| Research purpose and backgroundPrimary liver cancer is the second most malignant tumor with a fatality rate and its incidence is increasing year by year.More than half of the new cases worldwide are in China.At present,it is not only a major "killer" that threatens the health of our citizens,but also has a major impact on our country.Economic development has caused tremendous pressure.Among them,Hepatocellular carcinoma(HCC)is the most important pathological type,accounting for about 90%.Due to the development of interdisciplinary research,major advances have been made in surgical techniques,local ablation,conformable targeted radiotherapy,and targeted therapy for liver cancer in recent years,but the 5-year survival rate has not substantially improved,and treatment Consumption has gradually increased with the increase of treatment methods.It allows clinical and scientific researchers to recognize the heavy price paid for "repairing dead sheep" and realize the importance of studying the relevant mechanisms in the process of liver cancer to early warning and intervention.It also makes the node of tumor research more urgent in advance.At present,inflammation has been recognized as the seventh major feature of malignant tumors.The immune microenvironment formed by chronic inflammation can not only promote tumor invasion and metastasis,but also an indispensable condition for tumor development.More and more eyes are focused on On the mutual regulation between tumor cells and interstitial cells,and with the emergence of tumor immunotherapy,the inflammatory microenvironment has also become a research area of anti-tumor therapy “emerging”.And hepatocellular carcinoma is a typical manifestation of the mechanism of "inflammatory cancer transformation".The occurrence and development of most liver cancers(about 90%)are accompanied by the "reconstruction of the inflammatory microenvironment such as liver fibrosis or cirrhosis".And the degree of liver fibrosis and cirrhosis is positively correlated with the incidence of tumors,which also fully illustrates that liver fibrosis and cirrhosis play an important bridge role in "transition of inflammation and cancer".Even if the liver disease pedigree has gradually changed in recent years,manifesting as metabolic liver damage such as diabetes and fatty liver is increasing year by year,but "all the same," regardless of the etiology of chronic hepatitis infection,diabetes,non-alcoholic fatty liver,autoimmune hepatitis,etc.They all cause compensatory liver regeneration through chronic necrotizing inflammation of the liver,eventually leading to the occurrence of fatal liver diseases such as decompensation of liver cirrhosis or liver cancer.In addition,the progression of liver inflammation to cirrhosis can lead to decompensation of liver function,which not only makes the treatment of primary liver cancer more difficult,but also cirrhosis can lead to direct death from liver failure.It is disappointing that there is currently no effective method to reverse the process of liver fibrosis,and it is difficult to make breakthroughs in the prevention and treatment of liver cancer.Therefore,the key regulatory mechanism in the continuous process of examining "transition of inflammatory cancer" is to prevent liver cirrhosis,Frontiers and hotspots of liver cancer.Long noncoding RNA(Lnc RNA)is a type of noncoding RNA longer than 200 nt.In recent years,a lot of evidence has confirmed that long noncoding RNA can be used as a broad-spectrum regulatory factor.It can be used in chromosome modification,gene transcription and post-transcription.It can also play a regulatory role at multiple levels,and can also stably exist in extracellular exosomes,participate in the mutual regulation of cells,and transmit information such as cell metastasis and chemotherapy resistance.The role of long-chain non-coding RNA in the process of "inflammatory transformation" of malignant tumors has also gradually received attention.The expression microarray results in breast cancer cells revealed that lnc RNA-NKILA was significantly up-regulated after LPS stimulation,and that NKILA combined with the inhibitory NF-?? / I?-B? complex,which masked the I?-B? phosphorylation site and led to the release of NF-??.Inhibition of activation and translocation ultimately inhibits the occurrence of breast cancer.The inflammatory factor TNF-? can stimulate the up-regulation of long-chain non-coding RNA HOTAIR(HOX Antisense Intergenic RNA's)in ovarian cancer cells.HOTAIR can induce DNA damage response(DDR)to promote tumor formation,and can also reduce I?B? protein levels,leading to NF-?? nuclear translocation and activation increase,which further induces the exacerbation of the inflammatory response and the continuous upregulation of HOTAIR.The results show that the positive feedback loop of HOTAIR-NF-??