FOXK1 Regulates The Resistance To The Chemoradiotherapy In The Rectal Cancer

Colorectal cancer is one of the most common cancer in the clinic,Rectum cancer accounts for 50?60% of the total number of patients with colorectal cancer.According to the 2020 "standard of colorectal cancer diagnosis and treatment" in China and 2020 NCCN guidelines suggest that for the high-risk ?/? stage locally advanced rectal cancer(LARC)recommended preoperative neo-chemoradiotherapy(NCRT)followed TME surgery,It has obvious advantages in tumor shrinkage of rectal cancer,decreasing stage,improving local control rate,improving anal preservation rate and reducing local recurrence.However,differential patients have differential sensitivities to NCRT.And even though,tumor progression in some patients.Therefore,how to improve the efficacy of NCRT is still an urgent problem to be solved.The gene chip was performed to detected the LARC patients' samples before the NCRT.The weighted gene co-expression network analysis(WGCNA)was used to screen molecular biological markers related to the sensitivity of NCRT.The transcription factor forkhead K1(FOXK1)gene was finally screened.Firstly,we examined the expression of FOXK1 in LARC patients receiving NCRT,and the predictive power on NCRT response and prognosis.Secondly,constructing of the knock down and overexpression FOXK1 in colorectal cancer cell lines.To validation the differential FOXK1 gene expression whether affected the cell apoptosis,proliferation,stemness and sensitive of the NCRT in colorectal cancer cell lines.At the same time using nude mice to validate the proliferation of the knock down and overexpression FOXK1 cell lines.To explore the FOXK1 function in the rectal cancer cell lines,the RNA-seq was performed in the knock down and overexpression FOXK1 colorectal cancer cell lines,and the ChIP-seq data of the GSM1239397 from the GEO database was downloading and analysis.The result was demonstrated that the MAPK15 was the reliable downstream molecule,which was verified by the Luciferase analysis.Based on this,we will further explore the FOXK1/MAPK15 regulates the NCRT response of the colorectal cancer cells.Based on the above objectives,we divided the research into three parts:PART ONE Established oxaliplatin-resistance human colon carcinoma cell line,explored the FOXK1 expression in oxaliplatin-resistance cell line and patientsProjective To assess the role of the expression levels of FOXK1 in predicting response to neo-chemoradiotherapy(NCRT)and prognosis in locally advanced rectal cancer(LARC).Methods The WGCNA analysis was performed to screened the relative biomarker for the 31 LARC patients accepting NCRT which has cpmplished the genechip analysis.A total of 256 LARC patients who underwent NCRT and radical resection between 2011 and 2017 were enrolled in the present study.The patients were divided into a training dataset(n=169,2011-2015)and a validation dataset(n=87,2016-2017).Tumor tissues were collected before NCRT and post-surgery and were used for immunohistochemical analysis.Results The WGCNA anlaysis screened the Darkturquoise module relative with the NCRT response.And the FOXK1 as the hub geen in the Darkturquoise module was screened.The Oncomine database analysis revealed that FOXK1 were overexpressed in most cancers especially in colorectal cancer.Additionally,overexpression of FOXK1 was associated with poorer prognosis by the R2 database.In both our training and validation datasets the expression of FOXK1 was lower in the pathological complete response(pCR)group compared with the non-pCR group(P<0.05).Cox regression analysis demonstrated that pathological N stage((HR=1.810,95%CI 1.259 ? 2.827,P=0.009),and FOXK1 expression(HR=5.831,95%CI 2.925 ? 