Application of circM-TMEFF1 in diagnosis, treatment and prognosis evaluation of hepatocellular carcinoma

Abstract

The invention belongs to the technical field of biological detection and treatment, and particularly relates to an innovative application of novel circRNA: circM-TMEFF1 in diagnosis and treatment of hepatocellular carcinoma. The circM-TMEFF1 forms a ring in hepatocellular carcinoma cells, and the specificity of the circM-TMEFF1 is in low expression. The invention specifically provides application of circM-TMEFF1 as a marker in preparation of a hepatocellular carcinoma diagnosis or prognosis kit, and compared with a normal control person, the expression quantity of circM-TMEFF1 in hepatocellular carcinoma tissues and tumor recurrent tissues is remarkably reduced, so that circM-TMEFF1 can be used as a biomarker for diagnosis and prognosis of hepatocellular carcinoma. Besides, promotion of circM-TMEFF1 expression can effectively inhibit proliferation and growth of hepatocellular carcinoma cell lines, a new target is provided for improvement of treatment of hepatocellular carcinoma, and the circM-TMEFF1 has great clinical value.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection and treatment technology, and relates to the application of circM-TMEFF1 as a biomarker and therapeutic target for hepatocellular carcinoma, and specifically to the application of circM-TMEFF1 in the preparation of diagnostic or prognostic evaluation reagents or kits and therapeutic drugs for hepatocellular carcinoma. Background Art

[0002] Primary liver cancer (PHC) is the sixth most common cancer worldwide and the third leading cause of cancer death. Hepatocellular carcinoma (HCC) accounts for 75%–85% of PHC cases. Chronic hepatitis B virus (HBV) infection is the leading cause of HCC in my country. Currently, the main treatment options for PHC are surgical resection, liver transplantation, and radiofrequency ablation of the liver. However, PHC is prone to metastasis and postoperative recurrence, resulting in a poor prognosis for patients with HCC. Factors currently believed to influence the prognosis of PHC include tumor size, tumor capsule, microsatellite nodules, vascular invasion, high HBV load, elevated serum alpha-fetoprotein (AFP), and systemic inflammation. However, with the exception of HBV load and its associated inflammation, most known prognostic factors are not suitable as predictive biomarkers for HCC recurrence.

[0003] Circular RNAs (circRNAs) are a group of single-stranded, covalently closed RNA molecules formed by reverse splicing of pre-mRNA. Their expression is tissue-specific and conserved, and their functions are diverse, including acting as miRNA sponges and protein scaffolds, regulating gene transcription, and serving as translation templates. However, the role of circRNAs as potential biomarkers or therapeutic targets in the development and recurrence of HBV-related HCC (HBV-HCC) has not yet been reported. Summary of the Invention

[0004] The present invention is based on the above research and aims to provide new biomarkers for the diagnosis and prognosis of hepatocellular carcinoma, and also to provide new uses of the newly discovered circRNA molecule circM-TMEFF1, namely, its use in the preparation of diagnostic or prognostic evaluation kits or therapeutic drugs for hepatocellular carcinoma.

[0005] In a first aspect, the present invention provides a circular RNA molecule circM-TMEFF1, the corresponding nucleotide sequence of which is:

[0006] AAUUAAAUGUGAGGGAGUCUGACGUAAGAGUUUGUGAUGAGUCAUCAUGUAAAUAUGGAGGAGUCUGUAAAGAAGAUGGAGATGGUUUGAAAUGUGCAUGCCAAUUUCAGUGCCAUACAAAUUAUAUUCCUGUCUGUGGAUCAAAUGGGGACACUUAUCAAAAUGAAUGCUUUCUCAGAAGGGC UGCUUGUAAGCACCAGAAAGAGAUAACAGUAAUAGCAAGAGGACCAUGCUACUCUGAUAAUGGAUCUGGAUCUGGAGAAGGAGAAGGAAGGGUCAGGGGCAGAAGUUCACAGAAAACACUCCAAGUGUGGACCCTGCAAAUAUAAAGCUGAGUGUGAUGAAGAUGCAGAAAAUGUUGG(SEQ IDNO.1).

[0007] Furthermore, the present invention also provides a cyclization site of circM-TMEFF1, see Figure 1 .

