Relationships Of The Polymorphisms Of MALAT1 And MicroRNA-328-3p With Susceptibility To Lung Cancer And Mechanism Of Regulating Target Gene YWHAZ

Objective: The incidence rate and mortality rate of lung cancer are the highest malignant tumors.Global data(GLOBOCAN 2018)predicts that the number of cases in Asia accounts for nearly half of the world’s new malignant tumors(18.1 million),and nearly70% of cancer deaths are Asians.The number of new cases of lung cancer in the world is2 million 207 thousand,accounting for 11.4%,and the proportion of deaths is 1 million796 thousand,accounting for 18%,accounting for first of the second incidence rate of cancer worldwide and first of deaths worldwide.The National Cancer Center of China released that the number of new cases of malignant tumors in China was about 3.929 million,including about 787000 cases of lung cancer.The number of deaths from lung cancer was about 631000,with a mortality rate of 45.87/100000,ranking the first in China.By 2025,the number of new lung cancer deaths in China will exceed 1 million every year.Smoking is an important pathogenic factor of lung cancer.Most lung cancer is related to smoking.The risk rate of lung cancer in long-term smokers is 10 ～ 30 times that of non-smokers.But there are still cases of lung cancer that are not caused by smoking.It shows that there may be differences in pathogenic mechanism between smoking lung cancer patients and non-smoking lung cancer patients,which should be distinguished clinically.Studies have shown that genetic factors also play a crucial role in the occurrence of lung cancer.The molecular mechanism of lung cancer is very complex and unclear.Identifying new biomarkers for diagnosis and prognosis of lung cancer and looking for new treatment strategies are the focus of lung cancer research.Noncoding RNA is an endogenous RNA transcript that does not encode protein.It has tissue-specific expression and plays an irreplaceable role in the complex physiological and pathological processes of cells.According to the chain length of transcripts,non coding RNAs are divided into short chain non coding RNAs with a length of less than200 nucleotides,such as small interfering RNA(si RNA),microRNA(miRNA)and long chain non coding RNA(lncRNA)with a length of more than 200 nucleotides.Studies have confirmed that long-chain non-coding RNA plays an indispensable role in important activities in the life process of cells,such as epigenetic regulation,cell cycle control,transcriptional regulation,translation,post transcriptional shearing and cell differentiation.There is evidence that the changes of long-chain non-coding RNA and its expression level in cells may be closely related to tumor progression.It may play the role of proto oncogene or tumor suppressor gene in different cell environments,and then may promote or inhibit the process of tumor cell proliferation,invasion or metastasis.The length of microRNA is about 19 ～ 25 nucleotides.It affects gene expression through targeted binding with messenger RNA3 ’untranslated regions(3’-UTR).Specific miRNAs are highly enriched in some specific tissues,while they are low or even not expressed in other tissues.Regulate the physiological activities of cells,including cell proliferation,apoptosis and so on.Single nucleotide polymorphism(SNP)is a DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level.When the SNP on lncrna or microRNA occurs in the gene coding region or gene regulation region,it will have a great impact on gene expression and gene function.Competitive endogenous RNA(ce RNA)refers to the mutual regulation between RNAs through competitive binding of common microRNA response element(MRE),so as to affect the process of microRNA regulating downstream genes.Ce RNA has been found to include protein coding m RNA and non coding RNA,in which the latter includes pseudogene transcripts,long-chain non coding RNA,circular RNA(circ RNA),etc.There is evidence that long-chain non-coding RNA MALAT1 plays the role of protooncogene in lung cancer and can promote the occurrence and development of lung cancer,but the relationship between SNP polymorphism on MALAT1 and lung cancer is still unclear.miR-328-3p plays a role of tumor suppressor gene in lung cancer,but the relationship between SNP polymorphism on miR-328-3p and susceptibility to lung cancer has not been reported.This study investigated the relationship between the polymorphisms of lncRNA MALAT1 and mir-328-3p genes and lung cancer susceptibility,predicted the targeted binding relationship between MALAT1 and mir-328-3p,and YWHAZ was the downstream target gene regulated by mir-328-3p,and verified the competitive endogenous RNA mechanism of MALAT1,mir-328-3p and their target gene YWHAZ in the process of lung cancer.Methods: Part I: a case-control study was used to explore the relationship between the single nucleotide polymorphisms of rs619586 and rs3200401 on the long-chain non-coding RNA MALAT1 sequence and the susceptibility to lung cancer.Among them,444 cases of lung cancer and 460 healthy controls.The cases came from three class III first-class hospitals,the Fourth Affiliated Hospital of China Medical University,the General Hospital of the northern theater of the Chinese people’s Liberation Army and the First Affiliated Hospital of China Medical University,5ml venous blood was collected before treatment.The control group was selected from healthy people who underwent physical examination in the physical examination center in the same period.Genomic DNA was extracted from all blood samples by phenol chloroform method,and the genomic DNA of the subjects was SNP typed by Taqman probe.Student-t test and chi square test were used for the distribution difference between the two groups.Logistic regression model was used to estimate the relationship between different genotypes of different SNP sites and the risk of lung cancer.The interaction between SNP and smoking risk factors was investigated by cross-sectional analysis.The study was approved by the ethics committee.Part II: To explore the relationship between the single nucleotide polymorphism of rs12923138 in miR-328-3p sequence and the susceptibility to lung cancer.A hospital-based case-control study