CN113186289A - Application of lncRNA in kidney cancer urine screening and kidney cancer treatment - Google Patents
Application of lncRNA in kidney cancer urine screening and kidney cancer treatment Download PDFInfo
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Abstract
The invention discloses application of lncRNA in kidney cancer urine screening and kidney cancer treatment. The invention specifically discloses an application of a reagent and an apparatus for detecting an lncRNA molecular marker LINC00158 in preparation of a product for screening or diagnosing tumors, and an application of a reagent for inhibiting or reducing LINC00158 gene expression in preparation of a medicine for treating or preventing tumors. The lncRNA LINC00158 is used as a molecular marker for screening kidney cancer by urine, and can accurately predict, screen and diagnose kidney cancer. The method has high screening efficiency and guaranteed reliability, can realize rapid screening of the kidney cancer, can be used for judging the kidney cancer at the early stage of occurrence of the kidney cancer, provides a powerful molecular biology tool for further developing medicines and methods for treatment, diagnosis and prognosis of the kidney cancer, and has profound clinical significance and important popularization and application prospects.
Description
Technical Field
The invention relates to the field of biomedicine, and particularly relates to application of lncRNA in kidney cancer urine screening and kidney cancer treatment.
Background
Renal cancer, also known as Renal Cell Carcinoma (RCC), is one of the most common malignancies in the urinary system. Renal cancer can be classified into clear cell carcinoma of the kidney, papillary renal cell carcinoma, chromophobe cell carcinoma, and the like according to the histopathology. Of these, renal clear cell carcinoma is the most common type. The current screening and diagnosis methods of kidney cancer include ultrasound, computed tomography, magnetic resonance imaging, tissue biopsy, and the like. It is still not certain from imaging techniques that histological examination by renal biopsy is required. But kidney tissue biopsy is invasive. Therefore, the development of a simple, effective and non-invasive renal cancer screening method is of great significance.
Liquid Biopsy (Liquid Biopsy) refers to the analysis and diagnosis of diseases such as cancer by collecting and detecting biomarkers from non-solid biological tissues such as blood, urine, saliva, etc., and has the advantages of non-invasiveness, simple operation, continuous sampling, low risk, low cost, etc., and has extremely wide clinical application prospect.
Long non-coding RNAs (lncRNAs) are a class of RNA that do not code for proteins greater than 200 nucleotides in length. lncRNA is closely associated with many diseases, especially tumors, is expressed in a variety of cancers, and can regulate gene expression in cancers through a variety of pathways, including transcriptional, post-transcriptional, and translational regulation, among others. In addition, several studies have shown that lncRNA has shown potential as a molecular marker in cancer screening for bladder, prostate, stomach, pancreas, breast, and the like.
Disclosure of Invention
The technical problem to be solved by the invention is how to utilize liquid biopsy to screen kidney cancer at early stage and/or assist in diagnosing kidney cancer, and/or how to inhibit proliferation and migration of kidney cancer cells and/or apply the method to treatment of kidney cancer.
In order to solve the technical problems, the invention firstly provides application of a reagent and/or an apparatus for detecting LINC00158 in preparation of products for screening, auxiliary screening and/or auxiliary diagnosis of kidney cancer.
The LINC00158 is long non-coding RNA (lncRNA) related to kidney cancer, namely lncRNA LINC 00158. The gene for coding lncRNA LINC00158 is a LINC00158 gene, and the nucleotide sequence of the LINC00158 gene is shown in SEQ ID No. 1.
The reagent and/or apparatus for detecting LINC00158 can be a reagent and/or apparatus for detecting LINC00158 gene expression.
In the above application, the reagent and/or apparatus for detecting LINC00158 comprises a reagent and/or apparatus for detecting LINC00158 by reverse transcription-polymerase chain reaction (RT-PCR), real-time fluorescent quantitative PCR (RT-qPCR), transcriptome sequencing (RNA-seq), Northern blot, In Situ Hybridization (ISH), gene chip technology, Nanopore sequencing technology or PacBio sequencing technology.
In the above application, the product for screening, auxiliary screening and/or auxiliary diagnosis of kidney cancer includes a kit, a gene chip, a high-throughput sequencing platform or a biosensor.
In the application, the kit comprises a reagent for detecting the LINC00158 gene;
the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting LINC00158 genes;
the high-throughput sequencing platform comprises reagents for detecting the LINC00158 gene;
the biosensor includes a reagent for detecting the LINC00158 gene.
