CN115873945A - Application of Fidgetin like2 in preparation of tumor treatment medicine - Google Patents

Abstract

The invention discloses the application of Fidgetin like 2 in the preparation of tumor treatment drugs, and belongs to the technical field of biomedicine. According to the present invention, the expression of Fignl2 tends to increase during the occurrence and development of tumors by consulting the online database GEPIA; further, by designing substances that interfere with the expression of Fignl2 to reduce the expression of Fignl2 in HUVECs, it is verified that knocking down Fignl2 can significantly reduce the expression of Fignl2. Angiogenesis of HUVEC cells is inhibited, thus indicating that Fidgetin like 2 can be used for screening or preparing tumor therapeutic drugs.

Description

Application of Fidgetin like 2 in the preparation of tumor therapeutic drugs

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of Fidgetin like 2 in the preparation of tumor treatment drugs.

Background technique

Angiogenesis is the development of new blood vessels from existing capillaries or postcapillary veins. Induction of angiogenesis is one of the ten hallmark features of tumors proposed by Drs. Weinberg and Hanahan. Studies have found that angiogenesis is an important part of tumor progression and plays a very important role in tumor growth and metastasis. Tumor cells continue to divide and proliferate, consuming large amounts of oxygen and nutrients. When the volume of a solid tumor is less than ^2mm3 , oxygen and nutrients can be obtained by diffusion. But as the tumor tissue grows gradually, the tumor needs to form new blood vessels to obtain nutrients and oxygen to ensure the exponential growth of the tumor. In the 1970s, Professor Folkman proposed that tumor growth and metastasis depend on angiogenesis, and inhibiting angiogenesis can be used as one of the strategies for treating tumors. In recent years, targeting pro-angiogenic genes has become a research hotspot in tumor therapy and prevention of tumor expansion.

Fidgetin and its family members Fidgetin like 1 (Fignl1) and Fidgetin like 2 (Fignl2) belong to the AAA (ATPase associated with various cellular activities) protein superfamily and play an important role in mammalian development. The research on members of the Fidgetin family mainly focuses on mitosis, centrosome microtubule growth and attachment, axon growth and other aspects related to microtubule function. It has been reported in the literature that the high expression of Fidgetin is related to the progression of liver cancer, suggesting that the prognosis of liver cancer is poor. However, there are few studies on Fignl2, mainly related to the regulation of axon growth, skin repair and zebrafish development, etc. It has not been reported in the field of cancer, especially angiogenesis.

Contents of the invention

The purpose of the present invention is to provide the application of gene Fidgetin like 2 in screening or preparing tumor treatment drugs.

The nucleotide sequence of the CDS region of the Fignl2:

Homo sapiens fidgetin like 2 (FIGNL2), transcript variant 3, mRNA

NCBI Reference Sequence: NM_001013690.5

GenBank Graphics

>NM_001013690.5:173-2134 Homo sapiens fidgetin like 2 (FIGNL2), transcript variant 3, mRNA

