CN117106912B - Enhancer RNA functional site related to colorectal cancer high-risk group identification and early screening and application thereof - Google Patents
Enhancer RNA functional site related to colorectal cancer high-risk group identification and early screening and application thereof Download PDFInfo
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Abstract
The invention relates to an enhancer RNA functional site related to colorectal cancer high risk group identification and early screening and application thereof, and based on early large-scale crowd data and biological functional experiments, the rs3094296 site is found to be related to colorectal cancer susceptibility of Chinese crowd, and the action mechanism is that the rs3094296 site promotes expression of a target gene SENP7 through transcription of eRNA SENP7e, and finally proliferation of cancer cells is inhibited. By accurately designing the primer and the probe for the site rs3094296, the detection of the site rs3094296 can be carried out on normal people by means of fluorescence quantitative PCR, so that high risk people of colorectal cancer can be identified. The technical method has ingenious, simple and feasible design and accurate and reliable result, provides assistance for early screening and diagnosis of colorectal cancer patients, and is beneficial to early intervention of the crowd clinically.
Description
Technical Field
The invention relates to the fields of genetic engineering and oncology medicine, in particular to an enhancer RNA functional site related to colorectal cancer high risk group identification and early screening and application thereof.
Background
Colorectal cancer is a common malignant tumor of the digestive tract, seriously threatens the health of people in China, and has incidence and mortality rate respectively listed as the third and second in malignant tumors in China. Along with the aggravation of population aging and the change of resident life style and dietary structure, the incidence rate and death rate of colorectal cancer in China show an annual rising trend, and become a major public health problem to be solved urgently in China. Currently, the most effective screening method for colorectal cancer is fecal occult blood examination (FIT) in combination with colonoscopy. However, these methods have drawbacks such as low detection rate and low patient acceptance. Therefore, a more convenient and accurate method is needed to carry out screening and early diagnosis on high-risk individuals of colorectal cancer, and the acceptance of patients is improved, so that a great deal of medical expenditure is saved for the country.
In recent years, a personalized screening scheme based on molecular genetics is an important point for screening colorectal cancer high-risk groups. Single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNPs), which are the most common type of genetic variation, reflect differences in genetic background of individuals, have been shown to be associated with risk of tumor development. To date, a series of genetic variation sites closely related to colorectal cancer susceptibility in the chinese population have been identified by Genome-wide association studies (Genome-Wide Association Study, GWAS). However, the majority of colorectal cancer-associated sites identified are located in non-coding regions of the genome, revealing the truly functional genetic variations and their underlying biological mechanisms behind these statistical associations remains very difficult, for which researchers have exploited truly functional SNPs using a quantitative trait locus (Quantitative Trait Loci, QTL) based analysis approach system. The non-coding region contains a number of cis-regulatory elements, where enhancers play a critical role in the regulation of gene expression. With the rapid development of high throughput sequencing technology, active enhancers have been found to transcribe a non-coding RNA, called enhancer RNA (eRNA), which activates enhancer activity, promotes the formation of enhancer-promoter loops, and thus downstream gene expression, and many cancers and diseases in humans are associated with aberrant expression of eRNA. Furthermore, studies report that genetic variation on enhancers further affects disease development by affecting the production of eRNA. Therefore, the genetic variation (enhancer RNA quantitative trait locus, eRNAQTL) affecting eRNA expression is systematically identified in the whole genome range, and is an important entry point for analyzing the biological function of the susceptibility site, and meanwhile, the identified SNP marker can be applied to identification, early screening and auxiliary diagnosis of individuals at high risk of colorectal cancer of Chinese population.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and aims to provide an enhancer RNA functional site related to colorectal cancer high-risk group identification and early screening and application thereof.
In particular to application of a detection reagent of an enhancer RNA functional site rs3094296 in preparing a colorectal cancer auxiliary diagnosis kit.