included 442 lung cancer cases and 502 controls.The cases came from three class III first-class hospitals,the Fourth Affiliated Hospital of China Medical University,the General Hospital of the northern theater of the Chinese people’s Liberation Army and the First Affiliated Hospital of China Medical University,5ml venous blood was collected before treatment.The control group was selected from healthy people who underwent physical examination in the physical examination center in the same period.Taqman probe was used for SNP typing of genomic DNA.Student-t test and chi square test were used to compare continuous variables and categorical variables between the two groups.Logistic regression model was used to estimate the relationship between different genotypes of different SNP sites and the risk of lung cancer.The interaction between SNP and smoking risk factors was investigated by cross-sectional analysis.The study was approved by the ethics committee.Part III: Firstly,the lung cancer related data in TCGA database were mined to analyze the expression and prognostic correlation of MALAT1,miR-328-3p and YWHAZ in lung cancer.Biological information websites(Targetscan,miRwalk,miRDB)were used to screen and verify the target gene of miR-328-3p.Map the mutual aid network of MALAT1,miR-328-3p and YWHAZ proteins.Secondly,to study the biological functions of MALAT1 and miR-328-3p in lung cancer cell lines.RT-q PCR was used to detect the expression of background MALAT1 and miR-328-3p in cell lines A549,H1299,SK-MES-1 and BEAS-2B.The small interfering RNA of MALAT1 was constructed and transfected with miR-328-3p mimics,inhibitor and NC plasmid.The effects of MALAT1 and miR-328-3p on the expression level of target gene MALAT1 protein were analyzed.Transwell experiment was used to detect the effects of MALAT1 and miR-328-3p on the migration and invasion of lung cancer cells.The effects of MALAT1 and miR-328-3p on cell proliferation were analyzed by MTS.The effects of MALAT1 and miR-328-3p on apoptosis were studied by flow cytometry.Double luciferase reporter gene was used to verify the targeted binding relationship between MALAT1 and miR-328-3p.Results: Part I: There was no significant association between MALAT1 rs619586 and rs3200401 polymorphisms and the risk of lung cancer.rs3200401 polymorphism is associated with susceptibility to non-small cell lung cancer.rs3200401 ct genotype carriers can reduce the risk of non-small cell lung cancer.In the pathological subtypes of lung adenocarcinoma and lung squamous cell carcinoma,the polymorphism of rs3200401 was significantly associated with the risk of lung squamous cell carcinoma,but there was no significant association with lung adenocarcinoma.In the interaction between genes and smoking exposure,the combination of additive model and multiplication model showed that the interaction between gene polymorphisms at rs619586 and rs3200401 and smoking exposure had no significant statistical significance on the risk of lung cancer.Part II: There was no significant association between miR-328-3p rs12923138 polymorphism and the risk of lung cancer.Stratified analysis showed that rs12923138 polymorphism was not significantly associated with the incidence of lung adenocarcinoma and lung squamous cell carcinoma.The results showed that there was a multiplicative interaction between rs12923138 AC + CC genotype and smoking exposure,which was statistically significant.Part III: The expression of MALAT1 was up-regulated in lung adenocarcinoma in TCGA database.The expression of miR-328-3p was down regulated in lung adenocarcinoma and lung squamous cell carcinoma.The expression of YWHAZ was significantly increased in lung adenocarcinoma and lung squamous cell carcinoma.The differential expression of YWHAZ was statistically correlated with the prognosis of patients with stage I and II lung squamous cell carcinoma.The informatics website predicts that YWHAZ is the downstream target gene of miR-328-3p.The protein mutual aid network diagram shows that MALAT1 has a mutual aid and cooperation relationship with miR-328-3p and YWHAZ through RPS9 and PIKFYVE.RT-q PCR showed that MALAT1 was highly expressed in lung cancer cell line H1299 and miR-328-3p was low expressed in lung cancer cell line H1299.The results of cell function experiments such as cell proliferation,cell migration and apoptosis showed that low expression of MALAT1 could inhibit the proliferation and migration of lung cancer cells,and up regulating the expression of miR-328-3p could significantly inhibit the proliferation and migration of lung cancer cells H1299 and sk-mes-1 and promote cancer cell apoptosis.The results of RT-q PCR and Western blot showed that YWHAZ was highly expressed in lung cancer cells H1299 and sk-mes-1.The protein expression level of YWHAZ was positively correlated with the expression of MALAT1 and negatively correlated with the expression of miR-328-3p.Conclusion: 1.There was no statistical significance between MALAT1 rs619586 and rs3200401 sites and the risk of lung cancer.MALAT1 rs3200401 is associated with susceptibility to lung squamous cell carcinoma.2.The association between mir-328-3p rs12923138 locus and the risk of lung cancer was not statistically significant.There was a multiplicative interaction between rs12923138 AC + CC genotype and smoking exposure,which was statistically significant.3.In TCGA database,MALAT1 was up-regulated in lung adenocarcinoma,miR-328-3p was down regulated in lung adenocarcinoma and lung squamous cell carcinoma,and YWHAZ was significantly increased in lung adenocarcinoma and lung squamous cell carcinoma.4.YWHAZ differential expression was statistically correlated with the prognosis of patients with stage I and II lung squamous cell carcinoma.5.MALAT1 can inhibit the proliferation and migration of lung cancer cells miR-328-3p can inhibit the proliferation and migration of lung cancer cells and promote apoptosis.6.YWHAZ is the downstream regulation target gene of miR-328-3p.The protein expression level of YWHAZ is positively correlated with the expression of MALAT1 and negatively correlated with the expression of miR-328-3p.miR-328-3p has a competitive endogenous RNA mechanism with MALAT1 and YWHAZ.