A biomarker for diagnosis, treatment and prognosis of microvascular invasion of liver cancer and its application

Abstract

The present invention belongs to the technical field of disease molecular biology, and specifically relates to a biomarker for diagnosis, treatment and prognosis of microvascular invasion of liver cancer and its application. The biomarker includes IQGAP3 protein, which serves as a target for diagnosis, treatment and prognosis of microvascular invasion of liver cancer, and also provides the application of a reagent, test paper or kit for detecting the expression amount of IQGAP3 in the preparation of a reagent for diagnosis, treatment and prognosis of microvascular invasion of liver cancer. The IQGAP3 can be used as a target for intervention of microvascular invasion, and the delivery of IQGAP3 siRNA by nanomaterials can be used as a treatment method for intervention of microvascular invasion of liver cancer, thereby inhibiting the recurrence of intrahepatic and distant metastasis of liver cancer and improving the prognosis of liver cancer patients; and the cytokines FGF1, CTGF and IL8 are highly expressed in the serum of clinical liver cancer patients with positive microvascular invasion, which can be used for diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease molecular biology, and specifically relates to a biomarker for diagnosis, treatment and prognosis of microvascular invasion of liver cancer and its application. Background Art

[0002] Intrahepatic metastasis and recurrence are the main reasons for poor prognosis in patients with liver cancer. Clinical statistics show that patients with hepatocellular carcinoma (HCC; hereinafter referred to as liver cancer) are very likely to relapse 3 to 5 years after radical resection, and the recurrence rate is as high as 60-70% 5 years after surgery. The 5-year overall survival rate of recurrent patients is 34%, which is significantly lower than that of the corresponding non-recurrent patients. Therefore, inhibiting liver cancer recurrence is the key to improving the survival rate of liver cancer patients. More than 80%-90% of postoperative recurrences in patients with liver cancer are caused by intrahepatic recurrence. Recurrent liver tumors mainly come from residual cancer growth caused by surgical residues or intrahepatic metastasis. Most intrahepatic metastases originate from vascular invasion of liver cancer cells, and vascular invasion is the main adverse prognostic factor for postoperative recurrence, intrahepatic and distant metastasis in patients with liver cancer. Vascular invasion of liver cancer is mainly divided into large vascular invasion (MaVI) and microvascular invasion (MVI). Large vascular invasion refers to the cancer thrombus formed by the portal vein and its main branches, with an incidence of about 35%, which can be diagnosed by preoperative imaging. Microvascular invasion is defined as the presence of tumor cell clusters in the lumen of blood vessels lined with endothelial cells around the tumor. Once the intravenous microtumor thrombus increases in size, it will retrogradely spread along the lumen to the branches and trunk of the portal vein, and eventually invade the liver parenchyma to form intrahepatic metastases. There are many studies on the use of serum biomarkers to predict MVI before surgery. The specificity and sensitivity of using a single serum biomarker to predict MVI are not ideal, and microvascular invasion of liver cancer can only be detected through pathological diagnosis of postoperative tissue samples. In addition, the molecular mechanism and molecular markers of microvascular invasion of liver cancer have not yet been elucidated. Therefore, there is an urgent need to identify predictive markers of microvascular invasion to accurately assess the risk of liver cancer metastasis and recurrence, and to find intervention targets targeting microvascular invasion and formulate the best treatment strategy, which will provide an important basis for the diagnosis and treatment of intrahepatic metastasis and recurrence of clinical liver cancer.

[0003] Microvascular invasion is a multi-step process, including ① local infiltration into adjacent tissues; ② invasion of blood vessels; ③ formation of invasive tumor thrombi in veins. Therefore, the growth pattern of liver cancer cells invading blood vessels is a prerequisite for microvascular invasion. Studies have shown that tumors can "hijack" existing blood vessels in normal tissues around the cancer during invasion and infiltration. For example, in liver metastasis of breast cancer and colorectal cancer, cancer cells induce cytoskeleton rearrangement through the Arp2 / 3 complex to invade the liver plate and interact with the intrahepatic sinusoidal vessels to obtain blood supply, and cancer cells can migrate along the endothelial cell wall of the interacting blood vessels. In addition, clinical pathological analysis showed that the increase in the invasive growth of cancer cells into the hepatic sinusoidal vessels was positively correlated with the incidence of vascular invasion in patients with advanced liver cancer. This suggests that invasive cancer cells may interact with vascular endothelial cells in the form of vascular interactions, which is conducive to invasion of blood vessels. Therefore, clarifying the relationship between the tumor growth pattern of vascular interactions and microvascular invasion of liver cancer will further clarify the molecular mechanism of vascular invasion.

