CN113957152A - Detection kit for SNHG6 gene and application thereof - Google Patents
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Abstract
The invention discloses an SNHG6 gene detection kit and application thereof, wherein the kit consists of a sample RNA extraction reagent, a cDNA reverse transcription reagent, an SNHG6 primer, a GAPDH internal reference primer and a fluorescent quantitative PCR reaction solution; the invention provides a kit which has good sensitivity and strong specificity and can conveniently and quickly monitor the proliferation state of the liver cancer cells by using a small amount of samples; the kit can convert the proliferation state of the liver cancer cells into a quantitative index, has objective and effective results, and is convenient for statistical research; the kit provides 2 pairs of SNHG6 detection primers, can effectively reduce the randomness of detection results, and improves the statistical accuracy of the detection results.
Description
(I) technical field
The invention relates to a kit for detecting the expression level of lncRNA SNHG6 nucleic acid and indicating the malignant proliferation state of liver cancer cells and application thereof.
(II) background of the invention
Cell proliferation is the basis for the reproduction and development of organisms and is one of the major vital activities of cells. To ensure that the life process of cell proliferation is strictly and orderly, a series of regulatory mechanisms are developed in the cell to closely monitor the process. The cancer cells escape from the restriction of a cell proliferation monitoring mechanism due to high-frequency gene mutation and change of a gene expression mode, and show the characteristic of uncontrolled cell proliferation. Therefore, the method for monitoring the expression level change of the gene related to the cell proliferation abnormality is a method for efficiently, conveniently and accurately indicating the malignant progression degree of the tumor.
Primary liver cancer is the fourth leading cause of cancer-related death worldwide, with increasing incidence. The mortality rate of liver cancer is high due to the lack of accurate and effective early diagnosis methods and prognostic biomarkers. Therefore, the screening of appropriate molecular markers for indicating the growth state of liver cancer cells as a diagnostic basis for malignant proliferation state of liver cancer has a very high application prospect and research value.
SNHG6(small nuclear tumor RNAhost gene 6), a gene stably expressed in hepatoma cells, promotes malignant proliferation of tumor cells by mediating activation of proto-cancer signaling pathways. Moreover, the high expression of SNHG6 in liver cancer is in positive correlation with the recurrence and poor prognosis of the cancer of patients. Therefore, using RNA of cell or patient sample as template, SNHG6 detection primer and internal reference primer are selected to perform real-time fluorescence quantitative PCR detection reaction according to 2ΔΔCqCalculating relative abundance, wherein the value reflects the transcription activity level of SNHG6 in tumor cells or tissues, and the growth condition of the liver cancer cells can be judged and used as an auxiliary diagnosis basis for the proliferation state of the liver cancer cells.
Based on the theory, the invention develops the liver cancer diagnosis kit based on the SNHG6 molecular marker, detects liver cancer pathological samples according to the provided method, and can provide basis for clinical liver cancer occurrence and prognosis effect diagnosis. The kit provides a new molecular detection marker for preoperative accurate diagnosis of liver cancer, and evaluation of recurrence and prognosis of liver cancer, and has high clinical application value.
Disclosure of the invention
The invention provides a detection kit of an SNHG6 gene and application thereof, wherein the kit comprises an internal reference primer and two pairs of SNHG6 expression level detection primers for assisting in indicating the malignant proliferation state of liver cancer. The kit has the characteristics of high specificity, stability and sensitivity, can accurately indicate the growth state of the liver cancer cells, is used for assisting in diagnosing the poor prognosis of a liver cancer patient, and has great potential application value.
