Molecular target for preparing medicine for treating TFE3 gene rearrangement renal cell carcinoma and application of molecular target

Abstract

The invention provides a molecular target for preparing a medicine for treating TFE3 gene rearrangement renal cell carcinoma and application of the molecular target, and relates to the technical field of biological medicine. The molecular target is an LGI3 gene, and the base sequence of the LGI3 gene is as shown in SEQ ID No. 1. After the LGI3 is interfered, the proliferation, migration and invasion capabilities of the TFE3 rearrangement renal cell carcinoma can be effectively inhibited, so that the LGI3 gene is expected to be used as a molecular target to be applied to clinical treatment of patients with the TFE3 rearrangement renal cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a molecular target for preparing a drug for treating TFE3 gene rearrangement renal cell carcinoma and a use thereof. Background Art

[0002] TFE3-rearranged renal cell carcinoma (TFE3-RCC) is a rare subtype of renal cancer. It was first reported in 1986. It is also called Xp11.2 translocation / TFE3 gene fusion-related renal cell carcinoma (Xp11.2 translocation renal cell carcinoma, Xp11.2 tRCC) because the TFE3 gene in the first band and second subband of the X chromosome of the patient is translocated and forms a fusion gene. In 2004, the WHO classification of renal tumors officially listed it as an independent subtype of renal cancer. Since the TFE3 gene belongs to the microphthalmia transcription factor family (MiTF), Xp11.2 tRCC was classified as MiTF translocation renal cell carcinoma in 2016. The fifth edition of the WHO classification of renal tumors in 2022 renamed Xp11.2 tRCC as TFE3 rRCC and classified it as renal cell carcinoma diagnosed based on molecular technology.

[0003] TFE3-rearranged renal cell carcinoma is caused by the fusion of the TFE3 gene with different partner genes. It is highly malignant and has a poor clinical prognosis. However, the direct downstream signaling pathway that mediates the function of the TFE3 fusion gene is still unclear, resulting in a lack of universal and effective treatment for recurrent and advanced patients.

[0004] At present, the research on TFE3 rearrangement renal cell carcinoma mainly focuses on exploring the direct downstream target molecules of TFE3 fusion protein. According to the published literature, TRIM63 and GPNMB are highly expressed in TFE3 rearrangement renal cell carcinoma and may serve as special markers of TFE3 rearrangement renal cell carcinoma, but whether these molecules play a specific role in promoting cancer in TFE3 rearrangement renal cell carcinoma has not been reported. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a molecular target for preparing a drug for treating TFE3 gene rearrangement renal cell carcinoma and its use. The technical scheme is as follows:

[0006] A molecular target for preparing a drug for treating TFE3 gene rearrangement renal cell carcinoma, wherein the molecular target is the LGI3 gene, and the base sequence of the LGI3 gene is: SEQ ID No.1.

[0007] A use of a molecular target in preparing a drug for treating TFE3 gene rearrangement renal cell carcinoma, wherein the molecular target is the LGI3 gene, and the base sequence of the LGI3 gene is: SEQ ID No.1.

[0008] Optionally, the LGI3 gene is used to prepare a therapeutic agent for TFE3 gene rearrangement renal cell carcinoma.

[0009] Optionally, the LGI3 gene is used to prepare a treatment kit for TFE3 gene rearrangement renal cell carcinoma.

[0010] Use of LGI3 gene inhibitor in the preparation of drugs for treating TFE3 gene rearrangement renal cell carcinoma.

[0011] A medicine or a medicine composition for treating TFE3 gene rearrangement renal cell carcinoma, comprising an LGI3 gene inhibitor.

[0012] The beneficial effects brought about by the technical solution provided by the embodiment of the present invention include at least:

[0013] Interference with LGI3 in the present invention can effectively inhibit the proliferation, migration and invasion of TFE3 rearrangement renal cell carcinoma. Therefore, the LGI3 gene is expected to be used as a molecular target in the treatment of clinical TFE3 rearrangement renal cell carcinoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 The graphs of cell proliferation at different time points in the LGI3-interference group compared with the control group were provided, and it was concluded that interfering with LGI3 could significantly inhibit the proliferation of cells;

[0016] Figure 2 A graph showing the ability of cells to form clones in the LGI3-interference group compared to the control group was provided, showing that the proliferation ability of cells was significantly weakened after LGI3 interference;

[0017] Figure 3 A graph showing the inhibitory effect of the LGI3-interference group on cell migration compared to the control group was provided. The cell migration rate of the LGI3-interference group was about 40%, while the cell migration rate of the control group was about 18%. It was concluded that the cell migration ability could be significantly inhibited after LGI3 interference;

[0018] Figure 4A graph showing the inhibitory effect of the LGI3-interference group on cell migration and invasion compared with the control group was provided, and it was concluded that LGI3-interference could significantly inhibit cell migration and invasion.

[0019] Figure 5 A graph is provided showing the subcutaneous tumor-forming ability of the LGI3-interference group relative to the control group in severely immunodeficient mice (NSG mice). It is concluded that after interfering with the LGI3 group, the subcutaneous tumors in NSG mice were significantly reduced, and tumor growth was significantly inhibited. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is described below in conjunction with the accompanying drawings.

