KR102025005B1 - Biomarker for early diagnosis of hepatocellular carcinoma in precancerous lesion and use thereof - Google Patents

Abstract

The present invention relates to the use of BANF1, PLOD3 and SF3B4 as biomarkers capable of diagnosing and predicting early liver cancer in precancerous lesions. Specifically, the present invention relates to a composition and diagnostic kit for diagnosing early liver cancer comprising one or more markers for diagnosing early liver cancer selected from the group consisting of BANF1, PLOD3, and SF3B4 and a detection agent for the marker. The present invention also relates to a method for detecting one or more early liver cancer diagnostic markers selected from the group consisting of BANF1, PLOD3 and SF3B4 from a patient's sample, and a method for providing information for early liver cancer diagnosis using the marker.

Description

Biomarker for early diagnosis of hepatocellular carcinoma in precancerous lesion and use according to biomarkers for diagnosis and prediction of early liver cancer in precancerous lesions

The present invention relates to the use of BANF1, PLOD3 and SF3B4 as biomarkers that can diagnose and predict early liver cancer in precancerous lesions.

Cancer is a major threat to human health and the single leading cause of death in industrialized countries. The cause of cancer is still unknown, but it is considered to be a combination of carcinogens that act as internal and hereditary factors and external factors as cancer-causing factors, continuous inflammation and damage, and cancer-causing viral infections. .

However, cancer is not desperate enough to be determined to be an incurable disease and can be cured by early diagnosis and active treatment. Therefore, early detection, early diagnosis, and treatment are crucial to increase the effectiveness of cancer treatment, and even if advanced cancer is used with multiple and active methods, it can be cured or prolonged life and improve painful symptoms.

Among cancers, liver cancer is known as one of the most deadly cancers in the world, and more than half a million people die of liver cancer each year in Asia and sub-Saharan Africa. Liver cancer can be classified into primary liver cancer (hepatocellular carcinoma) originating from the liver cells itself and metastatic liver cancer in which cancers of other tissues have spread to the liver. More than 90% of liver cancers are primary liver cancers. Although most of the causes of liver cancer are reported to be acute or chronic infections caused by hepatitis B virus or hepatitis C virus, the molecular mechanisms within the cells regarding the development and progression of liver cancer are still unclear. . Conventional studies have shown that protooncogenes, such as various growth factor genes, are mutated to oncogenes for various reasons and are overexpressed or overactive, or tumors such as Rb protein or p53 protein. Tumor suppressor genes have been reported to cause the development and progression of various cancers, including liver cancer, when they are mutated for various reasons and underexpressed or lost function. In recent years, the onset and progression of most cancers, including liver cancer, is not caused by a few specific genes, but is caused by the complex interaction of various genes related to cell cycle, signaling, etc., and therefore, individual genes or proteins. Rather than focusing only on the expression and function of, the need for comprehensive research on various genes and proteins is emerging.

On the other hand, the biomarker test that can accurately detect liver cancer early in the normal population has not been developed yet, and the serum alpha fetoprotein test is used to diagnose non-invasive early liver cancer in high-risk groups. At the time of development, AFP was presented at 20 ng / mL as a baseline for achieving both good sensitivity and specificity.However, in this case, sensitivity is only 60% and is currently 200 ng / mL according to the international guidelines for liver cancer diagnosis. Based on the diagnosis of liver cancer, the specificity increases, but the sensitivity is only 22%. Previous studies have shown that AFP has an overall sensitivity of about 66% and a specificity of 82%, limiting the diagnosis of all liver cancer patients.

Serum markers for diagnosis of liver cancer, although not established by other diagnostic criteria, include Descarboxyprothrombin (DCP), Prothrombin Induced by Vitamin K Absence II (PIVKA-II), glycosylated AFP to total AFP (L3 fraction) distribution, alpha fucosidase, glypican 3 and HSP-70. However, each of them has a meaning as a prognostic factor, and when used alone, its accuracy is low and it is not yet used as a screening test. Early diagnosis of liver cancer seems to have reached its limit. In addition, only 30% of patients with liver cancer are diagnosed at the stage of curable treatment such as actual surgery or radiofrequency ablation.

Therefore, there is an urgent need to develop new diagnostic markers with improved specificity and sensitivity that allow for early diagnosis as early as possible in the stage of fundamental cancer treatment.

Republic of Korea Patent Publication No. 10-2016-0072027

The present inventors completed the present invention by first confirming that early diagnosis of liver cancer can be accurately diagnosed by measuring the expression level of BANF1, PLOD3 or SF3B4 at the precancerous lesion site.

Accordingly, an object of the present invention is to provide a marker composition for diagnosing early liver cancer, which comprises at least one selected from the group consisting of BANF1, PLOD3, and SF3B4.

Another object of the present invention is to provide an early liver cancer diagnostic composition comprising an agent for measuring mRNA expression level of at least one early liver cancer diagnostic marker protein selected from the group consisting of BANF1, PLOD3 and SF3B4 or a gene encoding the same. .

Another object of the present invention is to provide an early liver cancer diagnostic kit comprising the composition of the present invention.

Still another object of the present invention is to provide a method for detecting at least one early liver cancer diagnosis marker selected from the group consisting of BANF1, PLOD3 and SF3B4 from a patient's sample in order to provide information necessary for diagnosing liver cancer.

Another object of the present invention is to measure the expression level of mRNA or protein for at least one gene selected from the group consisting of BANF1, PLOD3 and SF3B4 from biological samples isolated from patients; And comparing the measured expression level of BANF1, PLOD3 or SF3B4 with the expression level of the gene of the control sample, to provide an information providing method for early liver cancer diagnosis.

In order to achieve the above object, the present invention is selected from the group consisting of BANF1 (barrier to autointegration factor 1), PLOD3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) and SF3B4 (splicing factor 3b subunit 4) Provided is a marker composition for early liver cancer diagnosis, comprising one or more.

In one embodiment of the present invention, the BANF1 is composed of the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, the PLOD3 is composed of the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4, SF3B4 may be composed of the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6.

The present invention also provides a composition for detecting early liver cancer, comprising an agent for measuring mRNA expression level of at least one early liver cancer diagnostic marker protein selected from the group consisting of BANF1, PLOD3 and SF3B4 or a gene encoding the same.

In one embodiment of the present invention, the BANF1 gene consists of the nucleotide sequence of SEQ ID NO: 1 and the BANF1 protein consists of the amino acid sequence of SEQ ID NO: 2, the PLOD3 gene consists of the nucleotide sequence of SEQ ID NO: 3 and the PLOD3 protein is the sequence It consists of the amino acid sequence of No. 4, SF3B4 gene is composed of the nucleotide sequence of SEQ ID NO: 5 and SF3B4 protein may be composed of the amino acid sequence of SEQ ID NO.

In one embodiment of the invention, the agent may be a primer, probe, aptamer, antibody or substrate specific for BANF1, PLOD3 or SF3B4.

In one embodiment of the present invention, the measurement may be to measure in a precancerous lesion sample.

In one embodiment of the present invention, the measurement is reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, DNA microarray method Northern blot, Western blot, Enzyme Linked Immuno Sorbent Assay (ELISA), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry and protein micro It may be performed by a method selected from the array method.

The present invention also provides an early liver cancer diagnosis kit comprising the composition of the present invention.

In another aspect, the present invention provides a method for detecting one or more early liver cancer diagnosis markers selected from the group consisting of BANF1, PLOD3 and SF3B4 from the sample of the patient in order to provide information necessary for the diagnosis of liver cancer.

In one embodiment of the present invention, the detection may be to measure the mRNA expression level of at least one marker protein selected from the group consisting of BANF1, PLOD3 and SF3B4 or a gene encoding the same in a precancerous lesion sample.

In another aspect, the present invention comprises the steps of measuring the expression level of mRNA or protein for one or more genes selected from the group consisting of BANF1, PLOD3 and SF3B4 from biological samples isolated from patients; And comparing the measured expression level of BANF1, PLOD3 or SF3B4 with the expression level of the gene of the control sample, provides an information providing method for early liver cancer diagnosis.

In one embodiment of the invention, the sample may be precancerous lesion tissue.

In one embodiment of the present invention, when the expression level of one or more genes or proteins selected from the group consisting of BANF1, PLOD3 and SF3B4 increases than the expression level of the control sample, it can be determined that the liver cancer.

The present invention relates to the use of BANF1, PLOD3, and SF3B4 as biomarkers for the diagnosis and prediction of early liver cancer in precancerous lesions.The present invention relates to liver cancer by measuring the expression level of these markers in precancerous lesions before the development of liver cancer. Early detection can prevent early detection, prevention, and improve the treatment rate. Also, the expression or activity inhibitors of these markers can inhibit the growth, proliferation, migration and metastasis of liver cancer. It can be used as a therapeutic agent.

