CN111909943A - Application of PRDX6 as target to enhancing sensitivity of colorectal cancer to oxaliplatin - Google Patents
Application of PRDX6 as target to enhancing sensitivity of colorectal cancer to oxaliplatin Download PDFInfo
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Abstract
The invention provides a drug resistance gene, wherein the sequence of PRDX6 is GCTTCTTCGCCAGAACCAACCGGTTGCTTGCTGTCCCAGCGGCGCCCCCTCATCACCGTCGCCATGCCCG. The invention firstly detects the expression of PRDX6 gene in drug-resistant cell HCT116OxR by RNA-seq technologyAnd higher. Reduction of PRDX6 expression in HCT116OxR evaluation of PRDX6 IC on drug-resistant cells50Influence of the values, the use of PRDX6 as a target gene for increasing the sensitivity of colorectal cancer to oxaliplatin is proposed.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of enhancing sensitivity of colorectal cancer to oxaliplatin by taking peroxide reductase 6(PRDX6) as a target.
Background
Colorectal cancer is one of the largest contributing factors to cancer-related mortality. Although 5-year survival rates for patients with early colorectal cancer (stages I and II) exceed 60%, when distant metastasis has occurred, more than 50% of patients are diagnosed as being in stage III or higher, in which case 5-year survival rates drop to 10%. Chemotherapy in combination with surgery is the basis of treatment of metastatic colorectal cancer and the only means to improve survival.
Oxaliplatin, the first platinum-based drug active in colorectal cancer, is capable of binding to DNA to form cross-linked and bulky adducts. Approximately 50% of colorectal cancer patients benefit from oxaliplatin treatment, so an oxaliplatin-containing chemotherapy regimen is usually the first line treatment regimen for patients with advanced colorectal cancer. Unfortunately, most patients suffer from chemotherapy failure due to acquired resistance. Therefore, finding new drug resistance targets, improving and optimizing therapeutic approaches, and thus reversing drug resistance, is critical for patients with advanced colorectal cancer.
Peroxiredoxin 6(PRDX6) belongs to the family of antioxidant enzymes other than seleno-Peroxidase (PRDX). It is the only member of the PRDX family, having both glutathione peroxidase and phospholipase a2 activity. PRDX6 can regulate phospholipid metabolic switching and protect cells from oxidative damage. Studies have shown that reduced levels of Reactive Oxygen Species (ROS) may be a significant cause of chemotherapy resistance, and it is not known whether PRDX6 promotes colorectal cancer against oxaliplatin by reducing ROS.
Since PRDX6 has not been reported in colorectal cancer resistance, we knocked down the expression of PRDX6 in drug-resistant cells HCT116OxR by detecting IC50It was verified that low expression of PRDX6 significantly increased the sensitivity of resistant cells to oxaliplatin.
Disclosure of Invention
The invention provides application of improving the sensitivity of colorectal cancer to oxaliplatin by taking PRDX6 as a target point aiming at the phenomenon that the colorectal cancer is insensitive to the oxaliplatin clinically.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
a tumor therapeutic agent for improving oxaliplatin sensitivity, which takes PRDX6 as a target and PRDX6 sequence as GCTTCTTCGCCAGAACCAACCGGTTGCTTGCTGTCCCAGCGGCGCCCCCTCATCACCGTCGCCATGCCCG.
Preferably, the tumor is colorectal cancer.
Preferably, the tumor treatment reagent is used for detecting the expression of PRDX6 in the sample by adopting a high-throughput sequencing method and/or a quantitative PCR method.
Preferably, the high throughput sequencing method is a second generation sequencing technology introduced by the company illumina.
Preferably, the method for quantitative PCR comprises primers that specifically amplify PRDX 6. The primer sequence is as follows: forward 5, -GCCACCCCAGTTGATTGGAA-3, reverse 5, -GGTGAAGACTCCTTTCGGGA-3.
Preferably, the method for reducing expression of LEF1 comprises an RNA interference (RNAi) technique. The RNA sequence is: 5, -GCAAUGAACUGUUGUAUG-3.
Preferably, the sample is colorectal cancer cell HCT116 and colorectal cancer anti-oxaliplatin cell line HCT116OxR cell line (purchased from the cell resource center of the institute of basic medicine of Chinese medical sciences).
The application of the gene in preparing colorectal cancer preparations.
(1)RNA-seq
Most known mrnas are found and identified by cDNA clone sequencing. The method requires the construction of a cDNA library of mRNA, followed by PCR amplification, and subsequent cloning of the amplified product into an expression vector for sequencing. High-throughput sequencing, also known as next-generation sequencing technology, is a revolutionary change to conventional sequencing, and performs sequence determination on hundreds of thousands to millions of DNA molecules at a time, thereby greatly improving the sequencing efficiency. High throughput sequencing at the same time makes it possible to perform a detailed global analysis of the transcriptome and genome of a species and is therefore also referred to as deep sequencing.
