Correlation Of MALAT1 In Risk Factors Of Gallstone Disease And Its Biological Significance In Gallbladder Mucosal Hyperplasia To Malignant Transformation

Objective: Gallstone disease(GD)is a common and frequently-occurring disease in hepatobiliary surgery.In China,with the change of lifestyle,the incidence of GD has gradually increased,especially in people over 50 years old.With the formation of gallstones,concurrent GD can lead to serious complications such as fatal cholangitis and acute pancreatitis.GD risk factors can be divided into environmental risk factors,population risk factors and genetic risk factors.However,most risk factors have been reported to vary among populations in different countries and regions.Long noncoding RNAs(lnc RNAs)are noncoding RNAs about 200 nucleotides in length.RNA polymerase II is involved in the transcription of most known lnc RNAs.These highly conserved and ubiquitous transcripts play important biological regulatory roles in many diseases.However,the correlation between lnc RNAs and the evolution of gallbladder precancerous lesions to gallbladder carcinoma in situ and cholelithiasis has not been reported at home and abroad.After reviewing and analyzing relevant literature on risk factors for cholelithiasis,the following lnc RNAs that may be associated with cholelithiasis were screened and detected: including H19(H19 Imprinted Maternally Expressed Transcript),MEG3(Maternally Expressed 3),GAS5(Growth Arrest Specific 5),uc.333,NEAT1(Nuclear Paraspeckle Transcript 1)and MALAT1(Metastasis Associated Lung Adenocarcinoma Transcript 1).The differential expression of the above candidate lnc RNAs in the serum of GD patients and healthy controls was compared and analyzed;the downstream target genes of MALAT1 were predicted and verified using GEO and TCGA RNA sequencing data and STARBASE database.And the function of MALAT1 and its downstream target genes in tumors was analyzed by bioinformatics;at the same time,the m RNA expression levels of MALAT1 and its downstream target genes and their correlation in gallbladder hyperplasia tissue and gallbladder carcinoma in situ tissue were verified and analyzed;The effect of MALAT1 and its downstream target genes on the proliferation and invasion of gallbladder cancer cells was analyzed by level validation.To clarify and verify the relationship between the risk factors of cholelithiasis and MALAT1;to describe the correlation and biological significance of MALAT1 and its regulated target genes with the evolution of gallbladder mucosal epithelial hyperplasia to gallbladder cancer.The aim of this study was to further explore the relationship between cholelithiasis and gallbladder cancer and its impact on the biological behavior of gallbladder cancer from the perspective of lnc RNA MALAT1.Methods:1.Case collection and data analysis: 3202 age and sex-matched eligible participants were recruited from January 2008 to July 2018 in Nancheng Hospital,Dongguan City,Guangdong Province to form the study population.The population was divided into GD group(patients with gallstones,N=1134)and healthy control group(N=2068).At the same time,257 tissue samples with complete clinical data were collected from pathological diagnosis of gallbladder mucosal epithelial hyperplasia,atypical hyperplasia and gallbladder carcinoma in situ.Demographic information includes age,gender,ethnicity,occupation and education.Clinical data included familial inheritance,alcohol exposure,smoking exposure,physical activity,body mass index(BMI),diastolic blood pressure,systolic blood pressure,alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride Esters(TG),Total Cholesterol(TC),Low Density Lipoprotein Cholesterol(LDL-C)and High Density Lipoprotein Cholesterol(HDL-C).2.Detection and analysis of lnc RNAs: Quantitative polymerase chain reaction(q PCR)was used to detect the expression levels of lnc RNA H19,MEG3,GAS5,uc.333,NEAT1 and MALAT1 in GD group and healthy control group.The correlation between GD and risk factors was estimated by logistic regression model.By detecting MALAT1 expression levels in serum samples of relevant cases at different stages during the evolution of gallbladder mucosal epithelial hyperplasia to canceration,the diagnostic value was determined by receiver operating characteristic(ROC)curve.3.Bioinformatics analysis: Using R language,analyze the m RNA transcriptome data of gallbladder pathological tissue of patients with gallstone.From the GEO database gallbladder RNA-seq dataset was downloaded,data number: GSE152419.The STARBASE database was used to predict the protein coding genes downstream of the lnc RNA,and the intersection of the difference analysis results of the above databases was used to obtain the corresponding downstream protein coding genes.The TCGA database was used for subsequent prediction of the function and correlation analysis of proteincoding genes downstream of lnc RNAs.The main analyses included: differential analysis,GSEA enrichment analysis,KEGG analysis,etc.4.Use