阅读说明：本技术 用于结直肠癌早期检测和复发监测的生物标志物及其应用 (Biomarker for early detection and recurrence monitoring of colorectal cancer and application thereof ) 是由 邱芳华 刘道利 于 2021-01-08 设计创作，主要内容包括：用于结直肠癌早期检测和复发监测的生物标志物及其应用,涉及生物技术领域；所述生物标志物为环状RNA circ-HIPK3。circ-HIPK3表达的下调抑制了大肠癌细胞的迁移、侵袭和存活,而circ-HIPK3的过度表达则显著促进了肿瘤细胞的迁移、侵袭和体外存活。(A biomarker for early detection and recurrence monitoring of colorectal cancer and application thereof, relating to the field of biotechnology; the biomarker is circular RNA circ-HIPK 3. The down-regulation of the expression of circ-HIPK3 inhibits the migration, invasion and survival of colorectal cancer cells, while the over-expression of circ-HIPK3 significantly promotes the migration, invasion and survival of tumor cells in vitro.)

1. Biomarkers for early detection and recurrence monitoring of colorectal cancer characterized by: the biomarker is circular RNA circ-HIPK 3.

2. The biomarker for early detection and recurrence monitoring of colorectal cancer according to claim 1, characterized in that: the sequence of the circ-HIPK3 is SEQ ID NO. 1 or SEQ ID NO. 2; wherein the content of the first and second substances,

sequence No. 1 is 5'-CAATCTCGGTACTACAGGTATG-3';

the sequence of SEQ ID NO. 2 is 5'-TCACATAGGTCCGTGGATAG-3'.

3. The biomarker for early detection and recurrence monitoring of colorectal cancer according to claim 1, characterized in that: the expression of the circ-HIPK3 is positively correlated with the migration, invasion and proliferation of colorectal cancer cells.

4. The biomarker for early detection and recurrence monitoring of colorectal cancer according to claim 3, characterized in that: when the expression of circ-HIPK3 is down-regulated, the activity of corresponding colorectal cancer cells is inhibited; the up-regulation of the expression of circ-HIPK3 corresponds to the increase of the activity of colorectal cancer cells.

5. The biomarker for early detection and recurrence monitoring of colorectal cancer according to claim 3, characterized in that: the colorectal cancer cell is at least one of Lovo cell, SW620 cell, HT29 cell and SW480 cell.

6. Use of the biomarker for early detection and recurrence monitoring of colorectal cancer according to any one of claims 1 to 5 in a drug for colorectal cancer detection.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a biomarker of colorectal cancer.

Background

Colorectal cancer is one of the most important problems threatening human health worldwide, ranked third among all cancers, with over 120 million new cases each year worldwide and over 600,000 cancer-related deaths. In china, the incidence and mortality of colorectal cancer has been rising dramatically, and two thirds of colorectal cancers are clinically diagnosed in the late stages of the disease. Early stage colorectal cancer can be treated by surgery, and chemoradiotherapy is used for adjuvant treatment of patients with advanced stage colorectal cancer. Recently, targeted therapies and immunotherapies for colorectal cancer are also under way. At present, the 5-year survival rate of colorectal cancer patients is about 50-70%. Therefore, in order to effectively control colorectal cancer and improve patient survival, early detection and preventive studies are urgently needed.

Recent studies on circular non-coding rnas (circrnas) have shown that it is likely to be a monitoring and prognostic biomarker for cancer recurrence and progression in early cancer detection or after treatment. circrnas are a class of non-protein coding RNAs, which are naturally occurring endogenous ncrnas, with single-stranded covalently closed circular molecules with neither 5'-3' polarity nor polyadenylation tails. circRNA is abundant and stable in the bloodstream and therefore potentially useful as a non-invasive biomarker. In addition, they can provide useful information to study tumor biology and represent a new targeted therapeutic strategy. To date, a large number of circRNAs have been identified in various cancer cells, which, despite limited knowledge of the biology of circRNAs, can target mirnas and mrnas.

Therefore, we expect that research on circRNA in colorectal cancer provides a new idea for the development of tumor biomarkers for early detection of colorectal cancer.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a biomarker for early detection and recurrence monitoring of colorectal cancer, wherein the expression of circ-HIPK3 is down-regulated and corresponds to the inhibition of colorectal cancer cell activity; the up-regulation of the expression of circ-HIPK3 corresponds to the increase of the activity of colorectal cancer cells.

