Effects of Tim-3 on Invasion and Migration of Cervical Cancer Cells and Its Mechanism

The aim of this study was to investigate effects of Tim-3 (T-cell immunoglobulin mucin-3) mediated by lentivirus on cervical cancer cells and its related mechanism. Tim-3 fragments were amplified by PCR and cloned into a pCDH vector for enzyme digestion and sequencing. Firstly, the recombinant positive expression vectors pCDH-Tim-3/ pCDH-NC/pCDH-GFP, psPAX2, and pMD2.G were co-transfected into 293T cells, forming three types of lentivirus groups. Next, three groups of lentiviruses were respectively infected with HeLa cells to obtain stable cell lines (HeLaTim-3, HeLa-NC, HeLa-GFP). HeLa-NC was set as the control group. Finally, the efficiency of infecting lentivirus wasobserved in HeLa-GFP group preliminarily. The relative expression levels of Tim-3 were detected by flow cytometry, Western blot, and cellular immunofluorescence. Abilities including migration and invasion were detected by cell scratch and Transwell. Expression of EMT (epithelial-to-mesenchymal) related to proteins (E-cadherin, N-cadherin, Snail) was detected by Western blot. The apoptosis rate was detected by flow cytometry assay. We successfully constructed a lentivirus plasmid containing Tim-3 and then packaged it as lentivirus particles. The stable cell lines of HeLa-Tim-3, HeLaNC, and HeLa-GFP were obtained, separately. Comparing with HeLa-NC group, the expression of Tim-3 in HeLa cells was increased after infection in the HeLa-Tim-3 group (P<0.05). The ability of invasion and metastasis were significantly enhanced in HeLa cells with high expression of Tim-3 (P<0.01). The observed effect was associated with downregulation of E-cadherin and upregulation of N-cadherin and snail (P<0.05). Meanwhile, the apoptosis rate was also markedly inhibited (P<0.001). Our findings suggest that the expression of Tim-3 in cervical cancer may facilitate the tumor invasion and metastasis by promoting EMT transformation and inhibiting apoptosis.

CSTR: 32200.14.cjcb.2020.04.0002