The detection reagent comprises a specific amplification primer and a specific probe of an enhancer RNA functional site rs3094296, wherein the sequence of the specific amplification primer is SEQ ID NO:1 and SEQ ID NO:2, the specific probe sequence is SEQ ID NO:3 and SEQ ID NO:4.
the beneficial effects of the invention are as follows: the invention provides a technical method for screening colorectal cancer high risk groups from the aspects of molecular biology and gene diagnosis. Through combining with colorectal cancer eRNAQTLs genetic map and large-scale population verification, the rs3094296 locus is related to colorectal cancer susceptibility of Chinese population. Through ingenious primer and probe design aiming at the site of rs3094296, the rs3094296 mutation detection can be carried out on normal people by means of fluorescence quantitative PCR, so that high-risk people of colorectal cancer are identified, and early screening and diagnosis of colorectal cancer patients are assisted. The technical method has ingenious, simple and feasible design, accurate and reliable results, can be popularized in hospitals at all levels, provides assistance for evaluating the risk of colorectal cancer, and is beneficial to clinically screening and early intervention of colorectal cancer for the crowd.
Drawings
FIG. 1 is a flow chart of eRNAQTLs profiling colorectal cancer susceptibility locus rs 3094296;
FIG. 2 is a schematic representation of crowd outcome and potential regulatory target eRNA for eRNAQTL rs 3094296;
FIG. 3 is a graph showing an AUC curve of a colorectal cancer risk prediction model based on SNP locus rs 3094296;
FIG. 4 is a schematic diagram of the experimental result of rs3094296 dual-luciferase reporter;
FIG. 5 is a schematic diagram of the allele-specific binding of transcription factor HOXA5 to rs3094296 upstream and downstream;
FIG. 6 is a schematic diagram of the regulation of the expression level of the target gene SENP7 by the rs3094296 genotype;
FIG. 7 is a schematic representation of the effect of SENP7e and SENP7 on the proliferative capacity of colorectal cancer cell lines.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
Combining with the research of colorectal cancer functional eRNAQTLs and multi-center case control of Chinese people, an enhancer RNA functional site marker related to early colorectal cancer screening and auxiliary diagnosis is identified, wherein the marker is rs3094296.
The specific amplification primer sequence of rs3094296 is SEQ ID NO:1 and SEQ ID NO:2.
the specific probe sequence of rs3094296 is SEQ ID NO:3 and SEQ ID NO:4.
the detection reagent of the enhancer RNA functional site marker related to colorectal cancer susceptibility is applied to the preparation of colorectal cancer early screening and auxiliary diagnosis kits.
An early colorectal cancer screening and auxiliary diagnosis kit is used for detecting rs3094296 in peripheral blood DNA.
The colorectal cancer early screening and auxiliary diagnosis kit comprises a specific amplification primer and a specific probe of a functional site marker rs3094296.
The technical scheme for solving the problems comprises the following steps: (1) establishing a unified standard specimen library and database: standard-compliant subject blood samples were collected with standard procedures (SOP) and the system collected complete demographic and clinical data. (2) genotype detection: selecting colorectal cancer cases and healthy controls, finding functional site markers related to colorectal cancer morbidity of Chinese people in sites with obvious eRNAQTL, and (3) verifying the screened positive associated functional site markers in independent samples to judge the associated stability. (4) development of colorectal cancer auxiliary diagnosis kit: and (3) developing an early screening and auxiliary diagnosis kit according to genetic functional site marks with obvious differences in genotype distribution frequency in colorectal cancer cases and healthy controls.