-DDR is an important regulatory mechanism in the process of "inflammatory cancer transformation" in ovarian cancer.Huang M et al recently reported that long non-coding RNA-MALT1 can play a role in the proliferation and metastasis of liver cancer cells caused by inflammation.In addition,liver-enriched long-chain non-coding RNA-LFAR1(liver fibrosis-associated lnc RNA1)can promote the activation of hepatic stellate cells by participating in the TGF-b signaling pathway,while c RNA-p21 can be used as ce RNA to inhibit liver The activation of stellate cells also preliminary suggested that long-chain non-coding RNA may be an indispensable key molecule in liver "inflammatory cancer transformation".The research team screened the expression profiles of long-chain non-coding RNA and messenger RNA in "normal liver tissue,chronic hepatitis tissue,liver cirrhosis tissue,liver cancer tissue" by high-throughput method in the early stage,and we targeted by bioinformatics analysis " DANCR(Anti-differentiated non-coding RNA)in the group of tumor-related long-chain non-coding RNAs may play a key role in the process of "inflammatory cancer transformation".Later,we found that DANCR activates the downstream pathway of B-catenin in hepatocellular carcinoma cells,which enhances the dryness of hepatocellular carcinoma cells and promotes the occurrence and development of tumors.It is further confirmed that the high expression of DANCR in the liver can be used as an independent predictor of the prognosis of hepatocellular carcinoma after radical operation.The risk factors suggest that DANCR can be used as a potential molecular marker for liver cancer.In addition,we found that the degree of liver inflammation and fibrosis was positively correlated with the expression level of DANCR in liver tissue.The expression of DANCR in liver tissue with high GS score was significantly higher than that of liver tissue with low GS score(see Research Foundation).In vitro studies have shown that inflammatory factors TNF-? and IL-6 can up-regulate the expression of DANCR in cell lines,and the expression of Dancr in mice is also induced by inflammatory factors.It is particularly important that we construct DANCR knockout mice,and through the DEN cancer-inducing model,we found that DANCR knockout mice not only significantly reduced the incidence of liver cancer,but also significantly reduced the degree of chronic liver injury including liver fibrosis.DANCR may play a core or key role in the development of "chronic hepatitis-cirrhosis-liver cancer"(see Research Basics).Although the aforementioned Hepatology has explained in detail the role of up-regulated expression of DANCR in hepatocytes in the development of liver cancer,its role in the progression of chronic liver injury through liver fibrosis to liver cancer,and a bidirectional regulatory network of liver interstitial cells and hepatocytes The functions in it are still unclear and worthy of further study.Research methods and resultsPart One: To investigate the expression of DANCR in the tissues of hepatitis,fibrosis and cirrhosis and to examine its correlation with the degree of inflammation and fibrosis.The clinical samples were expanded to detect the expression of DANCR in normal liver,hepatitis B,and cirrhotic tissues.It was found that DANCR showed an increasing trend in the pathological progress of "normal liver-chronic hepatitis-cirrhosis";according to the postoperative liver tissue GS score(G(Score is the degree of inflammation,S is the degree of fibrosis).Comparison of DANCR expression in liver samples with different inflammatory states.The results show that DANCR expression is higher in liver tissues with a higher overall GS score,and the higher the inflammation score or fibrosis score,the DANCR The higher the expression.DANCR in situ hybridization immunofluorescence staining in fresh clinical tissues confirmed that DANCR had relatively high expression abundance in hepatocytes,and that in tissues with higher GS scores,DACNR showed higher expression in hepatocytes.In addition,by collecting specimens of liver cirrhosis caused by different etiologies and examining their DANCR expression,it was found that in addition to HBV cirrhosis,DANCR expression was also increased in metabolic \ alcoholic \ HCV cirrhosis tissues.In the previous research,the research group used bioinformatics analysis to find that DANCR