CN116516002A - SLC24A2 and SLCO1B3 gene markers and shRNA silencing interference sequences and application thereof - Google Patents
SLC24A2 and SLCO1B3 gene markers and shRNA silencing interference sequences and application thereof Download PDFInfo
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Abstract
The invention discloses SLC24A2 and SLCO1B3 gene markers, and a shRNA silencing interference sequence and application thereof, and relates to the technical field of biology. According to the invention, after the shRNA is proved to interfere with the expression of SLC24A2 and SLCO1B3, the proliferation capacity and invasion capacity of non-small cell lung cancer cells can be obviously inhibited, so that the shRNA interference SLC24A2 and SLCO1B3 genes can be used as new drugs and targets for treating the non-small cell lung cancer; the invention designs shRNA silencing interference sequences of SLC24A2 and SLCO1B3, and verifies the functions of the interference sequences through cell experiments; the SLC24A2 and SLCO1B3 can be used as markers for diagnosing the non-small cell lung cancer, and shRNA interference SLC24A2 and SLCO1B3 can also be used as potential small nucleic acid drugs and targets for treating the non-small cell lung cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to SLC24A2 and SLCO1B3 gene markers, and shRNA silencing interference sequences and application thereof.
Background
Non-small cell lung cancer (non-small cell lung cancer, NSCLC) is one of lung cancer, accounting for about 85% of the number of lung cancer-causing diseases, which includes squamous cell carcinoma (squamous carcinoma), adenocarcinoma, large cell carcinoma, which has slower growth division and relatively late spread metastasis compared to small cell carcinoma. Early lung cancer patients have better prognosis, but many lung cancer patients have advanced diagnosis, with an overall five-year survival rate of only about 20%. The traditional treatment method mainly comprises chemotherapy, has poor treatment effect and strong toxic and side effects. About 75% of patients with non-small cell lung cancer have found middle and late stages with very low survival rates of 5 years. Thus, it is highly necessary to identify potent NSCLC biomarkers and oncogenes and to explore their potential regulatory mechanisms.
Soluble carrier (SLC) uptake transporter is an important component of the transporter family. SLC uptake transporters are the most bulky class of transporters within cells, mainly comprising organic anion transporters, organic cation transporters, organic anion transport polypeptides, sodium-taurocholate cotransporters. The SLC transporter family is mainly an uptake transporter, which is transported by co-transport and/or ion exchange and plays an important role in the absorption, distribution and excretion of some oncologic drugs in vivo. The organic anion transporter can also be used as a drug target to transfer drugs to specific tumor cells (breast cancer cells, prostate cancer cells and the like are found at present) so as to realize targeted treatment of diseases. The SLC24A2 gene encodes a member of the calcium/cation-reversal transport protein superfamily and the SLCO1B3 gene encodes a liver-specific member of the organic anion transporter family, and is a gene that plays an important role in the transporter family. However, the biological role of SLC24A2 and SLCO1B3 in the development of NSCLC remains to be elucidated. By researching the potential of SLC24A2 and SLCO1B3 as nucleic acid medicaments, the technical problem of NSCLC treatment difficulty is hopefully overcome, and a new treatment direction is further provided for NSCLC treatment.
Disclosure of Invention
The invention provides SLC24A2 and SLCO1B3 gene markers, shRNA silencing interference sequences and application thereof, and solves the problems.
In order to solve the technical problems, the invention is realized by the following technical scheme:
according to the invention, the SLC24A2 and SLCO1B3 are obviously increased in lung squamous carcinoma and lung adenocarcinoma samples, so that the SLC24A2 and SLCO1B3 are closely related to NSCLC. The inventors designed shRNA silencing interference sequences of SLC24A2 and SLCO1B3, and verified the function of the interference sequences by cell experiments: silencing SLC24A2 and SLCO1B3 by interfering sequences inhibits the proliferation, migration and invasiveness of non-small cell lung cancer cells, also resulting in increased apoptosis. These results demonstrate that the interfering sequences of SLC24A2 and SLCO1B3 can be used as potential nucleic acid drugs for NSCLC tumor treatment.