(2) Real-time fluorescent quantitative PCR technology (Real-time PCR, RT-PCR)
The fluorescence detection PCR instrument can draw a dynamic change curve for the accumulation rate of the amplified sequence in the whole PCR process. The greater the initial concentration of target sequence in the reaction mixture, the fewer PCR cycles (typically expressed in terms of a particular threshold cycle number Ct) are required to obtain a particular yield of amplified product. RT-PCR has many advantages such as the specificity is high, the sensitivity is good, fast simple.
Compared with the prior art, the invention has the advantages and positive effects that:
the present invention was first tested by RNA-seq to detect the increased expression of LEF1 in HCT116OxR cells and verified by RT-PCR (FIG. 1). The expression of PRDX6 was reduced in HCT116OxR cells by RNAi technology (FIG. 2), and the effect of PRDX6 on drug resistance of HCT116OxR was evaluated (FIG. 3), suggesting the application of PRDX6 in drug resistance treatment of colorectal cancer.
Drawings
FIG. 1 shows the expression of PRDX6 in HCT116 and HCT116 OxR. FIG. 2 shows the expression of PRDX6 in HCT116OxR and si-HCT116OxR cells. FIG. 3 is IC of HCT116OxR and si-HCT116OxR cells on oxaliplatin50The value is obtained.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
The concrete operation steps
(one) RNA-seq
Total RNA sample detection: total RNA amount and fragment distribution were determined on an Agilent 2100 pic 600.
Library construction: after the sample is qualified, the mRNA of the eukaryote is enriched by binding the pIyA tail of the mRNA through the complementary pair of A-T by using magnetic beads with oligo (dT). Fragmentation buffer was then added to break the mRNA into short fragments, single-stranded cDNA was synthesized using a hexabasic random primer (random hexamers) using the mRNA as a template, and double-stranded cDNA was then synthesized by adding buffer, dNTPs and DNA polymerase I, followed by purification of the double-stranded cDNA using AMPure XP beads. And (3) carrying out end repair on the purified double-stranded cDNA, adding an A tail and connecting a sequencing adaptor, then carrying out fragment size selection by using AMPure XP beads, and finally carrying out PCR enrichment to obtain a final cDNA library.
And (4) library inspection: and (3) quantifying the cDNA by using the qubit2.0, diluting to 1ng/ul, detecting the insert size of the library by using Agilent 2100, and quantifying the effective concentration of the library by using a PCR method (the effective concentration of the library is more than 2 nM) after the detection result accords with the prediction result, thereby ensuring the quality of the library.
And (3) machine sequencing: HiSeq sequencing was performed on the different libraries after posing as required for effective concentrations and target off-machine data volumes.
(II) Total RNA sample preparation in HCT116 and HCT116OxR cells
Extracting total RNA in cells according to the instructions of the Feijie total RNA rapid extraction kit.
Taking HCT116 and HCT116OxR cells in good growth states, digesting and dropping, centrifuging and collecting, leaving cell masses and 100 mu l of supernatant, and fully shaking until no cell masses exist; adding 500 mul of RA2 solution into the treated sample tube, fully reversing and uniformly mixing for 5-10 times, and standing for 1min at room temperature; sucking the lysate into the inner casing, and centrifuging at 12000rpm for 1 min; taking out the inner sleeve, sucking and removing the liquid in the outer sleeve, then putting the inner sleeve back, adding 500 mu l of Wash buffer, and centrifuging at 12000rpm for 1 min; washing again; taking out the inner sleeve, and centrifuging again without adding washing liquid after absorbing and discarding the liquid in the outer sleeve; the inner cannula was transferred to a new 1.5ml EP tube and 50 μ l of eluent was added to the center of the membrane; after standing at room temperature for 1min, the mixture was centrifuged at 12000rpm for 1min to obtain total RNA.
(III) RT-PCR detection of PRDX6 expression in HCT116 and HCT116OxR cells
Reverse transcription was performed according to the PrimeScript TM RT reagent Kit instructions, reverse transcription reaction 15min at 37 ℃ and inactivation reaction 5sec at 85 ℃. The reaction system was set as follows:
5×PrimeScriptTM Buffer (for real time) 2µl
PrimeScriptTM RT Enzyme Mix Ⅰ 0.5µl
Oligo dT Primer(50µM) 0.5µl
Random 6 mers(100µM) 0.5µl
Total RNA 500ng
RNase Free ddH2O added to 10 μ l
RT-PCR was then performed using the synthesized cDNA as template according to the TB Green TM Premix Ex Taq TM II kit instructions. The reaction conditions were 40 cycles of 42 ℃ for 5min,95 ℃ for 30s of pre-denaturation, 95 ℃ for 5s, 60 ℃ for 30 s. The reaction system is as follows:
TB Green 5µl
ROX Reference Dye Ⅱ 0.2µl
PCR Forward Primer (10µM) 0.2µl
PCR Reverse Primer (10µM) 0.2µl
ddH2O 3.4µl
template cDNA 1 mu l
The total volume is 10 mu l
In the PCR experiments, the replicates and negative control experiments (no cDNA template added in the negative control experiments) were performed, and each sample was repeated 3 times in the quantification experiments. Relative expression level of PRDX6 with GAPDH as internal reference 2-ΔΔCtAnd (4) calculating.
