| Previous studies had revealed that the prevalence of thyroid carcinoma(TC)had been increasing worldwide,as wellas in China.The growth comes mainly from the increase of incidence of papillary thyroid carcinoma(PTC).PTC,as an indolent tumor,has a relatively favorable prognosis,with 20-year postoperative survival rate>90%.But it influences the quality of life seriously and continuously,especially accounts for high medical costs and psychological burden.Moreover,about20%of the patients’ tumor size are bigger witha poor prognosis,suchas lymph node metastasis.It follows thatwith the increasing trend of the incidence of TC,the research on the most common and multiple histological type of thyroid cancer,namely PTC,has important clinical and social significance.Forkhead box(FOX)protein transcription factor family was first found in 1990,which can regulate gene expression during transcription.Studies had shown that FOXK1,a member of fox protein family,plays an important role in tumor progression,including promoting tumor proliferation and metastasis,regulating the process of Epithelial-Mesenchymal Transition(EMT)and angiogenesis.However,the biological function and molecular mechanism of FOXK1 in PTC remain unclear.Cysteine rich 61(CYR61),also known as cellular communication network factor 1(CCN1),is a member of the CCN gene family.Studies had shown that CYR61 can promote the malignant progression of a variety of tumors.It had been reported that CYR61 can enhances the abilities of migration and invasion of thyroid undifferentiated cancer(ATC)cells,suggesting that CYR61 may play a role in the tumorigenesis and development of TC.However,the function and mechanism of Cyr61 in PTC have not been studied.It’s worth noting that a study on colorectal cancer showed that CYR61 is the target of FOXK1 which can regulate the transcription of CYR61.Studies showed that CYR61 can regulate the expression of connective tissue growth factor(CTGF),which is also closely related to tumor progression.Nevertheless,there is no report on whether FOXK1 can regulate the transcription of CYR61 in PTC,and how the expression of FOXK1,CYR61 and CTGF affect the proliferation,migration and invasion of PTC.Therefore,we propose that FOXK1 may promote the malignant progression of PTC,and the mechanism of FOXK1 may promote the malignant progression of PTC by regulating the expression of CYR61and CTGF.And knockdown of FOXK1 can inhibit the expression of CYR61 and CTGF,which finally inhibit the growth of tumor.Studies in this topic are helpful for clarifying the role and mechanism of FOXK1,CYR61 and CTGF in PTC,deepening the understanding of FOXK1,CYR61,CTGF and PTC,and providing theoretical evidence for the targeted therapy of PTC.Part 1 Basic Biological Function of FOXK1 in PTC cellsObjectivesTo explore the basic biological functions of FOXK1 in PTC cells,and to clarify its effects on the proliferation,migration and invasion of PTC cells.Methods1.The expression of FOXK1 in PTC cell lines TPC-1,KTC-1,snu-790,K1 and human normal thyroid cell HTori-3 were measured by qRT-PCR and Western blot.2.For overexpression of FOXK1,fulllength cDNAs were constructed into pcDNA4.1.For knockdown of FOXK1,shRNAswere constructed to interfere expression of FOXK1.The overexpression and knockdown efficiency were measured by qRT-PCR and Western blot.For the overexpression experiment,FOXK1 group was recorded as FOXK1 overexpression group and vector group as control group.For the knockdown experiment,shFOXK1#1 and shFOXK1#2 were recorded as knockdown groups,while the sequences used for knockdown were different.Scramble group was the control group of knockdown group.3.Cell viability was detected by MTT,proliferation was detected by BrdU staining,migration was detected by wound healing assay,and cell invasion was detected by transwell assay.Results1.Compared with HTori-3 cells,the expression of FOXK1 in TPC-1,KTC-1,snu-790 and K1 cells was significantly higher(p<0.05).Compared with PTC cell lines,the expression of foxkl was the highest in TPC-1 cells and the lowest in SNU-790 cells.Therefore,these two cell lines were selected for follow-up experiments.2.SNU-790 cells were selected for FOXK1 overexpression,and TPC-1 cells for knockdown.Expression