CN111172283A - Application of detecting linc00673 expression quantity in esophageal cancer targeted therapy prognosis evaluation kit - Google Patents

Application of detecting linc00673 expression quantity in esophageal cancer targeted therapy prognosis evaluation kit Download PDF

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CN111172283A
CN111172283A CN202010100207.1A CN202010100207A CN111172283A CN 111172283 A CN111172283 A CN 111172283A CN 202010100207 A CN202010100207 A CN 202010100207A CN 111172283 A CN111172283 A CN 111172283A
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esophageal cancer
linc00673
detecting
expression
prognosis
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CN111172283B (en
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樊红
周梦晗
李懿萍
徐颖
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Southeast University
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Abstract

The invention relates to application of a reagent for detecting LINC00673 expression quantity in preparation of an esophageal cancer prognosis evaluation kit, belongs to the technical field of biology, and particularly relates to the field of medical diagnosis; the invention discloses application of a reagent for detecting LINC00673 expression quantity in preparation of an esophageal cancer prognosis evaluation kit; the prediction of esophageal cancer prognosis and the evaluation of esophageal cancer gene targeted therapy can be marked, and the expression level of the LINCRNA gene is detected by utilizing an RNA in-situ hybridization technology, so that the situations of the prediction of esophageal cancer prognosis and gene targeted therapy can be obtained, the introduction mode of the LINC00673 gene is simple, the whole prediction evaluation is simple and easy to implement, and the feasibility is high.