1 is a schematic diagram showing the procedure for identifying a novel liver cancer diagnostic marker of the present invention in hepatocellular carcinoma.
Figure 2 relates to the results of marker identification to diagnose early stage hepatocellular carcinoma, 2a is a heat map (top) of 3,628 genes showing a significant difference in expression in various stages of hepatocellular carcinoma and premalignant lesions (top) Welch's t-test p <0.05 with 1.5-fold differences in expression from normal samples) and heatmaps and phylograms for the mean value of each disease (bottom). 2b shows a heat map of 1,730 genes (top) that showed significantly aberrant expression (one way ANOVA p <0.005) in LGDN, HGDN, eHCC and G1 (top), and a hierarchical analysis of the mean values of LGDN, HGDN, eHCC and G1. Results are shown (bottom). 2c is a vennogram result of genes representing differences in expression between specific groups (left) and heatmaps for 690 genes with differential expression in all groups (middle), progressive in early stage hepatocellular carcinoma This shows a strategic model for the identification of 85 genes upregulated (right). 2d shows the ROC curve analysis for 13 genes, showing a statistically significant difference in AUC compared to the baseline. 2e shows the results of immunochemical analysis of the 10 candidate gene markers in the HCC TMA by the photograph (top) and the expression rate (bottom).
Figure 3 shows the expression profile of the novel cancer determination (diagnosis) marker, 3a shows the analysis of the expression level of the 10 marker genes for non-tumor tissue and tumor tissue of HCC patients derived from TCGA_LIHC dataset, 3b shows the analysis of the expression level of 10 marker genes in staged HCC patient tissue from Cohort 1.
4 shows the results of analysis of the possibility of BANF1, PLOD3 and SF3B4 as early stage HCC diagnostic markers in precancerous lesions. 4a shows immunohistochemical staining for 10 candidate markers in all sections of early stage HCC. 4b is a bar graph showing the degree of positive expression for each of the existing marker (CM) and the novel marker (NM) of the present invention (left), and an image of the immunohistochemical staining result. Right side). 4c shows the positive expression level of each of the existing marker (CM) and the novel marker (NM) of the present invention (left), and the positive expression rate of the novel marker of the present invention or the novel marker of the present invention in the HCC of the existing marker negative expression The positive expression rate of the existing marker in the HCC of negative expression is shown as a bar graph (middle), and the results of negative expression of all conventional markers are already shown as immunohistochemical staining results (right). 4d graphically shows NM negative expression rate in CM-positive RN tissue and CM negative expression rate in NM-positive RN tissue. 4e shows the ROC curve for each marker, and 4f shows the Kaplan-Meier survival curve analysis of the protein expression rate for the three new markers of the present invention in hepatocellular carcinoma patients.
FIG. 5 shows the results of ROC analysis for 10 new candidate gene markers, showing the results of ROC analysis for 3 existing markers and 3 new markers in Cohort 2 HCC patient group.
Figure 6 confirmed the abnormal expression patterns of BANF1, PLOD3 and SF3B4 in HCC and their inhibitory effect on hepatocellular carcinoma by their inactivation in vitro, 6a shows the expression level of BANF1, PLOD3 and SF3B4 in TCGA_LIHC HCC 50 pairs (Each left figure) and expression levels in 13 randomly selected pairs of HCC tissue (each right figure). 6b shows western blot results of BANF1, PLOD3 and SF3B4 expression levels in 6 pairs of early hepatocellular carcinoma (top) and 3 pairs of early hepatocellular carcinoma (bottom). 6c is a result confirming the expression level of BANF1, PLOD3 and SF3B4 in mice (top) and mice (bottom) induced hepatocellular carcinoma. 6d shows Western blot expression of BANF1, PLOD3 and SF3B4 expression levels in H-ras-transformed mice (top) and DEN-induced hepatocellular carcinoma mice (bottom). 6e shows the cell viability (left) and cell growth rate (right) of SNU-449 cells transfected with control si-RNA (si-Cont) and siRNA for each gene (si-BANF1, si-PLOD3 and si-SF3B4). The results measured by MTT analysis and BruD analysis are shown. 6f shows the result of analyzing the degree of colonization of the transfected SNU-449 cells.
Figure 7 shows the results of tumor tumor suppression and metastasis inhibition of hepatocellular carcinoma cell line targeting BANF1, PLOD3 and SF3B4, 7a is a control si-RNA (si-Cont) and siRNA (si-BANF1 for each gene) , si-PLOD3 and si-SF3B4) and the degree of migration and invasion of the SNU-449 cell line transfected with the cell migration (left) and the number of cells to move (right) are shown, and 7b is in the same experimental group as 7a Wound healing analysis results for the cell image (left) and the rate of movement (right). 7c confirms Western blot EMT regulatory protein in SNU-449 cell line transfected with control si-RNA (si-Cont) and siRNA for each gene (si-BANF1, si-PLOD3 and si-SF3B4), 7d Shows the results of measuring mouse pictures and tumor size of a mouse model transplanted with a control si-RNA and a SNU-449 cell line transfected with si-BANF1, si-PLOD3 or si-SF3B4. 7e is a schematic diagram showing the time point of MSNs administration to mice transformed with H-ras (top), and the number of tumors formed over time after administration of MSNs alone and MSNs_siRNA to mice transformed with H-ras was measured. Results are shown (bottom). 7f shows western blot results of BANF1, PLOD3 and SF3B4 expression levels in liver tissues of mice transformed with H-ras.
Figure 8 shows the results using mesoporous silica nanoparticles (MSN) containing siRNA, 8a is transfected with siRNA for each gene, and then BANF1, PLOD3 and SF3B4 expression levels in Hepa-1c1c7 mouse liver cancer cells 8b shows the expression levels of BANF1, PLOD3, and SF3B4 in Hepa-1c1c7 mouse liver cancer cells transfected with mesoporous silica nanoparticles (MSN) and siRNAs of each gene, and 8c is mesoporous silica nanoparticles. MSN) and BANF1, PLOD3 and SF3B4 expression levels in Hep3B cells transfected with siRNA of each gene.
FIG. 9 shows the clinical feasibility of SF3B4 as a liver cancer marker. 9a shows the expression levels of BANF1, PLOD3 and SF3B4 mRNA in the TCGA_LIHC data set, and 9b shows the total (left) and disease-free survival of HCC patients. Kaplan-Meier survival curves with mRNA expression of three new markers in the patient (right) are shown. 9c shows Kaplan-Meier survival curves for BANF1, PLOD3 and SF3B4 mRNA expression across HCC patients. 9d shows Kaplan-Meier survival curves for a combination of SF3B4 copy number amplification and mRNA upregulation in all HCC patients (left) and disease-free patients (right), and 9e shows the gene copy number of SF3B4 in the TCGA_LIHC data set. The frequency of variation was shown (left), and the correlation between copy number variation and mRNA expression (right). 9f shows the results of qRT-PCR analysis of gene copy number (left) and mRNA expression level (right) of SF3B4 for 20 pairs of HCC patient tissues.
10 shows cell cycle inhibition and EMT progression inhibition of liver cancer cells following the destruction of SF3B4, 10a shows genetic variation of SF3b complex subunit in 366 HCC patients in TCGA_LIHC data set, and 10b indicates designated HCC The GEO database shows a comparison of the relative expression levels of SF3b complex subunits for tumor and non-tumor, and 10c shows SFRT3PC4 mRNA expression in qRT-PCR (top) and Western in 2 normal hepatocyte and 8 HCC cell lines. The result of analysis by blot (bottom) is shown. 10d shows a heatmap for autonomic hierarchical clustering analysis of 342 SF3B4 correlation genes (left) and Kaplan-Meier survival curves for the SF3B4 high cluster patient group and the SF3B4 low cluster patient group of the GSE16757 dataset. 10e shows a set of genes in GSEA related to SF3B4, with a total of 11 gene lists identified by MSigDB (top). The growth-related gene set (KEGG_CELL_CYCLE) has been shown to be a feature associated with SF3B4, with the barcode indicating the location of the gene (bottom). 10F shows FACS analysis of two HCC cell lines after transfection with si-SF3B4, the left side shows the histogram and the right side shows the percentage of each cell cycle stage (p <0.05, p <0.01, p <0.001; t test).
Figure 11 analyzes the effect of knockdown of SF3B4 in HCC cell line, 11a is transfected with si-RNA Cont (control) and si-RNA SF3B4 for SNU-449 and SNU-368 cell line, respectively, cell survival rate (Left) and cell proliferation rate (right) are shown, 11b is the result of analysis of the degree of cell colonization, 11c is the transwell migration and invasion analysis to image the degree of migration and invasion of liver cancer cells And left (left) and moving cell numbers (right). 11d shows the results of analysis of wound healing effects at 0 and 24 hours after knocking down SF3B4, 11e shows Western blot expression of EMT regulatory proteins, and 11f shows 48 hours after knocking down SF3B4. The histogram results for the PI stained cells using FASC.
Figure 12 shows the analysis of the regulators of SF3B4 during liver cancer development, 12a shows the analysis of Western blot analysis of the expression of each protein in HeLa cells and hepatocellular carcinoma cell lines treated with siRNA and SSA for SF3B4, respectively. (Top), mRNA levels of the genes were analyzed by qRT-PCR. 12b shows Western blot expression of cell cycle regulators for each experimental group of 12a, and 12c shows a pipeline of gene expression analysis and selective splicing event analysis using the GSE80861 RNA-seq data set ( Left, pie charts show the distribution of genes with different differential expression (DEG, upper right) and selective splicing event (AS) types. 12d shows the relative expression levels of GRIP1, PLP1, KLF4 and KRT80 in the TCGA_LIHC dataset, 12e shows the results of qRT-PCR analysis of KLF4 expression in cells transfected with SF3B4 siRNA, and 12f shows the Sashimi plot. Shows selective splicing of KLF4 and skipped exons (SE) (p <0.05, p <0.01, p <0.001; t test).
FIG. 13 is an analysis of KLF4 regulation ability of tumor suppressor of SF3B4 in liver cancer cell line, and 13a is MTT analysis of cell viability of SNU-449 and SNU-368 cell lines treated with SF3B4 siRNA (left) or SSA (right). 13b shows the relative expression changes and selective splicing (comparison of SF3B4 knocked down versus control) for eight genes in the GSE80861 dataset. 13c shows the analysis of the expression levels of GRIP1, PLP1, KLF4 in SNU-449 and SNU-368 cell lines transfected with SF3B4 siRNA, and 13d and 13e were derived from TCGA_LIHC dataset (13d) and GSE77314 dataset (13e). Analysis of expression correlations between SF3B4 and 8 genes in 50 pairs of HCC patients.
Figure 14 shows the function of SF3B4, 14a p27 ^Kip1 in SF3B4 knockdown or KLF4 overexpressed HCC cell line And Slug expression level, 14b p27 ^Kip1 in SF3B4 knockdown or KLF4 overexpressed SNU-449 cells Or luciferase activity of the reporter plasmid containing the Slug promoter sequence, 14c showing RT-PCR product images of KLF4 mRNA and two KLF4 mRNA isoforms in SF3B4 knockdown cells (left) and in HCC cell lines. The two KLF4 mRNA isoforms identified and the splicing site are marked at the bottom of each diagram (middle) and the red lines represent splicing junctions (right). 14d represents tumor growth curve (left) and fluorescence image (right) in nude mouse model transplanted with SF3B4 knockdown SNU-449 cells, and 14e represents cancer tissue obtained from 14d mouse animal model. The results of analyzing the expression level of SF3B4 and KLF4 is shown, 14f shows a model of the carcinogenic action of SF3B4 through the inhibition of wild-type KLF4 expression in HCC.

The present invention provides novel biomarkers for early detection and diagnosis of liver cancer, such as barrier to autointegration factor 1 (BANF1), procedure-lysine (PLO3), 2-oxoglutarate 5-dioxygenase 3 (PLOD3), and splicing factor 3b subunit 4 (SF3B4). It is characterized by the fact that it can be used.

Liver cancer is known as the most fatal cancer in the world, and when it develops, it is a cancer with a high mortality rate due to a poor prognosis. Therefore, if liver cancer can be detected at an early stage, liver cancer can be prevented at an early stage and appropriate treatment can be applied to enhance the therapeutic effect.

However, most of the liver cancer is judged in the state of advanced cancer progression, it was necessary to develop a marker that can determine the early onset of liver cancer.

Therefore, the inventors of the present invention, while researching to find a biomarker for early diagnosis of liver cancer, confirmed the possibility of using BANF1, PLOD3 and SF3B4 as an early diagnosis marker of liver cancer, and if inhibited, can prevent or treat liver cancer. By confirming the presence of BANF1, PLOD3 and SF3B4 as a new target for the treatment of liver cancer.

  Barrier to autointegration factor 1 (BANF1) is a conserved human chromatin protein known to be primarily involved in nuclear assembly and chromatin decondensation, and BANF1 protects retroviruses from integrating between molecules. It was first identified as a viable protein, and its function is known to promote intermolecular integration into host cell genomes. However, the relationship between BANF1 and cancer has not been revealed.

PLOD3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) is a hydroxylysine-linked carbohydrate due to the activity of galactosyl- and glucosyl-transferases, which are important for procollagen intermolecular crosslinking and stabilization of the supramolecular collagen structure of fibers. It is known to be the only isoenzyme that produces it. In addition, PLOD3 is known to be involved in recruiting MMP9, and there is no known progression of cancer and its association with cancer. Splicing factor 3b subunit 4 (SF3B4) encodes a key protein of the mammalian SF3b complex and is known for binding U2 snRNP to the linking site as part of the U2-type spliceosome. The relevance of is unknown.

  On the other hand, the present inventors discriminate in normal liver tissue, early liver cancer tissue, and advanced liver cancer tissue using whole transcriptome-expressing microarrays to discover novel biomarkers that can predict and diagnose liver cancer early. Screening for genes showing expression levels confirmed that BANF1, PLODS3 and SF3B4 were all strongly overexpressed compared to normal tissue, and that these genes were found in all six early hepatocellular carcinoma sections in which cancer was transitioned from HGDN. It was found to be overexpressed.

  In particular, the markers discovered in the present invention are characterized by the ability to detect and diagnose early liver cancer in precancerous lesions.

Hepatocarcinoma has the development of multi-stage lesions ranging from normal liver to chronic hepatitis, cirrhosis and liver cancer. With recent advances in radiological examination techniques, small nodules are found in the liver, and large nodules are likely to progress to liver cancer. In particular, dysplastic nodule (DN), which is difficult to distinguish from liver cancer and has some characteristics of liver cancer, is a part of multi-stage development of liver cancer and is known as a representative form of precancerous lesion of liver cancer.

In addition, the nodular lesions observed in cirrhosis are regenerative nodules, dysplastic nodules, hepatocellular carcinomas, and focal fatty lesions. The regenerative nodules are 1-10 mm in size, spread throughout the liver, and uniform in size. Large regenerative nodules larger in size than the surrounding remodeling nodules have no cell orientation and have a normal context within the nodules. Dysplastic nodules are mostly 1-1.5 cm in size and have no encapsulation. Low grade dysplastic nodules are caused by the proliferation of monoclonal cells, which may be accompanied by a single artery without any cellular or structural abnormalities. High grade dysplastic nodules show cell dysplasia and dendritic abnormalities and polymorphisms of the nucleus of small cell changes.

The markers of the present invention can detect premature liver cancer in precancerous lesions. The precancerous lesions refer to the stage before the cancer is passed and do not require treatment comparable to liver cancer. Dysplastic nodule (DN) including LGDN; low-grade DN) or high-grade dysplastic nodule (HGDN), and in one embodiment of the present invention, the HGDN tissue is a precancerous lesion. Three genes were overexpressed compared to normal tissue.

Further, to confirm that the markers of the present invention are better than conventional markers, GPC3, GS, which is known to be able to distinguish early tumor cancer from other tumor lesions, including dysplastic nodules (DN), hepatocellular adenoma and local nodular hyperplasia And HSP70 markers and markers of the present invention, the expression patterns in hepatocellular carcinoma tissue were analyzed.

As a result, the positive expression rate of the conventional marker expressed in the hepatocellular carcinoma tissue was 50.9%, while the positive expression rate of the marker of the present invention was 72.7%. In addition, the negative expression rate of the conventional marker was 49.1% in hepatocellular carcinoma tissue, while the negative expression rate of the novel marker of the present invention was 27.3%.

Therefore, these results indicate that the BANF1, PLODS3 and SF3B4 markers identified in the present invention can diagnose liver cancer with better efficiency and accuracy than the conventional markers GPC3, GS and HSP70.

In addition, in one embodiment of the present invention, the analysis of Kaplan-Meier survival curves of hepatocellular carcinoma patients, disease-free survival rate of patients showing high expression of the novel markers of the present invention than patients with low expression level of the markers of the present invention It was found to be significantly lower, especially when the combination of the markers of the present invention was used alone, the accuracy was higher.

This suggests that the novel markers of the present invention have a carcinogenic effect that induces the development of hepatocellular carcinoma, and it is possible to diagnose early liver cancer by measuring the expression level of these markers, and further predict the survival prognosis of the patients. Could know.

Therefore, the present invention includes early liver cancer comprising at least one selected from the group consisting of barrier to autointegration factor 1 (BANF1), procedure-lysine (2-oxoglutarate 5-dioxygenase 3) and PL3B4 (splicing factor 3b subunit 4). Diagnostic marker compositions may be provided.