In the application, the reagent for detecting the LINC00158 can be a reagent for detecting the expression level of the LINC00158 gene.
In the application, the reagent for detecting the expression level of the LINC00158 gene comprises a primer for specifically amplifying the LINC00158 gene and/or a probe for specifically recognizing the LINC00158 gene.
The probe may be a DNA probe, an RNA probe, a cDNA probe, a cRNA probe, or an oligonucleotide probe.
The reagent for detecting the expression level of the LINC00158 gene can comprise a primer pair for amplifying the LINC00158 gene.
The primer pair for amplifying the LINC00158 gene consists of a forward primer and a reverse primer, wherein the sequences of the forward primer and the reverse primer are respectively as follows:
a forward primer LINC 00158-QF: 5'-TTAAGAAAGCGGTCGTCGCC-3' (SEQ ID No.4)
Reverse primer LINC 00158-QR: 5'-TCTGCCACCTTGTGGTTGTT-3' (SEQ ID No.5)
The test sample of the product may be a body fluid such as urine, blood or saliva.
Further, the detection sample of the product is urine.
The invention also provides application of the agent for inhibiting or reducing the expression of the LINC00158 gene in preparing a medicament for treating or preventing renal cancer.
The medicament for treating or preventing kidney cancer inhibits proliferation and/or migration of kidney cancer cells.
Further, the agent that inhibits or reduces the expression of LINC00158 gene comprises a nucleic acid molecule, or a recombinant microorganism that expresses the nucleic acid molecule.
The nucleic acid molecule is selected from: antisense oligonucleotides, small interfering RNAs (siRNAs), microRNAs (miRNAs), or short hairpin RNAs (shRNAs). The antisense oligonucleotide refers to a nucleic acid fragment which can be complementary with a target gene (LINC00158 gene) and can be combined with the target gene through a base complementary principle so as to inhibit, block or reduce the expression of the LINC00158 gene, and the antisense oligonucleotide can be antisense DNA or antisense RNA, can be artificially synthesized or expressed in vivo through a method known by a person skilled in the art; the small interfering RNA (siRNA), micro RNA (miRNA) or short hairpin RNA (shRNA) refers to RNA that inhibits or reduces the expression of the LINC00158 gene through RNA interference (RNAi), and can be prepared by a method known by a person skilled in the art.
In the above application, the recombinant microorganism may be a recombinant lentivirus expressing the shRNA or siRNA.
In the above application, the agent for inhibiting or reducing the expression of the LINC00158 gene may be a nucleic acid molecule.
The nucleic acid molecule can be short hairpin RNA (shRNA), and the target sequence of the shRNA can be a sequence shown in SEQ ID No.2 or SEQ ID No. 3.
The present invention also provides a medicament for treating or preventing renal cancer, which contains the agent that inhibits or reduces the expression of LINC00158 gene.
The present invention also provides an apparatus for screening and/or diagnosing kidney cancer, which includes the reagent for detecting LINC00158 described herein and a computer-readable storage medium storing a computer program for causing a computer to execute the step of screening and/or diagnosing kidney cancer disease according to the expression amount of LINC00158 gene in a specimen.
Herein, the sample is not particularly limited as long as it is suitable for the detection of the present invention, and for example, the sample may be blood, urine, saliva, or the like.
The invention discovers lncRNA LINC00158 related to kidney cancer, and the extensive and intensive research shows that the lncRNA LINC00158 can be used as a molecular marker for screening the kidney cancer by urine, so that the kidney cancer can be accurately predicted, screened and diagnosed. The method has high screening efficiency and guaranteed reliability, can realize the rapid screening of the kidney cancer, and can be used for judging the kidney cancer in the early stage of occurrence of the kidney cancer. The molecular marker LINC00158 can be used for preparing a reagent or a kit for screening kidney cancer and preparing a medicine for treating the kidney cancer. The potential of LINC00158 in kidney cancer urine screening and the influence on the occurrence and development of kidney cancer are researched, the screening and treatment of kidney cancer are of great significance, a powerful molecular biological tool is provided for further developing medicines and methods for treating and diagnosing kidney cancer, and the method has profound clinical significance and important popularization and application prospects.
Drawings
FIG. 1 shows the expression of LINC00158 gene in renal cancer tissue and renal cancer cells.
FIG. 2 is a graph showing the real-time fluorescent quantitative PCR detection of the expression level of LINC00158 gene in urine of renal cancer patients and healthy controls, respectively, and the corresponding working curves of subjects.
