CN113122627A

CN113122627A - Application of IGFL4 gene as marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer

Info

Publication number: CN113122627A
Application number: CN201911391131.6A
Authority: CN
Inventors: 赵杨; 陈说; 关雪; 江汝琪
Original assignee: Third Affiliated Hospital of Guangzhou Medical University
Current assignee: Third Affiliated Hospital of Guangzhou Medical University
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2021-07-16
Anticipated expiration: 2039-12-30
Also published as: CN113122627B

Abstract

The invention relates to the technical field of molecular biology and tumor marker medicine, in particular to application of an IGFL4 gene as a marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer. The IGFL4 gene can be used as a biomarker for diagnosing gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer or used for preparing a product for diagnosing the cancer. The IGFL4 gene shows specific high expression in gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer tumors, but does not show the specific high expression in normal tissues; by detecting the expression of the IGFL4 gene in a subject, the occurrence of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer can be determined quickly, accurately and clearly. The IGFL4 gene is used as a diagnostic marker of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, provides a new target point for clinical treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and can be applied to preparation of anti-tumor drugs.

Description

Application of IGFL4 gene as marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer

Technical Field

The invention relates to the technical field of molecular biology and tumor marker medicine, in particular to application of an IGFL4 gene as a marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer.

Background

Gastric cancer (gastric carcinoma) refers to a malignant tumor of epithelial origin that originates in the stomach. The incidence of gastric cancer is second to that of lung cancer in China, and the mortality rate is third. About 120 million new cases of stomach cancer occur every year in the world, and China accounts for about 40 percent of the cases. The early gastric cancer accounts for a very low percentage of only about 20 percent in China, most of the gastric cancers are developed, and the overall 5-year survival rate is less than 50 percent. In recent years, the proportion of early-stage gastric cancer has increased year by year with the spread of gastroscopy.

Colorectal cancer is one of the most common malignant tumors worldwide, and in recent years, the incidence rate of colorectal cancer in China is on a remarkable rising trend. Colorectal cancer has a high recurrence rate due to late discovery, and causes high mortality of patients, and the 5-year survival rate of colorectal cancer is highly dependent on the stage of tumor at the time of diagnosis. Early detection of colorectal cancer is crucial to improve clinical efficacy and prolong survival of patients. Meanwhile, lymph node metastasis is also an important factor influencing prognosis and survival of patients, and accurate prediction of lymph node state before colorectal cancer operation is the key for making a reasonable treatment scheme. However, the conventional detection methods of imaging, laboratory examination, enteroscopy, etc. currently used in clinical practice generally have the disadvantages of low sensitivity and specificity, high cost, invasiveness, discomfort to patients, etc. Therefore, there is a great clinical need to develop a sensitive, specific, economical and noninvasive method to facilitate colorectal cancer screening, improve the efficiency of disease diagnosis and the efficiency of pre-operative lymph node metastasis prediction. In addition, the TNM staging system, which is the most common method for predicting the survival of colorectal cancer patients, is also deficient, and even patients in the same stage have highly complex heterogeneity, which provides very limited information for clinical prognosis. The novel marker is excavated and used for establishing a colorectal cancer prognosis evaluation method superior to a TOM staging system, and the method is helpful for making a reasonable individualized treatment scheme for patients, so that the method has important clinical value.

Endometrial cancer is a common gynecological malignant tumor, and the incidence rate of the endometrial cancer tends to rise year by year. Because the disease usually shows abnormal bleeding of vagina in early stage, the diagnosis can be easily obtained in time, and after standard treatment of total uterine adnexal excision and lymph node excision, the prognosis is good. However, despite the increasing means of surgery and radiotherapy and chemotherapy, the prognosis of patients with recurrent or advanced endometrial cancer is still poor, fewer options are faced in treatment, the median survival time is only 7-10 months, and a new treatment mode needs to be discovered urgently. For malignant tumors that have progressed after standard therapy, currently, a promising therapeutic approach is targeted therapy, which acts on a well-defined carcinogenic site to specifically kill tumor cells without affecting normal tissues around the tumor, thereby reducing toxic and side effects on normal cells while exerting antitumor activity. With the deep research on the tumorigenesis mechanism, drugs aiming at cancer cell survival molecular pathway therapeutic targets are developed successively, and the drugs relate to various aspects such as angiogenesis, DNA repair, apoptosis and the like. Molecularly targeted drugs to the tumor gene pathway may also have utility in the treatment of endometrial cancer.