In another aspect, the present invention may provide a composition for detecting early liver cancer, comprising an agent for measuring mRNA expression level of at least one early liver cancer diagnostic marker protein selected from the group consisting of BANF1, PLOD3 and SF3B4 or a gene encoding the same.

 In the present invention, the term "diagnosis" refers to confirming a pathological condition, and for the purpose of the present invention, the diagnosis means confirming the presence or absence of expression of a liver cancer diagnostic marker and confirming the occurrence of liver cancer, and liver cancer. It also includes determining whether the diagnostic markers are expressed and the extent of their expression, thereby determining whether liver cancer develops, develops, and alleviates. In particular, the present invention can be used to diagnose early liver cancer.

In addition, the marker of the present invention can be used as a means for determining the prognosis of liver cancer, in the present invention, the "prognosis" is a cancer, such as the possibility of cure of cancer, the possibility of recurrence after treatment, the patient's survival after cancer is diagnosed According to predict the various conditions of the patient. The prognosis of cancer can be estimated from various points of view, but can typically be determined in terms of recurrence, viability, and disease-free survival. Prognosis for the purposes of the present invention may mean the prognosis of survival after diagnosis of liver cancer. By using the biomarkers provided in the present invention, it is possible to more easily predict the survival prognosis of liver cancer patients, which may be utilized to classify patients at high risk or to determine whether to use additional treatment methods. It can contribute to increase the survival rate later.

In addition, the "prediction" relates to whether and / or the likelihood that the patient will survive the treatment of the patient by preferentially or unfavorably responding to the therapy. The estimation method of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment modality for patients with cancer. In addition, the predictive methods of the present invention determine whether a patient preferentially responds to a treatment regimen, such as, for example, a treatment regimen, including administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc. It can be used to predict whether long term survival is possible.

In addition, the "diagnosis marker" refers to a substance capable of diagnosing cells or tissues of liver cancer by distinguishing them from normal cells or tissues, and a polypeptide showing an increase or decrease in cells of liver cancer compared to normal cells or Organic biomolecules such as nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. The marker for diagnosing liver cancer, preferably the marker for early diagnosis of liver cancer, provided by the present invention is BANF1, PLOD3 or SF3B4, in which the amount of expression is increased in cells (or tissues) or cells (or tissues) prior to developing liver cancer compared to normal cells Can be.

In the present invention, the BANF1 gene consists of the nucleotide sequence of SEQ ID NO: 1 and the BANF1 protein consists of the amino acid sequence of SEQ ID NO: 2, the PLOD3 gene consists of the nucleotide sequence of SEQ ID NO: 3, and the PLOD3 protein consists of the amino acid sequence of SEQ ID NO: 4 SF3B4 gene is composed of the nucleotide sequence of SEQ ID NO: 5 and SF3B4 protein may be composed of the amino acid sequence of SEQ ID NO: 6.

In the present invention, the level of the marker gene, preferably means the mRNA level, that is, the amount of mRNA, the expression of the marker gene, the material capable of measuring the level of the marker of the present invention BANF1, PLOD3 or SF3B4 gene It may include primers, probes, aptamers, antibodies or substrates specific for the.

In the present invention, the term “primer” refers to a single which can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers. -Refers to stranded oligonucleotides. Suitable lengths of primers can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can hybridize with the template to perform the primer-specific action. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the BANF1, PLOD3, or SF3B4 gene, which is a template. Do.

The “amplification reaction” refers to a reaction for amplifying a nucleic acid molecule, and amplification reactions of such genes are well known in the art, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR). , Ligase chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), and the like.

In the present invention, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, which is a natural It exists or artificially synthesized. The probe according to the invention may be single chain, preferably oligodioxyribonucleotides. Probes of the invention can include natural dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probes of the present invention may also comprise ribonucleotides. For example, the probes of the present invention can be used in the backbone modified nucleotides such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohex Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Tityl-, propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro- , Iodo-, me -, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine.

In addition, in the present invention, the level of the BANF1, PLOD3 or SF3B4 protein, preferably means a polypeptide for each marker produced through the translation process from mRNA expressed BANF1, PLOD3 or SF3B4 gene, and measure the level of the protein Substances that can be used include “antibodies” such as polyclonal antibodies, monoclonal antibodies, and recombinant antibodies that can specifically bind to BANF1, PLOD3, or SF3B4 proteins.

In addition, in the present invention, as BANF1, PLOD3 or SF3B4 protein has been identified as a marker protein for diagnosing liver cancer as described above, a method for producing an antibody using the protein is known to those skilled in the art. It can be produced easily using the technique described. For example, in the case of polyclonal antibodies, BANF1, PLOD3 or SF3B4 antigens can be injected into the animals and collected from the animals to obtain a serum containing the antibody, which is well known in the art, and such polyclonal antibodies can be produced by goats. , Rabbits, sheep, monkeys, horses, pigs, cattle, dogs and the like. Monoclonal antibodies can be prepared using hybridoma methods well known in the art (Kohler et al., European Jounral of Immunology , 6, 511-519, 1976) or phage antibody libraries ( Clackson et al, Nature , 352, 624-628, 1991, Marks et al, J. Mol. Biol ., 222: 58, 1-597, 1991). In addition, the antibodies according to the invention may comprise functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

In addition, the present invention may provide a kit for early liver cancer diagnosis or prognostic diagnosis comprising a composition containing an agent capable of measuring the expression level of a BANF1, PLOD3 or SF3B4 protein or a gene encoding the same.

Liver cancer diagnosis or prognostic diagnostic composition included in the kit of the present invention may include a primer, a probe, an aptamer, an antibody or a substrate capable of measuring the expression level of a BANF1, PLOD3 or SF3B4 protein or a gene encoding the same, these The definition of is as described above.

If the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermal stability DNA polymerase obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs, and when the liver cancer diagnostic kit of the present invention is applied to an immunoassay, Kits of the invention may optionally comprise a secondary antibody and a substrate of the label. Furthermore, the kits according to the invention can be produced in a number of separate packaging or compartments comprising the reagent components described above.

The present invention also provides a microarray for early liver cancer diagnosis comprising a composition for diagnosing early liver cancer capable of measuring the expression level of a BANF1, PLOD3 or SF3B4 protein or a gene encoding the same.

In the microarray of the present invention, primers, probes or antibodies capable of measuring the expression level of a BANF1, PLOD3 or SF3B4 protein or a gene encoding the same are used as a hybridizable array element and used on a substrate. Is immobilized on. Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. The hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by a chemical bonding method or a covalent binding method such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine).

On the other hand, when the sample to be applied to the microarray of the present invention is a nucleic acid can be labeled (labeled), it can be hybridized with the array element on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

In addition, the present invention may provide a method for detecting one or more early liver cancer diagnosis markers selected from the group consisting of BANF1, PLOD3 and SF3B4 from the sample of the patient in order to provide information necessary for diagnosing liver cancer.

In addition, the present invention comprises the steps of measuring the expression level of mRNA or protein for at least one gene selected from the group consisting of BANF1, PLOD3 and SF3B4 from biological samples isolated from patients; And comparing the measured expression level of BANF1, PLOD3 or SF3B4 with the expression level of the gene of the control sample, it can provide a method for providing information for early liver cancer diagnosis.

The method for measuring the expression level or protein level of the BANF1, PLOD3 or SF3B4 gene in the above may be carried out including a known process for detecting or separating mRNA or protein from biological samples using known techniques.

In the present invention, the "biological sample" refers to a sample collected from a living body in which the expression level or protein level of the BANF1, PLOD3 or SF3B4 gene according to the incidence or progression of liver cancer is different from a normal control group, and examples of the sample include: For example, but not limited to, tissue, cells, blood, serum, plasma, saliva and urine and the like can be included. Preferably, it may be a precancerous lesion, and the precancerous lesion may be included in all stages before progressing to liver cancer, and in one embodiment of the present invention, the precancerous lesion may be a high grade dysplastic node (HGDN; high-grade DN). Samples were used.

The measurement of the expression level of the marker gene is preferably to measure the level of mRNA, the method of measuring the level of mRNA reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blots and DNA chips, but are not limited thereto.

The marker protein level may be measured using an antibody, in which case the marker protein in the biological sample and the antibody specific thereto form a conjugate, i.e., an antigen-antibody complex, and the amount of antigen-antibody complex formed is a detection label. It can be measured quantitatively through the magnitude of the signal of the (detection label). Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement fixation assays, FACS, protein chips, and the like.

Therefore, the present invention can determine the amount of mRNA expression or protein expression of the marker gene of the control group and the amount of the marker gene mRNA expression or protein in liver cancer patients or suspected liver cancer patients through the above-described detection methods, the expression By comparing the amount of the sheep with a control, that is, a sample of a normal person, it is possible to predict and diagnose the onset, progression or prognosis of liver cancer.

According to one embodiment of the present invention, a tissue lysate is obtained from liver cancer tissues and normal liver tissues obtained from liver cancer patients, and then subjected to western blot using an antibody against BANF1, PLOD3 or SF3B4 from each sample. After measuring the amount of protein, the measurement was performed through a process of comparative analysis with the normal control.

In addition, the inventors have found that BANF1, PLOD3 or SF3B4 inhibitors can be used as a treatment for liver cancer. According to one embodiment of the present invention, as a BANF1, PLOD3 or SF3B4 inhibitor by knocking down using siRNA that inhibits the expression of these genes, and after analyzing the growth and proliferation of hepatocellular carcinoma, when inhibiting the expression or activity of these genes It was found that the growth and proliferation of cancer cells was inhibited, cell cycle abnormality was induced, and cell death was induced to ultimately prevent and treat liver cancer.

Therefore, a substance capable of inhibiting the activity and expression of BANF1, PLOD3 or SF3B4 can be used as a therapeutic agent for liver cancer, the present invention includes an inhibitor of BANF1, PLOD3 or SF3B4 as an active ingredient, pharmaceutical for inhibiting metastasis and recurrence of liver cancer A composition can be provided.

The pharmaceutical composition according to the present invention may include any material capable of inhibiting the expression or activity of BANF1, PLOD3 or SF3B4, preferably chemical, nucleotide, antisense, siRNA oligonucleotide or natural extract as an active ingredient. It may include, more preferably may comprise an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to the nucleotide sequence of the BANF1, PLOD3 or SF3B4 gene of the present invention as an active ingredient, even more preferably siRNA can be used.

In the present invention, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a specific mRNA, and binds to a complementary sequence in the mRNA to bind BANF1, PLOD3 or SF3B4. The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to BANF1, PLOD3 or SF3B4 mRNA and capable of binding to BANF1, PLOD3 or SF3B4 mRNA, and which is capable of binding to BANF1, PLOD3 or SF3B4. It can inhibit the translation of mRNA, translocation into the cytoplasm, maturation or any other essential activity on all other biological functions.

In addition, the antisense nucleic acid may be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol ., 5, 3, 343-55, 1995 ). The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may comprise one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides comprising fat-soluble moieties and methods of preparation are already well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

Antisense oligonucleotides can be synthesized in vitro in conventional manner to be administered in vivo or to allow antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is to use RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the recognition site (MCS). Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.

As used herein, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Patent Nos. 00/44895, 01/36646, 99/32619, 01/29058, 99/07409, and 00 SiRNA is provided as an efficient gene knock-down method or gene therapy method because it can inhibit the expression of a target gene.

The siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to a TCIRG1 mRNA sequence) and an antisense strand (a sequence complementary to a TCIRG1 mRNA sequence) are positioned opposite to each other to form a double-stranded structure. The siRNA molecule of can have a single chain structure with self-complementary sense and antisense strands. Furthermore, siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but can be paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. Parts that do not achieve may be included. In addition, the siRNA terminal structure can be either blunt or cohesive ends as long as the expression of the TCIRG1 gene can be suppressed by the RNAi effect, and the adhesive end structures are 3'-terminal protrusion structures and 5'-terminal structures. Both protruding structures are possible.

In addition, the siRNA molecules of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is subjected to intramolecular hybridization. As a result, a hairpin structure is formed, and as a whole, a stem-and-loop structure is formed. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The method for preparing siRNA is to directly synthesize siRNA in vitro and then introduce it into the cell through a transformation process, and to siRNA expression vector or PCR-derived siRNA expression cassette prepared to express siRNA in the cell. There is a method of conversion or infection.

In addition, the compositions of the present invention comprising gene specific siRNAs may include agents that promote intracellular influx of siRNAs. Agents that promote the influx of siRNA into the cell generally can be used agents that promote the influx of nucleic acids. Examples of these include the use of liposomes or the lipophilic of one of many sterols, including cholesterol, cholate and deoxycholic acid. It can be combined with a carrier. Poly-L-lysine, spermine, polysilazane, polyethylenimine, polydihydroimidazolenium, polyallylamine Cationic polymers such as chitosan), succinylated PLL, succinylated PEI, polyglutamic acid, and polyaspartic acid. anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulfate, heparin, hyaluronic acid, etc. It is available.

