CN115851931A - Application of liver cancer molecular marker in preparation of liver cancer detection or curative effect evaluation product - Google Patents

Abstract

The invention discloses application of liver cancer molecular markers in preparing liver cancer detection or curative effect evaluation products, wherein the liver cancer molecular markers are hsa _ circ _0002490 and hsa _ circ _0101802, and the nucleic acid sequence of the hsa _ circ _0002490 is shown as SEQ ID NO 1; the nucleic acid sequence of hsa _ circ _0101802 is shown in SEQ ID NO 3. The molecular marker has a correlation with liver cancer, can quickly, simply and accurately realize auxiliary diagnosis of the liver cancer, and provides a basis for diagnosis of the liver cancer and selection of treatment effect evaluation indexes; in addition, the molecular marker can also be used as a liver cancer drug treatment target.

Description

Application of liver cancer molecular marker in preparation of liver cancer detection or curative effect evaluation product

Technical Field

The invention relates to the field of biotechnology, in particular to application of a liver cancer molecular marker in preparation of a liver cancer detection or treatment effect evaluation product.

Background

The Primary Liver Cancer (PLC) is listed as the seventh most common cancer in the world, wherein hepatocellular carcinoma (HCC) caused by malignant transformation of liver cells accounts for about 75 percent of the Primary Liver Cancer (PLC). Intrahepatic cholangiocarcinoma is another major PLC, originating in bile duct cells, accounting for 12-15% of liver malignant tumors. Due to the aggressiveness of these liver malignancies, primary liver cancer has become the fourth most common cause of cancer death. It is statistically estimated that more than 90% of HCC cases occur in the context of chronic liver disease, and cirrhosis of the liver by any etiology is a high risk factor for HCC, including chronic alcohol consumption, non-alcoholic steatohepatitis (NASH) associated with diabetes or obesity, and HBV or HCV infection. HCC is the leading cause of death in patients with cirrhosis, with an incidence of HCC of 1-6% per year. The risk factors for liver cancer are currently shifting due to improvements in public health measures, the prevalence of chronic HBV and HCV infection is decreasing in many regions, and diseases associated with obesity, diabetes, and metabolic syndrome have become major factors that contribute to an increase in the incidence of liver cancer in many low-risk countries. Some socio-demographic characteristics are associated with liver cancer, where aging is a strong risk factor and reports indicate that the highest incidence of the relevant population is those over the age of 70. In addition, liver cancer has a strong male dominance (male-female ratio of 2-3. Despite advances in diagnosis, surgical techniques and liver transplantation, long-term survival of liver cancer patients has been poor to date. The relevant data show that the prognosis of liver cancer is closely related to the stage of disease progression: after the very early (Barcelona stage 0, BCLC 0) and early (BCLC A) liver cancer patients receive active surgical treatment, the 5-year survival rate is respectively close to 90 percent and 50 to 70 percent; however, the five-year survival rate of the middle and late stage liver cancer patients is only 14%. Therefore, effective early diagnosis and early treatment of liver cancer are one of the key factors for improving the prognosis of liver cancer patients.

Although early diagnosis and early treatment of liver cancer are necessary, in clinical practice, early diagnosis still faces many difficult difficulties. First, early stage liver cancer lacks specific symptoms, resulting in a low hospitalization rate for patients with early stage liver cancer. About 2/3 of liver cancer patients are diagnosed as advanced liver cancer for the first time, and the chance of surgical treatment is lost; secondly, the existing liver cancer screening means in clinic has defects. The diagnosis efficiency of liver ultrasound is affected by factors such as tumor size, subjective judgment of an operator, review frequency, instrument accuracy and the like, so that only less than half of early liver cancer patients can be found in the ultrasound examination. Alpha-fetoprotein (AFP) is a liver cancer marker widely used in clinic, but the detection rate of early liver cancer is only 66%. Although most HCCs have characteristic features in imaging, about 10% of tumors (up to 30% of tumors up to 1-2cm in diameter) have atypical manifestations, lacking the imaging characteristics of HCC. In addition, the prognosis after radical resection of liver cancer is still unsatisfactory due to the high incidence of postoperative recurrence. Identification of early diagnostic and prognostic biomarkers for metastatic liver cancer is therefore crucial to overcome these ongoing challenges for human health.

Disclosure of Invention

The invention aims to provide application of a liver cancer molecular marker in preparation of a liver cancer detection or curative effect evaluation product, and the molecular marker can quickly, simply and accurately realize auxiliary diagnosis of liver cancer.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a molecular marker for detecting or evaluating the curative effect of liver cancer, which is characterized in that the molecular marker comprises at least one of hsa _ circ _0002490, hsa _ circ _0005197 and hsa _ circ _0101802, and the nucleic acid sequence of hsa _ circ _0002490 is shown as SEQ ID NO. 1; the nucleic acid sequence of hsa _ circ _0005197 is shown as SEQ ID NO 2; the nucleic acid sequence of hsa _ circ _0101802 is shown in SEQ ID NO 3.