FIG. 3 shows the real-time fluorescent quantitative PCR method for detecting the expression level of LINC00158 after transfecting specific shRNA of LINC00158 in 769-P and 786-O cells respectively. In a, sh-NC, sh-LINC00158-1 and sh-LINC00158-2 respectively represent 769-P + sh-NC, 769-P + sh-LINC00158-1 and 769-P + sh-LINC00158-2, and in b, sh-NC, sh-LINC00158-1 and sh-LINC00158-2 respectively represent 786-O + sh-NC, 786-O + sh-LINC00158-1 and 786-O + sh-LINC 00158-2.
FIG. 4 shows that the CCK8 method is used to detect 769-P and 786-O cells respectively for the change of proliferation capacity after the expression level of LINC00158 gene is stably knocked down. In a, sh-NC, sh-LINC00158-1 and sh-LINC00158-2 respectively represent 769-P + sh-NC, 769-P + sh-LINC00158-1 and 769-P + sh-LINC00158-2, and in b, sh-NC, sh-LINC00158-1 and sh-LINC00158-2 respectively represent 786-O + sh-NC, 786-O + sh-LINC00158-1 and 786-O + sh-LINC 00158-2.
FIG. 5 shows that the change in the migration ability of 769-P and 786-O cells, respectively, was detected by the Transwell migration assay (magnification 400) after stably knocking down the expression level of LINC00158 gene. sh-NC, sh-LINC00158-1 and sh-LINC00158-2 in a and b respectively represent 769-P + sh-NC, 769-P + sh-LINC00158-1 and 769-P + sh-LINC00158-2, and sh-NC, sh-LINC00158-1 and sh-LINC00158-2 in c and d respectively represent 786-O + sh-NC, 786-O + sh-LINC00158-1 and 786-O + sh-LINC 00158-2.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 analysis of expression amount of LINC00158 Gene in renal cancer tissue and renal cancer cell
Expression of LINC00158 gene in kidney cancer tissue
The whole genome Expression profile of kidney cancer was downloaded from a Gene Expression integrated database (Gene Expression Omnibus, GEO, https:// www.ncbi.nlm.nih.gov/GEO /), and analyzed, and the Expression level of LINC00158 Gene in 101 clear cell renal carcinoma tissues and 101 paracarcinoma tissues was analyzed.
The experimental results are shown in a in fig. 1, and compared with the tissue beside the cancer, the expression level of the LINC00158 gene in the kidney cancer tissue is remarkably up-regulated. Data were processed using GraphPad Prism 6, and the results of the experiment were expressed as mean ± standard deviation, and P < 0.001(×) by t-test showed a very significant difference in the expression level of LINC00158 gene in renal cancer tissue compared to the expression level of LINC00158 gene in tissue adjacent to the cancer.
Second, expression of LINC00158 gene in renal cancer cell
The human tubular epithelial cell line HK-2 and the human renal cancer cell lines 769-P and 786-O were purchased from Shanghai institute of Life sciences cell Bank. HK-2 was cultured with DMEM/F-12(1:1) containing 10% fetal bovine serum, while 769-P and 786-O were cultured in 1640 medium (both media purchased from Thermo Fisher Scientific, USA). The cell culture conditions were 37 ℃ and 5% carbon dioxide.
Total RNA from cells was extracted using TRIzol reagent (Thermo Fisher Scientific, USA), and the expression level of LINC00158 gene in cells was detected by using real-time fluorescent quantitative PCR (RT-qPCR). Mu.g of total RNA was reverse transcribed into cDNA using a reverse transcriptase kit (Toyobo, Osaka, Japan). Beta-actin was used as a control gene. qPCR conditions were as follows: PCR amplification was performed in triplicate at 95 ℃ for 1 min, 95 ℃ for 15s, 60 ℃ for 45s, 40 cycles. By CT (2)-ΔΔCT) The method calculates a change in threshold Cycle (CT) value.
As shown in b in FIG. 1, the expression level of LINC00158 gene was significantly up-regulated in the renal cancer cell lines 769-P and 786-O, as compared to HK-2. The data were processed using GraphPad Prism 6 statistical software, and the results of the experiments were expressed as mean. + -. standard deviation, and P < 0.05 (. times.) represents a significant difference in the relative expression level of LINC00158 gene in the renal cancer cell line 769-P as compared with the relative expression level of LINC00158 gene in the tubular epithelial cells HK-2 using t-test, and P < 0.01 (. times.) represents a significant difference in the relative expression level of LINC00158 gene in the renal cancer cell line 786-O as compared with the relative expression level of LINC00158 gene in the tubular epithelial cells HK-2.
