CN112760321A - Detection method of pan-cancer inhibition non-coding RNA and pan-cancer inhibition attribute thereof - Google Patents
Detection method of pan-cancer inhibition non-coding RNA and pan-cancer inhibition attribute thereof Download PDFInfo
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Abstract
The invention provides PAn-cancer inhibiting Non-Coding RNA which is PANC-cancer Non-Coding RNA 246 and is named as PANC 246. Also provided is a method for detecting a pan-cancer suppressing property of a pan-cancer suppressing non-coding RNA, comprising the steps of: 2.1, constructing plasmids and culturing tumor cell lines; 2.2, detecting the proliferation capacity of a tumor cell strain over-expressing PANC246 by a CCK-8 experiment; 2.3, detecting the apoptosis rate of the PANC246 cancer cells by flow cytometry; 2.4, detecting the tumor cell migration inhibition ability of PANC246 by a scratch test; 2.5, detecting the tumor cell invasion inhibition ability of PANC246 by Transwell chamber experiment; 2.6, detecting the situation of the mRNA level of the protooncogene inhibited by the up-regulated expression of the PANC246 by fluorescent quantitative PCR. According to the invention, a brand-new PAn-cancer inhibiting ncRNA is discovered through PAn-cancer data mining in a TCGA database, and is named as PANC246(Pan-cancer Non-Coding RNA 246). The patent confirms the pan-cancer inhibition attribute of PANC246 by bioinformatics and functional research, and provides a new non-coding gene target for treating tumors.
Description
Technical Field
The invention belongs to the field of medicine and health, and particularly relates to a pan-cancer inhibition non-coding RNA and a detection method of pan-cancer inhibition attribute thereof.
Background
Non-coding RNA (ncRNA) is a class of RNA molecules that are not translated into protein, including transfer RNA (tRNAs), ribosomal RNA (rRNAs), microRNA (microRNAs), small interfering RNA (siRNAs), Piwi protein-interacting RNA (piRNAs), nucleolar RNA (snornas), extracellular RNA (exoRNAs), small nucleolar RNA (snRNAs), small cajal body-associated RNA (small cajal body associated RNAs, scarRNAs), circular RNA (circRNAs), and long non-coding RNA (lncRNAs). Early researches suggest that the ncRNA is 'garbage' of genome, but more and more evidences indicate that the ncRNA gene is deeply involved in the occurrence and development of diseases, so that the research on the role of the ncRNA in disease pathogenesis is particularly important, and the research on the ncRNAs with cancer inhibition and the mechanism thereof are particularly needed.
Oncogenes and cancer suppressor genes (TSGs) are two major types of genes involved in the development of tumorigenesis. Oncogenes cause uncontrolled growth of cells, while TSGs often act as negative regulators of oncogenes, cell cycle checkpoints or gene products, inhibiting the development of cancer. Traditional tumor suppressor gene screening is mainly limited to genes encoding proteins, and extension of this screening to non-protein encoding genes or regions allows discovery of new TSGs to discover new pan-cancer suppressor ncrnas, in order to provide new non-coding gene targets for tumor therapy.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a pan-cancer suppressing non-coding RNA and a detection method of pan-cancer suppressing attribute thereof, which are used for determining the pan-cancer suppressing attribute of PANC246 through bioinformatics and functional research and providing a new non-coding gene target for treating tumors.
In order to solve the above technical problems, the present invention provides a PAn-cancer suppressing Non-Coding RNA, which is named as PANC246, of PAN-cancer Non-Coding RNA 246.
The PANC246 has the following identification steps:
1.1, collecting transcriptome sequencing data of all tumors in a TCGA database;
1.2, extracting 11,529 non-coding RNAs from 11,093 RNA-seq data, and performing genome-wide meta analysis to identify new pan-cancer regulatory ncRNAs;
1.3, by using a stochastic effect model, 1615 ncRNAs were found to be significantly up-or down-regulated, wherein Pan-cancer Non-Coding RNA 246 is a novel ncRNA encoded by ENSG00000231246 and PANC246 is significantly down-regulated in 23 human cancer tissues.
