CN113337606A - Gene marker for liver cancer detection and targeted therapy - Google Patents
Gene marker for liver cancer detection and targeted therapy Download PDFInfo
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Abstract
The invention discloses a Gene marker for liver cancer detection and targeted therapy, wherein the Gene marker is a TINCR (terminal activating response) and a TINCR inhibitor of long-chain non-coding RNA (lncRNA), and the TINCR Gene ID: 257000, respectively; TINCR includes TINCR genes and homologues encompassing splice variants or allelic variants, including TINCR genes, gene sequences of TINCR; transcripts of TINCR include: NR _027064.3 (sequence shown in SEQ ID NO. 1). Use of a genetic marker for detection and targeted therapy of liver cancer in the development and manufacture of a liver cancer medicament comprising the TINCR inhibitor of claim 1. Has the advantages that: the invention provides a gene marker for diagnosing liver cancer cells, which can judge the canceration condition of the liver cancer cells by detecting the expression level of a marker TINCR and provides a molecular marker for detecting and judging the liver cancer cells with high specificity and high sensitivity.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a gene marker for liver cancer detection and targeted therapy.
Background
Liver cancer is one of the most common malignant tumors worldwide, with the incidence rate fifth worldwide and the mortality rate third. China is a big liver cancer country, the incidence and the death rate of liver cancer in China exceed half of the whole world, and the life and the health of people in China are seriously threatened. Liver cancer is mainly related to hepatitis B virus infection, alcoholic hepatitis or cirrhosis. The early liver cancer has no typical symptoms and is easy to be overlooked, and the liver cancer has the characteristics of fast progress, high mortality rate, poor prognosis and the like.
The effective molecular marker is vital in the detection and judgment process of the liver cancer cells, the specificity and the sensitivity of the existing marker are insufficient, and the research on the liver cancer cells is bottleneck due to the lack of effective cancer cell diagnosis markers, prognosis indexes and effective detection targets. Therefore, establishing the diagnostic marker of the liver cancer cell has great significance for the research of the canceration of the cell.
Most of the genes in the human whole genome can be transcribed into RNA, but only 1-2% of the genes can be translated into protein finally, and the rest of the RNA molecules transcribed from the genome but not encoding protein are named as non-coding RNA, wherein the long-chain non-coding RNA (incRNA) with the transcript length of more than 200nt is formed. Non-coding RNA was originally considered as "noise" of genome transcription, however, recent studies have shown that non-coding RNA exerts complex and precise regulatory functions in development and gene expression, and is involved in the development of various diseases, even in some human complex diseases, and is only in iceberg. With the development of high-throughput sequencing, gene chips and bioinformatics, more and more lncRNA is discovered; along with the improvement of tumor gene spectrum, more and more researches reveal the function of lncRNA, which indicates that the lncRNA is an indispensable part in a tumor occurrence development regulation network, the abnormal expression of the lncRNA is closely related to cancer, and the abnormally expressed lncRNA is different at different stages of diseases, thus suggesting that the lncRNA can be used as a gene diagnosis target and a treatment target of the cancer, and provide information for early diagnosis and targeted treatment of the cancer. Based on the above, the method finds the lncRNA gene marker related to the liver cancer, further elaborates the molecular mechanism in the liver cancer generation process, and has important significance for realizing early diagnosis and accurate treatment of the liver cancer.
Disclosure of Invention
The invention aims to provide a non-coding RNA gene marker TINCR related to liver cancer research, and a method for detecting and researching a hepatocellular carcinoma process by detecting the expression level of the TINCR.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a Gene marker for detection and targeted therapy of liver cancer, said Gene marker being TINCR of long non-coding rna (lncrna) and a TINCR inhibitor, said TINCR Gene ID: 257000, respectively; TINCR includes TINCR genes and homologues encompassing splice variants or allelic variants, including TINCR genes, gene sequences of TINCR; transcripts of TINCR include: NR _027064.3 (sequence shown in SEQ ID NO. 1).
Further, the TINCR is highly expressed in liver cancer.
Furthermore, a specific primer used for detecting the expression level of the TINCR is detected by a sequencing technology, a reverse transcription polymerase chain reaction (RT-PCR), a TaqMan probe and a hybridization probe, and the gene sequence of the specific primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.