[0004] Hepatic sinusoids are usually composed of vascular endothelial cells, pericytes (also known as hepatic stellate cells; HSCs), etc. Pericytes, as perivascular stromal cells, interact with vascular endothelial cells through chimeric structures such as tight junctions, gap junctions and adhesion junctions, providing structural support for the stability of blood vessels. As unique pericytes in the liver, hepatic stellate cells often extend several stellate processes close to the hepatic sinusoidal endothelial cells to stabilize the barrier connection of hepatic sinusoidal endothelial cells. The interaction between pericytes and vascular endothelial cells is closely related to tumor metastasis. For example, the low coverage of pericytes around blood vessels is significantly associated with lung metastasis in breast cancer patients. For example, the neural cell adhesion molecule NCAM enhances vascular stability by promoting the interaction between pericytes and endothelial cells, thereby inhibiting the metastasis of tumor cells to distant organs and local lymph nodes. Although the preliminary steps for tumor cells to locally infiltrate adjacent tissues through epithelial-mesenchymal transition have been intensively studied, little is known about the molecular mechanism by which cancer cells break through the barrier between endothelial cells and surrounding stromal cells or endothelial cells. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a biomarker for diagnosis, treatment and prognosis of microvascular invasion of liver cancer and its application.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides a biomarker for diagnosing microvascular invasion of liver cancer, wherein the biomarker is IQGAP3.

[0008] The present invention also provides an application of an IQGAP3 marker in the preparation of a diagnostic, therapeutic and prognostic product for microvascular invasion of liver cancer;

[0009] The product includes a reagent, a test strip or a test kit;

[0010] The kit includes a qPCR or ELISA kit.

[0011] The present invention also provides an application of an IQGAP3 marker as a target for diagnosis, treatment and intervention in microvascular invasion of liver cancer;

[0012] The IQGAP3 marker promotes the microvascular invasion of liver cancer by promoting tumor cells to adopt a growth pattern of vascular interaction and promoting hepatic stellate cells to detach from hepatic sinusoidal vessels by secreting cytokines. It can predict the risk of microvascular invasion, intrahepatic and distant metastasis in patients, thereby preventing the recurrence of intrahepatic and even extrahepatic metastasis of liver cancer.

[0013] The present invention also provides a biomarker for diagnosing microvascular invasion of liver cancer, wherein the biomarker further comprises cytokines FGF1, CTGF and IL8;

[0014] Cytokines FGF1, CTGF, and IL8 are highly expressed in the serum of clinical liver cancer patients with positive microvascular invasion, and the high expression level of IQGAP3 is positively correlated with the levels of FGF1, CTGF, and IL8;

[0015] The present invention also provides an application of IQGAP3 siRNA in the preparation of a drug for treating IQGAP3 highly expressed tumors;

[0016] The RNA sequence of the IQGAP3 siRNA is shown in SEQ ID NO. 1, which is GAGCCAACCAGGACACUAA, and is a double-stranded RNA molecule with a length of 19 nucleotides, which mainly plays a role in interfering with the expression of IQGAP3.

[0017] The present invention also provides an application of a nanomaterial to deliver the above-mentioned drug for treating IQGAP3 high-expression tumors, which helps to inhibit microvascular invasion of liver cancer, thereby achieving the effect of inhibiting intrahepatic and distant metastasis;