In particular, the experimental method of the invention mainly comprises the following steps:
first, we collected 10 pairs of fresh colorectal cancer surgically excised and its paracancerous normal tissue samples from the affiliated homozygous hospital of the university of Huazhong science and technology, and performed ATAC-seq and H3K27ac ChIP-seq experiments, respectively. A set of active enhancer elements was annotated by integrating the ATAC-seq and H3K27ac ChIP-seq data, further removing the region overlapping the protein encoding gene to give the eRNA region. Subsequently, we systematically identified and identified functional eRNAQTLs for colorectal cancer in the whole genome range using transcriptome data of 154 Chinese colorectal cancer samples in the eRNA region and genotype data of their corresponding samples for identification of colorectal cancer high risk populations. Next, the biological mechanisms of the eRNAQTLs affecting colorectal cancer occurrence were explored through a series of molecular biology experiments, by two-stage, multi-center case-control studies to identify eRNAQTLs affecting colorectal cancer risk.
Crowd verification included a two-stage case-control study in which colorectal cancer case-control samples at the discovery stage were recruited to Beijing (China medical science college) and to the Siraitia hospitals (both the same-Ji hospital, the university of Siraitia, and the university of Siraitia in the south China), collecting a total of 4,293 colorectal cancer patients and 7,176 healthy control blood samples. The basic demographic characteristics are shown in table 1. Using the unconditional Logi st ic regression model, we calculated the relationship of eRNAQTLs to colorectal cancer susceptibility in the additive model after correction of age, sex, smoking and drinking confounding factors, and thus drawn the Manhattan plot, the results of which are shown in figure 2, panel a. We identified 2,901 eRNAQTLs associated with colorectal cancer risk, with P values < 0.05 as the significance threshold, with the 3q12.3 region population most significant. In this region we found that the rs3094296 functional site was significantly correlated with expression of the target enona ENSR00000155786 in colorectal cancer tissue, and the results are shown in figure 2, panel b. The case control research results show that individuals carrying the rs3094296 risk genotype respectively reduce the risk of colorectal cancer by 9% compared with normal individuals in Chinese crowd samples (OR=0.88, 95% CI:0.83-0.94, P=2.69×10) -5 ) The results are shown in Table 2.
TABLE 1 basic data of the sample of the case control study of the China crowd at the discovery stage
Calculating by adopting independent t-test on two sides; * By using double-sided χ 2 Inspection calculation
TABLE 2 analysis results of risk correlation of rs3094296 colorectal cancer in Chinese crowd samples
Verification phase we co-recruited 6,024 colorectal cancer patients with complete medical history data from two areas of the Beijing, wuhan, and 10,022 normal control Chinese populations without a history of tumor disease, and genotyped these individuals. Colorectal cancer patients and normal controls were both in the han group. The patient is subjected to histopathological diagnosis without age limitation; normal controls had no history of tumor and no signs of tumor upon physical examination. And information of sex, age and the like of the study subjects is collected. Each subject informed consent was given to the study and 2ml of peripheral venous blood was donated for isolation of lymphocyte genomic DNA. The basic demographic characteristics are shown in table 3.
TABLE 3 basic data of Chinese crowd case control study sample at the verification stage
* By using double-sided χ 2 Checking and comparing;a two-sided independent sample t-test comparison was used.
We used two-place recruited crowd samples for crowd verification by employing unconditional logistic returnsAfter model establishment and correction of gender, age, smoking and drinking conditions, the association of SNP locus rs3094296 and colorectal cancer susceptibility is calculated for two-stage crowd samples respectively. Analysis results of two-stage case control of Chinese crowd show that rs3094296[ T ] is carried]Individuals of risk genotype have 16% and 6% reduced risk of colorectal cancer (or=0.84, 95% ci:0.75-0.92, p=3.74×10, respectively -4 ；OR＝0.94，95％CI:0.90-0.99，P＝1.76×10 -2 ). To enhance the test efficacy of the case control study, two-stage samples were pooled and rs3094296 was tested for association with colorectal cancer susceptibility. The result of the case control research of the Chinese crowd is consistent with the result of the sample in the discovery stage, and rs3094296[ T ] is carried]Individuals of risk genotype were at 9% reduced risk of developing colorectal cancer (or=0.93, 95% ci:0.89-0.97, p=4.97×10 -4 ) The detailed results are shown in Table 4.