The invention also provides a detection method of pan-cancer inhibition attribute of pan-cancer inhibition non-coding RNA, which comprises the following steps:
2.1, constructing plasmids and culturing tumor cell lines;
2.2, detecting the proliferation capacity of a tumor cell strain over-expressing PANC246 by a CCK-8 experiment;
2.3, detecting the apoptosis rate of the PANC246 cancer cells by flow cytometry;
2.4, detecting the tumor cell migration inhibition ability of PANC246 by a scratch test;
2.5, detecting the tumor cell invasion inhibition ability of PANC246 by Transwell chamber experiment;
2.6, detecting the situation of the mRNA level of the protooncogene inhibited by the up-regulated expression of the PANC246 by fluorescent quantitative PCR.
Wherein, the step 2.1 comprises the following specific steps:
2.1.1 Place six cell lines of esophageal cancer cell line Eca109, liver cancer cell lines HepG2 and Bel7402, gastric cancer cell line MGC803, colorectal cancer cell lines SW480 and DLD1, and plasmid pcDNA3.1 in DMEM medium (Kaikyi Bio, Nanjing) containing 10% fetal bovine serum (ExCell, USA) at 37 deg.C and 5% CO2An incubator;
2.1.2, chorus ENSG00000231246.1 was synthesized by Kinsley Biotechnology Ltd (Nanjing, China) and cloned into pcDNA3.1 plasmid, passed through Lipofectamine3000(Invitrogen, USA) and then transfected into the above cancer cell lines.
Wherein, the step 2.2 comprises the following specific steps: 2x105The cells are cultured in a 24-hole culture plate, pcDNA3.1 empty plasmid and PANC246 overexpression plasmid are transfected, the cells are collected for 0 hour, 24 hours, 48 hours and 72 hours respectively, and the proliferation capacity of the tumor cell strain overexpressing the PANC246 is detected by adopting a CCK-8 experiment.
Wherein, the specific steps of the step 2.4 are as follows:
2.4.1, inoculating the cells into a 6-well culture plate when the cells are 70-80% fused after culturing the cells for 24 hours;
2.4.2, lightly and slowly scratching the cell layer by using a 200-microliter pipette tip, after scratching, lightly washing the cell layer by using a culture medium for 2 times, removing the exfoliated cells, and measuring the gap distance as the gap distance at 0 h;
2.4.3 to determine the cell migration capacity, the gap distances were measured at 24h and 48h after scratching and the migration distance was calculated as:
the migration distance is equal to the gap distance at 0 h-t;
wherein t is 24h and 48 h.
Wherein, the specific steps of the step 2.5 are as follows:
2.5.1 cell suspensions (0.5X 10) to be transfected with pcDNA3.1-PANC246, untransfected controls and pcDNA3.15Individual cells/mL) was added to the Transwell chamber while adding 200 μ L of serum-free medium, and 500 μ L of medium containing 10% fetal bovine serum was added to the lower chamber as a stimulus for invasive transfer;
2.5.2, placing the Transwell chamber in a cell culture box for incubation for 24h, then carrying out crystal violet staining on invasive cells on the lower surface of the chamber membrane for 10min, taking a picture under a microscope at 40 times of high-power visual field, and detecting the tumor cell invasion inhibition capacity of PANC 246.
Wherein, the specific steps of the step 2.3 are as follows:
2.3.1 cells were trypsinized and washed twice with pre-cooled Phosphate Buffered Saline (PBS). Add 500. mu.L of binding buffer (KeyGen) to resuspend the cells to 5X 105(ii) individual cells;
2.3.2 Add 5. mu.L Annexin V-FITC (KeyGen) to the cell suspension and counterstain the cell suspension with 5. mu.L Propidium Iodide (PI);
2.3.2, the mixture was incubated at room temperature in the dark for 10min and then subjected to apoptosis analysis using a FlowSight flow cytometer (Merck, Germany).
Wherein, the specific steps of the step 2.6 are as follows:
2.6.1, using TRIzol (Invitrogen, USA) to collect the total RNA, using the first strand cDNA synthesis kit (Vazyme Biotech, China) according to the instructions to convert it into cDNA;
2.6.2, adopting SYBR Green Master Mix (Vazyme Biotech) to carry out real-time fluorescence quantitative PCR on an ABI 7500PCR instrument (ABI, USA), and detecting the situation that the upregulated expression of PANC246 inhibits the mRNA level of protooncogene.
