The Regulatory Role And Mechanism Of LncLHC1 In The Biological Process Of Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC)is one of the most common types of liver cancer in clinical practice,accounting for more than 80% of primary liver cancer.The incidence of HCC accounts for more than 80% of primary liver cancers.Due to its insidious development,difficulty in detection and few clinical treatments,the mortality rate of patients with HCC is high,and the five-year survival rate of patients is less than 10%.At present,surgical resection and liver transplantation are the best treatments for early stage hepatocellular carcinoma,however,the prognosis of patients with advanced hepatocellular carcinoma is poor due to its high invasiveness,strong metastatic ability and difficulty in early clinical diagnosis.Epithelial-mesenchymal transition(EMT)is one of the important cellular phenotypes in the development of invasive and metastatic hepatocellular carcinoma.EMT mainly refers to the gradual loss of polarity of epithelial cells to a motile mesenchymal phenotype.EMT activation leads to the development of migration and invasive ability of cancer cells.Many molecules have been reported to regulate EMT occurrence and development and regulate the invasive metastasis of tumour cells through the regulation of EMT-related transcription factors.Epigenetic changes and aberrant genomic transcription perform an important role in the regulation of multiple biological behaviours in malignant tumour cells with EMT as an important phenotype,especially in the progression of hepatocellular carcinoma.The discovery of non-coding RNAs and their important regulatory role on proteins has provided a novel research direction for studying the mechanism of hepatocellular carcinoma.Long non-coding RNAs(lncRNAs)are a class of endogenous RNA molecules greater than 200 nt in length which could not be translated into proteins.Lnc RNAs are conserved,tissue-specific and disease-specific.Lnc RNAs regulate the expression of downstream target proteins and promote m RNA degradation through binding to micro RNAs;Lnc RNAs can also bind directly to target proteins and participate in protein degradation.Important regulatory roles for lncRNAs have been identified in a variety of cancer cells.Lnc RNAs have been reported to be highly expressed in a variety of cancers and associated with poor prognosis,including breast cancer,colorectal,bladder cancer,epithelial ovarian cancer,pancreatic cancer and non-small cell lung cancers.lncRNAs are involved in the regulation of cellular phenotypes in a variety of malignancies,including cell proliferation,apoptosis,invasion and metastasis,epithelial mesenchymal transition,angiogenesis and tumour drug resistance.The molecular sponge mechanism with mi RNA adsorption is the most important mechanism in the regulation of lncRNAs.However,the expression and molecular markers of lncRNAs in HCC still need to be explored in depth,while their regulatory role on the biological behavior of HCC also needs to be experimentally verified.This study aims to identify key lncRNA molecules that regulate the malignant phenotype of hepatocellular carcinoma and explore their mechanisms of action,providing a scientific basis for finding new diagnostic indicators and therapeutic targets.Methods: In the first part of this study,lncRNAs with up-regulated expression in hepatocellular carcinoma were first screened through analysing lncRNA microarray data from the TGCA database,combined with R language analysis methods.q RT-PCR was used to validate the conclusions of the bioinformatics analysis and to detect the up-regulated lncRNAs in hepatocellular carcinoma tissues,paraneoplastic tissues and multiple hepatocellular carcinoma cell lines,respectively.The expression levels of lncRNAs molecules in hepatocellular carcinoma tissues,paraneoplastic tissues and multiple hepatocellular carcinoma cell lines were examined.The subcellular localization of the candidate lncRNA molecule(lncLHC1)was also determined by in situ fluorescence hybridization(FISH)experiments as well as nucleus and cytoplasm separation assays.The regulatory role and molecular mechanism of lncLHC1 on hepatocellular carcinoma cells were further investigated.A relatively high lncLHC1-expressing hepatocellular carcinoma cell line,HUH7 cells,was selected and lncLHC1 was knocked down in HUH7 cells using si RNA.lncLHC1 was selected from a relatively low-expressing hepatocellular carcinoma cell