CN113122627B

CN113122627B - Application of IGFL4 gene as marker in diagnosis and treatment of ovarian cancer

Info

Publication number: CN113122627B
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: 2023-08-11
Anticipated expiration: 2039-12-30
Also published as: CN113122627A

Abstract

The invention relates to the technical fields 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 can be used for preparing a product for diagnosing cancers. The IGFL4 gene shows a high specific expression phenomenon in gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer tumors, and has no high specific 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 rapidly, accurately and clearly determined. The IGFL4 gene is used as a diagnosis mark of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, provides a new target point for clinically treating gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and can be applied to the preparation of antitumor drugs.

Description

Application of IGFL4 gene as marker in diagnosis and treatment of ovarian cancer

Technical Field

The invention relates to the technical fields 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 an epithelia-derived malignant tumor that is primary to the stomach. In China, the incidence rate of gastric cancer is second only to lung cancer, and the death rate is third. The new gastric cancer cases worldwide are about 120 ten thousand per year, and about 40% of them are in China. The early gastric cancer in China has a very low proportion of only about 20%, most of the early gastric cancer is in an advanced stage, and the survival rate of the early gastric cancer in 5 years is less than 50%. In recent years, with the popularization of gastroscopy, the proportion of early gastric cancer is increasing year by year.

Colorectal cancer is one of the most common malignant tumors worldwide, and in recent years, the incidence of colorectal cancer in China has a remarkable rising trend. Colorectal cancer is found to be late, and has high recurrence rate, so that the death rate of patients is also high, and the survival rate of 5 years is highly dependent on tumor stage at the time of diagnosis. Early detection of colorectal cancer is critical to improving clinical efficacy and prolonging survival of patients. Meanwhile, lymph node metastasis is also an important factor affecting prognosis and survival of patients, and accurate prediction of colorectal cancer preoperative lymph node status is a key to formulating a reasonable treatment scheme. However, the conventional detection methods such as imaging, laboratory examination, enteroscopy and the like in clinical application at present generally have the defects of low sensitivity and specificity, high cost, invasiveness, discomfort to patients and the like. Therefore, there is a need in the clinic to develop sensitive, specific, economical, non-invasive methods to facilitate screening of colorectal cancer, to improve disease diagnosis efficiency and pre-operative lymph node metastasis prediction efficacy. Furthermore, the TNM staging system, which is the most common method of predicting survival in colorectal cancer patients, is also deficient, and even patients of the same stage are highly complex heterogeneous, which provides very limited information for clinical prognosis. The development of novel markers for establishing a prognosis evaluation method for colorectal cancer that is superior to the TOM staging system would help to formulate a reasonable personalized treatment regimen for patients and thus would have significant clinical value.

Endometrial cancer is a common gynecological malignancy, and the incidence rate tends to rise year by year. Because the disease usually manifests as abnormal vaginal bleeding in early stage, the disease is easy to be diagnosed in time, and the disease has good prognosis after standard treatment of total uterine double-accessory excision and lymph node excision, and the postoperative auxiliary radiotherapy or chemotherapy if necessary. However, although the surgical and chemoradiotherapy means are continuously improved, the prognosis of patients with recurrent or advanced endometrial cancer is still poor, fewer choices are faced in treatment, the median survival time is only 7-10 months, and new treatment modes are urgently needed to be discovered. For malignant tumors which progress after standard treatment, a promising treatment method is currently targeted treatment, and the targeted treatment acts on the already defined cancerogenic sites to enable tumor cells to specifically die without affecting normal tissues around the tumor, so that the toxic and side effects on the normal cells are reduced while the antitumor activity is exerted. With the intensive research of the tumorigenesis mechanism, medicines aiming at cancer cell survival molecular pathway treatment targets are developed successively, and the medicines relate to various aspects such as angiogenesis, DNA repair, apoptosis and the like. Molecular targeted drugs to tumor gene pathways may also have great 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 from gynecological malignant diseases. Many types of OC are known, with Epithelial Ovarian Cancer (EOC) being the most common. EOC has no obvious symptoms at the early stage, most patients are in the late stage at the time of primary diagnosis, the treatment effect is poor, and the survival rate of 5 years is extremely low. If the OC can be discovered and diagnosed early in clinic, and comprehensive treatment means such as radical surgery or targeted molecular drug treatment can be reasonably performed in time, the survival rate and the survival quality of OC patients can be greatly improved, and the survival time can be prolonged. Therefore, intensive research on related molecules and signaling pathway mechanisms of OC is of great clinical significance.

Although tumors of different tissue origin have their specificity, they share a common role in biological behavior and mechanisms of occurrence, as are adenocarcinomas. The genes and proteins which play a role in searching stomach cancer, colorectal cancer, endometrial cancer and ovarian cancer have important roles in searching for co-diagnosis and treatment targets of the diseases.

The IGFL gene encodes a protein of approximately 100 amino acids that contains 11 conserved cysteine residues at fixed positions, including the two CC motifs. In humans, this family consists of four genes and two pseudogenes, called IGFL1 through IGFL4 and IGFL1P1 and IGFL1P2, respectively. Human IGFL gene is clustered on chromosome 19 at 35 kb intervals, and structural considerations and sequence comparisons indicate that IGFL proteins are closely related to the IGF4 superfamily of growth factors. IGFL4mRNA is rarely expressed in tissues, but exhibits a specific expression pattern. Furthermore, the inventors found that the IGFL4 gene exhibits abnormally high expression in gastric, intestinal, ovarian, lung and intimal cancers, which may have a key role in the development and progression of these malignant tumors. However, the effect of the gene in tumors is not clear, no related literature reports the effect of the gene, effective biomarkers for early onset are found, and related molecular mechanisms are of great significance for 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 the clinical intervention and pre-cancer early warning, further researching the effect of the IGFL gene in malignant tumors, particularly in the occurrence and development of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and has very important help for early diagnosis, treatment and prognosis of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer.

Disclosure of Invention

In view of the defects existing in the prior art, in order to find diagnostic molecular markers and therapeutic targets of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, the invention aims to provide application of an IGFL4 gene as a marker in diagnosing and treating gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer. The IGFL4 gene is used as a diagnosis mark of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, provides a molecular target for treating gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and can be applied to the preparation of antitumor drugs.

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

A cancer marker, which is an IGFL4 gene.

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

The use of the IGFL4 gene as a diagnostic marker for cancer or for the preparation of a product for the diagnosis of cancer.

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

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

Further, the cancer diagnostic product includes 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 SEQ ID No. 1; the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2.

An anticancer medicine contains an IGFL4 gene inhibitor as an active component, wherein the inhibitor is one or more of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compounds, peptides, antibodies and the like.

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

The application of the IGFL4 gene in preparing medicines 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 that the IGFL4 gene can be used as a biomarker for diagnosing gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer for the first time, and has a high specificity expression phenomenon in gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer tumors, and has no high specificity 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 rapidly, accurately and clearly determined.

According to the invention, the expression level of the IGFL4 in a gastric cancer cell line (MGC-803, SGC 7901), an intestinal cancer cell line (HCT 116, SW480 and SW 620), an endometrial cancer cell line (H1C-1A, H C-1B, ishikuwa) and an ovarian cancer cell line (A2780, CAOV3, OVCAR3, SKOV3 and HO 8910) is detected, and one cell strain with high expression of the IGFL4 is selected from the 4 malignant tumors to carry out relevant tumor phenotype research, so that compared with a control group, the cell proliferation inhibition capability is obviously inhibited; inhibiting cell migration; obviously inhibiting the invasion capacity of tumor cells; increase the apoptosis level of tumor cells. Provides a new target for clinically treating cancers, gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer, and can be applied to the preparation of antitumor drugs.

Drawings

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

FIG. 2 shows the results of experiments in which IGFL4 was assayed for tumor cell proliferation by the MTT method in ovarian cancer cell line HO8910, endometrial cancer cell line Ishikawa, gastric cancer cell line MGC-803, and intestinal cancer cell line HCT 116.

Fig. 3 is an experimental result of determining the tumor cell migration ability of IGFL4 in intestinal cancer cell line HCT116 by cell scoring experiment.

Fig. 4 is an experimental result of determining the ability of IGFL4 to migrate tumor cells in ovarian cancer cell line HO8910 by cell scoring experiments.

FIG. 5 is an experimental result of determining the migration ability of tumor cells by cell scoring experiments in the endometrial cancer cell line Ishikawa by IGFL 4.

FIG. 6 shows the results of experiments for determining the invasiveness of tumor cells by the Transwell method in the gastric cancer cell line MGC-803 by IGFL 4.

FIG. 7 shows the results of experiments for determining the invasion ability of IGFL4 into tumor cells by the Transwell method in intestinal cancer cell line HCT 116.

FIG. 8 is a graph showing the results of an experiment for determining the invasive capacity of tumor cells by Transwell method in ovarian cancer cell line HO8910 by IGFL 4.

FIG. 9 is a graph showing the results of experiments for determining the invasive capacity of tumor cells by Transwell method in the endometrial cancer cell line Ishikawa by IGFL 4.

FIG. 10 shows the determination of tumor cell apoptosis by flow cytometry in gastric cancer cell line MGC-803.

Fig. 11 shows apoptosis of IGFL4 in tumor cells as determined by flow cytometry on intestinal cancer cell line HCT 116.

Fig. 12 is an illustration of apoptosis of tumor cells as determined by flow cytometry in ovarian cancer cell line HO8910 by IGFL 4.

FIG. 13 is a flow cytometry determination of tumor cell apoptosis in the endometrial cancer cell line Ishikawa by IGFL 4.

Detailed Description

The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are given for the purpose of illustration of the invention, but are not intended to be limiting. The methods of operation not specifically described in the examples are all conventional in the art or are in accordance with the manufacturer's instructions.

Example the expression of the IGFL4 gene in malignant (including gastric, colorectal, endometrial and ovarian) cell lines and related methods for experimental detection of cell function.

Cell strain sources referred to in the examples: gastric cancer cell lines (MGC-803, SGC 7901), intestinal cancer cell lines (HCT 116, SW480, SW 620), endometrial cancer cell lines (H1C-1A, H C-1B, ishikuwa), ovarian cancer cell lines (A2780, CAOV3, OVCAR3, SKOV3, HO 8910) Guangzhou Ji Ni European Biotech Co.

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

1.1, extracting total RNA of tumor cells by using a Trizol reagent, adding chloroform with the volume of Trizol of 1/5, shaking vigorously for 15 seconds, and standing for 5 minutes at room temperature after the solution is fully emulsified; centrifuging at 4deg.C for 20 min at 12,000g, transferring supernatant to another new centrifuge tube, adding equal volume of isopropanol into supernatant, mixing completely with upside down centrifuge tube, and standing at 30deg.C for 10 min; centrifuging at 4deg.C for 20 min, discarding supernatant, adding lml 75% ethanol along the tube wall, washing the tube wall upside down, centrifuging at 4deg.C for 10 min at 12,000g, discarding ethanol, drying the precipitate at room temperature for 5-10 min, adding appropriate amount (10-20 ul) of RNase-free water to dissolve the precipitate, and measuring RNA concentration and purity (quantitative RNA concentration 1ug/ul, OD260/OD280 1.8-2.0 indicates higher RNA purity) with UV-2800 type UV-visible spectrophotometer.

1.2 reverse transcription to cDNA.

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

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

RNA ( 1µg/ul) 5μl。

Random Primers (0.5 µg /ul) 1 μl。

Oligo(dT)15 Primer (0.5 µg/ul) 1 μl。

Nuclear-Free Water (added to 10. Mu.l) 3. Mu.l.

And a second step of: a mixture of template RNA and reverse transcription Primer (Random Primers and Oligo (dT) 15 Primer) was subjected to pre-denaturation at 70℃for 5min, and after completion, the mixture was taken out and placed on ice.

And a third step of: preparation of RT-Mix, 10. Mu.l of each sample tube was added

。

Fourth step: setting reverse transcription program, including three steps of annealing, extending and inactivating reverse transcriptase (annealing at 25 deg.c for 5min, extending at 42 deg.c for 60 min, inactivating at 70 deg.c for 15min and 4 deg.c++ infinity.

1.3 Real-time PCR。

(1) The PCR reaction mixture was prepared as follows (the reaction mixture was prepared at room temperature), and divided into reaction tubes, and then 2ul of template was added

。

(2) The PCR standard amplification procedure was performed using an ABI PRISM [ 7500 Real-Time ] PCR System, two-step method.

(3) The data were exported and the real time PCR results were analyzed by the 2-DeltaCT method.

2. Preparation of specific siRNA and primer.

The siRNA used in this experiment was designed based on Rosetta siRNA Design Algorithm and was purchased from Sigma AldrichShanghai Trading co. The primers used in the experiment were synthesized by a solid phase phosphoramidite triester method and purchased from Beijing Liuhua macrogene technologies Co.

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

Collecting cells in log phase, adjusting cell suspension concentration, spreading 3000 cells per well in 96-well plate, wherein the volume of each well is 100 microliters, adding PBS solution into edge hole to prevent experimental group liquid from evaporating, transfecting cells with specific siRNA after cell adhesion, and measuring cell proliferation condition at 0h, 24h,48h and 72h after transfection. 20. Mu.l MTT solution was added to each well, the culture was continued for 4 hours, the medium was then dried by blotting, 150. Mu.l dimethyl sulfoxide was added to each well, and shaking was performed for 6 minutes, and absorbance was measured on an microplate reader. Data were recorded and cell viability curves were plotted.

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

Selecting cells with good growth state, adding into 6-well plate, culturing to 5×10 cells per well ⁵ The individual cells are preferably cultured normally overnight, the next day the wells are scored with the same 200. Mu.l gun head, then washed 2-3 times with PBS to remove the scored cells, transfected with specific siRNA, and cultured with 5ml serum-free medium. And observing the growth and migration conditions of the scratched cells under a microscope, taking photos at 0, 24 and 48 hours after transfection, selecting the same position after the photos, washing with PBS to remove dead cells before the photos, and finally measuring the bare area by using Image J software (National Institutes of Health, bethesda, MD, USA). Scratch healing rate = (original scratch area-area of actual scratch at different times)/original scratch area x% of original scratch.

5. Tumor cell invasion assay (Transwell experiments).

Firstly, diluting matrigel (1:10) with serum-free culture solution, evenly spreading 30-40 mu l of matrigel into a small chamber, and incubating at 37 ℃ for 4 hours. 5X 10. DELTA.4 cells were counted, plated, 200. Mu.l of cells diluted in serum-free medium in the upper chamber, 600. Mu.l of medium with normal 10% FBS in the lower chamber, and cells were transfected with specific siRNA. After further culturing for 48 hours, staining is carried out, the cell-containing chamber is washed 3 times with PBS, 4% paraformaldehyde is fixed at room temperature for 15min or stored at 4 ℃ for a long time, formaldehyde is discarded, the cell-containing chamber is washed 3 times with PBS for 5min each time, 0.1% crystal violet is stained for 15min, the cell-containing chamber is washed 3 times with PBS for 5min each time, matrigel in the upper chamber is wiped off with a cotton swab, a film is gently cut off by a blade, one side containing the cell faces upwards, after drying, the resin gel is sealed, photographing and counting are carried out under a normal microscope, and the invasion capacity of the cell is observed and measured.

6. Tumor apoptosis assay (flow cytometry detection).

Counting 3×10 ⁵ Cells were plated in 6-well plates and transfected after attachment of the cells. After 48h cells were digested with EDTA-free trypsin, the time of trypsin digestion should be appropriate to prevent false positives. 1500 After centrifugation for 5min, the cells were collected and washed twice with pre-chilled PBS. Cells were resuspended in 100. Mu.L of 1 Xbinding buffer and 5. Mu.L of Annexin V-FITC and 5. Mu.L of 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 detects the rate of apoptosis.

7. Experimental results.

As shown in FIG. 1, the expression level of the IGFL4 gene in 13 malignant tumor cell lines, wherein IGFL4 is highly expressed in 5 lines of HO8910, OVCAR3, HEC-1A, MGC-803 and SW620, and is lowly expressed in 4 lines of CAOV3, HEC-1B, SW and SGC 7901.

As shown in fig. 2, cell proliferation ability was significantly inhibited compared to the control group after knocking down the expression level of IGFL4 using specific siRNA in ovarian cancer cell line HO8910, endometrial cancer cell line Ishikawa, gastric cancer cell line MGC-803, and intestinal cancer cell line HCT 116.

As shown in fig. 3-5, cell migration was inhibited compared to the control group after knocking down the expression level of IGFL4 using specific siRNA in ovarian cancer cell line HO8910, endometrial cancer cell line Ishikawa, and intestinal cancer cell line HCT 116.

As shown in fig. 6-9, tumor cell invasion ability was significantly inhibited after knocking down the expression level of IGFL4 using specific siRNA in ovarian cancer cell line HO8910, endometrial cancer cell line Ishikawa, gastric cancer cell line MGC-803, and intestinal cancer cell line HCT 116.

As shown in fig. 10-13, apoptosis levels of tumor cells increased compared to control after knocking down IGFL4 expression levels using specific siRNA in ovarian cancer cell line HO8910, endometrial cancer cell line Ishikawa, gastric cancer cell line MGC-803, and intestinal cancer cell line HCT 116.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Guangzhou medical university affiliated third hospital (Guangzhou serious pregnant and lying-in woman treatment center, guangzhou soft-aid hospital)

<120> application of IGFL4 gene as marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer

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CATGCCTGGG ACCTTG 16

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GUGUCAUCCU AGACUUGAA 19

Claims

The application of an IGFL4 gene inhibitor in preparing a medicament for treating ovarian cancer is characterized in that the IGFL4 gene inhibitor is an siRNA sequence capable of inhibiting the expression of the IGFL4 gene, and the sequence is as follows: 5'-GUGUCAUCCUAGACUUGAA-3'.