| Gastric cancer is a gastrointestinal malignant tumor caused by the cancerization of epithelial cells of gastric mucosa,and its morbidity and mortality rate ranks very high among all malignant tumors.Compared with other regions in the world,the incidence and mortality of gastric cancer in East Asia are significantly higher due to the unique geographical environment.While China,with a large population base,ranks the first in the world in terms of morbidity and mortality due to unbalanced medical conditions and relatively backward medical concepts of some people,and is truly a big country with gastric cancer.In the majority with advanced gastric cancer patients in China,also have quite a proportion of patients with late-stage,most of these patients need preoperative chemotherapy,postoperative treatment or chemotherapy alone,the chemotherapy drug resistance is a crucial independent factor which affects the prognosis of gastric cancer patients,and also a serious threat to the people’s lives and health of China.The mechanisms of chemoresistance mainly include decreased uptake and/or increased efflux of drugs by tumor cells,changes in the expression level and/or structure of intracellular target substances,decreased intracellular activation and/or increased inactivation of drugs,and DNA damage repair Wait.While striving to improve surgical techniques,it is also important explore oncogenes and/or tumor suppressor genes that may contribute to its occurrence and development,to discover new markers in order to provide the reference for the diagnosis of gastric cancer,the choice of treatment,chemotherapy drug resistance evaluation and the judgment of prognosis.Krüppel like factors(KLFs)were first discovered in the regulation of embryonic development in drosophila.Subsequent studies showed that this family of zinc finger protein like factors is widely expressed in mammalian eukaryotic cells.It also plays an important role in the occurrence and development of cardiovascular diseases,obesity,inflammation and tumors.Decades of research have brought the total number of KLF members to 17.KLF17,a new member of the KLF family discovered in recent years,has been found to be closely related to the occurrence and development of multiple tumors,including esophageal squamous cell carcinoma,breast cancer,liver cancer and colorectal cancer,and is involved in the regulation of biological behaviors such as proliferation,invasion and migration of tumor cells.However,studies on the relationship between the abnormal expression level of KLF 17 and the occurrence of gastric cancer have rarely been reported,so the specific mechanism needs to be further explored.In this study,RNA and protein extraction techniques,real-time quantitative polymerase chain reaction(RT-QPCR),Western Blot,immunohistochemical staining,plasmid extraction,cell transfection,cell proliferation assay(MTS method),flow cytology and double luciferase reporter gene detection were applied.First,the differences at mRNA level and protein level of KLF 17 between the gastric cancer tissues and adjacent tissues of gastric cancer patients were verified to be statistically significant.Then the expression levels of drug resistance and apoptosis-related genes in gastric cancer tissues were detected,and analyzed their correlations with KLF 17 expression level in corresponding gastric cancer tissues.Subsequently,the mRNA and protein expression differences and trends of KLF 17 in human normal gastric mucosa epithelial cell lines and human gastric cancer cell lines with different levels of differentiation at the cellular level were measured.Subsequently,the plasmid containing KLF 17 was constructed and gastric cancer cell lines BGC-823 and SGC-7901 were transfected to detect the effects of KLF 17 overexpression on the chemotherapy sensitivity and apoptosis to 5-fluorouracil(5-FU)by flow cytometry and MTS assay.Next,the changes of the drug resistance-related and apoptosis-related proteins in the above gastric cancer cell lines after the up-regulation of KLF 17 expression were measured.Finally,BGC-823 gastric cancer cell line was used as the experimental object,dual luciferase reporter gene detection technology was applied to explore the downstream targets of KLF17,which clarified the potential biological mechanism of KLF17 in regulating the chemotherapy resistance of gastric cancer cells,and opened