CN114002431B - Application of TNNT1 in preparation of virus type hepatocellular carcinoma diagnosis kit and preparation or screening of anti-liver cancer drugs - Google Patents

Abstract

The invention provides an application of TNNT1 in preparing a virus type hepatocellular carcinoma diagnosis kit and preparing or screening anti-liver cancer drugs, and also provides a detection primer of a TNNT1 molecular marker, the sequence of which is shown in SEQ ID NO:3 and SEQ ID NO: 4. The virus type hepatocellular carcinoma diagnosis kit comprises the detection primer. Also provided is an anti-liver cancer drug, including a drug for inhibiting TNNT1 expression. The cytoplasmic protein marker TNNT1 obtained by screening of the invention expresses liver cancer tissues and paracancerous tissues higher than liver-edge virus negative in liver cancer cytoplasm positive to hepatitis virus, can be used as potential markers for liver cancer development and cancer metastasis induced by hepatitis virus, and can also provide a new potential drug target for treating liver cancer.

Description

Application of TNNT1 in preparation of virus type hepatocellular carcinoma diagnosis kit and preparation or screening of anti-liver cancer drugs

Technical Field

The invention relates to the technical field of biomedicine, in particular to an application of TNNT1 in preparing a viral hepatocellular carcinoma diagnosis kit and preparing or screening an anti-liver cancer drug.

Background

Viral hepatitis caused by hepatitis b and hepatitis c viruses (HBV, HCV) is a common infectious disease. HBV and HCV infection causes cirrhosis, liver cancer and complications thereof. Hepatitis C Virus (HCV) is an enveloped single-stranded positive-strand RNA virus. A variety of factors have been shown to be risk factors for liver cancer onset, mainly including hepatitis b virus, hepatitis c virus, alcohol, aflatoxin, etc. The cause of global liver cancer mainly comes from chronic diseases caused by hepatitis B virus infection and hepatitis C virus infection, and is secondarily caused by long-term alcoholism and nonalcoholic fatty liver. In the aspect of the change trend of liver cancer incidence, researches indicate that the liver cancer incidence caused by the hepatitis C virus increases most rapidly in the global scope, and the liver cancer caused by alcohol is at a stable level.

HCV currently lacks an effective vaccine; HBV lacks effective therapeutic agents. Although the highly effective direct acting antiviral drug DAA (direct-acting antivirals) has been developed at present, the cure rate is more than 95% under the best clinical conditions. However, these drugs are expensive and because HCV has a high degree of variability, resistant mutant strains may develop during treatment. Even after successful treatment, the patient has a high risk of hepatocellular carcinoma.

Therefore, the development of new targets and strategies for HBV infection, and the development of new tumor markers and therapeutic drugs for hepatocellular carcinoma caused by two viruses are of great significance for diagnosis and treatment of HCC.

Disclosure of Invention

The invention aims to provide an application of TNNT1 as a molecular marker in preparing a virus type hepatocellular carcinoma diagnostic kit and an application of TNNT1 as a target in preparing or screening anti-hepatoma drugs, wherein the expression level of the cytoplasmic protein TNNT1 of a patient can be detected by using the hepatocellular carcinoma diagnostic kit, and the expression level of the cytoplasmic protein TNNT1 is independently detected to distinguish a hepatic carcinoma patient positive with hepatitis C from a hepatic carcinoma patient negative with hepatitis C, so that the sensitivity reaches 91% and the specificity reaches 68%; the sensitivity of the kit can reach 91% and the specificity can reach 52% for distinguishing patients with liver cell cancer positive with hepatitis B and patients with liver cell cancer negative with hepatitis B.

In a first aspect of the invention, there is provided the use of TNNT1 as a molecular marker in the preparation of a viral hepatocellular carcinoma diagnostic kit.

Further, the TNNT1 is used as a molecular marker for distinguishing hepatitis B virus-positive liver cancer patients from hepatitis virus-negative liver cancer patients, and the hepatitis includes hepatitis B and hepatitis C.

In a second aspect of the present invention, there is provided a primer for detecting a TNNT1 molecular marker, wherein the primer has a sequence as shown in SEQ ID NO:3 and SEQ ID NO: 4.

In a third aspect of the present invention, there is provided a viral hepatocellular carcinoma diagnostic kit comprising the detection primer.

In a fourth aspect of the invention, there is provided the use of TNNT1 as a target in the preparation or screening of an anti-liver cancer drug.

In a fifth aspect of the present invention, there is provided an anti-liver cancer drug comprising a drug that inhibits TNNT1 expression.

Further, the anti-liver cancer drug comprises shRNA for inhibiting TNNT1 expression, and the sequence of the shRNA is shown as SEQ ID NO:5-NO: shown at 6.

Further, the anti-liver cancer drug also comprises an inducer which can induce the shRNA to express in vivo.

Further, the anti-liver cancer drug comprises an anti-liver cancer lentiviral vector containing an interference TNNT1 gene.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

1. the cytoplasmic protein marker TNNT1 obtained by screening of the invention has higher expression than that of liver cancer tissues and paracancerous tissues which are positive to hepatitis C-HCV in liver cancer cytoplasm, can be used as a marker for diagnosis or prognosis of viral liver cancer, and can also provide a new potential drug target for treating liver cancer; TNNT1 is expressed in liver cancer cells positive for hepatitis B-HBV.

