| Objective:The previous research of the research group found that the increase of serum palmitic acid(PA)after obesity is closely related to the occurrence of endometrial cancer(EC).KLF7 plays an important role in regulating the process of lipid metabolism,and PA can up-regulate the expression of KLF7.Based on the collection of endometrial tissues of EC patients and non-cancer individuals,the cultivation of EC cells Ishikawa in vitro,and the construction of an obese EC tumor-bearing mouse model,combined with RNASeq technology,this study preliminarily screened and verified the oncogenes that may be regulated by KLF7.Through the above research,we clarify the molecular mechanism of PA up-regulating the expression of transcription factor KLF7 and promoting the occurrence and development of EC,providing a theoretical basis and experimental basis for clinical treatment and prognosis of EC.Methods:(1)The general data of EC patients(n=50)and non-cancer individuals(n=29),as well as paraffin embedded tissue samples of endometrial,were gathered in Shihezi People’s Hospital from September 2017 to December 2019.Immunohistochemistry was used to detect KLF7 expression in the endometrium.(2)GPR40/120 and KLF7 expression levels in human EC cells were determined using q RT-PCR and Western blot.CCK-8 and colony formation assays were used to evaluate the effects of PA treatment on cell proliferation,while Transwell and scratch assays were used to detect the effects on cell invasion and migration.(3)KLF7 overexpression plasmid and interference fragment were constructed to up/down regulate the expression of KLF7 in Ishikawa cells respectively,and the effects on the proliferation,invasion and migration of tumor cells were observed.(4)After up/down regulating the expression of KLF7 in Ishikawa cells,the oncogenes that may be regulated by KLF7 were screened by RNA Seq technology combined with bioinformatics analysis.(5)Seventeen BALB/C nude mice were fed with normal diet and divided into Ishikawa blank group(n=6),Ishikawa Ad-NC group(n=6)and Ishikawa Ad-KLF7 group(n=5);21 BALB/C nude mice were fed with high-fat diet and divided into Ishikawa blank group(n=10),Ishikawa Si-NC group(n=6)and Ishikawa Si-KLF7 group(n=5).Nude mice’s general information was gathered.Each group’s tumor tissues were compared in terms of tumor size and weight.GPR40/120,KLF7,and downstream oncogenes’ mRNA and protein expression levels in tumor tissues were determined using q RT-PCR and Western blot.(6)SPSS 20.0 software was used for data analysis.The t-test was used to compare differences between two groups when the data conformed to a normal distribution,the one-way analysis of variance(One WayANOVA)was used to compare data between multiple groups,and the rank sum test was used when the data did not conform to a normal distribution.The sample data were analysed by the above methods and differences were considered statistically significant when P<0.05.Results:1.The detection rate of obesity and the expression level of KLF7 in EC patients were significantly higher than those in non-cancer individuals.(1)After comparing the general data and biochemical indicators of the subjects,it was found that weight,BMI,TC,LDL,GLU,and obesity detection rates were significantly higher in EC patients(n=50)than in non-cancerous individuals(n=29),and the differences mentioned above were statistically significant(P<0.05).(2)Immunohistochemical results showed that the protein expression levels of GPR40 / 120 and KLF7 in the endometrium of EC patients were significantly higher than those of non-cancerous individuals.The differences mentioned above were statistically significant(P<0.05).2.PA increases Ishikawa cell proliferation,invasion,and migration via upregulating GPR40/GPR120 and KLF7 expression.(1)Ishikawa cells were treated with 0,20μM,50μM,200μM and 500μM PA for 24 h,48h,72 h and 96 h,respectively.Cytotoxicity test results showed that 200μM and 500μM PA had inhibition effect on cell activity at each time point.PA treatment with 20μM and 50μM for 24 h had the most significant promoting effect on cell activity,which could be used for follow-up research;(2)In Ishikawa cells treated with 20μM and 50μM PA for 24 hours,the mRNA and protein expression levels of GPR40/GPR120 and KLF7 were much higher than in the control group,and the proliferation,invasion,and migratory abilities of Ishikawa cells were significantly improved.The differences mentioned above were statistically significant(P<0.05).3.PA can promote proliferation,invasion and migration of Ishikawa cells by up-regulating KLF7 expression.