CN111084781B - Application of ARHGEF19 antisense nucleotide sequence in preparing medicine for inhibiting tumor cell growth and expression vector thereof - Google Patents
Application of ARHGEF19 antisense nucleotide sequence in preparing medicine for inhibiting tumor cell growth and expression vector thereof Download PDFInfo
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Abstract
The invention provides an application of an ARHGEF19 antisense nucleotide sequence in preparing a medicament for inhibiting the growth of tumor cells. The targeting sequence of the ARHGEF19 antisense nucleotide sequence is a sequence shown as SEQ ID NO.1 or SEQ ID NO.2 positioned on ARHGEF 19. The positive and negative sequences of the ARHGEF19 antisense nucleotide sequence are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6. Also provided is an expression vector of the ARHGEF19 antisense nucleotide sequence. The invention provides a targeted treatment strategy aiming at the condition of poor treatment effect of the existing small cell lung cancer, and reduces the expression of ARHGEF19 through the antisense nucleotide sequence of ARHGEF19, thereby inhibiting the growth of small cell lung cancer cells.
Description
Technical Field
The invention relates to the technical field of tumor cell growth inhibition, in particular to application of an ARHGEF19 antisense nucleotide sequence in preparing a drug for inhibiting tumor cell growth and an expression vector thereof.
Background
ARHGEF19(Rho guanine nucleotide exchange factor 19, also called WGEF) belongs to the Rho guanylate exchange factor (RhoGEFs, also called ARHGEFs) family, RhoGEFs proteins can activate Rho-GTP by catalyzing Rho-GDP to activate it, thereby activating Rho proteins, and Rho proteins participate in various intracellular signal transduction pathways, and play an important role in gene transcription, cell cycle, vesicle transport and angiogenesis. Research reports that RhoGEFs family members participate in the generation and development of tumors, for example, ARHGEF2 plays a promoting role in the metastasis process of breast cancer, melanoma, liver cancer and other tumors, and the high expression of ARHGEF5 can promote the generation and metastasis of lung adenocarcinoma. ARHGEF19 is taken as a member of RhoGEFs family, and we have reported that ARHGEF19 is significantly highly expressed in non-small cell lung cancer, and the processes of proliferation, metastasis and the like of the non-small cell lung cancer can be inhibited when ARHGEF19 is knocked down.
Lung cancer is the most common malignancy worldwide, with the incidence and mortality rates ranked first. The lung cancer is divided into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), the small cell lung cancer accounts for 15% -20% of the lung cancer incidence cases, although the proportion is lower than that of the non-small cell lung cancer, compared with the non-small cell lung cancer, the small cell lung cancer has the characteristics of higher tumor proliferation speed, higher malignancy degree, earlier metastasis generation period and the like, and the small cell lung cancer is difficult to cure due to the fact that the small cell lung cancer has the tendency of wide spread during diagnosis.
Because the small cell lung cancer is transferred quickly, only a few patients can be subjected to surgical resection, the main treatment means is combined chemotherapy and chest radiotherapy, but the small cell lung cancer patients are often poor in prognosis along with toxic and side effects and drug resistance reaction.
Aiming at the characteristics of small cell lung cancer, the specificity treatment is carried out to improve the specificity and the sensitivity of the small cell lung cancer treatment, and the method is an important strategy for achieving radical treatment. The expression of the ARHGEF19 is inhibited through RNA interference (RNAi), so that the growth of the small cell lung cancer is inhibited, and the method can be applied to the preparation of the medicine for inhibiting the growth of the small cell lung cancer.
Disclosure of Invention
The invention aims to provide application of an ARHGEF19 antisense nucleotide sequence in preparing a medicament for inhibiting tumor cell growth, provides a targeted treatment strategy aiming at the condition of poor treatment effect of the existing small cell lung cancer, and reduces the expression of the ARHGEF19 through the ARHGEF19 antisense nucleotide sequence so as to inhibit the growth of the small cell lung cancer cells.
In order to solve the technical problems, the invention provides an application of an ARHGEF19 antisense nucleotide sequence in preparing a medicament for inhibiting the growth of tumor cells.
Preferably, the ARHGEF19 antisense nucleotide sequence achieves the purpose of inhibiting the growth of the small cell lung cancer by reducing the expression of the ARHGEF 19.
Preferably, the targeting sequence of the ARHGEF19 antisense nucleotide sequence is a sequence shown as SEQ ID NO.1 or SEQ ID NO.2 positioned on ARHGEF 19.
Preferably, the positive and negative two sequences of the ARHGEF19 antisense nucleotide sequence are respectively shown as SEQ ID NO.3 or SEQ ID NO. 4.
Preferably, the positive and negative two sequences of the ARHGEF19 antisense nucleotide sequence are respectively shown as SEQ ID NO.5 or SEQ ID NO. 6.