[0008] Based on the newly discovered circular RNA molecule circM-TMEFF1, the present invention first found that circM-TMEFF1 was differentially expressed in HCC tissues and normal tissues, and in recurrent tumor tissues and non-recurrent tumor tissues through genomic analysis of the circRNA expression profile, and then verified in cohort samples that low circM-TMEFF1 expression levels were closely associated with poor prognosis in patients with hepatocellular carcinoma. After overexpressing circM-TMEFF1 in the form of a recombinant plasmid, it was found that circM-TMEFF1 significantly inhibited tumor cell growth and colony formation ability in HepG2, Huh7 and SK-Hep1 cells, and induced cell arrest in the G0 / G1 phase. At the same time, circM-TMEFF1 can significantly reduce cell migration ability. After knocking down circM-TMEFF1 using lentiviral infection, the proliferation ability of HepG2, Huh7 and SK-Hep1 cells was significantly enhanced, the proportion of S phase cells was significantly increased, the migration level of cells was significantly upregulated, and the anchorage-independent growth potential of liver cancer cells was significantly increased. In a tumorigenic experiment in Nod-SCID mice, downregulation of circM-TMEFF1 significantly increased tumor weight and size. Furthermore, upregulating circM-TMEFF1 expression in HepG2.215 and HepAD38 cells, which express HBV, significantly inhibited HBV replication. Therefore, circM-TMEFF1 inhibits the proliferation and migration of HBV-associated liver cancer cells and has a favorable prognostic effect.

[0009] These findings suggest that circM-TMEFF1 may be a promising option for the diagnosis, treatment, and prognosis of HBV-HCC. CircM-TMEFF1 expression levels in patients can be used to diagnose HCC, develop targeted drugs for treatment, and analyze survival and postoperative recurrence in HCC patients. This makes it a novel biomarker and a potential new therapeutic target, providing scientific, precise, and personalized diagnostic and treatment options for HCC patients, particularly those with HBV-HCC.

[0010] Specifically, based on the above research, the present invention provides the following technical solutions:

[0011] The first aspect of the present invention provides the use of circM-TMEFF1 as a diagnostic marker, and specifically provides the use of a reagent for detecting circM-TMEFF1 in the preparation of a product for diagnosing or evaluating the prognosis of hepatocellular carcinoma.

[0012] Preferably, the hepatocellular carcinoma is HBV-HCC.

[0013] Preferably, the reagent for detecting circM-TMEFF1 is a reagent for detecting the expression level of circM-TMEFF1 in a biological sample at the gene level; the kit contains the reagent for detecting the expression level of circM-TMEFF1 in a biological sample.

[0014] More preferably, the reagent for detecting the expression of circM-TMEFF1 in a biological sample comprises PCR primers, probes, or gene chips that are specific for the circM-TMEFF1 gene. The PCR primer sequences specific for the circM-TMEFF1 gene are shown in SEQ ID NOs. 2-3, and the primer sequences for the control GAPDH are shown in SEQ ID NOs. 4-5.

[0015]

[0016] In a second aspect, the present invention provides a product comprising the above-mentioned components or reagents for detecting circM-TMEFF1, wherein the product has any one or more of the following uses: diagnosis or auxiliary diagnosis of hepatocellular carcinoma; prognosis assessment or auxiliary prognosis assessment of hepatocellular carcinoma.

[0017] The product is preferably a kit for detecting at the gene level, which is composed of a reverse transcription system, a primer system and an amplification system, wherein the primer system includes the PCR primers shown in SEQ ID NOs. 2-3 and SEQ ID NOs. 4-5 above.

[0018] In the preliminary experiments of this invention, sequencing was used to discover the differentially expressed circRNA molecule circM-TMEFF1 in HCC tissues and normal tissues, and in recurrent HCC tissues and non-recurrent HCC tissues. In the cohort samples, the expression of circM-TMEFF1 in primary hepatocellular carcinoma tissues of recurrent patients was also significantly downregulated compared with HCC tissues of non-recurrent patients (P=0.033) ( Figure 2 Low expression of circM-TMEFF1 in tumor tissues of patients with hepatocellular carcinoma is closely associated with poor prognosis of patients. Increased expression of circM-TMEFF1 in patients significantly predicted good overall survival (OS) (P=0.031) and recurrence-free survival (RFS) (P=0.015) ( Figure 3 ).