2. The expression level of the cytoplasmic protein TNNT1 of the patient can be detected by using the hepatocellular carcinoma diagnostic kit, and the single detection of the expression level of the cytoplasmic protein TNNT1 can distinguish the hepatocellular carcinoma patient with positive hepatitis C from the hepatocellular carcinoma patient with negative hepatitis C, the sensitivity reaches 91 percent, and the specificity reaches 68 percent; the sensitivity of the kit can reach 91% and the specificity can reach 52% for distinguishing patients with liver cell cancer positive with hepatitis B and patients with liver cell cancer negative with hepatitis B.

3. The shRNA of the TNNT1 gene and the slow virus thereof can inhibit the expression of TNNT1 by taking TNNT1 as a novel target, so the shRNA can be used as an anti-liver cancer drug.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the detection of TNNT1 expression level in liver cancer tissue and other tissues; wherein, the figure 1A and the figure 1B are respectively the dyeing intensity of TNNT1 in the cells of the hepatitis C virus positive liver cancer tissue, the hepatitis C virus negative liver cancer tissue and the paired cancer side tissue and the significance analysis is carried out by adopting the unpaired t test; FIG. 1C is a receiver operating profile (ROC) curve of TNNT1 alone to distinguish between hepatitis C virus positive hepatocellular carcinoma patients and paracancerous controls;

FIG. 2 shows the results of detecting the expression level of TNNT1 in hepatoma cells under infection with hepatitis C virus; wherein, FIG. 2A is a graph showing the mRNA change level and expression level of TNNT1 in human hepatoma cell Huh7.5.1 under infection of hepatitis C virus by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and immunoblotting (Western blot); FIG. 2B is a graph showing the expression level of TNNT1 in human hepatoma cell Huh7.5.1 under HCV infection by confocal experiments; FIG. 2C shows the mRNA level variation of TNNT1 in Peripheral Blood Mononuclear Cells (PBMC) of hepatitis C virus positive and hepatitis C virus negative patients by qRT-PCR;

FIG. 3 shows the detection of TNNT1 expression level under HBV infection; wherein, FIG. 3A shows the protein expression level in Western blot verification TNNT1 human hepatoma cell HepG2 and stable transgenic hepatitis B virus HepG2 cell (HepG2.215); FIG. 3B shows qRT-PCR analysis of mRNA level variation in human hepatoma cell Huh7.5.1 after transfection of eukaryotic expression plasmids stably expressing the core gene of hepatitis C virus, the full-length gene of hepatitis B virus and the X gene of hepatitis B virus;

FIG. 4 shows the results of detecting the expression level of TNNT1 in different liver cancer cells; wherein, fig. 4A shows the expression level and mRNA variation level of TNNT1 in human hepatoma cell huh7.5.1 and human hepatoma cell HepG2, low-metastatic hepatoma cell mhc 97L and high-metastatic hepatoma cell HCCLM3 total cell proteins by Western blot experiment and qRT-PCR experiment; FIG. 4B is a graph showing the confocal experiment to verify the expression level of TNNT1 and E-cadherin (E-cadherin) and Vimentin (Vimentin) associated with the process of epithelial mesenchymal transition in human hepatoma cells Huh7.5.1 and HCCLM 3;

FIG. 5 shows the results of TNNT1 knockdown and over-expression cell line construction; wherein, FIG. 5A shows the efficiency of Western blot to verify the knockout and knockdown of TNNT1 in human hepatoma cell Huh7.5.1, and FIG. 5B shows the efficiency of Western blot to verify the overexpression of TNNT1 in human hepatoma cell Huh7.5.1;

FIG. 6 is a cell counting kit-8 (CCK 8) experiment to verify the effect of TNNT1 on the proliferative capacity of human hepatoma cells Huh7.5.1;

FIG. 7 shows the effect of TNNT1 on the cell cycle of human hepatoma cell Huh7.5.1 in a cell flow assay;

FIG. 8 is a graph showing the effect of TNNT1 on the ability of human hepatoma cells Huh7.5.1 to migrate and invade in a transfer chamber (Transwell) assay.

FIG. 9 is a graph showing the effect of TNNT1 on the ability of human hepatoma cells, huh7.5.1, to migrate.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

according to the invention, the immunohistochemical technology shows that TNNT1 expression in liver cancer tissues positive to hepatitis virus is obviously up-regulated compared with liver cancer tissues negative to hepatitis virus and cancer tissues beside the liver cancer, and TNNT1 can be obviously distinguished from liver cancer patients positive to hepatitis virus. The mRNA nucleic acid sequence of the TNNT1 is shown as SEQ ID NO. 1; the amino acid sequence of TNNT1 is shown as SEQ ID NO. 2.

Compared with the human liver cancer cell line Huh7.5.1, the expression of TNNT1 in human liver cancer cells HepG2, low-metastasis liver cancer cells MHCC97L and high-metastasis liver cancer cells HCCLM3 is found to be increased through western blot, qRT-PCR and confocal experiments, and the expression is proportional to the metastasis capacity of the TNNT 1.