Ovarian Cancer (OC) is a relatively common malignancy in the female reproductive system and has the highest mortality rate of gynecological malignancies. There are many types of OCs known, of which Epithelial Ovarian Cancer (EOC) is the most common. EOC has no obvious symptoms in the early stage, most patients are in the late stage at the time of initial diagnosis, the treatment effect is poor, and the 5-year survival rate is extremely low. If OC can be found and diagnosed at an early stage clinically and comprehensive treatment means such as radical operation or targeted molecular drug therapy can be reasonably carried out in time, the survival rate and the survival quality of OC patients can be greatly improved and the survival time can be prolonged. Therefore, the intensive research on related molecules of OC and signal transduction pathway mechanism has very important clinical significance.

Although tumors of different tissue origins have specificity, they also share some commonality in biological behavior and mechanisms of occurrence, as are adenocarcinomas. The gene and protein which play a role in gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer are searched, and the important role in searching the joint diagnosis and treatment targets of the diseases is played.

The IGFL gene encodes a protein of approximately 100 amino acids that contains 11 conserved cysteine residues at fixed positions, including two CC motifs. In humans, this family consists of four genes and two pseudogenes, referred to as IGFL1 through IGFL4 and IGFL1P1 and IGFL1P2, respectively. The human IGFL gene is clustered at 35 kb intervals on chromosome 19, and structural considerations and sequence comparisons indicate that the IGFL protein is closely related to the IGF4 superfamily of growth factors. IGFL4mRNA is rarely expressed in tissues, but exhibits a specific expression pattern. In addition, the inventors found that the IGFL4 gene exhibits abnormally high expression in gastric cancer, intestinal cancer, ovarian cancer, lung cancer and intimal cancer, which may play a key role in the development of these malignancies. However, the effect of the gene in tumors is still unclear, no relevant documents report the effect of the gene, and effective early-onset biomarkers are found, and relevant molecular mechanisms have important significance in guiding clinical diagnosis, treatment and prognosis of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer. In general, the research and identification of effective molecular markers by using a molecular biological method is a key means for assisting the existing clinical diagnosis, guiding clinical intervention and precancerous early warning, further researches the effect of the IGFL gene on malignant tumors, particularly on the occurrence and development of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and has very important help for the early diagnosis, treatment and prognosis of the gastric cancer, the colorectal cancer, the endometrial cancer and the ovarian cancer.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide application of an IGFL4 gene as a marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and provides a novel diagnostic marker for gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, which can clearly and clearly represent the occurrence and development of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer and shows a specific high-expression phenomenon in human clinical gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer tissues compared with corresponding normal tissues. The IGFL4 gene is used as a diagnostic marker of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, provides a molecular target for treatment of the gastric cancer, the colorectal cancer, the endometrial cancer and the ovarian cancer, and can be applied to preparation of anti-tumor drugs.

In order to achieve the purpose, the invention adopts the following technical scheme.

A cancer marker which is the IGFL4 gene.

Further, the cancer includes any one of gastric cancer, colorectal cancer, endometrial cancer, and ovary cancer.

The IGFL4 gene can be used as a cancer diagnosis marker or used for preparing a cancer diagnosis product.

Further, the use of the IGFL4 gene as a diagnostic marker for cancer or for the preparation of a product for the diagnosis of cancer, including any one of gastric cancer, colorectal cancer, endometrial cancer and ovary cancer.

Further, the cancer diagnosis product is selected from a preparation, a chip or a kit.

Further, the cancer diagnostic product comprises a primer pair for amplifying a nucleic acid sequence specifically recognizing the IGFL4 gene.

Specifically, the primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQID No. 1; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.

An anticancer medicine contains IGFL4 gene inhibitor as effective component, and the inhibitor is one or more of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compound, peptide, antibody, etc.

Furthermore, the IGFL4 gene inhibitor is siRNA, and the sequence of the siRNA is 5'-GUGUCAUCCUAGACUUGAA-3'.

The IGFL4 gene is used in preparing medicine for treating gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer.

Compared with the prior art, the invention has the following beneficial effects.

The invention discovers for the first time that the IGFL4 gene can be used as a diagnosis biomarker of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and has a specific high expression phenomenon in tumors of the gastric cancer, the colorectal cancer, the endometrial cancer and the ovarian cancer, but has no specific high expression phenomenon in normal tissues; by detecting the expression of the IGFL4 gene in a subject, the occurrence of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer can be determined quickly, accurately and clearly.