[0019] Therefore, the expression level of circM-TMEFF1 in the sample was detected by this kit. Based on the experimental conclusion that circM-TMEFF1 was significantly low expressed in HCC tissues and normal tissues, and that low expression of circM-TMEFF1 in tumor tissues of hepatocellular carcinoma patients was closely related to the patients' poor prognosis, and that the recurrence-free survival time and overall survival time of patients with low expression were shorter than those of patients with high expression, HCC can be diagnosed or the patient's prognosis can be evaluated.

[0020] Furthermore, the biological sample is selected from any one of tumor tissue obtained by puncture or circulating tumor cells collected from the patient's blood. By collecting and detecting the level of circM-TMEFF1 in tumor tissue or cells, early diagnosis and prognosis assessment can be achieved.

[0021] A third aspect of the present invention provides a method for detecting the expression level of circM-TMEFF1 in a biological sample, comprising the following steps:

[0022] (A) Total RNA was extracted from tissue using Trizol and reverse transcribed

[0023] The 10 μl reaction system is as follows:

[0024]

[0025] Reaction conditions: 37°C, 15 min; 85°C, 5 min; 4°C, ∞.

[0026] (B) RT-PCR quantitative amplification

[0027] Set up 3 replicate wells for each sample, with 20 μl per well. The required system is as follows:

[0028]

[0029] The above reaction system was added to a 96-well plate and placed in an RT-PCR instrument for amplification.

[0030] The specific procedure is: step 1: 95°C, 30 sec; step 2: 95°C, 5 sec; 60°C, 15 sec; step 3: 50 cycles (95°C, 15 sec; 60°C, 30 sec; 95°C, Quatification); step 4: 50°C, 30 sec.

[0031] (C) Result analysis: After RT-PCR quantification, the CT values ​​of the internal reference GAPDH and circM-TMEFF1 were obtained and calculated as follows: circM-TMEFF1 gene expression level = 2 -(ΔΔCT) .

[0032] Using the above method, the expression level of circM-TMEFF1 can be detected.

[0033] A fourth aspect of the present invention provides the use of circM-TMEFF1 as a therapeutic target, and specifically provides the use of a circM-TMEFF1 promoter in the preparation of a drug for treating hepatocellular carcinoma or an HBV virus inhibitor.

[0034] Preferably, the drug for treating hepatocellular carcinoma is a drug for treating HBV-related hepatocellular carcinoma; the drug for treating liver cancer is a substance that increases the expression level, activity or activity of circM-TMEFF1 or its encoded protein; the main mechanism of action of the drug for treating liver cancer is to inhibit HBV replication.

[0035] Preferably, the circM-TMEFF1 promoter is selected from any one of the following:

[0036] (1) Exogenous circM-TMEFF1 gene or protein;

[0037] (2) liposomes or nanomaterials encapsulating exogenous circM-TMEFF1;

[0038] (3) recombinant expression vector of circM-TMEFF1;

[0039] Among them, circM-TMEFF1 has the nucleic acid sequence shown in SEQ ID NO.1.

[0040] In addition to the above sequences, the following situations also fall within the scope of protection of the present invention:

[0041] (i) a molecule that hybridizes to the nucleotide sequence defined by SEQ ID NO. 1 under stringent conditions;

[0042] (ii) a nucleic acid molecule that is homologous to or has sequence identity with the nucleotide sequence shown in SEQ ID NO. 1 and has similar functions;

[0043] (iii) A nucleic acid molecule having similar functions, wherein one or more nucleotides are substituted, deleted or added to the nucleotide sequence shown in SEQ ID NO. 1.

[0044] Further preferably, the circM-TMEFF1 recombinant vector comprises an expression vector and a circM-TMEFF1 nucleic acid molecule inserted into the expression vector, and the sequence thereof is as described above.

[0045] The expression vectors include viral vectors and non-viral vectors.

[0046] The "viral vector" includes adenovirus, adeno-associated virus, lentivirus, coxsackie virus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus and vesicular stomatitis virus, etc. Suitable viral vectors are well known to those of ordinary skill in the art.

