Function Of Midkine On The Proliferation And Migration Of Thyroid Cancer

Objective: Human serum midkine(MDK)is highly expressed in a variety of malignant tumors,usually playing the role of oncogenes,participating in various biological behaviors such as tumor growth and migration.There are few reports on the function and mechanism of MDK in thyroid cancer.This article aims to explore the proliferation and migration functions of MDK on thyroid cancer and lymphatic endothelial cells of different differentiation types by establishing overexpression and knockout of thyroid cancer stable cell lines.And further study the molecular mechanism behind it.Methods: Part I: 6 pairs of thyroid cancer patient tissues were collected,and the expression of MDK protein in 6 pairs of cancer tissues and corresponding adjacent tissues was detected by immunoblotting.At the same time,Western blotting(WB)was used to detect MDK expression levels in normal thyroid cells(NTHY)and different types of thyroid cancer cells(K1,TPC1,THJ16 T,BCPAP,ARO,T238);Using enzyme-linked immunosorbent assay(Elisa)assay to detect extracellular MDK expression levels in normal thyroid cells(NTHY)and different types of thyroid cancer cells(K1,TPC1,THJ16 T,BCPAP,ARO,T238).Part II: According to the extracellular expression level of MDK,K1 and BCPAP cells with high expression were selected in papillary cancer cell lines,and THJ16 T cells were selected in anaplastic cancer cell lines.The Crisp-cas9 technology was used to construct stable MDK knockout cell lines Down-regulate MDK expression.Further use WB to verify the effect of stable system construction from the protein level.Part III: According to the extracellular expression level of MDK,in papillary cancer cell lines,select TPC1 cells that express low levels,and in anaplastic cancer cell lines,select THJ16 T cells to construct stable lentiviral stable overexpressing MDK stable cell lines and upregulate MDK expression..Elisa and WB were used to verify the effect of stable system construction from the protein expression levels inside and outside the cell.Part IV: To explore the effect of MDK on the proliferation and migration of thyroid cancer cells.(Cell Counting Kit-8,cck8)and clone formation experiments were used to analyze the proliferation phenotype;Trans-well and scratch experiments were used to analyze the migration phenotype.Further,the changes of protein molecules related to proliferation and migration were detected by WB.Part V: To explore the effect of MDK protein expression on proliferation and migration of lymphatic endothelial cells.Stable cells overexpressing MDK were selected and the proliferation and migration phenotypes of lymphatic endothelial cells were analyzed using co-incubation experiments.Results: Part I: MDK is highly expressed in human thyroid cancer tissues,and low or even not expressed in adjacent tissues.Consistent with this,MDK is highly expressed in thyroid cancer cell lines compared to normal thyroid epithelial cell.Part II:Through plasmid construction and lentivirus infection,successfully constructed MDK knockout stable cell lines K1-MDK-KO,BCPAP-MDK-KO,THJ16T-MDK-KO,compared with stable line cells transfected with corresponding empty plasmids,knocked out group cells The MDK intracellular and extracellular proteins were significantly down-regulated.Part III: Through plasmid construction and lentivirus infection,MDK overexpression stable cell lines TPC1-MDK-OVER and THJ16T-MDK-OVER were constructed.Compared with corresponding stable cell lines transfected with empty plasmids,intracellular and extracellular proteins of overexpression group cells Significantly increased.Part IV: MDK selectively affects the proliferation ability of thyroid cancer cell lines.In vitro experiments showed that after stable knockout / overexpression of MDK protein in thyroid papillary carcinoma cell lines,there was no significant change in cell proliferation ability,but in anaplastic cancer,cell proliferation was significantly slower / faster.WB found that the anti-apoptotic protein Bcl2 was up-regulated and the apoptotic protein Bax was down-regulated.MDK selectively affects the migration ability of thyroid cancer cell lines.In vitro experiments showed that after stable knockout / overexpression of MDK protein in thyroid carcinoma papillary carcinoma cell lines,there was no significant change in cell migration ability,but in anaplastic cancer,cell migration ability was significantly weakened / enhanced.In anaplastic cancer cell lines with phenotypic changes,WB found that the migration proteins vmintin,snail and N-ca were significantly up-regulated,while some migration proteins β-catenin and twist were slightly down-regulated.Part V: The secretion of MDK by anaplastic thyroid cancer cell lines affects the proliferation and migration of lymphatic endothelial cells.For lymphatic endothelial cells,in vitro co-incubation experiments showed that after over-expressing MDK protein in thyroid cancer anaplastic cancer cell lines,lymphatic endothelial cells proliferate and migrate more.Conclusion: MDK is highly expressed in thyroid cancer tissues and cell lines.In papillary cancer cell lines,the expression of MDK has no significant effect on proliferation and migration ability;in anaplastic cancer cell lines,MDK promotes proliferation and migration by affecting protein molecules related to proliferation and migration.MDK secreted by anaplastic cancer cell lines promotes the proliferation and migration of lymphatic endothelial cells.