TABLE 4 analysis results of risk correlation of rs3094296 colorectal cancer in Chinese crowd samples
* And (5) calculating by using a Logistic regression model, and correcting gender, age, smoking and drinking conditions.
Early-stage multi-stage crowd association analysis verifies that the site is a colorectal cancer risk site, so that we further use the SNP site to establish a colorectal cancer risk prediction model. Chinese population from the discovery phase GWAS study (4,293 cases and 7,176 controls), european population from the GECCO project (17,789 cases and 19,951 controls). A formula is constructed, and three genotypes and sexes of SNP, age, smoking and drinking conditions are comprehensively considered. Wherein, for SNP genotyping, wild homozygous= "1", heterozygous= "2", mutant homozygous= "3"; for gender, male is "1" and female is "0"; for age, greater than or equal to 60 years old is "1", less than 60 years old is "0". In the analysis, a multi-factor logistic regression coefficient beta is taken as a weight, and a formula for obtaining the rs 3094296-based typing risk score is as follows:
chinese population risk score = (-0.0692 x gender score) + (0.0023 x age score) + (0.1203 x smoking score) + (-0.0139 x drinking score) + (-0.1244 x rs3094296 scoring).
European population risk score = (-0.1891 x sex score) + (0.0296 x age score) +(-0.0654 x rs3094296 typed score)
By drawing the AUC curve, the area under the model curve is 0.532 and 0.571 for European and Chinese people respectively, see specifically FIG. 3.
To functionally resolve colorectal cancer risk associations at the rs3094296 functional site, we found that the different genotypes of the pathogenic site were significantly correlated with the expression of the target eRNA ENSR00000155786 using eRNAQTL analysis. And then, the reporter gene experiments prove that the reporter gene activity of the recombinant plasmid transfected with the rs 3094296T allele is obviously higher than that of the empty vector pGL3-Promoter group in two colorectal cancer cell lines HCT116 and SW480, and the DNA fragment with the target site has the enhancer activity. Compared with the rs3094296[ T ] genotype group, the reporter gene activity of the rs3094296[ C ] genotype group is obviously reduced. The experimental result shows that the region of rs3094296[ T ] has enhancer activity, and the enhancer of rs3094296[ T ] has stronger transcriptional activation effect than that of rs3094296[ C ], and the result is shown in figure 4. Mechanically, we found that the binding capacity of the rs3094296[ T ] allele to the transcription factor HOXA5 was stronger, resulting in stronger enhancer activity of rs3094296[ T ], thereby promoting expression of ENSR00000155786, see specifically FIG. 5.
Given that determining the downstream target gene regulated by eRNA ENSR00000155786 helps to clarify its role in colorectal cancer development, we combine co-expression and eQTL analysis. As a result, rs3094296 was found to be significantly associated with the expression of SENP7 in colorectal cancer tissues, as shown in fig. 6, so we determined that SENP7 is the target gene for this edrna and named SENP7e as ENSR 00000155786. Next, we examined the effect of SENP7e and SENP7 on colorectal cancer cell growth using the cell proliferation assay CCK-8 and the clonogenic assay. Experimental results show that knocking down SENP7e and SENP7, respectively, can enhance proliferation and clonogenic capacity of colorectal cancer cells, and that knocking down both genes simultaneously can make cell growth change more remarkable, as shown in FIG. 7. The cell phenotype experiments show that SENP7e and SENP7 play a role in inhibiting cancer in colorectal cancer, and can play a certain synergistic effect to inhibit the growth of colorectal cancer cells.
