The Relationship Between MicroRNA-618/HMGA2 Gene Polymorphism And Lung Cancer Susceptibility,and Its Regulatory Mechanism

Objective: At present,lung cancer related death has become an important part of cancer death causes worldwide.The 5-year survival rate is very low due to two main factors: the low early diagnosis rate and lack of late treatment.As one of the most common and highly malignant solid tumors,there are about one million new cases in the world every year,and the trend is increasing year by year.According to the current epidemiological data analysis,the incidence and mortality of lung cancer in China are in the forefront of all cancer rankings.At present,the main histopathological types of lung cancer are non-small cell lung cancer(NSCLC)and small cell lung cancer.According to research reports,the incidence of NSCLC can account for up to 80% of lung cancer.Diagnosis of NSCLC is difficult because there are no common symptoms in patients at an early stage.At the same time,most patients with lung cancer have been diagnosed in the middle and late stage,losing the opportunity of surgical treatment,so some patients with advanced stage can only be treated with combined radiotherapy and chemotherapy.However,despite this,the five-year survival rate for lung cancer patients remains low.At present,with the gradual advance of basic science,more and more tumor-related oncogenes and tumor suppressor genes have been proved to play a key role in the progression of lung cancer,which also provides new understanding and possible exploration direction for tumor targeted therapy.Therefore,in-depth exploration of the mechanism of lung cancer occurrence and development will provide theoretical guidance for future targeted therapy of lung cancer.With the development of high-throughput sequencing,microarray analysis and bioinformatics,basic research on lung cancer has been gradually explored,including the important protein-coding domain and the non-protein-coding RNA domain(ncRNA).ncRNAs can be classified into two categories according to the transcript length,namely microRNAs(miRNAs)and long non-coding RNAs(lncRNAs).People have paid little attention to non-coding research until now,but with the gradual discovery of its function,it has been clear that it can be used as a target to intervene in tumor progression.Mi RNAs are a unique group of regulatory molecules that inhibit messenger RNA(m RNA)translation and inhibit the m RNA translation process through base pairing to complementary sites of target m RNA.A single miRNA may have multiple m RNA regulatory functions,and multiple miRNAs may also bind and regulate the same target protein.Therefore,miRNAs are involved in a variety of biological processes,including gene regulation,apoptosis,hematopoietic development and cell differentiation.Data show that one-third of human genes are regulated by miRNAs.Importantly,with further research,there is increasing evidence that miRNAs are extensively involved in post-transcriptional regulation of oncogenes or tumor suppressor genes in lung cancer.This study intends to further advance the mechanism of lung cancer by exploring the relationship between miR-618 single nucleotide polymorphism and lung cancer risk factors.By exploring the relationship between miR-618 and its target gene HMGA2,the regulatory mechanism of miR-618 in the malignant progression of lung cancer is clarified,providing theoretical guidance for subsequent targeted therapy.Methods: Part I: We conducted in-depth analysis and correlation assessment of rs2682818 and rs1716543 of miR-618 through manual screening of data collected in db SNP database on NCBI website,combined with a large number of literature screening,and SNP function prediction website.DNA was extracted from venous blood by phenol-chloroform method,followed by genotyping of genomic DNA from 460 lung cancer patients and 490 non-cancer controls by Taq Man probe method.Logistic regression was used to calculate OR values adjusted for age,sex,and smoking exposure and 95% confidence intervals to evaluate the association between miR-618 polymorphism and lung cancer susceptibility.The interaction between miR-618 rs2682818 and rs1716543 and smoking exposure was studied by cross-sectional analysis.Part II: This study explored the correlation between rs2272047 and rs8756 polymorphisms of HMGA2 and the risk of lung cancer,lung adenocarcinoma and lung squamous cell carcinoma.A case-control study was adopted in this study,and inclusion and exclusion criteria were established.Genomic DNA of all subjects was genotyped by Taq Man probe method.Logistic regression analysis was performed to evaluate the relationship between HMGA2 polymorphism and the risk of lung cancer,lung adenocarcinoma and lung squamous cell carcinoma.Logistic regression was used to calculate OR values adjusted for age,sex,and smoking exposure and 95% confidence interval to evaluate the association between HMGA2 polymorphism and lung cancer susceptibility.The interaction between HMGA2 rs2272047,rs8756 and smoking exposure was studied by cross-bioassay.Part III: In this study,miR-618 and HMGA2 expression profiles and relevant clinical sample information were obtained from the TCGA database in lung cancer and adjacent non-cancer