atgcactggacaccagaacacgcccagcccctcaaccagtggccagagcagcacctggacgtctcctccaccaccccgtcgccggcccacaagttggagttgccccctgggggtcgccaacgctgccactacgcttgggcacacgacgacatctcagccctcactgcctccaacctcctaaagcgctatgcagagaagtactctggggtcttggattctccctacgagcgtccggccctgggcgggtacagcgacgcctccttcctcaacggcgccaaaggggatcccgagccctggccagggccggagccaccctaccccttggcctcactccacgaaggcctcccaggaaccaaatcgggcggtggcggcggttccggggccctggggggctccccagttttagccgggaacctccctgaacccctctacgccggcaatgcgtgcgggggcccatcggcggcgcccgagtacgcggccggctacggcggggggtacctggcgccgggttactgcgcgcagacgggcgccgcgctgcccccgccgcccccggccgcgctcctgcagcccccaccgcctccggggtacgggccctcagcgccgctgtacaactatcccgcagggggctacgcagcgcagcccggctatggcgcgctcccgccgcccccaggcccacccccggccccctacctgaccccgggcctgcccgcgcccacgcccctgcccgcgccggcaccgcccaccgcctatggcttccccacggccgcgccgggtgccgaatccgggctgtcgctgaagcgcaaggccgccgacgaggggcccgagggccgctaccgcaagtacgcgtacgagcccgccaaggcccccgtggctgacggagcctcctaccccgccgcggacaacggcgaatgtcggggcaacgggttccgggccaagccgccaggagccgcggaggaggcgtcgggcaagtacggtggcggcgtccccctcaaggtcctgggctcccccgtctacggcccgcaactggagccctttgaaaagttcccggagcgggccccggctcctcgtggggggttcgccgtgccgtcgggggagactcccaaaggcgtggaccctggggccctggagctggtgacgagcaagatggtggactgcgggcccccggtgcagtgggcggatgtggcgggccagggcgcgctcaaggcggcgctggaggaggagctggtgtggcccctgctcaggccgcccgcctacccgggcagcctgcgcccgccgcggaccgtcctgctctttgggccgcggggcgcgggcaaagcgctgctgggccgctgcctcgccacgcagctgggcgccacgctgttgcgcctgcgcggcgcgaccctggctgcgcccggcgccgccgagggcgcgcgcctcctccaggccgccttcgcggccgcgcgctgccgcccaccctccgtactcctcatcagcgagctagaggcgctgctccccgcccgggacgacggcgcggcggcagggggcgcgctgcaggtgccgctcctggcctgcctggacgggggctgcggcgcgggggctgacggcgtgctggttgtgggcaccacctcgcggcccgcggctctggacgaggcgacccgccggcgcttctctctccgcttctacgtggcgctgcccgacagcccggcccgcgggcagatcctgcagcgggcgctggcccagcagggctgcgcgctcagtgagcgggaactggcggcgctggtgcagggcacgcagggcttctctgggggcgagctggggcagctgtgccagcaggcggcggccggggcgggcctcccggggctgcagcgccccctctcctacaaggacctggaggcggcgctggccaaggtgggccctagggcctctgccaaggaactggactcgttcgtggagtgggacaaaatgtacggctccggacactga(SEQ IDNO.7)

The protein sequence of the Fignl2:

fidgetin-like protein 2 [Homo sapiens]

NCBI Reference Sequence: NP_001013712.4

GenPept Identical Proteins Graphics

>NP_001013712.4 fidgetin-like protein 2[Homo sapiens]

MHWTPEHAQPLNQWPEQHLDVSSTTPSPAHKLELPPGGRQRCHYAWAHDDISALTASNLLKRYAEKYSGVLDSPYERPALGGYSDASFLNGAKGDPEPWPGPEPPYPLASLHEGLPGTKSGGGGGSGALGGSPVLAGNLPEPLYAGNACGGPSAAPEYAAGYGGGYLAPGYCAQTGAALPPPPPAALLQPPPPPGYGPSAPLYNYPAGGYAAQPGYGALPPPPGPPPAPYLTPGLPAPTPLPAPAPPTAYGFPTAAPGAESGLSLKRKAADEGPEGRYRKYAYEPAKAPVADGASYPAADNGECRGNGFRAKPPGAAEEASGKYGGGVPLKVLGSPVYGPQLEPFEKFPERAPAPRGGFAVPSGETPKGVDPGALELVTSKMVDCGPPVQWADVAGQGALKAALEEELVWPLLRPPAYPGSLRPPRTVLLFGPRGAGKALLGRCLATQLGATLLRLRGATLAAPGAAEGARLLQAAFAAARCRPPSVLLISELEALLPARDDGAAAGGALQVPLLACLDGGCGAGADGVLVVGTTSRPAALDEATRRRFSLRFYVALPDSPARGQILQRALAQQGCALSERELAALVQGTQGFSGGELGQLCQQAAAGAGLPGLQRPLSYKDLEAALAKVGPRASAKELDSFVEWDKMYGSGH(SEQ ID NO.8)

In the present invention, the online database GEPIA (http://gepia.cancer-pku.cn/index.html) is consulted to know that the expression of Fignl2 is on the rise during the occurrence and development of tumors, especially in cholangiocarcinoma, esophageal cancer, and liver cells. Liver cancer, ovarian serous cystadenocarcinoma, gastric cancer, testicular cancer. Furthermore, by designing substances that interfere with the expression of Fignl2 to reduce the expression of Fignl2 in HUVEC, it was verified that knocking down Fignl2 can significantly inhibit HUVEC cell angiogenesis, thus indicating that the gene Fidgetin like2 can be used to screen or prepare tumor therapeutic drugs.