The technical scheme of the invention has the following beneficial effects: according to the invention, a brand-new PAn-cancer inhibiting ncRNA is discovered through PAn-cancer data mining in a TCGA database, and is named as PANC246(Pan-cancer Non-Coding RNA 246). The patent confirms the pan-cancer inhibition attribute of PANC246 by bioinformatics and functional research, and provides a new non-coding gene target for treating tumors.
Drawings
FIG. 1 is a graph showing the results of genome-wide analysis for identifying abnormally expressed non-coding RNAs in accordance with the present invention;
FIG. 2 is a schematic representation of the broad down-regulation of expression of PANC246 in 23 human cancers in accordance with the present invention;
FIG. 3 is a diagram showing the result of CCK-8 experiment for detecting the proliferation capacity of tumor cell line over-expressing PANC246 in the present invention;
FIG. 4 is a graph showing the results of flow cytometry detection of apoptosis of PANC 246-promoting cancer cells in the present invention;
FIG. 5 is a graph showing the results of the scratch test of the present invention for detecting the ability of PANC246 to inhibit tumor cell migration;
FIG. 6 is a graph showing the results of a Transwell chamber assay of the ability of PANC246 to inhibit tumor cell invasion in accordance with the present invention;
FIG. 7 is a graph showing the results of up-regulating the mRNA level of the protooncogene expressed by PANC246 in the fluorescent quantitative PCR assay of the present invention;
FIG. 8 is a graph showing the results of flow cytometry detection of apoptosis in PANC 246-facilitated cancer cells in accordance with one embodiment of the present invention;
FIG. 9 is a scratch test conducted in example one to examine the inhibition of the migration of colorectal cancer cell DLD1 by PANC246 and its statistical histogram;
FIG. 10 is a scratch test of the first embodiment to detect the migration of the esophageal cancer cell Eca109 inhibited by PANC246 and its statistical histogram;
FIG. 11 is a statistical histogram of the inhibition of migration of liver cancer cell HepG2 by PANC246 in the scratch test of the first embodiment;
fig. 12 is a statistical histogram of the inhibition of the migration of MGC803 of gastric cancer cells by PANC246 in the scratch test according to the first embodiment;
FIG. 13 is a scratch test performed to examine the inhibition of migration of colorectal cancer cells SW480 by PANC246 and statistical histograms thereof in example one;
FIG. 14 is a graph showing the results of a Transwell chamber assay in one example to examine the ability of PANC246 to inhibit tumor cell invasion.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The invention provides PAn-cancer inhibiting Non-Coding RNA which is PANC-cancer Non-Coding RNA 246 and is named as PANC 246.
The recognition steps of the PANC246 are:
1.1, collecting transcriptome sequencing (RNA-seq) data of all tumors in TCGA database;
1.2, extracting 11,529 non-coding RNAs from 11,093 RNA-seq data, and performing genome-wide meta analysis to identify new pan-cancer regulatory ncRNAs;
1.3 Using the random Effect model, 1615 ncRNAs were found to be significantly up-or down-regulated (FIG. 1, bonferroni-corrected P)<0.05), wherein PAn-cancer Non-Coding RNA 246(PANC246) is a novel ncRNA encoded by ENSG00000231246, PANC246 was significantly down-regulated in 23 human cancer tissues (SMD ═ 4.4, P in the fixed effect model<1.0x10-227Random effect model P3.28 x10-21Fig. 2).
FIG. 1 is a graph of the results of genome-wide analysis for the identification of abnormally expressed non-coding RNA (ncRNA), and Manhattan plots (Manhattan plot) show the differentially expressed ncRNA of pan-cancerous RNA-seq in the TCGA dataset found by genome-wide meta analysis. Random effect model P values showed that there was a significant difference in expression of 1615 ncRNA genes between cancerous and paracancerous control tissues.
Fig. 2 is a schematic representation of PANC246 widely down-regulated expression in 23 human cancers, sample data from the TCGA project (N-10,490). Prior to meta-analysis, the gene expression level was log2 transformed. Then, a fixed effect model and a random effect model are used for analysis. 95% CI is used to represent the mean and 95% interval of expression. To show more details of the different studies, any normalized mean difference (SMD) above-15 and below 5 is indicated by an arrow. The purple filled rectangles represent SMDs of the fixed effect model and the random effect model.
