Application of circm-tmefr1 in diagnosis, treatment and prognosis evaluation of hepatocellular carcinoma

Abstract

This invention belongs to the field of biodetection and treatment technology, specifically relating to the innovative application of a novel circRNA, circM-TMEFF1, in the diagnosis and treatment of hepatocellular carcinoma (HCC). circM-TMEFF1 forms a circular structure and is specifically low-expressed in HCC cancer cells. Specifically, this invention provides the application of circM-TMEFF1 as a biomarker in the preparation of diagnostic or prognostic kits for HCC. Compared with normal controls, the expression level of circM-TMEFF1 is significantly downregulated in HCC tissues and tumor recurrence tissues, thus serving as a biomarker for the diagnosis and prognosis of HCC. Furthermore, promoting circM-TMEFF1 expression can effectively inhibit the proliferation and growth of HCC cancer cell lines, providing a new target for improving HCC treatment and possessing significant clinical value.

Description

Technical Field

[0001] This 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. Specifically, it relates to the application of circM-TMEFF1 in the preparation of diagnostic or prognostic reagents or kits for hepatocellular carcinoma and in therapeutic drugs. Background Technology

[0002] Primary liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer death, with hepatocellular carcinoma (HCC) accounting for 75%–85% of all primary liver cancers. Chronic hepatitis B virus (HBV) infection is the main cause of HCC in my country. Current treatments for primary liver cancer primarily include surgical resection, liver transplantation, and radiofrequency ablation of the liver. However, primary liver cancer is characterized by its high rate of metastasis and recurrence after surgery, resulting in a generally unsatisfactory prognosis for patients. Factors currently considered to influence the prognosis of primary liver cancer include tumor size, tumor capsule, microsatellite nodules, vascular invasion, high HBV viral load, high serum alpha-fetoprotein (AFP), and systemic inflammation. However, apart from HBV viral 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 the reverse splicing of pre-mRNAs. Their expression is tissue-specific and conserved, and they have diverse functions, 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 occurrence and recurrence of HBV-related HCC (HBV-HCC) has not yet been reported. Summary of the Invention

[0004] This invention is based on the above research and aims to provide new biomarkers for the diagnosis and prognosis of hepatocellular carcinoma, as well as new uses for the newly discovered circRNA molecule circM-TMEFF1, namely, its application in the preparation of diagnostic or prognostic 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, this invention also provides a circularization site for circM-TMEFF1, see details below. Figure 1 .

[0008] Based on the newly discovered circular RNA molecule circM-TMEFF1, this invention first used genomic analysis of circRNA expression profiles to discover differential expression of circM-TMEFF1 between HCC and normal tissues, and between recurrent and non-recurrent tumor tissues. Further validation in cohort samples revealed a close correlation between low circM-TMEFF1 expression levels and poor prognosis in hepatocellular carcinoma patients. Overexpression of circM-TMEFF1 in recombinant plasmid form significantly inhibited tumor cell growth and colony formation in HepG2, Huh7, and SK-Hep1 cells, inducing cell arrest in the G0 / G1 phase, and significantly reducing cell migration. Knockdown of circM-TMEFF1 via lentiviral infection significantly enhanced the proliferation of HepG2, Huh7, and SK-Hep1 cells, significantly increased the proportion of cells in S phase, significantly upregulated cell migration, and significantly increased the anchored independent growth potential of liver cancer cells. In Nod-SCID mouse tumorigenesis experiments, downregulation of circM-TMEFF1 significantly increased tumor weight and size. Furthermore, upregulation of circM-TMEFF1 expression significantly inhibited HBV replication in HBV-expressing HepG2.215 and HepAD38 cells. Therefore, circM-TMEFF1 inhibits the proliferation and migration of HBV-related liver cancer cells and shows good predictive value.