According to the invention, the expression level of IGFL4 in gastric cancer cell lines (MGC-803 and SGC7901), intestinal cancer cell lines (HCT 116, SW480 and SW 620), endometrial cancer cell lines (H1C-1A, H1C-1B, Ishikuwa) and ovarian cancer cell lines (A2780, CAOV3, OVCAR3, SKOV3 and HO 8910) is detected, and one cell strain with high expression of IGFL4 is selected from the 4 malignant tumors for carrying out related tumor phenotype research, so that the cell proliferation inhibition capability is obviously inhibited compared with a control group; inhibiting cell migration; obviously inhibit the invasion capacity of tumor cells; increasing the level of apoptosis of tumor cells. Provides a new target spot for clinical treatment of cancer, gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and can be applied to preparation of anti-tumor medicaments.

Drawings

FIG. 1 shows the results of experiments on the expression level of IGFL4 in each of the 4 malignant tumors.

FIG. 2 is a graph showing the results of experiments in which tumor cell proliferation was measured by the MTT method in the ovarian cancer cell line HO8910, the endometrial cancer cell line Ishikawa, the gastric cancer cell line MGC-803, and the intestinal cancer cell line HCT 116.

FIG. 3 is the experimental result of the determination of the tumor cell migration ability by the cell scratch experiment in the ovarian cancer cell line HO 8910.

FIG. 4 is an experimental result of measuring the migration ability of tumor cells by a cell scratch experiment in the endometrial cancer cell line Ishikawa.

FIG. 5 is an experimental result of measuring the migration ability of tumor cells by a cell scratch experiment in a gastric cancer cell line MGC-803.

FIG. 6 is an experimental result of measuring the migration ability of tumor cells by a cell scratch experiment in the intestinal cancer cell line HCT 116.

FIG. 7 shows the results of experiments for determining the invasive potential of tumor cells by the Transwell method in the ovarian cancer cell line HO 8910.

FIG. 8 is the results of experiments for determining the invasiveness of tumor cells by the Transwell method in the endometrial cancer cell line Ishikawa.

FIG. 9 is the result of an experiment for measuring the invasive ability of tumor cells by the Transwell method in the gastric cancer cell line MGC-803.

FIG. 10 is a result of an experiment for measuring the invasion ability of tumor cells in the intestinal cancer cell line HCT116 by the Transwell method.

FIG. 11 is a flow cytometry determination of tumor cell apoptosis in ovarian cancer cell line HO 8910.

FIG. 12 is a graph showing the determination of apoptosis of tumor cells by flow cytometry at the endometrial cancer cell line Ishikawa.

FIG. 13 is a graph showing the determination of apoptosis of tumor cells by flow cytometry in a gastric cancer cell line MGC-803.

FIG. 14 is a flow cytometry determination of apoptosis of tumor cells in the intestinal cancer cell line HCT 116.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are provided to illustrate the present invention, but these examples are only for illustrating the present invention and the present invention is not limited to these. The procedures not specifically described in the examples are those conventionally employed in the art or are in accordance with the manufacturer's instructions.

Example IGFL4 gene expression in malignant tumor cell line including gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer and relevant cell function test method.

The cell lines referred to in the examples originate: gastric cancer cell lines (MGC-803, SGC7901), intestinal cancer cell lines (HCT 116, SW480, SW 620), endometrial cancer cell lines (H1C-1A, H1C-1B, Ishikuwa), ovarian cancer cell lines (A2780, CAOV3, OVCAR3, SKOV3, HO 8910) Guangzhou Jinie European Biotech Co., Ltd.

1. Identification of IGFL4mRNA levels in gastric, colorectal, endometrial, and ovarian cancer cell lines.

1.1 extracting total RNA of tumor cells by using Trizol reagent, adding chloroform with the volume of Trizol 1/5, violently shaking for 15 seconds, and standing for 5 minutes at room temperature after the solution is fully emulsified; centrifuging at 4 deg.C for 20 min at 12,000g, transferring supernatant to another new centrifuge tube, adding isopropanol with equal volume into supernatant, turning the centrifuge tube upside down, mixing, and standing at 30 deg.C for 10 min; centrifuging at 4 deg.C for 20 min at 12,000g, discarding supernatant, adding lml 75% ethanol along the tube wall, washing the tube wall upside down, centrifuging at 4 deg.C for 10 min at 12,000g, discarding ethanol, drying the precipitate at room temperature for 5-10 min, adding a proper amount (10-20 ul) of RNase-free water to dissolve the precipitate, and measuring RNA concentration and purity (RNA purity is higher between quantitative RNA concentration of 1ug/ul and OD260/OD 2801.8-2.0) with UV-2800A type ultraviolet-visible spectrophotometer.