[0021] The purpose of the present invention is to explore the target molecule that plays a cancer-promoting function in TFE3 rearranged renal cell carcinoma. In order to achieve the above purpose, the inventors combined the transcriptome sequencing results of the database and the transcriptome sequencing results after the inventors interfered with TFE3 in TFE3 rearranged renal cell carcinoma cell lines, screened out the highly expressed molecule leucine-rich glioma inactivator 3 (LGI3) in TFE3 rearranged renal cell carcinoma, and conducted functional studies on the molecule, confirming that it can promote the progression of TFE3 rearranged renal cell carcinoma, and interfering with LGI3 can significantly inhibit the progression of TFE3 rearranged renal cell carcinoma.

[0022]

[0023] In this article, interfering with LGI3 refers to interfering with LGI3 at the gene level, thereby reducing its expression at both the mRNA and protein levels.

[0024] Example 1. Cell proliferation assay:

[0025] 1). TFE3 rearranged renal cell carcinoma cell line with good normal culture status. Among them, TFE3 rearranged renal cell carcinoma cell line was obtained by primary isolation and culture of renal carcinoma tissue and immortalization at the Urology Department of the Third Medical Center of the General Hospital of the People's Liberation Army. The specific steps are as follows: 1. Prepare collagenase digestion solution: Mix equal volumes of type I collagenase and type II collagenase with a concentration of 2 mg / ml to prepare collagenase digestion solution for digesting tissue cells. 2. Prepare primary cell culture medium: 20% fetal bovine serum, 2% penicillin-streptomycin and DMEM / F12 culture medium are mixed to prepare primary cell culture medium. 3. Disrupt tumor tissue: Take a soybean-sized tumor tissue in a 1.5 ml centrifuge tube, take 500 µl of collagenase digestion solution and add it to the centrifuge tube, and then use autoclaved tissue scissors to repeatedly cut the tissue into pieces until the diameter is about the size of a sesame seed. 4. Initial tissue cell digestion: Place the centrifuge tube containing tissue cells and collagenase digestion solution in a 37 °C incubator for 5 min, centrifuge at 3000 rpm for 3 min, discard the upper layer of collagenase digestion solution, and obtain the digested tissue cells. 5. Re-crushing tissue cells and digestion: Resuspend the tissue cells obtained in the above steps with 1 ml of collagenase tissue digestion solution, and use autoclaved tissue scissors to repeatedly chop the tissue until there is no obvious resistance when using a 1 ml pipette to absorb the liquid. Place the centrifuge tube in a 37 °C incubator for 10 min, centrifuge at 3000 rpm for 3 min, discard the upper layer of collagenase digestion solution, and resuspend the tissue cell group with the prepared culture medium. 6. Pass the suspended tissue cell suspension through a 100 µm sterile filter and inoculate it into a T25 culture flask for culture. 7. Immortalization of primary cells: Observe the cell growth status daily, and when the fusion rate of normally growing adherent cells reaches 50%, transfect the primary cells with SV40 large T antigen stable lentivirus (purchased from Hanyin Biotechnology Co., Ltd.), and continue to passage for about 20 generations to establish a stable immortalized cell line. 8. Identification of TFE3 rearranged renal cell carcinoma immortalized cell line: Identify the expression of renal tissue marker PAX8 and TFE3 protein by immunohistochemistry, and identify it as a TFE3 rearranged renal cell carcinoma cell line by FISH, and identify the isolated primary immortalized cell line TFE301-1 cell line as NONO-TFE3 fusion type by genome sequencing technology.

[0026] The stable cell line and control cell line of LGI3 interference were constructed. The specific construction steps are as follows: the plasmid of LGI3 interference (pLenti-U6-shLGI3-CMV-GFP-2A-Neo) and the control plasmid (pLenti-U6-shRNA-CMV-GFP-2A-Neo) were purchased from abm, and the plasmid and the tool plasmid: VSVG and PAX2 were co-transfected into the tool cell HEK293T using a transfection reagent. The tool plasmid was provided by the global plasmid sharing platform (Addgene), and the tool cell was provided by the National Laboratory Cell Resource Technology Platform after quality inspection. After 48 hours, the lentivirus was collected, concentrated and transfected into the TFE3 rearranged renal cell carcinoma cell line. After 48 hours of lentivirus transfection, the red fluorescence of the cells was observed under a microscope to confirm that the plasmid was integrated into the host cell genome. The cell screening reagent neomycin was used to kill the cells that did not integrate the plasmid with neomycin resistance. The stable cell line that was successfully transfected was finally obtained by continuous screening for culture.

[0027] The stable cell lines and control cell lines after LGI3 interference were digested with trypsin, and about 2000 cells / well were counted on a cell counting plate after digestion. The cells were plated into a 96-well plate and cultured in an incubator at 37 degrees Celsius. Cell activity detection reagent (CCK-8) was added at 0 h, 24 h, 48 h, and 72 h, and the incubation culture was continued. The cell absorbance value was measured 2 hours after the addition of the reagent.