[0009] The above evidence suggests that circM-TMEFF1 may be a potential option for the diagnosis, treatment, and prognosis analysis of HBV-HCC. By analyzing the expression level of circM-TMEFF1 in patients, hepatocellular carcinoma can be diagnosed, targeted drugs can be developed for treatment, and the survival time and postoperative recurrence of hepatocellular carcinoma patients can be analyzed. It serves as a novel biomarker and a potential therapeutic target, providing scientific, precise, and personalized diagnostic and treatment plans for hepatocellular carcinoma, especially HBV-HCC patients.

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

[0011] In a first aspect, the invention provides the application of circM-TMEFF1 as a diagnostic biomarker. Specifically, it provides the application of reagents for detecting circM-TMEFF1 in the preparation of products for the diagnosis or prognostic assessment 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 biological samples at the gene level; the kit contains the reagent for detecting the expression level of circM-TMEFF1 in biological samples.

[0014] Further preferred, the reagent for detecting the expression level of circM-TMEFF1 in biological samples includes PCR primers, probes, or gene chips with detection specificity for the circM-TMEFF1 gene. The sequences of the PCR primers with detection specificity for the circM-TMEFF1 gene are shown in SEQ ID NO. 2–3, and the primer sequences for the control GAPDH are shown in SEQ ID NO. 4–5.

[0015]

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

[0017] The product is preferably a kit for detection at the gene level, consisting of a reverse transcription system, a primer system, and an amplification system. The primer system includes the PCR primers shown in SEQ ID NO. 2–3 and SEQ ID NO. 4–5 above.

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

[0019] Therefore, by detecting the expression level of circM-TMEFF1 in samples using this kit, and based on the experimental conclusions that circM-TMEFF1 is significantly lowly expressed in HCC tissues and normal tissues, and that low expression of circM-TMEFF1 in tumor tissues of hepatocellular carcinoma patients is closely related to poor prognosis, and that patients with low expression levels have shorter recurrence-free survival and overall survival than patients with high expression levels, HCC can be diagnosed or the prognosis of patients can be assessed.

[0020] Furthermore, the biological sample is selected from either tumor tissue obtained by puncture or circulating tumor cells collected from the patient's blood. Early diagnosis and prognostic assessment are achieved by collecting and detecting the level of circM-TMEFF1 in tumor tissue or cells.

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

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

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

[0024]

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

[0026] (B) RT-PCR quantitative amplification

[0027] Each sample was prepared in triplicate, with 20 μl of the required system per well as follows:

[0028]

[0029] Add the above reaction mixture to a 96-well plate and place it in an RT-PCR instrument for amplification.

[0030] The specific procedure is as follows: Step 1: 95℃, 30 seconds; Step 2: 95℃, 5 seconds; 60℃, 15 seconds; Step 3: 50 cycles (95℃, 15 seconds; 60℃, 30 seconds; 95℃, Quatification); Step 4: 50℃, 30 seconds.

[0031] (C) Results Analysis: After RT-PCR quantification, the CT values ​​of the internal control GAPDH and circM-TMEFF1 were obtained. The calculation method is as follows: circM-TMEFF1 gene expression level = 2 -(ΔΔCT) .

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

[0033] In a fourth aspect, the invention provides the application of circM-TMEFF1 as a therapeutic target. Specifically, it provides the application of circM-TMEFF1 promoters in the preparation of drugs for treating hepatocellular carcinoma or HBV virus inhibitors.

[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 accelerator is selected from any 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 sequence, the following situations also fall within the scope of protection of this invention:

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

[0042] (ii) Nucleic acid molecules that are homologous to or have the same sequence as the nucleotide sequence shown in SEQ ID NO.1 and have similar functions;

[0043] (iii) Nucleic acid molecules that have undergone substitution, deletion or addition of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1 and have similar functions.

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

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

[0046] The term "viral vector" includes adenovirus, adeno-associated virus, lentivirus, Coxsackievirus, 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 skilled in the art.

[0047] The term "non-viral carrier" includes liposomes or lipid complexes, cationic polymers, chitosan polymers, and nanoparticle carriers. Suitable non-viral carriers 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 component and a pharmaceutically acceptable carrier, said active component comprising the circM-TMEFF1 promoter described above.

