The Effects Of Gene Silencing LMTK3 Expression On Malignant Biological Behavior Of Thyroid Papillary Carcinoma Cells And Its Mechanism

Objective: To retrospectively analyze the risk factors of recurrence and metastasis after surgery of patients who with Papillary thyroid cancer(PTC)in our hospital in the past 5 years.To investigate the effect of gene silencing Lemur tyrosine kinase-3(LMTK3)on the malignant biological behavior of PTC cells and to explore the relationship between serum Thyroid Stimulating Hormone(TSH)and LMTK3.Maybe relevant clinical values will be found after our reaserch.Methods:1.438 patients who met the criteria after PTC surgery visited our hospital from January 2014 to December 2018 were analyzed.The correlation between postoperative recurrence and tumor size,lymph node metastasis and hyperthyroidism index TSH was retrospectively analyzed.2.qRT-PCR was used to detect whether the expression levels of LMTK3 in PTC cell lines K1 or IHH4 were different,and the cell lines with high expression of LMTK3 would be screened for the subsequent experiments.3.At the mRNA level,we explored the malignant biological regulatory signaling pathways of PTC cells that LMTK3 may be involved in.The designed fluorescent and resistant lentiviral silencing LMTK3 gene(shLMTK3)was stably transferred into the selected cell line,the LMTK3 expression was silenced in the end.According to the degree of gene knockdown,the experiment was divided into three groups: lentiviral silencing group(shRNA-LMTK3),empty lentivirus control group(shRNA-Control)and Blank Control group.Fluorescence microscopy was used to observe the fluorescence expression of the cells after infection and qRT-PCR was used to detect the transfection efficiency of the experimental group and the control groups.After successful transfection,qRT-PCR was used to investigate the expression changes of LMTK3,RelA and NF-?B1 mRNAs related to the experiment.4.The differential expression of shLMTK3 transcriptome gene from RNA-seq technology was used to analyze the regulation of LMTK3 on the occurrence and development of PTC,and to explore its association with TSH,a risk factor for recurrence and metastasis of PTC.Results:1.Tumor size and lymph node metastasis were independent risk factors for postoperative recurrence and distant metastasis(P=0.020,P=0.026,respectively),at the same time,TSH was highly expressed in the serum of patients with recurrence and metastasis,and the difference was statistically significant(P=0.018).The results indicated that patients with tumors diameter>1cm and/or lymph node metastasis and TSH level more than 0.1mIU/L were prone to postoperative recurrence and metastasis.2.The expression level of LMTK3 in K1 cell mRNA was significantly higher than that in IHH4 cells,and the difference was statistically significant(P=0.009).Therefore,the K1 cell line with highly expression of LMTK3 was selected for subsequent experiments.3.Firstly,lentivirus-silenced LMTK3 infected K1 cells,and under the fluorescence microscope we observated the morphology of the cells was not changed too much after fluorescenced.After 72 hours of infection,the fluorescence expression reached more than 80%,and the initial transfection was successful.Secondly,the results of qRT-PCR showed that the mRNA expression of LMTK3 in shRNA-LMTK3 group was obviously lower than those in the two control groups(P=0.030,P=0.042,respectively),nevertheless,there was no statistical expression in shRNA-Control group and Blank Control group(P=0.094).Therefore,it was speculated that the empty lentiviral vector has little effect on K1 cells,and there was no statistical difference.The subsequent experiments would be carried out in the shRNA-LMTK3 group and the shRNA-Control group.Thirdly,the expression levels of NF-?B1 and RelA mRNA were not significantly different between shRNA-LMTK3 group and shRNA-Control group(P=0.189,P=0.393,respectively),indicating that LMTK3 has no regulatory effect on NF-?B1/RelA.4.211 differentially expressed genes were screened from 26,000 genes,including 122 genes with up-regulated expression and 89 genes with down-regulated expression.Through KEGG signal pathway enrichment analysis,lentivirus-silencedLMTK3 gene in K1 cells by binding to related targets not only up-regulated TNF signaling pathway,but also led to down-regulation of cAMP pathway(P<0.05).Among the genes whose expression was down-regulated by GO function,the?-catenin target site was significantly regulated by LMTK3(P=0.013).Conclusion:1.Tumor diameter,cervical lymph node metastasis and TSH level were high risk factors for recurrence and metastasis after PTC surgery,it with statistical significance(P<0.05).2.Wnt/?-catennin signaling pathway may be involved in the regulation of K1 cells by LMTK3.3.LMTK3 may affect the binding of TSH and TSHR by down-regulating cAMP or up-regulating TNF signaling pathway,and promote the recurrence and metastasis of PTC.