Research On The Mechanism Of LncRNA-HOTAIR On Proliferation,Invasion,Metastasis And Apoptosis Of Hepatocellular Carcinoma Cells

According to the analysis of global cancer statistics in 2018,the morbidity of hepatocellular carcinoma(HCC)ranks sixth and the mortality rate ranks fourth.Tumor metastasis,frequent intrahepatic reproduction and extrahepatic progression are the main causes of poor prognosis of HCC.In the past 20 years,although surgical treatment and liver-protecting drugs had made great progress,the survival of liver cancer patients is largely threatened by cancer recurrence and cancer metastasis.Advances in genomic research have increased our understanding of the pathogenesis of liver cancer.Genetic events in the development and progression of HCC can be divided into genomes(somatic mutations and changes in genomic structure,such as gene fusion or copy number variation),epigenetics(methylated acetylated modification,chromatin remodeling,microRNA and long non-coding RNA)and gene transcription abnormalities.Long non-coding RNA-HOTAIR is highly expressed in HCC cells and has attracted increasing attention as an important molecule regulating tumor invasion and metastasis.At present,the clinical biomarkers for HCC detection are not clear,but multivariate analysis shows that HOTAIR is an independent prognostic factor for predicting the overall survival of HCC patients,which indicates that HOTAIR may be a potential biomarker for HCC progression.Therefore,clarifying the molecular mechanism of HOTAIR in regulating the proliferation,invasion and metastasis of hepatocellular carcinoma cells,and apoptosis is of great significance for the early prevention,diagnosis and targeted therapy of HCC.The epithelial to mesenchymal transition(EMT)process is a driving factor for tumor metastasis by giving cancer cells the ability to migrate and invade.EMT process has been linked as a driver of metastatic dissemination by conferring migratory and invasive capacity to cancer cells thus EMT is widely thought to be a critical switch for tumor-cell invasiveness.Decreased expression of the epithelial marker E-cadherin during EMT results in a loss of cell adhesion and subsequently cell dissociation,increased mobility and invasiveness.In this process,a series of EMT transcriptional factors(EMT-TF)including Snail1,Snail2,ZEB1,ZEB2 and Twist regulate EMT by inhibiting the transcription of epithelial genes and activating mesenchymal genes,but the specific mechanism unknown.In addition to the EMT process,clinical and epidemiological studies have also shown that some enzymatic biomarkers,such as matrix metalloproteinases(MMPs),play an important role in tumor invasion,neoangiogenesis,and metastasis.MMPs are zinc-dependent proteolytic enzymes used by cells for degradation of the extracellular matrix(ECM)and basement membrane during invasion and migration.Among them,MMP2 and MMP9 can both degrade type IV collagen of basement membrane and thus helps in invasion.Apoptosis plays a vital role in cell fate and tissue homeostasis.Previous studies have shown that microRNAs targeting the mitochondrial family,death receptor-mediated pathway and drug-induced autophagy can induce apoptosis in HCC cells.Few studies have focused on the role of long non-coding RNA in mitochondrial-induced apoptosis.According to De Paepe B,HOTAIR has become an effective regulator of mitochondrial-induced apoptosis.Among them,Bcl-2 family proteins play an important role in the apoptotic pathway of the endogenous / mitochondrial pathway.Bcl-2 family proteins can be divided into pro-apoptotic proteins,anti-apoptotic proteins and BH3-only proteins according to their functions."Activating factors"(BIM and BID)induce the structural changes of Bax and Bak effectors and then activate them.Activated Bax and Bak oligomerize the outer mitochondrial membrane to form channels,triggering the permeable transition of the outer mitochondrial membrane(MOMP),leading to apoptosis.We try to provide a reference for the treatment of HCC by targeting the HOTAIR-mediated mitochondrial-induced apoptosis pathway.Aims:The purpose of this experiment is to clarify the role of HOTAIR on the progression of HCC by investigating its function on the proliferation,invasion,metastasis,and apoptosis of HCC cells(HepG2,SNU-387)and its mechanism to clarify the molecular mechanism of HOTAIR to promote the occurrence and development of HCC,and to make preliminary exploration for targeted therapy of HCC.Method:1.q-PCR was used to detect the basic expression of HOTAIR in different types of HCC cells,and after that the ideal experimental cell line was selected.2.Real-Time Cell Analysis(RTCA)was used to detect the effects of HOTAIR on the growth rate of HCC cells(HepG2,SNU-387);Clone formation assay was used to detect the effects of HOTAIR on proliferation of HCC cells(HepG2,SNU-387).3.Transwell(Matrigel-)and scratch test to detect the effects of HOTAIR on the vertical and horizontal migration