[0050] The dosage forms of the drugs for treating hepatocellular carcinoma include, but are not limited to, tablets, capsules, granules, aerosols, or injections. Treatment methods include, but are not limited to, the use of the drug alone, or its preparation or combination with any other active or inactive ingredients. Routes of administration include, but are not limited to, oral, intravenous, nasal inhalation, subcutaneous or intramuscular injection, and sublingual 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 route of administration. The dosage can be determined with reference to the results of animal experiments and various other factors, but the total dosage should not 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] This invention found that overexpression of circM-TMEFF1 in plasmid form significantly inhibited cell growth, colony formation, and migration in Huh7 cells. Figure 4 .AC). The same phenomenon was observed after upregulating circM-TMEFF1 in HepG2 and SK-Hep1 cells. Figure 4 .DF and GI). Upregulation of circM-TMEFF1 in Huh7, HepG2, and SK-Hep1 cells resulted in cell cycle arrest at the G0 / G1 phase. Figure 5 .AC). Therefore, circM-TMEFF1 inhibits cell proliferation, migration, and cell cycle progression.

[0054] We used lentiviruses that downregulated circM-TMEFF1 expression to infect cells and constructed stable cell lines with knocked-down circM-TMEFF1 expression levels: Huh7, HepG2, and SK-Hep1. The knockout sequences are shown in Table 1 (siRNA-2 sequences were used). After downregulation of circM-TMEFF1, the proliferation and migration abilities of Huh7, HepG2, and SK-Hep1 cells were significantly enhanced. Figure 6 .AI). Downregulation of circM-TMEFF1 accelerated cell cycle progression by Huh7, HepG2, and SK-Hep1. Figure 7 .AC).

[0055] In the Nod-SCID mouse tumorigenesis experiment, the tumor weight and size were significantly increased in the xenograft group injected with circM-TMEFF1 knockdown cells. Figure 8 AC).

[0056] In HepG2.215 and HepAD38 cells expressing HBV, upregulating circM-TMEFF1 expression significantly inhibited HBV replication. Downregulating circM-TMEFF1 expression through co-transfection with two siRNAs resulted in more active HBV replication.

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

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows:

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

[0060] This invention discloses for the first time the application of circM-TMEFF1 in the diagnosis or prognostic assessment of hepatocellular carcinoma, providing evidence of the correlation between circM-TMEFF1 and the diagnosis and prognosis of hepatocellular carcinoma, and confirming that it can serve as a novel biomarker for the diagnosis and prognostic analysis of hepatocellular carcinoma patients.

[0061] This invention provides a method for detecting circM-TMEFF1. It utilizes RT-PCR to quantitatively determine the expression level of circM-TMEFF1 in patient tissues and, combined with postoperative follow-up information, establishes a correlation between circM-TMEFF1 expression and prognosis in liver cancer patients. circM-TMEFF1 can serve as a biomarker for predicting the prognosis of liver cancer patients; liver cancer patients with high circM-TMEFF1 expression have a better prognosis, while those with low expression have a poorer prognosis. The detection of circM-TMEFF1 is essentially a quantitative PCR detection based on the gene expression status of blood cells. It features simple operation, high sensitivity, good specificity, and high reproducibility, and is increasingly being used in clinical laboratory techniques. The basic detection method used in this invention is real-time quantitative PCR, a technology with high sensitivity and accuracy, widely used clinically, and a mature experimental technique.

