Modulatory Effect And Clinical Significance Of Long Non-coding RNA HPX206 Mediating Immune Infiltration Of Hepatic Alveolar Echinococcosis

Background:Echinococcus muitilocularis infection can cause Alveolar Echinococcus（AE）,which is a wide spread zoonotic parasitic disease with a long history.Alveolar echinococcosis is a life-threat disease due to the infiltration growth pattern to liver and adjacent organs.Studies on exploring the interaction mechanism between Echinococcus multilocularis,human immune system and hepatocytes at the molecular level could help to further study the pathogenesis of alveolar echinococcosis and develop specific anti-parasite drugs or vaccines.However,there is a lack of researches on long no-coding RNA（Lnc RNA）related to immune infiltration of alveolar echinococcosis.In this study,we established an animal model of Echinococcus multilocularis infection in thymectomized BALB/c mice and explore the growth and proliferation feasibility of the second generation of Echinococcus multilocularis in normal mice and immunodeficient mice.Search for the differential Lnc RNA expression profiles between the para-lesion liver tissue and normal liver tissue in AE patients.Explore the mechanism and clinical significance of Lnc RNA-HPX206 in mediating immune infiltration in AE via co-culturing Echinococcus multilocularis with human hepatocytes and monocytes in vitro.Materials and Methods:i)Echinococcus multilocularis were extracted and counted from infected Meriones unguiculatus annimal model.Then inject Echinococcus multilocularis protoscolex suspension into the abdominal cavity of 10 adult BALB/c mice with0.2ml per mouse.Two mice were killed at 21 days,1 month,2 months,3 months and4 months after inoculation to measure the Ig G antibody of Echinococcus multilocularis and measured the size,diameter,number,local invasion and Echinococcus multilocularis counts of lesions.ii)Twenty-four adult BALB/c mice were randomly transformed into experimental group and pseudo-opereations groups.Beyond anesthesia,objects in the experimental group were treated with endotracheal intubation and ventilator assisted ventilation.With the assistance of microscope,both sides thymus was excised,while in the sham operation group,only open the thorax but no thymectomy.Two weeks later,the mice were inoculated with Echinococcus multilocularis protoscolex.iii)Echinococcus multilocularis protoscolex were extracted and injected into the abdominal cavity of thymectomized mice and control mice from Echinococcus multilocularis infected BALB/c animal model established in step 1.The lesion were observed by ultrasound at 1,2 and 3 months after inoculation.At 4^thweek,all mice were sacrificed and their blood was collected to detect the inflammatory factors and measured the size,diameter,number,local invasion and Echinococcus multilocularis counts of lesions in the two groups.iv)Ten liver samples from 5 AE patients were divided into experimental group（1cm near to lesion）and control group（5-7cm distant to lesion）.Candidate Lnc RNA of two groups were explored by high-throughput sequencing and verified by RT-PCR.The correlation between the clinical indexes of AE patients and Candidate Lnc RNA was analyzed.GO enrichment analysis and KEGG function analysis were used to predict the corresponding target pathways.Mi RNAs with complementary sequences were found by mircode,lnceding and Diana lncbase,and the corresponding target genes were predicted by targetscan and mi RBasev)Protoscolex was extracted and purified from infected BALB/c mice.And protoscolex was co-cultured with human normal hepatocytes LX-2 and monocytes THP-1 in groups in vitro.In one group,si RNA of candidate Lnc RNA was added to silence the expression of candidate Lnc RNA in THP-1.The proliferation activity of hepatocytes was dettected by CCK8 test,and the migration abilities of hepatocytes were detected via scratch tests,the apoptoisis of hepatocytes and the deformabilities of hepatocytes were detected by TUNEL tests and Transwell tests.vi)The expression of candidate Lnc RNA and Caspase 3 in same specimen was det_tected by RT-PCR and Immunohistochemistry（IHC）,and the correlation between the expression of Lnc RNA and Caspase 3 was compared by Pearson test.vii)Echinococcus multilocularis was co-cultured with LX-2 and THP-1 for 48hours.THP-1 was isolated by flow cytometry and dettected the key markers of TGF-βpathway by Western blotting.viii)Double luciferase assay was used to verify the correlation between candidated Lnc RNA and micro RNAs.Reverse transecription PCRs as well as Westernn bloting test were both used to verify whether the candidated Lnc RNA and micro RNA formed a network and affected the proteins’expressions.Continuous variables were displayed as mean±SD.Student’s t test was applied for comparing the statistical differences between groups in continuous variables.Chi square test was applied for countable data.Wilcoxon test was adapted for skewed-distribution data.Correlation analysis use Pearson correlation analysis.All statistical analysis was in SPSS software,version 20.0（IBM Corporation,Armonk,NY,USA）and the R software,version 4.0.0（the R Foundation）.Results:i)About 14000 living protoscolex were obtained from infected Meriones unguiculatus annimal model.The infection rate of 10 BALB/c mice was 100%.The diameter of the lesions reached 17.2mm and the number of living Echinococcus multilocularis protoscolex was about 3800 per mouse at 4^thmonths after inoculation.ii)Four BALB/c mice in the thymectomized group died while two died in the sham operation group during the perioperative period with a perioperative mortality of the two