[0018] The drug includes the above-mentioned siRNA that inhibits IQGAP3 expression and a nanomaterial for transporting the siRNA; the nanomaterial is an acid-sensitive nanocarrier material that responds to the tumor microenvironment (such as acid response, hypoxia response, enzyme response, etc.), specifically a block polymer polyethylene glycol-b-poly (2-(diisopropylamino) ethyl methacrylate) (Meo-PEG-b-PDPA), which is a nanomaterial selected from the article Li Q, Qin T, Bi Z, Hong H, Ding L, Chen J, et al. Rac1activates non-oxidative pentose phosphate pathway to induce chemoresistanceof breast cancer. Nature communications 2020;11(1):1456 doi 10.1038 / s41467-020-15308-7. The pKa of the acid-sensitive polymer material is 6.24, and the siRNA targeting the IQGAP3 gene is encapsulated at the same time. The nanomaterial carrier is a high molecular polymer that responds to acidic pH, and its response pH value is 6.0-6.5. Studies have found that the pH of normal human blood circulation and tissues is about 7.4, while the tumor area has excessive lactic acid metabolites produced in a hypoxic environment due to its vigorous metabolism, making the tumor tissue microenvironment weakly acidic (pH 6.5). In addition, the pH value of the matrix environment of the cell endosome is about 5.5-6.0. When the nanocarrier prepared by the present invention uses the material to deliver siRNA, it can respond to the acidic pH of the endosome, accelerate the intracellular release of siRNA, silence the expression of the IQGAP3 target gene, significantly improve the gene therapy effect and reduce toxic side effects.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention discloses a biomarker for diagnosis, treatment and prognosis of microvascular invasion of liver cancer, including IQGAP3 protein, which serves as a target for diagnosis, treatment and prognosis of microvascular invasion of liver cancer. It also provides a reagent for detecting the expression of IQGAP3, a test paper or a kit for use in preparing a reagent for diagnosis, treatment and prognosis of microvascular invasion of liver cancer. The IQGAP3 can promote the invasion of liver cancer cells into blood vessels by promoting the common growth pattern of liver cancer blood vessels and inducing hepatic stellate cells to detach from blood vessels, thereby predicting the risk of microvascular invasion in liver cancer patients, thereby achieving the effect of preventing microvascular invasion; the IQGAP3 can be used as a target for intervention in microvascular invasion, and the delivery of IQGAP3 siRNA through nanomaterials can be used as a treatment method for intervention in microvascular invasion of liver cancer, thereby inhibiting the recurrence of intrahepatic and distant metastasis of liver cancer and improving the prognosis of liver cancer patients; and the cytokines FGF1, CTGF and IL8 are highly expressed in the serum of clinical liver cancer patients with positive microvascular invasion, and can be used for diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the experimental results of liver cancer cells using a blood vessel-sharing growth model to promote microvascular invasion;

[0022] Figure 2 This is an experimental result showing that hepatic stellate cells detaching from blood vessels and mediating vascular remodeling contribute to tumor invasion of blood vessels;

[0023] Figure 3 This is an experimental result diagram showing that cytokines FGF1, CTGF and IL8 are highly expressed in the serum of clinical liver cancer patients with positive microvascular invasion;

[0024] Figure 4 This is the experimental result of the cytokines FGF1, CTGF and IL8 derived from microvascular invasion positive liver cancer cells inducing hepatic stellate cells to detach from blood vessels;

[0025] Figure 5 This is an experimental result diagram showing that IQGAP3 is highly expressed in patients with microvascular invasion-positive liver cancer, and that high expression of IQGAP3 protein is positively correlated with the recurrence and survival prognosis of liver cancer patients;

[0026] Figure 6 This is an experimental result diagram showing that the high expression of IQGAP3 protein is positively correlated with the shared growth pattern of blood vessels, the detachment of hepatic stellate cells from blood vessels, and the levels of cytokines FGF1, CTGF, and IL8;

[0027] Figure 7 To explore the effect of high expression of IQGAP3 protein on microvascular invasion, intrahepatic metastasis and distant metastasis of liver cancer in vivo;

[0028] Figure 8To investigate the effects of nanomaterial-delivered IQGAP3 siRNA on the shared growth pattern of blood vessels, the detachment of hepatic stellate cells from blood vessels, and the levels of cytokines FGF1, CTGF, and IL8, and to investigate the effects on inhibiting microvascular invasion, intrahepatic metastasis, and distant metastasis of liver cancer.

[0029] Fig. 9 This figure is to explore the expression results of IQGAP3 at the RNA transcription level in clinically derived microvascular invasion-negative and -positive liver cancer tissues. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0031] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0032] Example 1

[0033] (1) Microvascular invasion-positive liver cancer samples showed a predominantly shared vascular growth pattern:

[0034] Methods: The interaction pattern between tumor cells and blood vessels was detected by double immunohistochemistry (dIHC) of tissues from patients with HCC with negative or positive microvascular invasion.