The technical scheme adopted by the invention is as follows:
the invention provides an SNHG6 gene detection kit, which consists of a sample RNA extraction reagent, a cDNA reverse transcription reagent, an SNHG6 primer, a GAPDH internal reference primer and a fluorescent quantitative PCR reaction solution; the SNHG6 primer is one or two groups of SNHG6F # 1/SNHG 6R # 1 or SNHG6F #2/SNHG 6R # 2;
the primer sequences are as follows:
GAPDH F：5’-AATGGGCAGCCGTTAGGAAA-3’；
GAPDH R：5’-GCGCCCAATACGACCAAATC-3’；
SNHG6 F#1：5’-GTTAGTCATGCCGGTGTGGT-3’；
SNHG6 R#1：5’-AATACATGCCGCGTGATCCT-3’；
SNHG6 F#2：5’-GCTCTGCGAGGTGCAAGAA-3’；
SNHG6 R#2：5’-ATGCTGCATGCCACACTTGA-3’。
furthermore, the sample RNA extraction reagent adopts TRIzol (Ambion) lysate; the cDNA reverse transcription reagent adopts
II Q Select RT SuperMix (Vazyme); the fluorescent quantitative PCR reaction solution adopts
Universal SYBR qPCR Master Mix(Vazyme)。
The invention also provides an application of the SNHG6 gene detection kit in detecting the expression quantity of the SNHG6 gene, and the application method comprises the following steps: extracting sample RNA by using a sample RNA extraction reagent, and performing reverse transcription on the RNA into cDNA by using a cDNA reverse transcription reagent; and (3) carrying out fluorescent quantitative PCR detection by using cDNA as a template and adopting an SNHG6 primer and a GAPDH internal reference primer, obtaining the expression level of the SNHG6 gene according to the Cq value, and comparing with a normal control, if the Cq value is higher than the normal control, the liver cancer cell is low in expression.
Further, the fluorescent quantitative PCR detection conditions are as follows: pre-denaturation, 30sec at 95 ℃; circulating for 40 times at 95 deg.C for 3-10sec and 60 deg.C for 10-30 sec; dissolution, 95 ℃ for 15sec, 60 ℃ for 60sec, and 95 ℃ for 15 sec.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a kit which has good sensitivity and strong specificity and can conveniently and quickly monitor the proliferation state of the liver cancer cells by using a small amount of samples; the kit can convert the proliferation state of the liver cancer cells into a quantitative index, has objective and effective results, and is convenient for statistical research; the kit provides 2 pairs of SNHG6 detection primers, can effectively reduce the randomness of detection results, and improves the statistical accuracy of the detection results.
(IV) description of the drawings
Fig. 1 is a graph of SNHG6 primer dissolution.
FIG. 2 shows the sensitivity verification of the detection of SNHG6 expression level by the primer pair provided by the kit.
FIG. 3 shows the expression level of SNHG6 in liver cancer cells.
Fig. 4 is the expression level of SNHG6 in liver cancer tissue samples.
FIG. 5 is a graph of the relationship between SNHG6 expression and survival time in liver cancer tissue samples.
Fig. 6 shows the expression of SNHG6 in different liver cancer patients.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 SNHG6 primer selection and primer quality identification
According to the sequence NR _002599.2 of the SNHG6 nucleic acid transcript in NCBI database, designing detection primers according to the principle that Tm value is about 60 and self-complement is low, and screening to obtain three pairs of detection primers, wherein one pair is internal reference primers (GAPDH F and GAPDH R), and two pairs are detection primers (SNHG 6F # 1+ SNHG6R #1, SNHG6F #2+ SNHG6R #2), and the sequence information of the primers is shown in Table 1:
TABLE 1 primers
Primer specificity was further tested using NCBI BLAST. As shown in the result of the lysis curve in FIG. 1, the primer lysis curve provided by the kit has a single peak and a single amplification sequence of the primer, and can be specifically and sensitively used for detecting the expression level of SNHG6 in liver cancer cells.
Example 2 verification of sensitivity of detection of SNHG6 expression level by primer pair provided by kit
The kit comprises the following components: a sample RNA extraction reagent, a cDNA reverse transcription reagent, a SNHG6 primer, a GAPDH internal reference primer and a fluorescent quantitative PCR reaction solution.
Grouping experiments: selecting wild type HepG2 liver cancer cell line as a control group, and adopting Hieff trainsTMLiposomal Transfection Reagent (assist in san) small interfering RNA (short interfering RNA, siRNA) was transfected into HepG2 cells, and HepG2 cell lines (SNHG 6#1 knocked-down, SNHG6# 2 knocked-down) specifically knocking down SNHG6 gene were used as experimental groups.