In addition, the invention also provides anti-liver cancer drugs such as shRNA and lentiviral vectors for inhibiting TNNT1 expression; pharmaceutical compositions comprising a therapeutically effective amount of the shRNA or TNNT1 gene interfering lentivirus, together with a pharmaceutically acceptable carrier, diluent or excipient, are also within the scope of the invention.

The application of TNNT1 as a molecular marker in preparing a hepatitis C virus positive hepatocellular carcinoma diagnostic kit and the application of TNNT1 as a target in preparing or screening an anti-liver cancer drug will be described in detail below with reference to examples and experimental data.

Example 1 comparison of TNNT1 expression differences in liver cancer tissue and paired paracancerous tissues under infection with hepatitis C Virus

1. Experimental objects

The paraffin embedded slices and clinical information of the tissues of 16 hepatitis C virus positive, 21 hepatitis B virus positive and 12 hepatitis C virus double negative liver cancer patients and the paired 17 cases of paracancerous tissue samples are obtained through the liver cancer tissue chip purchased by the Pinocraft biological technology limited company of Shanghai core super biotechnology limited company and Wuhan city.

2. Experimental method

1. Placing the paraffin tissue in a 65 ℃ oven for slicing for 1h;

2. dewaxing with 100% xylene twice for 10min each time until completely dewaxed;

3. sequentially placing in 100%, 95%, 85%, 75% gradient alcohol for 2min for hydration, and soaking in double distilled water for 5min;

4. placing the processed slice in a slice rack, placing in 500mL sodium citrate antigen retrieval solution containing 0.05% Tween 20, heating at 100deg.C for 30min, naturally cooling to room temperature, and washing with Phosphate Buffered Saline (PBS) for 2 times each for 3min;

5. dropwise adding 100 mu L of 3% catalase solution into each slice, incubating for 10min at room temperature, and washing with PBS for 2 times after incubation is finished, wherein each time is 3min;

6. adding primary antibody rabbit anti-TNNT1 (1:100 dilution), incubating overnight at 4deg.C, and using PBS instead of primary antibody as negative control;

7. washing with PBS for 3min for 2 times after the primary antibody incubation is finished; 100 mu L of goat anti-rabbit IgG secondary antibody (1:500) is dripped into each slice, and incubated for 30min at 37 ℃;

8. after the secondary antibody incubation is finished, the secondary antibody is washed by PBS for 2 times for 3min each time; 100 mu L of freshly prepared DAB color development liquid is dripped into each slice, and the reaction is stopped by flushing with flowing water immediately when the slice appears brown yellow;

9. then counterstaining with hematoxylin for 2min, adding hydrochloric acid alcohol for color separation, and immediately flushing with tap water to turn blue after 2-3 s;

10. sequentially dehydrating in 75%, 85%, 95% and 100% gradient alcohol for 2min, adding xylene for 10min for transparency, drying overnight, and dripping neutral gum for room temperature storage.

11. The intensity of staining of TNNT1 was examined with a scanning microscope.

3. Experimental results

As can be seen from fig. 1, the intensity of staining of TNNT1 in liver cancer tissue positive for hepatitis virus and negative for hepatitis virus and in cytoplasm of paired paracancestral tissue was significantly analyzed by the unpaired t test. The immunohistochemical experiment is used for verifying the TNNT1 expression level of tissues of 21 hepatitis C virus positive, 20 hepatitis B virus positive and 20 hepatitis C virus hepatitis B virus double negative liver cancer patients and paired 21 paracancerous tissues, and the result shows that TNNT1 expression is obviously up-regulated in liver cancer tissues and TNNT1 can obviously distinguish hepatitis (including hepatitis B and/or hepatitis C) virus positive liver cancer patients from hepatitis (including hepatitis B and/or hepatitis C) virus negative liver cancer patients.

Example 2, verifying the effect of hepatitis Virus on TNNT1 expression and comparing the expression differential of TNNT1 in human hepatoma cells Huh7.5.1 and in hepatoma cell lines with different metastatic potential

1. western blot: each cell was collected about 1X 10 ⁷ Is dissolved in 2X SDS loading buffer and the protein is denatured into linear cells at high temperature. The total protein was subjected to a 10% SDS-PAGE gel separation experiment, transferred to PVDF membrane, blocked with 5% nonfat milk powder for 2h, followed by incubation with primary TNNT1 (1:1000 dilution) at 4deg.C overnight, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000 dilution) as an internal reference protein. After the end of the primary antibody incubation, the membranes were washed 4 times with Trsi-HCl buffer+Tween (TBST) for 10min each. Secondary anti-HRP-goat anti-rabbit IgG (1:10000) was added and incubated at 37℃for 45min. After the secondary antibody incubation is finishedTBST washes the membrane 6 times for 5min each. Developing solution A and developing solution B are mixed according to the following ratio of 1: preparing a developing solution in a volume ratio of 1 in a dark place, and adding the developing solution onto a PVDF film to perform ECL color development.