ability of HCC cells(HepG2,SNU-387);Transwell(Matrigel +)evalutes the impact of HOTAIR for HCC(HepG2,SNU-387)cells invasive capabilities.4.Western blot and q-PCR were used to detect the changes of molecules related to the invasion,metastasis,and apoptosis-related pathways both in protein and transcription levels of HCC cells(HepG2,SNU-387)upon different HOTAIR expression.5.Immunofluorescence and nuclear / plasma protein isolation kit were used to detect the effect of HOTAIR on the nuclear expression of transcription factor Snail2 during EMT.At the same time,the effect of HOTAIR on the nuclear morphology of HCC cells(HepG2,SNU-387)was detected by Immunofluorescent Hoechst staining.6.The effects of HOTAIR on the apoptosis rate and mitochondrial membrane potential(MMP)of HCC cells(HepG2,SNU-387)were detected by flow cytometry.7.Correlation analysis of HOTAIR and E-cadherin(CDH1)as well as HOTAIR and Snail2 in mRNA levels,data results from TCGA database biological information.Results:1.Compared to normal HCC cell named changliver,among the five HCC cell types,the highest HOTAIR basal expression was found in HepG2(4.2-fold),followed by SNU-387(3.5-fold).2.The proliferation rate of SNU-387 cells in overexpression HOTAIR(LZRS-HOTAIR)group was faster than that in CON group,and the doubling time was shortened.The proliferation rate of HepG2 and SNU-387 cells in sh-HOTAIR group was slower and the doubling time was prolonged than that in the sh-NC group.The cloning ability of SNU-387 cells in overexpression HOTAIR(LZRS-HOTAIR)group was stronger than that in CON group;the cloning ability of HepG2 and SNU-387 cells in sh-HOTAIR group was weaker than that in sh-NC group.3.The invasion and migration ability of SNU-387 cells in HOTAIR overexpression(LZRS-HOTAIR)group was stronger than that in CON group;The invasion and metastasis ability of HepG2 and SNU-387 cells in the sh-HOTAIR group was weaker than that in the sh-NC group.4.Compared to CON group,after overexpression of HOTAIR,the expression of MMP2 and MMP9 was significantly increased both in protein and mRNA level in SNU-387;Besiedes,the level of epithelial cell markers E-cadherin was significantly reduced both in protein and transcriptional level;the expression of mesenchymal markers N-cadherin was significantly up-regulated in protein level,but mRNA transcription level was not significantly changed;we found that Snail2 was significantly increased both in protein and transcription level.Compared to sh-NC group,MMP2 and MMP9 levels of HepG2 and SNU-387 cells in sh-HOTAIR group were significantly reduced both in protein and transcriptional level.In sh-HOTAIR group of HepG2,E-cadherin significantly increased in protein and mRNA transcription level;N-cadherin decreased significantly only in protein level,but there was no significant change in transcription levels;Snail2 significantly decreased both in protein and mRNA transcription level.In sh-HOTAIR group of SNU-387,the elevated expression of E-cadherin was identified both in protein and transcription level;N-cadherin and Snail2 only decreased significantly in protein level,but there was no significant change in mRNA transcription levels.Compared to CON group,the expression of Snail2 in the nucleus of SNU-387 increased after HOTAIR overexpression,and the fluorescence intensity of Snail2 increased.Compared to sh-NC group,the expression of Snail2 in the nucleus reduced both in HepG2 and SNU-387 cells upon HOTAIR knockdown,and the protein fluorescence intensity of Snail2 decreased.5.The mitochondrial membrane potential(MMP)of HepG2 and SNU-387 in sh-HOTAIR group was significantly lower than that in sh-NC group,and the apoptosis rate was significantly increased.Besides,the expression of mitochondria pro-apoptotic protein Bak increased while the expression of mitochondrial anti-apoptotic proteins Bcl-2 and Mcl-1 decreased.6.Compared to sh-NC group,abnormal nuclear morphology such as nuclear chromatin agglutination,nuclear concentration,and even nuclear fragmentation were observed in HepG2 and SNU-387 in sh-HOTAIR group.7.TCGA database analysis showed that there was no significant negative regulatory relationship between HOTAIR and E-cadherin(CDH1)at mRNA transcription levels;At the same time,there was no significant positive regulatory relationship between HOTAIR and Snail2 at mRNA transcription levels.Conclusion:1.The basic expression level of HOTAIR in five HCC cell lines was higher than that in normal liver cells.2.HOTAIR promotes the proliferation of HCC cells and shortens their doubling time.3.HOTAIR promotes invasion and metstasis of HCC cells through mediating EMT occurrence,regulating the nuclear localization of Snail2 and increasing the expression of matrix metalloproteinases MMP2 and MMP9.4.HOTAIR knock down promotes mitochondria induced apoptosis in HCC cells.5.For the TCGA database,there is no significant correlation between HOTAIR and E-cadherin(CDH1)and HOTAIR and Snail2 at the transcription level...