| Background and purposeEsophageal cancer is one of the most common cancers in the world.Due to very minor symptoms in early stage,esophageal cancer is often diagnosed in clinic after onset of progressive dysphagia in patients.However,the esophageal cancer patients often,at advanced stage,lose the best time for surgical treatment.As the most widely using of chemotherapeutic drugs,the current cisplatin-based chemotherapy regimens is the preferred treatment of advanced esophageal cancer patients in clinical.However,during the course of cisplatin-based chemotherapy,the occurrence of chemoresistance reduces the treatment effect and poses a real challenge in anticancer chemotherapy.MicroRNAs(miRNAs)are a class of endogenous single-stranded,non-coding RNAs containing about 18-25 nucleotides.They are expressed in many organisms and highly conserved.Various miRNAs exert different regulatory roles due to their different target molecules.Furthermore,a miRNA can interact with a serious of target molecules to play distinct regulatory roles in same or different types of cells.Because miRNAs can regulate a large number of genes including tumor-related genes,the extensive studies showed that the abnormal expression of miRNAs is closely related to tumorigenesis and prognosis.DNA polymerase ?(pol?)is a key enzyme in maintaining genomic stability through base excision repair(BER),which can fill in the single nucleotide gap caused by DNA base excision and deoxyphosphate elimintion.Normally,pol? has consistently low level expression in most cell cycle phases and in most types of tissues.Aberrant pol? expression and mutants can cause aberrant BER,spontaneous mutation,genomic instability and chromosomal aberrations,and thus play an important role in the development of tumors.Our microarray data showed that the expression level of miR-499 in esophageal cancer tissues was significantly lower than that in paracancerous tissues,and the expression of miR-499 in cancer tissues of patients with chemotherapy-insensitive esophageal cancer was significantly lower than those with chemotherapy-sensitive esophageal cancer.By bioinformatics software prediction,we demonstrated that pol?was the potential target gene of miR-499.By detecting the mutation of the pol? gene,177-234 nt deletion was found in 12 patients out of 538 patients with esophageal cancer(2.23%).The purpose of this study was to investigate parallelly the effect and mechanism of miR-499 and 177-234 nt deletion pol? mutant on chemosensitivity of esophageal cancer cells.This study is divided into the following two parts.Part 1 Effect of miR-499 Targeting Pol? on the Chemosensitivity of Esophageal Cancer CellsMethods1.The qRT-PCR and Western blot methods were used to detect the expression of miR-499 and pol? in 538 cases of esophageal cancer tissues and adjacent tissues;analyze the correlation between the expression level of pol? and the expression level of miR-499 in esophageal cancer tissue.2.The EC9706 and KYSE30 cells with stable miR-499 expression were established to detect the effects of miR-499 on the chemosensitivity of EC9706 to KYSE30 by MTT,clone formationassay,flow cytometry,immunofluorescence and comet assays.3.The EC9706-Luc cells with stable miR-499 expression was established and then inoculated in the right armpit of nude mice to produce xenograft model.Each exprimentanimal was intraperitoneally injected with cisplatin saline solution(3mg/kg,once every three days)for a total of 9 times.The in vivo imaging of nude mice xenografts was performed once every 7 days after inoculation,and the luciferase activity in xenografts of nude mice was detected.4.The bioinformatics software was used to predict the target gene of miR-499.The pol? 3'UTR wild type and mutant reporter vectors were constructed and co-transfected into esophageal cancer cells EC9706 and KYSE30 with miR-499 mimic/negative control.Dual luciferase reporter assay was used to validate the interaction between pol? mRNA and miR-499.5.The recombinant plasmid pcDNA3.1-pol? without pol? 3'UTR was constructed and transfected into EC9706 and KYSE30 cells that stably expressing miR-499.MTT and flow cytometry assays were used to demonstrate whether pol?can rescue the effect of miR-499 on the chemosensitivity of EC9706 and KYSE30 cells.Results1.The relative expression level of miR-499 in esophageal cancer tissues was significantly lower than that in adjacent tissues(P<0.05).The expression levels of pol? mRNA and protein in esophageal cancer tissues were significantly higher than those in adjacent tissues(P<0.05).The correlation analysis showed that there was a negative correlation between the expression of miR-499 and the expression of pol? in esophageal cancer tissues.2.The results of cytological experiments showed that compared to NC and Blank groups,EC9706 and KYSE30 cells transfected with miR-499 mimic had significantly enhanced cisplatin-caused effects of proliferation inhibition and apoptosis promotion(P<0.05).3.The results of Xenograft tumor in vivo imaging showed that the absolute photon number of transplanted tumor in miR-499 group mice were significantly