The second aspect of the invention provides an application of the molecular marker in preparation of a liver cancer detection or treatment effect evaluation product.

Preferably, the product includes, but is not limited to, a kit.

The third aspect of the present invention provides a product for detecting or evaluating the therapeutic effect of liver cancer, wherein the product comprises an amplification primer for amplifying cDNA complementary to a molecular marker;

the molecular marker comprises at least one of hsa _ circ _0002490, hsa _ circ _0005197 and hsa _ circ _0101802, and the nucleic acid sequence of hsa _ circ _0002490 is shown as SEQ ID NO 1; the nucleic acid sequence of hsa _ circ _0005197 is shown as SEQ ID NO 2; the nucleic acid sequence of hsa _ circ _0101802 is shown in SEQ ID NO 3.

Preferably, the product further comprises an RNA extraction reagent.

Preferably, the upstream amplification primer and the downstream amplification primer corresponding to hsa _ circ _0002490 are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5;

the upstream amplification primer and the downstream amplification primer corresponding to hsa _ circ _0005197 are respectively shown as SEQ ID NO 6 and SEQ ID NO 7;

the upstream amplification primer and the downstream amplification primer corresponding to hsa _ circ _0101802 are respectively shown as SEQ ID NO 8 and SEQ ID NO 9.

The fourth aspect of the invention also provides an application of SiRNA of hsa _ circ _0005197 and/or hsa _ circ _0101802 in preparing a medicament for treating liver cancer.

Preferably, the SiRNA sequence of hsa _ circ _0101802 is as follows:

the sequence of the sense strand is shown as SEQ ID NO. 10, and the sequence of the antisense strand is shown as SEQ ID NO. 11; or, the sequence of the sense strand is shown as SEQ ID NO. 12, and the sequence of the antisense strand is shown as SEQ ID NO. 13.

In a fifth aspect, the invention provides a medicament comprising sirnas hsa _ circ _0005197 and/or hsa _ circ _0101802 for use in the treatment of liver cancer.

Preferably, the SiRNA sequence of hsa _ circ _0101802 is as follows:

the sequence of the sense strand is shown as SEQ ID NO. 10, and the sequence of the antisense strand is shown as SEQ ID NO. 11; or, the sequence of the sense strand is shown as SEQ ID NO. 12, and the sequence of the antisense strand is shown as SEQ ID NO. 13.

Compared with the prior art, the invention has the beneficial effects that at least:

the molecular marker has a correlation with liver cancer, can quickly, simply and accurately realize auxiliary diagnosis of the liver cancer, and provides a basis for diagnosis of the liver cancer and selection of treatment effect evaluation indexes; in addition, the molecular marker can also be used as a liver cancer drug treatment target.

The amplification primer adopted by the invention has excellent specificity, can stably amplify the molecular marker, and improves the detection accuracy of the molecular marker.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a bar graph showing the expression level of circPNN in liver cancer cells in example 1 of the present invention;

FIG. 2 is a bar graph showing the expression level of circPTPN12 in hepatoma cells in example 1 of the present invention;

FIG. 3 is a bar graph showing the expression level of circFCHO2 in hepatocarcinoma cells in example 1 of the present invention;

FIG. 4 shows the results of successful transfection observed under a fluorescence microscope after silencing of CircpNN expression in example 2 of the present invention;

FIG. 5 is a bar graph of PCR assay of the expression level of CircPNN after silencing of CircPNN expression in example 2 of the present invention;

FIG. 6 is a bar graph of cell proliferative capacity following silencing of CircPNN expression in example 2 of the invention;

FIG. 7 shows the result of the flow analysis of SK-HEP-1 apoptosis after silencing the expression of CircPNN in example 2;

FIG. 8 is a bar graph of the rate of apoptosis of SK-HEP-1 cells following silencing of CircPNN expression in example 2 of the invention;

FIG. 9 shows the results of the Western Blot detection of Caspase-3 protein in example 2 of the present invention;

FIG. 10 is a bar graph of the detection of Caspase-3 protein expression level by Western Blot in example 2 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

At present, the diagnosis efficiency of liver ultrasound is influenced by factors such as tumor size, subjective judgment of operators, recheck frequency and instrument precision, so that only less than half of early liver cancer patients can be found in the ultrasound examination. Alpha-fetoprotein (AFP) is a liver cancer marker widely used in clinic, but the detection rate of early liver cancer is only 66%. While most HCCs have characteristic features in imaging, about 10% of tumors (up to 30% of tumors up to 1-2cm in diameter) have atypical manifestations, lacking the imaging characteristics of HCC. In addition, the prognosis after radical resection of liver cancer is still unsatisfactory due to the high incidence of postoperative recurrence. Identification of early diagnostic and prognostic biomarkers for metastatic liver cancer is therefore crucial to overcome these ongoing challenges for human health.