2. Reverse transcription (PrimeScript TMRT kit-Toyobo Co., japan):

(1) RNA extraction: six well plate cells were collected, 1mL of LTrilzol was added to each well, incubated on ice for 3-5min, and transferred to a 1.5mL EP tube for RNase removal. 200 mu L of chloroform was continuously added to the EP tube, the tube was vigorously shaken back and forth for 15s, and left standing at room temperature for 2-3min. Centrifuge at 12000rpm,4℃for 15min, at which time the tube liquid separated into an upper inorganic aqueous phase, an intermediate layer and a lower organic phenol chloroform phase, and transfer the upper aqueous phase to a new RNase enzyme-free EP tube. Then, an equal volume of isopropyl alcohol was added, and the mixture was turned upside down to be thoroughly mixed, and the mixture was allowed to stand at room temperature for 5 minutes at 12000rpm and centrifuged at 4℃for 15 minutes. The white precipitate was washed with 1mL of pre-chilled 75% ethanol, centrifuged at 12000rpm for 5min, and then repeated 1 time. After the washing is finished, the supernatant is sucked and removed, and the white sediment is placed in a fume hood for drying until the sediment at the bottom becomes semitransparent. mu.L of DEPC-ddH preheated at 65℃in advance was added ₂ O dissolves the precipitate and then the RNA concentration is measured by a micro-spectrophotometer.

(2) Reverse transcription reaction: adding 2 μl of 5×RT Master Mix II (on ice operation) into the reaction solution, mixing, immediately separating for 10-15s, running the following procedures on a common PCR instrument at 37deg.C for 15min, 98deg.C for 5min, 4deg.C for infinity, and immediately cooling on ice after the operation is completed; at this point the RNA has been inverted into cDNA, which can be used as is or stored at-20 ℃.

3. qRT-PCR detects the expression effect of TNNT 1.

(1) The primer design is shown in Table 1.

TABLE 1 primers for qRT-PCR

(2) The reaction system: the kit used in qPCR was SYBR-Green I Premix Ex Taq from Vazyme company, and the reaction system was as follows: 1 (i.e., 18. Mu.L of reagent and 2. Mu.L of cDNA template). For each sample, the internal reference GAPDH and the TNNT1 of interest were tested simultaneously and 3 multiplex wells were made. The apparatus used in the experiment was ABI Q6, and the specific reaction system is shown in Table 2 below:

TABLE 2

Composition of the components	Dosage of
		SYBR-GreenIPremixExTaq	10μL
Forwardprimer(2μM)	1μL
		Reverseprimer(2μM)	1μL
RNase-freewater	6μL
		cDNA	2μL

(3) qRT-PCR: care was taken to operate on ice throughout the course and to avoid contamination with exogenous DNA. After sample addition was completed, the sample was centrifuged for 10 to 15 seconds, and the following procedure was performed on a qPCR apparatus at 95℃1min,95℃15sec,55℃20sec, and 72℃45sec, for a total of 40 cycles, except for the first step. After completion of the run, it was judged whether primer dimer was formed and nonspecific amplification was performed by observing the dissolution curve. The Ct value refers to the number of cycles that the fluorescent signal in the reaction tube experiences when reaching a set threshold, reflecting the initial copy number contained in the sample. The fewer the initial copy number, the greater the Ct and vice versa. For each sample, the actual Ct value of TNNT1 is the average of three complex-pore Cts, the actual Ct value of GAPDH is the average of its three complex-pore Cts, and ΔCt is obtained by calculating the Ct TNNT1-Ct GAPDH. The 8-pipe after qPCR operation can be stored at 4 ℃.

4. Immunofluorescence detection of TNNT1 expression in cells: 2X 10 ⁵ Uniformly spreading Huh7.5.1 cells on a confcal cuvette, adding 200 μl of HCVcc into supernatant to infect the cells, removing cell culture supernatant after 24 hours of infection, washing the cells 1 time with PBS, fixing the cells 10min at room temperature with 4% paraformaldehyde, washing the cells 3 times with PBS, and permeabilizing the cells 10min with 0.5% Triton X-100; blocking cells with 5% BSA, incubating for 1 hour at 37 ℃; anti-TNNT1 mAb (1:1000) and FITC-goat anti-mouse secondary antibody TNNT1; for detecting epithelial mesenchymal transition progress (EMT) related proteins, anti-E-cadherin mAb (1:1000) is utilized to dye E-cadherin (E-cadherin) with anti-E-cadherin mAb-Cy 5 goat anti-rabbit secondary anti-dye Vimentin (Vimentin); 4', 6-diamidino-2-phenylindole (DAPI) stained nuclei for 5min, washed twice with PBS and examined under confocal microscope for changes in TNNT1 protein expression.

5. Experimental results

From FIGS. 2A-C, western blot and confocal experiments prove that HCV can improve the expression level of protein and mRNA of TNNT1 in human hepatoma cells Huh 7.5.1;

as shown in fig. 3A, western blot verifies that the protein expression level of TNNT1 in human hepatoma cell HepG2 is lower than that in human hepatoma cell HepG2 (HepG 2.215) stably transformed with hepatitis b virus;

as shown in FIG. 3B, qRT-PCR verifies that the mRNA expression level of TNNT1 is increased after transfection of eukaryotic expression plasmids of full-length hepatitis B virus gene and X hepatitis B virus gene in human hepatoma cell Huh7.5.1;

