Associations Of P53-related MicroRNA Polymorphisms With Risk And Survival Of Lung Cancer In Chinese Non-smoking Females

Objective: Lung cancer is the most common malignant tumor and the leading cause of cancer death worldwide,has become a major public health problem to be solved urgently.The occurrence and development of lung cancer are multi-step and multi-step processes with the involvement of multi-factor and multi-gene,and the molecular mechanisms have not yet fully understood.Smoking is an independent risk factor for lung cancer,while women are more likely to suffer from non-smoking related lung cancer.In China,the smoking rate is 2.7% in females,while the incidence of lung cancer in female has reached up to 31.93 per 100,000.This incidence is far higher than the expected value caused by the corresponding smoking rate in females.It suggests that Chinese women may have a high genetic susceptibility to lung cancer.Therefore,in Chinese nonsmoking female lung cancer patients,exploring new occurrence and development related genes of lung cancer excluding the influence of smoking,and clarifying their mechanisms will be helpful to the early diagnosis and accurate treatment,as well as the improvement of prognosis.MicroRNAs(miRNAs)are a kind of highly conserved and endogenous non-protein coding small molecule RNAs that range in size about from 19-23 nucleotides.Some tumor related microRNAs can regulate the expression of target genes(oncogenes and anti-oncogenes)in the way of sequence specific regulation,thus affect the occurrence and development of tumor.MicroRNAs are widely involved in the malignant biological behavior of lung cancer,such as cell proliferation and metastasis,and are related to the prognosis of lung caner,such as pathological classification,TNM stage and survival.p53 is a multifunctional transcription factor that can regulate the expression of some microRNAs,whereas,microRNAs can regulate the expression of p53 directly or indirectly at post transcriptional level,thus forming a feedback loop.MiR-34 is the first confirmed p53 target microRNA,and then more p53 signal regulation network related microRNAs(such as miR-26,miR-145,miR-605,miR-31,etc.)was found.p53 can induce or inhibit the expressions of these microRNAs,and then the later can regulate many downstream target genes,affecting cell cycle and cell apoptosis signal pathways.MicroRNAs gene mutation may occur in the process of maturation,resulting in single nucleotide polymorphism(SNP).MicroRNA-SNP may influence the transcription of pri-microRNA,the processing of pre-microRNA and the ability to regulate target gene of mature microRNA,leading to individual difference in cancer susceptibility.Also,MicroRNA-SNP is associated with cancer prognosis,including the effects on recurrence,treatment outcomes and survival.At present,only two studies investigated the relationship between mi R-26a1 rs7372209 and the risk of lung cancer that found there was no association;in male and female lung cancer patients,the study conclusions about the relationship between miR-605 rs2043556 and the risk of lung cancer were inconsistent;the relationships between miR-34 rs4938723,miR-145 rs353291 and miR-31 rs13283671 and the risk of lung cancer have not been reported yet.In terms of prognosis,one study showed that miR-26a1 rs7372209 did not affect the recurrence time of complete surgical resection lung cancer patients;another study explored the relationships between miR-145 rs353291 and miR-31 rs13283671 and the prognosis of lung cancer in different populations of two stage and got an opposite conclusion;the relationships between miR-34 rs4938723 and miR-605 rs2043556 and the prognosis of lung cancer have not been reported yet.In this study,we first performed a systematic review,retrieving literatures about the abnormal expressions of miR-26,miR-34,miR-145,miR-605 and miR-31 in lung cancer and its relationship with prognosis,and clarifying the relationships between the expressions of these p53 associated microRNAs and the risk and prognosis of lung cancer;subsequently,we selected Chinese non-smoking female patients with lung cancer to explore the relationships between the genotypes of miR-26a1 rs7372209,miR-34b/c rs4938723,miR-145 rs353291,miR-605 rs2043556 and miR-31 rs13283671 and the risk and overall survival of lung cancer,as well as gene-gene interaction,further identifying non-smoking female lung cancer related microRNAs,in order to provide scientific basis for early diagnosis,accurate treatment and prognosis improvement in non-smoking female lung cancer.Methods: In the first part of this study,a systematic review was performed.In the PubMed,web of science,Wanfang and CNKI databases,literatures about the abnormal expressions of miR-26,miR-34,miR-145,miR-605 and miR-31 in lung cancer and its relationship with prognosis were retrieved.These literatures were screened in strict accordance with the inclusion and exclusion criteria.The names of authors,publication year,sample size,micro RNA's name,the detection method of microRNA's expression,microRNA's expression status and other related information were extracted exactly.In the second part of this study,a case-control study method was adopted.There were 450 non-smoking female inpatients from cooperation hospitals during July 2010 and December 2012 with histologically confirmed new lung cancer.At the same time,450 non-smoking healthy women were collected from general population with frequency matching method according to age ±5 years as controls.All subjects are Han population in Shenyang.After obtaining informed consent,blood sample were collected.Information about demographic and environmental exposure factors was collected by face to face interview.TaqMan Real-time PCR method was used to detect and determine genotype.Statistical analyses were performed using SPSS 19.0 software.Chi-square test or t-test was used to compare categorical or continuous variables between the case group and the control group.Univariate or multivariate logistic regression model was used to assess the odds ratios(OR)with 95% confidence interval(CI)for