Differential Expression And Clinical Significance Of Serum LncRNA NEAT1 In Patients With Pulmonary Tuberculosis And Patients With Liver Injury During Anti-tuberculosis Treatment

Research background and purpose:Tuberculosis(TB)is a chronic infectious disease caused by the entry of mycobacterium tuberculosis(MTB)into the respiratory tract.After entering the human body,mycobacterium tuberculosis can accumulate damage to multiple organs of the whole body,with the lung as the main affected organ,which seriously threatens human life and health.Therefore,tuberculosis is one of the important causes of human death due to infectious diseases worldwide.At present,the diagnostic methods of tuberculosis in clinical work are still very limited,mainly including sputum culture,sputum smear examination,tuberculin test,bronchoscopy,imaging examination,detection of mycobacterium tuberculosis DNA,RNA and pathological biopsy.However,the positive rate of a sputum smear is low,and a negative result does not rule out tuberculosis infection.Mycobacterium tuberculosis culture usually takes 6-8 weeks,which is limited in real-world application.Bronchoscopy and pathological biopsy are invasive examinations,and pathological biopsy is an invasive operation with low patient acceptance,so it is not used as a routine examination method.The nucleic acid test of mycobacterium tuberculosis can be affected by the operation of PCR,and the results can not be used as a basis for diagnosis independently.Therefore,it is necessary to find a simple,specific,sensitive,less invasive and shorter detection method.Long non-coding RNA(lnc RNA)is a class of non-coding RNA molecules with a length of more than 200 bases.Some researchers have found that lncrnas may be related to the occurrence,development and prognosis of tuberculosis.Lnc RNAs are differentially expressed in CD4~+T cells of patients infected with mycobacterium tuberculosis,suggesting a potential role of lnc RNAs in the diagnosis,evaluation of treatment effect and prognosis of tuberculosis.Therefore,this study analyzed the differential expression of lnc RNA in PBMC of tuberculosis patients to determine its diagnostic value in tuberculosis.At present,the first-line drugs for tuberculosis treatment are mainly composed of isoniazid(INH),ethambutol(EMB),pyrazinamide(PZA)and rifampicin(RFP).These drugs have a certain degree of damage to the liver,and the damage to the liver is more obvious when they are used in combination.According to the current short-term treatment regimen recommended by the World Health Organization,some patients have not fully achieved clinical cure,so they have to prolong the treatment time,leading to a variety of adverse reactions,among which drug-in-duced liver injury(DILI)is the most serious adverse reaction.It seriously damages the organ health of patients and leads to the interruption of anti-tuberculosis treatment,which is extremely unfavorable to the life and health development of patients.In current clinical work,glutamic oxaloacetic transaminase(AST)and alanine aminotransferase(ALT)are often used as indicators to evaluate liver function.However,when AST and ALT are elevated,liver function is often irreversibly damaged.Therefore,it is necessary to find a biomarker with higher specificity and sensitivity,intervene in time before irreversible liver damage occurs,and protect the life and health of patients to the greatest extent.Lnc RNA nuclear enriched transcript1(NEAT1)is a kind of long non-coding RNA.Studies have found that lnc RNA NEAT1 is expressed in many diseases,especially in inflammatory and neoplastic diseases.Previous researchers have found that lnc RNA NEAT1 is up-regulated in peripheral blood monocytes of patients with viral hepatitis,cirrhosis and primary liver cancer,and the degree of gene up-regulation is related to the severity of the disease.However,there is no study on the relationship between the differential expression of lnc RNA NEAT1 in PBMC of tuberculosis patients and anti-tuberculosis drug-induced liver injury or its diagnostic value.Therefore,this study analyzed the relationship between the differential expression of lnc RNA NEAT1 in PBMC and liver function in patients with tuberculosis during treatment,and evaluated whether it has diagnostic value.In summary,this study was divided into two parts.The first part was to study the differential expression and diagnostic value of lnc RNA NEAT1 in PBMC of pulmonary tuberculosis patients.The second part is the correlation between the differential expression