CN110628909B - miRNA molecule miR-4500 related to endometrial cancer and application thereof - Google Patents
miRNA molecule miR-4500 related to endometrial cancer and application thereof Download PDFInfo
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Abstract
The invention provides a miRNA molecule miR-4500 related to endometrial cancer and application thereof, belonging to the field of biological gene therapy. The expression rate of the miRNA molecule miR-4500 in endometrial cancer tissues is obviously lower than that of normal endometrial tissues, and the miRNA molecule miR-4500 can be used for preparing an endometrial cancer auxiliary diagnosis or endometrial cancer auxiliary treatment/prognosis evaluation reagent or kit.
Description
Technical Field
The invention belongs to the field of molecular biology and oncology, and particularly relates to a miRNA molecule miR-4500 related to endometrial cancer and application thereof.
Background
Endometrial cancer is a malignant tumor that occurs on the endometrium and originates from the epithelium, most commonly adenocarcinoma that originates from the endometrial glands, is one of three common malignant tumors of the female reproductive tract, and in recent years the incidence has a tendency to rise worldwide and to become younger. Endometrial cancer is currently believed to be of two types. Type I is estrogen-dependent and may occur as a result of endometrial hyperplasia (which may or may not be simple or complex, with or without atypical hyperplasia) which progresses to cancer with the long-term estrogen deprivation of progestogen antagonism. Type II is non-estrogen dependent, and the onset of this type is not clearly related to the level of estrogen. Early diagnosis of endometrial cancer, assessment of patient prognosis, and treatment of patients with advanced or recurrent endometrial cancer remain significant problems.
Micrornas (mirnas) are a class of endogenous small non-coding mature forms of RNA of about 18-22 nucleotides in length that can regulate a set of target genes and cause mRNA degradation or translational inhibition. miRNAs play an important role in the development and progression of cancer, for example, in various biological processes such as cell growth and differentiation, apoptosis, proliferation, embryonic development, and stem cell renewal. miRNAs can play a role in inhibiting and promoting tumors, and the expression imbalance of some miRNAs in endometrial cancer is reported in documents, so that the miRNAs play an important role in the occurrence and development of the endometrial cancer, for example, miR-29c, miR-200b, miR-873, miR-141 and miR-15 are abnormally expressed in the endometrial cancer. However, no report is provided about the expression of miR-4500 in endometrial cancer tissues.
Disclosure of Invention
In order to solve the problems, the invention provides miRNA molecule miR-4500 related to endometrial cancer and application thereof. The miRNA molecule miR-4500 can be used as a diagnosis marker of endometrial cancer, provides a molecular target for treatment of the endometrial cancer, and the mimics designed aiming at the miRNA molecule can be applied to preparation of antitumor drugs.
In order to achieve the aim, the invention provides a miRNA molecule miR-4500 related to endometrial cancer, and the nucleotide sequence of the miRNA molecule miR-4500 is shown in SEQ ID NO. 1.
The miRNA molecule miR-4500 can be applied to auxiliary diagnosis of endometrial cancer or treatment/prognosis evaluation of endometrial cancer.
miRNA molecule miR-4500 in preparation of endometrial cancer auxiliary diagnosis or endometrial cancer auxiliary treatment/prognosis evaluation
The reagent or the kit.
Application of a reagent for detecting miRNA molecule miR-4500 in preparation of an endometrial cancer auxiliary diagnosis or endometrial cancer treatment/prognosis evaluation reagent or kit.
The reagent for detecting the miRNA molecule miR-4500 comprises a specific primer for amplifying the miRNA molecule miR-4500, and the primer sequence is as follows:
F：5’-CGG CGG TGA GGT AGT AGT T-3’(SEQ ID NO .2)。
application of a kit for detecting miRNA molecules miR-4500 related to endometrial cancer in preparation of endometrial cancer auxiliary detection or auxiliary diagnosis tools.
Application of a kit for detecting miRNA molecule miR-4500 related to endometrial cancer in preparation of endometrial cancer treatment/prognosis evaluation tools.
A kit for the aided diagnosis or prognostic evaluation of endometrial cancer, comprising:
(1) A specific primer for amplifying miR-4500;
(2) A standard DNA template;
(3) And (3) PCR reaction liquid.
A method of detecting a miRN, comprising the steps of:
(1) Extracting total RNA of a sample;
(2) Preparing sample cDNA;
(3) And (4) carrying out quantitative amplification on the miR-4500.