(1)after transfection of 0,2μg,4μg,6μg and 8μg KLF7 overexpressed plasmids into Ishikawa cells for24 h,the transfection efficiency of 4μg KLF7 overexpressed plasmids was the highest,which could be used for follow-up studies;CCK-8,colony formation,and Transwell assay results demonstrated that following transfection of Ishikawa cells with 4μg KLF7 overexpressed plasmid for 24 hours,proliferation,invasion,and migration of Ishikawa cells were considerably enhanced compared to the NC group.The differences mentioned above were statistically significant(P<0.05);(2)Following transfection of 0,25 n M,50 n M,75 n M and 100 n M KLF7 interfering fragments into Ishikawa cells for 24 h,respectively,the 75 n M KLF7 interfering fragment had the highest transfection efficiency and could be used for subsequent studies;The results of CCK-8,colony formation and Transwell assays after transfection of Ishikawa cells with 75 n M KLF7 interference fragment for 24 h showed that the proliferation,invasion and migration ability of Ishikawa cells were significantly and significantly reduced after down-regulation of KLF7 compared to the NC group.The differences mentioned above were statistically significant(P<0.05).(3)PA treatment with concomitant downregulation of KLF7 using 75 n M interfering fragments for 24 h resulted in diminished proliferation,invasion and migration of Ishikawa cells compared to the PA treatment group alone.The differences mentioned above were statistically significant(P<0.05).4.PA can promote KLF7 expression through GPR40/GPR120,and enhance the proliferation,invasion and migration ability of Ishikawa cells.(1)After 50μM PA treatment and 10μM GPR40 antagonist GW1100 was added,the mRNA and protein expression levels of KLF7 in Ishikawa cells were significantly decreased compared with PA treatment alone.At the same time,the proliferation,invasion and migration ability of Ishikawa cells were significantly reduced by the addition of the GPR40 antagonist.The differences mentioned above were statistically significant(P<0.05);(2)After 50μM PA treatment and 100μM GPR120 antagonist AH7614 were added,the mRNA and protein expression levels of KLF7 in Ishikawa cells were significantly decreased compared with PA treatment alone.Meanwhile,the proliferation,invasion,and migration ability of Ishikawa cells were significantly reduced by the addition of the GPR120 antagonist.The differences mentioned above were statistically significant(P<0.05).5.Screening and validation of downstream oncogenes of KLF7.(1)Total RNA was extracted from 4 pairs of EC patients’ cancer tissues and corresponding para cancer tissues,as well as up/down KLF7 and Ishikawa cells of their respective control groups.RNA-Seq technology combined with bioinformatics was used to analyze and screen differentially expressed genes.The results showed that: Compared with paracancer tissues,1697 genes were overexpressed and 754 genes were underexpressed in EC patients’ cancer tissues.Compared with NC group,639 genes were highly expressed and 103 genes were low expressed in Ishikawa cells after up-regulation of KLF7.After downregulation of KLF7,313 genes were low expressed and 405 genes were highly expressed in Ishikawa cells.The intersection of differentially expressed genes in the above groups showed that the four genes IRX3,HEY1,RHBDF2 and FAM171A2 showed corresponding changes in cells and tissues.(2)After up-regulating KLF7 in Ishikawa cells,mRNA expression levels of IRX3,HEY1,RHBDF2,and FAM171A2 were detected,and corresponding changes in HEY1 expression were the most significant and stable(P<0.05);Bioinformatics software was used to predict that KLF7 binding sites were found in the promoter region of HEY1.After 24 h co-transfection of Wild type(WT)and Mutation type(MUT)luciferase reporter gene plasmids in HEY1 promoter region with KLF7 overexpression plasmid into Ishikawa cells,the results showed that: KLF7 significantly promoted the activity of the WT HEY1 promoter,while mutations at key binding sites in the HEY1 promoter eliminated this promotion.The differences mentioned above were statistically significant(P<0.05);(3)After transfection of Ishikawa cells with 4μg KLF7 overexpression plasmid for 24 h,the mRNA and protein expression levels of KLF7 and HEY1 in the cells were significantly increased and the mRNA and protein expression