Combining the above bioinformatics analysis, large-scale crowd data and biological function experiments, the results show that compared with individuals carrying the rs3094296[ C ] allele, individuals carrying the rs3094296[ T ] allele have lower colorectal cancer disease risk, and the action mechanism of the individuals has stronger enhancer activity by influencing the binding capacity of rs3094296 and the transcription factor HOXA5, so that the expression of eRNA SENP7e in the region is increased, the expression of a downstream target gene SENP7 is further promoted, and the growth of colorectal cancer cells is finally inhibited. Therefore, detection of rs3094296 variation in the population helps identify the high risk population for colorectal cancer, thereby assisting early intervention in colorectal cancer patients.
The experimental method comprises the following steps:
1. peripheral blood DNA extraction:
the DNA is extracted by a conventional phenol-chloroform method, and the specific steps are as follows:
1) About 3mL of anticoagulated blood was centrifuged at 5,000Xg for 15min at room temperature, and the upper layer was discarded, leaving about 0.3mL of blood cells. 0.5mL of freshly prepared extraction buffer with a final concentration of 20. Mu.g/mL RNase was added and incubated at 37℃for 1h after mixing.
2) Proteinase K was added at a final concentration of 100. Mu.g/mL and incubated overnight at 37℃after mixing.
3) 0.7mL of Tris buffer-equilibrated phenol (pH=7.0) was added to each tube, thoroughly mixed, and centrifuged at 8,000Xg for 15min at room temperature.
4) Transferring the upper liquid into another 1.5mL centrifuge tube, adding 0.7mL of equal volume phenol-chloroform (1:1), and fully and uniformly mixing for 15min; centrifuge at 8,000Xg for 15min at room temperature.
5) The supernatant was transferred to another clean 1.5mL centrifuge tube, 10% volume of 10M ammonium acetate solution was added, 2 volumes of pre-chilled absolute ethanol was added, and the mixture was allowed to stand at-20℃for 2h to precipitate DNA.
6) Washing the precipitated DNA with 75% ethanol, centrifuging at 12,000Xg for 15min, and discarding the upper layer liquid; the supernatant was discarded after centrifugation at 12,000Xg for 15min with 75% ethanol.
7) The tubes were inverted over absorbent paper, and after ethanol was evaporated, appropriate TE buffer was added to each tube, and stored at-20℃for one week at 4 ℃.
2. Genotyping
The typing platform adopted is TaqMan genotyping technology (ABI 7900HT Real Time PCR system,Applied Biosystems), and a5 mu L PCR reaction system is shown in Table 5:
TABLE 5 TaqMan genotyping PCR reaction System
The reaction conditions are as follows: pre-denaturation at 95℃for 10min, followed by 45 cycles of 15sec at 95℃and 1min at 60℃and cooling to 4 ℃.
The primers and probes used for the reaction were as follows:
rs3094296 primer:
forward primer: ACCCATCTCTCACATCCAGATT (SEQ ID NO: 1)
Reverse primer: GTGAGGGTATCTTAGTCTTTGTCTTTC (SEQ ID NO: 2)
rs3094296 probe:
forward probe: FAM-CTTAGTAATCTAACTGCATTCC-MGB (SEQ ID NO: 3)
Reverse probe: VI C-TTAGTAATTTAACTGCATTCCT-MGB (SEQ ID NO: 4)
3. Double luciferase reporter experiments
1) Reporter gene plasmid vector
The present study selected pGL3-Promoter vectors to construct reporter plasmids to verify whether the region of the site of interest has enhancer activity. pGL3-Promoter contains a firefly luciferase gene and its upstream SV40 Promoter, and can efficiently express firefly luciferase in various cell lines. Insertion of the fragment of interest upstream or downstream of the SV40 promoter allows detection of whether the region has transcriptional regulatory effects. Furthermore, we selected pRL-SV40 vector as an internal control. pRL-SV40 vector contains a Renilla luciferase gene and early enhancer and promoter elements, and can efficiently express Renilla luciferase in various cell lines. In the study, pRL-SV40 and pGL3-Promoter plasmids are transfected into cells together to normalize firefly luciferase activity, so that inherent changes in experiments are eliminated, and the accuracy of results is improved.