line,Hep G2 cells,and overexpressed by transfection with lncLHC1 overexpression plasmids.After PCR was performed to confirm the transfection efficiency,the cell proliferation changes were examined by CCK-8 assay,the effect on apoptosis by A/P label method by flow cytometry,the cell migration ability by scratch healing assay,and the cell invasion ability by transwell assay.Western blot were performed for epithelialmesenchymal transition-associated proteins E-cadherin,N-cadherin and Vimentin.To further explore the mechanism of regulatory action of lncLHC1 on hepatocellular carcinoma.Firstly,bioinformatics was used to predict several target mi RNA molecules which could bind with lncLHC1.q RT-PCR assay was performed to identify the downstream mi RNA molecules including mi R-139-5p.The bioinformatics software was used to predict the target protein of mi R-139-5p is Lysophosphatidylcholine acyltransferase 1(LPCAT1),and the expression of LPCAT1 m RNA was determined by q RT-PCR.Fluorescence in situ hybridization(FISH)assay was performed to detect the subcellular localization of mi R-139-5p to lncLHC1.Dual luciferase assays were performed to confirm whether exogenous mi R-139-5p could bind to lncLHC1 or LPCAT1 m RNA.The target binding of mi R-139-5p to lncLHC1 was further examined using RNA pull down assays.The effects of lncLHC1 and mi R-139-5p on the biological behaviour of HCC cells were compared by knock-down lncLHC1 or mi R-139-5p respectively and both knockdown simultaneously.We confirmted that lncLHC1 regulates the cellular behavior of HCC cells by modulating the level of mi R-139-5p by overexpressing lncLHC1 or mi R-139-5p,respectively,and comparing their regulatory effects on the biological behavior of HCC cells through overexpressing lncLHC1 and mi R-139-5p simultaneously.The proliferation,apoptosis,migration,invasion and epithelial mesenchymal transition of hepatocellular carcinoma cells were subsequently examined by overexpression of lncLHC1 and after simultaneous overexpression of lncLHC1 and knockdown of LPCAT1 using CCK-8,A/P double-labeled flow cytometry,scratch assay and shuttle cell assay as well as Western blot,respectively.To identify the effect of lncLHC1 in vivo,a HUH 7 cell line with knockdown lncLHC1 was prepared by lentiviral transfection,and the cells were injected subcutaneously into nude mice,where tumour volumes were periodically examined.After execution and sampling,they were photographed,weighed and immunohistochemically examined for expression of LPCAT1,Ki-67,E-calcineurin and N-calcineurin.RESULTS: In the first part of the study,the TCGA database was first downloaded and screened for differential expression of multiple long-stranded non-coding RNA molecules,50 in total,in hepatocellular carcinoma tissue after R language.Among them,10 lncRNAs were selected and validated by RT-q PCR in clinical tissues,and lncLHC1 was found to be more highly expressed in hepatocellular carcinoma than in paraneoplastic tissues and more highly expressed than other candidate lncRNA molecules.The high expression of lncLHC1 was also found to be associated with lymph node metastasis after clinical analysis.PCR results after fluorescence in situ hybridization and nucleoplasmic separation showed that lncLHC1 was mainly distributed in the cell plasma.The proliferation rate of hepatocellular carcinoma cells was significantly downregulated after knockdown of lncLHC1 and increased after overexpression of lncLHC1 in CCK-8 assay.The results of flow cytometry assay for apoptosis showed that the apoptosis rate of hepatocellular carcinoma increased after knockdown of lncLHC1 and decreased after overexpression of lncLHC1.Scratch healing assay showed that the migration ability of hepatocellular carcinoma cells was reduced after knockdown of lncLHC1,while the migration ability of hepatocellular carcinoma cells was enhanced after overexpression of lncLHC1.The results of the shuttle cell assay showed that the invasive ability of hepatocellular carcinoma cells was reduced after knockdown of lncLHC1,while the invasive ability of hepatocellular carcinoma cells was significantly up-regulated after overexpression of lncLHC1.Western blot of epithelial mesenchymal transition-related proteins showed that knockdown of lncLHC1 inhibited the expression of N-cadherin and vimentin and promoted the expression of E-cadherin protein.Overexpression of lncLHC1 promoted the conversion of epithelial cells to mesenchymal cells,i.e.the expression of