In view of the above, the present invention provides a molecular marker for detecting or evaluating the therapeutic effect of liver cancer, wherein the molecular marker comprises at least one of hsa _ circ _0002490, hsa _ circ _0005197 and hsa _ circ _0101802,

wherein, the nucleic acid sequence of hsa _ circ _0002490 is shown as SEQ ID NO:1, and specifically comprises:

GUGUGUCACGGGGUCCCAGCCCUGUCAGCCUUGGAAAUCAGGAUACCUUACCUGUGGCAGUUGCCCUUACAGAAUCUGUUAAUGCCUACUUUAAAGGAGCAGAUCCCACCAAGUGUAUUGUGAAGAUCACUGGUGAUAUGACAAUGUCAUUUCCAAGUGGAAUUAUUAAAGUCUUCACCAGCAAUCCAACUCCAGCUGUGUUGUGCUUCAGGGUGAAAAAUAUCAGCAGACUAGAGCAGAUUCUUCCAAAUGCACAGCUUGUGUUCAG；

the nucleic acid sequence of hsa _ circ _0005197 is shown in SEQ ID NO:2, and specifically comprises:

UUGAUCACAGCCGAGUUAAAUUGACAUUAAAGACUCCUUCACAAGAUUCAGACUAUAUCAAUGCAAAUUUUAUAAAGGGCGUCUAUGGGCCAAAAGCAUAUGUAGCAACUCAAGGACCUUUAGCAAAUACAGUAAUAGAUUUUUGGAGGAUGAUAUGGGAGUAUAAUGUUGUGAUCAUUGUAAUGGCCUGCCGAGAAUUUGAGAUGGGAAGGAAAAAAUGUGAGCGCUAUUGGCCUUUGUAUGGAGAAGACCCCAUAACGUUUGCACCAUUUAAAAUUUCUUGUGAGGAUGAACAAGCAAGAACAGACUACUUCAUCAGGACACUCUUACUUGAAUUUCAAAAU；

the nucleic acid sequence of hsa _ circ _0101802 is shown in SEQ ID NO:3, and specifically comprises:

CAAAAGCGGCGCCAGGAAAUUGAACAAAAACUUGAAGUUCAGGCAGAAGAAGAGAGAAAGCAGGUUGAAAAUGAAAGGAGAGAACUGUUUGAAGAGAGGCGUGCUAAACAGACAGAACUGCGGCUUUUGGAACAGAAAGUUGAGCUUGCGCAGCUGCAAGAAGAAUGGAAUGAACAUAAUGCCAAAAUAAUUAAAUAUAUAAGAACUAAGACAAAGCCCCAUUUGUUUUAUAUUCCUGGAAGAAUGUGUCCAGCUACCCAAAAACUAAUAGAAGAGUCACAGAGAAAAAUGAACG。

the molecular marker of the invention has a correlation with liver cancer, wherein hsa _ circ _0005197 and hsa _ circ _0101802 are positively correlated with liver cancer, and hsa _ circ _0002490 is negatively correlated with liver cancer; the molecular marker can quickly, simply and accurately realize the auxiliary diagnosis of the liver cancer and provide a basis for the diagnosis of the liver cancer and the selection of the evaluation index of the curative effect; in addition, the molecular marker can also be used as a liver cancer drug treatment target.

The invention also provides application of the molecular marker in preparation of liver cancer detection or curative effect evaluation products.

Further, the above products include, but are not limited to, kits.

In another embodiment, the present invention provides a product for detecting or evaluating the efficacy of liver cancer, which comprises an amplification primer for amplifying cDNA complementary to a molecular marker;

the molecular marker comprises at least one of hsa _ circ _0002490, hsa _ circ _0005197 and hsa _ circ _0101802, wherein the nucleic acid sequence of hsa _ circ _0002490 is shown as SEQ ID NO: 1.

Further, the product also comprises an RNA extraction reagent.