(IV) RNAi technique
Will 5 generate105The individual cells were seeded in 6-well plates to reach 70% confluence the next day; adding 5 μ l Lipofectamine 2000 into 250 μ l Opti-MEM, and standing at room temperature for 5 min; add 5. mu.l siRNA to 250. mu.l Opti-MEM; mixing the diluted Lipofectamine 2000 and siRNA gently, and standing for 20min at room temperature; adding the mixed solution into the well plate paved the previous day, incubating for 4-6h, removing the mixed solution, and replacing with a normal culture medium containing serum. After 48h total RNA was extracted and RT-PCR was used to detect the expression of PRDX 6.
(V) IC50Measurement of (2)
The HCT116OxR and si-HCT116OxR cells with good growth status were collected, digested, counted, and counted at 5X 10 per well3The individual cells were seeded in 96-well plates, and the medium was added to 100. mu.l per well, and cultured in an incubator for 48 hours. Oxaliplatin was added to each well in a final concentration of 0, 5, 10, 15, 20, 25, 30 μ M in sequence and the culture was continued for 48 h. Add 10. mu.l of CCK-8 to each well in the dark and incubate for 2h in the incubator. The light absorption of each well was measured using a microplate reader at a wavelength of 450 nm. Growth curves for each group were plotted using GraphPad Prism software.
Secondly, analyzing the results
The PRDX6 gene was detected to be expressed in HCT116OxR cells to be elevated by RNA-seq and confirmed by RT-PCR, see FIG. 1.
The expression of PRDX6 was reduced by RNAi technology in HCT116OxR cells and verified by RT-PCR, see figure 2.
IC of HCT116OxR cells on oxaliplatin following decreased expression of LEF150The value was 10. mu.M, see FIG. 3.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
SEQUENCE LISTING
<110> secondary Hospital of Shandong university
<120> application of PRDX6 as target to enhancing sensitivity of colorectal cancer to oxaliplatin
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 70
<212> DNA
<213> Artificial sequence
<400> 1
gcttcttcgc cagaaccaac cggttgcttg ctgtcccagc ggcgccccct catcaccgtc 60
gccatgcccg 70
<210> 2
<211> 40
<212> DNA
<213> Artificial sequence
<400> 2
gccaccccag ttgattggaa ggtgaagact cctttcggga 40
<210> 3
<211> 18
<212> RNA
<213> Artificial sequence
<400> 3
gcaaugaacu guuguaug 18
Claims (10)
1. An oxaliplatin drug resistance related target gene for treating tumors is characterized in that the nucleotide sequence of the target gene is shown as SEQ ID NO. 1.
2. Target gene according to claim 1, characterized in that said tumor is colorectal cancer.
3. The target gene of claim 1, wherein the expression of peroxiredoxin 6 in the sample cells is detected by high throughput sequencing and/or quantitative PCR.
4. A target gene according to claim 3, wherein the sample is colorectal cancer cell HCT116 and colorectal cancer anti-oxaliplatin cell line HCT116OxR cell line.
5. The target gene of claim 3, wherein the high throughput sequencing method is a second generation sequencing technique.
6. A target gene according to claim 3, characterized in that the method for quantitative PCR comprises primers specifically amplifying oxidoreductase 6.
7. A target gene according to claim 3, wherein the primer sequence is 5'-GCCACCCCAGTTGATTGGAA-3' in forward direction and 5'-GGTGAAGACTCCTTTCGGGA-3' in reverse direction.
8. Application of RNAi for reducing expression of oxidoreductase 6 in preparation of drugs or preparations for treating colorectal cancer.
9. The use of claim 8, wherein the RNAi sequence is 5'-GCAAUGAACUGUUGUAUG-3'.
10. Application of oxidoreductase 6 as a target gene in improving sensitivity of colorectal cancer to oxaliplatin.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113005174A (en) * | 2021-03-01 | 2021-06-22 | 中国医学科学院肿瘤医院 | Marker for predicting cancer patient response to chemotherapy and application |
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| US20150056194A1 (en) * | 2013-08-21 | 2015-02-26 | Georgia Regents Research Institute, Inc. | Modified green tea polyphenols and methods thereof for treating liver disease |
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Application publication date: 20201110 |