is more conserved in humans and mice in key functional regions.Models of acute and chronic hepatitis in mice were constructed and the expression of Dancr was detected.The results showed that Dancr expression was significantly increased in acute and chronic inflammation of mouse liver.Part Two: Investigate the regulatory relationship between inflammatory activation and DANCR up-regulation,and clarify the regulatory mechanismFirst,we explored the possibility of DANCR regulating inflammation.We observed the activation of NF-?B and STAT3 in hepatitis through the overexpression of lentivirus in human liver cancer cell lines and interference with DANCR.It was found that changes in DANCR expression could not change the two.The pathway was activated,and the inflammatory factors in the cell line culture medium were detected.It was found that there was no significant change in the expression of inflammatory factors after overexpression and interference with DANCR.Secondly,the possibility of DANCR was regulated by inflammation.Human hepatocellular carcinoma cell lines and primary mouse hepatocytes were cultured in vitro,and inflammatory factors TNF-? and IL-6 that activated NF-?B and STAT3 pathways were added.It was found that DANCR / Dancr expression was significant.Increased;through the lentivirus overexpression of NF-?B and STAT3 pathway transcription factors P65 and STAT3 in liver cancer cell lines,the expression of DANCR was found to increase significantly.Finally,the specific mechanism of NF-?B and STAT3 pathways up-regulating DANCR expression was studied.The DANCR promoter region(2000 bp upstream of the transcription initiation site)was inserted into the reporter plasmid.The double fluorescence report experiment confirmed that the transcription factors P65 and STAT3 can bind to DANCR.Promoter region promotes transcription;predicts the binding site of DANCR's promoter region based on the binding sequence of the transcription factor and designs specific primers,and performs chromatin immunoprecipitation + PCR experiments under normal conditions and inflammatory factor stimulation The results show that P65 and STAT3 are enriched in the promoter region of DANCR,and more enrichment can be observed in the state of inflammatory factor activation;mutations in the positive site of the chip experiment and access to the dual fluorescence reporting system The plasmid was tested and confirmed that P65 and STAT3 bind to specific sites in the DANCR promoter region.Part Three: Validation of DANCR in the transformation of inflammatory cancer through the inflammation model of transgenic mice with Dancr knockoutThe sequence of DANCR is highly conserved,and the sequence homology between mouse and human is high.We constructed Dancr knockout mice.Identified and multiplied to the number of models,and used DEN + CCL4 intraperitoneal injection to construct a model of "inflammatory cancer transformation" of chronic inflammation-cirrhosis-liver cancer.The results showed that the mice in the knockout group could reduce the degree of liver fibrosis compared with wild-type mice.Compared with the wild type mice,the mice in the group had a lower percentage of Sirius red staining at the same time,and the expression of fibrosis markers ACTA2 and COL1A1 was lower.In addition,the mice in the knockout group had lower liver tumor formation rate and shorter tumor formation period.At the same time,in wild-type mice,Dancr expression was detected at different time points and it was found to be related to the expression of other liver fibrosis marker molecules such as ACTA2 and COL1A1.At the same time,it was found that the increase of Dancr during the "inflammatory cancer transformation" process of wild-type mice was mainly present in hepatocytes by the original immuno-hybrid fluorescence experiment.Hepatocytes and stellates of wild-type model mice at different time points were separated by differential centrifugation.The cells were detected for Dancr expression,and it was found that Dancr was up-regulated in both cells,but mainly in hepatocytes.At the same time,Dancr detected in mouse serum correlated with Dancr expression in the liver.Part Four: The process of hepatocytes activating stellate cells and promoting cirrhosis by paracrine exosomes containing DANCRUltracentrifugation was used to isolate and purify exosomes in the supernatant of liver cancer cell culture medium and the serum of model mice,and confirmed and verified by particle size detection,electron microscopy,flow cytometry,and immunoblotting experiments.The exosomes isolated from the culture medium were detected and found to