The invention also provides a detection method of pan-cancer inhibition attribute of pan-cancer inhibition non-coding RNA, which comprises the following steps:
2.1, constructing plasmids and culturing tumor cell lines;
2.2, detecting the proliferation capacity of a tumor cell strain over-expressing PANC246 by a CCK-8 experiment;
to investigate the functional role of PANC246 in cancer cell phenotype, PANC246 plasmid was transfected in tumor cell lines and the CCK-8 method analyzed the effect of PANC246 overexpression on tumor cell proliferation. Compared with the control group or pcDNA3.1 group, the growth curves of the stomach cancer cell line MGC803 and the esophageal cancer Eca109 cells transfected by PANC246 were remarkably slowed down, and the proliferation capacity was remarkably inhibited, especially after 48h and 72h (P ═ 0.0004, P ═ 0.0002; FIGS. 3E and 3C). Similar proliferation patterns were also observed for the two liver cancer cell lines HepG2 and Bel-7402 (P0.00002, P0.00004; fig. 3D and 3A) and the two CRC cell lines SW480 and DLD1 (P0.00023 and P0.0007) (fig. 3F and 3B).
FIG. 3 is a diagram showing the result of CCK-8 experiment for detecting the proliferation potency of tumor cell line overexpressing PANC246, and FIGS. 3A and 3D show the proliferation potency of liver cancer cell line overexpressing PANC 246; FIG. 3B and FIG. 3F colorectal cancer cell lines overexpress the proliferative capacity of PANC 246; FIG. 3C the proliferative capacity of esophageal cancer cell lines over-expressing PANC 246; FIG. 3E gastric cancer cell line overexpresses the proliferative capacity of PANC 246; p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
2.3, detecting the apoptosis rate of the PANC246 cancer cells by flow cytometry;
to determine whether overexpression of PANC246 induced an increase in tumor cell apoptosis, the rate of tumor cell apoptosis was measured using flow cytometry. As shown in fig. 4, the apoptosis rate of the CRC cell strain SW480 after transfection of PANC246 was significantly increased compared to the control group and pcdna3.1 group (P ═ 0.0022). The same trend of increased apoptosis was also detected in Eca109(P ═ 0.001), HepG2(P ═ 0.0045), Bel-7402(P ═ 0.0018), MGC803(P <1.0x10-4) and DLD1(P ═ 5.0x10-4) cell lines.
FIG. 4 is a graph showing the results of detecting apoptosis of PANC 246-promoting cancer cells by flow cytometry, detecting early apoptosis by Annexin V-FITC staining and detecting late apoptosis by PI staining; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
2.4, detecting the tumor cell migration inhibition ability of PANC246 by a scratch test;
through scratch test experiments, as shown in FIGS. 5A and 5B, after the Bel7402 liver cancer cells transfected with pcDNA3.1-PANC246 (abbreviated as PANC246) are cultured for 24h and 48h, the migration distance is obviously shorter than that of the control group and pcDNA3.1 group, and P is less than 0.001 and P is less than 1.0x 10-4. Similar results were also observed in Eca109, HepG2, MGC803, SW480 and DLD1 cells when cultured for 48h (FIG. 5C).
FIG. 5 is a graph showing the results of a scratch test for detecting the ability of PANC246 to inhibit tumor cell migration, and FIG. 5A is a graph showing the ability of PANC246 to inhibit the migration of hepatoma carcinoma cell Bel 7402; FIG. 5B is a statistical histogram of PANC246 inhibition of migration of hepatoma cell Bel7402 detected by the scratch test; FIG. 5C statistical histogram of scratch test assay for PANC246 inhibition of colorectal cancer cell DLD1 migration; p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
2.5, detecting the tumor cell invasion inhibition ability of PANC246 by Transwell chamber experiment;
the effect of overexpression of PANC246 on tumor cell invasion was examined by Transwell experiments. It was found that after 48h of culture of Bel7402 hepatoma cells, there was a significant decrease in the cells invading the membrane of the Transwell chamber in the PANC246 group compared to the control group and pcdna3.1 group (P ═ 0.0003, fig. 6A and 6B). Meanwhile, the studies also found that the invasive ability of Eca109(P ═ 1.87x10-5), HepG2(P ═ 0.0004), MGC803(P ═ 0.0002), SW480(P ═ 0.0011), and DLD1(P ═ 0.0007) cells was significantly reduced (fig. 6B).