of FOXK1 in overexpression group was significantly higher compared to vector group(p<0.01).The expression of FOXK1 in knockdown group was significantly lower compared to scramble group(p<0.05).shFOXK1#1 was selected for subsequent experiments because of better knockdown efficiency compared with shfoxk1#2.3.Th cell viability of FOXK1 group was significantly higher compared to vector group(p<0.01),and the cell viability of shFOXK1#1 group was significantly compared to scramble group(p<0.01).BrdU positive rate of FOXK1 group was significantly higher compared to vector group(p<0.01),and the BrdU positive rate of shFOXK1#1 group was significantly lower compared to scramble group(p<0.01).Scratch width of FOXK1 group was significantly smaller than that in vector group(p<0.01),and the scratch width of shFOXK1#1 group was significantly larger than that in scramble group(p<0.001).The number of invasive cells of FOXK1 group was significantly more than that of vector group(p<0.01),and the number of invasive cells of shFOXK1#1 group was significantly less than that in scramble group(p<0.01).Conclusions1.The expression of FOXK1 can enhance the proliferation,migration and invasion of PTC cells in vitro.Part 2 Mechanism of FOXK1 promoting malignant phenotype of PTC cellsObjectivesTo clarify the regulations of FOXK1 on the expression of CYR61,CTGF and the regulations of CYR61 on the expression of CTGF.To clarify the effects of these regulations on the proliferation,migration and invasion of PTC cells.Methods1.SNU-790 cells were selected for FOXK1 overexpression,and TPC-1 cells for FOXK1 knockdown.The expression of CYR61 and CTGF were measured by qRT-PCR and Western blot.FOXK1 group was recorded as FOXK1 overexpression group and vector group as control group.shFOXK1#1 was the knockdown group and scramble group was the control group.2.Double luciferase report assay was carried out to detect the luciferase activities between FOXK1 and CYR61 wild-type(WT)or mutant(MUT)promoter.3.SNU-790 cells were selected for CYR61 overexpression experiment,and TPC-1 cells for CYR61 knockdown.The expressions of CYR61 and CTGF were measured by qRT-PCR and Western blot.CYR61 group was recorded as CYR61 overexpression group and Control group as its control group.Both shCYR61#1 and shCYR61#2 were CYR61 knockdown group,and shNC was control group.4.SNU-790 cells were transfected by FOXK1+shNC,FOXK1+shCYR61#2 and Vector+shNC respectively.The expression of CYR61 was detected by qRT-PCR and Western blot.5.TPC-1 cells were transfected by Scramble+Control,shFOXK1#1+Control and shFOXK1#1+CTGF respectively.SNU-790 cells were transfected by Vector+shNC,FOXK1+shNC and FOXK1+shCTGF respectively.qRT-PCR and Western blot were used to detect genes expression.Cell viability was detected by MTT,proliferation by BrdU staining,migration by wound healing assay,and invasion by transwell assay.Results1.The expression of CYR61 mRNA(p<0.01)and protein(p<0.001)in FOXK1 group were significantly higher compared to Vector group.The expression of CYR61 mRNA(p<0.01)and protein(p<0.05)in TPC-1 cells in shFOXK1#1 group were significantly lower compared to Scramble group.The mRNA expression of CTGF in FOXK1 group was not significantly different from that in vector group(p>0.05),but protein expression was significantly higher compared to Vector group(p<0.001).The mRNA expression of CYR61 in shFOXK1#1 group was not significantly different from that in scramble group(p>0.05),but the protein expression was significantly compared to scramble group(p<0.01).2.Compared with Vector group,there was no significant difference in luciferase activity between mutant CYR61 group(p>0.05),while wild-type CYR61 group luciferase activity was significantly higher(p<0.01).3.The expression of CYR61 in CYR61 group was significantly higher compared to ontrol group(p<0.01).Compared with shNC group,the expression of CYR61 in shCYR61#1 group(p<0.01)and shCYR61#2 group(p<0.001)was significantly lower.In contrast,the expression of CYR61 in shCYR61#2 group was lower than that in shCYR61#1 group.The above showed that the overexpression and silencing efficiency of CYR61 meet the expectations.The shCYR61#2 of knockdown group was selected for follow-up experiment qRT-PCR and Western