Further, the upstream amplification primer corresponding to hsa _ circ _0002490 is shown as SEQ ID NO. 4, and specifically CTGTGTTGTGCTTCAGGGTG; the downstream amplification primer is shown as SEQ ID NO. 5, and is specifically TGACACACCTGAACAAGC;

the upstream amplification primer corresponding to hsa _ circ _0005197 is shown as SEQ ID NO. 6, and specifically is CAAAATTTGATCACAGCCGAGT; the downstream amplification primer is shown as SEQ ID NO. 7, and specifically is GCCCATAGACGCCCTTTAAA;

the upstream amplification primer corresponding to hsa _ circ _0101802 is shown as SEQ ID NO:8, and specifically is AGAATGTGTCCAGCTACCA; the downstream amplification primer is shown as SEQ ID NO. 9, and is specifically CAAAGCCGCAGTTCTGTCT.

The amplification primer has excellent specificity, can stably amplify the molecular marker, and further effectively improves the detection accuracy of the molecular marker.

The embodiment of the invention also provides application of SiRNA of hsa _ circ _0005197 and/or hsa _ circ _0101802 in preparation of a medicament for treating liver cancer.

Further, the SiRNA sequence of hsa _ circ _0101802 is as follows:

the sense strand sequence is shown as SEQ ID NO. 10, specifically GAGAAAAAUGAACGCAAAA (TT), and the antisense strand sequence is shown as SEQ ID NO. 11, specifically UUUUGCGGUUCAUUUUUCC (TT); or, the sense strand sequence is shown as SEQ ID NO:12, specifically AAUGAACGCAAAAGCGGCG (TT), and the antisense strand sequence is shown as SEQ ID NO:13, specifically CGCCGCUUUGCGUUCAUU (TT).

The SiRNA can inhibit the expression level of hsa _ circ _0101802 in cells, further can inhibit the proliferation of liver cancer cells, and achieves the purpose of treating liver cancer.

The embodiment of the invention further provides a medicament for treating liver cancer, which comprises SiRNA of hsa _ circ _0005197 and/or hsa _ circ _ 0101802.

Further, the SiRNA sequence of hsa _ circ _0101802 is as follows:

the sense strand sequence is shown as SEQ ID NO. 10, specifically GAGAAAAAUGAACGCAAAA (TT), and the antisense strand sequence is shown as SEQ ID NO. 11, specifically UUUUGCGGUUCAUUUUUCC (TT); or, the sense strand sequence is shown as SEQ ID NO:12, specifically AAUGAACGCAAAGGCGGCG (TT), and the antisense strand sequence is shown as SEQ ID NO:13, specifically CGCCGCUUUGCGGUCUCUAUU (TT).

The technical solution of the present invention is further described in detail by the following specific examples.

Example 1

This example shows the expression of circPNN (hsa _ circ _ 0101802), circFCHO2 (hsa _ circ _ 0002490) and circPTPN12 (hsa _ circ _ 0005197) in human liver cancer cell lines and human normal liver cell lines:

human normal liver cell line HL-7702 cells FBS Fetal Bovine Serum (FBS) using RPMI-1640 medium + 10%; and Huh-7 using DMEM medium +10% FBS; SK-HEP-1 uses a special culture medium purchased from the cell bank of the Committee for the preservation of type cultures of the Chinese academy of sciences (Shanghai). The cells are cultured in a cell incubator with 37 ℃ and 5% of CO2 saturation humidity and grow in an adherent manner.

(1) Cell resuscitation

Quickly shaking the cells back and forth in a 37 ℃ water bath from a liquid nitrogen tank or an ultra-low temperature refrigerator at minus 80 ℃ to quickly melt the cells. And transferring the cell suspension of the cryopreserved tube into a 15ml centrifuge tube in a super clean bench by using a 1ml pipette gun, blowing, uniformly mixing, and centrifuging at 1000rpm for 5min. The supernatant was discarded in a clean bench and 1ml of culture medium was added to resuspend the cells and blow them well. The cell suspension was transferred to a flask, 5ml of culture medium was added, and the cells were evenly distributed at the bottom of the flask by gentle shaking. The cell state was observed under an inverted microscope. Marking cell information, date and culture person information, and placing the cells in a carbon dioxide incubator for culture.

(2) Cell exchange liquid

Observing the growth state of the cells under a mirror, and changing the cell culture medium once according to 1-3 days. In a clean bench, cells were washed 2-3 times with 1ml of the culture medium in a pipette cell flask and 1 XPBS buffer, and then 5ml of a new 10% FBS-containing cell culture medium was added.

(3) Cell passage

Cells were observed under the mirror and subcultured when 80% -90% was reached. In a clean bench, the old medium was aspirated with a 1ml pipette, followed by washing the cells 2-3 times with 1 × PBS, and then adding 1.5ml of a 0.25% trypsin solution. The flask was then tipped, shaken gently to bring the pancreatin into full contact with the cells, and digested in a carbon dioxide incubator for 3 minutes. When the cells were completely digested into spheres and changed from adherent to floating, 2.0ml of the culture medium was added to stop the digestion by observation under an inverted microscope. Centrifuging at 1000rpm for 5 minutes at room temperature by using a low-speed centrifuge, discarding supernatant, adding 1ml of complete culture medium, blowing, resuspending cells, inoculating into a new culture flask according to the proportion of 1.

