| BackgroundHepatocellular carcinoma(HCC)is the sixth most common malignant tumor in the world and the fourth most common malignant tumor in China.The overall prognosis of HCC patients is poor due to its insidious onset,rapid progression,advanced stage at the first diagnosis,high tumor heterogeneity,and high risk of recurrence and metastasis after treatment.Therefore,it is important to find new diagnostic markers,explore the mechanism of HCC development and metastasis,and discover new targets for intervention and treatment to improve the prognosis of HCC patients.Alpha-fetoprotein is the most commonly used diagnostic marker for HCC in clinical practice,but its greatest drawback is its low sensitivity.Exosomes,which are extracellular vesicles with a diameter of approximately 40-160 nm,are highly stable and are an emerging,minimally invasive liquid biopsy technique.Recent studies have shown that some circulating exosomal lnc RNAs have better diagnostic value than traditional biomarkers in the diagnosis of gastric,lung,and bladder cancers,so circulating exosomes may be feasible as diagnostic markers.However,there are few such studies in HCC,so it is necessary to further clarify the diagnostic value of circulating exosomal lnc RNA in HCC.The first part of this study focused on exploring the circulating exosomal lnc RNAs that could be used for the diagnosis of HCC by next-generation sequencing technology and q RT-PCR.Numerous studies have confirmed that lnc RNAs are widely dysregulated and play a fundamental regulatory role in the development and metastasis of HCC.As the research on lnc RNA mechanism continues,increasing lnc RNAs are found to be pivotal in transcriptional regulation,chromatin modification,and post-transcriptional regulation of HCC,which are potential target molecules for HCC treatment.More than 70% of the human genome is transcribed into non-coding RNAs,of which 4%-9% of the human genome is transcribed into lnc RNAs,which shows the large number of lnc RNAs.However,only a small number of lnc RNAs have been fully elucidated,and the functions and mechanisms of most lnc RNAs remain unclear.The second part of this study focuses on the function of lnc RNA THEMIS2-211,a new lnc RNA identified in the first part of the study,in HCC,while the thirdpart further reveals the mechanism of lnc RNA THEMIS2-211 in HCC.Part I Diagnostic value of circulating exosomal lnc RNA in HCCPurpose: To find valuable circulating exosomal lnc RNA for the diagnosis of HCC.Methods: The differentially expressed lnc RNAs in plasma exosomes of HCC patients and normal healthy volunteers were screened by next-generation sequencing,further validated by q RT-PCR in training cohort and validation cohort.The area under the receiver operating characteristic curve(AUC)is used to evaluate the value of exosomal lnc RNA in the diagnosis of HCC.Results: Compared with healthy controls,1446 down-regulated lnc RNAs(Fold Change ≤0.5)and 305 up-regulated lnc RNAs(Fold Change ≥2)were present in plasma exosomes of HCC patients,of which 5 down-regulated lnc RNAs and 4 up-regulated lnc RNAs were statistically significant(Fold Change ≤0.5 or ≥2 and p<0.05).q RT-PCR verified that exosomal lnc RNA H19-204,THEMIS2-211,and PRKACA-202 were up-regulated in HCC patients(p<0.05)and stable in circulation.In the training cohort,the AUC of exosomal lnc RNA H19-204,THEMIS2-211,PRKACA-202,and Alpha fetoprotein(AFP)distinguishing HCC patients from healthy normal volunteers were 0.701,0.832,0.804,and 0.838.In the validation cohort,the AUC of exosomal lnc RNA H19-204,THEMIS2-211,PRKACA-202,and AFP distinguishing HCC patients from healthy normal volunteers were 0.67,0.816,0.812,and 0.824.The optimal diagnostic combination based on exosomal lnc RNA THEMIS2-211 and PRKACA-202 was constructed in the training cohort.The AUC of exosomal lnc RNA THEMIS2-211 and PRKACA-202 for the diagnosis of HCC was 0.881,not inferior to exosomal lnc RNA H19-204 + THEMIS2-211 + PRKACA-202(AUC=0.887),and superior to