lentivirus to construct MALAT1 interference vector(sh-MALAT),KPNA2 gene interference plasmid(sh-KPNA2)and overexpression plasmid(KPNA2-OE).The transfection efficiency was detected by q PCR;the protein expression was detected by Western blot;the changes of cell proliferation were detected by CCK-8 method,and the migration and invasion abilities of cells were detected by Transwell chamber.Results:1.Analysis of high-risk factors for the formation of GD: Multivariate Logistic regression analysis: BMI,TG,HDL-C were high-risk factors for the formation of GD,and moderate and high-intensity physical activity was a protective factor.2.GD formation was correlated with serum lnc RNA H19,MEG3,GAS5,uc.333,NEAT1 and MALAT1 expression: MALAT1 was significantly correlated with GD,while lnc RNA H19,MEG3,GAS5,uc.333,NEAT1 had no correlation with GD.obvious.Combined analysis showed that high expression of MALAT1 was an independent risk factor for GD,and there was a significant interaction product between high expression of MALAT1 and low HDL-C in GD.3.MALAT1 expression in serum of patients with gallbladder mucosal epithelial hyperplasia,dysplasia or carcinoma in situ: Compared with simple hyperplasia of gallbladder mucosal epithelium,MALAT1 in patients with grade II or III dysplasia or carcinoma in situ was significantly increased.There was no significant difference between patients with grade I dysplasia and epithelial simple hyperplasia.With the aggravation of mucosal hyperplasia pathological lesions,the expression of MALAT1 gradually increased,and the expression of MALAT1 was the highest in carcinoma in situ,indicating that MALAT1 was significantly related to the pathological process of atypical hyperplasia of gallbladder epithelium to cancer.4.Using the m RNA transcriptome data of gallbladder pathological tissue of patients with gallstone for GSEA enrichment analysis,GD differential genes are mainly enriched in lipid metabolism and cell proliferation-related pathways;STARBASE prediction combined with tissue samples to verify that KPNA2(Karyopherin Subunit Alpha 2)may be a downstream target gene of MALAT1.5.Use the GEO dataset GSE152419 to analyze the correlation between KPNA family genes and lipid metabolism,cell cycle-related genes,KPNA family genes and lipid metabolism-related genes such as LPIN1(),ACSL4(),MYC(),TGFB2()There is a significant correlation between the expression levels of other cell proliferation-related genes.6.Using the RNA sequencing data of 33 solid tumors in the TCGA database,pan-cancer analysis of KPNA2 and lipid metabolism,cell cycle,tumor grade and functional enrichment,KPNA2 and FASN(),ACLY(),FADS1(),etc.The expression of genes regulating lipid metabolism has a significant correlation;KPNA2 is also significantly correlated with the expression of CDC45,CDC7,CDC6 and other genes involved in regulating the cell cycle;KPNA2 expression is significantly different in different stages of malignant tumors;KPNA2 is mainly enriched in MYC TARGET V1,E2F()TARGET and G2 M CHECKPOINT and other cell proliferation-related gene sets and MTOR1()signaling pathway.7.Knockdown of MALAT1 in gallbladder cancer cells(GBC-SD)found that the expression of KPNA2 protein decreased,and the cell proliferation,migration and invasion ability decreased;silencing KPNA2,the proliferation,migration and invasion ability of gallbladder cancer cells decreased;the recovery experiment showed that overexpression of KPNA2,Changes in proliferation,migration and invasion of gallbladder cancer cells partially reversed by knockdown of MALAT1.Conclusions:1.For the first time,it is clear that MALAT1 is an independent risk factor for the occurrence of GD,and the high expression of MALAT1 and low HDL-C have an interactive product effect on the occurrence of GD;2.The serum levels of patients with simple hyperplasia of the gallbladder,atypical hyperplasia of the mucosa,and carcinoma in situ of patients with different mucosal lesion stages.The expression level of MALAT1 increased gradually;the serum MALAT1 expression increased significantly in severe dysplasia and carcinoma in situ;3.The expression levels of MALAT1 and KPNA2 in gallbladder carcinoma in situ were significantly positively correlated.student credit score.Analysis predicts that the two have mutual binding sequences and play an interaction.4.Bioinformatics analysis showed that MALAT1 target gene KPNA2 is involved in lipid metabolism and cell proliferation regulation.5.It is proved for the first time that MALAT1 positively regulates the expression of KPNA2 in gallbladder cancer cell line,and promotes the proliferation,migration and invasion of gallbladder cancer cells.The above research results interpret the relationship between GD and gallbladder cancer from the dimension of lnc RNAs,and provide important biological information for the prevention and treatment of gallbladder cancer and cholelithiasis.MALAT1 is a potential serum biomarker for gallbladder precancerous lesions and early-stage gallbladder cancer.