(4) Cell cryopreservation

Prophase manipulations such as cell passaging. And mixing FBS and DMSO according to a volume ratio of 4. The digested cells were placed in a 10ml centrifuge tube and centrifuged at 1000rpm for 5min. And after centrifugation, adding cell freezing solution for resuspension, blowing, mixing uniformly, transferring to a cell sterile freezing tube with 1.6ml of tube, sealing the tube opening, and clearly marking the cell type and the freezing date. And (3) performing gradient cooling and freezing on the freezing tube for 24 hours in an ultra-low temperature refrigerator at the temperature of 4 ℃ for 30min and the temperature of 20 ℃ below zero for 30min, and finally putting the freezing tube into liquid nitrogen for long-term freezing.

(5) Cellular RNA extraction

After aspirating the medium, the medium was washed 1 time with PBS, 500. Mu.l of Trizol was added thereto and allowed to stand at room temperature for 5 minutes, and after transferring the Trizol to a new 1.5ml EP tube, 100. Mu.l of chloroform was added thereto and sufficiently mixed to form a chyle state and allowed to stand for 10 minutes. Centrifuging at 4 ℃ and 12000g for 15 minutes, taking the upper clear liquid and putting the upper clear liquid into a new 1.5ml centrifuge tube; adding isopropanol (same volume as the supernatant and precooled at-20 deg.C), standing at room temperature for 20min (or overnight at-80 deg.C), centrifuging at 4 deg.C for 10 min, and discarding the supernatant; adding 500 μ l precooled 75% ethanol, mixing well, centrifuging at 4 deg.C 8500g for 5 minutes, discarding the supernatant, drying at room temperature for 5 to 10 minutes, adding 10 μ l RNase-, DNase-free Water after the ethanol is completely volatilized, and measuring the RNA concentration and purity.

(6) Reverse transcription reaction

Based on the RNA concentration measurements, total RNA was diluted to 500 ng/. Mu.l with enzyme-free water, and the reverse transcription reaction solution was prepared on ice throughout the course of the procedure of TAKARA RR037A reverse transcription kit (Table 1). After preparation, the solution is immediately put into a PCR instrument for reverse transcription, and the reverse transcription reaction conditions are 15min at 37 ℃ and 5s at 85 ℃. The total cDNA concentration is obtained after the reaction is finished, and the total cDNA concentration can be stored in a refrigerator at-80 ℃ or immediately subjected to real-time fluorescent quantitative PCR.

TABLE 1 preparation of reverse transcription reaction solution

(7) Real-time fluorescent quantitative PCR

Light shielding, the whole process is carried out on ice, according to TAKARA RR820A kit instruction, real-time fluorescence quantitative PCR (qRT-PCR) reaction solution is prepared according to the table 2, and can be diluted properly according to cDNA concentration, and the reaction system is 20 mu l per well and at least 3 multiple wells.

TABLE 2 preparation of qRT-PCR reaction solution

The conditions of the amplification reaction are as follows: melting curves were obtained by fluorescent melting at 95 ℃ for 30s (pre-denaturation) → (95 ℃ for 5s,60 ℃ for 30 s). Times.40 cycles → melting. At the end of each cycle in Stage 2, the instrument will automatically collect and analyze the fluorescence intensity of each sample.

(8) Real-time fluorescent PCR data processing

After the reaction is completed, the information such as the melting curve and melting temperature of the target gene is checked. And (3) extracting the Ct value of the sample, performing relative quantitative analysis on the gene differential expression level by using a log 2-delta Ct (delta Ct = Ct target gene-Ct reference gene, delta Ct = delta Ct hepatocyte-delta Ct normal human hepatocyte) method, and setting U6 as the reference gene. Data analysis using GraphPad Premier 9 software, statistical analysis using a one-way variance test.

