MiR-146b Sponged By LncRNA-MALAT1 In THP1 Cells Regulates Inflammatory Responses Induced By Cryptococcus Neoformans

Objective:Cryptococcus is a kind of pathogenic fungi in the environment,which is common among the patients with immune deficiency.It is one of the three major causes of deep fungal infections in epidemiological studies.The most common type of cryptococcal disease is cryptococcal meningitis,which is often caused by Cryptococcus neoformans.It is usually insidious in onset and rapid development.In addition,therapeutic options are limited with major side effects so far.That’s the reason why we need to look for a novel method of early diagnosis and treatment.As a famous non-coding RNA,miRNA has been investigated the function of regulating gene expression since the 1990s.Although monocytes are important components of innate immune system,finding out the mechanism of the role of miRNA playing in regulating inflammation and immunity is very important.Recently,there is a growing interest in the relationship between lncRNA and miRNA.This study is about lncRNA-MALAT1 as a sponge to sequester miR-146b,which negatively regulates TRAF6/p-Akt pathway,which may provide insights into a new approach of earlier diagnosis and less-toxic effects.Methods:In the first step,we culture the THP-1 cells and Cryptococcus neoformans(WM148).The THP-1 cells are converted to macrophages with PMA.There are six groups infected THP-1 cells with WM148(0h,3h,6h,12 h,24h,48h).0h is setting as the control group.The cell pellets and supernatant are collected at the corresponding time point.After the total RNAs are extracted and reverse transcription is finished,real-time fluorescence quantitative PCR(qRT-PCR)are used to detect the expression changes of TNF-αand IL-6.Using ELISA to detect expression changes in TNF-αand IL-6,which can find that the best time point for the most obvious response of cryptococcal infection in THP-1 cells.The second part is about the regulation of the TRAF6/p-Akt signaling pathway by mir-146b.1,The relationship between mir-146b and TNF receptor associated factor 6(TRAF6)is detected by luciferase assays.2,qRT-PCR is used to verify the suppressive effects of miR-146b to TRAF6 and MMP9(the downstream gene of TRAF6/ p-Akt signaling pathway).And then,we add miR-146b mimic and inhibitor to test the result.3,The relationship between miR-146b and TRAF6 and MMP9 is verified by western blotting technique,and then use miR-146b mimic and inhibitor to test the result again.In the third part,we verify the MALAT1 suppressive effect of miR-146b with qRT-PCR and western blotting technique.Similarly,we silence MALAT1 and test the result again.Results:In the first part: the PCR result shows that TNF-α expression increased after the WM148 induced,and reach the peak in 6h group(P < 0.05),and then decreased in 12 h,24h and 48 h group.The trend of IL-6 is the same as TNF-α,with the highest expression level in 6h group,but the elevated level is not statistically significant.However,the ELISA test finds that TNF-αdecreased after rapid increase,and the expression level is highest at 6h(P < 0.01).The expression trend of IL-6 is consistent with that of TNF-α(a "inverted V" trend),but there is a large difference in expression between different batches,so the elevated level is not statistically significant(P>0.05).In the second part: 1,hsa-miR-146b-5p can reduced the activity of TRAF6 3 ’UTR region.The mutant TRAF6’s 3 ’UTR activity is higher than the wild-type TRAF6’s(P<0.01),indicating that the mutation site is the seed region of mir-146b.2,PCR result shows that the expression of TRAF6 and MMP9 is increased in the experimental group.The increase of TRAF6 and MMP9 in the miR-146b mimic group is inhibited,while in the miR-146b inhibitor group increased significantly(P < 0.01).3,In the western blotting experiment,the concentration of TRAF6 increased in the experimental group,decreased in the miR-146b mimic group,and significantly increased in the miR-146b inhibitor group.In the third part: In the PCR results,the expression of lncRNA-MALAT1 increases in the experimental group while it is obviously inhibited in the Smart Silencer group.The expression of miR-146b is increased,while the increase of TRAF6 and MMP9 is inhibited.The western blotting results show that the expression of TRAF6 in the experimental group was higher than that in the control group,and the increase could be inhibited in the Smart Silencer group.Conclusions:The expression of TRAF6 and MMP9 is increased when the THP-1 cells are infected with Cryptococcus neoformans,which results in the development of infection and the destruction of the blood-brain barrier.miR-146b can inhibit the expression of TRAF6.That’s the reason why it can negatively regulate the TRAF6/ p-Akt signaling pathway as well as reducing the expression of a downstream target MMP9.The increased expression of lncRNA-MALAT1 which act as sponges and inhibit miR-146b activity in cryptococcal infection may be the mechanism for the occurrence of cryptococcal meningitis.