[0047] The "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers and nanoparticle vectors, etc. Suitable non-viral vectors are well known to those skilled in the art.

[0048] Furthermore, the circM-TMEFF1 recombinant vector is an overexpression plasmid constructed by inserting the circM-TMEFF1 sequence into the pLCDH-ciR vector.

[0049] In a fifth aspect, the present invention provides a pharmaceutical composition for treating hepatocellular carcinoma or an anti-HBV viral inhibitor, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the circM-TMEFF1 promoter described above.

[0050] The dosage forms of the drug for treating hepatocellular carcinoma include, but are not limited to, tablets, capsules, granular preparations, aerosols, or injections. Treatment methods include, but are not limited to, the drug being used alone or in combination with any other active or inactive ingredients. Routes of administration include, but are not limited to, oral administration, intravenous administration, nasal inhalation, subcutaneous or intramuscular injection, and buccal administration.

[0051] When the composition of the present invention is administered to animals including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and administration route. The results of animal experiments and various circumstances can be referred to, and the total dosage cannot exceed a certain range.

[0052] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other pharmaceutical compositions for treating HCC.

[0053] The present study found that after overexpression of circM-TMEFF1 in the form of a plasmid, circM-TMEFF1 significantly inhibited cell growth, colony formation ability and migration ability in Huh7 cells ( Figure 4 .AC). The same phenomenon was observed after upregulation of circM-TMEFF1 in HepG2 and SK-Hep1 cells ( Figure 4 .DF and GI). After upregulation of circM-TMEFF1 in Huh7, HepG2, and SK-Hep1 cells, cell cycle arrest at the G0 / G1 phase was observed ( Figure 5 .AC). Therefore, circM-TMEFF1 inhibits cell proliferation, migration, and cell cycle progression.

[0054] We used lentivirus to infect cells and construct cell lines Huh7, HepG2, and SK-Hep1 that stably knocked down circM-TMEFF1 expression. The knockout sequences are shown in Table 1 (the sequence of siRNA-2 was used). After downregulating circM-TMEFF1, the proliferation and migration abilities of Huh7, HepG2, and SK-Hep1 were significantly enhanced ( Figure 6 .AI). After downregulation of circM-TMEFF1, Huh7, HepG2 and SK-Hep1 accelerated the cell cycle progression ( Figure 7 .AC).

[0055] In the Nod-SCID mouse tumor formation experiment, the tumor weight and size of the transplanted tumor group injected with circM-TMEFF1 knockdown cells increased significantly. ( Figure 8 AC).

[0056] In HepG2.215 and HepAD38 cells expressing HBV, upregulating circM-TMEFF1 expression significantly inhibited HBV replication. Downregulating circM-TMEFF1 expression by co-transfection with two siRNAs further activated HBV replication.

[0057] Therefore, we propose that "circM-TMEFF1 can be used as a new potential therapeutic target, and targeted treatment can be carried out for patients with low circM-TMEFF1 expression levels through circM-TMEFF1 liposome nanoparticles and other forms, providing scientific, precise and personalized diagnosis and treatment plans for patients with hepatocellular carcinoma."

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention discloses the application of a novel circRNA, circM-TMEFF1, in the diagnosis and treatment of hepatocellular carcinoma. The biomarker is a novel circRNA, and its corresponding DNA nucleotide sequence is shown in SEQ ID NO.1. The novel circRNA is stably circularized in hepatocellular carcinoma and is significantly lowly expressed in hepatocellular carcinoma.

[0060] The present invention discloses for the first time the application of circM-TMEFF1 in the diagnosis or prognosis evaluation of hepatocellular carcinoma, provides evidence that circM-TMEFF1 is associated with the diagnosis and prognosis of hepatocellular carcinoma, and confirms that it can be used as a new biomarker for the diagnosis and prognostic analysis of hepatocellular carcinoma patients.

