| Background:Breast cancer is a great threat to Chinese women.Its incidence rate continues to rise with an annual increase of 2.5%,and the mortality rate increases rapidly with an annual increase of 3%.The number of new cases and deaths accounts for 12.2% and 9.6%respectively in the world.Although with the development of medical technology,the diagnosis and treatment of breast cancer has been greatly improved,a high cure rate and survival rate.But breast cancer still has a high rate of recurrence and metastasis.The medical treatment of breast cancer includes chemotherapy,endocrine therapy and targeted therapy.In recent years,targeted therapy for breast cancer has become a focus of clinical research,and its clinical application has significantly improved the therapeutic effect of breast cancer.More and more molecular targeted therapeutic drugs have been developed for different subtypes of breast cancer.The clinical application of these drugs has improved the therapeutic effect of breast cancer and constantly changed the clinical practice process of breast cancer.How to overcome the drug resistance of tumors and develop new drugs that can surpass the efficacy of traditional targeted drugs is an important research direction in the future.MYC is a powerful proto-oncogene,initially expressed in burkitt lymphoma(t 8;14)(q24;Q32)was found in the chromosome.MYC is a member of the helical leucine zipper family,and is a transcription factor like other family members n-MYC and l-MYC.MYC functions on the basis of nuclear localization sequence,helical-loop-helical dimerization region,DNA binding region and transcriptional regulation region.In normal cells,MYC proto-oncogene is strictly regulated by various regulatory mechanisms and signal transduction pathways at the transcription level,post-transcription level,cell cycle test points and chromosome mitosis.These strict regulatory mechanisms prevent cells from becoming cancerous,and MYC is by far one of the most frequently deregulated oncogenes.In 50% of human cancers,the MYC family of oncogenes is out of control,which is often associated with poor prognosis and poor survival.MYC plays a central role in almost every aspect of the carcinogenic process,coordinating proliferation,apoptosis,differentiation and metabolism.Although MYC inhibition is an effective method for the treatment of many types of cancer,direct targeting of MYC has been a challenge due to the non-pharmacogenicity of its protein structure.Therefore,in order to achieve an ideal anti-tumor effect,alternatives to MYC blockade have been extensively explored,including MYC/Max complex blockade,MYC transcription and/or translation inhibition,MYC instability,and synthetic pathogenicity associated with MYC overexpression.MYC is an important regulatory mechanism,which plays a crucial role in preventing the proliferation of tumorigenic cells.Apoptosis induced by oncogene MYC occurs through a p53-dependent and independent mechanism,the mechanism of which remains unclear.P53 gene is mutated in about 50% of human tumors,and the loss of anti-tumor activity of p53 is related to cell cycle arrest and apoptosis defects.It has been reported that the deletion of DNA repair process caused by p53 deletion is associated with high cancer susceptibility observed in p53 deletion mice.MYC amplification rate in breast cancer tissues is about 30%.A large number of basic research data show that the amplification of proto-oncogene MYC plays an important role in the pathogenesis of breast cancer,but its amplification frequency is not consistent with the prognostic correlation in human studies.Studies have shown a significant but weak association between myc amplification and known prognostic factors(tumor grade,lymph node metastasis,hormone receptor negative).Amplification was significantly associated with the risk of recurrence and death.So we can see,MYC dysregulation can lead to the occurrence and progression of breast cancer and is associated with poor prognosis,especially in basal carcinoid subtypes.Therefore,targeting the MYC regulatory pathway may provide a promising therapeutic strategy for breast cancer.The purpose of our study is to understand the clinicopathology/prognosis of MYC and breast cancer,and its expression,function and mechanism in breast cancer need further discussion.Objective:1.The expression of MYC in breast cancer tissue was detected,and its correlation with clinicopathological factors and recurrence and metastasis was analyzed to explore the relationship between MYC and the occurrence,development,invasion and metastasis of breast cancer.2.Observe the effects of wild-type MYC and T58 A mutations on the proliferation and apoptosis of p53-deficient breast cancer cells,and further explore the causes;3.In vitro RNAi silencing gene technology was used to investigate the mechanism of MYC gene regulating Bim,an important factor in non-p53-dependent apoptosis,by regulating upstream genes p14 and p21.Materials and Methods:1.Detection of the relationship between MYC and clinicopathological features and recurrence and metastasis of breast cancer(1)From June 2013 to January 2014,100 invasive breast cancer patients admitted to the affiliated hospital of Qingdao university were selected and collected.(2)Immunohistochemical analysis of MYC expression in tissue samples.(3)Collect and sort out clinicopathological data,and analyze the correlation between MYC expression and clinicopathological factors;The relationship between MYC and 5-year DFS in breast cancer was analyzed.2.To observe the effect of wild-type MYC and T58 A mutations on the biological behavior of p53-deficient breast cancer cells(1)P53-deficient HCC1937 breast cancer cells were transfected by MYC wild-type and mutant T58 A lentivirus vectors respectively.(2)The effects of MYC wild-type and mutant T58 A on the proliferation of p53-deficient breast cancer cells were detected by colony formation and MTT methods.(3)The effects of MYC wild-type and mutant T58 A on apoptosis of p53-deficient breast cancer cells were detected by flow cytometry and TUNEL assay.3.To analyze the regulatory effect of MYC on Bim in the absence of p53(1)The expression levels of MYC and Bim were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot,in an attempt to find out the regulation mechanism of MYC wild-type and mutant T58 A on proliferation and apoptosis of p53-deficient breast cancer cells.(2)RT-PCR and Western blot were used to detect the mRNA and protein expression levels of p14 and p21 in MYC and Bim signaling pathways.(3)Interference with the expression of p21 by in vitro RNAi silencing gene technology.The expression levels of Bim and p21 after interference were detected by RT-PCR and Western blot.(4)Colony formation method and MTT method were used to detect the cell proliferation after p21 interference,and flow cytometry and TUNEL method were used to detect the cell apoptosis.(5)Interference with the expression of p14 by in vitro RNAi silencing gene technology.The expression levels of Bim and p14 after interference were detected by RT-PCR and Western blot.(6)Colony formation method and MTT method were used to detect cell proliferation after interference with p14,and flow cytometry and TUNEL method were used to detect cell apoptosis.Results:1.Immunohistochemical results showed that the positive rate of MYC in breast cancer tissues was 36%(36/100);The positive rate was 12%(12/100)in the paracancer normal tissues.The positive rate of MYC in cancer tissues was significantly higher than that in adjacent tissues,and the comparison between the two groups was highly statistically significant(p<0.01).2.The positive expression of MYC was not related to the age of breast cancer,and the positive rate was 37.2%(16/43)in patients younger than or equal to 50 years old,while35.1%(20/57)in patients older than 50 years old,with no statistical significance(p=0.827).3.The positive expression of MYC is related to the tumor diameter of breast cancer.The positive rate is 25%(12/48)in patients with less than or equal to 2cm,while 46%(24/52)in patients with more than 2cm.The positive rate of MYC was higher in patients with larger tumors,and the difference was statistically significant(p=0.028).4,MYC positive expression associated with the TNM staging of breast cancer,in patients with stage for ?+?,positive rate was 26.8%(15/56),and the installment for ?patients,the positive rate was 47.7%(21/44);The higher MYC staging was,the higher the positive rate was,and the difference was statistically significant(p=0.030).5,MYC positive expression associated with the cytological classification of breast cancer,in hierarchical ?+? patients,positive rate was 27.6%(16/58),and the classification of ? patients,positive rate was 47.6%(20/42),the difference was statistically significant(p= 0.039).6.Positive expression of MYC is correlated with ER status of breast cancer.In patients with positive ER,the positive rate is 25%(15/60),while in patients with negative ER,the positive rate is 52.5%(21/40).The higher the positive rate of MYC was in the patients with ER negative,and the difference was statistically significant(p=0.005).7.Positive expression of MYC is related to the status of HER in breast cancer.In patients with positive HER2,the positive rate is 48.6%(18/37),while in patients with negative HER2,the positive rate is 28.6%(18/63).The higher the positive rate of MYC was in patients with positive HER2,the difference was statistically significant(p=0.043).8.The positive expression of MYC is not related to the lymph node status of breast cancer.In the patients with positive lymph node,the positive rate is 43.1%(25/58),while in the patients with negative lymph node,the positive rate is 26.1%(11/42),with no statistical significance(p=0.082).9.Positive expression of MYC was associated with postoperative recurrence and metastasis of breast cancer.The positive rate was 60%(12/20)in patients with recurrence within 5 years,and 29.2%(24/80)in patients without recurrence within 5 years.MYC positive patients had a higher recurrence rate,and the difference was statistically significant(p=0.012).10.Positive expression of MYC was not related to p53 expression in breast cancer.In patients with positive expression of p53,the positive rate was 39.6%(21/53),while in patients with negative expression of p53,the positive rate was 31.9%(15/47),with no statistical significance(p=0.642).11.MYC wild-type(WT)and T58 A mutation(T58A)successfully transfected p53-deficient HCC1937 cells.Both of them can induce cell growth(p< 0.01).WT can also induce apoptosis(p< 0.01),while T58 A cannot(p> 0.05).12.RT-PCR and Western blot results showed that the expression level of Bim was increased in the WT group.There was no significant difference in the expression of Bim between T58 A group and control group(p> 0.05).These results indicated that the T58 A mutation could not up-regulate Bim and thus could not induce apoptosis.13.After WT successfully transfected HCC1937 cells,the expression level of p21decreased(p< 0.01)and the expression level of Bim increased(p< 0.01).After the silencing of p21 by RNAi,the expression level of Bim was higher than before the silencing(p< 0.01).The expression levels of p21 and Bim did not change after the successful transfection of T58 A into HCC1937 cells(p> 0.05),and there was no significant difference between the expression levels of Bim and pure silencing of p21 after T58 A transfection with RNAi silencing of p21(p> 0.05).14.After the successful transfection of WT into HCC1937 cells,the expression level of p14 increased(p< 0.01),while the expression level of Bim increased(p< 0.01).After the silencing of p14 by RNAi,the expression level of Bim was lower than that before the silencing(p< 0.01).The expression levels of p14 and Bim were not changed after the successful transfection of T58 A into HCC1937 cells(p> 0.05),and there was no significant difference between the expression levels of Bim and pure p14 after T58 A transfection and the silencing of p14 by RNAi(p> 0.05).Conclusion:1.The high expression of MYC in breast cancer tissue is related to tumor diameter,cytological grade,pathological stage,hormone receptor state and HER2 state.In addition,the risk of recurrence and metastasis in patients with MYC positive was increased within 5years,suggesting that MYC positive expression was related to the prognosis of breast cancer.2.In p53-deficient breast cancer cells,MYC can promote cell proliferation and apoptosis,while MYC mutation can only promote cell proliferation,but not apoptosis.3.In p53-deficient breast cancer cells,MYC plays a regulatory role in cell proliferation and apoptosis through two signaling pathways,MYC /p21/Bim and MYC /p14/Bim;After MYC mutation,p21 and p14 cannot be regulated,and Bim cannot be further regulated to participate in the regulation of apoptosis. |
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