CN117721109A - shRNA sequence for ENO1 gene and application thereof in colorectal cancer treatment - Google Patents
shRNA sequence for ENO1 gene and application thereof in colorectal cancer treatment Download PDFInfo
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Abstract
The invention relates to a shRNA sequence for ENO1 gene and application thereof in colorectal cancer treatment in the technical field of biological medicine, in particular to a shENO1 sequence which is used for silencing the ENO1 gene, cloning the shENO1 sequence to a lentivirus expression vector pLKO.1, packaging lentivirus, infecting human colorectal cancer cells, successfully reducing mRNA and protein expression level of the ENO1 in the human colorectal cancer cells, thereby obviously inhibiting growth rate of the colorectal cancer cells and having very important significance in colorectal cancer treatment. The shENO1 sequence designed by the invention is convenient to develop into a clinical therapeutic drug for treating colorectal cancer, and has wide market application prospect and potential social and economic benefits.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an shRNA sequence for an ENO1 gene and application thereof in colorectal cancer treatment.
Background
Colorectal cancer (colorectal cancer, CRC), including colon and rectal cancers, is the third largest cancer worldwide, and is one of the most common digestive tract malignancies, with both morbidity and mortality being among the common cancer progenitors. Since early symptoms of CRC are not obvious, most patients are already in advanced stages when obvious symptoms of rectal bleeding, anemia or abdominal pain occur, at which time tumor cells are invasive and metastatic, which results in a rapid increase in CRC mortality. Studies have shown that CRC has a 5-year survival rate of only about 50%. Currently, the treatment for CRC mainly includes surgical treatment, chemotherapy, targeted therapy, and immunotherapy. The targeted therapy has the characteristics of definite target point, strong specificity, high safety, small toxic and side effects and the like, and becomes a research hotspot for comprehensive treatment of advanced colorectal cancer. However, the available targeting therapeutic targets are very limited at present, so that the available targeting drugs are deficient, and only a very small number of patients can benefit from the targeting therapy, so that the development of novel tumor therapeutic targets is a problem to be solved at present.
The tumor cells have the Warburg effect, and the glycolysis mode is adopted preferentially under the aerobic condition to rapidly generate energy for the cancer cells, so that the rapid proliferation of the tumor is realized, and a great growth advantage is provided for the tumor. Meanwhile, the high lactic acid microenvironment caused by the transformation strengthens the invasiveness and metastasis of cancer cells. Therefore, the aim of inhibiting the growth of cancer cells can be achieved by blocking glycolysis.
Alpha-enolase 1 (eno 1) is one of 3 isoenzymes of the enolase family, is highly expressed in a variety of tumors, and promotes tumor growth. As an important rate-limiting enzyme in glycolysis, ENO1 catalyzes the dehydration of 2-phosphoglycerate of the glycolytic pathway to phosphoenolpyruvate, generates ATP, and affects tumor cell proliferation by promoting tumor cell energy metabolism. In addition to glycolysis, ENO1 is involved in a variety of processes such as growth control, hypoxia tolerance and allergic reactions.
In recent years, RNA interference technology has been developed at a high speed, and new dawn and revolutionary changes are brought to cancer treatment. RNA interference technology has great potential and development prospect as an emerging cancer treatment method. Short hairpin RNAs (shrnas) are a class of RNAs with tight hairpin turns that silence the expression of a target gene by the principle of RNA interference techniques. Therefore, to investigate the importance of a therapeutic target in tumor therapy, it is often evaluated using shRNA interference techniques. Designing an shRNA sequence aiming at a certain target point, utilizing the shRNA to interfere the expression of the target point so as to influence the function of the target point, and then evaluating whether the sequence has potential tumor treatment effect by examining the influence of the shRNA on the proliferation rate of cells.
The applicant designs a shRNA sequence which aims at the ENO1 gene and can inhibit malignant tumor cells, namely the shENO1 sequence according to the basic principle of the RNA interference technology, and provides a beneficial way for treating colorectal cancer and other related cancers.
Disclosure of Invention
Aiming at the demand of inhibiting tumor cells by an RNA interference technology in the prior art, the invention designs a shRNA sequence for an ENO1 gene, namely a shENO1 sequence, according to the basic principle of the RNA interference technology, and the shRNA sequence is used for inhibiting the proliferation rate of malignant tumor cells.
In order to achieve the aim, the invention firstly provides a shRNA sequence aiming at an ENO1 gene, namely a shENO1 sequence, wherein the shENO1 sequence is shown as SEQ ID NO. 1.
Further, the shENO1 sequence is used for silencing human ENO1 gene.