The invention is realized by the following technical scheme:
in a first aspect, the present invention provides a primer set of the above-described circRNA marker, the primer set comprising a forward primer and a reverse primer;
the nucleotide sequence of the SLC24A2 Forward Primer (Primer Forward) is:
CCAAGGAGACTACCCGAAAGA (SEQ ID NO: 1);
the nucleotide sequence of the SLC24A2 Reverse Primer (Primer Reverse) is CAGACAATGGCTAAGGCTATGAA (SEQ ID NO: 2);
SLCO1B3 Forward Primer (Primer Forward) the nucleotide sequence is TGGAGCAACAGTACGGTCAG (sequence 3);
the nucleotide sequence of the SLCO1B3 Reverse Primer (Primer Reverse) is TGCTTTCGCAGATTAGAGGGAA (SEQ ID NO: 4).
In a second aspect, the invention provides an application of the gene marker in preparing a product for diagnosing non-small cell lung cancer.
In a third aspect, the present invention provides the use of a reagent for detecting a gene marker as described above in the manufacture of a product for diagnosing cancer.
In a fourth aspect, the invention provides a nucleic acid sequence that specifically inhibits the expression of the SLC24A2 and SLCO1B3 genes, which is characterized by complementary, interfering with or inhibiting the translation of SLC24A2 and SLCO1B3 mRNA, the shRNA interfering sequence being as follows:
Sh-SLC24A2-1:
CCGGGCCGAAGAACTTGGATCATATCTCGAGATATGATCCAAGTTCTTCGGCTTTTTG (sequence 1);
Sh-SLC24A2-2:
CCGGCATTACCTGGATTGCAGTATTCTCGAGAATACTGCAATCCAGGTAATGTTTTTG (SEQ ID NO: 2);
Sh-SLC24A2-3:
CCGGGCAAATGATAAACCGCAATAACTCGAGTTATTGCGGTTTATCATTTGCTTTTTG (SEQ ID NO: 3);
Sh-SLCO1B3-1:
CCGGGGTAGTTGTAACTGCTAATAACTCGAGTTATTAGCAGTTACAACTACCTTTTTG (SEQ ID NO: 4);
Sh-SLCO1B3-2:
CCGGTGGTTAGTGTGTGATACAATACTCGAGTATTGTATCACACACTAACCATTTTTG (SEQ ID NO: 5);
Sh-SLCO1B3-3:
CCGGGCTTTAAGATTCCCAGCACTTCTCGAGAAGTGCTGGGAATCTTAAAGCTTTTTG (SEQ ID NO: 6);
in a fifth aspect, the application of the shRNA interference sequences expressed by two gene markers in preparing a medicament for treating non-small cell lung cancer is provided. Two shRNA interference technologies for inhibiting SLC24A2 and SLCO1B3 gene expression are characterized in that a nucleic acid sequence capable of specifically inhibiting SLC24A2 and SLCO1B3 gene expression is used as a core sequence to prepare a gene interference RNA fragment.
Preferably, two shRNA interference technologies for inhibiting SLC24A2 and SLCO1B3 gene expression have effective shRNA interference sequences of
Sh-SLC24A2-1:
CCGGGCCGAAGAACTTGGATCATATCTCGAGATATGATCCAAGTTCTTCGGCTTTTTG (SEQ ID NO: 7);
Sh-SLCO1B3-3:
CCGGGCTTTAAGATTCCCAGCACTTCTCGAGAAGTGCTGGGAATCTTAAAGCTTTTTG (SEQ ID NO: 8).
In a sixth aspect, two shRNA interference technologies targeting SLC24A2 and SLCO1B3 gene expression are characterized in that after the prepared gene interference RNA fragments are introduced into non-small cell lung cancer cells (H1299 and a 549) by a transgenic technology, specific recognition of SLC24A2 and SLCO1B3 mRNA is generated in the cells, so that the effects of inhibiting SLC24A2 and SLCO1B3 gene mRNA and protein translation and reducing the expression level of SLC24A2 and SLCO1B3 genes are achieved, and the purpose of treating non-small cell lung cancer is achieved.