contain DANCR.In the culture medium of hepatocellular carcinoma cells stimulated by the inflammatory factors TNF-? and IL-6(the number of cells is the same 1 * 107),they can be enriched in More DANCR was detected in exosomes.Similarly,the content of Dancr in serum exosomes(300ul)of cirrhotic mice was higher than that of ordinary mice.The isolated exosomes were co-cultured with hepatic stellate cells,and the uptake of stellate cells into exosomes was observed by laser confocal experiments,and the DANCR content in stellate cells increased after the exosomes were detected.DANCR mutants were transfected into hepatoma cells cultured in vitro.Exosomes were extracted and co-cultured with hepatic stellate cells.The expression of mutant DANCR in stellate cells was detected.The mutant form is present,indicating that it is derived from uptake exosomes.Co-culture of exosomes in the serum of wild-type model mice and Dancr-knockout model mice with the primary hepatic stellate cells of normal mice.Co-cultured stellate cells have a higher degree of activation,while co-cultured with sera exosomes of knockout mice has a lower degree of activation.Exosomes in the supernatant of the isolated liver cancer cell culture medium were fluorescently stained,and co-cultured with stellate cell lines in vitro,and then subjected to flow cytometry.It was found that stellate cells that took up more exosomes had activation markers.The higher the expression of ?-SMA,the higher the degree of activation.Part Five: Exploring the specific mechanism of DANCR's promotion after it enters stellate cellsOverexpression of DANCR in stellate cell line LX2 by lentivirus,detection of downstream altered genes by expression profiling chips,and screening of cell functions and cell signaling pathways enriched by differentially expressed genes by go / pathway analysis,It was found that related genes with differential expression were significantly enriched in interferon-related pathways.Later,different concentrations of interferon were added to LX2 cells that overexpressed DANCR and controls.It was found that overexpression of DANCR could reduce interferon's proliferation on stellate cells.With the inhibition of activation,the IC50 value of interferon overexpressing DANCR is higher,and the expression of activation markers in LX2 cells overexpressing DANCR is higher than that in the control group under the same concentration of interferon.Later,CCL4 intraperitoneal injection was used to establish a liver cirrhosis model of knockout Dancr mice and wild-type mice.Interferon treatment was also given during the model establishment period.The results showed that the degree of liver cirrhosis in the knockout group mice was lower,indicating that knockout Dancr can Significantly increases the sensitivity of interferon to cirrhosis.ConclusionBased on the above results in four parts,we can conclude that:1.Detection of DANCR in tissue specimens gradually increased during the pathological process of hepatitis cirrhosis and was significantly related to the degree of inflammation and fibrosis,and it was significantly up-regulated in cirrhotic tissues caused by various causes.At the same time,in mouse acute and chronic hepatitis models,Dancr expression also increased significantly.These results suggest that DANCR may play an important role in the process of liver inflammation and cancer transformation.2.Intracellular molecular biology tests clearly activated the inflammatory pathways NF-?B and STAT3 to up-regulate the expression of DANCR.The classic transcription factors P65 and STAT3 of the pathway bind to the promoter region of DANCR to promote the transcription of DANCR.3.By constructing Dancr knockout transgenic mice and establishing a model of mouse liver inflammation cancer transformation process,it was found that knocking out Dancr can reduce the degree of liver cirrhosis,reduce tumor incidence and tumor burden,indicating its role in liver cancer transformation Play an important role.4.Hepatocytes can secrete DANCR-containing exosomes and are taken up by stellate cells to promote the activation of stellate cells.Under inflammatory conditions,stellate cells can take up more DANCR through this pathway.5.The differentially expressed genes in stellate cells after knockdown of DANCR were detected in the interferon-related pathways by expression profile microarrays.It was confirmed through cell functional experiments and mouse in vivo experiments that DANCR can reduce the sensitivity of stellate cells to interferon Thereby promoting the progress of liver fibrosis. |
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