[0062] This invention discloses for the first time the application of promoting circM-TMEFF1 expression in the prevention and treatment of hepatocellular carcinoma. By targeting and upregulating the expression of circM-TMEFF1 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 indicator circM-TMEFF1 involved in this invention is specifically lowly expressed in hepatocellular carcinoma patients, and the difference is statistically significant (P<0.05), which can serve as a diagnostic and / or prognostic marker for hepatocellular carcinoma. Upregulation of circM-TMEFF1 can inhibit HBV replication and the proliferation of hepatocellular carcinoma cells, and promote the death of hepatocellular carcinoma cells. Therefore, it has high clinical reference value and reliability, and has important clinical significance and social benefits. Attached Figure Description

[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 quantitative PCR. In the validation cohort, the expression of circM-TMEFF1 was found to be significantly downregulated in recurrent tumor tissues (P<0.05).

[0066] Figure 3 The study demonstrated the correlation between circM-TMEFF1 expression and prognosis in patients with hepatocellular carcinoma (HCC). Low expression of circM-TMEFF1 in tumor tissues of HCC patients was closely associated with poor prognosis, while elevated circM-TMEFF1 expression levels in tumors significantly predicted good overall survival (OS) (P = 0.032) and recurrence-free survival (RFS) (P = 0.015).

[0067] Figure 4 The 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 were demonstrated. Overexpression of circM-TMEFF1 in recombinant plasmid form significantly inhibited cell growth, colony formation, and migration.

[0068] Figure 5 The effects of circM-TMEFF1 on the cell cycle of Huh7 (A), HepG2 (B), and SK-Hep1 (C) cells were shown. Overexpression of circM-TMEFF1 in recombinant plasmid form arrested the cell cycle at the G0 / G1 phase.

[0069] Figure 6 This study demonstrated the 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 constructed by infecting cells with lentivirus. Downregulation of circM-TMEFF1 expression levels enhanced cell proliferation, colony formation, and migration.

[0070] Figure 7 The effects of circM-TMEFF1 knockdown on the cell cycle of Huh7(A), HepG2(B), and SK-Hep1(C) cells were demonstrated. Stable knockdown cell lines were constructed by infecting cells with lentivirus, and cell cycle arrest at the S phase was achieved after downregulating circM-TMEFF1 expression.

[0071] Figure 8The images show that upregulation of circM-TMEFF1 can inhibit HBV replication in HBV-expressing cell lines HepG2.215 and HepAD38. A shows a comparison of tumor size after upregulation and inhibition of circM-TMEFF1 expression; B shows a comparison of tumor size after upregulation and inhibition of circM-TMEFF1 expression; and C shows a comparison of tumor weight after upregulation and inhibition of circM-TMEFF1 expression.

[0072] Figure 9 The study showed that upregulating circM-TMEFF1 expression levels can suppress HBV replication. Detailed Implementation

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

[0074] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or under conventional conditions, or as recommended by the manufacturer.

[0075] Example 1: circM-TMEFF1 showed significant differential expression between tumors and adjacent tissues, and between recurrent and non-recurrent patients.

[0076] Thirteen patients who underwent radical resection of hepatobiliary cancer (HCC) at Shanghai Eastern Hepatobiliary Surgery Hospital between February 2011 and September 2012 were selected as the study subjects. All enrolled patients had not received any drug treatment, had a surgical margin of 2 cm, and no intrahepatic or distant metastases. Immediately after surgery, tumor tissue, paired recurrent tumor tissue, and paired adjacent tissue were removed and stored at -80℃. Of the 13 patients, 4 relapsed within 2 years after the first radical resection, and 9 remained relapse-free within 5 years. Sequencing and multi-assembly analysis of mRNA, circRNA, and microRNA (miRNA) expression profiles were performed on the tissues from these 13 patients. The study protocol complied with the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the Naval Medical University. Each participant obtained a signed informed consent form. Analysis of the sequencing results revealed 53 circular RNAs, which showed differential expression between tumors and adjacent tissues, and between recurrent and non-recurrent patients. Among them, circM-TMEFF1 expression was significantly different, and its sequence and circularization sites are as follows: Figure 1 As shown.