The results verified in the kidney cancer tissues and the kidney cancer cell lines show that the LINC00158 gene is highly expressed in the kidney cancer tissues and the kidney cancer cell lines compared with the paracarcinoma tissues and the normal tubular epithelial cell lines, and the difference of the expression levels has statistical significance.
Example 2 detection of expression level of LINC00158 Gene in urine and screening of renal carcinoma
First, collection of clinical urine sample
Urine samples were collected from 14 human renal cancer patients and 10 human healthy controls. Among healthy controls, there were 8 males and 2 females.
The basic data of renal cancer patients are shown in the following table:
TABLE 114 renal cancer patient cases
Urine sampling is carried out in central hospitals in Xiangyang city, and an informed consent is required to be signed before sampling. One night before urine collection, kidney cancer patients and healthy volunteers were issued with disposable urine collection cups and instructed how to collect urine correctly. A total of 100 ml of the first urine mid-stream was collected in the morning, immediately frozen on ice, and centrifuged within 2h after collection.
The urine was dispensed into 50mL tubes, 50mL each, and centrifuged at 2500 Xg for 15 minutes at 4 ℃. The supernatant in the 50mL tube was removed by a waste liquid suction vacuum pump, and the urinary sediment was left. 1mL of PBS at 4 ℃ is added into the urinary sediment of each tube, gently sucked, beaten and mixed evenly, and then transferred into a 1.5mL enzyme-removing centrifuge tube. Centrifuge at 2500 Xg for 5 min at 4 ℃ and discard the supernatant. Washing with PBS for 2 times, and removing supernatant to obtain urinary sediment. Add 1mL TRIzol to each tube, vortex and mix well, and store at-80 ℃.
Secondly, detecting the expression of LINC00158 gene in urine
Total RNA from cells was extracted using TRIzol reagent (Thermo Fisher Scientific, USA), and the expression level of LINC00158 gene in cells was detected by using real-time fluorescent quantitative PCR (RT-qPCR). Mu.g of total RNA was reverse transcribed into cDNA using a reverse transcriptase kit (Toyobo, Osaka, Japan). The relative expression level of the LINC00158 gene (also called relative expression level of LINC 00158) is detected by using beta-actin as an internal reference gene. qPCR conditions were as follows: PCR amplification was performed in triplicate at 95 ℃ for 1 min, 95 ℃ for 15s, 60 ℃ for 45s, 40 cycles. Tong (Chinese character of 'tong')Over CT (2)-ΔΔCT) The method calculates a change in threshold Cycle (CT) value.
Wherein, the primer pair for PCR amplification of the LINC00158 gene is as follows:
LINC00158-QF：5’-TTAAGAAAGCGGTCGTCGCC-3’
LINC00158-QR，5’-TCTGCCACCTTGTGGTTGTT-3’
the primer pair for PCR amplification of the beta-actin gene comprises the following components:
Actin-F：5’-TGACGTGGACATCCGCAAAG-3’
Actin-R：5’-CTGGAAGGTGGACAGCGAGG-3’
the data were processed using GraphPad Prism 6, and the results of the experiment were expressed as mean ± standard deviation, and P < 0.05 (. sup.) (representing that the expression level of LINC00158 gene in the urine of renal cancer patient was significantly different from that of LINC00158 gene in the urine of healthy control) using t-test, and P < 0.01 (. sup.) (representing that the expression level of LINC00158 gene in the urine of renal clear cell carcinoma patient was significantly different from that of LINC00158 gene in the urine of healthy control).
The result of the urine RT-qPCR detection of 14 renal cancer patients and 10 healthy controls is shown in a in fig. 2, and the expression level of LINC00158 gene in the urine of renal cancer patients is significantly up-regulated compared to healthy controls.
Among them, urine test results of 10 patients with renal clear cell carcinoma and 10 healthy controls are shown in c in fig. 2, and the expression level of LINC00158 gene in urine of renal clear cell carcinoma patients is significantly increased compared to healthy controls.
At this time, the threshold value of the renal cancer patient is judged to be that the relative expression quantity of the LINC00158 gene is not less than 0.024247661, namely the relative expression quantity of the LINC00158 gene in the urine sample of the object to be detected is not less than 0.024247661, the object to be detected is the renal cancer patient, the relative expression quantity of the LINC00158 gene in the urine sample of the object to be detected is less than 0.024247661, and the object to be detected is a non-renal cancer patient. The above screening criteria are for reference only.