For the reporter gene plasmid of rs3094296, we downloaded a DNA sequence of 500bp on the upstream and downstream of the site as the center from NCBI, conventionally synthesized DNA fragments containing different alleles of the site, cloned the target sequence forward (adding MluI cleavage site at the 5 'end and XhoI cleavage site at the 3' end) into pGL3-Promoter vector, thereby constructing 2 recombinant reporter gene plasmids, as shown in FIG. 4. Plasmid was constructed by the same company of the Qinke biotechnology (Wuhan) and prepared in the form of a piercing bacterium, and fragment accuracy was verified by DNA sequencing and restriction enzyme cleavage.
2) Plasmid amplification and extraction
(1) Preparing a liquid culture medium in advance: taking 5g of LB culture medium powder and 200ml of deionized water, fully oscillating and dissolving, sterilizing under high pressure, cooling, sealing by a sealing film, and storing in a refrigerator at 4 ℃;
(2) 15ml of LB liquid medium and 30 mu l of ampicillin are added into a 50ml centrifuge tube, and the mixture is gently shaken and mixed evenly;
(3) the aseptic gun head is used for lightly selecting puncture bacteria, and the puncture bacteria are thrown into a centrifuge tube;
(4) fixing the centrifuge tube on a constant temperature shaking table, setting the temperature to 37 ℃, and oscillating for 12-16 hours (preferably 14 hours) at the rotating speed of 220rpm, and collecting bacterial liquid when the liquid is turbid;
(5) collecting fresh bacterial liquid after plasmid amplification, centrifuging for 15min at room temperature at 5g, and discarding the supernatant;
(6) plasmid was extracted using Omega endotoxin-free plasmid extraction kit, plasmid concentration and purity were checked using NanoDrop 1000, and qualified plasmid was packaged and stored in-20 ℃ refrigerator.
3) Transient transfection of reporter plasmids
(1) Spreading the cells to a 96-hole culture plate one day before transfection, culturing for 16-24 hours in a constant temperature incubator, and carrying out transfection when the growth of the cells reaches 70-90% of fusion degree;
(2) fresh culture medium is replaced 30min before transfection, and the culture medium is put back into a constant temperature incubator for incubation, and a transfection system is prepared during the incubation period;
(3) the transfection reagent formulation ratio per well is as follows:
a. 1.5ml EP tube was prepared as solution A: 5 μl Opti-MEM and 0.3 μl Lipofectamine 3000 reagent are added respectively, and the mixture is gently blown and mixed;
b. another 1.5ml EP tube was taken to prepare solution B: mu.l of Opti-MEM, 2ng of pRL-SV40 reference plasmid, 200ng of rs4810856 reporter plasmid and 0.2 mu l P3000 reagent are added respectively, and the mixture is gently beaten and mixed;
(4) fully and uniformly mixing the solution A and the solution B, and standing for 5-10 min at room temperature to obtain a DNA-liposome compound;
(5) adding the prepared compound into 10 mu l of each hole, shaking and mixing uniformly, and then placing the mixture into a constant temperature incubator for continuous culture.
4) Dual luciferase reporter activity assay
The present study uses the Promega dual luciferase reporter detection system to detect luciferase activity. Preparing LAR II initial solution and Stop & Glo Stop solution according to the description, subpackaging, and storing in a refrigerator at-80deg.C. The method comprises the following specific steps:
(1) harvesting cells after 24-36 h of transfection of the reporter gene plasmid, adding PBS for cleaning for 2 times, and discarding residual PBS;
(2) diluting PLB lysate (5X) to working concentration (1X) with ultrapure water, adding 30 μl of the PLB lysate into a 96-well culture plate per well, and mixing by shaking;
(3) sealing the culture plate, and placing in a refrigerator at-80deg.C overnight (more than 8 hr) to allow cells to be completely lysed;
(4) taking out the cell plate and the required amount of fluorescent reagent from the refrigerator at-80 ℃ before fluorescence measurement, and standing and melting at room temperature;
(5) taking 1.5ml of colorless transparent EP tube, respectively adding 20 μl of starting solution and 5 μl of cell lysate, gently blowing and mixing, taking care to avoid generating bubbles, and immediately placing into an instrument to detect firefly luciferase activity;
(6) adding 20 μl of stop solution, gently beating, mixing, and immediately detecting the luciferase activity of Renilla; and (3) calculating: normalized luciferase activity = firefly luciferase activity/renilla luciferase activity, relative to luciferase activity = experimental group normalized luciferase activity/control group normalized luciferase activity mean.