as shown in fig. 4A, western blot and qRT-PCR verify that the expression level and mRNA change level of TNNT1 in human hepatoma cell huh7.5.1 and human hepatoma cell HepG2, low-metastatic hepatoma cell mhc 97L and high-metastatic hepatoma cell HCCLM3 total cell proteins are increased compared with that of human hepatoma cell huh7.5.1, TNNT1 expression in human hepatoma cell HepG2, low-metastatic hepatoma cell mhc 97L and high-metastatic hepatoma cell HCCLM 3;

as can be seen from fig. 4B, the confocal experiment verifies that the confocal experiment verifies the expression levels of TNNT1 and the associated protein E-cadherin (E-cadherin) and Vimentin (Vimentin) in the cytoplasm of human hepatoma cells huh7.5.1 and high-metastatic hepatoma cells HCCLM 3; compared with human liver cancer cell Huh7.5.1, TNNT1 and Vimentin have lower expression and E-cadherin expression in human liver cancer cell Huh7.5.1 knocked down and TNNT1 knocked down and high metastasis liver cancer cell HCCLM3 knocked down.

In conclusion, the Western blot, qRT-PCR and confocal experiments are utilized to verify the expression quantity of TNNT1 of human liver cancer cells Huh7.5.1 infected with HCV, and the result shows that the HCV can promote the expression of TNNT1 in liver cancer cells; verification of TNNT1 expression level is carried out on human hepatoma cell HepG2 and stable transgenic HBV virus HepG2.215 cell protein by using Western blot and qRT-PCR experiments, and after eukaryotic expression plasmids of hepatitis B virus full-length gene and hepatitis B virus X gene are transfected, mRNA expression level of TNNT1 in human hepatoma cell Huh7.5.1 is verified, and the result shows that HBV can promote TNNT1 expression in hepatoma cells; the cell proteins of human liver cancer cells Huh7.5.1, human liver cancer cells HepG2, low-metastasis liver cancer cells MHCC97L and high-metastasis liver cancer cells HCCLM3 are verified by Western blot, qRT-PCR and confocal experiments, and the result shows that compared with Huh7.5.1 cells, the expression level of TNNT1 in total protein of other liver cancer cells is increased, and the expression level of TNNT1 in total protein is progressively increased along with the increase of the metastasis degree of liver cancer.

6. Hepatocellular carcinoma diagnostic kit

Comprising the following steps: the sequence of the primer is shown in SEQ ID NO:3 and SEQ ID NO: 4.

The kit is a real-time fluorescence quantitative PCR kit, comprises the detection primer and further comprises: internal reference primers (as shown in Table 1) for homogenization and qPCR reaction conventional reagents.

Meanwhile, reverse transcription is carried out on cDNA synthesized in a sample to be detected by using GAPDH as a homogenizing reference primer, and qPCR amplification is carried out, so that Ct GAPDH is obtained;

by calculating the Ct TNNT1-Ct GAPDH, the delta Ct is obtained, and the analysis and judgment principle of the delta Ct is as follows:

and comparing the delta Ct value of the sample to be detected with the delta Ct value of the sample of the healthy control person, and carrying out statistical analysis to obtain a P value, wherein the P value is statistically significant with the difference of less than 0.05.

If P is less than 0.05, diagnosing the sample to be tested as hepatocellular carcinoma;

if P is more than or equal to 0.05, the sample to be tested is not diagnosed as hepatocellular carcinoma;

the detection is carried out by adopting the method, and the result is as follows:

(1) The expression level of the cytoplasmic protein TNNT1 of the patient can be detected by using the hepatocellular carcinoma diagnostic kit, and the single detection of the expression level of the cytoplasmic protein TNNT1 can distinguish the hepatocellular carcinoma patient with positive hepatitis C from the hepatocellular carcinoma patient with negative hepatitis C, the sensitivity reaches 91 percent, and the specificity reaches 68 percent;

(2) The sensitivity of the kit can reach 91% and the specificity can reach 52% for distinguishing patients with liver cell cancer positive with hepatitis B and patients with liver cell cancer negative with hepatitis B.

Example 3, verification of the Effect of TNNT1 on proliferation potency, cell cycle and migration potency of human hepatoma cell Huh7.5.1

1. Construction of TNNT1 knockout and knockdown cell lines and overexpressing cell lines

(1) Construction of TNNT1 knockout cell line (manufactured by Jiangsu Ji Rui Bio Inc.): the specific method is as follows, company operation: TNNT1-gRNA bacteria solution was dipped with yellow gun head and transferred into a 1.5mL centrifuge tube containing 600. Mu.L LB medium, the specific sequences are shown in Table 3, and a large amount of extraction was performed. 1 day before transfection, cas9 KO huh7.5.1 cells with good growth state were grown at 1×10 ⁶ The concentration of each mL is inoculated into a 6-hole plate, 2mL of the cell culture medium is arranged in each hole, and the cell culture medium is in a 37 ℃ cell culture box overnight, so that the cell aggregation rate reaches about 90 percent. Using Neofect ^TM Transfection reagent plasmids gTNNT1-1 and gTNNT1-2 were transfected according to the instructions and wells not transfected with plasmid were used as blank. Fresh complete medium was changed 6h after transfection. After 48h of transfection, the expression level of blue fluorescence was observed under an inverted fluorescence microscope. Each well of cells was passaged into 12-well plates, and 2. Mu.g of the final concentration +.And (3) screening the mL puromycin, wherein after 2-3 times, the rounded death of the cells starts to float, the cells in the blank group are all dead, the cells in the control group are mostly dead, and only a small part of cells in a monoclonal form are left in an adherent state. After daily replacement of fresh complete medium, floating cells were washed off with PBS and resistance was maintained by adding puromycin to a final concentration of 1. Mu.g/mL. After 2-3 weeks, after the cell density in the hole reaches 90%, the cells are passaged, puromycin with half concentration is added every day to maintain the cell resistance, after the cell resistance is transferred to 3-4 generations, the western blot is used for detecting the gene knockout efficiency, and the cells are frozen