Plotting of ROC curves
And (3) utilizing GraphPad Prism 6 to draw a ROC curve for the relative expression data of the LINC00158 gene in the urine detected by RT-qPCR. If the area under the ROC curve is greater than 0.5 and closer to 1, the screening test performed by LINC00158 will be better.
The result is shown in b in fig. 2 and d in fig. 2, the area under the ROC curve for screening the kidney cancer by the LINC00158 is 0.9786, and the area under the ROC curve for screening the clear cell carcinoma of the kidney is 0.9800, which shows that the screening of the kidney cancer, especially the clear cell carcinoma of the kidney by the LINC00158 in the urine has good specificity and high sensitivity, and the LINC00158 has great potential value as a novel molecular marker of the kidney cancer, thereby providing a powerful molecular biological tool for screening, diagnosing or assisting in diagnosing the kidney cancer.
Example 3 application of knocking down the expression of LINC00158 Gene in tumor cells in treating tumors
Construction of 769-P and 786-O cell lines stably knocking down LINC00158 gene by shRNA technology
3.1769-P cell lentivirus infection assay
A lentiviral expression vector expressing shRNA designated sh-LINC00158-1 (targeting sequence 5'-GGATAAGGAGATCTGAAATTG-3' (SEQ ID No.2)) and a lentiviral expression vector expressing shRNA designated sh-LINC00158-2 (targeting sequence 5'-GGATGAGCACAGATCACATCA-3' (SEQ ID No.3)) were designed and synthesized by Suzhou Jima Biotechnology Co., Ltd.
The recombinant lentivirus expressing sh-LINC00158-1 was obtained by co-transfecting 293T cells with a transfection reagent RNAi-Mate from Gima using a lentiviral expression vector expressing shRNA named sh-LINC00158-1 and packaging plasmids (pGag/Pol, pRev, and pVSV-G) thereof.
The recombinant lentivirus expressing sh-LINC00158-2 was obtained by co-transfecting 293T cells with a transfection reagent RNAi-Mate from Gima using a lentiviral expression vector expressing shRNA named sh-LINC00158-2 and packaging plasmids (pGag/Pol, pRev, pVSV-G) thereof.
A lentivirus expression vector expressing shRNA (control non-target shRNA) named sh-NC and its plasmid-packaged plasmids (pGag/Pol, pRev and pVSV-G) were co-transfected into 293T cells using RNAi-Mate, a transfection reagent from Gilma to obtain a recombinant lentivirus expressing sh-NC.
The experiment was set up with two blank control groups (no transfection), two vector control groups (transfection of recombinant lentiviruses expressing NC-shRNA) and two LINC00158 knockdown groups (transfection of recombinant lentiviruses expressing LINC 00158-shRNA).
The specific experimental method is as follows: 769-P cells were packed at 2X 105Per cm2The density of (A) was inoculated in a 6-well plate and incubated at 37 ℃ with 5% CO2The cells are cultured in the constant temperature incubator, when the cells reach 80% confluence, recombinant lentivirus solution (MOI is 20) and polybrene (working concentration is 5mg/mL) are added, and the solution is changed after 12 hours. After the cells were confluent, they were transferred to a petri dish and the selection was started 3 days later. Puromycin is screened, the concentration is maintained at 2 mu g/mL, 769-P cells (recorded as 769-P + sh-LINC00158-1 and 769-P + sh-LINC00158-2) infected by the recombinant lentiviruses for expressing the LINC00158-shRNA and 769-P cells (recorded as 769-P + sh-NC) infected by the recombinant lentiviruses for expressing the NC-shRNA are obtained. After one week of selection with puromycin, the blank control group cells (769-P cells infected by recombinant lentivirus expressing NC-shRNA) die, and one month later, cells capable of normally living in the drug at the concentration are stably transfected cells, namely 769-P cells infected by recombinant lentivirus expressing LINC 00158-shRNA.
3.2786-O cell lentivirus infection test
The procedure was as in 3.1 except that the cells were replaced with 786-O. Obtaining a 786-O cell (marked as 786-O + sh-LINC00158-1 and 786-O + sh-LINC00158-2) infected by the recombinant lentivirus for expressing the LINC00158-shRNA and a 786-O cell (marked as 786-O + sh-NC) infected by the recombinant lentivirus for expressing the NC-shRNA. After one week of selection with puromycin, the blank control group cells (786-O cells infected by the recombinant lentivirus expressing NC-shRNA) die, and one month later, cells capable of normally living in the drug at the concentration are stably transfected cells, namely 786-O cells infected by the recombinant lentivirus expressing LINC 00158-shRNA.