In a seventh aspect, the use of shRNA sequences that interfere with SLC24A2 and SLCO1B3 gene expression is characterized by effective inhibition of proliferation activity, migration and invasion capacity of non-small cell lung cancer cells, and promotion of apoptosis.
Preferably, the use of shRNA sequences that interfere with SLC24A2 and SLCO1B3 gene expression is characterized by the use in the preparation of a medicament for inhibiting non-small cell lung cancer growth and tumor metastasis.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the research, the expression of SLC24A2 and SLCO1B3 in lung adenocarcinoma and lung squamous carcinoma tissues is obviously different, and the expression of SLC24A2 and SLCO1B3 in lung adenocarcinoma and lung squamous carcinoma tissues is obviously increased, so that the SLC24A2 and SLCO1B3 can be used as non-small cell lung cancer diagnosis markers, are obviously increased in non-small cell lung cancer tissues, and can well predict the occurrence of non-small cell lung cancer.
(2) According to the invention, after the shRNA is further researched and discovered to interfere with the expression of SLC24A2 and SLCO1B3, the proliferation capacity and invasion capacity of non-small cell lung cancer cells can be obviously inhibited, so that the shRNA interference SLC24A2 and SLCO1B3 genes can be used as new medicines and targets for treating the non-small cell lung cancer.
(3) The invention designs shRNA silencing interference sequences of SLC24A2 and SLCO1B3, and verifies the functions of the interference sequences through cell experiments: the influence study of SLC24A2 and SLCO1B3 on cell proliferation is detected through a CCK8 experiment, and the result shows that after interference inhibition expression is carried out on a lung cancer cell line A549 and H1299 by SLC24A2 and SLCO1B3, the growth of lung cancer cells is obviously inhibited; the influence study of SLC24A2 and SLCO1B3 on the migration capacity of cells is detected through a Transwell and cell scratch-healing experiment, and the result shows that the invasion and migration capacity of the cells are obviously inhibited after the SLC24A2 and SLCO1B3 are subjected to interference inhibition expression of lung cancer cell lines A549 and H1299; the influence study of SLC24A2 and SLCO1B3 on apoptosis is detected through a flow cytometry experiment, and the result shows that after interference inhibition expression of SLC24A2 and SLCO1B3 in lung cancer cell lines A549 and H1299 is carried out, the apoptosis capacity is increased.
(4) Experiments prove that SLC24A2 and SLCO1B3 can be used as markers for diagnosing non-small cell lung cancer, and shRNA can interfere SLC24A2 and SLCO1B3 and can also be used as potential small nucleic acid drugs and targets for treating non-small cell lung cancer. Reagents for detection and shRNA interference with SLC24A2 and SLCO1B3 may be used in products for diagnosis or treatment of cancer, such as chips, kits or nucleic acid strips, nucleic acid drugs and immunotherapy.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the expression of SLC24A2 and SLCO1B3 in lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) according to an embodiment;
FIG. 2 is a graph showing the knock-down efficiency data of PCR detection of interference sequences of SLC24A2 and SLCO1B3 in H1299 cells in the specific example;
FIG. 3 is a graph showing the effect of CCK8 assay to detect SLC24A2 and SLCO1B3 on cell proliferation in the examples;
FIG. 4 is a graph showing the effect of Transwell experiments on cell invasion by SLC24A2 and SLCO1B 3;
FIG. 5 is a graph showing the effect of cell scratch-healing assay (scratch-sealing) on the ability of SLC24A2 and SLCO1B3 to migrate cells in an exemplary embodiment;
FIG. 6 is a graph showing the induction of apoptosis by SLC24A2 and SLCO1B3 tested by flow cytometry in the specific examples.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Example 1
Expression of SLC24A2 and SLCO1B3 in lung adenocarcinoma and lung squamous carcinoma
(1) The website GEPIA online tool was analyzed interactively by gene expression data to see the expression of SLC24A2 and SLCO1B3 in lung adenocarcinoma (Lung adenocarcinoma, LUAD) and lung squamous carcinoma (Lung squamous cancer, lucc), respectively, of the TCGA database. Wherein, the lung adenocarcinoma has 483 tumor samples and 59 normal tissue samples; in lung squamous carcinoma, 486 cases of tumor samples and 50 cases of normal tissue samples are obtained.
