting results showed that the overexpression of TIM-1 in He La and Si Ha cells could significantly up-regulate the protein expression levels of cyclin D1 and Bcl-2,and down-regulate the protein expression levels of p53 and Bax,while there was no statistical significance between the MOCK and NC groups(P > 0.05).9.Western blotting results showed that the overexpression of TIM-1 in He La and Si Ha cells significantly up-regulated the protein expression levels of Ncadherin,Vimentin,Snail1,MMP-2 and VEGF,while down-regulated the protein expression levels of E-cadherin,while there was no statistical significance between the MOCK and NC groups(P > 0.05).10.Western blotting results showed that overexpression of TIM-1 in He La and Si Ha cells could significantly up-regulate the protein expression levels of PI3 K,p AKT and m TOR,but had no significant effect on the expression of total AKT,while there was no statistical significance between the MOCK and NC groups(P > 0.05).Conclusion:1.Compared with normal cervical epithelial tissue and CIN,TIM-1 was highly expressed in human cervical cancer tissue,and was negatively correlated with the involvement of vaginal stump;TIM-1 is expressed in He La(HPV-18positive),Si Ha(HPV-16 positive),and C-33A(HPV-negative)cells.2.Overexpression of TIM-1 down-regulates the expression of p53,Bax,and Ecadherin,and up-regulates the expression of cyclin D1,Bcl-2,Snail 1,Ncadherin,Vimentin,MMP-2,and VEGF;and the levels of PI3 K,p AKT and m TOR were also up-regulated,while the total AKT protein level remained unchanged.TIM-1 can induce EMT transformation by regulating intracellular PI3K/ AKT /p53 and PI3K/ AKT /m TOR signaling pathways,promote cell proliferation,migration and invasion,and inhibit cell apoptosis.3.TIM-1 may provide a new diagnostic and therapeutic target for cervical cancer.