The invention also aims to provide application of the biomarker in a colorectal cancer detection drug.

The purpose of the invention is realized by adopting the following technical scheme:

a biomarker for early detection and recurrence monitoring of colorectal cancer is provided, the biomarker being cyclic circ-HIPK3(has _ circ _ 0000284).

Further, the sequence of the circ-HIPK3 is SEQ ID NO. 1 or SEQ ID NO. 2; wherein the sequence of SEQ ID NO. 1 is 5'-CAATCTCGGTACTACAGGTATG-3';

the sequence of SEQ ID NO. 2 is 5'-TCACATAGGTCCGTGGATAG-3'.

Further, the expression of the circ-HIPK3 is positively correlated with the migration, invasion and proliferation of colorectal cancer cells.

Further, the down-regulation of the expression of circ-HIPK3 corresponds to the inhibition of the activity of colorectal cancer cells; the up-regulation of the expression of circ-HIPK3 corresponds to the increase of the activity of colorectal cancer cells.

Further, the colorectal cancer cell is at least one of a Lovo cell, a SW620 cell, a HT29 cell and a SW480 cell.

Use of biomarkers for early detection and recurrence monitoring of colorectal cancer in colorectal cancer detection drugs.

Compared with the prior art, the invention has the beneficial effects that:

the biomarker detection and prognosis of the invention have high reliability, and ROC analysis shows that the AUC of the circ-HIPK3 is 0.815 (95% reliability interval, 0.674-0.956); the down-regulation of the expression of circ-HIPK3 inhibits the migration, invasion and survival of colorectal cancer cells, while the over-expression of circ-HIPK3 significantly promotes the migration, invasion and survival of tumor cells in vitro.

Drawings

FIG. 1A is a comparison graph of circRNA sequencing in example 1 of the present invention.

FIG. 1B is a simplified structural diagram of circRNA in example 1 of the present invention.

FIG. 1C is a diagram of genomic distribution data of circRNA of example 1 of the present invention.

FIG. 1D is a schematic representation of the differential expression of circRNA in example 1 of the present invention.

Fig. 2A is a schematic diagram of the potential binding of circRNA to miRNA according to example 2 of the present invention.

FIG. 2B is a comparative graph of the gene pathway analysis of the circRNA-binding miRNA network of example 2 of the invention.

FIG. 2C is a graph comparing the gene pathway data of FIG. 2B of the present invention.

FIG. 3 is a chart comparing the expression of circRNA in example 3 of the present invention.

FIG. 4A is a graph comparing the expression levels of circ-HIPK3 in rectal tissue according to example 3 of the present invention.

FIG. 4B is a diagram showing ROC analysis of circ-HIPK3 in example 3 of the present invention.

FIGS. 4C and 4D are graphs showing the expression of circ-HIPK3 in example 3 of the present invention.

FIG. 5A is a table comparing the expression of circ-HIPK3 in highly aggressive colorectal cancer cell lines in example 4 of the present invention.

FIG. 5B is a schematic diagram of tumor cells in example 4 of the present invention.

FIGS. 5C and 5D are graphs comparing the survival rates of tumor cells in example 4 of the present invention.

FIG. 6 is a comparison of cell cycles in example 4 of the present invention.

FIGS. 7A and 7B are schematic views showing the changes in the distribution of tumor cells in example 4 of the present invention.

FIG. 7C is a table of the data statistics of FIGS. 7A and 7B according to the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

Example 1

Colorectal cancer and paracancerous tissue circRNA expression levels:

1. construction of cDNA library and DNA sequencing.

Total RNA was isolated from cells and tissues using Trizol reagent (Invitrogen, Carlsbad, Calif., USA) following the manufacturer's protocol and quantified using the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, Santa Clara, Calif., USA). Approximately 10. mu.g of total RNA was purified using Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA) and these poly (A) -or poly (A) + RNA or circRNA fractions were fragmented at high temperature using divalent cations into small pieces, then the cleaved RNA fragments were reverse transcribed into cDNA according to the mRNA-Seq sample preparation Kit (Illumina) for cDNA library construction, with an average insert size of 300bp (+ -50 bp) in the paired-end library, and DNA was sequenced using Illumina Hiseq 3000.