up a new way to further explore and research the biological mechanism in the occurrence,development and chemotherapy resistance of gastric cancer.Part one:The expression of KLF17 in gastric cancer and its correlations with expressions of drug resistance and apoptosis related genesObjective:To verify the mRNA and protein expression differences of KLF17 in tumor tissues and adjacent tissues of gastric cancer patients,GES-1 cell lines and gastric cancer cell lines with different degrees of differentiation,and analyze the correlations between KLF17 expression level and drug resistance or apoptosis-related genes in gastric cancer tissues.Methods:1.RT-qPCR,Western Blot and immunohistochemistry were used to detect the mRNA and protein expressions of KLF17 in gastric cancer tissues and adjacent tissues of 60 pairs of patients with gastric cancer.2.The mRNA and protein expressions of KLF17 in GES-1 cell line and four different differentiated gastric cancer cell lines were detected by RT-qPCR and Western Blot.3.RT-qPCR was used to detect the expression levels of drug resistance related genes MDR1,GST-71 and apoptosis related genes Cyclin D1,Bcl-2 and Bax in gastric cancer tissues,then analyzed their correlations with the KLF17 expression level in corresponding gastric cancer tissues.Results:1.Whether at the mRNA level or the protein level,the expression level of KLF17 in gastric cancer tissue was significantly lower than that in adjacent normal tissue,and the difference was statistically significant(P<0.05).2.The mRNA and protein expressions of KLF17 in HGC-27,BGC-823,SGC-7901,and NCI-N87 gastric cancer cell lines were significantly lower than that in GES-1 cell line,and the differences were statistically significant(P<0.05),which were negatively correlated with the degree of differentiation of gastric cancer cell lines.3.The mRNA expression of KLF17 in gastric cancer was negatively correlated with MDR1,Cyclin D1 and Bcl-2(P<0.05),but there was no significant correlation with GST-π and Bax.Summary:1.KLF17 is a tumor suppressor gene in gastric cancer,and its down-regulated expression plays an important role in the occurrence and progression of gastric cancer.2.Compared with normal gastric mucosa epithelial cell lines,the mRNA and protein expression levels of KLF17 in gastric cancer cell lines were significantly reduced,which proved that it could be used as a research object for subsequent cell function experiments.3.The expression level of KLF17 in gastric cancer tissues was negatively correlated with the expression levels of drug-resistance related gene MDR1 and apoptosis-related genes Cyclin D1 and Bcl-2,suggesting its potential as a new target for the treatment of chemotherapy resistance in gastric cancer.Part two:The effect of KLF17 on the chemoresistance of gastric cancer cell lines BGC-823 and SGC-7901Objective:To investigate the effect of KLF17 overexpression on the chemoresistance of gastric cancer cell lines SGC-7901 and BGC-823.Methods:1.Construct pcDNA3.1-KLF17 and pcDNA3.1 vector plasmids,and use them to transfect SGC-7901 and BGC-823 gastric cancer cell lines respectively.2.The mRNA and protein expressions of KLF17 in SGC-7901 and BGC-823 gastric cancer cell lines after transfection were detected by RT-QPCR and Western Blot to verify the overexpression of KLF17 in the gastric cancer cell lines after the transfection with pcDNA3.1-KLF17.3.The chemosensitivity and apoptosis level of SGC-7901 and BGC-823 gastric cancer cell lines were detected by MTS and flow cytometry after the transfection of pcDNA3.1-KLF17 plasmid.Results:1.RT-qPCR and Western Blot results showed that the expression levels of KLF17 in the SGC-7901 and BGC-823 gastric cancer cell lines in the pcDNA3.1-KLF17 plasmid transfection group were significantly higher than those in the original gastric cancer cell lines(Ctr group)(P<0.05).There was no significant change in the pcDNA3.1 vector plasmid transfection group(Vector group).2.The results of MTS showed that the curve of the growth inhibition rate of gastric cancer cell lines in the pcDNA3.1-KLF17 plasmid transfection group under different concentration gradients of 5-Fu was larger than that of the Vector group,and the IC50 value was also significantly lower than that of the Vector group,the difference was statistically significant(P<0.05).3.The results of flow cytometry showed that the apoptosis