Further, the TINCR inhibitor is a nucleic acid inhibitor and includes siRNA, shRNA, dsRNA, microrna or inhibitory small molecule compounds.
Furthermore, the gene sequence of the TINCR inhibitor is shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Use of a genetic marker for detection and targeted therapy of liver cancer in the development and manufacture of a liver cancer medicament comprising the TINCR inhibitor of claim 1.
Further, the administration routes of the liver cancer drug include oral administration, intravenous injection, subcutaneous injection and intramuscular injection.
Compared with the prior art, the invention has the advantages that: the invention provides a gene marker for diagnosing liver cancer cells, which can judge the canceration condition of the liver cancer cells by detecting the expression level of a marker TINCR and provides a molecular marker for detecting and judging the liver cancer cells with high specificity and high sensitivity.
Drawings
FIG. 1 high throughput sequencing analysis of representative differentially expressed lncRNA in liver cancer and paracancer tissues.
FIG. 2 expression of TINCR in liver cancer and tissues adjacent to the cancer.
FIG. 3 ROC curve analysis of expression of TINCR in liver cancer and tissues adjacent to the cancer.
FIG. 4 expression of infectious virus interfering TINCR RNA.
FIG. 5 shows interference of the effect of TINCR on the spheronization of hepatoma cells
Figure 6 interferes with the effect of TINCR on the sternness of liver cancer stem cells.
FIG. 7 shows interference of the effect of TINCR on hepatoma cell proliferation.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present invention and are intended to limit the scope of the invention. In the examples, unless otherwise specified, all experimental techniques are commonly used in cell biology or molecular biology, and are performed according to conventional conditions.
A Gene marker for detection and targeted therapy of liver cancer, said Gene marker being TINCR of long non-coding rna (lncrna) and a TINCR inhibitor, said TINCR Gene ID: 257000, respectively; TINCR includes TINCR genes and homologues encompassing splice variants or allelic variants, including TINCR genes, gene sequences of TINCR; transcripts of TINCR include: NR _027064.3 (sequence shown in SEQ ID NO. 1).
The TINCR is highly expressed in liver cancer. The specific primer used for detecting the expression level of the TINCR by a sequencing technology, a reverse transcription polymerase chain reaction (RT-PCR), a TaqMan probe and a hybridization probe is shown in SEQ ID NO.2 and SEQ ID NO. 3. The TINCR inhibitor is a nucleic acid inhibitor and includes siRNA, shRNA, dsRNA, microrna or inhibitory small molecule compounds. The gene sequence of the TINCR inhibitor is shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Use of a genetic marker for detection and targeted therapy of liver cancer in the development and manufacture of a liver cancer medicament comprising the TINCR inhibitor of claim 1. The administration routes of the liver cancer medicament comprise oral administration, intravenous injection, subcutaneous injection and intramuscular injection.
Example 1 screening of Gene markers associated with liver cancer
1. Tissue sample collection
Collecting the liver cancer and the tissues beside the cancer before the anti-tumor treatment, wherein the liver cancer tissues are proved to be primary liver cancer by pathology. All specimen source patients signed informed consent. Taking three pairs of tissue samples, carrying out high-throughput sequencing, and screening lncRNA with difference of more than 2 times.
2. Tissue RNA extraction and cDNA acquisition
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Collecting tissue specimens, taking mung beans to soybeans, putting the mung beans to soybeans in a mortar, grinding the mung beans to powder under the protection of liquid nitrogen, adding 1ml of Trizol into each sample, and fully cracking the samples on ice. 200ul of chloroform was added to each sample, shaken for 10 seconds and then kept on ice for 10 minutes. 12000g, 4 ℃ centrifugal 15min, the upper layer transfer to another centrifuge tube. Adding isopropanol with the same volume, mixing, and centrifuging at 12000g and 4 deg.C for 20 min; the precipitate was collected, washed with 75% ethanol, dissolved in 10-30ul of enzyme-free water, and the concentration and purity of total RNA were determined.
PrimeScript was obtained by TaKaRaTMKit (Bio Inc., Otsu, Japan) synthesized cDNA. The specific operation steps refer to the specification.