(2) Such asFIG. 1As shown, the differences in SLC24A2 and SLCO1B3 expression are seen by means of box plots. The results show that SLC24A2 exhibits a significant increase in both lung adenocarcinoma LUAD and lung squamous carcinoma luc compared to normal tissue; likewise, SLCO1B3 exhibited significant elevation in both lung adenocarcinoma and lung squamous carcinoma. These results suggest that SLC24A2 and SLCO1B3 are potential oncogenes in lung adenocarcinoma and lung squamous carcinoma, and can be studied as potential targets.
Example 2
(II) PCR detection of knock-down efficiency of interference sequences of SLC24A2 and SLCO1B3 in H1299 cells
(1) The mature sequences of SLC24A2 and SLCO1B3 described in this example 2 were designed to design primers for fluorescent quantitative PCR, the primer sequences were as follows (5 '-3'):
SLC24A2-Forward CCAAGGAGACTACCCGAAAGA (sequence 1)
SLC24A2-Reverse CAGACAATGGCTAAGGCTATGAA (SEQ ID NO: 2)
SLCO1B3-Forward TGGAGCAACAGTACGGTCAG (SEQ ID NO: 3)
SLCO1B3-Reverse TGCTTTCGCAGATTAGAGGGAA (SEQ ID NO: 4)
The shRNA interference sequences of SLC24A2 and SLCO1B3 are obtained by design as follows:
Sh-SLC24A2-1:
CCGGGCCGAAGAACTTGGATCATATCTCGAGATATGATCCAAGTTCTTCGGCTTTTTG (SEQ ID NO: 5);
Sh-SLC24A2-2:
CCGGCATTACCTGGATTGCAGTATTCTCGAGAATACTGCAATCCAGGTAATGTTTTTG (SEQ ID NO: 6);
Sh-SLC24A2-3:
CCGGGCAAATGATAAACCGCAATAACTCGAGTTATTGCGGTTTATCATTTGCTTTTTG (SEQ ID NO: 7);
Sh-SLCO1B3-1:
CCGGGGTAGTTGTAACTGCTAATAACTCGAGTTATTAGCAGTTACAACTACCTTTTTG (SEQ ID NO: 8);
Sh-SLCO1B3-2:
CCGGTGGTTAGTGTGTGATACAATACTCGAGTATTGTATCACACACTAACCATTTTTG (SEQ ID NO: 9);
Sh-SLCO1B3-3:
CCGGGCTTTAAGATTCCCAGCACTTCTCGAGAAGTGCTGGGAATCTTAAAGCTTTTTG (SEQ ID NO: 10).
(2) RNA extraction (Trizol method)
H1299 cells were added with 1ml TRIZOL LS reagent;
b. 200 mu L of chloroform is added, the mixture is vigorously oscillated for 10 seconds and is kept stand at room temperature for 10 minutes;
c.4℃centrifuging at 12,000g for 10min, dividing the solution into three layers, dissolving RNA in the aqueous phase, transferring the aqueous phase to another new RNase free EP tube;
d. adding 1-time volume of isopropanol, and fully and uniformly mixing by vortex;
centrifuging at e.4 ℃ for 15min at 12,000g, precipitating RNA at the bottom of the tube after centrifugation, and discarding the supernatant;
f. 1ml of 75% ethanol was added, gently inverted by hand, centrifuged at 12,000g for 5min, and the supernatant was discarded;
g. air-dried at room temperature, and add 20. Mu.L DEPC water to dissolve the precipitate.
(3) Genomic DNA removal
Removing the residue of genome DNA in total RNA, adopting DNA digestive enzyme, and adopting a specific reaction system and conditions as follows, wherein the total volume of the reaction solution is 10 mu L, and the composition is as follows:
table 1: reaction liquid composition meter
After digestion at 37℃for 40min, DNA digestive enzymes were inactivated at 85℃for 3 min.