[0061] The present invention provides a method for detecting circM-TMEFF1, which uses RT-PCR to quantitatively determine the expression of circM-TMEFF1 in patient tissues, and combines the patient's postoperative follow-up information to determine that the expression of circM-TMEFF1 is related to the prognosis of liver cancer patients. circM-TMEFF1 can be used as a biomarker for judging the prognosis of liver cancer patients. Liver cancer patients with high expression of circM-TMEFF1 have a better prognosis, while liver cancer patients with low expression have a poorer prognosis. The detection of circM-TMEFF1 is essentially a quantitative PCR detection based on the expression status of blood cell genes. It has the characteristics of simple operation, sensitive detection, good specificity, and high repeatability. It has been increasingly used in clinical testing technology. The basic detection method used in the present invention is real-time fluorescence quantitative PCR. This technology has high sensitivity and accuracy, is widely used clinically, and the test technology is already very mature.

[0062] The present invention discloses for the first time the application of promoting circM-TMEFF1 expression in the prevention and treatment of hepatocellular carcinoma. By targeted upregulation of circM-TMEFF1 expression in hepatocellular carcinoma, the proliferation, migration ability and cell cycle progression of hepatocellular carcinoma cells can be inhibited, and tumor cell death can be promoted, ultimately improving or treating hepatocellular carcinoma.

[0063] The marker circM-TMEFF1 involved in this study was specifically underexpressed in patients with hepatocellular carcinoma (HCC), with a statistically significant difference (P < 0.05), making it a diagnostic and / or prognostic marker for HCC. Upregulating circM-TMEFF1 inhibited HBV replication and HCC cell proliferation, promoting HCC cell death. Therefore, its clinical reference value and credibility are high, with important clinical significance and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The sequence of circM-TMEFF1 and its circularization site are shown.

[0065] Figure 2 The expression levels of circM-TMEFF1 in recurrent and non-recurrent tissues were detected by real-time fluorescence quantitative PCR. In the validation cohort, it was found that the expression of circM-TMEFF1 in recurrent tumor tissues was significantly downregulated (P<0.05).

[0066] Figure 3 The results showed a correlation between circM-TMEFF1 expression and the prognosis of patients with liver cancer. Low expression of circM-TMEFF1 in tumor tissues of patients with liver cancer was closely associated with poor prognosis. Increased circM-TMEFF1 expression in tumors significantly predicted good overall survival (OS) (P = 0.032) and recurrence-free survival (RFS) (P = 0.015).

[0067] Figure 4 Figure 3. Effects of circM-TMEFF1 on the proliferation, colony formation, and migration of Huh7 (A, B, C), HepG2 (D, E, F), and SK-Hep1 (G, H, I) cells. Overexpression of circM-TMEFF1 using a recombinant plasmid significantly inhibited cell growth, colony formation, and migration.

[0068] Figure 5 Figure 3 shows the effects of circM-TMEFF1 on the cell cycle of Huh7 (A), HepG2 (B), and SK-Hep1 (C) cells. Overexpression of circM-TMEFF1 using a recombinant plasmid resulted in cell cycle arrest at the G0 / G1 phase.

[0069] Figure 6 Figure 3. Effects of circM-TMEFF1 knockdown on the proliferation, colony formation, and migration of Huh7 (A, B, C), HepG2 (D, E, F), and SK-Hep1 (G, H, I) cells. Stable knockdown cell lines were generated using lentiviral infection. Downregulation of circM-TMEFF1 expression enhanced cell proliferation, colony formation, and migration.

[0070] Figure 7 Figure 3: Effects of circM-TMEFF1 knockdown on the cell cycle in Huh7 (A), HepG2 (B), and SK-Hep1 (C) cells. Stable knockdown cell lines were constructed using lentiviral infection, and downregulation of circM-TMEFF1 expression resulted in cell cycle arrest in the S phase.

[0071] Figure 8It shows that upregulation of circM-TMEFF1 can inhibit HBV replication in HBV-expressing cell lines HepG2.215 and HepAD38 cells, among which, A, comparison of tumor size after upregulation and inhibition of circM-TMEFF1 expression; B, comparison of tumor size after upregulation and inhibition of circM-TMEFF1 expression; C, comparison of tumor tissue weight after upregulation and inhibition of circM-TMEFF1 expression.

[0072] Figure 9 It was shown that upregulating circM-TMEFF1 expression levels could inhibit HBV replication. DETAILED DESCRIPTION

[0073] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings, but the implementation of the present invention is not limited thereto.