Referring to example 2, the expression level of LINC00158 gene in the constructed cell line was measured using RT-qPCR method.
As shown in FIG. 3, after sh-LINC00158-1 or sh-LINC00158-2 was transfected in 769-P and 786-O cell lines, respectively, the expression of LINC00158 gene was significantly reduced, which indicates that 769-P and 786-O cell lines with stable knockdown of LINC00158 gene were successfully constructed.
Second, cell proliferation assay
Cell Counting Kit-8(CCK-8) Kit (MCE, K0301, USA) was used to determine the proliferation potency of 769-P and 786-O cells after infection, respectively. 6 cells of 769-P + sh-NC, 769-P + sh-LINC00158-1, 769-P + sh-LINC00158-2, 786-O + sh-NC, 786-O + sh-LINC00158-1 and 786-O + sh-LINC00158-2 are respectively inoculated into a 96-well plate, and each well is 2 multiplied by 103And (4) cells. After culturing for 1 day, 2 days and 3 days, the cell proliferation ability was examined. 10 μ L of CCK-8 reagent was added to each well and after incubation at 37 ℃ for 2 hours, the absorbance was measured at 450nm using a microplate reader (BioTek, Epoch, USA). The experiment was repeated 3 times and the level of cell proliferation was counted according to absorbance.
The result is shown in figure 4, after the expression of the LINC00158 gene is stably knocked down, the proliferation capacities of 769-P and 786-O cells are obviously inhibited, which shows that LINC00158 can influence the proliferation of renal cancer cells and can be used as a drug target for treating renal cancer. Data were processed using GraphPad Prism 6 statistical software and experimental results were expressed as mean ± standard deviation, with P < 0.05(×) representing significant differences and P < 0.01(×) representing very significant differences using the t-test.
Third, in vitro cell migration experiment
Respectively about each 3 × 104Cells of 769-P + sh-NC, 769-P + sh-LINC00158-1, 769-P + sh-LINC00158-2, 786-O + sh-NC, 786-O + sh-LINC00158-1 and 786-O + sh-LINC00158-2 were suspended in 200. mu.L of serum-free medium, added to the upper chamber of the Transwell cell and 600. mu.L of medium containing 10% serum was added as a chemotactic agent to the lower chamber. Cells were seeded in 24-well plates and incubated for 24 hours. After carefully wiping the non-migrating cells in the upper chamber with a cotton swab, the cells on the membrane were fixed with methanol for 30min, stained with 0.5% crystal violet solution for 30min, and ddH2And cleaning, drying, and taking a picture for analysis by a microscope. And the number of migrated cells (number/chamber) was counted using ImageJ. The experiment was repeated 3 times.
The result is shown in figure 5, after the expression of the LINC00158 gene is stably knocked down, the number of migrating cells of 769-P and 786-O is obviously reduced, and the migration capacity is obviously inhibited, which indicates that LINC00158 can influence the migration of renal cancer cells and can be used as a drug target for treating renal cancer. Data were processed using GraphPad Prism 6 statistical software and experimental results were expressed as mean ± standard deviation, with P < 0.05(×) representing significant differences and P < 0.01(×) representing very significant differences using the t-test.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
Application of <120> lncRNA in renal cancer urine screening and renal cancer treatment
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1480
<212> DNA
<213> Homo sapiens
<400> 1
ggattaatct aagcttctgc tgtgtatgga cctgtggcta cattacaaaa ttcagttgtg 60