[0035] The following is a test of the interaction pattern between tumor cells and blood vessels in liver cancer patient tissues using double immunohistochemistry (dIHC):

[0036] 1) Formalin-fixed, paraffin-embedded liver resection specimens were sliced ​​into 4 μm sections and stained using standard hematoxylin-eosin (H&E) staining procedures;

[0037] After staining, two pathologists with extensive experience in identifying microvascular invasion independently identified negative and positive microvascular invasion, and then divided the samples into groups;

[0038] 2) Impress®Duet double staining polymer kit (MP-7724, Vector Laboratories) and blue alkaline phosphatase substrate (SK-5300, Vector Laboratories) were used for immunohistochemical double staining according to standard staining procedures, with staining indicators of PCNA (indicating liver cancer tumor cells) and CD31 (indicating blood vessels);

[0039] 3) After the immunohistochemical double staining is completed, two experienced pathologists will independently score and judge the samples. The scoring criteria are as follows:

[0040] Vascular co-positivity: At the tumor-paracancer interface, more than 70% of PCNA-positive liver cancer cells invade the adjacent normal liver tissue, and the adjacent blood vessels are morphologically characterized by one end located in the normal liver and the other end embedded in the tumor.

[0041] Negative vascular sharing: At the tumor-paracancerous liver interface, the vascular structure in the paracancerous liver is not destroyed, there is no direct contact between cancer cells and normal liver tissue, or there is contact between cancer cells and normal liver tissue but the cancer cells have not invaded the liver plate, and the binding rate of CD31-positive liver blood vessels and PCNA-positive tumor cells is less than 20%.

[0042] Results: Figure 1 As shown, Figure 1 Figure A shows that the microvascular invasion-negative liver cancer tissue is clearly demarcated from the normal tissue, while the liver cancer cells in the microvascular invasion-positive liver cancer tissue infiltrate into the liver parenchyma and interact more with the blood vessels in the normal tissue around the cancer. Figure 1 B shows that the shared vascular growth pattern is positively correlated with microvascular invasion of liver cancer.

[0043] (2) Analysis of the impact of vascular co-occurrence negative and positive on the survival prognosis of patients with microvascular positive liver cancer:

[0044] Methods: Among 130 clinical samples of microvessel-positive liver cancer, the samples were divided into two groups: vascular common negative and vascular common positive according to the experimental results of IHC in (1) above, and the survival prognosis and recurrence of the two groups of samples were analyzed.

[0045] Results: Figure 1 As shown in C, compared with liver cancer patients with negative vascular sharing, patients in the vascular sharing positive group had a shorter overall survival time and recurrence-free survival time, indicating that the vascular sharing growth pattern is conducive to the occurrence of microvascular invasion.

[0046] Example 2

[0047] (1) In the microvascular invasion-positive liver cancer samples, hepatic stellate cells appear to be detached from blood vessels:

[0048] Methods: The distance and position of hepatic stellate cells and endothelial cells were detected by multiplex immunofluorescence (mIF) staining of tissues from patients with microvascular invasion-negative and -positive HCC.

[0049] The following is the detection of the distance and position status between hepatic stellate cells and vascular endothelial cells in liver cancer patient tissues using multiple immunofluorescence staining:

[0050] 1) Formalin-fixed, paraffin-embedded liver resection specimens were sliced ​​into 4 μm thick sections and stained according to the standard hematoxylin-eosin (H&E) staining procedure. After staining, two pathologists with rich experience in microvascular invasion identification independently performed microvascular invasion negative and positive identification, and then divided the sections into groups;

[0051] 2) Multiplex immunofluorescence staining was performed using the four-color TSA-RM-275 kit (Benuo Panorama, 10001100020) according to standard staining procedures. The staining indicators were α-SMA / desmin (indicating hepatic stellate cells) and CD31 (indicating vascular endothelial cells);

[0052] 3) After the immunohistochemical double staining is completed, two experienced pathologists will independently score and judge;