Preferably, the tumor cell is a small cell lung cancer cell with high ARHGEF19 expression.
Preferably, the small cell lung cancer cell is a NCI-H69 cell or a NCI-H526 cell.
Preferably, the ARHGEF19 antisense nucleotide sequence is cloned into a silent expression vector for preparing a medicament for inhibiting the growth of small cell lung cancer cells.
The invention also provides an expression vector of the ARHGEF19 antisense nucleotide sequence, which is constructed by taking pLKO.1-TRC cloning vector as a basic vector.
Aiming at the small cell lung cancer with high ARHGEF19 expression, the NCI-H69 and NCI-H526 cell lines are selected as research objects, the gene expression of the ARHGEF19 is inhibited by combining the antisense nucleotide sequence of the ARHGEF19 with messenger RNA (mRNA) of a target gene, and the result shows that the antisense nucleotide of the ARHGEF19 can reduce the expression of the RNA and protein thereof, thereby inhibiting the growth of the small cell lung cancer cells.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
The application of the ARHGEF19 antisense nucleotide sequence in the preparation of drugs for inhibiting the growth of tumor cells and the expression vector thereof are further described in detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 shows the sequencing result of ARHGEF19-shRNA-1 plasmid, which indicates that the vector construction is successful;
FIG. 2 is a sequencing result of the ARHGEF19-shRNA-2 plasmid, which shows that the vector construction is successful;
FIG. 3 is a sequencing result of the Scramble-shRNA plasmid, which shows that the vector construction is successful;
FIG. 4 is a qPCR assay for mRNA expression of ARHGEF19 in NCI-H69 knockdown cells;
FIG. 5 is a western blot to detect protein expression levels of ARHGEF19 in NCI-H69 knockdown cells;
FIG. 6 is a qPCR assay for mRNA expression of ARHGEF19 in NCI-H526 knockdown cells;
FIG. 7 is a western blot for detecting protein expression levels of ARHGEF19 in NCI-H526 knockdown cells;
FIG. 8 is a growth curve of NCI-H69 knockdown cells;
FIG. 9 is a growth curve of NCI-H526 knockdown cells;
FIG. 10 is a flow cytometry analysis of the cell cycle distribution of NCI-H69 cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein can be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 construction of pLKO.1-ARHGEF19-shRNA lentiviral vector
(1) The following positive and negative oligonucleotide sequences (oligos, 5 '→ 3') containing the antisense nucleotide sequence of ARHGEF19 were synthesized:
ARHGEF19-shRNA-1-Forward:
CCGGGCCCAGTGTGTCTGGACCTTTCTCGAGAAAGGTCCAGACACACTGGGCTTTTTG (shown in SEQ ID NO. 3);
ARHGEF19-shRNA-1-Reverse:
AATTCAAAAAGCCCAGTGTGTCTGGACCTTTCTCGAGAAAGGTCCAGACACACTGGGC (shown in SEQ ID NO. 4);
ARHGEF19-shRNA-2-Forward:
CCGGCCAGTCTCGATTCCTCCTTAACTCGAGTTAAGGAGGAATCGAGACTGGTTTTTG (shown in SEQ ID NO. 5);
ARHGEF19-shRNA-2-Reverse:
AATTCAAAAACCAGTCTCGATTCCTCCTTAACTCGAGTTAAGGAGGAATCGAGACTGG (shown in SEQ ID NO. 6);
and simultaneously synthesizing the following two oligonucleotide sequences (5 '→ 3') in the sense of a scrambled sequence without any corresponding sequence on the genome as controls:
Scramble-shRNA-Forward:
CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG (shown in SEQ ID NO. 7);
Scramble-shRNA-Reverse:
AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG (shown in SEQ ID NO. 8).
(2) Annealing of oligonucleotides
The synthesized oligonucleotides were dissolved with sterile deionized water to a final concentration of 100nM and the following reaction system was prepared in a microcentrifuge tube:
boiling in water bath at 100 deg.C for 5min, and slowly cooling to room temperature in water bath.
(3) Cleavage of pLKO.1-TRC cloning vector (hereinafter, pLKO.1) lentiviral vector
The lentivirus vector can effectively integrate exogenous genes onto a host genome so as to realize persistent expression, and the lentivirus is widely applied to the research of RNAi at present and has the advantages of high-efficiency gene knockdown, capability of blocking the expression of genes for a long time and the like, so that the lentivirus vector has wide application prospect. The pLKO.1 lentiviral vector has a Puromycin (Puromycin) resistance gene, and can quickly obtain a stable cell strain. pLKO.1 vector (Addgene, cat #10878, plasmid full length 8901bp) containing Age I and EcoR I restriction sites, and double-digesting the vector with these two enzymes to obtain a linearized vector containing sticky ends, the double-digesting reaction system is shown below:
incubating in water bath at 37 ℃ for 2-3h, running agarose gel electrophoresis, and purifying the enzyme digestion product by using a gel purification recovery kit to obtain a linearized vector fragment.