Description of drawings

Figure 1 shows the expression of Fignl2 in different tumors. Among them: CHOL refers to cholangiocarcinoma, ESCA refers to esophageal carcinoma, HNSC refers to head and neck squamous cell carcinoma, LAML refers to acute myeloid leukemia, LIHC refers to hepatocellular carcinoma, OV refers to ovarian serous cystadenocarcinoma, PAAD refers to pancreatic cancer, and PCPG refers to Pheochromocytoma and paraganglioma, STAD for gastric cancer, TGCT for testicular cancer, THYM for thymus cancer; in each group, the tumor tissue on the left and the normal tissue on the right.

Figure 2 shows the interference efficiency results of Fignl2 siRNA.

Figure 3 shows the results of angiogenesis in HUVEC cells after knocking down Fignl2. Among them: A is a representative diagram of angiogenesis after Fignl2 siRNA and CtrlsiRNA treatment of HUVEC cells; B is a statistical histogram of the number of nodes; C is a statistical histogram of the number of grids; D is a statistical histogram of the number of branches.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention. The experimental methods and unspecified formulations of the reagents in the examples that do not indicate the specific conditions are all in accordance with the conventional conditions in this field.

Example 1

Expression patterns of Fignl2 in tumors

Check the expression of Fignl2 in different tumors through the online database GEPIA (http://gepia.cancer-pku.cn/index.html), the results are shown in Figure 1, Fignl2 in cholangiocarcinoma, esophageal cancer, head and neck squamous cells Carcinoma, acute myeloid leukemia, hepatocellular carcinoma, ovarian serous cystadenocarcinoma, pancreatic cancer, pheochromocytoma and paraganglioma, gastric cancer, testicular cancer, thymic carcinoma, etc. .

Example 2

Functional verification of Fignl2

Induction of angiogenesis is one of the ten hallmark features of tumors, so this example is designed to study whether knocking down the expression of Fignl2 in vascular endothelial cells (HUVEC) will affect angiogenesis.

1. Culture of human umbilical vein endothelial cells (HUVEC)

Take out the frozen HUVEC cells from the -80°C refrigerator, thaw them quickly in a 37°C water bath, suck out the cell suspension in an ultra-clean bench, add 1ml of ECM complete medium, centrifuge, and wash once with an appropriate amount of ECM complete medium , the cells were evenly planted in a 60mm culture dish, placed in a 37°C cell culture incubator for routine culture, and the medium was replaced every other day. When the cells grew to 80%-90%, they could be used for subsequent experiments.

2. Extraction of cellular RNA

Reagent(Thermo fisher)，然后放入1.5mL RNase-free EP管，冰上裂解5min。加入三氯甲烷200μL，涡旋剧烈振荡20s，室温静置5min；13000rpm，4℃离心15min。小心仔细吸取上清，加入异丙醇500μL，上下颠倒轻柔混匀，室温静置10min，13000rpm，4℃离心15min，弃除上清。加入1mL75％乙醇，轻轻洗涤沉淀，4℃离心13000rpm，5min；去除上清，吹干。加入适量RNase-freeH₂O，65℃促溶10min；检测RNA的OD值及浓度，-80℃保存备用。Wash HUVEC normally cultured in a 35mm Petri dish with PBS 3 times, add 1mL

Reagent (Thermo fisher), then put into 1.5mL RNase-free EP tube, and lyse on ice for 5min. Add 200 μL of chloroform, vortex vigorously for 20 s, let stand at room temperature for 5 min; centrifuge at 13000 rpm at 4°C for 15 min. Carefully aspirate the supernatant, add 500 μL of isopropanol, mix gently up and down, let stand at room temperature for 10 minutes, centrifuge at 13000 rpm for 15 minutes at 4°C, and discard the supernatant. Add 1mL of 75% ethanol, gently wash the precipitate, centrifuge at 13000rpm at 4°C for 5min; remove the supernatant, and blow dry. Add an appropriate amount of RNase-freeH ₂ O, promote dissolution at 65°C for 10 minutes; detect the OD value and concentration of RNA, and store at -80°C for later use.

3. Synthesis of cDNA by reverse transcription of RNA

Using a reverse transcription kit (Vazyme R312-01), 500 ng of RNA was reverse transcribed into cDNA.

Operate on ice, 20 μL of each reaction system, as follows:

The reaction program is: 37°C for 15 min, 85°C for 5 sec, 4°C∞.

4. Real-time fluorescence quantitative PCR (qRT-PCR)

The primer sequences of Fignl2 qRT-PCR were designed according to the primer design principles.