3. qPCR detection of expression of TINCR in liver cancer tissue and para-cancer tissue
Reaction reagents: SYBR Green method, forward and reverse primers, cDNA template and ddH2O were added as described. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 2 min, 95 ℃ for 15 sec, sequentially at 60 ℃ for 60 sec, with 40 cycles. DNA is subjected to several stages of template denaturation, annealing, primer extension, etc., to synthesize a strand complementary to the template. Calculating relative RNA expression: 2- Δ Ct [ Δ Ct ═ Ct (gene of interest) -Ct (gapdh) ], fold change between different treatments was calculated from 2- Δ Δ Ct.
Primer sequences
TINCR
F:5’-TGCTGCCTGGATCGACCTCTG-3’(SEQ ID NO.2)
R:5’-GTGTCCTCCTGTTCTGTTCGTAGC-3’(SEQ ID NO.3)
GAPDH
F:5’-CAGGAGGCATTGCTGATGAT-3’(SEQ ID NO.4)
R:5’-GAAGGCTGGGGCTCATTT-3’(SEQ ID NO.5)
4. Results
The results of qPCR are shown in fig. 2, and the relative expression level of TINCR in the liver cancer tissue is significantly higher than that in the tissue adjacent to the cancer, and the difference is statistically significant (p < 0.05). The result is consistent with the high-throughput sequencing result, namely the TINCR in the liver cancer tissue is highly expressed, which indicates that the TINCR can be used as a gene marker for diagnosing liver cancer.
An ROC curve of the expression of TINCR is drawn, as shown in FIG. 3, the area under the ROC curve is 0.92, the 95% confidence interval is 0.833-1.000, and p is less than 0.0001, which indicates that the differential expression of TINCR in liver cancer and paracarcinoma has excellent value for the early diagnosis of liver cancer, the sensitivity and the specificity are higher, whether a subject suffers from liver cancer can be judged by detecting the expression level of TINCR, and the liver cancer can be treated by designing and inhibiting the expression of TINCR in view of the TINCR and the risk of suffering from liver cancer.
Example 2 TINCR Gene knockdown
1. TINCR gene interference lentivirus transfection
The lentivirus RNA interference vector adopts GV248, and the interference RNA and negative control aiming at TINCR are constructed by a Kjeldahl gene. Interfering RNA and control RNA viruses are infected into cells through an infection enhancer, and the specific operation is shown in a product instruction.
The transfection reagent was Lipofectamine 2000 (Invitrogen). The introduction method comprises the following steps: liver cancer cells HCCLM3 and HepG2 in logarithmic growth phase, inoculating 4-5104 cells in a 24-well plate the day before transfection, and preparing a transfection compound when the cells are fused to 70-80 percent: DNA (0.8g) was diluted with 50. mu.l serum-free medium DMEM, and Lipofectamine 20002. mu.l was diluted with 48. mu.l serum-free medium DMEM. The diluted DNA and Lipofectamine 2000 were gently mixed, and the mixture was allowed to stand at room temperature for 15 minutes to prepare a DNA-Lipofectamine 2000 complex for transfection. The cells in the well plate were discarded from the old medium, and 100. mu.l of the above complex was added dropwise to each well and gently mixed. After culturing in an incubator for 24-48h, the culture medium is replaced with fresh medium, subcultured, and puromycin (10. mu.g/mL) pressure screening is performed.
The interference sequences against TINCR are as follows:
siRNA-1 design:
Target Seq：caGCTTGGAACTAGATACAGA(SEQ ID NO.6)
siRNA-2 design:
Target Seq：TAGCTTCAATACCTGCTACTT(SEQ ID NO.7)
siRNA-3 design:
CAGCGCTGGCATGTTCTGAAA(SEQ ID NO.8)
2. qPCR detection of TINCR inhibition efficiency
The procedure is as in example 1
The results show that the interference efficiency of siRNA-1 and siRNA-2 is higher than 70%, and subsequent experiments can be carried out.
Example 3 Effect of TINCR inhibitors on liver cancer cell function
1. Test for detecting cell dryness by balling-up experiment
Digesting si-TINCR and negative control cells with good growth state, centrifuging, resuspending the cells by using a stem cell culture medium (DMEM/F12+ B27+20ng/ml bFGF +20ng/ml EGF), plating the cells on an ultra-low adsorption cell culture six-well plate for culture, and observing the spheroidization state; collecting the cell balls obtained by the primary balling, digesting, washing by PBS, re-suspending by using a stem cell culture medium, and then plating for secondary balling. The results are shown in FIG. 4, where si-TINCR cells had decreased spheroids, indicating that inhibition of TINCR expression could inhibit the self-renewal ability of hepatoma cells.