(2) Construction of TNNT1 knockdown cell line: at Life Technologies website: http:// rnaidesign. Lifetechnologies. Com/rnaexpress/two shRNA sequences were designed for TNNT1, the specific sequences are shown in table 3.

TABLE 3 shRNA sequences

The pLKO.1-EGFP plasmid was digested, water-bath was performed at 37℃for 3 hours, and the specific system was as follows (50. Mu.L system):

TABLE 4 Table 4

The synthesized primers were sterilized with ddH ₂ O was dissolved and diluted to 100. Mu.M, and annealed as follows:

TABLE 5

Oligo1	10μL
		Oligo2	10μL
Total volume of	20μL

The PCR instrument was slowly annealed, naturally cooled to room temperature (95 ℃ C., 5 min), and then the annealed product was ligated with the pLKO.1-EGFP double enzyme section, the reaction procedure was as follows, the ligation conditions were 16 ℃ C., 30min:

TABLE 6

Oligo1 and Oligo2 annealed products	8μL
		pLKO.1-EGFP plasmid double enzyme section	2μL
Ligationhigh	5μL
		Total volume of	15μL

The ligation product was then transformed and plasmids were extracted. And transfected into 293T cells for lentiviral packaging. Huh7.5.1 cells were then plated at 2X 10 ⁵ Inoculating into 6-well plate at a concentration of one/mL, culturing at 37deg.C overnight, and culturing until the cells grow to a density of 60% -70%Infection. The split-charged virus was added dropwise to the cells, and 500. Mu.L of virus solution per well was added to 2mL of complete DMEM medium containing 10% Gbico serum, followed by addition of polybrene at a ratio of 1:1000 to give a final concentration of 10. Mu.g/mL. Cells were changed after overnight infection, and after 48 hours of infection, the GFP expression efficiency was observed under a fluorescence microscope, followed by addition of 10% complete DMEM medium containing puromycin 2. Mu.g/mL for cell culture. The complete DMEM culture medium containing puromycin is replaced every 2 days, the positive monoclonal is passaged after the whole visual field is full, 1 mug/mL puromycin is added to maintain the resistance, the Western blot is used for detecting the gene knockdown efficiency after 3-4 times of passaging, and the stable transgenic cell strain is frozen.

(3) Construction of TNNT1 overexpressing cell line: the primer sequences at both ends were designed for TNNT1 and PCR amplification was performed, and specific sequences are shown in Table 7.

TABLE 7 TNNT1 primer sequences

The specific PCR system and the flow are as follows:

TABLE 8

F(10μM)	1μL
		R(10μM)	1μL
Huh7.5.1cDNA	1μL
		dNTP	2μL
2×PhantaMix	25μL
		polymerase	2μL
Sterilizing ddH 2 O	18μL
		Total volume of	50μL

TABLE 9

95℃	4min
		95℃	15s
65℃	15s
		72℃	30s
72℃	4min
		Total system	30cycle

The PCR amplification product and pcDNA3.1 plasmid were digested, water-bath was performed at 37℃for 3 hours, and the specific system was as follows (30. Mu.L system):

table 10

And (3) carrying out rubber cutting recovery on the PCR amplification product subjected to enzyme digestion and the pcDNA3.1 plasmid, and connecting the PCR amplification product and the pcDNA3.1 plasmid, wherein a connecting system is as follows:

TABLE 11

pcDNA3.1 enzyme cutting vector	2μL
		Gene PCR product enzyme section	8μL
LigationHigh	5μL
		Total volume of	15μL

And (3) converting the enzyme linked product, extracting plasmids, and transferring the obtained product to Huh7.5.1 cells after sequencing is successful, and detecting the gene knockdown efficiency by using western blot.

2. CCK8 experiment: huh7.5.1TNNT1 KO and Huh7.5.1 cells at 2X 10 ⁵ The concentration of each mL is inoculated into a 12-well plate, and each well is 1mL; after overnight incubation at 37 ℃, the cell aggregation rate reached about 70%. Using Neofect ^TM Transfection reagent, according to the instruction, pcDNA3.1 empty vector and pcDNA3.1-TNNT1 were transfected, and the cells not transfected were used as blank control; 48h after transfection, the above groups of cells were mixed at 1X 10 ⁴ The concentration of each/well was inoculated into 96-well plates, 100 μl per well; culturing at 37 ℃ and carrying out CCK8 detection according to different time points; the method comprises the following steps of: 100 proportion CCK8 reagent and DMEM complete culture medium are mixed uniformly, 100 mu L of mixture is added in each hole in a dark place after the liquid is changed into a fresh culture medium, the mixture is incubated for 1h in a 37 ℃ incubator, and the absorbance at 450nm is measured by an enzyme-labeled instrument.

