| Background Colon cancer is a malignant tumor with the highest incidence and mortality in the world.The treatment options for colon cancer mainly include endoscopic therapy,conventional surgery,adjuvant chemotherapy,targeted therapy and immunotherapy.Although the diagnosis and treatment of colon cancer have greatly improved,most colon cancer patients are in the advanced stage once diagnosed,the development of chemotherapy resistance,as well as occurrence of metastasis,making the prognosis of colon cancer patients poor.Current researches on targeted therapies or biomarker driven therapies for this malignancy are not promising.Therefore,it is urgent to discover the critical biomarkers for the early diagnosis and development of colon cancer,elucidate the molecular mechanism of colon cancer and develop more effective therapeutic methods.FEZ family zinc finger 1-antisense RNA 1(FEZF1-AS1)is an antisense long noncoding RNA(lnc RNA)with a length of 2653 bp.FEZF1-AS1 is highly expressed in gastric cancer,esophageal cancer,and ovarian cancer,which is correlated with prognosis and clinicopathological features of tumor patients(such as tumor size,TNM stage),and is involved in the occurrence and development of tumors.Autophagy recycles unwanted or damaged cellular organelles and proteins,regenerates various precursors to maintain cell biosynthesis and viability.In normal cell,autophagy is the “brake” that prevents the oncogenesis.However,once the tumor is formed,tumor cell usually uses autophagy as a survival mechanism to promote its growth and proliferation,and acquires drug resistance.It has been reported that FEZF1-AS1 promotes the progression of gastric cancer by activating autophagy and induces multiple drug resistance.Studies have shown that activation of autophagy can inhibit the apoptosis of colon cancer cells and increase the resistance of colon cancer cells to chemotherapy drugs.Based on the above researches,we hypothesize that FEZF1-AS1 may be involved in the progression of colon cancer through autophagy,reducing the sensitivity of colon cancer cell to chemotherapy drugs.Objectives To explore the function of FEZF1-AS1 in colon cancer and its possible molecular mechanism,and to provide new method and theoretical basis for the early diagnosis and treatment of colon cancer.Methods 1.We downloaded the RNA sequencing data and corresponding clinicopathological information of colon cancer patients from TCGA database,and obtained differentially expressed lnc RNA through the “limma” package in R language.Through comprehensive analysis,we selected FEZF1-AS1 as the target molecule of this study.2.In NCBI,NONCODE and UCSC databases,we analyzed the expression of FEZF1-AS1 in various normal tissues.In TCGA,GEPIA and ln CAR databases,we visualized the expression of FEZF1-AS1 in colon cancer and normal colon tissues.The expression of FEZF1-AS1 in 36 colon cancer and paired normal colon tissues was further detected by q RT-PCR.Mutations in FEZF1-AS1 in colon cancer were analyzed using the cbioportal database.3.We used the “cluster Profiler” package in R language,Gene Set Enrichment Analysis(GSEA)and ln CAR database to conduct functional enrichment analysis of FEZF1-AS1 and its co-expressed genes.Therefore,we preliminarily acquired the related signaling pathways that FEZF1-AS1 may participate in the development of colon cancer.4.The “p ROC” package in R language was used to analyze the receiver operating characteristic curve(ROC)of FEZF1-AS1 in colon cancer.Mann-Whitney U test and Kruskal-Wallis test were used to analyze the relationship between FEZF1-AS1 and clinicopathological parameters of colon cancer patients.In the TCGA database,COX regression was used to analyze the risk factors associated with colon cancer.The association between FEZF1-AS1 and survival of colon cancer patients was analyzed using the GEPIA database.5.We cultured colon cancer cells(RKO,caco2,SW620,SW480)and colon epithelial cell(NCM460),detected the expression of FEZF1-AS1 in the cells by q RT-PCR.Based on the result,caco2 and RKO were selected to construct FEZF1-AS1 overexpressed and knocked down cell lines lentivirus transfected,respectively.We used q RT-PCR to detect the expression of FEZF1-AS1 in cell lines lentivirus transfected to verify the efficiencies of overexpression and knockdown.The effects of FEZF1-AS1 on cell proliferation,migration,invasion,cycle and apoptosis were analyzed by CCK8,plate cloning assay,scratch assay,transwell assay and flow cytometry.6.RKO cell knocked down FEZF1-AS1 and control cell were performed RNA sequencing,each group was repeated three times.According to the sequencing results,differential genes and pathway enrichment before and after FEZF1-AS1 knocked down were analyzed.The related signaling pathway of FEZF1-AS1 might involve in colon cancer development were confirmed.7.The expression of critical proteins in the signaling pathway of FEZF1-AS1 might involve in the development of colon cancer was verified by western blot and immunofluorescence methods.Then,the above cells were treated with a certain concentration of autophagy inhibitor(chloroquine)and m TOR inhibitor(rapamycin)to verify the expression of critical proteins in the signaling pathway again.8.Coco2 cell overexpressed FEZF1-AS1,RKO cell knocked down FEZF1-AS1 and corresponding control cells were injected into the subcutaneous skin of nude mice to construct tumor transplantation models of tumor-bearing mice.The tumor volumes were measured to verify the effect of FEZF1-AS1 on tumorigenic ability of colon cancer cells.Immunohistochemistry was used to detect the expression of critical proteins of the related signaling pathway of FEZF1-AS1 might involve in colon cancer development in transplanted tumors.9.Coco2 cell overexpressed FEZF1-AS1,RKO cell knocked down FEZF1-AS1 and corresponding control cells were treated with or without chloroquine and rapamycin at certain concentrations,respectively.Then,a certain concentration of the chemotherapy drug(oxaliplatin)was applied to the above cells.CCK8 and flow cytometry were used to detect drug sensitivity.Results 1.In the TCGA database,we obtained RNA sequencing data and corresponding clinicopathological information of 371 patients with colon cancer.507 lnc RNA were differentially expressed in colon cancer and normal colon tissues.We finally selected FEZF1-AS1 as the target molecule.2.The results of NCBI,NONCODE and UCSC databases showed that FEZF1-AS1 was in a low expression mode in normal colon tissues.The results of TCGA,GEPIA,ln CAR databases and q RT-PCR showed that FEZF1-AS1 was highly expressed in colon cancer compared with normal colon tissues.The cbioportal database showed that FEZF1-AS1 presented an amplified mutation in colon cancer.3.Functional enrichment showed that FEZF1-AS1 and its co-expressed genes were enriched into extracellular matrix,ABC transporters,VEGF signaling pathway,PI3K/AKT signaling pathway,MAPK signaling pathway,lysosome,and tumor-related signaling pathways.4.In colon cancer,the area under ROC curve of FEZF1-AS1 was 0.949.The expression of FEZF1-AS1 was different among different clinical parameter groups of colon cancer patients,such as T stage,age,tumor site and race.COX regression analysis showed that the age,pathological T stage,N stage,clinical stage and lymph node metastasis of patients with colon cancer were correlated with overall survival,and could be used as independent prognostic factors for colon cancer.GEPIA database showed that FEZF1-AS1 was not significantly associated with the survival of colon cancer patients.5.We used q RT-PCR to detect the expression of FEZF1-AS1 in colon cancer cell lines.It showed that compared with NCM460 cell,the expression of FEZF1-AS1 in caco2 cell was relatively lower,and in RKO cell was relatively higher.Therefore,caco2 was selected to successfully construct FEZF1-AS1 overexpressed cell line lentivirus transfected,and RKO was selected to successfully construct FEZF1-AS1 knocked down cell line lentivirus transfected.Compared with the control cell,the proliferation,migration,invasion and cycle(S phase cell ratio)of caco2 cell overexpressed FEZF1-AS1 were significantly increased,and the apoptosis was significantly decreased.Compared with control cell,the proliferation,migration,invasion and cycle(S phase cell ratio)of RKO cell knocked down FEZF1-AS1 were significantly reduced,and apoptosis was significantly increased.6.RKO cell knocked down FEZF1-AS1 and control cell were analyzed by RNA sequencing,and the obtained differentially expressed genes were enriched into PI3K/AKT signaling pathway,lysosome,autophagy,extracellular matrix and other tumor-related pathways.We suggested that FEZF1-AS1 may promote the development of colon cancer by regulating the PI3K/AKT signaling pathway to mediate autophagy.7.The results of western blot showed that compared with control cell,the proteins expression of PI3 K,AKT,p-AKT,m TOR,p-m TOR,p62 was decreased in caco2 cell overexpressed FEZF1-AS1,while LC3 II expression was increased.On the contrary,compared with control cell,the proteins expression of PI3 K,AKT,p-AKT,m TOR,pm TOR,and p62 was increased in RKO cell knocked down FEZF1-AS1,while LC3 II expression was decreased.After 12 h treatment with 300μmol/L chloroquine,compared with control cell,the proteins expression of PI3 K,AKT,p-AKT,m TOR,p-m TOR,and p62 was decreased in caco2 cell overexpressed FEZF1-AS1,while LC3 II expression was increased.The protein expression of LC3 II in chloroquine-treated caco2 cell overexpressed FEZF1-AS1 and control cell was significantly higher than that in non-chloroquine-treated caco2 cell overexpressed FEZF1-AS1 and control cell.After 5h treatment with 0.62nmol/L rapamycin,compared with control cell,the proteins expression of PI3 K,AKT,p-AKT,m TOR,p-m TOR,and p62 was increased in RKO cell knocked down FEZF1-AS1,while LC3 II expression was decreased.The protein expression of p-m TOR was significantly decreased and LC3 II expression was significantly increased in rapamycin-treated RKO cell knocked down FEZF1-AS1 and control cell,compared with non-rapamycin-treated RKO cell knocked down FEZF1-AS1 and control cell.The results of immunofluorescence detection of LC3 were consistent with those of western blot.We believed that FEZF1-AS1 may inhibit PI3K/AKT/m TOR signaling pathway to activate autophagy and promote the development of colon cancer.8.The results of tumor-bearing mice experiment showed that the tumor formation volumes of caco2 cell overexpressed FEZF1-AS1 in nude mice were significantly larger than those in control mice.The tumor formation volumes of RKO cell knocked down FEZF1-AS1 in nude mice were significantly smaller than those in control mice.The results of immunohistochemistry were consistent with those of western blot.9.The results of drug sensitivity test indicated that caco2 cell overexpressed FEZF1-AS1 had significantly lower mortality than that of control cell.Compared with nonchloroquine-treated caco2 cell overexpressed FEZF1-AS1,the mortality of caco2 cell overexpressed FEZF1-AS1 treated with chloroquine was significantly higher.RKO cell knocked down FEZF1-AS1 had significantly higher mortality than that of control cell.Compared with non-rapamycin-treated RKO cell knocked down FEZF1-AS1,the mortality of RKO cell knockdown FEZF1-AS1 treated with rapamycin was significantly lower.Conclusions FEZF1-AS1 was highly expressed in colon cancer,which may be caused by the amplification mutation of FEZF1-AS1 at the gene level.FEZF1-AS1 had the potential to be a biomarker in the diagnosis of colon cancer.FEZF1-AS1 may activate autophagy through inhibiting PI3K/AKT/m TOR signaling pathway to promote the development of colon cancer,and reduce the drug sensitivity of colon cancer cells to oxaliplatin.In conclusion,FEZF1-AS1 was expected to be a biomarker for the diagnosis,progression and efficacy evaluation of colon cancer. |
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