estimating the association between risk factors and lung cancer risk,and gene-gene interaction was also examined.In the third part of this study,450 cases of non-smoking female lung cancer patients were included.The starting time of included patients of this study was July 2010,and the follow-up time was terminated in February 2016.Time of death was obtained using one of the following ways: death registration reporting system of Shenyang Centers for Disease Control and Prevention,telephone follow-up and so on.During the following period there were 26 patients lost to follow-up,and the lost rate in this study was 5.78%.Statistical analyses were performed using SPSS 19.0 software.Kaplan-Meier and Logrank test were used to perform survival analysis.Univariate and multivariate Cox regression models were carried out to calculate the crude hazard ratio(HR),adjusted HR and 95% CI.Results : The first part of this study we performed a systematic review,retrieving literatures about the abnormal expressions of miR-26,miR-34,miR-145,miR-605 and miR-31 in lung cancer and its relationship with prognosis.We found that the expression of miR-26 b,miR-34,miR-145 and mi R-605 were down regulated and miR-31 was up regulated in lung cancer.They may be act as either tumor suppressors or oncogenes.The relationship between mi R-26 a and lung cancer was not consistent.Two studies showed that miR-26 a was over expressed in lung cancer,but the other two studies had the opposite results.The patients with reduced mi R-145 expression or elevated miR-31 expression exhibited poor survival.There were three studies explored the relationship between miR-34 family and prognosis of lung cancer.The results were different among these studies.The relationship between miR-26 and miR-605 expression and prognosis of lung cancer had not been reported.The second part of this study explored the influences of miR-26a1,mi R-34b/c,mi R-145,miR-605 and mi R-31 single nucleotide polymorphisms on lung cancer susceptibility in non-smoking females.The results showed that after adjusting for age,compared with miR-34b/c TT genotype carrier or TT+TC genotype carrier,lung cancer risk of CC genotype carrier was significantly increased(OR=1.838,95%CI: 1.092-3.092;OR=1.758,95%CI: 1.055-2.929;respectively).Participants carrying miR-34b/c CC genotype had a significant increased risk in lung squamous cell carcinoma(OR=2.557,95%CI: 1.206-5.422).The dominant model and recessive model showed similar results.Compared with miR-605 AA or AA+AG genotype carrier,lung squamous cell carcinoma risk was increased in GG genotype carrier(OR=2.115,95%CI: 1.028-4.518;OR=2.079,95%CI: 1.013-4.267;respectively).MiR-26a1 rs7372209 TT genotype was associated with an increased risk of lung adenocarcinoma compare with CC+CT genotype(OR=1.789,95%CI: 1.022-3.131).Multivariate analysis showed that miR-34b/c CC genotype was an independent risk factor for lung cancer;miR-34b/c CC genotype and mi R-605 GG genotype were independent risk factor of lung squamous cell carcinoma.Compared with miR-26a1 CC and miR-34b/c TT genotype carrier,lung cancer risk of miR-26a1 TT and miR-34b/c CC genotype carrier was significantly increased(OR=2.648,95%CI: 1.200-5.845;OR=2.292,95%CI: 1.056-4.978;respectively).Combined miR-26a1 TT and miR-34b/c CC genotypes had a relatively higher risk of lung cancer(OR=3.362,95%CI: 0.872-12.961).Compared with miR-26a1 CC and miR-605 AA genotype carrier,lung cancer risk of mi R-26a1 TT and mi R-605 GG genotype carrier was increased(OR=2.250,95%CI: 1.010-5.011;OR=1.607,95%CI: 0.775-3.331;respectively).Combined miR-26a1 TT and miR-605 GG genotypes had a higher risk of lung cancer(OR=3.000,95%CI: 0.777-11.585).The third part of this study explored the influences of miR-26a1,miR-34b/c,miR-145,miR-605 and miR-31 polymorphisms on lung cancer prognosis in non-smoking females.Univariate Cox regression analysis showed that the relationship between miR-26a1,miR-34b/c,mi R-145,miR-605,miR-31 polymorphisms and lung cancer prognosis was not statistically significant.After adjusted for age,clinical stage,chemotherapy and surgical operation status,compared with miR-26a1 CC genotype,TT genotype carriers had a shorter median survival time and increased the death risk of lung cancer(HR=1.573,95%CI: 1.013-2.442).Compared with CC or CC+CT genotype,miR-26a1 TT genotype was significantlly associated with lung adenocarcinoma survival(HR=1.682,95%CI: 1.057-2.678;HR=1.660,95%CI: 1.061-2.597;respectively).There was no association between SNPs and lung squamous cell carcinoma survival.Multivariate Cox regression analysis showed that miR-34b/c CC genotype was independently associated with lung cancer and lung adenocarcinoma survival(HR=0.609,95%CI: 0.377-0.985;HR=0.550,95%CI: 0.315-0.963;respectively).In the combined analysis,there was a negative interaction between mi R-34b/c and miR-26a1 polymorphisms,and the effect of miR-34b/c polymorphism was more obvious in the influence of prognosis.Compared with miR-34b/c TT genotype and miR-145 AA genotype carriers,miR-34b/c CC genotype and miR-145 AA genotype carriers had a decreased death risk of lung cancer(HR=0.402,95%CI: 0.191-0.849).Carrying both miR-605 GG genotype and miR-26a1 CC genotype had better outcome(HR=0.431,95%CI: 0.229-0.814).Combined analysis between mi R-605 and miR-145 polymorphisms,we found that miR-605 polymorphism also decreased the risk of death(HR=0.403,95%CI: 0.191-0.850).Conclutions: 1.MiR-26 b,miR-34 family,miR-145 and miR-605 may act as tumor suppressors and mi R-31 may play a role as oncogene in lung cancer.The patients with reduced miR-145 expression or elevated miR-31 expression exhibited worse survival of lung cancer.2.MiR-34b/c and miR-605 polymorphisms may be independent genetic risk factors of non-smoking female lung cancer susceptibility.The interaction between miR-26a1,miR-34b/c and miR-605 polymorphisms may influence lung cancer susceptibility.3.MiR-34b/c polymorphism may be a biomarker affecting prognosis of nonsmoking female lung cancer.Carrying miR-34b/c CC genotype may improve the outcome of non-smoking female lung cancer.