4. Cell proliferation assay
1) Cell count
Before CCK-8 proliferation and clonogenic experiments, cell counting is required to ensure that appropriate numbers of cells are seeded in different groups of well plates. The experiment used a blood cell counting plate consisting of two equally sized cells, each cell consisting of nine 1mm x 1mm squares, each square accommodating 0.1mm 3 Liquid volume. The specific counting steps are as follows:
(1) and (3) digesting the cell pore plate after being cultured for a period of time to obtain a cell suspension, and uniformly mixing and scattering the cells as much as possible.
(2) The counting plate is sterilized and then covered with a cover glass.
(3) 10. Mu.L of the cell suspension was aspirated and slowly dropped into the counting plate along the edge of the slide, ensuring no air bubbles were generated between the cover glass and the counting plate).
(4) The plate was left to stand for 1min, and the square at the four corners was counted under a microscope as follows: a. for cells pressed against the square edges, only the upper and left cells are counted, b. If the cells are clumped, they are counted as one cell.
(5) Cell suspension concentration = total number of cells in square/4×10 4 And each mL.
2) CCK-8 cell proliferation assay
The present study uses CCK-8 kit (Dojindo, japan) to examine colorectal cancer cell proliferation capacity.
(1) 100. Mu.L of the cell suspension was aspirated and diluted to a cell size of about 2,000 at the concentration counted, and plated in 96-well plates.
(2) Cell activity assays were performed at four time points, 24h, 48h, 72h and 96 h. To each well, 10. Mu.L of CCK-8 reagent was added, and the mixture was shaken up and down and left and right to mix gently and returned to the cell incubator for further cultivation for 1.5 hours.
(3) Absorbance measurement was performed using a microplate reader, the wavelength was set at 450nm, and cell proliferation curves of different experimental groups were plotted according to absorbance values at four time points to compare differences.
3) Cloning formation experiments
(1) After 36h of transfection of cells, cell counts were performed and diluted to appropriate concentrations. 2mL of the cell suspension was added to each well of the 6-well plate to give a cell amount of about 1,000, and the culture was continued for about 2 weeks.
(2) During which the cell morphology was observed every 2-3 days and fresh medium was changed. If cell clones are observed under a microscope, a fixation stain may be performed.
(3) The 6-well plate was removed from the incubator, the waste liquid was aspirated and slowly washed 2 times with PBS. After each well of PBS was blotted, 2mL of methanol solution was added and left at room temperature for 30min.
(4) After methanol was removed by suction, a 0.25% crystal violet solution was added and the mixture was left in the dark for 30min to dye.
(5) After the dyeing is finished, waste liquid in the hole is sucked, the hole plate is washed by double distilled water until the visual field is clear, and photographing and storage are carried out.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (2)
1. Application of a detection reagent of enhancer RNA functional site rs3094296 in preparation of colorectal cancer auxiliary diagnosis kit.
2. The use according to claim 1, characterized in that the detection reagent comprises a specific amplification primer of enhancer RNA functional site rs3094296, the specific amplification primer sequence being SEQ ID NO:1 and SEQ ID NO:2, the specific probe sequence is SEQ ID NO:3 and SEQ ID NO:4.
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