Fig. 6 is a graph of the results of a Transwell chamber experiment testing the ability of PANC246 to inhibit tumor cell invasion, fig. 6A showing significant inhibition of tumor cell invasion by PANC246 through tumor cell staining of the membrane of the Transwell chamber; FIG. 6 statistical histogram of BTranswell cell experiment; p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
2.6, detecting the mRNA level condition of the protooncogene inhibited by the up-regulated expression PANC246 by fluorescent quantitative PCR;
some protooncogenes, such as cyclin dependent kinase 2(CDK2), murine sarcoma virus oncogene (KRAS, P21), titin (TTN), mucin 16(MUC16, CA125), phosphatidylinositol-4, 5-bisphosphatase catalytic subunit α (PIK3CA), CUB and Sucshi multi-domain complement regulator protein 3(CSMD3), are significantly highly expressed in tumors and promote tumorigenesis. It is hypothesized that PANC246, a cancer suppressor ncRNA, may suppress the expression of these proto-oncogenes. As shown in fig. 7, the overexpressed PANC246 significantly suppressed the expression levels of CDK2 gene, KRAS gene, TTN gene, MUC16 gene, PIK3CA gene, and CSMD3 gene mRNA, compared to the control group and pcdna3.1 group. Therefore, the PANC246 can directly or indirectly regulate the expression of protooncogene and play a role in inhibiting cancers.
FIG. 7 is a graph showing the results of fluorescent quantitative PCR assays for up-regulating the level of proto-oncogene mRNA expressed by PANC246, FIG. 7A proto-oncogene CDK 2; FIG. 7B protooncogene CSMD 3; FIG. 7C protooncogene KRAS; FIG. 7D protooncogene MUC 16; FIG. 7E proto-oncogene PIK3 CA; FIG. 7F protooncogene TTN; p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
The technical scheme of the invention is further illustrated by the following specific examples.
A detection method for pan-cancer inhibition attribute of pan-cancer inhibition non-coding RNA comprises the following specific experimental methods:
I. cell lines and plasmids
The esophageal cancer cell strain Eca109, the liver cancer cell strains HepG2 and Bel7402, the gastric cancer cell strain MGC803, the colorectal cancer cell strains SW480 and DLD1 and the plasmid pcDNA3.1 are stored in the laboratory. The six cell lines were placed in DMEM medium (Kaikyi organism, Nanjing) containing 10% fetal bovine serum (ExCell corporation, USA) at 37 deg.C and 5% CO2An incubator. Full-length ENSG00000231246.1 was synthesized by kasuga biotechnology ltd (south kyo, china) and cloned into pcdna3.1 plasmid, which was transfected into the above cancer cell lines by Lipofectamine3000(Invitrogen, usa).
II. Cell proliferation assay
2x105The cells are cultured in a 24-hole culture plate, pcDNA3.1 empty plasmid and PANC246 overexpression plasmid are transfected, the cells are collected for 0 hour, 24 hours, 48 hours and 72 hours respectively, and the cell proliferation capacity is calculated by adopting a CCK-8 method.
III, cell migration capability detection by cell scratching experiment
When the cells were in 70-80% confluency after 24h of culture, they were plated in 6-well plates. The cell layer was gently and slowly crossed with a 200. mu.L pipette tip. After scratching, the exfoliated cells were removed by gently rinsing with the medium 2 times, and the gap distance was measured as the gap distance at 0 h. To determine cell migration ability, the gap distance was measured at 24h and 48h after scratching. The migration distance is calculated by the formula:
the migration distance is 0 h-t (t is 24h or 48 h).