TABLE 2 siRNA target sequences
The experimental steps are as follows:
firstly, respectively taking a proper amount (about 50-100 ten thousand) of cells from each group, adding an equal amount of Trizol (Ambion) reagent to crack the cells and extracting cell RNA; use of
II Q Select RT SuperMix (Vazyme) kit reverse transcribes equal amounts of RNA into cDNA; two pairs of SNHG6 primers (SNHG 6F # 1 for knocking down SNHG6# 1, SNHG6R # 1, SNHG6F # 2 for knocking down SNHG6# 2, SNHG6R #2) and GAPDH internal reference primers screened in example 1 were used, with
The Universal SYBR qPCR Master Mix (Vazyme) kit is used for carrying out fluorescent quantitative PCR detection on the cDNA template, and the PCR reaction conditions are shown in the following table 3:
TABLE 3 PCR reaction conditions
According to the Cq value of each group of samples, carry out 2ΔΔCqAnd calculating, performing statistical analysis, and drawing a table. As shown in fig. 2, SNHG6 was expressed in knock-down cell lines significantly less than wild-type cells (. P)<0.01), the statistical error (SD, standard deviation) is small. The detection result shows that the primer provided by the invention has high sensitivity, strong specificity and good accuracy for detecting the expression level of the SNHG6 gene in the cell, and is suitable for detecting the expression level of SNHG6 in the liver cancer cell.
Example 3 expression level of SNHG6 in hepatoma cells has a significant positive correlation with malignant proliferation of hepatoma
The wild type and two liver cancer cell strains HepG2 with the specific knockdown SNHG6 constructed in the example 2 are subjected to cell proliferation capacity detection, and the specific steps are as follows:
wild-type HepG2 cells and specifically knockdown cells (SNHG 6#1 knockdown) were inoculated into DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2The culture was carried out for 24 hours. 3000 cells per group were then seeded in 96-well plates in DMEM medium containing 10% fetal bovine serum in a volume of 100. mu.l per group, 6-8 replicates per group, and a blank control group (containing no cells) was set. At 37 ℃ with 5% CO, respectively2After 24, 48 and 72 hours of culture, 10. mu.l of thiazole blue (Methylzolylpentyl-tetrazolium bromide, MTT) working solution (5mg/mL) was added to each well and incubated at 37 ℃ for 4 hours; the supernatant medium was discarded, 100. mu.l DMSO was added, and the mixture was mixed on a horizontal shaker at low speed for 10min, and then absorbance at a wavelength of 570nm was read with a microplate reader for cell proliferation analysis. Subsequently, the absorbance of each group was subtracted by the number of blank wells, and the mean and standard deviation were calculated, respectively, and the difference between groups was analyzed using t-test<0.01. The result is shown in fig. 3, the proliferation capacity of the cell line with the knockdown SNHG6 is obviously lower than that of the control group, which indicates that the higher the detection index (i.e., expression level) of SNHG6 is, the stronger the proliferation capacity of the liver cancer cell is, and the feasibility of SNHG6 as the proliferation state index of the liver cancer cell is shown.
Example 4 statistical analysis of expression levels of SNHG6 in liver cancer tissue samples and patient survival
After informed consent of patients with liver cancer was obtained, 48 clinical tissue samples of liver cancer and matched tissue samples beside the cancer from the center for tumor prevention and treatment (SYSUCC) of Zhongshan university were collected. Shearing a proper amount of sample tissue, placing the sample tissue into a 1.5ml centrifuge tube, adding 1ml of Tizaol lysate and 3-4 grinding beads, and placing the sample into a tissue grinder (60 Hz) for grinding for 15-20 min. Then, the kit of embodiment 1 is adopted to extract tissue RNA, SNHG6F # 1+ SNHG6R # 1 detection primers and GAPDH internal reference primers provided by the kit are utilized to respectively carry out real-time fluorescence PCR detection, and the expression quantity of SNHG6 in liver cancer tissues and paracancerous tissues is obtained according to Cq values. The statistical result is shown in fig. 4, the expression level of SNHG6 in the liver cancer tissue is much higher than that of the tissue beside the cancer (t-test, P <0.01), and this result shows that SNHG6 not only can effectively characterize the proliferation capacity of the liver cancer cells, but also can be used for assisting in judging and distinguishing the cancer and the tissue beside the cancer, and the applicability is wide.
And calculating the correlation between the SNHG6 expression level of the liver cancer tissue and the survival time of the patient by using GraphPad Prism 8.0. As shown in fig. 5, the survival time of the liver cancer patients with high expression of SNHG6 was short, indicating that the expression level of SNHG6 was significantly correlated with the survival time of the patients.