[0074] All reagents and starting materials used in the present invention are commercially available or can be prepared according to literature methods. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions or conditions recommended by the manufacturer.

[0075] Example 1: circM-TMEFF1 is significantly differentially expressed between tumors and adjacent tissues, and between relapsed and non-relapsed patients

[0076] Thirteen patients who underwent curative resection for HCC pathologically diagnosed at Shanghai Oriental Hepatobiliary Surgery Hospital between February 2011 and September 2012 were enrolled in this study. All enrolled patients received no medical treatment, had a 2-cm surgical margin, and were free of intrahepatic and distant metastases. Tumor tissue, paired recurrent tumor tissue, and paired adjacent tissue were immediately removed and stored at -80°C. Of the 13 patients, four experienced recurrence within 2 years of the initial curative resection, while nine remained relapse-free within 5 years of curative resection. Tissues from these 13 patients were sequenced and analyzed for mRNA, circRNA, and microRNA (miRNA) expression profiles, along with multi-omics analyses. The study protocol adhered to the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the Naval Medical University. Signed informed consent was obtained from each participant. Analysis of the sequencing results revealed 53 circular RNAs that were differentially expressed between tumor and adjacent tissues, and between patients with recurrence and those without recurrence. Among them, circM-TMEFF1 expression was significantly different, and its sequence and cyclization site were as follows Figure 1 shown.

[0077] Example 2: The expression level of circM-TMEFF1 is significantly downregulated in recurrent tumor tissues

[0078] A validation cohort of 101 patients pathologically diagnosed with HCC and undergoing radical resection at Shanghai Oriental Hepatobiliary Surgery Hospital between February 2011 and September 2012 was selected. All enrolled patients received no medical treatment, had a 2-cm surgical margin, and were free of intrahepatic and distant metastases. Tumor and adjacent tissues were immediately removed postoperatively and stored at -80°C. The study protocol adhered to the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the Naval Medical University. Signed informed consent was obtained from each participant. CircM-TMEFF1 expression levels in various tissues from these 101 patients were assessed by RT-PCR. Total RNA was extracted from human cells or tissues using an RNA extraction kit according to the kit's instructions. RNase R (4 U / μL) was added to the total RNA and digested at 37°C for 15 minutes. cDNA was then obtained using a reverse transcription kit. CircM-TMEFF1 primers were designed and used to amplify the cDNA. The amplified products were subjected to agarose gel electrophoresis.

[0079] The specific steps are as follows:

[0080] (A) Total RNA was extracted from tissue using Trizol and reverse transcribed

[0081] The 10 μl reaction system is shown in Table 1 below:

[0082] Table 1 Reverse transcription reaction system

[0083]

[0084] Reaction conditions: 37°C, 15 min; 85°C, 5 min; 4°C, ∞.

[0085] (B) RT-PCR quantitative amplification

[0086] Each sample was set up in 3 replicate wells, with 20 μl per well. The required system is shown in Table 2 below, and the PCR used is shown in Table 3:

[0087] Table 2 Summary of amplification systems

[0088]

[0089] Table 3 Summary of PCR primers

[0090]

[0091] The above reaction system was added to a 96-well plate and placed in an RT-PCR instrument for amplification.

[0092] The specific procedure is: step 1: 95°C, 30 sec; step 2: 95°C, 5 sec; 60°C, 15 sec; step 3: 50 cycles (95°C, 15 sec; 60°C, 30 sec; 95°C, Quatification); step 4: 50°C, 30 sec.

[0093] (C) Result analysis: After RT-PCR quantification, the CT values ​​of the internal reference GAPDH and circM-TMEFF1 were obtained and calculated as follows: circM-TMEFF1 gene expression level = 2 -(ΔΔCT) .

[0094] The results showed that the expression level of circM-TMEFF1 was significantly downregulated in recurrent tumor tissues (P<0.05) ( Figure 2 ).

