| Objective: This study intends to take papillary thyroid cancer cell line infected by recombinant lentivirus vector bearing LMTK3 gene silencing as a research object and to explore the role of LMTK3 in papillary thyroid cancer cell biological behavior,including proliferation,invasion,metastasis and apoptosis.To understand whether LMTK3 is involved in the regulation of thyroid papillary cancer cell signal transduction pathways of proliferation,invasion,metastasis and apoptosis.Methods: 1.To investigate the expression of LMTK3 in the two PTC cell lines: we detected the expression of LMTK3,ERα,ERβ and AR in the two PTC cell lines by q RT-PCR and Western blot,and selected the PTC cell line in which the expression level of LMTK3 is high and ERα and ERβ are present in the expression level of gene and protein.2.Lentiviral vector infected PTC cell line selected along with its validation experiments: Lentivirus particles loading short hairpin LMTK3(sh LMTK3)or its corresponding control respectively infected PTC cell line in which the gene and protein expression level of LMTK3 is high,and ERα and ERβ are present in the gene and protein expression level,which were taken as the LV-sh LMTK3 groups(three silenced sequences were denoted as LV-sh LMTK3-1,LV-sh LMTK3-2 and LV-sh LMTK3-3 respectively)and the LV-Control group,and the blank control group(Control)without any treatment.After 72 hours,Western blot and CCK-8 assay were used to detect the expression level of LMTK3 protein and cell activity of the PTC cells.Finally,the reasonable experimental group and control group were selected to carry out the followup experiment.3.The research about the influence of LMTK3 on PTC cell proliferation,colony formation,invasion,metastasis and apoptosis: The proliferation ability of the selected cells was detected by CCK-8 assay.Cloning capacity of the selected cells was evaluated by clone formation assay.Scratch test and Transwell assay were used to evaluate the infected cells migration and invasion capabilities.The apoptosis rate of the infected cells was detected by flow cytometry.4.Investigation of signaling pathways about LMTK3 participating in the proliferation,invasion,metastasis and apoptosis of PTC cell line on the m RNA expression level: We first observed the effects of LMTK3 on morphological alterations of PTC cell after lentiviral vector infection by inverted phase contrast microscopy.The m RNA expression level of LMTK3,ERα,ERβ,AR,CDK2,CDK3,CDK4,CDK6,E-cadherin,N-cadherin,Vimentin,MMP-2,MMP-3,MMP-9,TGF-β1,MEK,ERK,AKT,PKC,Bcl2,Bax,Capsase3,Snail,Twist1,Twist2 in the infected cells was detected by q RTPCR.CDK2,CDK3,CDK4 and CDK6 are the growth cycle-associated proteindependent kinases.E-cadherin is the epithelial marker,and N-cadherin and Vimentin are the interstitial markers.MMP-2,MMP-3 and MMP-9 are the metastasis-related factors.Bcl2,Bax and Capsase3 are the apoptosis-related factors.Twist,Snail1 and Snail2 are the transcription-related factors.5.Investigation of signaling pathways about LMTK3 participating in the proliferation,invasion,metastasis and apoptosis of PTC cell line on the protein expression level: The total protein level of its key downstream signaling effectors,including ERa,ERβ,β-catenin,ERKl/2,AKT and protein phosphorylation level of ERKl/2 and AKT on LMTK3 interfered and Control cells were detected by Western blot.Results: 1.The m RNA level of LMTK3 expression was approximately equal in BCPAP and TPC-1 cell,but the protein level of LMTK3 expression was obviously high(P=0.0275)and ERα and ERβ expression was present in BCPAP cell which was selected as the research object.2.The following experiments were performed using the cells with an interference efficiency of more than 80%.Western blot demonstrated that there was no significant difference in protein expression between in the LV-Control group and the blank Control group(P=0.2677),while the expression level of LMTK3 protein in the LV-sh LMTK3 group was significantly lower than that in the LV-Control group(P=0.0055,P=0.0024 and P=0.0003,respectively).The result of cell viability were consistent with the result of Western blotting experiments(P=0.4987,P=0.3086,P=0.0030 and P=0.0046,respectively).Therefore,we considered that the Control lentiviral vector had little effect on the cell and there was no statistical difference.Then the following experiments proceeded on in the LV-sh LMTK3 group and the LV-Control group.3.The results of CCK-8 assay showed that there was no obvious difference in the proliferation rate of cell between in the LV-sh LMTK3 group and the LV-Control group at 24 hour(P=0.4730).The proliferation rate of the LV-sh LMTK3 cell was significantly lower than that of the LV-Control cell at 48,72,96 and 120 hour(P=0.0084,P=0.0005,P=0.0005 and P=0.0006,respectively).The colony formation number of the cells in the LV-sh LMTK3 group was prominently less than that in the LV-Control group(P=0.0032).The scratch test result showed that the migration ability of the LVsh LMTK3 group cell was notably lower than that of the LV-Control group at 48 hour(P=0.0038).The same result was detected by transwell migration assay(P=0.0010).Transwell invasion test result showed that the invasive ability of the LV-sh LMTK3 group cell was distinctively lower than that of the LV-Control cell at 48 h(P=0.0055).Flow cytometry result showed that the apoptosis rate of the LV-sh LMTK3 cell was observably higher than that of the LV-Control cell at 48 h(P=0.0078).4.After BCPAP cells were infected by lentiviral vectors bearing gene silencing LMTK3,the morphological change of BCPAP cells was not obvious.The m RNA expression level of ERα,CDK6,E-cadherin,Twist,MEK and PKC in the LV-sh LMTK3 group was remarkably higher than that in the LV-Control group(P=0.0033,P=0.0389,P=0.0151,P=0.0251,P=0.0050 and P=0.0049,respectively).The expression level of LMTK3,ERβ,AR,N-cadherin,Vimentin,Snail2,MMP-2,MMP-9,ERK,TGF-β1,Bcl2 and Capsase3 m RNA in the LV-sh LMTK3 group was prominently lower than that in the LV-Control group(P=0.0008,P<0.0001,P=0.0288,P=0.0005,P<0.0001,P=0.0480,P=0.0221,P=0.0239,P=0.0463,P=0.0197,P=0.0008 and P=0.0044,respectively).But the m RNA expression level of CDK2,CDK3,CDK4,MMP-3,Bax,Snail1 and AKT was not dramatically different in the two groups(P=0.2159,P=0.3582,P=0.9184,P=0.0880,P=0.8941,P=0.1508 and P=0.1554,respectively).5.After LMTK3 gene silencinglentiviral vector infected BCPAP cells,in which ERα protein expression was signally higher than that in the LV-Control group(P=0.0005).ERβ,p-ERK1/2,p-AKT and β-catenin protein expression was noticeably lower than that in the LV-Control group(P=0.0021,P=0.0006,P=0.0033 and P=0.0009,respectively),but the protein expression level of the total ERK1/2 and the total AKT did not change memorably(P=0.0986 and P=0.6428,respectively)..Conclusion: 1.LMTK3 gene silencingcan particularly inhibit the proliferation,invasion and metastasis of BCPAP cell line,and can promote its apoptosis.2.The estrogen receptor,PI3K/AKT,MEK/ERK,TGF-β1,Wnt/β-catenin signal pathways and EMT process all participated in the regulation of LMTK3 on the proliferation,invasion,metastasis and apoptosis of BCPAP cell line. |
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