aagctataaa atcaattagt ctagacctca aatcttattt cctatgaaaa gcctcaaact 120
tgtagctgta tgaacaagtg tttttggaat tttttttttt ttgctagaat ggagagaaca 180
cactggagat cacaaatgca tttattcttt ttcaccaact gtttcattgg ccttaaaaaa 240
tactaggcta aacttaagtg gtaaagatct gaaataaaaa gatgcatctt tatgtctgaa 300
cagcaatttt tcataggtct ctaataactt gcatttcctt atctatggat aaggagatct 360
gaaattgatc tacattttct ttagtaatgt tttctctgtt tattgagaac agatatcttc 420
atctccttca tgcattatcc ttagtaacag atgtcagtag gatccagagc cattttggaa 480
ctttaccaag gattaaggat gagcacagat cacatcaaga gtctaaatat cactttggag 540
gccatgtgca gatcagattt ggaagtgacc aagactggag gcagggacac cagagttttc 600
tgttaaaaac tggtccatgc aagaaaagat gactcctgaa ataagacagt gatcattagg 660
atgaagagga agagaagatt tgaggaatac ttagaacaga atttactgaa tttactggtt 720
gaattgaatg tgaagaggag atgaataaag aagaaaacac cctcaaagga acaaatatga 780
aacaatctct ggaaaatgga atttgatttt gattcaacaa caggagaatt ctactgtttt 840
tctccagcag ctccacgata aaagggaacc ctgagcagaa cacatgcaca aactcatgcc 900
ataccctaag ggacaaggtt ccccacatct acttggaaat ctttgggatg ctcaatggag 960
aaaaacaagg agagaaaaat ctaaacccag cagaagagaa agtttaagaa agcggtcgtc 1020
gccaatacca aaattagcta tgatattcag gaagaaaaaa atggaaacac gataaccaca 1080
gtcaataaag accaaggcca cattccattg accagagctc agccacgcgc cacacctggc 1140
taaagagact ctggctgaag aattccctga atattttaat gtagctctag gttataacac 1200
agaacaacca caaggtggca gaagaactcc attcttgtaa atctcttagt tgcttcttta 1260
tttcatcttc cctattccat ctaatgttcc ctgaatttta ctattttgtt ggtttgaaaa 1320
tatgtgtgac ttacgtcctt taaaggattt ggttcgtaag gttttgaata gactggaata 1380
taaaacattt atagaaaaga aaaataccta cagtatgatg ttagtgaact attaataatc 1440
caattaaata aaatatattt gctttggatt aatacaaaaa 1480
<210> 2
<211> 21
<212> DNA
<213> Homo sapiens
<400> 2
ggataaggag atctgaaatt g 21
<210> 3
<211> 21
<212> DNA
<213> Homo sapiens
<400> 3
ggatgagcac agatcacatc a 21
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
Claims (10)
1. The application of the reagent and/or the apparatus for detecting LINC00158 in preparing products for screening, auxiliary screening and/or auxiliary diagnosis of kidney cancer.
2. The use of claim 1, wherein said reagents and/or apparatus for detecting LINC00158 comprise reagents and/or apparatus for detecting LINC00158 by reverse transcription-polymerase chain reaction, real-time fluorescence quantitative PCR technique, transcriptome sequencing technique, Northern blot, in situ hybridization technique, gene chip technique, Nanopore sequencing technique, or PacBio sequencing technique.
3. The use according to claim 1 or 2, wherein the product for screening, assisted screening and/or assisted diagnosis of kidney cancer comprises a kit, a gene chip, a high throughput sequencing platform or a biosensor.
4. The use of any one of claims 1 to 3, wherein the agent for detecting LINC00158 is an agent for detecting the expression level of LINC00158 gene.
5. The use of claim 4, wherein the reagent for detecting the expression level of LINC00158 gene comprises a primer for specifically amplifying the LINC00158 gene and/or a probe for specifically recognizing the LINC00158 gene.
6. The use according to any one of claims 1 to 5, wherein the test sample of the product is urine.
7. Application of the agent for inhibiting or reducing LINC00158 gene expression in preparing a medicament for treating or preventing renal cancer.
8. The use of claim 7, wherein the agent that inhibits or reduces the expression of the LINC00158 gene is a nucleic acid molecule.
9. A medicament for treating or preventing renal cancer, which comprises the agent for inhibiting or reducing the expression of LINC00158 gene according to claim 6, 7 or 8.