In addition, when an antibody specific for the BANF1, PLOD3 or SF3B4 protein is used as a substance for reducing the expression and activity of the BANF1, PLOD3 or SF3B4 protein, the antibody is directly coupled to an existing therapeutic agent or indirectly through a linker or the like. For example, covalent bonds). Therapeutic agents that can be combined with the antibody include, but are not limited to, radionuclide such as 131I, 90Y, 105Rh, 47Sc, 67Cu, 212Bi, 211At, 67Ga, 125I, 186Re, 188Re, 177Lu, 153Sm, 123I, 111In, etc. ); Biological response variants or drugs such as methotrexate, adriamycin, and lympokine such as interferon; Toxins such as lysine, abrin, diphtheria and the like; Heterofunctional antibodies, ie antibodies that bind to other antibodies so that the complex binds to both cancer cells and agonist cells (eg, K cells such as T cells); and -May be associated with associated or non-complexed antibodies.

  In addition, in the present invention, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, and the like. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as an injectable formulation as a typical parenteral dosage form. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner may be administered in an effective amount via a variety of routes including oral, transdermal, subcutaneous, intravenous or intramuscular. The term “administration” introduces certain substances into a patient in any suitable manner. The route of administration of the substance may be administered via any general route as long as it can reach the target tissue.

In addition, the above "effective amount" refers to an amount that exhibits a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the disease type and severity of the patient, age, sex, weight, sensitivity to the drug, type of current treatment, administration method, target cell, and the like. It can be easily determined by experts. In addition, the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be single or multiple administrations. Preferably all of the above factors can be administered in an amount that can achieve the maximum effect in a minimum amount without side effects, more preferably 1 to 10000 ㎍ / weight kg / day, even more preferably 10 to 1000 mg It may be administered repeatedly several times a day at an effective dose of / kg body weight / day.

The present invention further comprises the steps of (a) contacting a sample to be analyzed with a cell comprising a BANF1, PLOD3 or SF3B4 gene or a protein thereof; (b) measuring the expression amount, protein amount or protein activity of the BANF1, PLOD3 or SF3B4 gene; And (c) when the amount of expression of the BANF1, PLOD3 or SF3B4 gene, the amount of protein or the activity of the protein is reduced as a result of the measurement in step (b), the sample is determined to be a substance for preventing or treating liver cancer. It may include a method for screening a substance for preventing or treating liver cancer.

According to the method of the present invention, first, a sample to be analyzed may be contacted with a cell containing or expressing a BANF1, PLOD3 or SF3B4 gene or a protein thereof. Here, the "sample" refers to an unknown substance used in screening to test whether the expression of the BANF1, PLOD3 or SF3B4 gene, the amount of protein or the activity of the protein is affected. The sample may include, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts. Thereafter, the expression amount of the BANF1, PLOD3 or SF3B4 gene, the amount of protein or the activity of the protein in the cells treated with the sample can be measured, and as a result of the measurement, the amount of expression of the BANF1, PLOD3 or SF3B4 gene, the amount of protein or protein When the decrease in activity is measured, the sample can be determined to be a substance that can treat or prevent liver cancer.

The activity measurement method in the above may be carried out through various methods known in the art, for example, but not limited to reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction ( real time-polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay can do.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Preparation Example>

Sample Preparation

Three independent liver disease patient cohorts were used for the following experiments. Cohort 1 (107 tissues from 81 patients, snap-frozen tissue) was used for gene expression microarrays, cohort 2 (546 tissues from 546 patients, tissue microarray) was used for tissue microarrays, and cohort 3 ((160 tissues from 70 patients, full section) were used for immunohistochemistry The clinical pathologic features of these three groups of cohorts are shown in Table 1 below.

Progression free survival was defined as the interval between the date of surgery and the date when a particular type of relapse was diagnosed. In addition, all subjects obtained written informed consent based on the declaration, and this study was approved by the Institutional Review of Board (IRB) of the Catholic University Medical College (IRB Approved Numbers: MC12EISI0106, MC12SNMI0184).

Experimental Method

① Tissue microarray and immunohistochemical staining

To construct a tissue microarray (TMA), hepatic cancer tissues of cohort 2 (n = 546) patient groups immobilized with formalin and then embedded in paraffin were mapped for pathologists to check their scores. Liver tissue was punched out of each block and cut into 5 mm thick sections using 0.6 mm diameter stilettos and placed in paraffin blocks. The sections were adhered to the slides using the adhesive transfer tape method and crosslinked by UV irradiation. Immunohistostaining was performed using the antibodies of Table 2 below for TMA and whole section samples to examine each protein level.

TABLE 2

List of Antibodies Used in Western Blot and Tissue Micro Arrays

② TCGA and NCBI GEO Data Analysis

To reconstruct the expression levels of HCC markers in HCC, data were obtained from the TCGA Hepatocellular Carcinoma Project and the NCBI GEO Database (Accession Number: GSE16757, GSE22058, GSE25097, GSE36376, GSE44106, GSE45436, GSE54236, GSE80861, GSE89377, and GSE93392). . TCGA RNA-seq data were first analyzed for replacement of log2 after all RPKM values were replaced.

③ Microarray analysis

Frozen liver tissue from Cohort 1 patient group was used for gene expression microarray analysis for large scale gene expression profiling. Three independent sets were selected from samples of Cohort 1 patient group, total RNA was extracted using TRIzol reagent, and RNA was isolated using Illumina Total-Prep RNA Amplification Kit column (Ambion Inc., Austin, TX). Quality control of RNA was performed using Experion ™ system (Bio-Rad, Hercules, CA), and microarray analysis was performed using HumanHT-12 V4 Expression BeadChips (Illumina, Inc., San Diego, CA). Biotinylated cRNA was purified using the MessageAmp kit (Ambion, Inc.), and the hybridization and scanning of chips were performed according to Illumina's standard method. Primary microarray data is available in the GEO database (GSE89377).

④ Western blot analysis

The patient's frozen tissues and cells were extracted with protein extract buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na2P2O7, 1 mM Na3VO4, 100 x Halt protease inhibitor cocktail). Lysates containing the same amount of protein were electrophoresed on SDS-PAGE, electrophoresed with PVDF membrane and then blocked with blocking buffer (5% skimmed milk in Tris-buffered saline, 0.1% Tween-20). Next, Western blot was performed by using the antibodies of Table 2 above.

⑤ Motility and invasion assay

Cell migration and infiltration assays were performed in vitro, cell migration was performed by a modified Boyden chamber assay (BD Bioscience), and infiltration analysis was performed using Matrigel (BD Biosciences). Matrigel was diluted with coating buffer to a concentration of 0.3 mg / ml for infiltration analysis. The diluted Matrigel solution dispensed with 100ul was then covered with a portion of the transwell cell culture insert, incubated at 37 ° C. for 1 hour, and then the insert (insert) was prepared for seeding. After transfecting the cells with si-SF3B4, the cells were applied to transwell inserts and cultured using a serum free medium containing 5% FBS as chemoattractant. After cell culture for each assay (cell migration: time incubation, infiltration assay: 12 hours), migrated or infiltrated cells were stained using a Diff-Quik staining kit (Sysmex, Japan) and Axiovert 200 Inverted Microscope (Zeiss, Jena, Germany) at 200-fold magnification.

⑥ Transfection of Mesoporous Silica Nanoparticles

Specific si-RNA for BANF1, PLOD3 and SF3B4 was added to 80 ul InViVojectionTM RNAi-nano reagent (mesoporous silica nanoparticle, Cat.No. DHMSN-vivoRNA; Lemonex Inc., Seoul, Korea) at a concentration of 3 nmol and 200ul PBS solution was prepared. Mixtures of MSNs and si-RNA were injected intravenously into the tail weekly for 9-23 weeks in H-ras transformed HCC mouse model. After that, the ultrasound image was taken on the 17th week, 19th week, and 21st week of the week, and image images were collected.

Cells were incubated overnight and then stained using Diff-Quik stain kit (Sysmex, Chuo-ku, Cobe, Japan). Cell images were observed using an Axiovert 200 inverted microscope (Zeiss, Oberkochen, Germany) at 200 × magnification and counted cell numbers in three random image fields.

⑦ Production of liver cancer transplant mouse model

For xenograft tumor formation assays, cells for transfection of 1 × 10 ⁷ cells were 0.2

Mix with ml PBS (pH 7.4) and 30% (v / v) Matrigel matrix (BD Biosciences). The cell suspension was injected subcutaneously into 6 week old male Balb / c nude mice. Mice were observed for tumor formation at the injection site twice a week. Tumor volume was calculated as 0.5 × length (L) × width ² (W ² ). Each experimental group consisted of 10 mice, and tumor growth was measured in three orthogonal directions using calipers. Results are expressed as mean tumor volume and 95% confidence intervals. H-ras12V-activated homozygous transgenic mice were obtained from Dr. Yu, Dae-Yeol (Human Genome Lab, Korea Lab, Korea Research Institute of Biotechnology, Daejeon). Transgenic mice are H-ras12V activated mice. Male mice began to develop spontaneously from 15 weeks of age. Thus, the present inventors obtained by resecting non-tumor tissue and HCC tissue from five 35-week-old mice, and selected three pairs of HCC tissue to perform a pathological investigation. Induction of HCC was performed using DEN (Diethylnitrosamine).

⑧ Statistical Analysis

Survival curves were plotted using the Kaplan-Meier product limit method, and differences in survival curves were determined using the log-rank test. All experiments were performed at least three times and all samples analyzed in triplicate. The results are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical differences between groups were evaluated by unpaired bilateral Student's t-test using Graphpad ™ 5.0 (GraphPad software Inc., San Diego, Calif., USA). It was judged to be statistically significant for p <0.05, and chi-square test (2-sided) was used to determine the association between parameters.

⑨ RNA and DNA isolation, RT-PCR and qRT-PCR analysis

Total RNA was isolated from frozen tissues and cell lines using TRIzol reagent. 1 ug of total RNA was synthesized cDNA by reverse transcription using the Tetro cDNA Synthesis Kit (Bioline, London, UK). RT-PCR reaction was performed using nTaq DNA polymerase (Enzynomics, Daejeon, Korea), and the DNA was confirmed by ethidium bromide reaction with Gel Doc XR image system (Bio-Rad, Hercules, CA). qRT-PCR was performed using SensiFAST SYBR No-ROX Kit (Bioline) and real-time monitoring using iQ ™ -5 (Bio-Rad). Mean Ct by triplicate analysis was used for further calculations. Normalized gene expression was determined using relative quantification methods. The results are shown as the average of three experiments. Genomic DNA of tissues and cells was extracted and isolated according to instructions using DNAzol reagent (Invitrogen). For copy number variation analysis, the SF3B4 genomic DNA region was genbank accession no. Amplified from 20 pairs (non-tumor and tumor) HCC tissue using primer sets covering exon-1 to intron 1 according to genome sequence of NC_000001.11. qRT-PCR was reacted as described above, and glycerol aldehyde-3-phosphate dehydrogenase was used as an endogenous loading control. The sequences of the primers used for RT-PCR and qRT-PCR are shown in the table below.

TABLE 3

Primer sequence

⑩ Cell culture

Human HCC cell lines (Hep3B, PLC / PRF / 5, SK-Hep-1, SNU-354, SNU-368, SNU-387, SNU-423, SNU-449) and Hepa-1c1c7 mouse hepatic cell lines Seoul, South Korea) was used. Immortalized liver cell lines MIHA and L-02 Provided and used by Jayanta Roy-Chowdhury (Albert Einstein College of Medicine, New York, NY). Each cell line was incubated in EMEM (American Type Culture Collection, Manassas, VA), RPMI-1640 or DMEM medium containing 10% FBS, 100units / mL penicillin-streptomycin (Invitrogen, Carlsbad, Calif.), All cultures 5% The culture was carried out under the condition that carbon dioxide and a temperature of 37 ° C were maintained.

⑪ transfection

SiRNAs for transfection were synthesized by Genolution (Seoul, Korea) or used by Bioneer (Daejeon, Korea). The base sequence of si-RNA is as shown in Table 4 below. KLF4 expressing plasmids with 1.4 Kb human KLF4 ORF (NM_004235) subcloned into pcDNA3.1 + / C- (K) -DYK plasmid were purchased from GenscriptTM (Piscataway, NJ). Transfection was performed using Lipofectamine RNAiMAX or Lipofectamine 2000 reagent (Invitrogen). Spliceostatin A (SSA), an SF3b complex inhibitor, was purchased from AdooQ Bioscience (Cat No. A12700; Irvine, CA, USA) and used.

TABLE 4

siRNA sequence

⑫ Cell growth and cell proliferation analysis

For cell growth analysis, cells were first seeded in 30-well plates in 30-well plates. After treatment with the transfection and inhibitor, 0.5 mg / mL MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was treated and reacted for 1 hour. Formazan crystals were dissolved in DMSO and absorbance was measured at 570 nm using a VICTOR3 ™ multilabel plate reader (PerkinElmer, Boston, Mass.).

For cell proliferation analysis, cells were aliquoted into 30-well plates in 24-well plates. After transfection, cells were treated with BrdU (5-bromo-2'-deoxyuridine) and reacted for 2 hours, and then fixed at room temperature for 30 minutes. The cells were reacted with the antibody against anti-BrdU for 1 hour at room temperature. Unreacted antibodies were removed by washing with wash buffer. Horseradish peroxidase-bound secondary antibody was added to each well, and then reacted with the addition of a substrate solution, followed by addition of the reaction stop solution to terminate the reaction. The final reaction product was measured at 490 nm absorbance using a VICTOR3 ™ multilabel plate reader (PerkinElmer).

