Effect Of KLF5 On The Growth And Sensitivity To Doxorubicin Of Anaplastic Thyroid Carcinoma Cells And Its Mechanism

Background:Thyroid cancer accounts for about 0.5%to 1%of all human malignant tumors,which is the most common cancer in the endocrine system.Anaplastic thyroid carcinoma(ATC)is an undifferentiated tumor in thyroid follicular epithelium,accounting for 1%-2%of all thyroid cancers.The prognosis of patients with ATC is poor due to its aggressive nature and resistance to treatment.Therefore,new therapeutic targets are needed to improve the clinical treatment of these patients.KLFs is a transcriptional regulator,which is associated with cell proliferation,migration,apoptosis and tissue remodeling.KLF5 is a member of the KLF family,which is translated to regulate cellular processes through phosphorylation,acetylation,and ubiquitination.Studies have shown that KLF5 is often upregulated in some human cancer,which plays a role as an oncogene.The JNK signaling pathway regulates a variety of physiological processes,including cell differentiation,cell proliferation,cell death,inflammatory response and protein expression.JNK disorders are associated with a variety of diseases,including cancer,diabetes,neurodegenerative diseases,autoimmune diseases,cardiac hypertrophy.JNK1,JNK2 and JNK3 are regulatory genes of JNK pathway.Studies have found that JNK signaling pathway can promote the proliferation,invasion and migration of thyroid cancer cells,which may provide a new molecular target for the treatment of thyroid cancer.However,the regulatory role of the JNK signaling pathway in ATC has not been known.Objective:This study to explore effect of KLF5 on the growth and sensitivity to doxorubicin of anaplastic thyroid carcinoma cells and its mechanism,first KLF5 expression in a variety of ATC cells was measured by qRT-PCR,and the effect of knockout KLF5 on cell proliferation,apoptosis,invasion and migration of ATC was determined by MTT,flow cytometry assay and Western blot,Transwell assay and scratch assay,studing preliminaryly the influence of the knockout KLF5 on ATC cell biology function;Effect of KLF5 overexpression and KLF5 interference on HTh74 and HTh74Rdox cell proliferation,clone formation,apoptosis and sensitivity to doxorucin was determined at the cell level,and the changes of the JNK signaling pathway related molecules were detected,further exploring the influence of KLF5 on sensitivity to doxorucin in ATC cells and its mechanism;In vivo,ATC transplanted tumor was build by using 8505c cells,then tumor-burdened mice were treated with adriamycin,the volume of the transplanted tumor was regularly tested,and mice were put to death after 30 days to detect the weight of the transplanted tumor,and KLF5 on the influence of transplanted tumor tissue morphology and KLF5 protein expression in ATC transplanted tumor were measured by HE staining and immunohistochemical assays respectively,and the JNK signaling pathway related molecular expression changes were determined by Western blot,and effect of KLF5 on growth of ATC transplantation tumor and susceptibility to adriamycin and its mechanism was discussed.This study is mainly divided into three parts:Part ?:Effect of KLF5 on biological characteristics of ATC cells;Part ?:Effect of KLF5 on adriamycin sensitivity in ATC cells and its mechanism;Part ?:Effect of KLF5 on the growth and sensitivity to adriamycin of ATC transplanted tumor in vivo.Methods:Part ?:Effect of KLF5 on Biological Characteristics of ATC Cells1.The expression of KLF5 in various ATC cell lines was measured by qRT-PCR.2.The expressions of KLF5,proliferation-related protein Ki67,apoptotic-related protein cleaved caspase-3,invasion and migration-related proteins MMP-2 and MMP-9 in 8505c,HTh74 and HTh74Rdox cells were detected by Western blot.3.Construction of KLF5 interference vector:pLenti-hU6-GFP-Puro lentivirus vector cut with AgeI and EcoRI enzyme was linked and transformed with KLF5 interference fragment.After PCR identification of single colony,the correct clone was sent to Shanghai shenggong biological engineering co.,ltd.for sequencing.And to package and titrate recombinant lentiviruses,after that,the recombinant lentivirus was infected with 8505c and HTh74 cells to obtain stable cell lines.4.The proliferation of 8505c and HTh74 cells was determined by MTT assay.5.The apoptosis of 8505c and HTh74 cells was mearsured by flow cytometry