(4) The oligonucleotide annealing product is connected and recombined with the linearized pLKO.1 vector
The ligation reaction system is shown below:
incubate at 16 ℃ for 1 h.
(5) Conversion of ligation products
Taking out DH5 alpha competent cells from an environment at minus 80 ℃, adding 10 mu l of the ligation product after the cells are melted, gently mixing the cells uniformly, and then placing the cells on ice for 30 min; heat shock is carried out for 90s in water bath at 42 ℃, and then the mixture is rapidly placed on ice for 2-3 min; adding 600 μ lLB liquid culture medium (without antibiotic resistance), and shake culturing in shaker at 37 deg.C for 45-60 min; centrifuging at 3000rpm for 2min, resuspending the precipitate with 100. mu.l of supernatant, uniformly spreading in LB solid culture dish containing Ampicillin (Ampicillin), inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing overnight in a 37 ℃ constant temperature incubator.
(6) Picking single clone and sequencing verification
After the culture dish is cultured for 14h, the single colony can grow, 2-3 single colony colonies are selected each and inoculated in 5ml LB liquid medium, and shaking culture is carried out on a shaker at 37 ℃ for 15h at 240 rpm; extracting plasmids, sending to a company for second-generation sequencing, performing Blast homology comparison analysis on a sequenced sequence and a designed sequence, and selecting a monoclonal with correct alignment for subsequent experiments, wherein a figure 1, a figure 2 and a figure 3 are respectively result graphs of sequencing of three plasmids.
Example 2 construction of Stable knockdown ARHGEF19 Small cell Lung cancer cells
(1) Packaging of lentiviruses
HEK293T cells were first plated at 5X 10 per well5The cells are spread in a six-well plate and cultured overnight; the next day when the cell density is 60-70%, the transfection is carried out, the proportion of the transfection plasmid is pLKO.1 vector to psPAX.2 to pMD2.G is 4: 3: 1, and the transfection process is carried out according to LipofectinTM2000 instructions for transfection reagents; and 8h after transfection, replacing the fresh culture medium, and collecting supernatant 48h after liquid replacement to obtain the slow virus liquid.
(2) Screening of lentivirus-infected small cell lung cancer cells and stable cell lines
And infecting the cells of the small cell lung cancer cell lines NCI-H69 and NCI-H526 by using the packaged lentivirus liquid, adding Puromycin (Puromycin) for stable screening after infecting for 48 hours, and screening for about 5 days to ensure that the cells of the blank control group are completely dead and the cells infected with lentivirus survive.
(3) Identification of stable cell lines
And respectively extracting RNA and protein from the screened cells, and verifying the knock-down effect of the mRNA level and the protein level by qPCR and Western blot.
Knockdown efficiency of qPCR assay for mRNA levels: the resulting cells were screened, total RNA was extracted using TRIzol Reagent (ThermoFisher Scientific), and cDNA was synthesized by reverse transcription using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TranGen Biotech) kit under the conditions of 42 ℃ for 15min, 85 ℃ for 5s, and 4 ℃ for infinity. The synthesized cDNA was diluted 10-fold with sterile deionized water and subjected to qPCR to detect mRNA level of ARHGEF 19. The Gene sequence of ARHGEF19 was obtained from NCBI Gene database, and Gene-specific primers were designed using Primer 5 and synthesized by Ruibo corporation, and the Primer sequences were as follows:
ARHGEF 19-qPCR-F: 5'-TGAAGAGGACAGAGGAAC-3' (shown in SEQ ID NO. 9)
ARHGEF 19-qPCR-R: 5'-GAAGAGGTGGAGGTAGAC-3' (shown in SEQ ID NO. 10)
qPCR results analysis GAPDH was used as an internal control, with-2ΔΔc(t)The relative expression of ARHGEF19 was calculated and statistically analyzed by SPSS software. Results as shown in fig. 4 and 6, both shrnas could significantly knock down ARHGEF19 at the mRNA level in both small cell lung cancer cells.
The efficiency of knocking down protein levels by Western blot detection is as follows: and collecting the total protein of the screened cells by using protein lysate (lysine), fully cracking, centrifuging to take supernatant for protein quantification, carrying out the quantification by a quantification method according to a method of a BCA protein content detection kit, and extracting 20 mu g of the total protein to carry out a Western blot experiment. As shown in fig. 5 and 7, two ARHGEF19 shRNA also significantly knocked down ARHGEF19 at the protein level in two small cell lung cancer cells compared to scarmble-shRNA, with GAPDH protein as the internal reference protein.
qPCR and Western blot verification show that compared with Scamble-shRNA, ARHGEF19-shRNA-1 and ARHGEF19-shRNA-2 can obviously knock down ARHGEF19 and can be used for subsequent experiments.