[0095] Example 3: circM-TMEFF1 as a prognostic marker for hepatocellular carcinoma

[0096] Combined with the prognostic information, the 101 patients in Example 2 were divided into a circM-TMEFF1 high expression group and a low expression group, and a survival analysis of the patients was performed to evaluate whether circM-TMEFF1 could be used as an independent prognostic factor. The survival curve analysis used the Kaplan-Meier method, and the comparison between the two groups was performed using the log-rank test. The results showed that the low expression of circM-TMEFF1 in the tumor tissue of patients with liver cancer was closely related to the poor prognosis of the patients, and the increased expression level of circM-TMEFF1 in the tumor significantly predicted a good overall survival (OS) (P = 0.032) and recurrence-free survival (RFS) (P = 0.015) ( Figure 3 ), which can serve as an independent prognostic factor for patients with hepatocellular carcinoma.

[0097] Example 4: Upregulation of circM-TMEFF1 inhibits the proliferation of hepatocellular carcinoma cells

[0098] To further clarify the function and mechanism of circM-TMEFF1 in the progression of hepatocellular carcinoma, we constructed a circM-TMEFF1 plasmid and studied the effects of overexpression of circM-TMEFF1 on cell cycle, proliferation and migration ability. The circM-TMEFF1 sequence was inserted into the pLCDH-ciR vector according to the restriction sites of EcoRI and BamHI to construct an overexpression plasmid. The plasmid overexpressing circM-TMEFF1 was introduced into HepG2, Huh7 and SK-Hep1 cells. In HepG2, Huh7 and SK-Hep1 cells, circM-TMEFF1 significantly inhibited cell growth, colony formation and migration ability ( Figure 4), inducing cell arrest in the G0 / G1 phase ( Figure 5 ). It can be seen that circM-TMEFF1 inhibits cell proliferation, migration and cell cycle progression.

[0099] Example 5: Downregulation of circM-TMEFF1 promotes the development of hepatocellular carcinoma

[0100] The following siRNAs were constructed according to the interfering RNA (siRNA) sequences of circM-TMEFF1 described in Table 4 below and lentiviral packaging was performed: siRNA-1 (nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7) and siRNA-2 (nucleotide sequences as shown in SEQ ID NO.8 and SEQ ID NO.9) and control nucleotide sequences (as shown in SEQ ID NO.10 and SEQ ID NO.11).

[0101] Table 4 circM-TMEFF1 siRNA sequences

[0102]

[0103]

[0104] The effects of knocking down circM-TMEFF1 on cell cycle, proliferation and migration were investigated. The constructed lentivirus for knocking down circM-TMEFF1 was used to infect HepG2, Huh7 and SK-Hep1 cells. It was found that after downregulating circM-TMEFF1, the proliferation, clone formation and migration abilities of HepG2, Huh7 and SK-Hep1 cells were significantly enhanced; the proportion of S phase cells in HepG2, Huh7 and SK-Hep1 cells was significantly increased ( Figure 6 and Figure 7 ).

[0105] The constructed stable expression cell line with knockdown of circM-TMEFF1 was used for subcutaneous tumor formation experiments, which showed that the weight and volume of tumors in the circM-TMEFF1 knockdown group were significantly increased ( Figure 8 ).

[0106] Example 6: Use of circM-TMEFF1 as an anti-HBV therapeutic drug

[0107] After HBV inhibition, the expression level of circM-TMEFF1 in Huh7 cells was detected before and after inhibition. The results showed that the expression level of circM-TMEFF1 in Huh7 cells was significantly upregulated after HBV inhibition ( Figure 9 A, 9B).

[0108] Plasmids overexpressing circM-TMEFF1 were introduced into the HepAD38 and HepG2.2.15 human liver cancer cell models used for HBV research. The results showed that overexpression of circM-TMEFF1 reduced the expression of HBeAg and cccDNA, important markers of HBV infection and disease progression. Figure 9 C, 9D); on the contrary, after inhibiting circM-TMEFF1 expression, the expression levels of HBeAg and cccDNA were significantly increased ( Figure 9 E, 9F), suggesting that upregulating circM-TMEFF1 expression levels can inhibit HBV replication.