[0028] 2). Plate cloning experiment: TFE3 rearranged renal cell carcinoma cell lines in good normal culture state were digested with trypsin and counted on a cell counting plate with about 2000 cells / well. The cells were plated into a six-well plate and divided into two groups (control group and LGI3 interference group). After culturing in an incubator at 37 degrees Celsius for 10 days, 4% tissue cell fixative was added and fixed for 10 minutes. The fixative was then discarded and crystal violet solution was added for staining to observe the cell colony formation.

[0029] Result analysis:

[0030] The experimental results are as follows Figure 1 and Figure 2 As shown. Figure 1 It can be seen that compared with the control group, the growth of TFE3 rearranged renal cell carcinoma cell line was significantly inhibited after interfering with LGI3. Figure 2 It can be seen that compared with the control group, the proliferation of TFE3 rearranged renal cell carcinoma cell line was significantly inhibited after interfering with LGI3.

[0031] Example 2. Cell migration and invasion assay:

[0032] 1). Cell scratch test: The constructed interfering LGI3 stable transfected cell line and the control cell line, and the cells in good normal culture state were plated into a six-well plate and placed in a 37-degree Celsius incubator for culture. The scratch test was performed after the two groups of cells filled the entire field of view. The specific steps of the scratch test are as follows: After the cells were plated, the cell growth density was observed. After the cells completely filled the field of view, a straight line was drawn with a 200µl yellow gun tip with appropriate force. Then, the cells that fell off the scratch were washed off with PBS and observed under a microscope. The coordinates and scratch width of the photo were recorded. After culturing in a 37-degree Celsius incubator for 24 hours, the scratch width at the same coordinate was recorded. The formula was used: cell migration rate = (scratch width at 0 h - scratch width after 24 hours) / scratch width at 0 h × 100%.

[0033] 2). Cell migration: TFE3 rearranged renal cell carcinoma cell lines with good normal culture status were divided into two groups (control group and LGI3 interference group). After trypsin digestion, the cells were resuspended in serum-free medium, and about 30,000 cells were counted and evenly spread in the upper chamber. 10% serum medium was added to the lower chamber and cultured in an incubator at 37 degrees Celsius for 24 hours. Then the medium was discarded, 4% tissue cell fixative was added, and fixed for 10 minutes. The fixative was discarded, and crystal violet solution was added for staining. The number of cells passing through the chamber was observed under a microscope and photographed to count.

[0034] 3). Invasion assay: Before the start of the experiment, matrigel was spread on the upper layer of the chamber (matrigel: serum-free medium = 1:8), and the chamber was placed in a 37°C incubator for 30 minutes before the same steps as the cell migration assay were performed.

[0035] Result analysis:

[0036] The experimental results are as follows Figure 3 and Figure 4 As shown. Figure 3 It can be seen that the migration rate of cells in the LGI3 interference group was about 40%, and the migration rate of control cells was about 18%. Compared with the control group, the migration ability of TFE3 rearranged renal cell carcinoma cell line decreased significantly after LGI3 interference.

[0037] from Figure 4 It can be seen that compared with the control group, the migration and invasion ability of the TFE3 rearranged renal cell carcinoma cell line was significantly reduced after interfering with LGI3.

[0038] Example 3. Mouse subcutaneous tumor model:

[0039] The stable cell line interfering with LGI3 and the control cell line were constructed, and the construction steps were as in Example 1. After counting, about 3×10 6 The cells were injected into the axilla of 4-week-old male NSG mice, with 4 mice in each group. The mice were killed 4 months later and the subcutaneous tumors were obtained, photographed and weighed.

[0040] The experimental results are as follows Figure 5 As shown. Figure 5 It can be seen that compared with the control group, the tumor size of TFE3-rearranged renal cell carcinoma was significantly reduced after interfering with LGI3.

[0041] Although several embodiments of the present invention have been given herein, those skilled in the art should understand that the embodiments of the present invention may be modified without departing from the spirit of the present invention. The above embodiments are merely exemplary and should not be used as a limitation on the scope of the present invention.

Claims

1. A molecular target for preparing a drug for treating TFE3 gene rearrangement renal cell carcinoma, characterized in that: The molecular target is the LGI3 gene, and the base sequence of the LGI3 gene is: SEQ ID No.

1.

2. Use of a molecular target in the preparation of a drug for treating TFE3 gene rearrangement renal cell carcinoma, characterized in that: The molecular target is the LGI3 gene, and the base sequence of the LGI3 gene is: SEQ ID No.

1.

3. The use according to claim 2, characterized in that: The LGI3 gene is used to prepare a therapeutic reagent for TFE3 gene rearrangement renal cell carcinoma.

4. The use according to claim 2, characterized in that: The LGI3 gene is used for preparing a therapeutic kit for TFE3 gene rearrangement renal cell carcinoma.

5. Use of LGI3 gene inhibitors in the preparation of drugs for treating TFE3 gene rearrangement renal cell carcinoma.

6. A drug or drug composition for treating TFE3 gene rearrangement renal cell carcinoma, comprising an LGI3 gene inhibitor.

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