The sequenced transcriptomes were paired in a million pairs, and reads yielded giga-DNA sequences approximately seven times the size of the genome. Prior to assembly, low quality reads (1, reads containing sequencing adapters; 2, reads containing sequencing primers; 3, nucleotides with a quality score of less than 20) were removed. The clean paired end reads were then submitted to NCBI Short Read Archive.

2. And (5) analyzing DNA sequence data.

To analyze differential circRNA expression, STAR was used to map reads to the genome and DCC to identify circRNA and estimate circRNA expression levels. The trimmed mean of M values (TMM) was used to normalize differential circRNA expression using the edgeR program. The miRanda tool is used to predict mirnas targeting circRNA.

As shown in fig. 1A, the circrnas differentially expressed in both colorectal cancer and paracarcinoma tissues, identified 17659 circrnas, of which 9466 were new circrnas, which were not recorded in the circRNA database.

These new circrnas were mostly generated by exon sequencing, and their median length was 359bp, see fig. 1B; as shown in fig. 1C, their genomic distribution data show that the circRNA is present in all human chromosomes.

As shown in FIG. 1D, differentially expressed circRNAs were selected in colorectal cancer and corresponding paracancerous tissues using criteria of ≧ 2-fold change and significant difference P <0.05, resulting in 724 differentially expressed circRNAs, 241 of which were upregulated and 483 downregulated.

Example 2

Binding miRNA and regulatory gene pathways:

microrna (miRNA) sponges are frequently reported functional models of circRNA for bioinformatics prediction of competitive binding of circRNA to endogenous mirnas. In this example, we first predicted potential binding of circRNA differentially expressed at 724 to miRNA in colorectal cancer tissues.

As shown in fig. 2A, genetic pathway analysis was performed on these differentially expressed circRNA-binding miRNA networks using the R language and Ingenuity pathway analysis IPA platform; a summary of the data for its relevant pathway is shown in figure 2B. Among them, as shown in fig. 2C, AMPK pathway, insulin resistance pathway, HIF-1 pathway and ErbB pathway play a major role in colorectal cancer.

Example 3

circRNA differentially expressed in colorectal cancer tissues:

blood samples were collected from colorectal cancer patients, centrifuged at 2,000g for 30 minutes, and the supernatant was transferred to a 50mL conical tube and centrifuged at 2,000g for 30 minutes. The supernatant was transferred to a new tube, and 0.5 volume of TEI reagent (Invitrogen) was added to the tube, or 1mL of ExoQuick reagent (SBI) was added per 5mL of medium, mixed well, and incubated overnight at 4 ℃. The next day, the mixture was centrifuged at 10,000g for 1 hour, and then the supernatant was aspirated. The resulting pellet was added in PBS and then the circRNA levels were analyzed using qRT-PCR.

To verify the expression of these differentially expressed circrnas in colorectal cancer and corresponding paracancerous tissues and exosomes, we first isolated the RNAs from the tissues or exosome samples using Trizol reagent (Invitrogen) and reverse transcribing them to cDNA using Geneseed II. qPCR was performed using ABI 7500 machine (ABI, USA). The qPCR conditions were: each SYBR Green reaction, in a total volume of 20. mu.l, contained 2. mu.l of DNA and each primer (10. mu.M). The PCR was performed for 5 minutes at 95 ℃ initially, followed by 40 cycles: 95 ℃ for 10 seconds, 60 ℃ for 34 seconds and 60 ℃ for 60 seconds. To verify whether SYBR Green dye was detected as specific for quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) products, we performed a thermal dissociation protocol on the samples after the last cycle of qRT-PCR to check if there was only one peak. And calculating the relative level of circRNA expression.

As shown in FIG. 3, in order to verify differentially expressed circRNAs, four circRNAs with major changes were selected in this example as circ-SATB2(has _ circ _0003915), circ-HIPK3(has _ circ _0000284), circ-ARHGAP12(has _ circ _0000231) and circ-AHNAK (has _ circ _ 0008194); to verify in 88 colorectal cancers and corresponding paracancerous tissues; among them, as shown in FIG. 3C, the expression of circ-HIPK3 was up-regulated in colon cancer tissues compared to the corresponding paracarcinoma tissues.

