LncRNA-HOTAIR Affects The Invasion And Metastasis Of Liver Cancer Cells Through The MiR-145/NUAK1 Axis | | Posted on:2022-07-17 | Degree:Master | Type:Thesis | | Country:China | Candidate:D X Chu | Full Text:PDF | | GTID:2504306329488714 | Subject:Pathology and pathophysiology | | Abstract/Summary: |  PDF Full Text Request | | Background: Hepatocellular carcinoma is one of the deadliest cancers in the world.Due to the hidden dangers of liver cancer and the lack of effective clinical treatment methods,the five-year average survival rate is less than 10%.The best treatment is surgical resection and liver transplantation.Because liver cancer is highly invasive and prone to metastasis,the prognosis of patients with advanced liver cancer is not ideal.Epigenetic regulation and genomic transcription abnormalities in the human body can affect the occurrence and development of hepatocellular carcinoma,such as methylation and acetylation modification,chromatin remodeling,histone modification,long non-codingRNA and microRNA and other influencing factors.The activation of epithelial-mesenchymal transition is an important sign of the invasion and metastasis of liver cancer,which mainly refers to the transformation of epithelial cells from loss of polarity to a motility mesenchymal phenotype;the activation of EMT enables cancer cells to have migration ability,invasion ability,and resistance to anaerobic disease.Of resistance.The degradation of extracellular matrix by secretase is the main feature of EMT,such as the degradation of ECM by matrix metalloproteinases;a variety of LncRNAs can affect the EMT process and the invasion and metastasis of cancer cells by regulating EMT-related transcription factors.LncRNA HOTAIR has been extensively studied in a variety of diseases in recent years.It can play a biological role in a variety of cancer cells.The 5’end of HOTAIR interacts with Polycomb inhibitory complex 2 and induces its genomewide redirection.The 3’end of HOTAIR interacts with lysine-specific histone demethylase 1A to regulate gene expression.Recent studies have reported that HOTAIR is highly expressed in a variety of cancers and is associated with poor prognosis,including breast cancer,epithelial ovarian cancer,pancreatic cancer,colorectal cancer,non-small cell lung cancer and endometrial cancer.MicroRNA is a type of non-codingRNA.It is a shortRNA molecule with a length of ~22 nt that is endogenously initiated.miRNA acts as a gene expression regulator by directly binding to mRNA,resulting in mRNA translation inhibition or target mRNA degradation.The two main types of non-codingRNA are competitive endogenous circularRNA and LncRNA,which can competitively bind to miRNA through miRNA response element(MRE),thereby reducing the amount of miRNA bound to mRNA and regulating downstream The post-transcriptional expression level of the target.Abnormal miRNA function can interfere with the expression of carcinogenic or tumor-inhibiting target genes,which is related to the pathogenesis of cancer.It has been found that a large number of miRNAs are down-regulated or up-regulated in human cancers,and have functions as oncogenes or tumor suppressor genes.It is worth noting that the molecular mechanism of miRNA expression dysregulation in cancer has recently been discovered.The genetic deletion or amplification of miRNA genome locus,epigenetic methylation and transcription factor-mediated primary miRNA regulation usually change miRNA in cancer.The distribution of expression and the dysregulation of multiple processing steps in the biogenesis of mature miRNAs may also lead to changes in miRNA expression in cancer.miR-145-5p is usually low-expressed in liver cancer cells,and participates in various biological processes of liver cancer cells by regulating different target genes.A detailed understanding of the regulatory mechanism of miRNAs in cancer is essential for understanding its physiological role and the meaning of cancer-related dysfunctions and regulatory abnormalities.NUAK1 is a new member of human AMP-activated protein kinase family.The abnormal regulation of NUAK1 is related to a variety of pathological diseases,including neurodegenerative diseases,metastatic cancer and diabetes;NUAK1 is involved in