(4) Reverse transcription of RNA into CDNA
The reaction system and conditions for reverse transcription of RNA into cDNA are as follows
Table 2: reverse transcription of RNA into cDNA reaction Components Table
The reaction conditions are as follows: 37℃for 10min,42℃for 20min,85℃for 5min and 4℃for 2min.
(5) Finally, the fluorescent quantitative PCR amplification is adopted for detection, and the reaction system and the reaction conditions for detecting the expression condition of SLC24A2 and SLCO1B3 molecules in H1299 cells by the fluorescent quantitative PCR amplification are as follows:
table 3: fluorescent quantitative PCR amplification detection SLC24A2 and SLCO1B3 molecules in H1299 cell expression reaction system composition table
The fluorescent quantitative PCR reaction conditions are as follows: denaturation at 95℃for 5 min; 95℃for 10 seconds and 60℃for 35 seconds; 40 cycles.
(6) Such asFIG. 2As shown in the figure, the interference knock-down effect of Sh-SLCO1B3-3F and Sh-SLC24A2-1F is found to be best through a knock-down efficiency experiment, and the interference knock-down effect can be studied as a potential nucleic acid drug, so that Sh-SLCO1B3-3F and Sh-SLC24A2-1F are selected as subsequent shRNA interferenceSequence.
Example 3
(III) CCK8 assay to detect Effect on cell proliferation after interference with SLC24A2 and SLCO1B3
The effect of interfering with SLC24A2 and SLCO1B3 on proliferation of NSCLC cell lines was next studied.
(1) Taking transfected H1299 and A549 cells in the logarithmic growth phase, digesting the transfected H1299 and A549 cells by using 0.25% pancreatin solution, centrifuging the digested H1299 and A549 cells to obtain cell sediment, and carrying out cell counting by using a cell counting plate after re-suspending the cells by using a culture medium;
(2) Diluting the concentration of the cell suspension to about 5000 cells/100. Mu.L according to the result of cell counting, and then adding the diluted cell suspension to each well in a 96-well plate;
(3) SLC24A2 and SLCO1B3 knockout vectors are constructed to effectively inhibit the expression of the RNA in a non-small cell lung cancer cell line H1299 or A549. Control (Control) transfected empty plasmid;
(4) The 96-well plates were incubated in an incubator at 37℃for appropriate periods of time (0, 24, 48 and 72 hours);
(5) mu.L of 10% CCK8 solution (i.e., 90. Mu.L of 10. Mu.L of CCK8 solution in basal medium) was added to each well plate in a 96-well plate;
(6) Placing the 96-well plate in a constant temperature incubator at 37 ℃ for continuous culture for 1-4h;
(7) Finally, the spectra 34 continuous spectrum spectrophotometer was tuned to 450nm wavelength, absorbance values for each well were measured and recorded, and comparative analysis was performed.
(8) Cell growth curves were drawn to assess the effect of SLC24A2 and SLCO1B3 knockdown on cell proliferation of non-small cell lung cancer cell lines.
(9) Results such asFIG. 3As shown, when SLC24A2 was silenced, growth of H1299 and a549 cells was significantly inhibited compared to the control, with inhibition rates of 72H: a549:28% and H1299:60 percent; since the inhibition rate was more pronounced in H1299, subsequent experiments were performed in the H1299 cell line. When SLCO1B3 was silenced, growth of H1299 cells was significantly inhibited compared to the control group, with inhibition rates of 25% for 72H, respectively. Thereby demonstrating the knock down of SLC24A2 and SLCO1B3 can obviously inhibit the cell proliferation of a non-small cell lung cancer cell line.
Example 4
(IV) Transwell experiments to detect the Effect on cell invasion after interference with SLC24A2 and SLCO1B3
(1) Coating a substrate film: matrigel was prepared at 1: the upper chamber side of the transwell chamber bottom membrane was coated after 8 dilutions (note the whole procedure on ice, otherwise Matrigel would solidify above 10 ℃), and air dried for 3 hours.
(2) Hydrating the basement membrane: the plates were aspirated for residual fluid, 50. Mu.L of 10g/LBSA serum-free medium was added to each well and incubated at 37℃for 30min.