of lnc RNA NEAT1 and liver damage during anti-tuberculosis treatment and its diagnostic value.It provides clues for the diagnosis,condition evaluation,prognosis of tuberculosis,and the diagnosis,treatment and prevention of hepatocellular injury during anti-tuberculosis treatment in the future.Materials and Methods:Specimens were collected between January 2022 and January 2023.The subjects included healthy people,newly diagnosed TB patients without treatment,TB patients with regular anti-TB treatment.Thirty-three healthy people were randomly selected from the health examination Center of the First Affiliated Hospital of Chengdu Medical College,including 19 males(57.58%)and 14 females(42.42%),with an average age of(52.36±18.20)years.A total of 33 patients with newly diagnosed and untreated pulmonary tuberculosis were recruited from the tuberculosis clinic of the First Affiliated Hospital of Chengdu Medical College,including 19 males(57.58%)and 14 females(42.42%),with an average age of(48.06±12.28)years.A total of 100 patients received regular anti-tuberculosis treatment,including 69 males(69%)and 31 females(31%),with an average age of(45.33±17.68)years.The age of the subjects was between 18 and80 years old.The subjects were selected by medical history,physical examination and outpatient follow-up.According to the 2019 Guidelines for the diagnosis and treatment of anti-tuberculosis drug-induced liver Injury,liver injury during anti-tuberculosis drug-induced treatment generally occurs within 1 week to 3 months after medication,and the peak period mostly occurs about 2 months after medication.Therefore,we chose to collect peripheral blood from patients with active tuberculosis who received regular anti-tuberculosis treatment(2 months±10 days)for this study.A total of 100patients with active pulmonary tuberculosis were regularly treated with anti-tuberculosis drugs(2 months±10 days),and their liver function indexes were detected(the normal range of liver function indexes was referred to the standard of the laboratory Department of the First Affiliated Hospital of Chengdu Medical College:ALT reference value:9-60U/L;AST reference value:15-45U/L).The active pulmonary tuberculosis patients were divided into liver injury group and non-liver injury group according to whether the liver function index was in the normal range.Fasting peripheral blood samples were collected from healthy people and untreated patients at the time of enrollment,and blood samples were collected at 2±10 days of treatment for patients with regular anti-tuberculosis treatment.RNA was extracted from peripheral blood by using Fast King c DNA first-strand synthesis reagent produced by Tiangen company,and then c DNA was finally obtained by reverse transcription.The expression of lnc RNA NEAT1 in peripheral blood mononuclear cells(PBMC)was determined by real-time quantitative PCR(RT-PCR).In this experiment,the measurement data conforming to normal distribution were tested by t test and expressed as mean±standard deviation(x±s).Non-normal distribution data were analyzed by rank sum test and expressed as median M(Q25,Q75).Count data were analyzed by chi-square x~2test and expressed as[n(%)].When P<0.05,the difference was statistically significant.To investigate the expression difference of lnc RNA NEAT1between healthy people and newly diagnosed untreated pulmonary tuberculosis patients in a cross-sectional study,and to evaluate whether lnc RNA NEAT1 expression is associated with Mycobacterium tuberculosis infection.Then,a comparative study was conducted between tuberculosis patients with liver injury and those without liver injury who were treated for the same time(2 months±10 days).To investigate the correlation between the expression of lnc RNA NEAT1 and liver damage in the process of anti-tuberculosis treatment,and to analyze the correlation between the expression of lnc RNA NEAT1 in peripheral blood and liver function in patients with liver injury.The value of Lnc RNA NEAT1 expression in the diagnosis of tuberculosis and the diagnosis of anti-tuberculosis drug-induced liver injury was expressed by the area under the ROC curve(AUC),and its sensitivity and specificity were analyzed to determine its value in clinical application.Result:1.To compare the expression of lnc RNA NEAT1 in peripheral blood monocytes between healthy people and newly diagnosed untreated pulmonary tuberculosis patients.The median of lnc RNA NEAT1 in healthy people was 0.487(0.185,0.809),and the median of lnc RNA NEAT1 in