C Colonogenic assay

In a 6-well plate Cells were aliquoted (1000 cells / well) and cells were transfected with si-control and si-SF3B4. Cell colonies formed after 2 weeks were fixed with formaldehyde (1% v / v) at room temperature for 30 minutes and crystallized. Staining with violet (0.5% v / v) The number of colonies formed was analyzed using the clone counter program.

⑭ Wound Healing Analysis

The transfected cells were dispensed into 6-well plates and the plates were filled to 100% and scratched using a micropipette tip. Afterwards, the image of the stretched region was observed using an IX70 fluorescence inverted microscope (Olympus, Tokyo, Japan).

⑮ apoptosis assay

Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) was used to analyze the degree of cell death. After transfection, liver cancer cells were washed with cold PBS buffer and suspended with 1 × binding buffer. 1 × 10 ⁵ cells were transferred to 5 ml culture tubes and mixed by adding 5 ul of annexin V-FITC and 10 ul propidium iodide solution. After reacting at room temperature for 20 minutes under dark conditions, 400ul of 1 × binding buffer was added to each tube, and then fractions in which apoptosis occurred were analyzed using a FACS Calibur flow cytometer (BD Biosciences).

 Cell cycle analysis

Liver cancer cell lines were transfected with si-SF3B4, incubated for 48 hours and then transfected cells were collected by trypsin treatment. After washing with cold PBS buffer and fixed with 70% ethanol, suspended in 200ul of PBS containing 1mg / ml RNase, and reacted for 30 minutes at a temperature of 37 ℃ in dark conditions. Nuclei were stained with 50 ug / ml propidium iodide (BD Biosciences), and the stained cell fractions were analyzed using Cell-Quest FACS analysis software (BD Biosciences).

<Example 1>

Identification of Potential Liver Cancer Diagnostic Markers in Precancerous Lesions

Clinical pathology data and gene expression were analyzed by the following non-biased analysis method to identify the expression characteristics of genes related to hepatocellular carcinoma (HCC) in premalignant lesions. Here, precancerous lesions refer to the stages before the liver cancer, and do not require treatment equivalent to liver cancer.

First, normal liver tissue (13 tissues) using whole transcriptome expressing microarrays; Chronic hepatitis tissue including eight low-grade fibrosis (LGCH) and twelve high-grade fibrosis (HGCH); 12 liver hard tissues; Dysplastic nodule (DN) comprising eleven low-grade heterogeneous nodes (LGDN) and eleven high-grade heterogeneous nodes (HGDN) high-grade DNs; Five early hepatocellular carcinoma tissues (eHCC); Hepatocellular carcinoma tissue comprising 9 Edmonson Grade 1 (G1), 12 G2, and 14 G3; A schematic diagram of the identification sequence of a potential cancer diagnostic marker according to the present invention is shown in FIG. 1.

Hierarchical clustering analysis shows a schematic tree divided into three distinct subclusters: chronic liver disease (LGCH, HGCH, cirrhosis and LGDN), early liver cancer (HGDN, eHCC, and G1), and liver cancer HCC (G2 and G3). (See FIG. 2A). In addition, as a result of reanalysis of LGDN, HGDN, eHCC and G1, it was confirmed that LGDN is an early stage of hepatocellular carcinoma (see FIG. 2B). Therefore, it can be seen that HGDN is a characteristic that indicates the onset of early stage hepatocellular carcinoma, and precancerous lesions can be a starting site of driver gene activity and then develop into hepatocellular carcinoma. Based on these results, we designed a gene selection strategy to identify a set of genes for predicting precancerous lesions, ie, potential hepatocellular carcinoma within HGDN.

The molecular characteristics of LGDN to G1 HCC were analyzed for LGDN vs HGDN, LGDN vs eHCC / G1 HCC, and HGDN vs eHCC / G1 HCC. Of the 690 genes, 8 genes were found to gradually increase in expression as they progress from LGDN to hepatocellular carcinoma (HCC) (see FIG. 2C).

Next, a comparative analysis of a receiver operating characteristic (ROC) curve was performed to select a molecule capable of confirming progression to HCC in HGDN, a precancerous lesion (see FIG. 2D).

As a result, 13 out of 85 genes exhibited an AUC (area under the curve) value of 0.8 or more, and thus could be used as markers of specificity and sensitivity (see Table 5 below), and Cancer Genome Atlas hepatocellular carcinoma (TCGA_LIHC). ) Data expression and expression of 13 genes in a large cohort of HCC patients provided in the Gene Expression Omnibus (GEO) database of the National Biotechnology Information Center (NCBI) were analyzed.

TABLE 5

13 candidate genes based on ROC analysis

As a result, three genes (ALKBH2, HSD3B7, PARP10) were excluded because they did not show sustained overexpression (> 1.5-fold) in HCC compared to non-tumor (<50% in six test cohorts, see Table 6 below). .

TABLE 6

Expression Levels of Early HCC Driver Gene Candidates in a Large Cohort of Liver Cancer Patients

    Thereafter, 10 candidate marker genes identified as overexpressed in both the TCGA_LIHC data cohort and 107 HCC sets were identified (see FIGS. 3A and 3B).

Tissue microarrays (TMA) containing 546 HCCs (140 G1, 297 G2 and 109 G3) were then used to confirm overexpression of these 10 genes in HCC compared to surrounding non-tumor tissues. Immunohistochemistry (IHC) was performed.

As a result, among 10 marker candidate genes, CYC1, FAM83H, MAD3 and MELK were not significantly overexpressed in hepatocellular carcinoma compared to normal liver tissue (NL) (see FIG. 2E), and clinical pathology data and expression of HCC patients Based on multivariate analysis of BANF1, PLOD3 and SF3B4 were selected as candidate markers (see Table 7 below).

TABLE 7

Analysis of Histopathological Characteristics and Expression of 10 Genes in Cohort 2 (n = 546)

<Example 2>

Verification as Hepatocellular Carcinoma Determining Markers for BANF1, PLODS3, and SF3B4 in Precancerous Lesions

To verify the availability of BANF1, PLODS3 and SF3B4 as decisive markers for hepatocellular carcinoma in precancerous lesions, immunohistochemical analysis (IHC) was performed using six early hepatocellular carcinoma full sections transitioning from HGDN. It was.

 As expected, BANF1, PLODS3 and SF3B4 were strongly overexpressed or positively expressed in both early hepatocellular carcinoma (eHCC) and HGDN among 10 candidate markers as expected. On the other hand, only one was positively expressed in the non-tumor site (see FIG. 4A). In addition, the remaining six markers did not show significant differences in non-tumor tissue and eHCC within regenerating nodules (RNs).

In addition, three genes, GPC3, GS and HSP70, are known to distinguish eHCC from other tumor lesions, including DN, hepatocellular adenoma and local nodular proliferation. The present inventors have identified three existing markers (CMs; GPC3, GS and HSP70) and 70 new sections including 55 early liver cancers and 105 RNs and novel markers (NMs; BANF1, PLOD1, Comparative IHC analysis was performed on SF3B4).

As a result, the expression positive rate (≥2 (+) / 3 CM or more) of two or more conventional marker genes in HCC was found to be 50.9% (28/55), whereas two or more novel markers identified in the present invention in HCC. The positive rate of expression of the gene (two or more (+) / 3 NM) was 72.7% (40/55) (see Figure 4b and Table 8). In addition, the negative expression rate (≤1 (+) / 3 CM) of the conventional marker in HCC was 49.1% (27/55), and the negative expression rate (≤1 (+) / 3 NMs) of the novel marker of the present invention was 27.3% ( 15/55).

TABLE 8

 Analysis of Expression Patterns in Early Hepatocellular Carcinoma of Existing Markers and New Markers of the Present Invention

   In addition, the positive expression rate of the new marker was 59.3% (16/27), especially in HCC negative expression of the conventional marker, and the positive expression rate of the conventional marker in the HCC negative expression of the new marker was 26.6% (4 / 15) (see FIG. 4C). However, there was no significant difference in the negative ratio between conventional markers and new markers in RNs (see FIG. 4D).

Furthermore, as a result of the comparative analysis of the ROC curve, it was confirmed that the new markers of the present application (BANF1, PLOD1, SF3B4) are better markers than the existing markers (GPC3, GS, and HSP70) (see Table 9 and FIG. 5).

TABLE 9

Diagnostic performance analysis of six markers for early HCC diagnosis by ROC curve evaluation

In addition, it was found that early diagnosis of hepatocellular carcinoma was most sensitive and specific for precancerous lesions, especially when two sets of combinations (HSP70, GPC3, BANF1 and SF3B4) were used (see Table 10 and FIG. 4E below). ).

TABLE 10

Results of ROC analysis between early liver cancer and regenerative nodules for existing markers, novel markers and combination markers of the present invention

In addition, the analysis of Kaplan-Meier survival curves (cohort 2) of HCC patients revealed that disease-free survival rate of hepatocellular carcinoma patients with high expression of the novel marker genes of the present invention was significantly higher than that of patients with low expression levels of the new marker genes of the present invention. It was found to be low (FIG. 4F), indicating that the novel marker genes of the present invention can induce the early stages of HCC.

<Example 3>

Analysis of anticancer effects through overexpression of BANF1, PLODS3 and SF3B4 in hepatocellular carcinoma and inhibition of their activity

<3-1> Confirmation of BANF1, PLODS3 and SF3B4 Overexpression in Hepatocellular Carcinoma

Abnormal regulation of BANF1, PLOD3 and SF3B4 in HCC was identified as a matched pair of 50 HCC patients in the TCGA_LIHC data set cohort, and further qRT-PCR analysis of 13 randomly selected sets of HCC tissues (FIG. 6A). Reference).

As a result, PLOD3 and SF3B4 were overexpressed in> 9-12 (69-92%) out of 13 HCCs (> 2 times overexpression) compared to adjacent non-tumor liver tissue (NL). In addition, Western blot analysis of three pairs of eHCCs with DN and NL adjacent to eHCC 6 pairs with NL showed significantly higher expression levels of BANF1, PLOD3 and SF3B4 proteins in eHCC (see FIG. 6B).

Next, Banf1, Plod3 and Sf3b4 expression was reconstituted in chemically induced mouse and rat HCC models available in the GEO database.

As a result, as shown in FIG. 6C, expression of Banf1, Plod3 and Sf3b4 showed upregulation of corresponding NL in epithelial tumor (AD), eHCC and progressive HCC (aHCC) in both mouse and rat models.

To verify these results, H-ras transgenic mice and a rat HCC model derived from diethylnitrosamine (DEN) were prepared, and the expression of BANF1, PLOD3 and SF3B4 in these animal groups was examined by Western blot.

As a result, as shown in FIG. 6D, the expression of all three markers was increased.

<3-2> Inhibition of proliferation and colonization of cancer cells by inhibition of BANF1, PLODS3 and SF3B4 Next, RNAi and 3- (4,5-dimethylthiazol- to examine the biological functions of BANF1, PLOD3, SF3B4 during liver cancer development 2-yl) -2,5-diphenyltetrazolium bromide (MTT) analysis was used to knock down each gene and measure cell growth rates. In addition, brodoxyuridine (BrdU) introduction and colonization assay were performed to measure the proliferation rate of SNU-449 HCC cells.

As a result, when KANF1, PLOD3, and SF3B4 were knocked down, the growth and proliferation of SNU-449 HCC cells were inhibited (FIGS. 6E and 6F), and BANF1, PLOD3, and SF3B4 were found in the clinicopathological characteristics of HCC. It has been shown to be associated with cancer metastasis such as vascular infiltration and differentiation (see Table 1).

<3-3> Analysis of inhibitory effect on cancer metastasis according to BANF1, PLODS3 and SF3B4 inhibition In vitro, cell mobility and invasion assays were performed to analyze whether the novel markers according to the present invention influence the cancer metastasis of HCC.

As a result, the modified Boyden chamber analysis of the groups knocked down BANF1, PLOD3 and SF3B4, respectively, showed that SNU-449 cells significantly inhibited cell migration and invasion (Fig. 7a). Similarly, in the wound healing analysis, the groups knocked down BANF1, PLOD3 and SF3B4, respectively, showed that the wound healing effect of SNU-449 cells by serum stimulation was reduced (see FIG. 7B).

Furthermore, in order to confirm whether the novel markers according to the present invention also affect EMT, the expression change for EMT regulators was analyzed through Western blot. N-cadherin, Fibronectin, Snail and Slug, typical markers of EMT, knocked down the genes of the present invention, respectively, and showed that their expression was significantly reduced. In contrast, Ecadherin, an epithelial cell marker, was found to be increased in the same cell (see FIG. 7C). From these results, the inventors found that BANF1, PLOD3 and SF3B4 according to the present invention selectively regulate the expression of EMT regulators in hepatocellular carcinoma and have cancer metastasis ability.