rate of the cells in the pcDNA3.1-KLF17 plasmid transfection group was significantly higher than that in the Vector group after being treated with the same concentration of 5-Fu for 24 hours,and the difference was statistically significant(P<0.05).Summary:1.SGC-7901 and BGC-823 gastric cancer cell lines overexpressing KLF17 were successfully constructed by plasmid transfection.2.Overexpression of KLF17 can significantly up-regulate the chemosensitivity and apoptosis of SGC-7901 and BGC-823 gastric cancer cell lines to 5-Fu,suggesting that the decreased expression of KLF17 is involved in the occurrence and development of gastric cancer chemotherapy resistance.Part three:The molecular mechanism of the effect of transcription factor KLF17 on chemotherapy resistance of gastric cancer cellsObjective:To explore the biological mechanism of KLF17 regulating the chemoresistance of gastric cancer cells.Methods:1.RT-qPCR and Western Blot techniques were used to detect the expression changes of Cyclin D1,drug resistance-related and apoptosis-related proteins in gastric cancer cell lines SGC-7901 and BGC-823 after KLF17 overexpression.2.The biological relationship between KLF17 and Cyclin D1 was detected by dual-luciferase reporter system.Results:1.RT-qPCR and Western Blot results showed that compared with the Ctr group and Vector group,the expression levels of Cyclin D1,P-gp and Bcl-2 in gastric cancer cell lines in the KLF17 overexpression group were significantly decreased,and the differences were statistically significant(P<0.05);the expression levels of GST-π and Bax did not change significantly.2.The results of dual-luciferase reporter gene technology showed that the up-regulation of KLF17 expression could significantly inhibit the transcriptional activity of Cyclin D1 gene promoter,and the difference was statistically significant(P<0.05).There was no obvious inhibitory effect after the sequence mutation.And the inhibitory effect of KLF17 on the transcriptional activity of Cyclin D1 gene promoter was significantly KLF17 dose-dependent,and the difference was statistically significant(P<0.05).Summary:1.In SGC-7901 and BGC-823 gastric cancer cell lines,KLF17 overexpression can significantly down-regulate the expression levels of Cyclin D1,drug resistance-related protein P-gp and apoptosis-related protein Bcl-2,suggesting that KLF17 may affect the chemotherapy resistance of gastric cancer cells by regulating the expressions of Cyclin D1,P-gp and Bcl-2.2.In BGC-823 gastric cancer cells,KLF17,as an upstream regulator of Cyclin D1,can regulate the occurrence and development of chemotherapy resistance in gastric cancer cells by directly binding to the Cyclin D1 promoter and inhibiting its transcriptional activity.Conclusions:1.The expression of KLF17 in human gastric cancer tissues was significantly lower than that in adjacent tissues,and was negatively correlated with the expressions of drug resistance related gene MDR1 and apoptosis related genes Cyclin D1 and Bcl-2;the expression of KLF17 in human immortalized gastric mucosal epithelial cell lines was higher than those in human gastric cancer cell lines,and the lower the differentiation degree of gastric cancer cells,the more obvious the difference,indicating that the decreased expression of KLF17 may be involved in the occurrence and development of gastric cancer and play an important role in the chemotherapy resistance of gastric cancer.2.Overexpression of KLF17 can significantly up-regulate the chemosensitivity and apoptosis of SGC-7901 and BGC-823 gastric cancer cell lines to 5-Fu,suggesting that KLF17 plays a relevant biological function in the occurrence and development of gastric cancer chemotherapy resistance.3.In SGC-7901 and BGC-823 gastric cancer cell lines,KLF17 overexpression can significantly down-regulate the expression levels of Cyclin D1,drug resistance-related protein P-gp and apoptosis-related protein Bcl-2,suggesting that KLF17 may affect the chemotherapy resistance of gastric cancer cells by regulating the expressions of Cyclin D1,P-gp and Bcl-2.4.In BGC-823 gastric cancer cells,KLF17,as an upstream regulator of Cyclin D1,can regulate the occurrence and development of chemotherapy resistance in gastric cancer cells by directly binding to the Cyclin D1 promoter and inhibiting its transcriptional activity. |
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