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

[0078] One hundred and one patients who underwent radical resection of hepatobiliary cancer (HCC) at Shanghai Eastern Hepatobiliary Surgery Hospital between February 2011 and September 2012 were selected as the validation cohort. All enrolled patients had not received any drug treatment, had a surgical margin of 2 cm, and no intrahepatic or distant metastases. Tumor tissue and adjacent tissues were removed immediately postoperatively and stored at -80℃. The study protocol complied with the 1975 Declaration of Helsinki and was approved by the Ethics Committee of Naval Medical University. Each participant obtained a signed informed consent form. The expression level of circM-TMEFF1 in different tissues of the 101 patients was detected using RT-PCR. Specifically, total RNA was extracted from human cells or tissues using an RNA extraction kit according to the kit instructions. RNase R (4 U / µL) was added to the total RNA, and the mixture was digested at 37℃ for 15 min. cDNA was obtained using a reverse transcription kit. Primers for circM-TMEFF1 were designed to amplify the obtained cDNA by PCR. The amplified products were subjected to agarose gel electrophoresis.

[0079] The specific steps are as follows:

[0080] (A) Total RNA was extracted from tissues 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℃, 15 min; 85℃, 5 min; 4℃, ∞.

[0085] (B) RT-PCR quantitative amplification

[0086] Each sample was prepared in triplicate, with 20 μl per well. The required system is shown in Table 2 below. 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] Add the above reaction mixture to a 96-well plate and place it in an RT-PCR instrument for amplification.

[0092] The specific procedure is as follows: Step 1: 95℃, 30 seconds; Step 2: 95℃, 5 seconds; 60℃, 15 seconds; Step 3: 50 cycles (95℃, 15 seconds; 60℃, 30 seconds; 95℃, Quatification); Step 4: 50℃, 30 seconds.

[0093] (C) Results Analysis: After RT-PCR quantification, the CT values ​​of the internal control GAPDH and circM-TMEFF1 were obtained. The calculation method is as follows: circM-TMEFF1 gene expression level = 2 -(ΔΔCT) .

[0094] The results showed that circM-TMEFF1 expression 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] Based on prognostic information, the 101 patients in Example 2 were divided into a high-expression group and a low-expression group of circM-TMEFF1. Survival analysis was performed to assess whether circM-TMEFF1 could be used as an independent prognostic factor. Survival curve analysis was conducted using the Kaplan-Meier method, and the log-rank test was used for comparison between the two groups. The results showed that low expression of circM-TMEFF1 in liver cancer tumor tissue was closely related to poor prognosis. Increased circM-TMEFF1 expression levels in the tumor significantly predicted good overall survival (OS) (P = 0.032) and recurrence-free survival (RFS) (P = 0.015). Figure 3 ( ) can serve as an independent prognostic factor for patients with hepatocellular carcinoma.

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

[0098] To further clarify the function and mechanism of circM-TMEFF1 in hepatocellular carcinoma progression, we constructed a circM-TMEFF1 plasmid and investigated the effects of circM-TMEFF1 overexpression on cell cycle, proliferation, and migration. The circM-TMEFF1 sequence was inserted into the pLCDH-ciR vector according to the EcoRI and BamHI restriction sites to construct the overexpression plasmid. The circM-TMEFF1 overexpression plasmid 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. Figure 4), inducing cell arrest in the G0 / G1 phase ( Figure 5 Therefore, circM-TMEFF1 inhibits cell proliferation, migration, and cell cycle progression.

[0099] Example 5: Downregulation of circM-TMEFF1 can promote the development of hepatocellular carcinoma.

[0100] The following siRNAs were constructed based on the interfering RNA (siRNA) sequences of circM-TMEFF1 described in Table 4 below, and then coated with lentiviruses: 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 sequence

[0102]

[0103]

[0104] This study investigated the effects of circM-TMEFF1 knockdown on cell cycle, proliferation, and migration. HepG2, Huh7, and SK-Hep1 cells were infected with a lentivirus containing circM-TMEFF1 knockdown. Downregulation of circM-TMEFF1 significantly enhanced the proliferation, colony formation, and migration abilities of HepG2, Huh7, and SK-Hep1 cells; the proportion of cells in S phase in HepG2, Huh7, and SK-Hep1 cells also significantly increased. Figure 6 and Figure 7 ).