1.2 reverse transcription to synthesize cDNA.

The cDNA obtained by reverse transcription was performed using the Promega GoScript reverse transcription system (A5000, A5001) according to the following procedure.

The first step is as follows: a certain amount of template RNA is taken and added into the primer.

RNA ( 1µg/ul) 5μl。

Random Primers (0.5 µg /ul) 1 μl。

Oligo(dT)15 Primer (0.5 µg/ul) 1 μl。

Nuclean-Free Water (added to 10. mu.l) 3. mu.l.

The second step is that: the mixture of template RNA and reverse transcription Primers (Random Primers and oligo (dT)15 Primer) was pre-denatured at 70 ℃ for 5min, and after completion, it was removed and placed on ice.

The third step: RT-Mix was prepared and 10. mu.l of each sample tube was added.

The fourth step: setting a reverse transcription program comprising three steps of annealing, extending and reverse transcriptase inactivation (annealing at 25 ℃ for 5min, extending at 42 ℃ for 60 min, inactivating at 70 ℃ for 15min, and obtaining cDNA at 4 ℃ + infinity).

1.3 Real-time PCR。

(1) The PCR reaction mixture was prepared as follows (the reaction mixture was prepared at room temperature), and distributed to each reaction tube, followed by addition of 2ul of template.

(2) The ABI PRISM 7500 Real-Time PCR System and a two-step method are adopted to carry out the PCR standard amplification program.

(3) The data were derived and the Realtime PCR results were analyzed by the 2- Δ CT method.

2. And preparing specific siRNA and primers.

The siRNA used in this experiment was designed based on Rosetta siRNA Design Algorithm and purchased from Sigma Aldrich Shanghai tracing co. Ltd (Shanghai, china) by chemical synthesis. The primers used in the experiment were synthesized by solid phase phosphoramidite triester method, purchased from Beijing Liuhe Huada Gene science and technology Co.

3. Tumor cell proliferation capacity assay (MTT method).

Collecting cells in logarithmic phase, adjusting the concentration of cell suspension, paving 3000 cells in each hole on a 96-well plate, wherein the hole volume is 100 microliters, adding PBS (phosphate buffer solution) into marginal holes to prevent the evaporation of experimental group liquid, transfecting the cells by using specific siRNA after the cells are attached to the wall, and measuring the cell proliferation conditions of 0h, 24h, 48h and 72h after transfection. Adding 20 microliters of MTT solution into each hole, continuously culturing for 4 hours, then sucking the culture medium, adding 150 microliters of dimethyl sulfoxide into each hole, shaking for 6 minutes, and measuring the absorbance on an enzyme-linked immunosorbent assay. Data were recorded and cell viability curves were plotted.

4. Tumor cell migration assay (cell scratch experiment).

Cells with good growth status were selected and cultured in 6-well plates at 5X 10 per well⁵The cells were cultured normally overnight, and the same 200. mu.l pipette tip was scratched in each well for the second day, followed by washing 2-3 times with PBS to remove the scratched cells, transfecting the cells with specific siRNA, and adding 5ml of serum-free medium to continue the culture. The growth and migration of the scratched cells were observed under a microscope, photographed at 0, 24, and 48h after transfection, photographed at the same position, washed with PBS before photographing to remove dead cells, and finally the bare area was measured using Image J software (National Institutes of Health, Bethesda, Md., USA). Scratch healing rate = (original scratch area-area of actual scratch at different time)/original scratch area × original scratch%.