groups reaching to 33.3%and 16.7%respectively.The rest of the mice survived well within one month after inoculation.iii)After inoculation of Echinococcus multilocularis protoscolex,no mice died in the perioperative period.Two mice in experimental group died of anaphylactic shock at second and third month after inoculation,respectively.Abdominal ultrasound in the first three months after inocu1ation showed that the diameter of echinococcosis 1esions in thymectomized group were significantl₁y higher than those in control₁group.Fourth months after operation,the weight of multilocular echinococcosis lesions in thymectomized set was statistically higher than pseudo-operation set（p==0.001）,as well as the number of living protoscolex（Pvalue<0.00l）.The lenvels of IL-10 and TGF-βl in thymectomized sets and pseudo-operation sets were statistically different（P<0.00l）.The levels of IL-l0 in two sets were 811.47±130.03 pg/ml and 389.51±74.58 pg/ml,respectively.iv)A total of 198 differential Lnc RNAs were found by high-throughput sequencing and 8 immune-pathway-related Lnc RNA were verified by RT-PCR.Finally,ENST0000529037,which was named by HPX-206,was chose for the further cytological experiment.Through GSEA analysis,we found HPX-206 has a significant correlation with TGF-βpathway,which may play as an immune regulatory molecular through TGF-βpathway.Mircode,LNCeding and DIANA-Lnc Base tools predicted that Lnc RNA-HPX206 and mi R-96 had obvious complementary sequences with a 78%complementarity degree.Furthermore,literature review and online databases targetscan and mi RBase predicted that Smad2 was the target gene of mi R-96.v)CCK8 tests showed that when the expression of Lnc RNA-HPX206 increased,the activity of hepatocytes decreased significantly in the presence of THP-1,while without THP-1,whether Lnc RNA-HPX206 was inhibited or not had no significant effect on the activity of hepatocytes,which suggest that lncrna-hpx206 may participate in the toxic effect of Echinococcus multilocularis on hepatocytes by inhibiting the immune protection mechanism nor in hepatocyte.TUNEL test showed that compared with LX-2+THP-1+Echinococcus multilocularis protoscolex group,the apoptosis of hepatocytes was significantly reduced in the si RNA-HPX206 group,suggesting that Lnc RNA-HPX206 may affect the apoptosis of hepatocytes by regulating the killing ability of monocytes against Echinococcus multilocularis;Scratch test showed there was no significant difference in migration distance between LX-2+THP-1 group and LX-2+THP-1+si RNA-HPX206 group after 36hours co-culture.However,compared with THP-1+LX-2+Echinococcus multilocularis protoscolex,the migration distance and proliferation range of hepatocytes were significantly longer in the group with si RNA-HPX206（P<0.05）.These results suggest that Lnc RNA-HPX206 may affect the migration activity of hepatocytes by regulating THP-1 of monocytes.Transwell test showed that compared with LX-2+THP-1+Echinococcus multilocularis protoscolex,the hepatocytes had stronger deformability and more transmembrane cells in added si RNA-HPX206 group.In the absence of THP-1,whether si RNA-HPX206 was added into the mixed culture medium of LX-2 and protoscolex had no significant effect on the number of transmembrane cells.vi)The expression of Lnc RNA-HPX206 in 20 AE patients was positively correlated with the count of Caspase 3（r=0.611,P<0.01）.This study further suggests that Lnc RNA-HPX206 may play a significant role in the toxic effect of protoscolex on hepatocytes by inhibiting immune response and promoting hepatocyte apoptosis in patients with AE.vii)The results of Western blotting showed that in the presence of THP-1,inhibition of Lnc RNA-HPX206 expression would lead to inhibition of TGF-βpathway expression and decrease hepatocytes apoptosis.At the same time,if THP-1was not involved,Lnc RNA-HPX206 expression had no significant effect on the pathway and hepatocyte apoptosis.viii)The luciferase reporter vectors of Lnc RNA-HPX206^wildand Lnc RNA-HPX206^mutantwere constructed and co-transfected with mi R-96 to THP-1cells.The results showed that when mi R-96 and Lnc RNA-HPX206^wildwere co-transfected,the luciferase activity decreased significantly,and when mi R-96 and Lnc RNA-HPX206^mutantwere co-transfected,the luciferase activity increased significantly,that is,there was a binding site between mi R-96 and Lnc RNA-HPX206^wildand the results showed that there was also a correlation between mi R-96 and Smad2.Conclusion:The second generation strain of Echinococcus multilocularis can successfully infect normal BALB/c mice and survived,but the proliferation ability of protoscolex is slightly lower than that in the first and second generation strains.More protoscoleces of Echinococcus multilocularis can be obtained by E.multilocularis infected thymectomized BALB/c animal model which the virulence and proliferation ability of protoscoleces of E.multilocularis are close to those of primary and second generation strains.The expression of Lnc RNA-HPX206 was verified by high-throughput sequencing and RT-PCR.And the expression of Lnc RNA-HPX206in the liver of AE patients was related to the lesion size,lesion number,local invasion and inflammatory infiltration.Lnc RNA-HPX206 can be used as a new biomarker in the diagnosis,prognosis prediction follow-up and vaccine synthesis of AE.It can also be used to evaluate the invasion ability and immune infiltration degree of AE patients.Lnc RNA-HPX206 can competitively bind to mi R-96 through ce RNA network,and regulate the expression of TGF-β/Smad2 pathway.And further basic experiments and animal experiments are still needed to confirm these findings.