(9) Results

The expression results of circPNN (hsa _ circ _ 0101802), circFCHO2 (hsa _ circ _ 0002490) and circPTPN12 (hsa _ circ _ 0005197) in human liver cancer cell lines and human normal liver cell lines are shown in tables 3 to 5; the expression histograms are shown in FIGS. 1-3;

TABLE 3 expression of circPNN in hepatoma cells (X native SD, n = 3)

VS HL-7702，*P<0.05，**P<0.01；

TABLE 4 expression of circPTPN12 in hepatoma cells (Xterr SD, n = 3)

VS HL-7702，***P<0.001

TABLE 5 expression of circFCHO2 in hepatoma cells (Xterr SD, n = 3)

VS HL-7702，*P<0.05,**P<0.01

As can be seen from tables 3 to 5 and FIGS. 1 to 3:

compared with a human normal liver cell line HL7702 (FC value is 1.000 +/-0.045), the circFCH02 has low expression in liver cancer cells (FC values are 0.785 +/-0.039 and 0.036 +/-0.004 respectively, and p-knot is 0.05); the circPNN shows high expression in the liver cancer cell line (FC values are HL-7702 0.950 +/-0.010, huh-7.206 +/-0.118, SK-HEP-1.822 +/-0.288 and p-woven layer is 0.05 respectively); circPTPN12 is also up-regulated in hepatoma cells (FC values HL-7702; it can be seen that the molecular markers have a correlation with liver cancer, and can be used as markers for liver cancer examination and therapeutic effect evaluation.

Example 2

This example is to silence the effect of circPNN (hsa _ circ _ 0101802) in the liver cancer cell SK-HEP-1 on cell biological behavior:

(1) Experiment grouping

(1) siRNA-1 group: circPNN-siRNA-1 (SEQ ID NO:10 and SEQ ID NO: 11) was added to the lentiviral complex at the time of transfection. (2) siRNA-2 group: circPNN-siRNA-2 (SEQ ID NO:12 and SEQ ID NO: 13) lentiviral complex was added at the time of transfection. (3) Blank control group: hepatoma carcinoma cells without any intervention.

(2) Cell transfection

Collecting SK-HEP-1 hepatocarcinoma cells with good growth state and logarithmic growth phase, digesting with trypsin, centrifuging, resuspending in culture medium, counting, adjusting cell concentration to 1.0 × 10 ⁵ The cells were seeded at a density of one/ml in a 6-well plate and placed in CO ₂ Culturing in an incubator until the cell density reaches 70-80%; old media was aspirated away and cells were washed 2 times with PBS. Using complete medium, 25 Xvirus infection solution is diluted to 1 ×, and 500. Mu.l of diluted 1 Xvirus infection solution is added to each well. Transfection was performed with MOI =30 and 15 μ l circPNN-siRNA-virus complex was added per well at a concentration of 1 × 108TU/ml as recommended by the instructions. And (4) slightly shaking the culture plate, fully and uniformly mixing, placing in an incubator, incubating for 8 hours, then replacing fresh culture solution, and continuing to culture for 24 hours until needed by the next experiment.

(3) Detection of transfection efficiency

After 24h of transfection, the cells are placed under a fluorescence microscope to observe transfection efficiency, the observation result is shown in figure 4, then total RNA of the cells is extracted, reverse transcription is carried out on the extracted total RNA, and the expression level of circPNN is detected by qRT-PCR.

As can be seen from FIG. 4, under the fluorescence microscope, green fluorescent cells were observed after successful transfection of the SiRNA-1 and SiRNA-2 groups.

The results of the expression level measurements of circPNN are shown in table 6, and the bar graph is shown in fig. 5;

TABLE 6 PCR detection of changes in the expression level of CircpNN after silencing: (

n＝3)

VS Control group，*P<0.05，**P<0.01

As can be seen from table 6 and fig. 5:

after SiRNA-1 and SiRNA-2 are transfected to a human liver cancer cell SK-HEP-1, the PCR result indicates that the SK-HEP-1 cell inner Control group is 1.005 soil 0.386; siRNA-1 group 0.366 soil 0.041, the expression level is reduced compared with that of Control group (P < 0.01); siRNA-2 group 0.449 soil 0.023, compared with Control group the expression level was decreased (P < 0.01), compared with SiRNA-1 group the expression level was increased (P > 0.05).

(4) CCK8 detection of cell proliferation after transfection

Taking SK-HEP-1 cells after successful transfection, digesting with pancreatin, centrifuging, and preparing into the product with the concentration of 2.0 × 10 ⁴ Cell suspension per ml. The 96-well plates were divided according to the experiment and 100. Mu.l of cell suspension was added to each well, 3 replicates of each group were emptied. To prevent cell culture medium evaporation from interfering with the results of the experiment, PBS was added to the surrounding blank wells. The 96-well plate is placed in a cell incubator, and is taken out after being cultured for 24 hours, and 10 mul of CCK8 reagent is added into each well. The 96-well plate was returned to the cell incubator, and the absorbance of each well was measured on a microplate reader after 2 hours. Cell proliferation rate = (experimental OD value/control OD value);

the results are shown in Table 7, and the bar graph is shown in FIG. 6;

TABLE 7 cell proliferation potency Change following silencing of Circpnn: (

n＝3)/>

VS Control group，*P<0.05，**P<0.01；VS SiRNA-1 group， ^# P<0.05， ^## P<0.01

As can be seen from table 7 and fig. 6:

control group 102.280 soil 5.399; siRNA-1 group 49.259 and 2.272, the cell proliferation rate is reduced compared with the Control group (P < 0.01); siRNA-2 group 82.628 and 4.013 showed decreased cell proliferation rate (P < 0.01) compared to Control group and increased cell proliferation rate (P < 0.01) compared to SiRNA-1 group.