[0053] All CD31-positive vessels surrounded by α-SMA-negative / desmin-positive hepatic stellate cells were counted as vessels covered by hepatic stellate cells, and necrotic areas were excluded from the analysis. Ten representative fields of each section were evaluated under a 200× light microscope, and the average number of vessels covered by hepatic stellate cells / average total number of vessels in each specimen were calculated;

[0054] The status of hepatic stellate cells and vascular endothelial cells in liver cancer specimens was divided into two phenotypes according to the following criteria:

[0055] Hepatic stellate cells without detachment from blood vessels (dHSC negative): greater than 70% of α-SMA negative / desmin positive hepatic stellate cells surrounded CD31 positive blood vessels;

[0056] Hepatic stellate cells detached from blood vessels (dHSC positive): less than 30% of α-SMA negative / desmin positive hepatic stellate cells surrounded CD31 positive blood vessels.

[0057] result: Figure 2 A shows that the hepatic stellate cells in the microvascular invasion-positive tissue are more likely to be detached from the blood vessels. According to statistics, the distance between the hepatic stellate cells and the hepatic sinusoidal vessels in the microvascular invasion-positive tissue is significantly increased compared with that in the microvascular negative liver cancer tissue ( Figure 2 B). Therefore, the two hepatic stellate cell-endothelial cell position states were defined as: hepatic stellate cells without detachment from the blood vessels (dHSC-negative) and hepatic stellate cells with detachment from the blood vessels (dHSC-positive) ( Figure 2 C)

[0058] (2) Analysis of the impact of dHSC negative and positive on the survival prognosis of liver cancer patients:

[0059] Methods: Among 130 clinical samples of microvessel-positive HCC, the samples were divided into two groups, dHSC-negative and dHSC-positive, according to the experimental results of dIHC in (1) above. The survival prognosis and recurrence of the two groups of samples were analyzed.

[0060] Results: Figure 2 As shown in D, dHSC positivity is positively correlated with microvascular invasion in patients with liver cancer. At the same time, the analysis of the shared growth pattern with blood vessels also showed that dHSC positivity is positively correlated with shared blood vessel positivity ( Figure 2 E). Compared with dHSC-negative liver cancer patients, dHSC-positive patients had shorter overall survival and recurrence-free survival time ( Figure 2 F) Hepatic stellate cells detaching from blood vessels to mediate vascular remodeling can contribute to tumor invasion of blood vessels.

[0061] Example 3

[0062] Cytokines FGF1, CTGF and IL8 are highly expressed in the serum of clinical liver cancer patients with positive microvascular invasion:

[0063] The cytokines in the serum of patients with negative and positive microvascular invasion HCC were analyzed by Bio-Plex Pro Human Cytokine Screening Panel (48-Plex #12007283). Figure 3 As shown in A, the volcano plot analysis of abnormal secretory proteins expressed in the serum of patients with microvascular invasion-negative and -positive liver cancer showed that compared with patients with microvascular invasion-negative liver cancer, the expression of cytokines FGF1, CTGF and IL8 in the serum of patients with microvascular invasion-positive liver cancer was significantly increased.

[0064] Figure 3 B is an ELISA enzyme-linked immunosorbent assay kit (human FGF1-ab219636; human CTGF-ab261851; human CTGF-ab214030) used to analyze the levels of cytokines FGF1, CTGF and IL8 in microvascular invasion negative / positive primary liver cancer cells. Compared with microvascular invasion negative primary liver cancer cells, the secretion levels of FGF1, CTGF and IL8 in the supernatant of microvascular invasion positive primary liver cancer cells were significantly increased.

[0065] Example 4

[0066] Cytokines FGF1, CTGF and IL8 derived from microvascular invasion-positive hepatocellular carcinoma cells induce hepatic stellate cells to detach from blood vessels

[0067] Figure 4A shows that in tumor-bearing mice inoculated with microvascular invasion-positive liver cancer primary cells, the mice were treated with FGF1, CTGF and IL8 neutralizing antibodies, respectively. Compared with the control group, FGF1, CTGF and IL8 neutralizing antibodies could significantly inhibit the detachment of hepatic stellate cells from blood vessels.

