| Objective: To investigate the effects of Flotilin-1 on proliferation, migration and invasion of SCLC(small cell lung cancer cells) and to explore the possible molecular mechanism of Flotilin-1 suppressing epithelial-mesenchymal transition of SCLC.Methods:1 We got Aptamers identificating to SCLC cells by using the method of SELEX.We choosed C12 to continue our later research after comparing the specificity and bonding strength of the sequences we got.2 We synthesized biotin labelled C12 in vitro, and then used the labeled C12 to enrich particular protein in SCLC ell lysate. We choosed the FLOT1 to the following research from the results of the MS.3 DNA-pulldown experiment was applied to prove C12 was combined with FLOT1 ecially.4 Immunofluoresence tested the location of FLOT1 in small cell lung cancer cells.5 Immunohistochemistry detected the expression of FLOT1 in tissue chip of lung disease.6 Immunohistochemistry detected the expression of FLOT1 in small cell lung cancer.7 The proliferation, migration and invasion abilities of SCLC cells with FLOT1 low-expression were detected by RTCA, transwell migration and matrigel invasion chamber assays,respectively.8 We constructed stably FLOT1 low expressed cells namely shNC,shFLOT1-1 and shFLOT1-2 through lentivirus infection, and constructed control vector stably expression cells, namely control.9 The effects of FLOT1 low-exoression on the cell cycle of NCIH446 cells were detected by FCM.10 The expression of apoptosis related proteins including the Bcl- 2, Caspase3, PAKT and Parp in NCIH446 cells FLOT1 low expression were tested by Western blot after cisplatin induced by different concentration.11 The effects of FLOT1 low-exoression on the cell apoptosis were detected by FCM after cisplatin induced by different concentration.12 Cells of shNC and shFLOT1-1 groups were injected to nude mice through subcutaneous, tail vein.13 The mRNA expression differences of shNC and shFLOT1-1 groups cellls were found in the Gene chip.14 The expression of CDC42, SOX11, Twist2 choosing from the gene chip were proved by Real- time PCR.15 The expression of TGF-β and H-Ras in NCIH446 of shNC,shFLOT1-1 and shFLOT1-2 was detected by Western-blot.Results:1 Mass Spectrometry analysis showed that FLOT1 could bind to C12.2 DNA pull-down combining with western blotting assay demonstrated that FLOT1 binded to C12 really.3 Immunofluorescence assay showed FLOT1 located on the cell membrane.4 Tissue microarray immunohistochemistry results showed FLOT1 expressed in most of the lung disease, and in healthy people was not expressed in the lung tissue.5 Small cell lung cancer patients with immunohistochemistry displayed FLOT1 expressed in cancerous tissue and were not expressed in normal tissues.6 RTCA assay demonstrated that FLOT1 low-expression could inhibit the proliferation of NCIH446(P<0.05).7 Transwell migration assays demonstrated that low-expression of FLOT1 inhibited migration abilities of NCIH446(P<0.05). Matrigel invasion chamber assays demonstrated that low-expression of FLOT1 inhibited invasion abilities of NCIH446(P<0.05).8 Flow Cytometry showed that after knocking down FLOT1, the cell cycle of small cell lung cancer cell line NCIH446 arrested in G0G1 phase.9 Western blot experiment found the expression of cyclinD1 was reduced in NCIH446 cells low-expression FLOT1.10 After the treatment of cis-platinum, the protein level of Bcl-2,PAKT and cleaved Parp and Caspase 3 increased.11 Flow Cytometry showed that the cell apoptosis in NCIH446 of low-expression Flotilin-1 was increased.12 Results demonstrated that, the ability of tumor formation and lung metastasis were repressed obviously, verifying low-expression FLOT1 could inhibit NCIH446 cells proliferation and migration in vivo(P<0.05).13 Compared with shNC group,the expression level of Twist2,CDC42,SOX11 in NCIH446 of shFLOT1-1 group was decreased.14 Compared with shNC group,the expression level of TGF-β and H-Ras in NCIH446 of shFLOT1-1 and shFLOT1-2 group was decreased. Conclusions:The expression level of FLOT1 in small cell lung cancer tissues was increased, FLOT1 low-expression could inhibit the proliferation, migration abilities and invasion abilities of NCIH446.Besides that, FLOT1 low-expression could promote the apoptosis of NCIH446 and arrested cell cycle in G0G1 of NCIH446. In addition, knowing down the expression of FLOT1 can inhibit the occurrence of EMT of small cell lung cancer cells, the process may be associated with inhibiting the expression of TGF-β.The malignant phenotype of small cell lung cancer cells may be associated with the activation of the H-Ras. |
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