10. An apparatus for screening and/or diagnosing kidney cancer, characterized in that the apparatus comprises the reagent for detecting LINC00158 as set forth in any one of claims 1 to 5 and a computer-readable storage medium having stored thereon a computer program for causing a computer to execute the step of screening and/or diagnosing a disease of kidney cancer based on an expression amount of LINC00158 gene in a specimen.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105256051A (en) * | 2015-11-11 | 2016-01-20 | 首都儿科研究所 | Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes |
| US20160283653A1 (en) * | 2013-11-06 | 2016-09-29 | The United States Of America, As Represented By The Secretary, De Partment Of Health And Human Ser | Method for subtyping lymphoma types by means of expression profiling |
| CN107435074A (en) * | 2017-08-31 | 2017-12-05 | 北京泱深生物信息技术有限公司 | Application of the CES8 genes in clear cell carcinoma of kidney diagnosis and treatment |
| US20180162942A1 (en) * | 2015-05-29 | 2018-06-14 | Bristol-Myers Squibb Company | Treatment of renal cell carcinoma |
| CN108251533A (en) * | 2018-04-11 | 2018-07-06 | 蒋灵锋 | A kind of gene detecting kit for being used to detect chromophobe cell tumor hypotype in kidney |
| CN108277283A (en) * | 2018-03-21 | 2018-07-13 | 中国人民解放军***南京总医院 | Application of the lncRNA combinations in preparing the product of prediction clear cell carcinoma of kidney prognosis and molecular targeted agents therapeutic sensitivity |
| CN108728547A (en) * | 2018-07-09 | 2018-11-02 | 郑州大学第附属医院 | Application, kit and the detection method of kidney prognosis novel molecular marker non-coding RNA LINC00158 |
| CN109576365A (en) * | 2018-11-02 | 2019-04-05 | 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） | It is a kind of predict head and neck neoplasm immunization therapy curative effect marker and application |
| CN109735545A (en) * | 2019-02-19 | 2019-05-10 | 上海交通大学医学院附属仁济医院 | A kind of long-chain non-coding RNA and its purposes as Diagnosis of Renal Cell Carcinoma and prognostic markers object |
| US20200078401A1 (en) * | 2016-12-07 | 2020-03-12 | La Jolla Institute For Allergy And Immunology | Compositions for cancer treatment and methods and uses for cancer treatment and prognosis |
| CN111630183A (en) * | 2017-09-05 | 2020-09-04 | 新加坡科技研究局 | Clear cell renal cell carcinoma biomarkers |
-
2021
- 2021-05-12 CN CN202110515711.2A patent/CN113186289B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160283653A1 (en) * | 2013-11-06 | 2016-09-29 | The United States Of America, As Represented By The Secretary, De Partment Of Health And Human Ser | Method for subtyping lymphoma types by means of expression profiling |
| US20180162942A1 (en) * | 2015-05-29 | 2018-06-14 | Bristol-Myers Squibb Company | Treatment of renal cell carcinoma |
| CN105256051A (en) * | 2015-11-11 | 2016-01-20 | 首都儿科研究所 | Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes |
| US20200078401A1 (en) * | 2016-12-07 | 2020-03-12 | La Jolla Institute For Allergy And Immunology | Compositions for cancer treatment and methods and uses for cancer treatment and prognosis |
| CN107435074A (en) * | 2017-08-31 | 2017-12-05 | 北京泱深生物信息技术有限公司 | Application of the CES8 genes in clear cell carcinoma of kidney diagnosis and treatment |
| CN111630183A (en) * | 2017-09-05 | 2020-09-04 | 新加坡科技研究局 | Clear cell renal cell carcinoma biomarkers |
| CN108277283A (en) * | 2018-03-21 | 2018-07-13 | 中国人民解放军***南京总医院 | Application of the lncRNA combinations in preparing the product of prediction clear cell carcinoma of kidney prognosis and molecular targeted agents therapeutic sensitivity |
| CN108251533A (en) * | 2018-04-11 | 2018-07-06 | 蒋灵锋 | A kind of gene detecting kit for being used to detect chromophobe cell tumor hypotype in kidney |
| CN108728547A (en) * | 2018-07-09 | 2018-11-02 | 郑州大学第附属医院 | Application, kit and the detection method of kidney prognosis novel molecular marker non-coding RNA LINC00158 |
| CN109576365A (en) * | 2018-11-02 | 2019-04-05 | 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） | It is a kind of predict head and neck neoplasm immunization therapy curative effect marker and application |
| CN109735545A (en) * | 2019-02-19 | 2019-05-10 | 上海交通大学医学院附属仁济医院 | A kind of long-chain non-coding RNA and its purposes as Diagnosis of Renal Cell Carcinoma and prognostic markers object |
Non-Patent Citations (5)
| Title |
|---|
| YI CHEN 等: "Comprehensive analysis of miRNA-mRNA-lncRNA networks in severe asthma", 《EPIGENOMICS》 * |
| 吕东晨 等: "长链非编码RNA与肾癌肿瘤标志物的研究进展", 《局解手术学杂志》 * |
| 程树林 等: "LINC00158通过促进脾酪氨酸激酶基因的表达抑制膀胱癌细胞增殖和迁移", 《实用医学杂志》 * |
| 陈宁 等: "《全科医生临床诊疗手册》", 30 June 2016, 中国医药科技出版社 * |
| 韩俊庆 等: "《肿瘤科诊疗思路点拨》", 31 March 2007, 山东科学技术出版社 * |
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