assay.6.The invasion of 8505c and HTh74 cells was detected by Transwell assay.7.The migration of 8505c and HTh74 cells was tested by scratch assay.Part ?:Effect of KLF5 on Adriamycin Sensitivity in ATC Cells and its Mechanism1.Construction of KLF5 overexpression vector:pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector was subjected to double enzyme digestion with EcoRI and Notl,and then the enzyme fragment and KLF5 gene fragment were linked and transformed,and colony PCR identification was carried out,which was sent to Shanghai shengong biological engineering co.,LTD for sequencing.After that,the recombinant lentivirus was infected with HTh74 and HTh74Rdox cells to obtain stable cell lines.2.The expressions of KLF5,Ki67,cleaved caspase-3,P-gp,ABCG2,and JNK signaling pathway related proteins p-jnk and JNK in HTh74 and HTh74Rdox cells were detected by Western blot.3.The proliferation of HTh74 and HTh74Rdox cells was detected by MTT assay.4.colony formation assay was used to detect the colony formation ability of HTh74 and HTh74Rdox cells.5.MTT was used to detect the drug sensitivity of HTh74 and HTh74Rdox cells to doxorubicin.6.Apoptosis of HTh74 and HTh74Rdox cells was detected by flow cytometry.7.Expression of MDR1 and ABCG2 in HTh74 and HTh74Rdox cells was detected by qRT-PCR.Part ?:Effect of KLF5 on the Growth and Sensitivity to Adriamycin of ATC Transplanted Tumor in Vivo1.The transplanted tumor of undifferentiated thyroid cancer was constructed with 8505c cells,and the tumor-bearing mice were treated with doxorubicin.The volume of the transplanted tumor was regularly tested,and the tumor-bearing mice and the weight of the transplanted tumor were killed 30 days later.2.The influence of KLF5 on the tissue morphology of transplanted tumors and the expression of KLF5 protein in transplanted tumors were detected by HE staining and immunohistochemistry.3.The expression changes of JNK signaling pathway related molecules were detected by Western blot.Results:Part ?:Effect of KLF5 on Biological Characteristics of ATC Cells1.Compared with the normal human thyroid follicular epithelium Nthy-ori-3-1,KLF5 was significantly overexpressed in 8505c,HTh74 and HTh74Rdox cells,among which the highest expression level was found in HTh74Rdox cells.2.Sequencing analysis showed that KLF5 interference vector was successfully constructed.The titration values of Lv-shRNA,Lv-sh1-KLF5 and Lv-sh2-KLF5 lentivirus were 4×108.38,4×108.21 and 4×107.93 TU/mL.In 8505c and HTh74 cells,the expression of KLF5 was apparently reduced in sh1-KLF5 and sh2-KLF5 groups compared with the control group.The sh1-KLF5 group had the lowest KLF5 expression and the most obvious interference efficiency,so the sh1-KLF5 group was selected for subsequent functional analysis.3.Compared with the control group,KLF5 knockout significantly inhibited the proliferation of 8505c and HTh74 cells,and significantly reduced the expression of Ki67.4.Compared with the control group,KLF5 knockout significantly promoted the apoptosis of 8505c and HTh74 cells,and significantly increased the expression of cleaved caspase-3 protein.5.Compared with the control group,knockout KLF5 significantly inhibited the invasion and migration of 8505c and HTh74 cells,and the expression of mmp-2 and mmp-9 proteins were significantly reduced.Part II:Effect of KLF5 on Adriamycin Sensitivity in ATC Cells and its Mechanism1.Sequencing analysis showed that KLF5 overexpression vector was successfully constructed.The titration values of Lv-Con and Lv-KLF5 viruses were 3×108.14 and 3×10.84 TU/mL,respectively.Compared with the control group,KLF5 expression was significantly increased in HTh74 cells and significantly decreased in HTh74Rdox cells.2.In HTh74 cells,compared with the control group,the overexpression of KLF5 significantly promoted the proliferation of HTh74 cells.In HTh74Rdox cells,KLF5 interference significantly inhibited the proliferation of HTh74Rdox cells compared with the control group.3.In HTh74 cells,KLF5 overexpression significantly promoted the cloning formation of HTh74 cells compared with the control group.In HTh74Rdox cells,KLF5 interference significantly inhibited the cloning formation of HTh74Rdox cells compared with the control group.4.In HTh74 cells,compared with the control group,the overexpression of KLF5 significantly promoted the expression of Ki67 in HTh74 cells.In HTh74Rdox cells,KLF5 interference