the metastasis and invasion of hepatocellular carcinoma.But at present,the mechanism of action of HOTAIR-miR-145-NUAK1 in liver cancer is not clear.Aims: Preliminary experiments have verified that HOTAIR can promote the invasion and metastasis of hepatocellular carcinoma cells.At the same time,it has been found that NUAK1 is highly expressed in hepatocellular carcinoma.There is a positive correlation between the two,but the specific molecular mechanism is not clear;by exploring whether HOTAIR induces EMT through the miR-145/NUAK1 axis Affect the invasion and metastasis of liver cancer cells,clarify the new mechanism of the influence of LncRNA-HOTAIR on the invasion and metastasis of liver cancer cells,and provide new ideas for preventing the metastasis of liver cancer.Methods: 1.Screening of differentially expressed genes by transcriptome sequencing analysis 2.Analyze the expression of miR-145 in liver cancer tissues by TCGA database 3.Detect the gene expression of NUAK1 in liver cancer tissues and five types of liver cancer cells by q-PCR 4.Detect the influence of miR-145 and NUAK1 on the invasion ability of Hep G2 and SNU-387 cells by Transwell experiment 5.Scratch test to detect the influence of miR-145 and NUAK1 on the lateral migration ability of Hep G2 and SNU-387 6.q-PCR experiment to detect the effects of miR-145 and NUAK1 on EMTrelated molecules in liver cancer cells(Hep G2 and SNU-387)at theRNA level 7.Western blot experiment to detect the effect of miR-145 and NUAK1 on EMTrelated molecules in liver cancer cells(Hep G2 and SNU-387)at the protein level 8.Analyze the effect of miR-145 and NUAK1 on the expression of N-cadherin by immunofluorescence technologyResults: 1.Through database analysis,it was found that the expression of miR-145 in liver cancer tissues was lower than that in HCC tissues.2.Compared with liver tissues,the expression of NUAK1 in liver cancer tissues is increased;among the five liver cancer cell lines,compared with normal liver cells,the expression of NUAK1 in HCC cells is increased.3.The increased expression of miR-145 weakens the migration and invasion ability of SNU-387 and Hep G2 cells.Western blot and q-PCR detected that theRNA level and protein level of EMT marker E-cadherin increased,while theRNA level and protein level of N-cadherin and Vimentin decreased;Transfection of miR-145 inhibitor promotes the lateral migration and invasion ability of SNU-387 and Hep G2 cells.TheRNA and protein levels of E-cadherin decreased,and theRNA and protein levels of N-cadherin and Vimentin increased.4.Add HTH-01-015 to inhibit the expression of NUAK1,weaken the lateral migration and invasion ability of SNU-387 and Hep G2 cells,increase theRNA level and protein level of EMT marker E-cadherin,and increase the level of Ncadherin and Vimentin.RNA levels and protein levels were reduced;HTH-01-015 was added while overexpressing HOTAIR,and theRNA levels and protein levels of EMT markers E-cadherin,N-cadherin and Vimentin did not change significantly.5.Transfection of miR-145 mimic,the detection found that the expression of NUAK1 at theRNA level and protein level decreased,and the transfection of miR-145 inhibitor increased theRNA level and protein level of NUAK1.6.Overexpression of HOTAIR can inhibit the gene expression level of miR-145 and promote the gene expression of NUAK1;inhibiting the expression of HOTAIR can increase the expression of miR-145,while the expression of NUAK1 decreases.Adding miR-145 analogs while overexpressing HOTAIR can reverse the effect of HOTAIR on NUAK1.Conclusion: 1.The low expression of miR-145 in HCC is negatively correlated with the expression of HOTAIR;HOTAIR is used as a molecular scaffold of PRC2 to regulate the gene expression of miR-145.2.NUAK1 is highly expressed in HCC and is positively correlated with the expression of HOTAIR;inhibiting NUAK1 can reverse the effect of HOTAIR on EMT.3.miR-145 induces EMT through NUAK1 to affect the invasion and metastasis of HCC cells.4.HORAIR can regulate the invasion and metastasis of HCC cells through the miR-145/NUAK1 axis... | | Keywords/Search Tags: | HOTAIR, miR-145, NUAK1, EMT, invasion, metastasis | | PDF Full Text Request | Related items |
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