(5) Detection of apoptosis by flow cytometry

Taking SK-HEP-1 cells transfected by siRNA of a blank control group and an experimental group to prepare single cell suspension, adjusting the cell density to be 2 multiplied by 10 ⁵ Inoculating each/ml of the cells into a 6-well plate, culturing the cells in serum-free DMEM culture solution for 48 hours, digesting the cells with trypsin, stopping digestion of complete culture medium, collecting cell digestion solution, centrifuging, collecting by centrifugation, discarding supernatant, then re-suspending the cell suspension by PBS, centrifuging the cell suspension, discarding supernatant, adding 100 mu l of 1 × loading buffer solution to gently re-suspend the cells, and transferring the cells to a flow tube; adding 5. Mu.l Annexin V and 5. Mu.l 7-AAD, mixing gently, incubating for 15min (on ice) in the dark at room temperature, adding 400. Mu.l of 1 × loading buffer solution, and detecting and analyzing by using a flow cytometer; and (4) judging a result: the lower left quadrant Q3 shows viable cells, the upper left quadrant Q1 is necrotic cells, the upper right quadrant Q2 is late apoptotic cells, and the lower right quadrant Q4 is early apoptotic cells. Total apoptosis rate = (Q2 + Q4)%.

The result of the SK-HEP-1 cell apoptosis flow analysis after the CircPNN expression is silenced is shown in figure 7; the apoptosis rate of SK-HEP-1 cells after silencing of the expression of CircPNN is shown in Table 8, and the histogram is shown in FIG. 8;

TABLE 8 silent Circpnn tablePost-expression SK-HEP-1 apoptosis rate ()

n＝3)

VS Control group，*P<0.05，**P<0.01；VS SiRNA-1 group， ^# P<0.05， ^## P<0.01

As can be seen from table 8 and fig. 7 to 8, control group 4.800 soil 0.608; siRNA-1 group 27.133 soil 0.751, and compared with Control group, the apoptosis rate is increased (P < 0.01); the apoptosis rate of SiRNA-2 group (16.633 th 0.404) was higher than that of Control group (P < 0.01) and lower than that of SiRNA-1 group (P < 0.01).

(6) Western blot detection of intracellular protein expression

Preparation of protein sample and determination of content

(1) The cells with good growth state are collected, cultured and passaged by the cells, inoculated into a 6-hole cell culture plate, observed to have good cell growth state and about 80 percent of confluence, rinsed twice by precooled PBS, digested conventionally, collected and transferred to 3 1.5ml EP tubes in groups. (2) To each EP tube, 300. Mu.l of the complex cell lysate (25 Xprotease inhibitor 12. Mu.l +100 XPMSF 3. Mu.l +10 Xphosphatase inhibitor 30. Mu.l + RIPA make-up volume to 300. Mu.l) was added and lysed by shaking on ice for 30min. (3) Transferring the EP tube to a high-speed refrigerated centrifuge for centrifugation at 4 ℃, 12000rpm for 20min, sucking the supernatant in the EP tube, and discarding the precipitate to obtain the total cell protein.

Preparation of SDS-PAGE gels

According to the molecular weight of the protein required by the experiment and the instruction of the kit, separating gel and concentrated gel with proper concentration are prepared, and during gel preparation, the phenomenon of gel leakage is noticed, and bubbles are prevented from being generated between the separating gel and the concentrated gel.

Sample loading

Putting an electrophoresis device into an electrophoresis tank, adding electrophoresis liquid to exceed a glue plane, pulling out a comb, taking protein samples with the same mass according to the measured protein concentration, adding the protein samples into each hole in sequence, wherein the sample loading amount is generally 25 mu l, the samples are added to the bottoms of the holes during sample loading, so that bubbles are prevented from being generated, overflowing protein into the electrophoresis liquid to influence an experimental result is avoided, and pre-dyed protein molecule markers with proper types are added into two holes at the outermost side according to the molecular weight of target protein.

Electrophoresis

Adopting a constant pressure method: 80V is set for 30min during gel concentration electrophoresis; the gel was separated by 120V and the electrophoresis was stopped when bromophenol blue reached the bottom of the gel.