[0068] Example 5

[0069] IQGAP3 is highly expressed in patients with microvascular invasion-positive HCC:

[0070] The results were as follows: Figure 5 As shown in the figure, the volcano plot analysis of abnormal protein expression in the tissues of liver cancer patients with negative and positive microvascular invasion showed that compared with liver cancer patients with negative microvascular invasion, the expression of IQGAP3 in liver cancer patients with positive microvascular invasion was significantly increased, indicating that IQGAP3 can be used in the diagnosis of liver cancer microvascular invasion;

[0071] Figure 5 B is the Kaplan-Meier analysis of the recurrence-free survival curves of HCC patients with low and high expression of IQGAP3 (n=365; P < 0.001, log-rank test) and HCC patients with positive microvascular invasion (n=130; P < 0.001, log-rank test). The results showed that high levels of IQGAP3 significantly promoted microvascular invasion of HCC.

[0072] Example 6

[0073] High expression of IQGAP3 protein was positively correlated with the shared growth pattern of blood vessels, the detachment of hepatic stellate cells from blood vessels, and the levels of cytokines FGF1, CTGF, and IL8:

[0074] Immunohistochemistry and multiplex immunofluorescence staining were performed on tissues from patients with HCC with and without microvascular invasion to detect the correlation between IQGAP3 expression and the shared vascular growth pattern, dHSC status, and FGF1, CTGF, and IL8 levels.

[0075] The results are as follows Figure 6 As shown in A, high expression levels of IQGAP3 were positively correlated with the shared growth pattern of blood vessels and dHSC positivity. At the same time, the analysis of cytokines also showed that high expression levels of IQGAP3 were positively correlated with the levels of FGF1, CTGF and IL8 ( Figure 6 B).

[0076] Example 7

[0077] In vivo experiments, high expression of IQGAP3 protein can promote microvascular invasion, intrahepatic metastasis and distant metastasis of liver cancer:

[0078] Methods: Mice were orthotopically inoculated with control and IQGAP3-overexpressing tumor cells in the liver, and tumor growth in the above designated groups of mice was monitored.

[0079] Results: Hepatocellular carcinoma cells with high expression of IQGAP3 significantly promoted microvascular invasion, intrahepatic metastasis and distant metastasis ( Figure 7 AB), resulting in worse survival time ( Figure 7 C).

[0080] Example 8

[0081] In vivo experiments, the use of nanomaterials to deliver IQGAP3 siRNA can inhibit liver cancer microvascular invasion, intrahepatic metastasis and distant metastasis:

[0082] The nanomaterial is an acid-sensitive nanocarrier material that responds to the tumor microenvironment (such as acid response, hypoxia response, enzyme response, etc.), specifically a block polymer polyethylene glycol-b-poly (2-(diisopropylamino)ethyl methacrylate) ((Meo-PEG-b-PDPA), which is selected from the article Li Q, Qin T, Bi Z, Hong H, Ding L, Chen J, et al. Rac1 activates non-oxidative pentose phosphate pathway to induce chemoresistance of breast cancer. Nature communications 2020;11(1):1456 doi10.1038 / s41467-020-15308-7. The pKa of the acid-sensitive polymer material is 6.24, and the siRNA targeting the IQGAP3 gene is encapsulated at the same time. The nanomaterial carrier is a polymer that responds to acidic pH, and its response pH value is 6.0-6.5. Studies have found that the pH of normal blood circulation and tissues in the human body is about 7.4, while the tumor area has excessive lactic acid metabolites produced in a hypoxic environment due to its vigorous metabolism, making the tumor tissue microenvironment weakly acidic (pH 6.5). In addition, the pH value of the matrix environment of the cell endosome is about 5.5-6.0. When the nanocarrier prepared by the present invention uses the material to deliver siRNA, it can respond to the acidic pH of the endosome, accelerate the intracellular release of siRNA, silence the expression of the IQGAP3 target gene, significantly improve the gene therapy effect and reduce toxic side effects.

[0083] like Figure 8 A shows the preparation of nanomaterials encapsulating IQGAP3 siRNA. The RNA sequence is shown in SEQ ID NO.1 and is synthesized by RiboBio Corporation.

[0084] Figure 8 B: After orthotopic inoculation of microvascular invasion-positive patient-derived tumor (microvascular invasion-positive / PDX#1) into the mouse liver, control, nanoparticles, and IQGAP3 siRNA-encapsulated nanoparticles were treated.