E-cadherin was down-regulated and the expression of Ncadherin and vimentin were up-regulated.Results:The expression of lncLHC1 was upregulated in hepatocellular carcinoma tissues,and this upregulation was associated with hepatocellular carcinoma metastasis.lncLHC1 was found to promote the proliferation of hepatocellular carcinoma cells,inhibit apoptosis,and promote cell migration,invasion and epithelial-mesenchymal transition of cells.Part II: The expression database analysis identified three mi RNAs that may bind to lncLHC1.PCR results showed that the expression of mi R-139-5p was most significantly regulated by lncLHC1.It was found that mi R-139-5p expression was low in cancer tissues and its expression was negatively correlated with survival of liver cancer patients.mi R-139-5p expression was negatively correlated with lncLHC1 expression.The results of the dual luciferase reporter gene assay showed that the fluorescence intensity of the mi R-139-5p mimics+lncLHC1 was significantly reduced.The results of the RNA pull down assay showed that the probe containing the lncLHC1 sequence was enriched for mi R-139-5p molecules.The dual luciferase assay and the RNA pull down assay were able to demonstrate that lncLHC1 was able to adsorb mi R-139-5p and regulate the level of mi R-139-5p in the cells.In functional rescue assays,it was found that knockdown of mi R-139-5p in cells could effectively reverse the altered biological behaviour of hepatocellular carcinoma induced by knockdown of lncLHC1,specifically the phenotypes of cell proliferation,apoptosis,cell migration and invasion,and epithelial-mesenchymal transition;overexpression of mi R-139-5p in cells could effectively reverse the biological behaviour of hepatocellular carcinoma induced by overexpression of lncLHC1.Overexpression of mi R-139-5p was able to effectively reverse the effects of overexpression of lncLHC1 on promoting the biological behaviors of hepatocellular carcinoma,specifically the phenotypes of cell proliferation,apoptosis,cell migration and invasion,and epithelial-mesenchymal transition.This result suggests that the regulation of lncLHC1 is dependent on downregulation of mi R-139-5p.LPCAT1 was subsequently predicted by bioinformatics sites and studied as a candidate target gene for mi R-139-5p.After RT-PCR assay,LPCAT1 was found to be highly expressed in cancer tissues and its expression level was negatively correlated with mi R-139-5p,while high LPCAT1 expression was positively correlated with poor patient prognosis.Further dual luciferase reporter gene assay and RNA pull down assay revealed that mi R-139-5p specifically bound to the 3’UTR on LPCAT1 m RNA.cell function rescue assay results showed that lncLHC1 overexpression and knockdown of LPCAT1 protein could partially rescue the suppression of overexpression lncLHC1-induced prohepatocellular carcinoma development.These results suggest that LPCAT1 is a downstream regulatory molecule of the lncLHC1/mi R-139-5p axis and involved in the regulation of cancer cell progression.The results of the subcutaneous tumor genesis assay in nude mice showed that the tumour volume and weight of the lncLHC1 knockdown were smaller than those of the control group,and the expression of Ki-67,LPCAT1,N-calcine mucin and Vimentin were lower than those of the control group,while the expression of E-cadherin was higher than that of the control group.The above results demonstrated in vivo that knockdown of lncLHC1 inhibited the growth and epithelial mesenchymal transition of hepatocellular carcinoma cells.Conclusions: 1.lncLHC1 is an oncogenic lncRNA molecule in hepatocellular carcinoma.lncLHC1 is one of the lncRNAs localized in the cytoplasm and significantly upregulated in hepatocellular carcinoma.lncLHC1 is more likely to develop lymph node metastasis in patients with high lncLHC1 expression.2.lncLHC1 promotes proliferation,migration and invasion of hepatocellular carcinoma cells and epithelial mesenchymal transition,and inhibits apoptosis of cancer cells.3.lncLHC1 acts as a molecular sponge that specifically adsorbs mi R-139-5p and reduces the targeting inhibitory effect of mi R-139-5p on LPCAT1.lncLH1 promotes proliferation,inhibits apoptosis,promotes cell migration and invasion as well as epithelial mesenchymal transition in hepatocellular carcinoma cells by down-regulating mi R-139-5p and promoting the level of LPCAT1,thus lncLHC1 is a key molecule in promoting the biological progression of hepatocellular carcinoma.