FIG. 9 is a scratch test performed to examine the inhibition of colorectal cancer cell DLD1 migration by PANC246 (left panel in FIG. 9) and its statistical histogram (right panel in FIG. 9); p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
FIG. 10 is a scratch test for detecting that PANC246 inhibits the migration of esophageal cancer cells Eca109 (left panel in FIG. 10) and a statistical histogram thereof (right panel in FIG. 10); p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
FIG. 11 is a scratch test for detecting that PANC246 inhibits the migration of hepatoma cell HepG2 (left panel in FIG. 11) and a statistical histogram thereof (right panel in FIG. 11); p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
Fig. 12 shows scratch test detection PANC246 inhibits the migration of the gastric cancer cell MGC803 (left panel in fig. 12) and its statistical histogram (right panel in fig. 12); p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
FIG. 13 is a scratch test assay to detect that PANC246 inhibits the migration of colorectal cancer cells SW480 (left panel in FIG. 13) and statistical histograms thereof (right panel in FIG. 13); p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
IV, Transwell chamber method for detecting cell invasion capacity
Cell invasion capacity assays were performed using Transwell chambers (Chemicon, usa). Cell suspensions (0.5X 10) transfected with pcDNA3.1-PANC246, untransfected control and pcDNA3.15Individual cells/mL) was added to the Transwell chamber along with 200 μ L of serum free medium, and 500 μ L of medium containing 10% fetal bovine serum was added to the lower chamber as a stimulus for invasive metastasis. After the Transwell chamber was placed in a cell incubator and incubated for 24h, the invasive cells on the lower surface of the chamber membrane were stained with crystal violet for 10min and photographed under a microscope at 40 times higher field.
FIG. 14 is a graph showing the results of a Transwell chamber assay for the ability of PANC246 to inhibit tumor cell invasion, and it can be seen in FIG. 14 that staining of tumor cells through the membrane of the Transwell chamber indicates that PANC246 significantly inhibits tumor cell invasion; p < 0.001; p < 0.0001; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
V, apoptosis detection
Cells were trypsinized and washed twice with pre-cooled Phosphate Buffered Saline (PBS). Adding 500 mu L knotThe confluent buffer (KeyGen) was resuspended to 5X 10 cell number5And (4) cells. mu.L Annexin V-FITC (KeyGen) was added to the cell suspension, and the cell suspension was counterstained with 5. mu.L Propidium Iodide (PI). The mixture was incubated in the dark at room temperature for 10min, and then apoptosis analysis was performed using FlowSight flow cytometer (Merck, germany).
FIG. 8 is a graph showing the results of the flow cytometry detection of apoptosis of PANC 246-promoted cancer cells in the present example, Annexin V-FITC staining for early apoptosis, and PI staining for late apoptosis; control: untransfected control group; pcDNA3.1: empty plasmid transfection group; PANC 246: pcDNA3.1-PANC246 plasmid transfection group.
VI, fluorescent quantitative PCR
The total RNA was collected using TRIzol (Invitrogen, USA), and converted into cDNA using a first strand cDNA synthesis kit (Vazyme Biotech, China) according to the instructions. Real-time fluorescent quantitative PCR was performed on an ABI 7500PCR instrument (ABI, USA) using SYBR Green Master Mix (Vazyme Biotech). The GAPDH gene served as an internal reference for this experiment.
VII, PANC246 screening and meta analysis
11,093 gene expression data were downloaded from the TCGA database (https:// portal.gdc.cancer. gov/repository) and the data source was transcriptome sequencing (RNA-seq) data. The RNA-seq data initially covered 32 cancer types, but excluded 9 of them due to the low sample size of the control group. Differential gene expression analysis was performed using the log2 of transcripts of HTSeq per kilobase for the quartile on the transformed fragment (FPKM-UQ). Differential gene expression analysis was performed using the Bayesian generalized linear model (bayesglm) of the ARM package (v 1.10-1). Meta analysis was performed on 23 cancer types using the Metacor software package (v 2.1-1).
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A PAn-cancer suppressing Non-Coding RNA, which is a Pan-cancer Non-Coding RNA 246 and is named PANC 246.
2. The pan-cancer suppressing non-coding RNA according to claim 1, wherein the recognition step of PANC246 is:
1.1, collecting transcriptome sequencing data of all tumors in a TCGA database;
1.2, extracting 11,529 non-coding RNAs from 11,093 RNA-seq data, and performing genome-wide meta analysis to identify new pan-cancer regulatory ncRNAs;
1.3, by using a stochastic effect model, 1615 ncRNAs were found to be significantly up-or down-regulated, wherein Pan-cancer Non-Coding RNA 246 is a novel ncRNA encoded by ENSG00000231246 and PANC246 is significantly down-regulated in 23 human cancer tissues.