<3-4> Analysis of anticancer activity according to inhibition of BANF1, PLODS3 and SF3B4 in vivo In order to confirm the anticancer activity according to inhibition of BANF1, PLODS3 and SF3B4, tumor cells knocked down by BANF1, PLODS3 and SF3B4 genes respectively Subcutaneous injection was performed subcutaneously in nude mice, and tumor growth and tumor volume were measured. As a result, the group injected with cells knocked down BANF1, PLODS3, and SF3B4, respectively, were injected with cells treated with si-Cont as a control group. Tumor growth and tumor volume were significantly reduced compared to the group.

In addition, after 50 days after the injection of the control cell line into the rat, the analysis showed that, out of 10, 6 of the tumor was formed, while the cell line knocked down BANF1, PLODS3 or SF3B4 was injected into the rat In 0, only 0 to 2 of 10 mice were found to have weak tumors (FIG. 7D).

In addition, as a method for inducing anticancer effect through specific si-RNA delivery to BANF1, PLOD3 and SF3B4, mesoporous silica nanoparticles (MSNs) having very large pores were used. First, H-ras transgenic mice with spontaneously developed hepatocellular carcinoma of 12-15 weeks were prepared. Mesoporous silica nanoparticles (MSN_si-RNA) containing si-RNAs targeting BANF1, PLOD3 and SF3B4 were injected into the prepared mice and confirmed, wherein the control group was injected with only MSN without si-RNA as a control group. Hepatocellular carcinoma in mouse liver was analyzed by ultrasonography from 16 weeks after birth to 23 weeks of sacrifice.

As a result, hepatocellular carcinoma was detected in 3 of 4 rats at 17 weeks in the control group injected with MSN, whereas small hepatocarcinoma was found in 2 of 4 rats at 19 weeks in the group injected with MSN_si-RNA (Fig. 7e). As a result of Western blot, it was confirmed that in the group injected with MSN_si-RNA, BANF1, PLOD3, and SF3B4 were knocked down compared to the control group, indicating that target genes were accurately suppressed by MSN_si-RNA (see FIG. 7F).

Therefore, through these results, the inventors have found that substances capable of inhibiting the expression or activity of BANF1, PLOD3 and SF3B4 can be used as therapeutic agents for liver cancer, in particular, hepatocellular carcinoma.

<Example 4>

Inhibition Analysis of Hepatocellular Carcinoma Tumors Inhibited by SF3B4

To investigate the clinical relevance in HCC, the frequency of changes in BANF1, PLOD3 and SF3B4 in 360 HCC patients in TCGA_LIHC was evaluated. Overexpression of BANF1, PLOD3 and SF3B4 was seen in 140 of 360 patients (see FIG. 9A). Kaplan-Meier survival curves showed lower overall survival (OS) and disease-free survival (DFS) in HCC patients with upregulation of BANF1, PLOD3, and SF3B43 compared to the hepatocellular carcinoma patients with normal expression (OS; Logrank P = 0.002, DFS; Logrank P = 0.001, see FIG. 9B).

Interestingly, the group overexpressing only SF3B43 among these genes showed very low overall survival (OS) and disease free survival (DFS) of HCC patients (see FIG. 9C). Amplification of SF3B43 has been shown to cause very bad results in patients with hepatocellular carcinoma of OS and DFS, which could be expected to activate the mechanism of SF3B43 in liver cancer development (FIGS. 9D-9F).

SF3B4 is one of six splicing factor 3b (SF3b) complexes and is an important protein in the U2 snRNP super-complex. Analysis of expression levels and frequency of changes in the six subunits of the splicing factor 3b (SF3b) complex in TCGA_LIHC and GEO databases revealed that only SF3B4 was specifically overexpressed and amplified in gene copy number in hepatocellular carcinoma (FIG. 10a and 10b).

  The expression levels of SF3B4 were analyzed by qRT-PCR in 10 different hepatocytes, MIHA immortalized normal hepatocytes, and 8 human hepatocellular carcinoma cell lines. As a result, SF3B4 was expressed in hepatocellular carcinoma cell lines compared with MIHA cell lines. Both mRNA and protein were overexpressed (see FIG. 10C). Further, as a result of suppressing the expression of SF3B4, it was shown that both cell growth and proliferation of SNU-449 and SNU-368 HCC cells were inhibited (FIGS. 11A and 11B).

In modified Boyden chamber analysis and wound healing analysis, SF3B4 knockdown also showed that all cancer metastatic features of the same cell line were suppressed (FIGS. 11C and 11D).

Western blot analysis of the effects on the expression of proteins involved in EMT revealed that N-cadherin, Fibronectin, Snail and Slug were all inhibited during SF3B4 knockdown in hepatocellular carcinoma cell lines, and only E-cadherin was overexpressed. (FIG. 11E).

According to the results of the previous experiment, as genes and proteins of SF3B4 were overexpressed in hepatocellular carcinoma, the association between SF3B4 expression and prognosis was analyzed in a cohort of 100 HCC patients (GSE16757).

Genes with an expression pattern highly correlated with SF3B4 expression (n = 342) were selected for cluster analysis (P <0.001, r> 0.5 or r <-0.5). Patients were divided into SF3B4 high cluster (SF3B4_high) group or SF3B4 low cluster (SF3B4_low) group. The Kaplan-Meier survival curve of HCC patients was significantly lower in patients with high SF3B4 expression than in patients with low SF3B4 expression (P = 0.04, FIG. 10D).

Next, gene set enrichment analysis (GSEA) was performed to identify signaling pathways related to SF3B4-related gene labels in the Molecular Signatures Database (MsigDB) to investigate the biological relevance of SF3B4-related gene labels.

KEGG_CELL_CYCLE was identified as a very meaningful gene set (FIG. 10E). In addition, the flow cytometry showed that the hepatocellular carcinoma cell line in which SF3B4 was knocked down significantly increased the number of G1 cells and decreased the number of S and G2 / M cells. Knockdown of SF3B4, on the other hand, did not affect the cell death process (FIGS. 10F and 11F).

Example 5

Abnormal Splicing Activity of SF3B4 Against KLF4 and Induction of Hepatocellular Carcinoma

To identify the biological role of SF3B4 in the development of HCC, spliceosome activity by SF3B4 knockdown was investigated. SF3B4 Knockdown While suppressing the expression of VEGF, a representative molecule regulated by the SF3b complex, the non-splicing form of VEGF mRNA (VEGF pre-mRNA) was increased by SF3B4 knockdown in HeLa cells (FIG. 12A).

Treatment with Spliceostatin A (SSA), an inhibitor of the SF3b complex, was found to be involved in the inhibition of splicing activity of the SF3b complex, with the same result in two different HCC cell lines. Treatment of SF3B4 knockdown SSA was shown to inhibit tumor cell growth in HCC cell lines (FIG. 13A).

Western blots for cell cycle regulatory proteins were performed to confirm the mechanism of cancer cell growth inhibition targeting SF3B4. SF3B4 knockdown inhibited the expression of CDK4, CDK2, cyclin D1 and cyclin E and simultaneously induced p27Kip1 in HCC cell lines, and SSA treatment repeated this result in the same cells (FIG. 12B). These results suggest that SF3B4 overexpression induces the inhibition of negative cell cycle regulators including p27Kip1 and at the same time induces the expression of CDK4, CDK2, cyclinD1, cyclin E, which are specific regulators in cell cycle transition of HCC cells. . SF3B4 knockdown in hepatocellular carcinoma induces hypophosphorylation of pRb.

Next, to investigate the direct target of abnormal expression SF3B4 in HCC cells, next generation sequencing (NGS) -RNA sequence data of SF3B4 knockdown HepG2 cells was analyzed. This dataset is available from the GEO database (GSE80861) and was part of the ENCODE project (the Encyclopedia of DNA Element). This analysis identified 3,182 genes differentially expressed genes (DEGs) that were specific in SF3B4 knockdown cells (more than 1.5-fold difference from control). To identify the AS3 associated with SF3B4 knockdown, paired-end reads were mapped to the ENCODE database and quantified exon inclusions using a Multivariate Analysis of Transcript Splicing Analysis (MATS) analysis tool.

As a result, six analyzes were performed: skipped exon (SE), retained intron (RI), alternative 3 'splicing site (A3SS), alternative 5' splicing site (A5SS), and mutually exclusive exon (MXE). Different splicing phenomena were analyzed to occur (FIG. 12C). In addition, combining these two assays with two or more AS events showed that eight genes significantly inhibited transcription in HepG2 cells knocked down SF3B4 (FIG. 13B).

To more clearly confirm this, eight genes were applied to the TCGA_LIHC dataset, of which four genes GRIP, PLP1, KLF4 and KRT80 were selected as target candidate genes of SF3B4 (FIG. 12D). Specifically, only KLF4 was shown to be activated in two hepatocellular carcinoma cell lines in which SF3B4 was knocked down (see FIGS. 12E and 13C).

Among the eight genes, KLF4 and SF3B4 showed a significant negative correlation with each other in hepatocellular carcinoma (FIGS. 13D and 13E). These results indicate that SF3B4 knockdown induces the recovery of transcriptional expression of wild type KLF4 and exhibits antitumor effect on HCC cells. Indeed, NGS-RNA analysis indicates that the transcript of wild type KLF4 is inhibited in HCC tissues. And KLF4 SE transcripts were increased in hepatocellular carcinoma patients (FIG. 12F).

Furthermore, the present inventors applied a plasmid with SF3B4 knockdown and KLF4 ectopic expression to hepatocellular carcinoma cell line to confirm whether SF3B4 overexpression induced SF3b complex to splice KLF4 transcript to become nonfunctional AS transcript. And analyzed.

Subsequently, the regulators of SF3B4, p27Kip1 and Slug, were observed in cells in which SF3B4 was knocked down and KLF4 was overexpressed.

As a result, ectopic expression of KLF4 showed the effect of SF3B4 knockdown in the same cells (FIG. 14A).

Next, to investigate whether SF3B4 activation directly regulates p27Kip1 and Slug expression via KLF4 transcriptional activity in HCC cells, a luciferase reporter plasmid with p27Kip1 or slug-promoter was prepared and transfected with SNU-449 cells, Simultaneously transfected with SF3B4 specific siRNA or KLF4 plasmid.

As a result, luciferase activity of the p27Kip1 promoter transfectant was significantly increased in both SF3B4 knockdown cell line and KLF4 overexpressing cell line, and luciferase activity of the Slug-promoter transfectant was significantly inhibited in the same cell (FIG. 14B).

Since SF3B4 knockdown induces the expression of KLF4, it was confirmed whether KLF4, Liver-KLF4-iso1 and Liver-KLF4-iso2 inhibited various splicing isomers.

Direct sequencing analysis confirmed the generation of skipped exon transcripts in wild-type KLF4 transcripts (FIG. 14C).

Finally, SF3B4 knockdown and KLF4 overexpressing cells were injected subcutaneously into thymus nude mice to determine whether inactivation of SF3B4 induced tumor suppressor effects through the recovery of tumor suppressor KLF4 in vivo.

As a result, the tumor growth rate and the mean volume at sacrifice were significantly reduced overall in both SNU-449 cells expressing SF3B4 knockdown and KLF4 (FIG. 14D). In addition, immunoblot analysis confirmed the induction of KLF4 in both SF3B4 knockdown and xenograft tissue expressing KLF4 (FIG. 14E).

Therefore, through the above experimental results, the present inventors found that BANF1, PLOD3, and SF3B4 can be used as biomarkers for accurately predicting early liver cancer, and in particular, two or more combinations of these markers compared to a single use. When used, it was found that the diagnostic accuracy is higher, in particular, these markers are characterized in that can be confirmed in the precancerous lesion sample.