lower than NC and Blank groups from day 14(P<0.05).4.Bioinformatics analysis showed that the pol? 3'UTR region contains a sequence complementary to miR-499,and indicted that pol? gene is the potential target of miR-499.The results of luciferase reporter assay showed that the luciferase activity of cells co-transfected with miR-499 mimic and pol? 3'UTR wild-type recombinant vectors was significantly lower than those co-transfected with miR-499 mimic and pol? 3'UTR mutants Recombinant vector,and cells transfected with miR-499 negative control(P<0.05).5.The IC50 of miR-499 cells transfected with pcDNA3.1-pol? was significantly higher than that of miR-499 alone(P<0.05),and the percentage of apoptotic cells after transfected with pcDNA3.1-pol? was significantly lower than that of miR-499 alone(P<0.05).These results indicated that expression of pol? can reverse the effect of miR-499 on chemosensitivity of EC9706 and KYSE30 esophageal cancer cells.Part 2 Effect of 177-234 nt deletion pol? on the chemosensitivity of esophageal cancer cellsMethods1.The complete follow-up data of esophageal cancer patients whose pol? has been sequenced were preserved and selected for downstream analysis.Kaplan-Meier method was used to analyze the survival time based on the data of 538 cases esophageal cancer patients.2.EC9706 cells with stable 177-234 nt deletion pol? expression was established and subjected to MTT and flow cytometry assays to detect the effect of 177-234 nt deletion pol? on the chemosensitivity of EC9706 cells.3.EC9706 cells with stable 177-234 nt deletion pol? or wt pol? expression were inoculated into the right armpit of nude mice.Each animal was intraperitoneally injected with cisplatin saline(3 mg/kg)for 3 days.Nude mice were euthanased 4 weeks after inoculation.The tumor tissue specimens were peeled for photographing and weighing.4.The biotinylated 177-234 nt deletion pol? proteins were conjugated with streptavidin beads and incubate with esophageal cancer cell lysate.The bound proteins were separated and analyzed by mass spectrometry.5.BIAcore and pull-down experiments were used to verify whether PIK3IP1 could bind to 177-234 nt deletion pol? protein.6.The Western blot assay was used to detect the effect of 177-234 nt deletion pol? on the expression of protein involved in PI3K-Akt pathway in EC9706 cells.7.MTT and flow cytometry assays were used to demonstrate the effects of PIK3IP1 knockdown and 177-234 nt deletion pol? on the chemosensitivity of EC9706 cells.8.PI3K inhibitor LY294002 was added into EC9706 cells with 177-234 nt deletion pol?,and MTT and flow cytometry assays were used to detect the rescue effect of PI3K inhibitor on the sensitivity of EC9706 cells with 177-234 nt deletion pol? to cisplatin and 5-FU..Results1.Kaplan-Meier survival analysis showed that patients with 177-234 nt deletion pol? had lower overall survival rate than those with wild type pol?,the difference was statistically significant(P<0.05).2.Compared with wild-type pol?,177-234 ntdeletion pol? weakened the proliferation inhibitory and apoptosis enhancement effect of cisplatin/5-FU in EC9706 cells.The difference has statistically significant(P<0.05).3.Results of xenograft tumor showed that transplanted tumor weightof 177-234nt deletion group nude mice was significantly greater than that of wild-type pol? animals transplanted tumor weight(P<0.05),the difference has statistically significant.4.According to the mass spectrometry results,protein may bind to 177-234 nt deletion pol? protein in EC9706 cells is PIK3IP1.5.The results of BIAcore and pull-down experiments validated the interaction between the PIK3IP1 and 177-234 nt deletion pol? protein.6.Western blot results showed that 177-234 nt deletion pol? could promote the phosphorylation of Akt protein,thereby inhibiting the activation of downstream apoptotic genes and promoting the activation of downstream proliferation genes.7.Compared with pol?-/-group,the proliferation inhibition and apoptosis promotion effect of cisplatin/5-FU in PIK3IP1 siRNA and 177-234 nt deletion pol?group were weaker,the difference has statistically significant(P<0.05).There was no significant difference between PIK3IP1 siRNA and 177-234 nt deletion pol? group(P>0.05).8.The results of MTT and flow cytometry assays showed that the PI3K inhibitor LY294002 could partially restore the effect of 177-234 nt deletion pol?onthe chemotherapy sensitivity of EC9706 cells by repressing the activity of PI3K and restoring the cytotoxic effects of cisplatin/5-FU.Conclusions1.miR-499 enhances cytotoxic effects of cisplatin on the EC9706 and KYSE30 cells through downregulating the expression of pol?,and thus,promotes the chemosensitivity of esophageal cancer cells.2.177-234 nt deletion pol? protein binding with PIK3IP1 molecules could rescue PI3K from inactivation and then activate various downstream genes through PIK3/Akt signaling pathway.In EC9706 cells,presence of 177-234 nt deletion pol?protein gave the esophageal cancer cells phenotype of weakened sensitivity to cisplatin via PIK3/Akt pathway. |
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