This example verifies the level of circ-HIPK3 expression in 18 other colorectal tissue samples as shown in figures 4A-B, which data further confirm the above data (significant difference P < 0.01).

As shown in fig. 4B, ROC analysis showed AUC of 0.815 (95% CI, 0.674-0.956), threshold of 0.0065, sensitivity and specificity of 88.9% and 61.1%, respectively.

As shown in fig. 4C, we correlated the expression level of circ-HIPK3 with the clinicopathological data of colorectal cancer patients, and found that the relative expression of circ-HIPK3 was higher in the tumor stage (higher in the a stage than in the B and C stages, significant difference P < 0.01).

As shown in fig. 4D, the expression of circ-HIPK3 was detected in exosomes of 30 healthy controls and colorectal cancer patients, and it was found that circ-HIPK3 expression was upregulated in exosomes of colorectal cancer patients (significant difference P < 0.001).

Example 4

The down-regulation of the expression of circ-HIPK3 has the inhibition effect on migration, invasion and proliferation of colorectal cancer cells.

Transwell tumor cell migration and invasion assay:

colorectal cancer cells were seeded at a density of 2 × 104/well in 24-well plates and incubated overnight. After 24 hours of transfection of HIPK3 siRNA and cDNA using Lipofectamine 2000(Invitrogen), cells were detached with 2.5% trypsin and 1X 10⁶The concentration of the solution/ml is mixed in serum-free DMEM. 200 μ l of the cell solution was added to each well of Transwell, and 600 μ l of DMEM containing 20% FBS was added to each well. Subsequently, Transwell filters with 8.0- μm pores were incubated at 37 ℃ for 48 hours, the filters being pre-coated with 50 μ L of matrix gel for the invasion assay. After the measurement, the cells remaining on the top surface of the filter were removed using a cotton swab, and the cells migrated to the lower surface of the membrane were fixed with 10% formalin, stained with a 0.5% crystal violet solution, and they were counted under an optical microscope for 5 random fields (× 200).

Flow cytometry Annexin V apoptosis assay:

to detect changes in apoptosis, cells 48 hours after HIPK3 siRNA or cDNA transfection were isolated using 2.5% trypsin and stained with FITC-conjugated annexin V and Propidium Iodide (PI) using the annexin V-FITC apoptosis detection kit (KeyGen, jiangsu). Analysis was performed by FACScan flow cytometry (DB Biosciences, San Jose, Calif., USA). The apoptosis rate was quantified using Cell Quest software (BD Biosciences).

As shown in FIG. 5A, the expression of circ-HIPK3 was detected in colorectal cancer cells, and it was found that circ-HIPK3 was highly expressed in highly aggressive colorectal cancer cell lines such as Lovo and SW620, but was less expressed in HT29, SW 480.

We reduced the expression of circ-HIPK3 in colorectal cancer Lovo cells and evaluated the effect of circ-HIPK3 on the cells. As shown in fig. 5B, tumor cell migration and invasion capacity decreased after decreasing circ-HIPK3 expression in colorectal cancer cells; as shown in fig. 5C, the survival rate of tumor cells did not change significantly.

As shown in FIG. 6, overexpression of circ-HIPK3 regulated the cell cycle of G1 (pre-DNA synthesis).

As shown in figure 7, down-regulation of circ-HIPK3 expression induced apoptosis in tumor cells, indicating that circ-HIPK3 is an oncogene or has oncogenic activity in colorectal cancer cells.

The above examples show that, compared with the tissue beside cancer, circ-HIPK3 is over-expressed in colorectal cancer cells, the migration, invasion and viability of colorectal cancer cells are promoted, and the tumor cell migration and invasion can be inhibited by knocking down the expression of circ-HIPK 3; therefore, the circ-HIPK3 is used as a biomarker, is suitable for early detection of colorectal cancer and monitoring of cancer recurrence and progression after treatment, has high authenticity and achieves an AUC value of 0.815.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Sequence listing

<110> Guangzhou college of medical sciences subsidiary traditional Chinese medical hospital

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