AFP(AUC=0.824).Lnc RNA THEMIS2-211 and PRKACA-202 were upregulated in exosomes derived from HCC cells(p<0.05).High expression of circulating exosomal lnc RNA THEMIS2-211 was associated with larger tumor diameter(≥5 cm)(p=0.014)and advanced TNM stage(III+IV)(p=0.006),while high expression of circulating exosomal lnc RNA PRKACA-202 was related to advanced TNM stage(III+IV)(p=0.001).Conclusions: Circulating exosomal lnc RNA THEMIS2-211 and lnc RNA PRKACA-202 are potential biomarkers for hepatocellular carcinoma.Part II The function of lnc RNA THEMIS2-211 in HCCPurpose: To elucidate the function of lnc RNA THEMIS2-211 in HCC.Methods: After knockdown and overexpression of lncRNA THEMIS2-211 in HCC cells by lentivirus,the proliferation ability of cells was detected by CCK-8 assay,migration ability by scratch assay,and transwell migration assay,invasion ability by transwell invasion assay,EMT related protein markers by q RT-PCR and Western blotting.The tumor size was evaluated in subcutaneous transplantation tumors in nude mice.Results: The expression of lncRNA THEMIS2-211 was significantly higher in HCC tissues than in paired adjacent tissues,and its expression level was positively correlated with tumor size and TNM stage.The expression of lnc RNA THEMIS2-211 was significantly higher in four HCC cell lines than in normal liver cell lines(p<0.05).For HCC cells with lnc RNA THEMIS2-211 knockdown,CCK-8 assay showed their proliferation ability was decreased,and the scratch assay and Transwell migration assay showed their migration ability was decreased,and transwell invasion assay showed their invasion ability was diminished;vice versa for lnc RNA THEMIS2-211 overexpression.For HCC cells with lnc RNA THEMIS2-211 knockdown,E-cadherin were upregulated and,however,N-cadherin and vimentin were downregulated;vice versa for lnc RNA THEMIS2-211 overexpression.In vivo animal experiment showed the tumor volume of subcutaneous transplantation tumors for HCC cells with lnc RNA THEMIS2-211 knockdown were smaller than those of the control(p<0.05);the mean positive staining area of Ki67 for lnc RNA THEMIS2-211 knockdown was significantly less than that of the control(p<0.05).Conclusion: Lnc RNA THEMIS2-211 exerts a pro-carcinogenic effect in HCC.Part III The mechanism of lncRNA THEMIS2-211 promoting the proliferation,migration,and invasion of HCC cellsPurpose: To elucidate the mechanism of lnc RNA THEMIS2-211 to function in HCC.Methods: Fluorescence in situ hybridization(FISH)was utilized to detect the subcellular localization of lnc RNA THEMIS2-211 in HCC cells,and an RNA pull-down assay was applied to detect the cytoplasmic proteins that may bind to lnc RNA THEMIS2-211.Bioinformatic methods predicted mi RNAs that may bind to lnc RNA THEMIS2-211 and m RNAs that may be acting as endogenous competitive RNAs;RIP assay,RNA pull-down assay,and dual luciferase reporter gene assays verified the target binding relationship between them.Rescue assay further verified the relationship among lnc RNA THEMIS2-211,mi RNA,and its target gene.Results: FISH assay showed that lncRNA THEMIS2-211 was mainly localized in the cytoplasm of HCC cells.RNA pull-down assay did not find any cytoplasmic proteins bound to lnc RNA THEMIS2-211.QRT-PCR and Western blotting verified the result of the bioinformatics prediction,showing that SPOCK1 was the ce RNA molecule of lnc RNA THEMIS2-211 and was positively correlated with lnc RNA THEMIS2-211 levels in HCC tissues(r=0.8303,p=0.009).RIP assay,RNA pull-down assay,and dual luciferase reporter gene assay verified the functional interaction and target binding relationship between lnc RNA THEMIS2-211 and mi R-940.Rescue assays showed that the regulation of SPOCK1 by lnc RNA THEMIS2-211 is dependent on mi R-940,and lnc RNA THEMIS2-211 promotes the proliferation,migration,and invasion of HCC cells dependent on miR-940.Conclusion: LncRNA THEMIS2-211 regulates SPOCK1 dependent on mi R-940 and promotes the proliferation,migration,and invasion of HCC cells. |