2. qPCR detection of cell dryness related index
The procedure is as in example 1
The results show that the mRNA expression levels of dryness indexes such as Oct4, SOX2 and OV6 are obviously reduced when TINCR is inhibited, and the inhibition of the dryness characteristics of hepatoma cells by TINCR is indicated.
3. Cell proliferation assay
Each of 5 × 103 si-TINCR and negative control cells was plated in 3 duplicate wells, inoculated in a 96-well plate and cultured for 0, 24, 48, and 72 hours, 10ul of CCK8 solution was added thereto, incubated at 37 ℃ for 1 hour in the dark, and the absorbance at OD 450nm was measured with a microplate reader to prepare a growth curve. As shown in the figure, inhibition of TINCR can significantly reduce the OD value of hepatoma cells, i.e., decrease cell proliferation.
The present invention and the embodiments thereof have been described above, and the description is not restrictive, and the embodiments shown in the detailed description are only a part of the embodiments of the present invention, not all embodiments, and the actual configuration is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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<120> a gene marker for liver cancer detection and targeted therapy
<130> 2021.06.28
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ccaggaggca cctgtgccca ccccctgggc tgtttccccc ttccccttaa atccggatgc 1200
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agaaggaggg gaagagagag gagccccact tcactccggc aggcagacct gctggagctg 1800
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tggcgggaac ccgggaggca gaggttgcag tgagctgaga tcacgccact gcactccagc 2520
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ctccacttgg tttgcatgtc ccaccttctt tgtttggcaa gctcctattc atccttcaaa 3480
gccctagctt caatacctgc tacttcatgc agccattgct tatccttcaa ggagagccta 3540
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Claims (7)
1. A gene marker for liver cancer detection and targeted therapy, which is characterized in that: the Gene markers are TINCR of long-chain non-coding RNA (lncRNA) and a TINCR inhibitor, and the TINCR Gene ID: 257000, respectively; TINCR includes TINCR genes and homologues encompassing splice variants or allelic variants, including TINCR genes, gene sequences of TINCR; transcripts of TINCR include: NR _027064.3 (sequence shown in SEQ ID NO. 1).
2. The genetic marker for liver cancer detection and targeted therapy according to claim 1, characterized in that: the TINCR is highly expressed in liver cancer.
3. The genetic marker for liver cancer detection and targeted therapy according to claim 1, characterized in that: the specific primer used for detecting the expression level of the TINCR by a sequencing technology, a reverse transcription polymerase chain reaction (RT-PCR), a TaqMan probe and a hybridization probe is shown in SEQ ID NO.2 and SEQ ID NO. 3.
4. The genetic marker for liver cancer detection and targeted therapy according to claim 1, characterized in that: the TINCR inhibitor is a nucleic acid inhibitor and includes siRNA, shRNA, dsRNA, microrna or inhibitory small molecule compounds.
5. The gene marker for liver cancer detection and targeted therapy according to claim 4, characterized in that: the gene sequence of the TINCR inhibitor is shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
6. The application of the gene marker for liver cancer detection and targeted therapy is characterized in that: the gene marker is applied to the development and production of liver cancer drugs, and the liver cancer drugs comprise the TINCR inhibitor in claim 1.
7. The use of the gene markers for liver cancer detection and targeted therapy according to claim 6, wherein: the administration routes of the liver cancer medicament comprise oral administration, intravenous injection, subcutaneous injection and intramuscular injection.
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| CN111187773A (en) * | 2020-03-20 | 2020-05-22 | 青岛思拓新源细胞医学有限公司 | Long non-coding RNA gene marker for detecting liver cancer and application thereof |
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| CN111187773A (en) * | 2020-03-20 | 2020-05-22 | 青岛思拓新源细胞医学有限公司 | Long non-coding RNA gene marker for detecting liver cancer and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 田小星等: "长链非编码RNA TINCR通过miR-7调控肝癌细胞系Huh7侵袭和迁移", 《基础医学与临床》 * |
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