5. Tumor cell invasion assay (Transwell experiment).

Firstly, 30-40 mul of matrigel (1: 10) is diluted by serum-free culture solution and evenly spread in a small chamber, and the small chamber is put into a room to be incubated for 4 hours at 37 ℃. Count and take 5X 10 ^ 4 cells, shop, the upper chamber for serum-free medium diluted cells 200 u l, the lower chamber added normal 10% FBS medium 600 u l, simultaneously with specific siRNA transfection cells. And (3) continuously culturing for 48h, then dyeing, washing the cell-containing chamber with PBS (phosphate buffer solution) for 3 times, fixing 4% paraformaldehyde at room temperature for 15min or storing at 4 ℃ for a long time, discarding formaldehyde, washing with PBS for 3 times, 5min each time, dyeing with 0.1% crystal violet for 15min, washing with PBS for 3 times, 5min each time, wiping off matrix glue in the upper chamber with a cotton swab, slightly cutting off the membrane with a blade, drying, sealing with resin glue, taking pictures and counting under an upright microscope, and observing and determining the invasion capacity of the cells.

6. Tumor apoptosis assay (flow cytometry detection).

Count 3X 10⁵Individual cells, plated in 6-well plates, were transfected after cell attachment. After 48h, cells were trypsinized without EDTA for an appropriate time to prevent false positives. After centrifugation at 1500 r for 5min, cells were collected and washed twice with pre-cooled PBS. The cells were resuspended in 100. mu.L of 1 Xbinding buffer and added5 μ L Annexin V-FITC and 5 μ L PI staining solution were added. After 20 minutes, 200. mu.L of 1 Xbinding buffer was added under dark conditions at room temperature. Flow cytometry was used to detect the rate of apoptosis. Results of the experiment

As shown in FIG. 1, the expression level of IGFL4 gene was high in 13 malignant tumor cell lines, among which IGFL4 was highly expressed in 5 of HO8910, OVCAR3, HEC-1A, MGC-803 and SW620, and low in 4 of CAOV3, HEC-1B, SW480 and SGC 7901.

As shown in fig. 2, after knocking down the expression level of SMCO2 using specific siRNA in the ovarian cancer cell line OVCAR3, the endometrial cancer cell line Ishikawa, the gastric cancer cell line MGC-803, and the intestinal cancer cell line HCT116, the cell proliferation ability was significantly inhibited compared to the control group.

As shown in fig. 3 to 6, cell migration was inhibited after knocking down the expression level of SMCO2 using specific siRNA in the ovarian cancer cell line HO8910, the endometrial cancer cell line Ishikawa, the gastric cancer cell line MGC-803, and the intestinal cancer cell line HCT116, as compared to the control group.

As shown in fig. 7 to 10, the tumor cell invasion ability was significantly inhibited after knocking down the expression level of SMCO2 using specific siRNA in the ovarian cancer cell line HO8910, the endometrial cancer cell line Ishikawa, the gastric cancer cell line MGC-803, and the intestinal cancer cell line HCT 116.

As shown in fig. 11 to 14, the tumor cells had increased levels of apoptosis compared to the control group after knocking down the expression level of SMCO2 using specific sirnas in the ovarian cancer cell line HO8910, the endometrial cancer cell line Ishikawa, the gastric cancer cell line MGC-803, and the intestinal cancer cell line HCT 116.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. A cancer marker which is the IGFL4 gene.

2. The cancer marker of claim 1 wherein said cancer comprises any one of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer.

3. The IGFL4 gene can be used as a cancer diagnosis marker or used for preparing a cancer diagnosis product.

4. The use of the IGFL4 gene as a diagnostic marker for cancer or in the manufacture of a product for use in the diagnosis of cancer according to claim 3, wherein the cancer comprises any one of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer.

5. The use of the IGFL4 gene as a diagnostic marker for cancer or in the preparation of a product for cancer diagnosis according to claim 3, wherein the diagnostic product for cancer is selected from the group consisting of a formulation, a chip or a kit.

6. Use of the IGFL4 gene according to claim 3 as a diagnostic marker for cancer or in the preparation of a product for cancer diagnosis comprising a primer pair for amplifying a nucleic acid sequence specifically recognizing IGFL4 gene.

7. The use of the IGFL4 gene as a cancer diagnostic marker or in the preparation of a product for cancer diagnosis according to claim 3, wherein the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.

8. The anti-cancer medicine is characterized in that the effective component of the anti-cancer medicine is an IGFL4 gene inhibitor, and the inhibitor is one or more of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compounds, peptides and antibodies.

9. The anti-cancer drug of claim 8, wherein the IGFL4 gene inhibitor is an siRNA having the sequence: 5'-GUGUCAUCCUAGACUUGAA-3' are provided.

The application of IGFL4 gene in preparing medicine for treating gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer.