| Background and ObjectivesCervical cancer is the most common gynecological malignant tumor,and in recent years it has a tendency to become younger.Human Kruppel-like factor 14(KLF14),also known as BTEB5,is one of the members of the Kruppel-like transcription factor family,which encoding intron-free genes.The encoded protein acts as a co-repressor of transcription and can be induced by TGF-? to suppress TGF-? receptor ? gene expression.It was reported that KLF14 is related to the level of multiple genes in subcutaneous fat and is the master switch controlling these genes.Recent studies found that KLF14 also plays an important role in the occurrence and development of a variety of tumors.However,the role and mechanism of KLF14 on proliferation and autophagy in cervical cancer has not been well characterized.Objective:1.To clarify the role of transcription factor KLF14 in cervical cancer cell proliferation,autophagy,and apoptosis;2.To explore whether KLF14 affects the proliferation and apoptosis of cervical cancer cells through autophagy;3.Explore the autophagy gene related to KLF14,and screen out the possible downstream target genes of KLF14 acting on cervical cancer cell autophagy.Methods 1.Construct a vector plasmid overexpressing KLF14,transiently transfect in human cervical cancer cell lines HeLa and C-33A cells,and verify the expression effect on gene level and protein level by RT-PCR and Western blot.2.Using CCK-8,plate clone formation experiments and EdU cell imaging system to study the effect of transcription factor KLF14 on the proliferation of human cervical cancer cells.3.Using Western blot technology to detect the autophagosome membrane marker protein LC3 and autophagy substrate p62 in HeLa cells to explore the effect of KLF14 on HeLa cell autophagy;using Western blot and cell immunofluorescence technology to explore the effect of KLF14on C-33A cells.Bite effect.4.Use flow cytometry to explore the effect of transcription factor KLF14 on cervical cancer cell apoptosis.5.After applying the autophagy inhibitor 3-MA to inhibit autophagy on the basis of up-regulating the expression of KLF14,use CCK-8,plate clone formation experiment,EdU detection,flow cytometry to explore whether KLF14 affects the proliferation and apoptosis of cervical cancer cells through changing the level of cell autophagy.6.Up-regulate the expression of the transcription factor KLF14,and analyze the differentially expressed genes between the overexpressed KLF14 and the control group using transcriptome high-throughput molecular sequencing technology.7.Combine the literature and GO analysis to screen for possible downstream genes of KLF14 in cell autophagy,and verify at the cellular level using PT-PCR technology.Results1.KLF14 inhibits cervical cancer cell proliferationThe effect of KLF14 on the proliferation of cervical cancer cells:The cervical cancer cell lines HeLa and C-33A up-regulated the expression of KLF14 and then conducted EdU,CCK-8 and plate clone formation experiments.The results showed that the transcription factor KLF14 inhibited the proliferation of cervical cancer cells.2.KLF14 promotes cervical cancer cell autophagyThe effect of KLF14 on the autophagy of cervical cancer cells:Western blot results showed that in HeLa cells,compared with the control group,the ratio of autophagosome membrane marker protein LC3-?/? was increased in the overexpression group,and the autophagy substrate p62 was obvious decreased,indicating that KLF14 promotes autophagy in HeLa cells;in C-33A cells,Western blot results showed that the ratio of autophagosome membrane marker protein LC3-II/I increased in the overexpression group,and the results of cellular immunofluorescence showed that the content LC3 was higher than that of the control group,indicating that KLF14 promoted autophagy of C-33A cells.3.KLF14 inhibits cervical cancer cell apoptosisThe effect of KLF14 on the apoptosis of cervical cancer cells:Cervical cancer cell lines HeLa and C-33A up-regulated the expression of KLF14 and tested by flow cytometry.The results showed that the transcription factor KLF14 inhibited the apoptosis of cervical cancer cells.4.KLF14 inhibits cell apoptosis through autophagy and has no effect on cell proliferationOn the basis of overexpressing KLF14,3-MA was used to inhibit the level of autophagy.The results of flow cytometry showed that the apoptosis rate increased after inhibiting autophagy;the results of CCK-8 and plate cloning experiments showed that after the level of autophagy was decreased cell proliferation did not change significantly.Overall,the results show that KLF14 inhibits apoptosis by promoting autophagy,but changing the level of autophagy has no effect on cell proliferation.5.Detect the downstream target genes of KLF14 in regulating cell autophagyHigh-throughput molecular sequencing screened a total of 862 differentially expressed genes downstream of KLF14,including 750 up-regulated genes and 112 down-regulated genes;by consulting the literature and combining GO function analysis,10 genes related to cell autophagy were selected from them,including DEPP1,EVAIA,FOXO3,LAMP3,IRGM,ATP6V1C2,TRIM17,TRIM22?WNT16?MAP1LC3A.RT-PCR verified the mRNA expression levels of FOXO3 and EVA1A,and the results showed that they were consistent with the sequencing results.Looking at the literature,based on the TCGA database,it was found that FOXO3 is down-regulated in cervical cancer,and low FOXO3 is positively correlated with the prognosis of cervical cancer patients.ConclusionsKLF14 inhibits the proliferation of cervical cancer cells and inhibits the apoptosis of cervical cancer cells by promoting autophagy.SignificanceThis study verified the role of transcription factor KLF14 in the proliferation,autophagy and apoptosis of cervical cancer cells.1.This research enriched and perfected the mechanism development in cervical cancer,provided theoretical basis for clinical diagnosis and treatment.2.This research provided the possibility of KLF14 as a target for cervical cancer's targeted treatment and explored to provide new ideas for the treatment of cervical cancer. |
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