Example 3 Effect of ARHGEF19 knockdown on growth of Small cell Lung cancer cells
The above-identified cells were counted and diluted to 1X 105One cell/ml, in six well plates, 1ml per well, i.e. 1X 105And (4) inoculating 7 cells in each group per well, and placing the cells in an incubator for continuous culture. The cells from one well of each group were counted three times the next day, and the average was taken as the number of cells in the group. Counting is continued, and each group of the fine powder is counted for 7 daysThe number of cells. The cell growth curves were plotted in triplicate, time (days) on the horizontal axis and cell number per day on the vertical axis, and statistically analyzed using SPSS software. FIGS. 8 and 9 are growth curves of cells after the cells NCI-H69 and NCI-H526 knock down ARHGEF19 respectively, which show that after the expression of ARHGEF19 is inhibited by shRNA, the proliferation capacities of the two cells are obviously reduced.
Example 4 Effect of ARHGEF19 knockdown on cell cycle
The constructed NCI-H69 knockdown cell line was collected, centrifuged at about 1000g for 3min to pellet the cells, carefully aspirated off the supernatant, 1ml of pre-cooled PBS was added to resuspend the cells, and the cells were transferred to a 1.5ml centrifuge tube, centrifuged again, carefully discarded. The cells were resuspended in 300. mu.l of precooled PBS, and then 700. mu.l of precooled absolute ethanol was slowly added to the cells while vortexing them at a low speed on a vortex shaker to mix them well and allowed to stand overnight at 4 ℃. The following day the cells were centrifuged at 1000g for 3min, then washed twice with pre-cooled PBS, then stained with Propidium Iodide staining solution (PI) for 30min, and then fluorescence was detected with a flow cytometer at an excitation wavelength of 488 nm. The ModFit software was used to analyze the cell distribution at various stages of the cell cycle, and the results are shown in FIG. 10 and the following table.
Cell cycle distribution of NCI-H69 cells following ARHGEF19 knockdown
The result shows that after the ARHGEF19 gene is knocked down, the proportion of the cells in the S phase is increased, and the cells are blocked in the S phase.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
- The application of the ARHGEF19 antisense nucleotide sequence in the preparation of a medicine for inhibiting the growth of small cell lung cancer cells, wherein the ARHGEF19 antisense nucleotide sequence achieves the purpose of inhibiting the growth of small cell lung cancer cells by reducing the expression of ARHGEF 19.
- 2. The application of the ARHGEF19 antisense nucleotide sequence in preparing the medicines for inhibiting the growth of the small cell lung cancer cells according to claim 1, wherein the targeting sequence of the ARHGEF19 antisense nucleotide sequence is the sequence shown as SEQ ID NO.1 or SEQ ID NO.2 positioned on the ARHGEF 19.
- 3. The application of the ARHGEF19 antisense nucleotide sequence in the preparation of the drugs for inhibiting the growth of the small cell lung cancer cells according to claim 1, wherein the positive and negative sequences of the ARHGEF19 antisense nucleotide sequence are respectively shown as SEQ ID No.3 or SEQ ID No. 4.
- 4. The application of the ARHGEF19 antisense nucleotide sequence in the preparation of the drugs for inhibiting the growth of the small cell lung cancer cells according to claim 1, wherein the positive and negative sequences of the ARHGEF19 antisense nucleotide sequence are respectively shown as SEQ ID No.5 or SEQ ID No. 6.
- 5. The use of an ARHGEF19 antisense nucleotide sequence of claim 1 in the preparation of a medicament for inhibiting the growth of small cell lung cancer cells, wherein the small cell lung cancer cells are high expressing ARHGEF 19.
- 6. The use of an ARHGEF19 antisense nucleotide sequence of claim 5 in the preparation of a medicament for inhibiting the growth of small cell lung cancer cells, wherein the small cell lung cancer cells are NCI-H69 cells or NCI-H526 cells.
- 7. The use of an ARHGEF19 antisense nucleotide sequence of any one of claims 1-6 in the preparation of a medicament for inhibiting the growth of small cell lung cancer cells, wherein the ARHGEF19 antisense nucleotide sequence is cloned into a silent expression vector for use in the preparation of a medicament for inhibiting the growth of small cell lung cancer cells.
- 8. The use of the ARHGEF19 antisense nucleotide sequence of claim 7 in the preparation of a medicament for inhibiting the growth of small cell lung cancer cells, wherein the expression vector of the ARHGEF19 antisense nucleotide sequence is constructed by using pLKO.1-TRC cloning vector as a basic vector.
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