The mimics of which overexpresses miR-4500 is designed by Suzhou Jima Gene GmbH. After a specific sequence is obtained, designing a mimics sequence for transfection; when the fragment is adopted to over-express the endometrial cancer cell line HEC-1A, the migration and invasion capacity of tumor cells is found to be reduced. The function of this molecule may therefore be related to the migratory invasive capacity of cancer cells.
The miRNA molecule with low expression in endometrial cancer provided by the invention can be used as a endometrial cancer molecular marker or target, is applied to clinical early diagnosis, prognosis judgment or targeted therapy of endometrial cancer, is beneficial to further elucidating the occurrence and development mechanism, signal path and the like of endometrial cancer, and has great application prospect and theoretical value.
Drawings
FIG. 1 is a dissolution curve and diffusion curve diagram corresponding to miR-4500 in real-time-PCR, wherein FIG. 1-1 is a corresponding dissolution curve, and FIG. 1-2 is a corresponding amplification curve diagram.
FIG. 2 is a graph showing the relative expression amount of miR-4500 in real-time PCR in cancer group and non-cancer group.
FIG. 3 is a ROC curve analysis plot of miR-4500 in endometrial cancer tissue.
FIG. 4 is a graph of the transfection efficiency of the PCR detection of the mimics miR-4500 in the HEC-1A cells.
FIG. 5 is a graph of the effect of miR-4500 overexpression on endometrial cancer cell migration detected by a scratch assay.
FIG. 6 is a graph of the effect of miR-4500 overexpression on endometrial cancer cell invasion detected using the transwell assay.
Detailed Description
The present invention is further illustrated by the following examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The skilled person will appreciate that appropriate modifications and adaptations of the primer sequences of the invention may be made to the sequences of the relevant markers, and that such modified primer sequences may still be used to detect the markers. The present invention also includes such equivalent solutions.
Example 1 expression of miR-4500 in endometrial cancer and normal endometrial tissue.
1. And (6) collecting the specimen.
Sample collection Normal endometrial tissue surgically removed during the period of 2016 to 2019 in the tumor Hospital, liaoning, university of medical sciences, of endometrial carcinoma and cervical intraepithelial neoplasia grade III (CIN III) of China. The group was divided into endometrial cancer group (n = 41) and normal control group (n = 22). The patients do not receive radiotherapy, chemotherapy and hormone treatment before operation and have been pathologically diagnosed. Clinical staging and pathological staging of endometrial cancer group surgery (fig 2009): early stage: stage i plus stage ii 29, late: 12 cases in stage III and IV; histological grading (WHO standard): high-differentiation endometrial cancer 17 cases, medium-differentiation endometrial cancer 14 cases, and low-differentiation endometrial cancer 10 cases; lymph node metastasis positive 34 cases, no lymph node metastasis 7 cases; 33 cases of the infiltrated muscle layer are less than 1/2, and 8 cases of the infiltrated muscle layer are more than or equal to 1/2. The study was approved by the ethical committee of the tumor hospital, liaoning province, affiliated to the university of medical science, china.
2. And (3) designing a primer.
And designing a primer according to the sequence of hsa-miR-4500. The specific sequence is as follows: reverse transcription primer sequence of hsa-miR-4500: f:5 'CGG CGG TGA GGT AGT AGT T-3'; (SEQ ID No. 2)
Forward primer sequence of U6 SEQ ID No.3: GGA ACG ATA CAG AGA AGA TTA GC;
reverse primer sequence of U6 SEQ ID No.4: TGG AAC GCT TCA CGA ATT TGC G.
The above primers were synthesized by Shanghai Biotechnology Ltd, wherein U6 is an internal reference calibration gene.
3. And detecting the expression quantity of the miR-4500 in endometrial cancer tissues and normal endometrial tissues by using a Real-time-PCR method.
3.1, extracting the total RNA of the collected tissue sample.
(1) The endometrial cancer samples (1 ml of trizol lysate had been added) were allowed to stand on ice for 5min.
(2) Adding 200 μ l chloroform, shaking, mixing, and standing at room temperature for 2-3min. Centrifuge at 12,000g for 15min at 4 ℃.
(3) The upper aqueous phase was aspirated into another centrifuge tube. Note: tens of millions do not draw in intermediate interfaces.
(4) Adding equal amount of isopropanol, mixing, and precipitating at-20 deg.C.