Further, the target sequence of the shENO1 is shown as SEQ ID NO. 2.
Still further, the oligonucleotide sequence that clones the shRNA sequence includes a sense strand and an antisense strand, the sense strand sequence being:
5’- CCGGCGCATTGGAGCAGAGGTTTACCTCGAGGTAAACCTCTGCTCCAATGCGTTTTTG -3’；
the antisense strand sequence is:
5’- AATTCAAAAACGCATTGGAGCAGAGGTTTACCTCGAGGTAAACCTCTGCTCCAATGCG -3’。
the invention also provides application of the shRNA sequence aiming at the ENO1 gene in treating colorectal cancer, aiming at ENO1 as a treatment target.
The shRNA sequence for the ENO1 gene designed by the invention can obviously reduce the expression level of mRNA and protein of ENO1 in colorectal cancer cells, thereby obviously inhibiting the growth rate of colorectal cancer cells, having very important significance for treating colorectal cancer, being convenient for further developing the shRNA sequence into clinical therapeutic drugs, having wide market application prospect and very great potential social and economic benefits.
Drawings
FIG. 1 shows the effect of real-time fluorescent quantitative PCR method for detecting the expression of the ENO1 gene silenced by the shENO1.
FIG. 2 is a graph showing the effect of immunoblotting for detecting the expression of the gene of the shENO1 silencing ENO1.
FIG. 3 is a graph comparing the inhibition of HCT116 colorectal cancer cell proliferation by shENO1 evaluated by cytometry.
Detailed Description
The sources of materials involved in this example:
1) 1×te buffer (ph=8.0) was purchased from beijing solebao technologies limited; pancreatin, opti-MEM ® Medium I and DMEM powder were purchased from Gibco corporation; mcCoy's 5A powder was purchased from Sigma; lipofectamine 2000 was purchased from Invitrogen corporation; restriction endonucleasesAgeI、EcoR I and is provided withBamH I from NEB company; t4 DNA ligase was purchased from Nanjinouzan Biotechnology Co., ltd; coli DH 5. Alpha. Was purchased from Beijing kang as century biotechnology Co., ltd; lenti-X293T cells were purchased from Clontech; puromycin is purchased from amerco corporation; plasmids pLKO.1, pCMV-VsVg and pCMV-deltaR8.2 were purchased from Addgene; HCT116 cells were purchased from the cell bank of the national academy of sciences;
2) The DMEM medium formulation was as follows: weighing DMEM powder 13.37 g, sodium bicarbonate 3.7 g, sodium pyruvate 110 mg, adding Milli Q ultra-pure water 800 mL, magnetically stirring, fully dissolving, adding 100 Xpenicillin-streptomycin stock solution (penicillin concentration is 10000U/mL, streptomycin concentration is 10000 μg/mL) 10 mL, adjusting pH to 7.2-7.4 with concentrated hydrochloric acid, fixing Milli Q water to 1L, filtering with double-layer 0.22 μm microporous filter membrane for sterilization, adding 10% fetal bovine serum, and storing in refrigerator at 4deg.C for use;
3) The McCoy's 5A medium formulation is as follows: weighing 11.9-g of McCoy's 5A powder, 2.2-g of sodium bicarbonate, adding 800 mL of Milli Q ultrapure water, magnetically stirring, fully dissolving, adding 10 mL of 100 Xpenicillin-streptomycin stock solution (penicillin concentration is 10000U/mL, streptomycin concentration is 10000 mug/mL), adjusting pH to 7.2-7.4 with concentrated hydrochloric acid, sterilizing by Milli Q water to 1L and 0.22 mu m microporous membrane filtration, adding 10% fetal bovine serum, and storing in a refrigerator at 4 ℃ for standby;
4) The remaining reagents and materials involved in this example are all commercially available and are not listed here.