tissues.After data cleaning and screening,R language of limma package was used for differential expression analysis.Lung cancer patients were divided into groups with high and low expression of miR-618 and HMGA2,respectively,to analyze the effects of miR-618 and HMGA2 on the prognosis of lung cancer patients.Target genes of miR-618 were predicted using Targetscan,miRWalk and miRDB target databases.Differential expression,prognostic analysis and enrichment analysis of miR-618 target gene were observed by the same method.Part IV: Firstly,the expression of miR-618 was detected by qRT-PCR in normal lung epithelial cell lines and lung cancer cell lines A549,H1299 and SK.We used cell transfection technology to construct miR-618 mimics and miR-618 inhibitors of H1299 and SK cell lines for functional studies in vitro.MTS assay was used to evaluate the proliferation of lung cancer cells,Transwell assay was used to evaluate the migration of lung cancer cells,and Annexin-APC/7-AAD was used to detect the apoptosis of lung cancer cells.The binding of miR-618 and HMGA2 was verified using a dual luciferase reporter assay.Finally,PCR and Western Blot were used to detect HMGA2 m RNA and protein expression in lung cancer cells.Results: Part I: There was no statistically significant difference in age and gender distribution between the case group and the control group(P < 0.05),but there was statistically significant difference in smoking status between the two groups(P < 0.001).In this study,rs1716543 and rs2682818 were not found to be statistically correlated with lung cancer susceptibility(P>0.05),and neither of them was associated with lung adenocarcinoma and lung squamous cell carcinoma in all gene models.In this study,there was no statistical significance in the dose-response relationship between the number of alleles rs2682818-A and rs1716543-A and the susceptibility to lung cancer(P >0.05).Stratified analysis showed that individuals with A allele at rs2682818 had A higher risk of lung cancer than those with C allele in patients older than 60 years.Among smokers,individuals with the homozygous AA genotype had a greater risk of lung cancer than individuals with the rs1716543 AC+CC genotype.Interaction results confirmed an increased risk of lung cancer in CC genotype smokers with rs2682818.Compared with CC genotype,smokers with rs2682818AC+AA genotype had a 1.201-fold increased risk of lung cancer.Individuals who smoke and carry the rs1716543 AC+CC genotype have an increased risk of lung cancer.Compared with AC+CC genotype,smoking individuals with rs1716543 AA genotype had a 2.079-fold increased risk of lung cancer.rs1716543 polymorphism and smoking in lung cancer may have additive model interaction.Part II: In this study,no statistical association was found between rs2272047 and rs8756 of HMGA2 and susceptibility to lung cancer.Stratified analysis found no statistical association between rs2272047 and rs8756 and susceptibility to lung adenocarcinoma and lung squamous cell carcinoma.The interaction found that smoking increased the risk of lung cancer in rs2272047 TT genotype carriers.Compared with rs2272047 TT genotype carriers,individuals with rs2272047 CC+CT genotype had a 1.405-fold increased risk of lung cancer.Individuals who smoked and carried the rs8756 AA genotype had a 1.378-fold increased risk of lung cancer compared with individuals with the rs8756 AC+CC genotype.Part III: TCGA data analysis suggested that miR-618 expression was significantly down-regulated and HMGA2 expression was significantly increased in lung cancer tissues compared with adjacent tissues(P<0.001).Mi R-618 and HMGA2 have good diagnostic efficacy in lung cancer(P<0.05),and miRNA-m RNA prediction database predicts that HMGA2 is the target gene of miR-618.Mi R-618 may be involved in dysregulation of transcription,RAS pathway,cytoplasmic transport,nicotine addiction,and sphingolipid biosynthesis affecting tumor metabolism(P<0.05).Enrichment analysis showed that HMGA2 plays a role in chemical carcinogenesis and epithelial mesenchymal transformation pathway of lung cancer(P<0.05).Part IV: Overexpression of miR-618 resulted in decreased proliferation ability(P<0.05),decreased migration ability(P<0.001)and increased apoptosis rate(P<0.05).Knockdown of miR-618 can lead to increased cell proliferation ability(P<0.05),enhanced migration ability(P<0.001)and decreased apoptosis rate(P<0.05).Mi R-618 can inhibit HMGA2 protein expression(P<0.01).Double luciferase reporter gene assay confirmed the existence of targeted binding between them.Conclusion: 1.No statistically significant association was found between miR-618 rs2682818 and rs1716543 polymorphisms and lung cancer susceptibility in this study.2.There was no statistically significant association between HMGA2 rs2272047 and rs8756 polymorphisms and the risk of lung cancer.3.TCGA database results showed that miR-618 was low expression in lung cancer,HMGA2 was high expression in lung cancer,and miR-618 was targeted to HMGA2.4.In vitro functional studies showed that miR-618 inhibited the proliferation and migration of lung cancer cells and accelerated apoptosis.HMGA2 is a target gene of miR-618,which regulates HMGA2 m RNA and protein expression levels in lung cancer cells.