(5) Centrifugation is carried out for 10min at 12,000g at 4 ℃, the supernatant is discarded, and RNA is deposited on the bottom of the tube.
(6) 1ml of 75% ethanol was added and the precipitate was suspended.
(7) Centrifuge at 12000g for 5min at 4 deg.C, and discard the supernatant as much as possible.
(8) Air drying at room temperature or vacuum drying for 5-10min. Note: the RNA sample is not dried too much, otherwise it is difficult to dissolve.
(9) With 20. Mu.l DEPC H 2 O lysis of RNA samples.
(10) Determination of RNA purity and quantitation of RNA with DEPC H 2 O is used as a control (Blank), and 2 μ l of RNA solution is taken to detect the concentration and the quality of the sample on a microplate reader.
3.2, reverse transcription synthesis.
(1) And (4) performing reverse transcription reaction.
First, miRNA was subjected to reverse transcription and pre-denaturation to prepare a pre-denaturation reaction solution, and the preparation method is shown in table 1.
TABLE 1 miRNA Pre-denaturation reaction solution
1H at 37 ℃ and 5min at 85 ℃ and then 90ul DEPC H was added to each tube 2 O to a total volume of 100ul, and storing at-20 ℃.
3.3、PCR。
Fluorescent quantitative PCR amplification, as in table 2.
TABLE 2 PCR reaction System
Wherein, the Forward primer is a Forward primer aiming at hsa-miR-4500, and the nucleotide sequence is shown as SEQ2.
Wherein, the nucleotide sequence of the forward primer of the internal reference U6 is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 4.
And (5) performing statistical analysis.
All results are presented as mean ± SEM values and tabulated. Statistical data analysis Software used was SPSS22.0, and mapping Software was Graphpad prism 5 (Graphpad Software, san Diego, calif.), P <0.05
And P <0.01 is the screening criteria for significant and very significant differences.
And (6) analyzing the result.
And (6) analyzing the data.
Quantitative PCR data were processed with 7500 Fast Software v2.3 Software configured with an amplificator to establish a threshold and baseline for each gene, and a lytic amplification curve was generated by the Software, as shown in FIG. 1, indicating good amplification results. And obtaining a CT value corresponding to each sample reaction. In order to ensure the reliability of the result data, the RNA quality of the sample with the CT value of the endogenous control gene being more than 30 is considered to be low, and the RNA is excluded from the analysis, and the relative expression quantity in the sample is 2 -ΔΔct The algorithm of (1).
Expression levels in endometrial cancer tissues and normal endometrial tissues using the Real-time PCR method, as shown in the results in fig. 2, the results indicate that miR-4500 is significantly lower in endometrial cancer tissues than in normal endometrium (P < 0.05), and the sensitivity and specificity of miR-4500 are examined by ROC curve analysis. And the relevant region under the curve (AUC) was used to confirm the diagnostic efficacy of miR-4500. As shown in FIG. 3, the AUC of miR-4500 reached 0.9557, (95% CI. These results indicate that miR-4500 can distinguish endometrial cancer and normal endometrial tissue.
The relationship between the expression level of miR-4500 in endometrial cancer and clinical pathological factors is shown in Table 3, wherein 63 patients in the group are divided into groups according to age, clinical stage, differentiation degree, infiltration depth and lymph node metastasis. The results show that the miR-4500 expression of the early-stage intima cancer (stages I-II) is obviously higher than that of the late-stage intima cancer (stages III-IV), and the differences have statistical significance (P is less than 0.05). The clinical pathological analysis result shows that the expression of miR-4500 is related to the clinical stage of the patient with endometrial cancer.
TABLE 3 relation of miR-4500 expression in endometrial cancer tissue and clinical pathological parameters thereof
Note:P=0.2036, High differentiationvs.Middle differentiation；P=0.3770, High differentiationvs.Low differentiation。
example 2 in vitro cell experiments.
HEC-1A human endometrial cancer differentiated cell lines were purchased from Shanghai cell bank of Chinese academy of sciences, respectively.
A cell culture medium McCoy'5A and fetal calf serum are purchased from Gibico company, overexpression mix synthesis is purchased from Gilmar technology, inc. Suzhou, 6-hole culture plates are used for culturing HEC-1A cells, transfection is carried out when the cells grow to about 80%, overexpression miR-4500 is mix-4500, miR-NC is a control group, HEC-1A cells are a blank control group, and lip3000 is used as a transfection reagent, and transfection is carried out. After transfection for 48h, cell RNA was extracted, RNA extraction reagent RNAioso Plus was purchased from Takara Bio Inc., and reverse transcription quantitative kit was purchased from Takara Bio Inc. The transfection efficiency was examined. As shown in FIG. 4, the results show that miR-4500 is successfully overexpressed in HEC-1A cells.