[0085] Figure 8 C shows that the microvascular invasion of mice treated with nanoparticles encapsulating IQGAP3 siRNA was significantly reduced, indicating that IQGAP3 is an important target for inhibiting microvascular invasion of liver cancer. The use of nanomaterials to deliver IQGAP3 siRNA can achieve the effect of targeted inhibition of IQGAP3 and inhibiting microvascular invasion.

[0086] Example 9

[0087] Fig. 9 In order to detect the RNA transcription level expression of IQGAP3 in clinically derived microvascular invasion-negative and -positive liver cancer tissues, the results showed that IQGAP3 was highly expressed in the tissues of microvascular invasion-positive liver cancer patients, suggesting that a kit containing a reagent for quantitatively detecting the expression level of IQGAP3 can be prepared to determine whether the patient has a higher risk of microvascular invasion.

[0088] The kit includes a qPCR kit or an ELISA kit for quantitatively detecting the transcription level of IQGAP3 RNA;

[0089] The qPCR kit used comprises a reagent (conventional reagent) for quantitatively detecting the RNA transcription level of IQGAP3 and a specific primer for the marker, and the nucleic acid sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0090] IQGAP3-F: 5'-AGGAACAAGTGCAGCTAGTTG-3' (SEQ ID NO. 2);

[0091] IQGAP3-R: 5'-GCTGGCACAGTAGACCTTATAGA-3' (SEQ ID NO. 3).

[0092] In summary, the IQGAP3 of the present invention can be used as a diagnostic, therapeutic and prognostic marker in liver cancer microvascular invasion and the application of the preparation kit, as well as the application of nanomaterial delivery of IQGAP3 siRNA in targeted intervention of liver cancer microvascular invasion.

[0093] There are many studies on the use of serum biomarkers to predict MVI before surgery. The specificity and sensitivity of using a single serum biomarker to predict MVI are not ideal, and liver cancer microvascular invasion can only be detected through pathological diagnosis of postoperative tissue samples. The present invention uses IQGAP3 to more specifically and sensitively predict the occurrence of liver cancer microvascular invasion, diagnose liver cancer microvascular invasion, predict disease progression, guide drug use and evaluate prognosis.

[0094] Compared with existing drugs / reagents that do not specifically target liver cancer microvascular invasion, IQGAP3 can promote the common growth pattern of liver cancer blood vessels, induce hepatic stellate cells to detach from blood vessels, and thus promote liver cancer cells to invade blood vessels, and can be used as a target for intervention in microvascular invasion. Therefore, nanomaterial delivery of IQGAP3 siRNA can be used as a treatment method for intervention in liver cancer microvascular invasion, thereby inhibiting the recurrence of liver cancer intrahepatic and distant metastasis, and improving the prognosis of liver cancer patients.

Claims

1. An application of an IQGAP3 marker in the preparation of a product for diagnosis, treatment and prognosis of microvascular invasion of liver cancer, characterized in that: The product includes a reagent, a test strip or a test kit; The kit includes a qPCR or ELISA kit.

2. The use of the IQGAP3 marker according to claim 1 in the preparation of diagnostic, therapeutic and prognostic products for microvascular invasion of liver cancer, characterized in that: The kit contains specific primers for the IQGAP3 marker, and the nucleic acid sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

3. A biomarker for diagnosing microvascular invasion of liver cancer, characterized in that: The biomarkers include IQGAP3, and also include cytokines FGF1, CTGF, and IL8; The high expression level of IQGAP3 in the serum of clinical liver cancer patients with positive microvascular invasion is positively correlated with the levels of FGF1, CTGF and IL8.

4. A use of IQGAP3 siRNA in the preparation of a drug for treating IQGAP3 overexpressing tumors, characterized in that: The RNA sequence of the IQGAP3 siRNA is shown in SEQ ID NO.

1.

5. Use of a nanomaterial as a delivery material for the IQGAP3 high-expression tumor drug according to claim 4, characterized in that: The drug comprises the siRNA of claim 4 that inhibits the expression of IQGAP3 and a nanomaterial for transporting the siRNA; The nano material is a block polymer polyethylene glycol- b -Poly (2-(diisopropylamino) ethyl methacrylate) (Meo-PEG- b -PDPA).

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