newly diagnosed untreated pulmonary tuberculosis patients was 1.41(0.934,1.776),z=5.495,(P=0.01),suggesting that there was a significant difference between the two groups.These results suggest that the up-regulation of lnc RNA NEAT1 in peripheral blood may be related to mycobacterium tuberculosis infection.The expression of lnc RNA NEAT1 in the peripheral blood of newly diagnosed untreated tuberculosis patients was significantly higher than that of healthy people.2.Comparison of lnc RNA NEAT1 expression between newly diagnosed untreated pulmonary tuberculosis patients and patients without liver injury after anti-tuberculosis treatment(2 months±10 days).The median value of lnc RNA NEAT1 in newly diagnosed untreated pulmonary tuberculosis patients was 1.41(0.934,1.776),and that in patients without liver injury after anti-tuberculosis treatment(2 months±10 days)was0.62(0.27,1.00),z=5.056,(P=0.01).The expression of lnc RNA NEAT1 in peripheral blood of patients after treatment was lower than that of untreated patients,which again suggested that the expression of lnc RNA NEAT1 in peripheral blood of patients was correlated with mycobacterium tuberculosis infection.During regular anti-tuberculosis treatment,the number of mycobacterium tuberculosis in patients is gradually decreased,and the expression of lnc RNA NEAT1 is down-regulated.3.To compare the expression of lnc RNA NEAT1 in peripheral blood monocytes between patients without and with liver injury during regular anti-tuberculosis drug treatment(2 months±10 days).The median value of lnc RNA NEAT1 in patients without liver injury was 0.62(0.27,1.00),and that in patients with liver injury was1.65(1.05,2.18),z=5.856,(P=0.001).There was a significant difference between the two groups(P<0.05).The expression of lnc RNA NEAT1 in peripheral blood of patients with liver injury is significantly higher than that of patients without liver injury.4.The receiver operating characteristic(ROC)curve,sensitivity and specificity were used to determine whether the expression of lnc RNA NEAT1 in peripheral blood had diagnostic value for mycobacterium tuberculosis infection.The area under the ROC curve was 0.893,which was greater than 0.7,and the sensitivity and specificity were0.939 and 0.758,respectively.It is suggested that the expression of lnc RNA NEAT1 in peripheral blood monocytes has a certain diagnostic value for tuberculosis with a high sensitivity.5.The receiver operating characteristic(ROC)curve and sensitivity and specificity analysis were used to determine whether the expression of lnc RNA NEAT1 in peripheral blood has a certain diagnostic value for liver injury in patients with anti-tuberculosis treatment.The area under the ROC curve was 0.845,which was greater than 0.7,and the sensitivity and specificity were 0.780 and 0.814,respectively.6.By Spearman correlation analysis,the relative expression of lnc RNA NEAT1 in PBMC was positively correlated with ALT and AST(ρ=0.484,0.532,both P<0.05).7.To compare the occurrence of liver function damage in patients with active pulmonary tuberculosis during treatment.Logistic regression results showed that lnc RNA NEAT1 was significantly associated with the risk of anti-tuberculosis drug-induced liver injury(OR=9.98,95%CI:3.83-26.00,P<0.01),while gender,age,smoking,drinking and anti-tuberculosis drug-induced liver injury were not statistically significant(P>0.05).Conclusions:1.Up-regulation of Lnc RNA NEAT1 in peripheral blood monocytes is associated with Mycobacterium tuberculosis infection in patients.The expression of lnc RNA NEAT1 in monocytes of tuberculosis patients was significantly higher than that of healthy people.2.The expression of lnc RNA NEAT1 in peripheral blood of tuberculosis patients with regular drug anti-tuberculosis treatment is lower than that of untreated tuberculosis patients.3.The up-regulation of lnc RNA NEAT1 in peripheral blood monocytes is associated with liver function impairment in tuberculosis patients during regular anti-tuberculosis treatment.The correlation between ALT and AST showed a positive correlation.4.Lnc RNA NEAT1 is an independent related factor for anti-tuberculosis drug-induced liver injury.5.The expression of lnc RNA NEAT1 in peripheral blood of patients has a certain diagnostic value for the diagnosis of tuberculosis and liver function damage during anti-tuberculosis treatment.The area under the ROC curve was greater than 0.7,which had relatively high sensitivity and specificity.It is expected to be used as a biomarker in the future.