2. The migration ability of the cells is detected by a scratch test, the cell culture state in a 6-well plate is good, no pollution is caused, the cell culture is performed until the fusion degree is ninety percent, a middle gun head is used for drawing a straight line with uniform force, the cells are continuously cultured by a serum-free culture medium, and during the period, pictures of scratch healing are taken every 12h,24h,48 and 60h under a microscope to detect the migration ability of the cells. The results show that compared with the miR-NC group, the HEC-1A cell migration capacity of the miR-4500 overexpression group is remarkably reduced (figure 5), and the fact that the migration capacity of endometrial cancer cells can be inhibited by overexpression of miR-4500 is shown.
3. The matrigel of the kit was diluted as indicated, 20. Mu.L of diluted matrigel was spread on the chamber, the chamber was placed on a 24-well plate, and 700. Mu.L of complete medium was added under the chamber. Observing the cell state under a microscope, digesting the liquid cells with 0.25 percent pancreatin, putting the mixed solution into a centrifuge tube, and centrifuging for 5 minutes at 1000 rpm; remove supernatant, use serum-free culture solution to wash cells, under the microscope cell count, the transwell chamber upper chamber into 200 u L cell suspension, cell number about 5 x 10 4 And (4) cells. The cells are put through an incubation incubator for 16 hours, then the transwell chamber is stopped to be taken out, the cells are lightly washed for 2 times by PBS, 4% paraformaldehyde is used for fixing the cells, 700ul of cell fixing solution is added into each hole, 60min is fixed in the fixing solution, the chamber is washed by PBS, the chamber is dried in the air, hematoxylin is used for staining for 50min, the chamber is placed into the staining chamber, then the chamber is washed for 3 times by PBS, the cells are observed under a 20-time inverted mirror, 3 visual fields are randomly selected, the number of the cells is counted, and the invasion capacity of the cells is detected. the transwell results show that compared with the control group, the invasion capacity of HEC-1A cells of the miR-4500 overexpression group is remarkably reduced, and the fact that the invasion capacity of endometrial cancer cells can be inhibited by overexpression of miR-4500 is shown (figure 6).
While the preferred embodiments of the present invention have been illustrated and described in detail, it should be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings without inventive faculty. Therefore, any technical solutions that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the present inventive concept should be within the scope of protection defined by the present claims.
<110> Liaoning province tumor hospital
<120> miRNA molecule miR-4500 related to endometrial cancer and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> human (Homo sapiens)
<400> 1
GUGCAUUGUA GUUGCAUUGC A 21
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<400> 2
CGG CGG TGA GGT AGT AGT T 19
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
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GGAACGATACAGAGAAGATTAGC 23
<210> 4
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<212> DNA
<213> Artificial sequence
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TGGAACGCTTCACGAATTTGCG 22
Claims (3)
1. Application of a reagent for detecting miRNA molecule miR-4500 in preparation of an endometrial cancer auxiliary diagnosis or endometrial cancer treatment/prognosis evaluation reagent or kit.
2. Application of a kit for detecting miRNA molecule miR-4500 related to endometrial cancer in preparation of endometrial cancer auxiliary detection or auxiliary diagnosis tools.
3. Application of a kit for detecting miRNA (micro ribonucleic acid) molecule miR-4500 related to endometrial cancer in preparation of endometrial cancer treatment/prognosis evaluation tools.
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| CN110205386A (en) * | 2019-07-11 | 2019-09-06 | 中国医科大学附属盛京医院 | One kind miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its application |
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| Title |
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| Down-regulation of miR-4500 promoted non-small cell lung cancer growth;Lei Zhang等;《Cellular Physiology and Biochemistry》;20140922;第34卷(第4期);1166-1174 * |
| microRNA-4500 inhibits human glioma cell progression by targeting IGF2BP1;Zheng-Wei Li等;《Biochemical and Biophysical Research Communications》;20190416;第513卷(第4期);800-806 * |
| miRNAs对293T细胞IL-10表达调控的研究;郭东更等;《宁夏医科大学学报》;20180930;第40卷(第09期);997-1000、1005 * |
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