blot results also showed that the protein expression of CTGF in CYR61 group was significantly higher compared to Control group(p<0.01),and the protein expression of CTGF in shCYR61#1 group(p<0.05)and shCYR61#2 group(p<0.01)was significantly lower compared to shNC group.4.The protein expression of CTGF in FOXK1+shNC group was significantly higher compared to Vector+shNC group(p<0.001)and FOXK1+shCYR61#2 group(p<0.001).5.The protein expression of CTGF in shFOXK1#1+ Control group was significantly lower compared to Scramble+Control group(p<0.001)and shFOXK1#1 CTGF group(p<0.001).MTT assay showed that the cell viability of TPC-1 in shFOXK1#1+Control group was significantly lower compared to Scramble+Control group(p<0.01)and shFOXK1#1+CTGF group(p<0.01);The cell viability of SNU-790 in FOXK1+shNC group was significantly higher compared to Vector+shNC group(p<0.05)and FOXK1+shCTGF group(p<0.01).BrdU staining showed that the BrdU positive rate of TPC-1 in shFOXK1#1+Control group was significantly lower compared to Scramble+Control group(p<0.01)and shFOXK1#1+CTGF group(p<0.001).The results of wound healing asssy showed that the scratch width of TPC-1 in shFOXK1#1+Control group was significantly larger compared to Scramble+Control group(p<0.01)and shFOXK1#1+CTGF group(p<0.01).The results of transwell showed that the number of invasive cells of TPC-1 in shFOXK1#1 control group was significantly lower compared to Scramble+Control group(p<0.01)and shFOXK1#1+CTGF group(p<0.01);The number of invasive cells of FOXK1+shNC group was significantly higher than compared to Vector+shNC group(p<0.01)and FOXK1+shCTGF group(p<0.01).Conclusions1.FOXK1 can upregulate the expression of CYR61 at mRNA and protein.The expression level of FOXK1 has no effect on the mRNA expression level of CTGF,but promoted the expression of CTGF at the protein.2.FOXK1 can bind to CYR61 promoter and is site-dependent.3.The expression of CYR61 can promote the expression of CTGF protein.4.FOXK1 regulates the expression of CTGF by regulating CYR61.5.The expression of CTGF can enhance the proliferation,migration and invasion of PTC cells in vitro.6.FOXK1 combines with CYR61 promoter to promote the expression of the latter,and CYR61 can promote the expression of CTGF.FOXK1 promotes the proliferation,migration and invasion of PTC cells by up regulating CTGF.Part 3 Bioinformatics and vivo experiments preliminarily verified the role and mechanism of FOXK1ObjectivesTo clarify the correlation between the expression levels of FOXK1,CYR61 and CTGF.To clarify the correlation between expression levels overall survival.To observe the effect of inhibiting the expression of FOXK1 on tumor growth and related factors.Methods1.Bioinformatics tool Timer 2.0 was used to analyze the expression of FOXK1 in different tumors.GEPIA was used to analyze the effect of the expression of FOXK1,CYR61 and CTGF on the overall survival of TC,and the relationship between the expression levels of FOXK1,CYR61 and CTGF.2.shFOXK1#1 group and Scramble group TPC-1 cells were injected into BALB/C nude mice to carry out mouse xenograft assay.The size and weight of the tumor were measured.3.FOXK1,Ki-67,CYR61 and CTGF in tumor tissues were detected by immunohistochemical staining,and E-cadherin and N-cadherin were measured by Western blot.Results1.Compared with normal tissues,FOXK1 was significantly overexpressed in a variety of tumors including TC(p<0.05).The expression levels of FOXK1,CYR61 and CTGF more higher,the overall survival time more shorter(p<0.05).The expression levels of FOXK1,CYR61 and CTGF showed a positive correlation.2.The tumor volume and weight in shFOXK1 group were significant less compared to Scramble group(p<0.01).3.The expressions of FOXK1,CYR61,CTGF,Ki-67 and N-cadherin protein in shFOXK1 group were lower than those in Scramble group,and E-cadherin was higher than those in scramble group.Conclusions1.The results of bioinformatics analysis are consistent with the function and mechanism of FOXK1 obtained in part 1 and part 2.2.Knockdown the expression of FOXK1 can downregulate the expression of CYR61 and CTGF,significantly inhibit tumor growth,and upregulate Ki-67 and E-cadherin expression. |
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