In addition, SF3B4 was found to inhibit the expression and activity of KLF4 in the onset of liver cancer to progress and induce liver cancer, and inhibiting SF3B4 expression may induce anticancer activity through increased expression of KLF4. Could.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Biomarker for early diagnosis of hepatocellular carcinoma in          precancerous lesion and use <130> PN1706-209 <160> 42 <170> KoPatentIn 3.0 <210> 1 <211> 1179 <212> DNA <213> Artificial Sequence <220> <223> BANF1 cDNA sequence <400> 1 ccagtgttcc cagttcccac cagtccaact gcgaggagtg cgacgtgagt ctgagtctga 60 tccctccgaa aaccgtactt ccggcgctgt ctcggaggcc tcccgtcccc tcccttgtcc 120 gtcttctaac tcttccccac gccaggtccg tcaagcctaa gtccttgagt tccgggtccg 180 ggcagcagag aaaggaagtc ctctccctgg aggcctatct ccctcagaac tgcgcgagaa 240 gcgagacctt agaaggcagg gcttcccgcg aaggaccgga aaggagcgcc tactaaggac 300 gccgtcgagg tccggggcgc ctcaactcta tagctctaac tggctagaag tgcccaacgt 360 ggaatgtttc ttttttaaag gcggctcttg aagcgacccg gaagcggaag tggaagaaag 420 ttctagtggc ttgaggtatc cgcaggagcg gccgggtggc gggaggaacc gttacgggaa 480 ctgaagttgc ggattaagcc tgatcaagat gacaacctcc caaaagcacc gagacttcgt 540 ggcagagccc atgggggaga agccagtggg gagcctggct gggattggtg aagtcctggg 600 caagaagctg gaggaaaggg gttttgacaa ggcctatgtt gtccttggcc agtttctggt 660 gctaaagaaa gatgaagacc tcttccggga atggctgaaa gacacttgtg gcgccaacgc 720 caagcagtcc cgggactgct tcggatgcct tcgagagtgg tgcgacgcct tcttgtgatg 780 ctctctggga agctctcaat ccccagccct catccagagt ttgcagccga gtagggactc 840 ctcccctgtc ctctacgaag gaaaagattg ctattgtcgt actcacctcc gacgtactcc 900 ggggtctttt gggagttttc tcccctaacc atttcaactt ttttttggat tctcgctctt 960 gcatgcctcc cccgtccttt ttcccttgcc agttccctgg tgacagttac cagctttcct 1020 gaatggattc ccggccccat ccctcacccc caccctcact ttcaatccgt ttgataccat 1080 ttggctcctt ttttggcaga acagtcactg tccttgtaaa gttttttaga tcaataaagt 1140 cagtggcttt caaaaaaaaa aaaaaaaaaa aaaaaaaaa 1179 <210> 2 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> BANF1 Amino acid sequence <400> 2 Met Thr Thr Ser Gln Lys His Arg Asp Phe Val Ala Glu Pro Met Gly   1 5 10 15 Glu Lys Pro Val Gly Ser Leu Ala Gly Ile Gly Glu Val Leu Gly Lys              20 25 30 Lys Leu Glu Glu Arg Gly Phe Asp Lys Ala Tyr Val Val Leu Gly Gln          35 40 45 Phe Leu Val Leu Lys Lys Asp Glu Asp Leu Phe Arg Glu Trp Leu Lys      50 55 60 Asp Thr Cys Gly Ala Asn Ala Lys Gln Ser Arg Asp Cys Phe Gly Cys  65 70 75 80 Leu Arg Glu Trp Cys Asp Ala Phe                  85 <210> 3 <211> 2995 <212> DNA <213> Artificial Sequence <220> <223> PLOD3 cDNA sequence <400> 3 taggcttcct ccctccctgt cgccgccagc ctctcgctct ccccgtcttc cgcccgcact 60 ctctgggcag ggctgcggcc aggacccgcc cccgcgtccc gcccgaccct ccgccagggg 120 tcacttcccc tgtccaggtt tcagcttcca catgtgtcaa gcggctggct cagcccagag 180 tccctgtctc ccgcccgccg gcccgagccg ccgcccctcc cccgcctccc gtgcgcccgg 240 gacaatcctc gccttgtctg tggcgccggc atctggagct ttctgtagcc tccggatacg 300 cctttttttc agggcgtagc cccagccaag ctgctccccg cggcggccgc acagcagccc 360 gagcgccccc tttccagagc tcccctccgg agctgggatc caggcgcgta gcggagatcc 420 caggatcctg ggtgctgtct gggcccgctc cccaccatga cctcctcggg gcctggaccc 480 cggttcctgc tgctgctgcc gctgctgctg ccccctgcgg cctcagcctc cgaccggccc 540 cggggccgag acccggtcaa cccagagaag ctgctggtga tcactgtggc cacagctgaa 600 accgaggggt acctgcgttt cctgcgctct gcggagttct tcaactacac tgtgcggacc 660 ctgggcctgg gagaggagtg gcgagggggt gatgtggctc gaacagttgg tggaggacag 720 aaggtccggt ggttaaagaa ggaaatggag aaatacgctg accgggagga tatgatcatc 780 atgtttgtgg atagctacga cgtgattctg gccggcagcc ccacagagct gctgaagaag 840 ttcgtccaga gtggcagccg cctgctcttc tctgcagaga gcttctgctg gcccgagtgg 900 gggctggcgg agcagtaccc tgaggtgggc acggggaagc gcttcctcaa ttctggtgga 960 ttcatcggtt ttgccaccac catccaccaa atcgtgcgcc agtggaagta caaggatgat 1020 gacgacgacc agctgttcta cacacggctc tacctggacc caggactgag ggagaaactc 1080 agccttaatc tggatcataa gtctcggatc tttcagaacc tcaacggggc tttagatgaa 1140 gtggttttaa agtttgatcg gaaccgtgtg cgtatccgga acgtggccta cgacacgctc 1200 cccattgtgg tccatggaaa cggtcccact aagctgcagc tcaactacct gggaaactac 1260 gtccccaatg gctggactcc tgagggaggc tgtggcttct gcaaccagga ccggaggaca 1320 ctcccggggg ggcagcctcc cccccgggtg tttctggccg tgtttgtgga acagcctact 1380 ccgtttctgc cccgcttcct gcagcggctg ctactcctgg actatccccc cgacagggtc 1440 acccttttcc tgcacaacaa cgaggtcttc catgaacccc acatcgctga ctcctggccg 1500 cagctccagg accacttctc agctgtgaag ctcgtggggc cggaggaggc tctgagccca 1560 ggcgaggcca gggacatggc catggacctg tgtcggcagg accccgagtg tgagttctac 1620 ttcagcctgg acgccgacgc tgtcctcacc aacctgcaga ccctgcgtat cctcattgag 1680 gagaacagga aggtgatcgc ccccatgctg tcccgccacg gcaagctgtg gtccaacttc 1740 tggggcgccc tgagccccga tgagtactac gcccgctccg aggactacgt ggagctggtg 1800 cagcggaagc gagtgggtgt gtggaatgta ccatacatct cccaggccta tgtgatccgg 1860 ggtgataccc tgcggatgga gctgccccag agggatgtgt tctcgggcag tgacacagac 1920 ccggacatgg ccttctgtaa gagctttcga gacaagggca tcttcctcca tctgagcaat 1980 cagcatgaat ttggccggct cctggccact tccagatacg acacggagca cctgcacccc 2040 gacctctggc agatcttcga caaccccgtc gactggaagg agcagtacat ccacgagaac 2100 tacagccggg ccctggaagg ggaaggaatc gtggagcagc catgcccgga cgtgtactgg 2160 ttcccactgc tgtcagaaca aatgtgtgat gagctggtgg cagagatgga gcactacggc 2220 cagtggtcag gcggccggca tgaggattca aggctggctg gaggctacga gaatgtgccc 2280 accgtggaca tccacatgaa gcaggtgggg tacgaggacc agtggctgca gctgctgcgg 2340 acgtatgtgg gccccatgac cgagagcctg tttcccggtt accacaccaa ggcgcgggcg 2400 gtgatgaact ttgtggttcg ctaccggcca gacgagcagc cgtctctgcg gccacaccac 2460 gactcatcca ccttcaccct caacgttgcc ctcaaccaca agggcctgga ctatgaggga 2520 ggtggctgcc gcttcctgcg ctacgactgt gtgatctcct ccccgaggaa gggctgggca 2580 ctcctgcacc ccggccgcct cacccactac cacgaggggc tgccaacgac ctggggcaca 2640 cgctacatca tggtgtcctt tgtcgacccc tgacactcaa ccactctgcc aaacctgccc 2700 tgccattgtg cctttttagg gggcctggcc cccgtcctgg gagttggggg atgggtctct 2760 ctgtctcccc acttcctgag ttcatgttcc gcgtgcctga actgaatatg tcaccttgct 2820 cccaagacac ggccctctca ggaagctccc ggagtccccg cctctctcct ccgcccacag 2880 gggttcgtgg gcacagggct tctggggact ccccgcgtga taaattatta atgttccgca 2940 gtctcactct gaataaagga cagtttgtaa gtcttgaaaa aaaaaaaaaa aaaaa 2995 <210> 4 <211> 738 <212> PRT <213> Artificial Sequence <220> <223> PLOD3 Amino acid sequence <400> 4 Met Thr Ser Ser Gly Pro Gly Pro Arg Phe Leu Leu Leu Leu Pro Leu   1 5 10 15 Leu Leu Pro Pro Ala Ala Ser Ala Ser Asp Arg Pro Arg Gly Arg Asp              20 25 30 Pro Val Asn Pro Glu Lys Leu Leu Val Ile Thr Val Ala Thr Ala Glu          35 40 45 Thr Glu Gly Tyr Leu Arg Phe Leu Arg Ser Ala Glu Phe Phe Asn Tyr      50 55 60 Thr Val Arg Thr Leu Gly Leu Gly Glu Glu Trp Arg Gly Gly Asp Val  65 70 75 80 Ala Arg Thr Val Gly Gly Gly Gln Lys Val Arg Trp Leu Lys Lys Glu                  85 90 95 Met Glu Lys Tyr Ala Asp Arg Glu Asp Met Ile Ile Met Phe Val Asp             100 105 110 Ser Tyr Asp Val Ile Leu Ala Gly Ser Pro Thr Glu Leu Leu Lys Lys         115 120 125 Phe Val Gln Ser Gly Ser Arg Leu Leu Phe Ser Ala Glu Ser Phe Cys     130 135 140 Trp Pro Glu Trp Gly Leu Ala Glu Gln Tyr Pro Glu Val Gly Thr Gly 145 150 155 160 Lys Arg Phe Leu Asn Ser Gly Gly Phe Ile Gly Phe Ala Thr Thr Ile                 165 170 175 His Gln Ile Val Arg Gln Trp Lys Tyr Lys Asp Asp Asp Asp Gln             180 185 190 Leu Phe Tyr Thr Arg Leu Tyr Leu Asp Pro Gly Leu Arg Glu Lys Leu         195 200 205 Ser Leu Asn Leu Asp His Lys Ser Arg Ile Phe Gln Asn Leu Asn Gly     210 215 220 Ala Leu Asp Glu Val Val Leu Lys Phe Asp Arg Asn Arg Val Arg Ile 225 230 235 240 Arg Asn Val Ala Tyr Asp Thr Leu Pro Ile Val Val His Gly Asn Gly                 245 250 255 Pro Thr Lys Leu Gln Leu Asn Tyr Leu Gly Asn Tyr Val Pro Asn Gly             260 265 270 Trp Thr Pro Glu Gly Gly Cys Gly Phe Cys Asn Gln Asp Arg Arg Thr         275 280 285 Leu Pro Gly Gly Gln Pro Pro Pro Arg Val Phe Leu Ala Val Phe Val     290 295 300 Glu Gln Pro Thr Pro Phe Leu Pro Arg Phe Leu Gln Arg Leu Leu Leu 305 310 315 320 Leu Asp Tyr Pro Pro Asp Arg Val Thr Leu Phe Leu His Asn Asn Glu                 325 330 335 Val Phe His Glu Pro His Ile Ala Asp Ser Trp Pro Gln Leu Gln Asp             340 345 350 His Phe Ser Ala Val Lys Leu Val Gly Pro Glu Glu Ala Leu Ser Pro         355 360 365 Gly Glu Ala Arg Asp Met Ala Met Asp Leu Cys Arg Gln Asp Pro Glu     370 375 380 Cys Glu Phe Tyr Phe Ser Leu Asp Ala Asp Ala Val Leu Thr Asn Leu 385 390 395 400 Gln Thr Leu Arg Ile Leu Ile Glu Glu Asn Arg Lys Val Ile Ala Pro                 405 410 415 Met Leu Ser Arg His Gly Lys Leu Trp Ser Asn Phe Trp Gly Ala Leu             420 425 430 Ser Pro Asp Glu Tyr Tyr Ala Arg Ser Glu Asp Tyr Val Glu Leu Val         435 440 445 Gln Arg Lys Arg Val Gly Val Trp Asn Val Pro Tyr Ile Ser Gln Ala     450 455 460 Tyr Val Ile Arg Gly Asp Thr Leu Arg Met Glu Leu Pro Gln Arg Asp 465 470 475 480 Val Phe Ser Gly Ser Asp Thr Asp Pro Asp Met Ala Phe Cys Lys Ser                 485 490 495 Phe Arg Asp Lys Gly Ile Phe Leu His Leu Ser Asn Gln His Glu Phe             500 505 510 Gly Arg Leu Leu Ala Thr Ser Arg Tyr Asp Thr Glu His Leu His Pro         515 520 525 Asp Leu Trp Gln Ile Phe Asp Asn Pro Val Asp Trp Lys Glu Gln Tyr     530 535 540 Ile His Glu Asn Tyr Ser Arg Ala Leu Glu Gly Glu Gly Ile Val Glu 545 550 555 560 Gln Pro Cys Pro Asp Val Tyr Trp Phe Pro Leu Leu Ser Glu Gln Met                 565 570 575 Cys Asp Glu Leu Val Ala Glu Met Glu His Tyr Gly Gln Trp Ser Gly             580 585 590 Gly Arg His Glu Asp Ser Arg Leu Ala Gly Gly Tyr Glu Asn Val Pro         595 600 605 Thr Val Asp Ile His Met Lys Gln Val Gly Tyr Glu Asp Gln Trp Leu     610 615 620 Gln Leu Leu Arg Thr Tyr Val Gly Pro Met Thr Glu Ser Leu Phe Pro 625 630 635 640 Gly Tyr His Thr Lys Ala Arg Ala Val Met Asn Phe Val Val Arg Tyr                 645 650 655 Arg Pro Asp Glu Gln Pro Ser Leu Arg Pro His His Asp Ser Ser Thr             660 665 670 Phe Thr Leu Asn Val Ala Leu Asn His Lys Gly Leu Asp Tyr Glu Gly         675 680 685 Gly Gly Cys Arg Phe Leu Arg Tyr Asp Cys Val Ile Ser Ser Pro Arg     690 695 700 Lys Gly Trp Ala Leu Leu His Pro Gly Arg Leu Thr His Tyr His Glu 705 710 715 720 Gly Leu Pro Thr Thr Trp Gly Thr Arg Tyr Ile Met Val Ser Phe Val                 725 730 735 Asp pro         <210> 5 <211> 2001 <212> DNA <213> Artificial Sequence <220> <223> SF3B4 cDNA sequence <400> 5 aaatcagggt tgtgaggcca aatctgcaag aaggctgcag gcgagaggga aggctccctc 60 tgcgcctgaa agcccccaag tcctcgggtt ggggggaact cttcagcaca tccttctctg 120 gacaacagaa gaagaatcac tgtccacaaa atatattaaa actgaaattt ttgttctttt 180 agctttcgaa tttacacttt tgagagcagt gtgattcttt caagtctagc agtgcattaa 240 gagggcctga gcgtttccgt tccgccctcc tcgctaagac aactagcagc ggcacctcag 300 gacgcgagcc gaggactcta ccgcccgacc caggttaagt ttcggaggct cctaagatgg 360 ctgcagccgc ttccaggcac ctcctcttcc gcggccccgc ccaccgctac gtccgtcttc 420 gccccggaag tggaagtcgt gctgaggtca gaaggcggaa ccgctgctgg gagacggcgg 480 gatctctttc gccatggctg ccgggccgat ctccgagcgg aatcaggatg ccactgtgta 540 cgtggggggc ctggatgaga aggttagtga accgctgctg tgggaactgt ttctccaggc 600 tggaccagta gtcaacaccc acatgccaaa ggatagagtc actggccagc accaaggcta 660 tggctttgtg gaattcttga gtgaggaaga tgctgactat gccattaaga tcatgaacat 720 gatcaaactc tatgggaagc caatacgggt gaacaaagca tcagctcaca acaaaaacct 780 ggatgtaggg gccaacattt tcattgggaa cctggaccct gagattgatg agaagttgct 840 ttatgatact ttcagcgcct ttggggtcat cttacaaacc cccaaaatta tgcgggaccc 900 tgacacaggc aactccaaag gttatgcctt tattaatttt gcttcatttg atgcttcgga 960 tgcagcaatt gaagccatga atgggcagta cctctgtaac cgtcctatca ccgtatctta 1020 tgccttcaag aaggactcca agggtgagcg ccatggctca gcagccgaac gacttctggc 1080 agctcagaac ccgctctccc aggctgatcg ccctcatcag ctgtttgcag atgcacctcc 1140 tccaccctct gctcccaatc ctgtggtatc atcattgggg tctgggcttc ctccaccagg 1200 catgcctcct cctggctcct tcccaccccc agtgccacct cctggagccc tcccacctgg 1260 gataccccca gccatgcccc caccacctat gcctcctggg gctgcaggac atggcccccc 1320 atcggcagga accccagggg caggacatcc tggtcatgga cactcacatc ctcacccatt 1380 cccaccgggt gggatgcccc atccagggat gtctcagatg cagcttgcac accatggccc 1440 tcatggctta ggacatcccc acgctggacc cccaggctct gggggccagc caccgccccg 1500 accaccacct ggaatgcctc atcctggacc tcctccaatg ggcatgcccc cccgagggcc 1560 tccattcgga tctcccatgg gtcacccagg tcctatgcct ccgcatggta tgcgtggacc 1620 tcctccactg atgccccccc atggatacac tggccctcca cgacccccac cctatggcta 1680 ccagcggggg cctctccctc cacccagacc cactccccgg ccaccagttc cccctcgagg 1740 cccacttcga ggccctctcc ctcagtaaat tcacattttc cttcctcctg ttacattttc 1800 ccaatatctt ttctattcct tggaccaatc agagatgctg tagctccttg gggcaaaggt 1860 actaatccct ttcagcaccc ccactccatt ccccttttta atgtaacttt ttccacagga 1920 ggtatttctt ttttatgttg gtcctgagta ttttgcaaat gcacagagaa aataaaacta 1980 aactccttgt ttaaaaaaaa a 2001 <210> 6 <211> 424 <212> PRT <213> Artificial Sequence <220> <223> SF3B4 Amino acid sequence <400> 6 Met Ala Ala Gly Pro Ile Ser Glu Arg Asn Gln Asp Ala Thr Val Tyr   1 5 10 15 Val Gly Gly Leu Asp Glu Lys Val Ser Glu Pro Leu Leu Trp Glu Leu              20 25 30 Phe Leu Gln Ala Gly Pro Val Val Asn Thr His Met Pro Lys Asp Arg          35 40 45 Val Thr Gly Gln His Gln Gly Tyr Gly Phe Val Glu Phe Leu Ser Glu      50 55 60 Glu Asp Ala Asp Tyr Ala Ile Lys Ile Met Asn Met Ile Lys Leu Tyr  65 70 75 80 Gly Lys Pro Ile Arg Val Asn Lys Ala Ser Ala His Asn Lys Asn Leu                  85 90 95 Asp Val Gly Ala Asn Ile Phe Ile Gly Asn Leu Asp Pro Glu Ile Asp             100 105 110 Glu Lys Leu Leu Tyr Asp Thr Phe Ser Ala Phe Gly Val Ile Leu Gln         115 120 125 Thr Pro Lys Ile Met Arg Asp Pro Asp Thr Gly Asn Ser Lys Gly Tyr     130 135 140 Ala Phe Ile Asn Phe Ala Ser Phe Asp Ala Ser Asp Ala Ala Ile Glu 145 150 155 160 Ala Met Asn Gly Gln Tyr Leu Cys Asn Arg Pro Ile Thr Val Ser Tyr                 165 170 175 Ala Phe Lys Lys Asp Ser Lys Gly Glu Arg His Gly Ser Ala Ala Glu             180 185 190 Arg Leu Leu Ala Ala Gln Asn Pro Leu Ser Gln Ala Asp Arg Pro His         195 200 205 Gln Leu Phe Ala Asp Ala Pro Pro Pro Ser Ala Pro Asn Pro Val     210 215 220 Val Ser Ser Leu Gly Ser Gly Leu Pro Pro Pro Gly Met Pro Pro Pro 225 230 235 240 Gly Ser Phe Pro Pro Pro Val Pro Pro Pro Gly Ala Leu Pro Pro Gly                 245 250 255 Ile Pro Pro Ala Met Pro Pro Pro Pro Met Pro Pro Gly Ala Ala Gly             260 265 270 His Gly Pro Pro Ser Ala Gly Thr Pro Gly Ala Gly His Pro Gly His         275 280 285 Gly His Ser His Pro His Pro Phe Pro Pro Gly Gly Met Pro His Pro     290 295 300 Gly Met Ser Gln Met Gln Leu Ala His His Gly Pro His Gly Leu Gly 305 310 315 320 His Pro His Ala Gly Pro Pro Gly Ser Gly Gly Gln Pro Pro Pro Arg                 325 330 335 Pro Pro Pro Gly Met Pro His Pro Gly Pro Pro Pro Met Gly Met Pro             340 345 350 Pro Arg Gly Pro Pro Phe Gly Ser Pro Met Gly His Pro Gly Pro Met         355 360 365 Pro Pro His Gly Met Arg Gly Pro Pro Pro Leu Met Pro Pro His Gly     370 375 380 Tyr Thr Gly Pro Pro Arg Pro Pro Pro Tyr Gly Tyr Gln Arg Gly Pro 385 390 395 400 Leu Pro Pro Pro Arg Pro Thr Pro Arg Pro Pro Val Pro Pro Arg Gly                 405 410 415 Pro Leu Arg Gly Pro Leu Pro Gln             420 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BANF1 Forward Primer <400> 7 gaaccgttac gggaactgaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BANF1 Reverse Primer <400> 8 cccggaagag gtcttcatct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PLOD3 Forward Primer <400> 9 cagctccagg accacttctc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PLOD3 Reverse Primer <400> 10 gagcgggcgt agtactcatc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SF3B4 Forward Primer <400> 11 ctcagatgca gcttgcacac 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SF3B4 Reverse Primer <400> 12 ggagggccag tgtatccat 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEGFA_exon1-2 Forward Primer <400> 13 tccaacttct gggctgttct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEGFA_exon1-2 Reverse Primer <400> 14 atgattctgc cctcctcctt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEGFA_intron1 Forward Primer <400> 15 tgtcaggtcc gcatgtcttg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEGFA_intron1 Reverse Primer <400> 16 acccgcatca gatcattcct 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KLF4 Forward Primer <400> 17 ttaccaagag ctcatgccac c 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KLF4 Reverse Primer <400> 18 ggtgtgcctt gagatgggaa 20 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KLF4-Full Forward Primer <400> 19 ttctgggccc ccacatta 18 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> KLF4-Full Reverse Primer <400> 20 tccactgtct gggatttaaa aatg 24 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GRIP1 Forward Primer <400> 21 tgagagtccc tacactaaat ccg 23 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GRIP1 Reverse Primer <400> 22 attcctcccg ataccgtcag a 21 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PLP1 Forward Primer <400> 23 acctatgccc tgaccgttg 19 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PLP1 Reverse Primer <400> 24 tgctggggaa ggcaatagac t 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KRT80 Forward Primer <400> 25 tgatgggtga gtcggaatta 20 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KRT80 Reverse Primer <400> 26 cttcccagga attccagata ca 22 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward Primer <400> 27 accaggtggt ctcctctgac 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse Primer <400> 28 tgctgtagcc aaattcgttg 20 <210> 29 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Control Sense siRNA <400> 29 ccuacgccac caauuucgu 19 <210> 30 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Control Antisense siRNA <400> 30 acgaaauugg uggcguagg 19 <210> 31 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> BANF1 Sense siRNA <400> 31 aagaagcugg aggaaagggg u 21 <210> 32 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> BANF1 Antisense siRNA <400> 32 accccuuucc uccagcuucu u 21 <210> 33 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> PLOD3 Sense siRNA <400> 33 gcaucuggag cuuucugua 19 <210> 34 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> PLOD3 Antisense siRNA <400> 34 uacagaaagc uccagaugc 19 <210> 35 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SF3B4 Sense siRNA <400> 35 gcaguaccuc uguaaccgu 19 <210> 36 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SF3B4 Antisense siRNA <400> 36 acgguuacag agguacugc 19 <210> 37 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Banf1 Sense siRNA <400> 37 ccucagcguu ucaaucuuu 19 <210> 38 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Banf1 Antisense siRNA <400> 38 aaagauugaa acgcugagg 19 <210> 39 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Plod3 Sense siRNA <400> 39 cgacugcaga aucuccucu 19 <210> 40 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Plod3 Antisense siRNA <400> 40 agaggagauu cugcagucg 19 <210> 41 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Sf3b4 Sense siRNA <400> 41 cugcuuuacg auacuuuca 19 <210> 42 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Sf3b4 Antisense siRNA <400> 42 ugaaaguauc guaaagcag 19