Fignl2 qRT-PCR Primers

Fignl2-F: CGCCCACCCCTCCGTACTCCT (SEQ ID NO.5)

Fignl2-R: CACAACCAGCACGCCGTCA (SEQ ID NO. 6)

The cDNA obtained from the reverse transcription reaction was diluted 1:5 and subjected to the following qRT-PCR reaction.

(1) Prepare qRT-PCR reaction solution according to the following components:

(2) The reaction solution is mixed evenly, and the reaction procedure of the Real-time PCR instrument is as follows:

Stage 1: 95°C for 5 minutes

Stage 2: Cycle: 40

95℃ 10sec

60℃ 30sec

Stage 3: 95°C 15sec

60℃ 1min

95℃ 15sec

(3) Three replicate wells were set up for the reaction, and the internal reference was GAPDH. After the program ended, check the melting curve and amplification curve, discard the experimental data with large errors, and perform statistical analysis on the data according to the specific experimental requirements.

5. siRNA electrotransfection of HUVEC

Count the HUVECs cultured to the P1 generation, absorb a certain amount of cells and mix them with siRNA, and mix well to make the final concentration reach 2.0×10 ⁶ cells + 200 nM siRNA in each tube of 100 μL mixed solution, in which the volume of cells is 90 μL. The siRNA volume was 10 μL. Then, electrotransfection was performed according to the electrotransfection operation procedure of NEPA21 (NEPAGENE Company) endothelial cell line (140V, 5ms).

Fignl2 siRNAs are as follows:

Sense strand: AGCGCCGCUGUACAACUAUTT (SEQ ID NO.1)

Antisense strand: AUAGUUGUACAGCGGCGCUTT (SEQ ID NO.2).

Ctrl siRNA:

Sense strand: UUCUCCGAACGUGUCACGUTT (SEQ ID NO.3)

Antisense strand: ACGUGACACGUUCGGAGAATT (SEQ ID NO.4).

6. Identification of siRNA interference efficiency

Reagent(Thermo fisher)说明书提取HUVEC细胞RNA，逆转录，进行qRT-PCR。结果如图2所示，siRNA处理48h后，Fignl2 siRNA可以显著降低HUVEC中Fignl2的表达，Fignl2 siRNA vs.Ctrl siRNA，**P<0.01。HUVEC cells were transfected with siRNA specific for Fignl2 and its control Ctrl (Control) siRNA respectively. After 48 hours, the cells were harvested and pressed

Reagent (Thermo fisher) instructions extract HUVEC cell RNA, reverse transcription, and perform qRT-PCR. The results are shown in Figure 2, after siRNA treatment for 48 hours, Fignl2 siRNA can significantly reduce the expression of Fignl2 in HUVEC, Fignl2 siRNA vs. Ctrl siRNA, **P<0.01.

7. Induction of HUVEC into angiogenesis and index measurement

Thaw Matrigel at 4°C from a -20°C refrigerator. For 96-well plates, pipette tips should be pre-cooled in a -20°C refrigerator. During the experiment, put the 96-well plate and Matrigel on ice, pipette 50 μL of Matrigel and evenly spread it on the bottom of the 96-well plate, and place it in a 37°C incubator to form a gel for 30 minutes. At the same time, 0.25% trypsin was used to digest the HUVEC cells transfected with Fignl2 siRNA and Ctrl siRNA for 48 hours, resuspended and counted, and planted 2×10 ⁴ cells in each well, and then put them in the incubator to continue culturing for 6 hours, took pictures and recorded them, and then used Image The J software counts the number of vascular nodes, grids and branches. The results are shown in Figure 3. Knockdown of Fignl2 can significantly inhibit HUVEC cell angiogenesis, Fignl2 siRNA vs. Ctrl siRNA, *P<0.05, **P<0.01.

Claims (5)

1. Application of Fidgetin like2 in screening of tumor treatment drugs.
2. 2. Use according to claim 1, characterized in that: the drug inhibits angiogenesis.
3. Application of the Fidgetin like2 inhibitor in preparation of tumor treatment medicines.
4. 4. Use according to claim 3, characterized in that: the Fidgetin like2 inhibitor is selected from one or more of compounds, proteins, polypeptides, polysaccharides, glycoproteins, glycopeptides and nucleic acids.
5. 5. Use according to claim 4, characterized in that: the nucleic acid is siRNA, and the sequence of the nucleic acid is as follows:

   a sense strand: AGCGCCGCUGUACAACUAUTT

   Antisense strand: AUAGUUGUACACGCGGCCUTT.

Images