(3) Cells were digested with conventional pancreatin, washed 1-2 times with PBS to remove serum effects, resuspended with serum-free medium, and cell density adjusted to 5 x 10 5 And each mL.
(4) 200. Mu.L of the cell suspension was added to the upper chamber of the Transwell chamber, and 600. Mu.L of 10% FBS-containing medium was added to the lower chamber of the 24-well plate. Note that no bubbles are generated between the lower culture solution and the cells.
(5) The plates were placed in a CO2 incubator at 37℃and incubated for 48h.
(6) Taking out the cell, leaching with PBS for 2 times, carefully wiping off cells in the upper layer of the microporous membrane of the cell with a cotton swab, fixing with 4% paraformaldehyde (or 95% alcohol) in a 24-well plate for 20min, and dyeing with crystal violet solution for 15min.
(7) Photographs were taken under an inverted microscope, 10 fields were counted at random for each sample, averaged, and statistically analyzed.
(8) Results such asFIG. 4As shown, when SLC24A2 was silenced, the invasion trend of H1299 cells was significantly inhibited compared to the control group, with inhibition rates of 48H: 80%; when SLCO1B3 was silenced, growth of H1299 cells was significantly inhibited compared to the control group, with inhibition rates of 85% for 48H, respectively. Thus, the SLC24A2 and SLCO1B3 knockdown can obviously inhibit the invasion capacity of the non-small cell lung cancer cell line.
Example 5
(V) cell scratch-healing test (scratch-sealing) test for influence on cell migration ability after interference with SLC24A2 and SLCO1B3
(1) Will be 1X 10 6 The H1299 cells were plated in 60mm dishes, 3mL 1640 complete medium was added, and the plates were incubated in a 5% CO2 air incubator at 37 ℃.
(2) After 24 hours, the confluence of cells is observed to reach more than 95 percent under a mirror, and a 200 mu L yellow gun head is used for scribing a line with 0.5 cm to 1cm behind a 6-hole plate to traverse through holes. Each hole passes through at least 5 lines. Then tumor cells infected by SLC24A2 and SLCO1B3 over-expressed lentivirus are inoculated into a 6-well plate, and the gun head is vertical and cannot be inclined as far as possible and perpendicular to the transverse line scratch behind the straight ruler by being compared with the gun head on the next day.
(3) The medium was discarded, the residual cell debris was washed away by adding 2mL of sterile PBS, and 3mL of 1640 complete medium containing 0.5% FBS was added.
(4) The initial cell position was observed under a microscope and photographed and recorded, counting at the 0h time point.
(5) After 24h, the cell migration sites were again photographed and recorded.
(6) Samples were taken at 0 and 24 hours, and cell migration areas were calculated and statistically mapped using Image J software. The cell mobility calculation formula was (1-24 h scratch area/0 h scratch area) ×100%.
(7) Investigation of changes in the invasive potential of cells by microscopy, results such asFIG. 5As shown, when SLC24A2 was silenced, the invasion trend of H1299 cells was significantly inhibited compared to the control group, with inhibition rates of 48H: 50%; when SLCO1B3 was silenced, growth of H1299 cells was significantly inhibited compared to the control group, with inhibition rates of 60% for 48H, respectively. Thus, it was demonstrated that interfering with the expression of SLC24A2 and SLCO1B3 significantly inhibited the ability of non-small cell lung cancer cells to migrate.
Example 6
Sixth flow cytometry to detect induction of apoptosis after interfering with SLC24A2 and SLCO1B3
Next, flow cytometry experiments were performed to investigate the apoptotic effects of SLC24A2 and SLCO1B3 on human non-small cell lung cancer cell line H1299. The experimental procedure is as follows:
(1) Culturing cells by using a 6-hole plate, sucking out old culture medium when the growth of the cells reaches 60% -70%, processing according to experimental requirements, and continuously culturing for 24 hours.
(2) Sucking the cell culture fluid into a proper centrifuge tube, washing the adherent cells once by PBS, and adding proper amount of pancreatin cell digestive fluid to digest the cells. Incubating at room temperature until the cells are gently blown off, and sucking out pancreatin cell digestive juice.