Rotary film

Selecting a PVDF membrane with a proper pore size, shearing the membrane with a proper size according to the width of the glue with the protein molecular weight, marking, and soaking in methanol; and (3) taking out the separated protein gel plate from the electrophoresis tank, separating the target protein from the internal reference according to the molecular weight markers of the pre-dyed protein, placing the protein gel plate on a membrane transferring plate, covering the membrane transferring plate with a corresponding PVDF membrane, and completely covering the PVDF membrane on the surface of the separation gel to avoid generating bubbles in the covering process. And (4) placing the film into a film rotating groove according to the principle of 'red to red and black to black', and paying attention to the placing direction of the film rotating plate. The membrane was rotated at constant voltage, 120V for 90min. The film rotating tank is placed in ice water, and the continuous low temperature is kept.

Sealing of

Washing PVDF membrane in TBST for 5min, blocking with 5% BSA solution, placing PVDF membrane in blocking solution, and blocking on shaking table at room temperature for 1h;

anti-incubation

Taking out the PVDF membrane from the confining liquid, putting the PVDF membrane into TBST, washing for 5min X3 times, after washing, putting the PVDF membrane into the diluted primary antibody solution, and shaking overnight at 4 ℃;

incubation with secondary antibody

Placing the PVDF membrane and the diluted primary anti-solution on a shaking table for room temperature rewarming for 1h, taking out the PVDF membrane from the primary anti-dilution, placing the PVDF membrane in TBST, shaking the table for 5minx4 times at room temperature, selecting a secondary antibody of horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse, adding the secondary antibody into TBS containing 5% according to a certain proportion, shaking uniformly, placing the PVDF membrane in a secondary antibody dilution, placing the PVDF membrane on the shaking table for incubation for 1h at room temperature

Development

After the secondary antibody incubation is finished, the strips are placed in TBST and washed for 5minx3 times, ECL developing solution is prepared by the luminous solution to the stabilizing solution according to the proportion of 1; the photographing result is shown in fig. 9;

the expression level of Caspase-3 protein detected by Western Blot is shown in Table 9, and a bar chart is shown in FIG. 10;

TABLE 9 Western Blot to detect Caspase-3 protein expression level ((S))

n＝3)/>

VS Control group，*P<0.05，**P<0.01；VS SiRNA-1 group， ^# P<0.05， ^## P<0.01

As can be seen from table 9 and fig. 9 to 10:

control group 0.553 Tu 0.067; siRNA-1 group 1.237 soil 0.110, compared with Control group, C3 protein expression level is increased (P < 0.01); siRNA-2 group 0.957 and 1.102, C3 protein expression level was higher than that of Control group (P < 0.01), and C3 protein expression level was lower than that of SiRNA-1 group (P < 0.05).

Statistical analysis

All data were the results of 3 independent experiments, statistical analysis using SPSS 25.0 software. Normal distribution data results are expressed as mean. + -. Standard error (X. + -. SD), differences between groups are compared by one-way anova, and P <0.05 is considered as statistically significant.

In conclusion, the molecular marker has a correlation with liver cancer, can quickly, simply and accurately realize auxiliary diagnosis of the liver cancer, and provides a basis for diagnosis of the liver cancer and selection of treatment effect evaluation indexes; in addition, the molecular marker can also be used as a liver cancer drug treatment target.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims (6)

1. An application of liver cancer molecular markers in preparing products for detecting or evaluating curative effects of liver cancer, wherein the liver cancer molecular markers are hsa _ circ _0002490 and hsa _ circ _0101802, and the nucleic acid sequence of the hsa _ circ _0002490 is shown as SEQ ID NO 1; the nucleic acid sequence of hsa _ circ _0101802 is shown in SEQ ID NO. 3.

2. The use of claim 1, wherein the product includes, but is not limited to, a kit.

Application of SiRNA of hsa _circ _0101802in preparing medicine for treating liver cancer.

4. The use of claim 3, wherein the SiRNA sequence of hsa _ circ _0101802 is as follows:

the sequence of the sense strand is shown as SEQ ID NO. 10, and the sequence of the antisense strand is shown as SEQ ID NO. 11; or, the sequence of the sense strand is shown as SEQ ID NO. 12, and the sequence of the antisense strand is shown as SEQ ID NO. 13.

5. A medicine for treating liver cancer comprises SiRNA of hsa _ circ _ 0101802.

6. The medicament of claim 5, wherein the SiRNA sequence of hsa _ circ _0101802 is as follows:

the sequence of the sense strand is shown as SEQ ID NO. 10, and the sequence of the antisense strand is shown as SEQ ID NO. 11; or, the sequence of the sense strand is shown as SEQ ID NO. 12, and the sequence of the antisense strand is shown as SEQ ID NO. 13.

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