3. Flow cytometry: huh7.5.1TNNT1 KO and Huh7.5.1 cells at 2X 10 ⁵ The concentration of each mL is inoculated into a 12-well plate, and each well is 2mL; the cells were cultured overnight at 37℃to achieve a cell aggregation rate of about 70%. Using Neofect ^TM Transfection reagent pcDNA3.1-TNNT1 was transfected according to the instructions, all with non-transfected cells as blank; 48h after transfection, cell pellet was collected, cells were washed 3 times with pre-chilled PBS, resuspended in 1mL of BS, and 3mL of pre-chilled absolute ethanol was slowly added dropwise to a final concentration of 75% and fixed overnight at-20 ℃. Washing with PBS for 3 times, adding 500 mu L PI dye, incubating for 30min at room temperature in dark place, filtering with 200 mesh filter screen, and detecting.

Transwell experiments: huh7.5.1 cells at 1X 10 ⁶ The concentration of each mL is inoculated into a 6-well plate, and each well is 2mL; culturing overnight at 37 ℃ to ensure that the cell aggregation rate reaches about 70%; using Neofect ^TM Transfection reagent pcDNA3.1-TNNT1 was transfected according to the instructions, all with non-transfected cells as blank; serum starvation was continued for 24h after 24h transfection, cell pellet was collected from each of the above groups of cells Huh7.5.1TNNT1 KO cell line and resuspended in 1mL serum-free DMEM medium, and cells counted at 5X 10 ⁴ The concentration of each mL was aspirated into the transwell upper chamber, and 500. Mu.L of DMEM complete medium containing 20% FBS was added to the lower chamber; after 24h the medium was discarded, washed 3 times with PBS and added500 μL of 4% paraformaldehyde is fixed for 15min at room temperature; washing 3 times by PBS, adding 500 mu L of 0.1% crystal violet solution, and dyeing for 20min at room temperature; the cells in the upper chamber were gently wiped clean with a cotton swab, stained under a microscope and counted.

5. Cell scratch assay: huh7.5.1 and Huh7.5.1TNNT1 KO cells at 1X 10 ⁶ The concentration of each mL is inoculated into a 6-well plate, and each well is 2mL; uniformly scribing transverse lines by using a marker pen behind the 6-hole plate, and crossing the through holes at intervals of about 0.5-1 cm by using a ruler; the gun head is vertical and cannot incline compared with the ruler in the next day, and the gun head is vertical to the transverse line scratch behind the ruler as much as possible; washing cells with PBS for 3 times, removing the scraped cells, and adding a serum-free culture medium; placing 37 degrees 5% CO ₂ And (5) culturing in an incubator. Samples were taken at 0, 24, 48 hours and photographed.

3. Experimental results

As shown in FIG. 5A, western blot verifies that TNNT1 is knocked out and knocked down in human hepatoma cell Huh7.5.1, compared with untransfected cells and transfected empty vector, TNNT1 is obviously inhibited in human hepatoma cell Huh7.5.1; as shown in FIG. 5B, western blot verifies that the over-expression efficiency of TNNT1 in human hepatoma cell Huh7.5.1 is obviously improved compared with that of untransfected cells and empty vector groups.

As can be seen from fig. 6, CCK8 experiments verify the effect of TNNT1 on human hepatoma cell huh7.5.1 proliferation; compared with the untransfected group and the empty group, the TNNT1 over-expression at 48 hours can obviously promote the proliferation capacity of Huh7.5.1 cells, and the proliferation capacity of Huh7.5.1 cells after TNNT1 is knocked out is obviously inhibited. The phenotype was increased after the expression of the supplemented TNNT 1.

As can be seen from fig. 7, the flow cytometry experiments verify the effect of TNNT1 on the huh7.5.1 cell cycle of human hepatoma cells; compared with the untransfected group, the over-expression of TNNT1 can promote Huh7.5.1 cells to enter S phase, the number of cells in S phase is reduced after TNNT1 is knocked out, the number of cells in G0/G1 phase is increased, and the phenotype is improved after the expression of the supplemented TNNT 1.

As can be seen from fig. 8, the Transwell experiment verifies the influence of TNNT1 on the migration ability of human hepatoma cells huh 7.5.1; compared with the untransfected group and the empty group, the over-expression of TNNT1 can promote the obviously increased migration quantity of Huh7.5.1 cells, the migration quantity of TNNT1 is obviously reduced after being knocked out, and the phenotype is improved after the expression of the supplemented TNNT 1.

As can be seen from fig. 9, the cell scratch experiment verifies the effect of TNNT1 on the migration ability of human hepatoma cells huh 7.5.1; compared with normal Huh7.5.1 cells, TNNT1 is knocked out, the cell migration capacity is reduced, the scratch width is slowly changed, and the phenotype is improved after the TNNT1 is supplemented again.

In summary, the effect of TNNT1 on the proliferation and migration capacity and the cell cycle of human hepatoma cells Huh7.5.1 was verified by CCK8, flow cytometry, transwell and cell scratch, and the result shows that TNNT1 can promote the proliferation and migration capacity of Huh7.5.1 cells and promote the Huh7.5.1 cells to enter S phase.