[0109] The above experimental results show that the present invention discovered a new circular RNA molecule - circM-TMEFF1 through sequencing. In a validation cohort of 101 liver cancer patients, it was demonstrated that the expression level of circM-TMEFF1 in the tissues of patients with recurrent liver cancer was significantly lower than that in the tumor tissues of non-recurrent patients. Low expression of circM-TMEFF1 in tumor tissues of patients with liver cancer is closely related to the patients' poor prognosis. The tumor-free survival and overall survival times of patients with low expression levels are shorter than those of patients with high expression. Cell experiments confirmed that the proliferation and migration abilities of cells overexpressing circM-TMEFF1 were significantly reduced; the opposite was true after knocking out circM-TMEFF1. Animal experiments confirmed that the weight and volume of tumors in the circM-TMEFF1 knockdown group were significantly increased. Overexpression of circM-TMEFF1 in cell models can inhibit HBV replication.

[0110] In summary, circM-TMEFF1 is a circular RNA with a stable circular structure that is specifically highly expressed in hepatocellular carcinoma. Its low expression is associated with a poor prognosis in hepatocellular carcinoma and is an independent prognostic factor for patients with hepatocellular carcinoma. Detecting the expression level of circM-TMEFF1 in patients with hepatocellular carcinoma can be used to diagnose hepatocellular carcinoma, analyze the survival time of patients with hepatocellular carcinoma, and determine whether they relapse after surgery. Upregulating circM-TMEFF1 can inhibit the proliferation of hepatocellular carcinoma cells and promote the death of hepatocellular carcinoma cells. These results suggest a close correlation between circM-TMEFF1 and the occurrence and development of hepatocellular carcinoma tumors. Therefore, it can be used as a marker for tumor diagnosis, tumor treatment plan selection, and tumor prognosis assessment. It can also serve as a new potential therapeutic target for the treatment of hepatocellular carcinoma, especially HBV-related hepatocellular carcinoma, providing scientific, precise, and personalized diagnosis and treatment plans for patients with liver cancer.

[0111] Any undescribed parts of the present invention are the same as or implemented using existing technologies. The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. Use of a reagent for detecting the expression level of circM-TMEFF1 in the preparation of a kit for diagnosing or evaluating prognosis of hepatocellular carcinoma, characterized in that: The nucleic acid sequence of circM-TMEFF1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The hepatocellular carcinoma is HBV-related hepatocellular carcinoma; the reagent for detecting the expression level of circM-TMEFF1 is a reagent for detecting the expression amount of circM-TMEFF1 in a biological sample at the gene level; the kit contains a reagent for detecting the expression amount of circM-TMEFF1 in a biological sample.

3. The use according to claim 2, characterized in that The reagent for detecting circM-TMEFF1 in a biological sample is selected from one or more detection techniques or methods: in situ hybridization, Northern blot, high-throughput sequencing, RT-PCR, and real-time quantitative PCR.

4. The use according to claim 3, characterized in that The reagent for detecting the expression level of circM-TMEFF1 in a biological sample comprises a PCR primer, a probe or a gene chip that has detection specificity for the circM-TMEFF1 gene. The PCR primer sequence that has detection specificity for the circM-TMEFF1 gene is shown in SEQ ID NOs. 2 to 3.

5. A kit for diagnosing, treating or evaluating prognosis of hepatocellular carcinoma, characterized in that: The kit contains reagents for detecting the content of circM-TMEFF1 in biological samples.

6. The kit according to claim 6, characterized in that The kit is composed of a reverse transcription system, a primer system and an amplification system. The primer system includes PCR primers shown as SEQ ID NOs. 2-3 and SEQ ID NOs. 4-5.

7. Application of circM-TMEFF1 promoter in the preparation of drugs for the treatment of hepatocellular carcinoma.

8. The use according to claim 7, characterized in that The circM-TMEFF1 promoter is selected from any of the following situations: (1) exogenous circM-TMEFF1 gene or protein; (2) liposomes or nanomaterials encapsulating exogenous circM-TMEFF1; (3) recombinant expression vectors of circM-TMEFF1, and the expression vectors include viral vectors and non-viral vectors.

9. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that: It comprises an active component and a pharmaceutically acceptable carrier, wherein the active component is the circM-TMEFF1 promoter according to claim 8.

10. An anti-HBV virus inhibitor, characterized in that: It comprises an active component and a pharmaceutically acceptable carrier, wherein the active component is the circM-TMEFF1 promoter according to claim 8.

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