(3) Adding the cell culture solution collected in the step (2), slightly mixing, transferring into a centrifuge tube, centrifuging for 5min at 1000g, discarding the supernatant, collecting cells, and lightly suspending the cells with PBS and counting.
(4) 5-10 ten thousand resuspended cells were taken, centrifuged at 200g for 5min, the supernatant was discarded, and 195. Mu.L of Annexin V-FITC conjugate was added to gently resuspend the cells.
(5) mu.L of Annexin V-FITC was added and gently mixed.
(6) Incubate at room temperature (20-25 ℃) for 10min in the dark. Light protection can be performed using aluminum foil.
(7) The cells were resuspended by centrifugation at 200g for 5min, discarding the supernatant and adding 190. Mu.L of Annexin V-FITC conjugate.
(8) Add 10 μl propidium iodide staining solution, mix gently, and place in ice bath in the dark.
(9) And (3) detecting by using a flow cytometer, wherein Annexin V-FITC is green fluorescence, and PI is red fluorescence.
(10) Results such asFIG. 6It is shown that knocking down SLC24A2 and SLCO1B3 significantly enhanced apoptosis in lung cancer cell line H1299. The enhancement effects are respectively as follows: compared with control, the apoptosis rate of SLC24A2 group is increased from 8% to 21%, and the apoptosis rate is remarkably different; compared with control, the apoptosis rate of SLCO1B3 group is increased from 6% to 16%, and the apoptosis rate has obvious difference; thus, the ability of knock-down SLC24A2 and SLCO1B3 to induce apoptosis in non-small cell lung cancer cell lines was demonstrated.
In summary, the present invention provides a gene marker, namely SLC24A2 and SLCO1B3 genes, for diagnosis and treatment of non-small cell lung cancer. The inventors found that SLC24A2 and SLCO1B3 were significantly elevated in lung squamous carcinoma and lung adenocarcinoma samples, suggesting that SLC24A2 and SLCO1B3 are intimately associated with the occurrence of NSCLC. Therefore, SLC24A2 and SLCO1B3 markers can be used for predicting occurrence of the non-small cell lung cancer and can be used as diagnostic markers of the non-small cell lung cancer. Further, the inventors designed shRNA silencing interfering sequences of SLC24A2 and SLCO1B3 and verified the function of the interfering sequences by cell experiments: the effect study of SLC24A2 and SLCO1B3 on cell proliferation is detected through CCK8 experiment, and the result shows that after interference inhibition expression is carried out on the lung cancer cell lines A549 and H1299 by SLC24A2 and SLCO1B3, the growth of the lung cancer cells is obviously inhibited. The influence study of SLC24A2 and SLCO1B3 on the migration capacity of cells is detected through Transwell and cell scratch-healing experiments, and the result shows that the invasion and migration capacity of the cells are obviously inhibited after the interference inhibition expression of the lung cancer cell lines A549 and H1299 is carried out on the SLC24A2 and SLCO1B 3. The influence study of SLC24A2 and SLCO1B3 on apoptosis is detected through a flow cytometry experiment, and the result shows that after interference inhibition expression of SLC24A2 and SLCO1B3 in lung cancer cell lines A549 and H1299 is carried out, the apoptosis capacity is increased. Therefore, SLC24A2 and SLCO1B3 can be used as markers for diagnosing the non-small cell lung cancer, and shRNA can interfere with SLC24A2 and SLCO1B3 and can also be used as potential small nucleic acid drugs and targets for treating the non-small cell lung cancer. Reagents for detection and shRNA interference with SLC24A2 and SLCO1B3 may be used in products for diagnosis or treatment of cancer, such as chips, kits or nucleic acid strips, nucleic acid drugs and immunotherapy.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
- SLC24A2 and SLCO1B3 gene markers are used as detection primers of a non-small cell lung cancer treatment target, and are characterized in that the detection primers comprise forward primers and reverse primers, and the nucleotide sequences of the forward primers are shown in the following sequence 1 and sequence 3; the nucleotide sequence of the reverse primer is shown in the following sequence 2 and sequence 4;SLC24A2-Forward CCAAGGAGACTACCCGAAAGA (sequence 1)SLC24A2-Reverse CAGACAATGGCTAAGGCTATGAA (SEQ ID NO: 2)SLCO1B3-Forward TGGAGCAACAGTACGGTCAG (SEQ ID NO: 3)SLCO1B3-Reverse TGCTTTCGCAGATTAGAGGGAA (SEQ ID NO: 4).