Taken together, it is shown that TNNT1 can provide a new potential drug target for treating liver cancer, and TNNT1 is used as a new target, and shRNA of TNNT1 gene and slow virus thereof can inhibit expression of TNNT1, so that the TNNT1 can be used as an anti-liver cancer drug.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Sequence listing

<110> university of Wuhan

<120> TNNT1 and its use in preparing diagnosis kit for viral hepatocellular carcinoma and preparing or screening anti-liver cancer medicine

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 756

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgtcggaca ccgaggagca ggaatatgag gaggagcagc cggaagagga ggctgcggag 60

gaggaggagg aagaagagga acgccccaaa ccaagccgcc ccgtggtgcc tcctttgatc 120

ccgccaaaga tcccagaagg ggagcgcgtt gacttcgatg acatccaccg caagcgcatg 180

gagaaagacc tgctggagct gcagacactc atcgatgtac atttcgagca gcggaagaag 240

gaggaagagg agctggttgc cttgaaggag cgcattgagc ggcgccggtc agagagagcc 300

gagcaacagc gcttcagaac tgagaaggaa cgcgaacgtc aggctaagct ggcggaggag 360

aagatgagga aggaagagga agaggccaag aagcgggcag aggatgatgc caagaaaaag 420

aaggtgctgt ccaacatggg ggcccatttt ggcggctacc tggtcaaggc agaacagaag 480

cgtggtaagc ggcagacggg gcgggagatg aaggtgcgca tcctctccga gcgtaagaag 540

cctctggaca ttgactacat gggggaggaa cagctccggg agaaagccca ggagctgtcg 600

gactggatcc accagctgga gtctgagaag ttcgacctga tggcgaagct gaaacagcag 660

aaatatgaga tcaacgtgct gtacaaccgc atcagccacg cccagaagtt ccggaagggg 720

gcagggaagg gccgcgttgg aggccgctgg aagtga 756

<210> 2

<211> 262

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Ser Asp Thr Glu Glu Gln Glu Tyr Glu Glu Glu Gln Pro Glu Glu

1 5 10 15

Glu Ala Ala Glu Glu Glu Glu Glu Ala Pro Glu Glu Pro Glu Pro Val

20 25 30

Ala Glu Pro Glu Glu Glu Arg Pro Lys Pro Ser Arg Pro Val Val Pro

35 40 45

Pro Leu Ile Pro Pro Lys Ile Pro Glu Gly Glu Arg Val Asp Phe Asp

50 55 60

Asp Ile His Arg Lys Arg Met Glu Lys Asp Leu Leu Glu Leu Gln Thr

65 70 75 80

Leu Ile Asp Val His Phe Glu Gln Arg Lys Lys Glu Glu Glu Glu Leu

85 90 95

Val Ala Leu Lys Glu Arg Ile Glu Arg Arg Arg Ser Glu Arg Ala Glu

100 105 110

Gln Gln Arg Phe Arg Thr Glu Lys Glu Arg Glu Arg Gln Ala Lys Leu

115 120 125

Ala Glu Glu Lys Met Arg Lys Glu Glu Glu Glu Ala Lys Lys Arg Ala

130 135 140

Glu Asp Asp Ala Lys Lys Lys Lys Val Leu Ser Asn Met Gly Ala His

145 150 155 160

Phe Gly Gly Tyr Leu Val Lys Ala Glu Gln Lys Arg Gly Lys Arg Gln

165 170 175

Thr Gly Arg Glu Met Lys Val Arg Ile Leu Ser Glu Arg Lys Lys Pro

180 185 190

Leu Asp Ile Asp Tyr Met Gly Glu Glu Gln Leu Arg Glu Lys Ala Gln

195 200 205

Glu Leu Ser Asp Trp Ile His Gln Leu Glu Ser Glu Lys Phe Asp Leu

210 215 220

Met Ala Lys Leu Lys Gln Gln Lys Tyr Glu Ile Asn Val Leu Tyr Asn

225 230 235 240

Arg Ile Ser His Ala Gln Lys Phe Arg Lys Gly Ala Gly Lys Gly Arg

245 250 255

Val Gly Gly Arg Trp Lys

260

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gagcgtaaga agcctctgg 19

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gctaccgatg ggacaaac 18

<210> 5

<211> 56

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gctgcagaca ctcatcgatg tttcaagctt acatcgatga gtgtctgcag cttttt 56

<210> 6

<211> 64

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aattaaaaag ctgcagacac tcatcgatgt aagcttgaaa catcgatgag tgtctgcagc 60

ggcc 64

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gaaggtgaag gtcggagtc 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gaagatggtg atgggatttc 20

<210> 9

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tagaattcta atgtcggaca ccgagga 27

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

taaagcttct tccagcggcc tccaa 25

Claims (1)

1. The application of a detection primer of TNNT1 in preparing a viral hepatocellular carcinoma diagnosis kit is characterized in that the sequence of the primer is shown as SEQ ID NO:3 and SEQ ID NO:4, wherein the TNNT1 is used as a molecular marker for distinguishing a hepatitis virus-positive liver cancer patient from a hepatitis virus-negative liver cancer patient, and the hepatitis comprises hepatitis B and/or hepatitis C.

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