11.625,P<0.001)were independent predictors of disease-free survival(DFS).Based on the Cox multivariate analysis,we constructed a risk score model that served as a prognostic biomarker and had a powerful ability to predict pCR in LARC patients upon NCRT in both training and validation groups.Conclusion Expression levels of FOXK1 was associated with chemoradiotherapy resistance and prognosis of LARC patients following NCRT and were used to construct a risk score model that is a promising biomarker for LARC.PART TWO The Effects of the knockdowning/overexpression of the FOXK1 expression on the function of rectal cancer cells in vitro and in vivoProjective The Effects of the knockdowning/overexpression of the FOXK1 expression on the function of rectal cancer cells in vitro and in vivo.Methods According to the sh RNA and overexpression plasmid design principle,3 interference fragments and overexpression plasmid were designed for the target gene FOXK1,and the colorectal cancer cell lines,DLD and SW620,were transfected with lentiviral vectors to establish the knockdown and overexpression FOXK1 cell lines.The expression of FOXK1 gene was detected by RT-q PCR and WB respectively,and FOXK1-sh RNA and FOXK1-OVER with the highest interference efficiency were selected.The effect of FOXK1 on 5-FU and radiotherapy was determined by CCK-8and clone formation.The proliferation of cells were detected by CCK-8 and clone formation.Flow cytometry(FCM)was used to analyze cell apoptosis,stem cell sphere formation assay was performed to analysis the cells' stemness.The subcutaneous tumorigenesis in nude mice to validate the proliferation of the knock down and overexpression FOXK1 cell lines.The Ki-67 expression was detected by the immunohistochemical.Results RT-q PCR and WB results show that FOXK1 expression in knockdown FOXK1 group significantly lower than control group(P <0.01),among them,Sh-FOXK1-02 and Sh-FOXK1-03 groups FOXK1 m RNA and protein expression of the minimum.And the overexpression of FOXK1 group significantly higher than control group(P < 0.01).FOXK1 knockdown cell lines significantly increased the sensitivity of 5-FU and radiotherapy(P<0.01),and the overexpression group significantly increased the resistance to the 5-FU and radiotherapy(P<0.01).Knockdown FOXK1 inhibited cell growth and increased apoptosis(P<0.01).On the contrary,the FOXK1 overexpression can induced the cell growth and inhibited apoptosis(P<0.01).The similarly result could be found in the stem cell sphere formation assay.Moreover,the subcutaneous tumorigenesis showed that the diameter of the low expression of the FOXK1 group was shorter than the control group,and the Ki-67 expression was also lower.Contrarily,the high expression of the FOXK1 group had longer diameter and higher Ki-67 expression.Conclusion We successfully constructed a FOXK1 gene expression modified cell lines,and the knockdowning/overexpression of the FOXK1 expression colorectal cancer cell lines affected the proliferation,apoptosis,stemness,and resistance to the NCRT.PART THREE FOXK1 regulated the sensitive to the NCRT by the MAPK15 in rectal cancerProjective To explore the underline mechanism of the FOXK1 affected the sensitive to the NCRT in rectal cancer.Methods The RNA-seq was performed to analysis the FOXK1 gene expression modified cell lines.The GEO database and TCGA database were enrolled to explore and verify the mechanism of the FOXK1.the WB and luciferase analysis were used to confirm the result.Results The RNA-seq result demonstrated that FOXK1 associated with the cytokines and metabolic disorder in the GO functional analysis.And the MAPK signal pathway and p53 signal pathway were correlated with the differential FOXK1 gene expression in the rectal cancer.And the differential expression genes among the knockdown FOXK1 groups,control groups,and overexpression FOXK1 groups were as follows,MAPK15?TGF-?1?IL32?SLC29A4 and LINC00973.Moreover,the result from the ChIP-seq data of the GSM1239397 demonstrated that the MAPK15 were associated with the FOXK1.And the Pearson analysis demonstrated that the MAPK15 expression was significant associated with the FOXK1 expression(R=0.81,P<0.001),The above result also confirmed by the WB analysis in the FOXK1 gene expression modified cell lines and luciferase analysis.Moreover,basing on the above GO function,KEGG pathway,and differential expression gene results,we hypothesis that the TGF-?1 was the target gene of the MAPK15.We verified the hypothesis by the WB and TCGA database.Conclusion FOXK1 associated with the cytokines and metabolic disorder in the GO functional analysis.And the MAPK signal pathway and p53 signal pathway were correlated with the differential FOXK1 gene expression in the rectal cancer.The FOXK1 protein bingding th promoter region of MAPK15 increased the MAPK15 expressiono.And the TGF-?1 expression and autocrine was also induced to increased the stemness of the rectal cancer,resulting in the NCRT resistance.