[0105] The constructed cell line with stable expression of circM-TMEFF1 knockdown was used in subcutaneous tumorigenesis experiments, showing 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 agent

[0107] After HBV inhibition, the expression level of circM-TMEFF1 in Huh7 cells was measured 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] In the HepAD38 and HepG2.2.15 human liver cancer cell models used for HBV research, plasmids overexpressing circM-TMEFF1 were introduced. The results showed that overexpression of circM-TMEFF1 reduced the expression levels of HBeAg and cccDNA, important markers of HBV infection and disease progression. Figure 9 C, 9D); conversely, inhibiting circM-TMEFF1 expression significantly increased the expression levels of HBeAg and cccDNA. Figure 9 E, 9F) suggest that upregulating circM-TMEFF1 expression levels can inhibit HBV replication.

[0109] The above experimental results demonstrate that this invention discovered a novel circular RNA molecule—circM-TMEFF1—through sequencing. In a validation cohort of 101 hepatocellular carcinoma (HCC) patients, the expression level of circM-TMEFF1 in recurrent HCC tissues was significantly lower than that in non-recurrent HCC tumor tissues. Low expression of circM-TMEFF1 in HCC tumor tissues is closely related to poor prognosis; patients with low expression levels had shorter disease-free survival and overall survival than those with high expression levels. Cellular experiments confirmed that overexpression of circM-TMEFF1 significantly reduced cell proliferation and migration; the opposite was true after circM-TMEFF1 knockout. Animal experiments confirmed that the tumor weight and volume were significantly increased in the circM-TMEFF1 knockdown group. In cell models, overexpression of circM-TMEFF1 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 (HCC). Its low expression is associated with poor prognosis in HCC and is an independent prognostic factor for patients. Detecting circM-TMEFF1 expression levels in HCC patients can be used to diagnose HCC, analyze patient survival time and postoperative recurrence. Upregulation of circM-TMEFF1 can inhibit HCC cell proliferation and promote cell death. These results suggest a close correlation between circM-TMEFF1 and the occurrence and development of HCC tumors, thus it can serve as a biomarker for tumor diagnosis, treatment selection, and prognostic assessment. Furthermore, it could also serve as a novel potential therapeutic target for HCC treatment, especially HBV-related HCC, providing patients with scientific, precise, and personalized diagnostic and treatment options.

[0111] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. The application of a reagent for detecting circM-TMEFF1 gene expression levels in the preparation of diagnostic or prognostic kits for hepatocellular carcinoma, characterized in that... The nucleic acid sequence of circM-TMEFF1 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The hepatocellular carcinoma mentioned is HBV-related hepatocellular carcinoma.

3. The application according to claim 1, characterized in that, The reagent used to detect the expression level of the circM-TMEFF1 gene is a substance used in real-time quantitative PCR detection methods.

4. The application according to claim 1, characterized in that, The reagent for detecting the expression level of the circM-TMEFF1 gene includes PCR primers, probes, or gene chips that are specific to the detection of the circM-TMEFF1 gene. The PCR primer sequences that are specific to the detection of the circM-TMEFF1 gene are shown in SEQ ID NO.2~3.

5. The application of the circM-TMEFF1 overexpression vector in the preparation of drugs for treating hepatocellular carcinoma, characterized in that, The nucleic acid sequence of circM-TMEFF1 is shown in SEQ ID NO.

1.

6. The application according to claim 5, characterized in that, The circM-TMEFF1 overexpression vector is selected from any of the following: (1) exogenous circM-TMEFF1 gene or protein expression plasmid; (2) liposomes or nanomaterials encapsulating exogenous circM-TMEFF1; (3) recombinant expression vector of circM-TMEFF1, the expression vector including viral vectors.

7. The application according to claim 5, characterized in that, The drug is used to treat HBV virus.

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