levels of E-Cadherin were significantly decreased compared to the NC group;after transfection of Ishikawa cells with 75 n M KLF7 interference fragment for 24 h,the mRNA and protein expression levels of KLF7 and HEY1 in the cells were significantly decreased and the mRNA and protein expression levels of E-Cadherin were significantly increased compared to the NC group.The mRNA and protein expression levels of KLF7 and HEY1 in Ishikawa cells were significantly decreased,while the mRNA and protein expression levels of E-Cadherin were significantly increased.The differences mentioned above were statistically significant(P<0.05).6.A high-fat diet enhanced the tumorigenic capacity of mouse Ishikawa cells,with significantly greater expression levels of GPR40/120,KLF7,and HEY1,and significantly lower expression levels of E-cadherin in tumor tissue.(1)The body weight,Lee’s index,GLU,TC,TG,and FFA of mice fed with a high-fat diet(n=5)for 10 weeks were significantly higher than those fed with a normal diet(n=5).6 weeks after subcutaneous injection of Ishikawa cells into mice,the tumor formation rate of Ishikawa cells in mice fed with a high-fat diet was(6/6,100%),and the tumor formation rate in mice fed with a normal diet was(3/6,50%).The weight and volume of tumor tissue were significantly higher in mice fed a high-fat diet than in mice fed a normal diet.The differences mentioned above were statistically significant(P<0.05);(2)Compared with the normal diet group,the protein expression levels of GPR40/120,KLF7,and HEY1 in tumor tissues of mice fed with high-fat diet group were significantly increased.In contrast,the protein expression level of E-cadherin was significantly reduced.The differences mentioned above were statistically significant(P<0.05).7.Overexpression of KLF7 enhanced the tumorigenic capacity of Ishikawa cells in normal diet mice,and the expression level of HEY1 was significantly increased in tumor tissues,while the expression level of Ecadherin was significantly decreased.(1)Ishikawa cells overexpressing KLF7(n=6)and control Ishikawa cells(n=6)were administered subcutaneously to mice after 4 weeks of feeding on a normal diet.No significant difference in body weight and Lee’s index between the two groups after 6 weeks of feeding.After the mice were sacrificed,the tumor formation rate of Ishikawa cells in both groups was(6/6,100%).The volume of tumor formation of KLF7-overexpressed Ishikawa cells in mice was significantly higher than that of control Ishikawa cells.The differences mentioned above were statistically significant(P<0.05);(2)HEY1 protein expression levels were significantly higher and E-cadherin protein expression levels were significantly lower in tumor tissues formed from mice overexpressing KLF7 Ishikawa cells compared to null control Ishikawa cells.The differences mentioned above were statistically significant(P<0.05).8.Interference with KLF7 attenuated the tumourigenic capacity of Ishikawa cells in mice on a high-fat diet,with significantly lower levels of HEY1 expression and higher levels of E-cadherin expression in tumor tissue.(1)After 4 weeks on the high-fat diet,the mice showed a significant increase in body weight and Lee’s index compared to the normal diet group.At this time,mice were given a subcutaneous injection of KLF7down-regulated Ishikawa cells(n=5)and no-load control Ishikawa cells(n=5)respectively.After 6 weeks of high-fat diet feeding,there was no significant difference in body weight and Lee’s index between the two groups,but both were higher than the normal diet group.After mice were executed,the tumor formation rate of Ishikawa cells was(5/5,100%)in both groups.The tumor-forming volume of KLF7 down-regulated Ishikawa cells in mice was significantly lower than that of empty vector control Ishikawa cells.The differences mentioned above were statistically significant(P<0.05);(2)Compared to empty vector control Ishikawa cells,HEY1 protein expression levels were significantly reduced and E-cadherin protein expression levels were significantly increased in tumor tissues formed by KLF7 down-regulated mouse Ishikawa cells.The differences mentioned above were statistically significant(P<0.05).Conclusion:Increased serum PA levels following obesity can upregulate KLF7 expression via GPR40/120,which is a downstream increase in the expression of the oncogene HEY1,ultimately leading to the development of endometrial cancer. |
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