Description

Application of detecting linc00673 expression quantity in esophageal cancer targeted therapy prognosis evaluation kit
Technical Field
The invention relates to application of detecting the expression quantity of linc00673 in a kit for evaluating prognosis of esophageal cancer targeted therapy, belongs to the field of biotechnology, and particularly relates to a clinical detection technology.
Background
Esophageal cancer is a (esophageal cancer) common malignant tumor in human, accounts for more than 90% of esophageal tumors, and is second only to gastric cancer in the review survey of all malignant tumor deaths, and is the 2 nd place. It is estimated that about 20 million people die of esophageal cancer every year worldwide, one of the most common malignancies with great harm to people's life and health. Esophageal cancer is a common tumor of the digestive tract, and about 30 million people die of esophageal cancer every year worldwide. The morbidity and mortality varies greatly from country to country. China is one of the high-incidence areas of esophageal cancer in the world, and the average death rate of people is about 15 ten thousand every year. In Chinese esophageal cancer, squamous carcinoma accounts for almost 95 percent, and the incidence rate of primary esophageal adenocarcinoma is very low.
The death rate of esophageal cancer has a rising trend in recent 20 years, which shows that the high incidence of esophageal cancer in China is very severe, but no better targeted drug exists so far, which causes poor treatment effect and poor prognosis of patients. It is important to find biomarkers closely related to the prognosis of esophageal cancer.
There is increasing evidence that long non-coding RNAs (lincRNAs) are closely related to the development and therapeutic effects of tumors. lincRNAs are involved in a wide range of cellular processes including regulation of epigenetic markers and gene expression, and maintenance and differentiation of pluripotency of embryonic stem cells. LINC00673 is a potential tumor suppressor, and our studies find that its high level expression in esophageal cancer tissues is closely related to poor prognosis of patients. The siRNA interference sequences of 2 LINC00673 are designed to knock down the expression level of the esophageal cancer LINC00673, and the result shows that the expression level of LINC00673 is reduced, the growth of esophageal cancer tumor cells is obviously inhibited, and the molecule is expected to become an evaluation marker for poor prognosis of the esophageal cancer and a target point of targeted therapy.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a reliable prognosis evaluation kit aiming at esophageal cancer, so that the prognosis condition of a patient with esophageal cancer can be monitored, and the patient can see a doctor in time; meanwhile, an individual treatment scheme is designated by using a prognosis evaluation result, and chemotherapy drugs are reasonably applied, so that ineffective chemotherapy is avoided, and the life cycle of a patient is effectively prolonged. And simultaneously, selecting molecules which are expected to become therapeutic targets.
In order to realize the aim, the application of a reagent for detecting the expression quantity of the linc00673 in preparing an esophageal cancer prognosis evaluation kit and the application of a linc00673 interference sequence in targeted therapy of esophageal cancer are disclosed.
The technical scheme is as follows:
the survival period of the esophageal cancer patient is related to the expression of linc00673, and the clinical esophageal cancer chemotherapy can be guided by detecting the expression level of the gene of the patient, so that the curative effect is improved. Therefore, the linc00673 gene test can be applied to esophageal cancer prognosis judgment and chemotherapy drug resistance evaluation. So that the prognosis of esophageal cancer can be accurately predicted, and an optimal individual treatment can be effectively established based on the predicted prognosis to significantly reduce death caused by esophageal cancer.
The detection kit is used for prognosis judgment of esophageal cancer through the following steps: (a) detecting the expression level of linc00673 in a biological sample, (b) classifying the result into high expression and low expression (or non-expression) according to the expression level of linc00673 detected in (a), (1) a linc00673 high expression patient: the expression level of the patient esophageal cancer tissue linc00673 is high, and the prognosis is poorer than that of a patient with low expression. (2) linc00673 no/low expression patients: the expression level of the esophageal cancer tissue linc00673 of the patient is low, and the prognosis is higher and the expression of the patient is good. Patients need to be followed up closely and the corresponding tumor indications are reviewed in time.
As used herein, low expression means negative, false positive and weak positive, and high expression means positive and strong positive.
The invention discloses 2 siRNA sequences for inhibiting the expression of linc00673 and an inhibition effect on the growth of esophageal cancer cells.
The sequence is an interference RNA sequence, is used for inhibiting the expression of non-coding RNA LINC00673 (LINC 00673) in tumor cells, and belongs to the RNA sequence
SiRNA and shRNAs Sequences 5’-3’
shLINC00673-1 CAGCCGGAUACAGAGUGAAUAGUUA
shLINC00673-2 UGUGCCUUUGUACUCAGCAAUUCUU
Has the advantages that:
as the expression level of the linc00673 gene of the esophageal cancer patient is related to the pathological state and prognosis of the patient, particularly the high expression of the linc00673 gene in the esophageal cancer can be used as an independent risk factor for poor prognosis of the esophageal cancer. Can guide the clinical formulation of individualized chemotherapy by detecting the expression level of the gene of the patient, thereby improving the curative effect.
By adopting the technical scheme disclosed by the invention, the expression level of linc00673 can be knocked down by predicting the prognosis of esophageal cancer and utilizing an siRNA interference experiment, so that the prognosis of esophageal cancer is predicted, the knocking-down mode of linc00673 is simple, the whole prediction and evaluation is simple and easy to implement, and the feasibility is high.
Drawings
FIG. 1 shows that the prognosis of a patient with LINC00673 high-expression esophageal cancer is poor;
FIG. 2 shows the knockdown efficiency of two siRNA interfering sequences;
FIG. 3 is an in vitro experiment: the proliferation capacity of esophageal cancer cells is reduced after the LINC00673 is knocked down;
FIG. 4 is an in vivo experiment: the growth of mouse esophageal cancer cell transplantation tumor is slowed down after the knockout of LINC 00673.
Detailed Description
For a better understanding of the present invention, the present invention will be further illustrated with reference to the following specific examples and drawings, but it should be noted that the present invention is not limited thereto.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. The test methods under specific conditions not specified in the examples of the present invention were carried out under the conventional conditions or conditions recommended by the manufacturers.
Example 1: relationship between linc00673 expression level and esophageal cancer prognosis
1) And (3) carrying out RNA in-situ hybridization on the expression quantity of the linc00673 in the tissues of the esophageal cancer patients, wherein negative, false positive and weak positive are low expression groups, positive and strong positive are high expression groups, and analyzing the correlation between the low expression and high expression groups of the linc00673 and prognosis according to Kaplan-Meier (KM), wherein the result is shown in figure 1. FIG. 1 shows the prognostic relevance of relative expression of LINC00673 in tumor tissue of esophageal cancer patients, and the survival period of the patients with cancer tissues under-expressing LINC 00673.
2) The detection of the transcription level of linc00673 was performed by means of real-time quantitative PCR.
The results show that linc00673 has obvious correlation with prognosis, namely the stronger the expression of linc00673 is, the poorer the prognosis of the patient is; conversely, the lower the linc00673 expression, the better the patient prognosis will be.
Example 2: experimental method for knocking down linc00673 by cell transfection
1. Adherent cells were planted 1 day ahead of time: cells were plated in 24-well plates one day in advance, preferably with a confluency of cells at transfection (Confluence) of about 30%, and a total volume of whole medium before transfection of 0.45 ml.
2. 0.67mg (50 pmol) of siRNA was added to a serum-free diluent and mixed well to prepare an RNA diluent of 25 ml in final volume.
3. 1ml of Entranster-R4000 was taken, and 24 ml of serum-free diluent was added thereto, followed by mixing well to prepare a final volume of 25 ml of Entranster-R4000 diluent. The mixture was allowed to stand at room temperature for 5 minutes.
4. The Entranster-R4000 dilution and the RNA dilution were mixed well (shaking with a shaker or pipetting with a sample applicator 10 times or more) and allowed to stand at room temperature for 15 minutes. The transfection complex preparation is complete.
5. 50 ml of the transfection complex was added dropwise to cells containing 0.45 ml of whole medium (which may contain 10% serum and antibiotics), and the plates were moved back and forth and mixed well.
6. And (3) observing the cell state 6 hours after transfection, if the cell state is good, continuously culturing for 24-96 hours without changing a culture medium, and detecting the expression level of the cell linc 00673.
The detection results are as follows:
FIG. 2 shows the knockdown efficiency of two siRNA interfering sequences; upon knock-down of LIN00673 by two siRNA interfering sequences, LIN00673 expression levels were significantly reduced. (paired t-test, double tail, P <0.05, P <0.01, because P < 0.001).
FIG. 3 is an in vitro experiment: the cell proliferation capacity is reduced after the knockdown of LINC 00673. The CCK-8 cell proliferation detection kit is used for detecting the light absorption value of the cell by using an enzyme-linked immunosorbent assay detector at the wavelength of 450 nm according to the operation of instructions, and the quantity of living cells can be indirectly reflected. The lower the OD, the lower the cell proliferation potency. (paired t-test, two tails, P <0.05, P < 0.01).
FIG. 4 is an in vivo experiment: the growth of the esophageal cancer cell transplantation tumor after the LINC00673 is knocked down is reduced. P <0.05, P < 0.01) by log rank test.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.
Sequence listing
<110> university of southeast
<120> application of detecting linc00673 expression quantity in esophageal cancer targeted therapy prognosis evaluation kit
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>25
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
cagccggaua cagagugaau aguua 25
<210>2
<211>25
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ugugccuuug uacucagcaa uucuu 25