Example 5 expression of SNHG6 can characterize recurrence in patients with liver cancer
21 clinical liver cancer recurrent tissue samples and 27 clinical liver cancer non-recurrent samples of the tumor prevention and treatment center (SYSUCC) of Zhongshan university are collected, and the samples are collected and informed consent of patients is obtained. The clinical specimens were crushed by grinding as shown in example 4. According to the method described in example 2, the RNA of the tissue sample is extracted, and real-time fluorescence PCR quantitative analysis is carried out by using a SNHG6F # 2+ SNHG6R # 2 detection primer and a GAPDH internal reference primer, and the expression information of SNHG6 in the sample, namely the expression level of SNHG6, is obtained according to the Cq value. As shown in fig. 6, SNHG6 was expressed at a significantly higher level in patients with recurrent liver cancer than in patients without recurrence, indicating that high expression of SNHG6 was closely linked to recurrence of liver cancer. This example reveals that the expression level of SNHG6 in liver cancer tissue is effective for indicating recurrence in patients with liver cancer.
It should be noted that the above experiments are only illustrative of several specific examples of the present invention, and obviously, many modifications are possible in the present invention, and all modifications that can be derived or suggested by those skilled in the art from the disclosure of the present invention should be considered as the protection scope of the present invention.
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Claims (4)
1. A SNHG6 gene detection kit is characterized in that the kit consists of a sample RNA extraction reagent, a cDNA reverse transcription reagent, an SNHG6 primer, a GAPDH internal reference primer and a fluorescent quantitative PCR reaction solution; the SNHG6 primer is one or two groups of SNHG6F #1/SNHG 6R #1 or SNHG6F #2/SNHG 6R # 2;
the primer sequences are as follows:
GAPDH F：5’-AATGGGCAGCCGTTAGGAAA-3’；
GAPDH R：5’-GCGCCCAATACGACCAAATC-3’；
SNHG6 F#1：5’-GTTAGTCATGCCGGTGTGGT-3’；
SNHG6 R#1：5’-AATACATGCCGCGTGATCCT-3’；
SNHG6 F#2：5’-GCTCTGCGAGGTGCAAGAA-3’；
SNHG6 R#2：5’-ATGCTGCATGCCACACTTGA-3’。
2. the SNHG6 gene detection kit of claim 1, and its application in detecting SNHG6 gene expression level.
3. The application according to claim 2, characterized in that the method of application is: extracting sample RNA by using a sample RNA extraction reagent, and performing reverse transcription on the RNA into cDNA by using a cDNA reverse transcription reagent; and carrying out fluorescent quantitative PCR detection by using the cDNA as a template and adopting an SNHG6 primer and a GAPDH internal reference primer, and obtaining the expression level of the SNHG6 gene according to the Cq value.
4. The use according to claim 2, wherein the fluorescent quantitative PCR assay conditions are: pre-denaturation, 30sec at 95 ℃; circulating for 40 times at 95 deg.C for 3-10sec and 60 deg.C for 10-30 sec; dissolution, 95 ℃ for 15sec, 60 ℃ for 60sec, and 95 ℃ for 15 sec.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120304318A1 (en) * | 2009-12-17 | 2012-11-29 | Cambridge Enterprise Ltd | Cancer diagnosis and treatment |
| CN108949988A (en) * | 2018-08-03 | 2018-12-07 | 武汉大学 | A kind of application of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120304318A1 (en) * | 2009-12-17 | 2012-11-29 | Cambridge Enterprise Ltd | Cancer diagnosis and treatment |
| CN108949988A (en) * | 2018-08-03 | 2018-12-07 | 武汉大学 | A kind of application of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment |
Non-Patent Citations (2)
| Title |
|---|
| 常磊: "长链非编码RNA SNHG6在肝癌发生、发展中的功能及机制研究", 中国博士学位论文全文数据库 医药卫生科技辑 * |
| 杨鹏生;宋黎明;段希斌;梁马可;梁占强;李学民;: "LncRNA SNHG6调控miR-186表达对肝癌细胞增殖、迁移和侵袭的影响及机制", 现代肿瘤医学 * |
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