3. A method for detecting a pan-cancer suppressing property of a pan-cancer suppressing non-coding RNA, comprising the steps of:
2.1, constructing plasmids and culturing tumor cell lines;
2.2, detecting the proliferation capacity of a tumor cell strain over-expressing PANC246 by a CCK-8 experiment;
2.3, detecting the apoptosis rate of the PANC246 cancer cells by flow cytometry;
2.4, detecting the tumor cell migration inhibition ability of PANC246 by a scratch test;
2.5, detecting the tumor cell invasion inhibition ability of PANC246 by Transwell chamber experiment;
2.6, detecting the situation of the mRNA level of the protooncogene inhibited by the up-regulated expression of the PANC246 by fluorescent quantitative PCR.
4. The method for detecting the pan cancer suppressing property of a pan cancer suppressing non-coding RNA according to claim 3, characterized in that the specific steps of step 2.1 are:
2.1.1 placing six cell strains of esophageal cancer cell strain Eca109, liver cancer cell strains HepG2 and Bel7402, gastric cancer cell strain MGC803, colorectal cancer cell strains SW480 and DLD1 and plasmid pcDNA3.1 in DMEM containing 10% fetal bovine serum to culture at 37 deg.C、5％CO2An incubator;
2.1.2, chorus ENSG00000231246.1 and cloning into pcDNA3.1 plasmid, and transfecting into the cancer cell strain.
5. The method for detecting the pan cancer suppressing property of a pan cancer suppressing non-coding RNA according to claim 3, characterized in that the specific steps of step 2.2 are: 2x105The cells are cultured in a 24-hole culture plate, pcDNA3.1 empty plasmid and PANC246 overexpression plasmid are transfected, the cells are collected for 0 hour, 24 hours, 48 hours and 72 hours respectively, and the proliferation capacity of the tumor cell strain overexpressing the PANC246 is detected by adopting a CCK-8 experiment.
6. The method for detecting the pan cancer suppressing property of a pan cancer suppressing non-coding RNA according to claim 3, characterized in that the specific steps of step 2.4 are:
2.4.1, inoculating the cells into a 6-well culture plate when the cells are 70-80% fused after culturing the cells for 24 hours;
2.4.2, scratching the cell layer by using a 200-microliter pipette tip, after scratching, slightly washing the cell layer for 2 times by using a culture medium, removing the fallen cells, and measuring the gap distance as the gap distance of 0 h;
2.4.3 to determine the cell migration capacity, the gap distances were measured at 24h and 48h after scratching and the migration distance was calculated as:
the migration distance is equal to the gap distance at 0 h-t;
wherein t is 24h and 48 h.
7. The method for detecting the pan cancer suppressing property of a pan cancer suppressing non-coding RNA according to claim 3, characterized in that the specific steps of step 2.5 are:
2.5.1, adding the cell suspension of the transfected pcDNA3.1-PANC246, the untransfected control and pcDNA3.1 into a Transwell chamber while adding 200. mu.L of a serum-free medium, and then adding 500. mu.L of a medium containing 10% fetal calf serum into the lower cavity as the stimulus for invasion and transfer;
2.5.2, placing the Transwell chamber in a cell culture box for incubation for 24h, then carrying out crystal violet staining on invasive cells on the lower surface of the chamber membrane for 10min, taking a picture under a microscope at 40 times of high-power visual field, and detecting the tumor cell invasion inhibition capacity of PANC 246.
8. The method for detecting the pan cancer suppressing property of a pan cancer suppressing non-coding RNA according to claim 3, characterized in that the specific steps of step 2.3 are:
2.3.1 cells were trypsinized and washed twice with pre-cooled phosphate buffer. Resuspend to 5X 10 cell number by adding 500. mu.L of binding buffer5(ii) individual cells;
2.3.2, adding 5 mu L Annexin V-FITC into the cell suspension, and then counterstaining the cell suspension by 5 mu L propidium iodide;
2.3.2, the mixture was incubated in the dark at room temperature for 10min, followed by apoptosis analysis using FlowSight flow cytometer.
9. The method for detecting the pan cancer suppressing property of a pan cancer suppressing non-coding RNA according to claim 3, characterized in that the specific steps of step 2.6 are:
2.6.1, collecting the total RNA by using TRIzol, and converting the total RNA into cDNA by using a first strand cDNA synthesis kit;
2.6.2, adopting SYBR Green Master Mix to carry out real-time fluorescence quantitative PCR on an ABI 7500PCR instrument, and detecting the mRNA level condition of the up-regulated expression PANC246 inhibition protooncogene.
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