| BackgroundTuberculosis(TB)is one of the top ten causes of human death,and millions of people are infected with it every year.Although the success rate of tuberculosis treatment is high and millions of deaths can be avoided every year,tuberculosis is also one of the serious infectious diseases that endanger human physical in this century.The epidemic of tuberculosis in the world has not been well controlled,on the contrary,it has become worse.The increase in immigration and the number of people infected with the human immunodeficiency virus(HIV)and TB have contributed to the TB epidemic,making it more difficult to control.Epidemiological studies have shown that tuberculosis has become a public health problem that threatens human health,with pulmonary tuberculosis(PTB)suffering from the largest proportion of patients.At present,TB detection methods mainly use sputum smears or culture to find Mycobacterium tuberculosis(MTB).The quality of the material directly affects the test results.There is much limitation in these methods.Moreover,the MTB has a longer growth cycle which takes a long time to make a clear diagnosis.In the early stages of a patient's infection with TB,doctors can only take empirical treatment.Therefore,in order to effectively control the TB epidemic,a timely and effective diagnostic method is urgently required.Long non-coding RNAs(lncRNAs),which is defined as transcripts longer than 200 nucleotides without protein-coding capacity,are characterized by high tissue specificity and low sequence conservation.In recent years,more and more evidence show that lncRNAs regulate gene expression through chromatin remodeling,transcriptional regulation,post-transcriptional processing and other methods to regulate various biological processes and thus participate in the occurrence and development of diseases.In cancer and metabolic diseases,Immunodeficiency disease and other diseases,it has received extensive attention.lncRNAs are involved in cell-mediated immunological mechanisms and play an important role in the tuberculosis immune protection system;at the same time,they are also involved in mediating apoptosis and autophagy of macrophages in vivo when infected with Mycobacterium tuberculosis.In the study of sputum and plasma samples to diagnose active PTB and obsolete PTB,it was found that lncRNAs have the potential to become biomarkers.However,at present,no maturely clinical biomarkers for diagnosing tuberculosis have appeared.ObjectiveTo lncRNAs in peripheral blood plasma between patients with active pulmonary tuberculosis and healthy people,and to explore its clinical application value.Materials and MethodData Mining:In order to identify the differential lncRNAs between active TB and healthy control(HC),We reanalyze the GEO dataset(GSE94907)to compare two groups of data of HC(GSM2491733,GSM2491734)and active TB(GSM2491737,GSM2491738),screen for significantly differentially expressed lncRNA(FC?2,q?0.05)and draw heat map,volcano plot,etc.In order to screen for IncRNAs that can be used for clinical detection and diagnosis,we picked FKPM?5 and localized lncRNAs on autosomes.As an important database of non-coding RNA,NONCODE provides the expression of lncRNA in the peripheral blood exosomes of active tuberculosis and healthy people.We further confirmed the selected lncRNAs in the NONCODE database and obtained IncRNAs consistent with the trend of NONCODE data.Experimental group and normal control group:In this experiment,a total of 69 patients with pulmonary tuberculosis in Shandong Chest Hospital from June 2018 to November 2019 were collected as the experimental group(active PTB),including primary and recurrent active PTB,aged between 15 and 79 years old.69 healthy donor plasma samples were taken from the Second Hospital of Shandong University as the control group(HC),aged between 17 and 69 years.According to the diagnostic criteria for tuberculosis in the "Internal Medicine"(8th edition)of People's Medical Publishing House.All patients with active PTB were diagnosed according to clinical manifestations and the results of auxiliary examinations such as typical chest CT,acid-fast staining of Mycobacterium tuberculosis in sputum or bronchoalveolar lavage fluid,nucleic acid detection,and tuberculosis antibodies.The HC group and the experimental group had no previous underlying diseases such as diabetes,hypertension,immunodeficiency diseases,and tumors.There was no significant difference in age and gender between the experimental group and the control group.lncRNAs sample extraction in plasma:138 samples of EDTA-K2 anticoagulated plasma were extracted,including 69 patients and 69 healthy specimens.All plasma samples were extracted with total RNA using viral RNA extraction kit(Tiangen).The RNA concentration was measured with an ultra-differential spectrophotometer(Tiangen)and stored at-80?.Extraction of lncRNAs in PBMC:Extract PBMC cells from EDTA-K2 anticoagulated whole blood,extract RNA by blood RNA extraction kit(day root),measure the RNA concentration with ultra-differential photometer(day root),and store at-80?.After RNA extraction,PrimeScriptTMRT Master Mix kit(Takara,China)was used to generate 500 ng RNA samples by reverse transcription PCR(RT-PCR)to generate complementary DNA(cDNA),and at-20? save.ABI-7500 was used to run the Real-time Quantitative PCR(qPCR)program to determine the expression of lncRNAs,SYBR Premix Ex TaqTM(Tli RNaseH Plus)(Takara,China)is the reaction system.SYBR as a fluorescent dye,and GAPDH gene as an internal reference.As measured,the expression level of lncRNAs was calculated using the following method.?Ct=Ct-CtGPADH??Ct=?CtPTB-?CtHCMeasurement data using independent sample T test by using SPSS 19.0 statistical analysis software,?2 was used for counting data.Results841 lncRNAs were downregulated in ATB samples,while 351 lncRNAs were upregulated.Among them,111 significantly under-expressed lncRNAs and 1 up-regulated lncRN A were localized on autosomes with FKPM>5.By confirming with the NONCODE database information,we obtained 9 lncRNAs consistent with the change trend ofNONCODE data:NONHSAT004992.2?NONHSAT036869.2?NONHSAT006652.2?NONHSAT078957.2?NONHSAT101518.2?NONHSAT148822.1?NONHSAT131809.2?NONHSAT067134.2?NONHSAT257478.1?Compared with the content of lncRNAs in plasma of HC group and PTB patients by qPCR:the difference of four down-regulated lncRNAs molecules(NONHSAT101518.2,NONHSAT067134.2,NONHSAT148822.1,NONHSAT078957.2)P?0.05,were statistically significant.According to qPCR detection,the content of lncRNAs in PBMC of HC group and PTB patients were compared:2 lncRNAs molecules:NONHSAT078957.2(t=4.836,p?0.001)and NONHSAT101518.2(t=6.969,p?0.0001)were significantly down-regulated.Draw the receiver operating characteristic curve(ROC curve)of the above 4 molecules.The sensitivity of NONHSAT101518.2 is 0.9502(95%CI,0.9153-0.9852);NONHSAT148822.1(0.7080,95%CI,0.6207-0.7954);NONHSAT078957.2(0.8994,95%CI,0.8443-0.9545);NONHSAT067134(0.8725,95%CI,0.8064-0.9386).ConclusionThe expression of lncRNAs in active PTB and healthy people of plasma and PBMCs has certain statistical differences,which has certain guiding significance for the timely and accurate diagnosis of active tuberculosis,and is expected to become a potential molecular biological marker for rapid diagnosis of tuberculosis. |
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