significantly inhibited the expression of Ki67 in HTh74Rdox cells compared with the control group.5.In HTh74 cells,KLF5 overexpression significantly promoted the activity of HTh74 cells with the increase of doxorubicin concentration compared with the control group.In HTh74Rdox cells,KLF5 interference significantly inhibited the activity of HTh74Rdox cells with the increase of doxorubicin concentration compared with the control group.6.Compared with the control group,the apoptosis of HTh74 cells was significantly promoted after doxorubicin treatment,while the overexpression of KLF5 significantly inhibited the apoptosis of HTh74 cells.In HTh74Rdox cells,compared with the control group,doxorubicin treatment significantly promoted the apoptosis of HTh74Rdox cells,and the interference of KLF5 significantly promoted the apoptosis of HTh74Rdox cells after doxorubicin treatment.7.In HTh74 cells,compared with the control group,the expression of cleaved caspase-3 was significantly increased when adriamycin was added,while the overexpression of KLF5 significantly inhibited the expression of cleaved caspase-3 in HTh74 cells.In HTh74Rdox cells,compared with the control group,the expression of cleaved caspase-3 was also significantly increased after the addition of adriamycin,while the interference of KLF5 significantly promoted the expression of cleaved caspase-3 in HTh74Rdox cells.8.In HTh74 cells,compared with the control group,the overexpression of KLF5 significantly promoted the expression of MDR1 and ABCG2 in HTh74 cells.In HTh74Rdox cells,KLF5 interference significantly inhibited the expression of MDR1 and ABCG2 in HTh74Rdox cells compared with the control group.9.In HTh74 cells,compared with the control group,the overexpression of KLF5 significantly promoted the expression of p-JNK protein in HTh74 cells,and there was no significant difference in the expression of JNK protein.In HTh74Rdox cells,compared with the control group,KLF5 interference significantly inhibited the expression of p-JNK protein in HTh74Rdox cells,and there was no significant difference in the expression of JNK protein.Part ?:Effect of KLF5 on the Growth and Sensitivity to Adriamycin of ATC Transplanted Tumor in Vivo1.Compared with the Saline group,the growth,volume and weight of the transplanted tumor were significantly reduced after adriamycin addition and KLF5 knockout.Compared with Dox+KLF5 group,Dox+KLF5 group showed significant decrease in tumor volume and body weight.2.HE staining showed that compared with Saline group,Dox+sh-KLF5 group showed the most reduction in malignant degree of transplanted tumor after addition of doxorubicin and deletion of KLF5,while Dox+sh-KLF5 group showed the most reduction in malignant degree of transplanted tumor compared with doxorubicin and deletion of KLF5 group.3.Immunohistochemistry results showed that compared with the Saline group,the positive expression rate of KLF5 in the transplanted tumor of Dox+sh-KLF5 group was significantly reduced in the addition of doxorubicin group and KLF5 knockout group,and compared with the the addition of doxorubicin group and KLF5 knockout group,the positive expression rate of KLF5 in the Dox+sh-KLF5 group was significantly reduced.4.Compared with the Saline group,the expression of p-JNK was significantly reduced after adriamycin addition and KLF5 knockout,and the expression level of JNK was not significantly changed.However,compared with the Dox+sh-KLF5 group,the expression of p-JNK was significantly reduced,and the expression level of JNK was not significantly changed.Conclusion:1.In vitro experiments results showed that knocking out KLF5 could significantly inhibit the proliferation,invasion and migration of 8505c and HTh74 cells,and promote apoptosis.2.In vitro studies further showed that KLF5 overexpression could promote the JNK signaling pathway activity in HTh74 cells,thereby promoting cell proliferation and clone formation,reducing cell sensitivity to adriamycin,and inhibiting apoptosis;KLF5 interference could inhibit the JNK signaling pathway activity in HTh74Rdox cells,thereby inhibiting cell proliferation and clone formation,improving cell sensitivity to adriamycin,and promoting apoptosis.3.In vivo studies have shown that KLF5 knockout could enhance the sensitivity of JNK to adriamycin by inhibiting the activity of the JNK signaling pathway,thereby inhibiting the growth of ATC transplanted tumor.