Claims (13)

delete delete A composition for diagnosing early liver cancer, comprising an agent for measuring mRNA expression level of at least one protein selected from the group consisting of BANF1, PLOD3, and SF3B4 or a gene encoding the same.
The measurement is characterized in that for measuring in precancerous lesions, early liver cancer diagnostic composition. The method of claim 3,
The BANF1 gene consists of the nucleotide sequence of SEQ ID NO: 1, the BANF1 protein consists of the amino acid sequence of SEQ ID NO: 2, the PLOD3 gene consists of the nucleotide sequence of SEQ ID NO: 3, and the PLOD3 protein consists of the amino acid sequence of SEQ ID NO: 4, SF3B4 gene is composed of the nucleotide sequence of SEQ ID NO: 5 SF3B4 protein, characterized in that consisting of the amino acid sequence of SEQ ID NO: 6, early liver cancer diagnostic composition. The method of claim 3,
The agent is a primer, probe, aptamer, antibody or substrate specific for BANF1, PLOD3 or SF3B4, characterized in that, for early liver cancer diagnostic composition. delete The method of claim 3,
The measurement was performed by reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real time polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, DNA microarray method, Northern blot, Western blot, ELISA ( Enzyme Linked Immuno Sorbent Assay), radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation, complement fixation, FACS, mass spectrometry and protein microarrays. Characterized in that, early liver cancer diagnostic composition. Comprising a composition according to claim 3, Early liver cancer diagnostic kit. delete delete Measuring the expression level of mRNA or protein for at least one gene selected from the group consisting of BANF1, PLOD3 and SF3B4 from precancerous lesion tissue isolated from the patient; And
Comparing the measured expression level of BANF1, PLOD3 or SF3B4 with the expression level of the control sample, Information providing method for early liver cancer diagnosis. delete The method of claim 11,
When the expression level of mRNA or protein for one or more genes selected from the group consisting of BANF1, PLOD3 and SF3B4 increases than the expression level of the control sample, it is determined that the liver cancer has occurred, information for early liver cancer diagnosis How to Provide.

Images