- 2. The use of SLC24A2 and SLCO1B3 gene markers according to claim 1 for the preparation of a product for diagnosis or treatment of non-small cell lung cancer, wherein said use is in the form of a reagent, further comprising an RNA extraction reagent, a reverse transcription reaction system and a fluorescent quantitative PCR reagent.
- 3. The shRNA silencing interference sequence corresponding to SLC24A2 and SLCO1B3 gene markers of claim 1, wherein the shRNA silencing interference sequence is capable of specifically inhibiting SLC24A2 and SLCO1B3 gene expression, is complementary to SLC24A2 and SLCO1B3 mRNA, and interferes with or inhibits protein translation thereof, and is as follows:Sh-SLC24A2-1:CCGGGCCGAAGAACTTGGATCATATCTCGAGATATGATCCAAGTTCTTCGGCTTTTTG (SEQ ID NO: 5);Sh-SLC24A2-2:CCGGCATTACCTGGATTGCAGTATTCTCGAGAATACTGCAATCCAGGTAATGTTTTTG (SEQ ID NO: 6);Sh-SLC24A2-3:CCGGGCAAATGATAAACCGCAATAACTCGAGTTATTGCGGTTTATCATTTGCTTTTTG (SEQ ID NO: 7);Sh-SLCO1B3-1:CCGGGGTAGTTGTAACTGCTAATAACTCGAGTTATTAGCAGTTACAACTACCTTTTTG (SEQ ID NO: 8);Sh-SLCO1B3-2:CCGGTGGTTAGTGTGTGATACAATACTCGAGTATTGTATCACACACTAACCATTTTTG (SEQ ID NO: 9);Sh-SLCO1B3-3:CCGGGCTTTAAGATTCCCAGCACTTCTCGAGAAGTGCTGGGAATCTTAAAGCTTTTTG (SEQ ID NO: 10).
- 4. The use of shRNA silencing interfering sequences corresponding to SLC24A2 and SLCO1B3 gene markers of claim 3 for the preparation of a medicament for inhibiting non-small cell lung cancer growth and tumor metastasis.
- 5. The shRNA targeting interference technology for inhibiting SLC24A2 and SLCO1B3 gene expression is characterized in that a segment of nucleotide sequence which can specifically inhibit SLC24A2 and SLCO1B3 gene expression is used as a core sequence to prepare a gene interference RNA fragment.
- 6. The shRNA targeting interference technology for inhibiting the expression of SLC24A2 and SLCO1B3 genes according to claim 5, wherein the nucleotide sequence capable of specifically inhibiting the expression of SLC24A2 and SLCO1B3 genes is:Sh-SLC24A2-1:CCGGGCCGAAGAACTTGGATCATATCTCGAGATATGATCCAAGTTCTTCGGCTTTTTG (SEQ ID NO: 5);Sh-SLCO1B3-3:CCGGGCTTTAAGATTCCCAGCACTTCTCGAGAAGTGCTGGGAATCTTAAAGCTTTTTG (SEQ ID NO: 10).
- 7. The shRNA targeted interference technology for inhibiting SLC24A2 and SLCO1B3 gene expression according to claim 5, wherein the prepared gene interference RNA fragment is introduced into non-small cell lung cancer cells (H1299 and A549) through a transgenic technology, and specific recognition of SLC24A2 and SLCO1B3 mRNA is generated in the cells, so that the effects of inhibiting SLC24A2 and SLCO1B3 gene mRNA and protein translation and reducing SLC24A2 and SLCO1B3 gene expression level are achieved, and the purpose of treating non-small cell lung cancer is achieved.
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