Claims (5)

1. Application of a reagent for detecting the expression quantity of linc00673 in preparation of a kit for evaluating prognosis of targeted therapy of esophageal cancer.
2. Use according to claim 1, characterized in that: and detecting the linc00673 by an RNA in-situ hybridization method.
3. Use according to claim 1, characterized in that: the detection reagent consists of a digoxin detection reagent and a linc00673 detection probe.
4. Use according to claim 1, characterized in that: the kit is a CCK-8 cell proliferation detection kit.
5. Use according to claim 2 or 3 or 4, characterized in that: the kit comprises a reagent for detecting the expression quantity of linc00673 in a biological sample, wherein the biological sample is an esophageal cancer operation tissue specimen and an esophageal cancer tissue fixed by formalin and/or embedded by paraffin.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113607946A (en) * 2021-08-06 2021-11-05 东南大学 Application of detecting transcription factor MAX gene expression level in liver cancer c-MYC targeted therapy prognosis evaluation kit
CN115992231A (en) * 2022-08-02 2023-04-21 内蒙古大学 Reagent and prevention and treatment medicine for early diagnosis of melanoma
CN116676385A (en) * 2023-02-22 2023-09-01 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Application of LINC02096 in serving as marker for predicting esophageal cancer immunotherapy effect and prognosis and treatment target

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113607946A (en) * 2021-08-06 2021-11-05 东南大学 Application of detecting transcription factor MAX gene expression level in liver cancer c-MYC targeted therapy prognosis evaluation kit
CN113607946B (en) * 2021-08-06 2023-12-08 东南大学 Application of detecting transcription factor MAX gene expression quantity in liver cancer c-MYC targeted therapy prognosis evaluation kit
CN115992231A (en) * 2022-08-02 2023-04-21 内蒙古大学 Reagent and prevention and treatment medicine for early diagnosis of melanoma
CN116676385A (en) * 2023-02-22 2023-09-01 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Application of LINC02096 in serving as marker for predicting esophageal cancer immunotherapy effect and prognosis and treatment target
CN116676385B (en) * 2023-02-22 2023-12-08 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Application of LINC02096 in serving as marker for predicting esophageal cancer immunotherapy effect and prognosis and treatment target

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