Nucleotide sequence information (NM-001428.5) of the human ENO1 gene was obtained by means of the GenBank database (http:// www.ncbi.nlm.nih.gov/GenBank). Then, according to the design principle of the shRNA, the shRNA sequence aiming at the human ENO1 gene is designed and is marked as shENO1 and used for silencing the human ENO1 gene. The target sequence of the shENO1 is CGCATTGGAGCAGAGGTTTAC (SEQ ID NO. 2), and two oligonucleotide sequences are designed according to the target sequence and used for cloning the shENO1 coding sequence (SEQ ID NO. 1) into a lentiviral vector pLKO.1. The synthetic oligonucleotide sequences were as follows:
the sense strand sequence is:
5’- CCGGCGCATTGGAGCAGAGGTTTACCTCGAGGTAAACCTCTGCTCCAATGCGTTTTTG -3’；
the antisense strand sequence is:
5’- AATTCAAAAACGCATTGGAGCAGAGGTTTACCTCGAGGTAAACCTCTGCTCCAATGCG -3’。
meanwhile, a Luciferase (Luciferase) gene is adopted as a control group, a control shRNA sequence (marked as shLuc) aiming at the Luciferase is designed, and a target sequence is CGCTGAGTACTTCGAAATGTC (SEQ ID NO. 4). Two oligonucleotide sequences were designed for the target sequence for cloning the shLuc coding sequence (SEQ ID No. 3) into lentiviral vector plko.1. The sequence of the synthesized oligonucleotide is shown below:
the sense strand sequence is:
5’-CCGGCGCTGAGTACTTCGAAATGTCCTCGAGGACATTTCGAAGTACTCAGCGTTTTT-3’
the antisense strand sequence is:
5’-AATTAAAAACGCTGAGTACTTCGAAATGTCCTCGAGGACATTTCGAAGTACTCAGCG-3’。
cloning the coding sequence of the shENO1 to a lentiviral expression vector pLKO.1 to construct a recombinant lentiviral plasmid pLKO.1-shENO1. The construction method was as follows, the synthesized sense strand and antisense strand were formulated with 1×TE buffer as solutions at a concentration of 100. Mu.M. 1. Mu.L of each of the above solutions was added to a PCR tube containing 8. Mu.L of 1 XTE buffer, and mixed well. The mixture was placed in a conventional PCR apparatus, treated at 94℃for 3 min, and then annealed slowly to 25℃at a rate of 0.5℃per min. After the reaction is finished, firstly diluting the reaction product by 100 times by adopting 1 xTE buffer solution; then the lentiviral expression vector pLKO.1 is subjected to the processAgeI/EcoR I double enzyme digestion and purification; and then the diluted reaction product is connected with a double enzyme cutting carrier plKO.1 by a T4 DNA ligase according to a conventional method, the connection product is transformed into competent escherichia coli DH5 alpha, and the single clone is selected for overnight culture, and plasmid is extracted, so as to obtain the recombinant plasmid. Warp yarnEcoR I/BamH I double cleavage and sequencing analysis, and identification of recombinant plasmids.
The construction method of the control plasmid pLKO.1-shLuc is the same as that of the above-mentioned pLKO.1-shENO1.
Packaging lentiviruses with recombinant plasmids: the lentiviruses were packaged by taking the recombinant lentivirus plasmids pLKO.1-shENO1 and pLKO.1-shLuc identified correctly, referring to the instructions for the Lipofectamine 2000 reagent. Taking a 6-well cell culture plate as an example, the method for packaging lentiviruses is as follows:
(1) inoculating Lenti-X293T cells into a 6-well cell culture plate at 37℃with 5% CO 2 Culturing overnight in an incubator, and starting experiments when the cell fusion degree (conflux) reaches 90% -95%;
(2) a1.5 mL centrifuge tube 1 was taken and 195. Mu.L Opti-MEM was added ® The culture medium I, according to the mass ratio of the plasmid to the pLKO.1-shENO1, pCMV-VsVg, pCMV-delta-R8.2=3:1:2, respectively adding three plasmids (the total amount of the plasmids is 2.34 mug) into a centrifuge tube, uniformly mixing, and standing at room temperature for standby;
(3) a new 1.5. 1.5 mL centrifuge tube 2 was taken and 195. Mu.L Opti-MEM was added ® Mixing the culture medium I and 4.68 mu L Lipofectamin 2000, upside down, and standing at room temperature for 5 min;
(4) adding all the mixed liquid in the centrifuge tube 2 into the centrifuge tube 1, slightly and reversely mixing the mixed liquid, and standing for 20 min at room temperature;
(5) transferring the mixed solution obtained in the step (4) into a 6-hole cell culture plate hole inoculated with Lenti-X293T cells, gently mixing, and heating at 37 ℃ with 5% CO 2 Culturing in an incubator, and changing the liquid after 11 h (using fresh DMEM culture medium);
(6) continuing to culture 48 h, collecting the cell culture supernatant in 15 mL centrifuge tube 3, and temporarily storing the supernatant at 4deg.C while supplementing 6-well cell culture plates with fresh DMEM medium at 37deg.C, 5% CO 2 Culturing in an incubator for 24 h;
(7) collecting the cell culture supernatant again in a 15 mL centrifuge tube 3, and gently mixing the supernatant collected twice by upside down;
(8) the mixed supernatant was centrifuged at 3000 rpm for 10 min at room temperature and then sub-packed into 1.5 mL centrifuge tubes, 1 mL/tube, and stored at-80℃for either use or direct use in cell infection. The method for packaging the lentivirus by the recombinant plasmid pLKO.1-shLuc is the same as above.
And detecting the effect of the shENO1 in silencing ENO1 gene expression by adopting a fluorescent quantitative PCR method and an immunoblotting method. Packaging lentivirus with recombinant plasmid pLKO.1-shENO1, and infecting HCT116 human colorectal cancer cells in logarithmic growth phase; then, mRNA and protein levels of intracellular ENO1 were detected by a fluorescent quantitative PCR method and an immunoblotting method, respectively, to evaluate the effect of the shENO1 on the inhibition of ENO1 gene expression. The specific method comprises the following steps:
human colorectal cancer cells HCT116 were seeded in 6 cm cell culture plates, 4X 10 per well 5 Individual cells, at 37 ℃, 5% CO 2 Culturing in an incubator for 24 hours. HCT116 cells in the culture wells were infected with pLKO.1-shENO1 and pLKO.1-shLuc lentiviruses, respectively, and at the time of infection, polybrene (polybrene) was added to the culture wells at a final concentration of 8. Mu.g/mL. After 16-18 hours the fresh McCoy's 5A medium containing serum was changed. Puromycin (puromycin) was added 2 days later for screening to obtain HCT116 cells stably infected with lentivirus. After 5 days of continued culture, stable cell lines infected with lentivirus were collected. Each cell line was divided into two parts: one part is used for extracting total RNA and synthesizing cDNA by reverse transcription, and the relative level of ENO1 mRNA in cells is detected by a fluorescent quantitative PCR method; the other was used to extract total protein and relative levels of intracellular ENO1 protein were detected by immunoblotting. Finally, the gene silencing effect of the shENO1 is comprehensively evaluated according to the detection results of a real-time fluorescence quantitative PCR method and an immunoblotting method (see fig. 1 and 2). The real-time fluorescent quantitative PCR results (fig. 1) showed that ENO1 mRNA was reduced to 9%, and the immunoblotting results (fig. 2) showed that the protein level of ENO1 in the ENO1 gene silencing group (shENO 1) was significantly reduced compared to the control group (shruc). Therefore, the shENO1 designed by the invention can effectively silence the expression of the ENO1 gene.
Inoculating HCT116 cells with silence of ENO1 gene into a 6-hole cell culture plate, and placing CO 2 The incubator was kept in culture, and cells were counted at different time points to evaluate the effect of the shENO1 on cell proliferation. The operation steps are as follows:
inoculating the cells to be tested into a 6-hole cell culture plate, wherein the number of the cells is 2 multiplied by 10 5 /well. At 37℃with 5% CO 2 Culturing in incubator for 2 days, digesting with pancreatin, counting, inoculating the cells again to 6-hole cell culture plate, and inoculating 2×10 cells 5 /well. The culture was continued and the above steps were repeated on days 4 and 6.
Cell growth curves were plotted according to the count results to evaluate whether silencing of the ENO1 gene inhibited proliferation of HCT116 cells. The results are shown in FIG. 3, in which the growth of cells in the shENO1 experimental group was significantly inhibited compared to the cells in the shLuc control group. Therefore, the shENO1 designed by the invention is hopeful to be developed into a drug molecule for treating colorectal cancer.
The foregoing is only one specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and those skilled in the art can make adaptation and substitution within the technical scope of the present invention disclosed herein.
Claims (5)
1. A shRNA sequence for ENO1 gene, namely a shENO1 sequence, is characterized in that the shENO1 sequence is shown as SEQ ID NO. 1.
2. The shRNA sequence for an ENO1 gene of claim 1, wherein said shrno 1 sequence is used to silence a human ENO1 gene.
3. The shENO1 according to claim 1, wherein the target sequence of the shENO1 is shown in SEQ ID No. 2.
4. The shRNA sequence for an ENO1 gene according to claim 2, wherein the oligonucleotide sequence that clones the shRNA sequence comprises a sense strand and an antisense strand, the sense strand sequence being:
5’- CCGGCGCATTGGAGCAGAGGTTTACCTCGAGGTAAACCTCTGCTCCAATGCGTTTTTG -3’；
the antisense strand sequence is:
5’- AATTCAAAAACGCATTGGAGCAGAGGTTTACCTCGAGGTAAACCTCTGCTCCAATGCG -3’。
5. use of the shRNA sequence directed against the ENO1 gene according to any one of claims 1 to 4 as a therapeutic target for ENO1 in the treatment of colorectal cancer.
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