CN117448335A - shRNA for HSPA8 and application thereof in colorectal cancer treatment - Google Patents
shRNA for HSPA8 and application thereof in colorectal cancer treatment Download PDFInfo
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Abstract
The invention relates to a shRNA sequence for an HSPA8 gene and application thereof in colorectal cancer treatment in the technical field of biological medicine, in particular to a shHSPA8 sequence which is used for silencing the HSPA8 gene, cloning the shHSPA8 sequence to a lentivirus expression vector pLKO.1, packaging lentivirus, infecting human colorectal cancer cells, successfully reducing mRNA and protein expression level of the HSPA8 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 shHSPA8 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 shRNA for an HSPA8 gene and application thereof in colorectal cancer treatment.
Background
Colorectal cancer is the third largest cancer worldwide, and is also one of the most common digestive tract malignancies, with morbidity and mortality all leading to the common cancer. At present, the treatment means of colorectal cancer mainly comprise operation treatment, chemotherapy, targeted treatment and immunotherapy. The targeted therapy has the characteristics of definite target point, strong specificity, high safety, small toxic and side effects and the like, becomes a research hot spot for comprehensive treatment of advanced colorectal cancer, and is also a model for accurate treatment of colorectal cancer. However, the available targeting therapeutic targets are limited at present, the available targeting drugs are deficient, and only a very small number of patients can benefit from the targeting therapy, so that the provision of a novel tumor therapeutic target is a problem to be solved at present.
Chaperones are a class of proteins that are widely distributed in prokaryotic and eukaryotic cells and can assist in folding/shirring, assembling/disentangling of other macromolecular structures. In general, proteins that maintain normal cell growth are dependent on chaperones for proper folding, whereas cancer cells divide rapidly and are more dependent on chaperones than normal cells. Therefore, the aim of inhibiting the growth of cancer cells can be achieved by regulating the expression level of molecular chaperones to block the correct folding of proteins.
Heat shock protein family a (Hsp 70) member 8 (HSPA 8) is a chaperone that plays an important role in the quality control system of proteins. It acts as a folding catalyst for the protein, can help refolding the misfolded conformation into the correct conformation, and acts as a controller for the subsequent degradation of the target protein. HSPA8, through interaction or cooperation with other molecular chaperones, constitutes a complex protein folding network involved in a variety of vital activities of cells, such as cell cycle, autophagy, viral replication, etc. In various cancer cells, the expression level of HSPA8 is high, such as breast cancer, liver cancer, etc. Research shows that when HSPA8 is deleted, the growth of solid tumor cells is inhibited, apoptosis is induced, and the cell cycle is stopped.
In recent years, RNA interference technology has undergone rapid development, and has also brought about new dawn and revolutionary changes in 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 artificially synthesized RNAs with tight hairpin turns that silence the expression of a target gene by the principle of RNA interference technology. 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.
Therefore, how to use RNA interference technology to design a shRNA sequence which is directed against HSPA8 gene and can inhibit malignant tumor cells, namely shHSPA8, and provide a beneficial way for treating colorectal cancer and other related cancers becomes a research direction.
Disclosure of Invention
Aiming at the defects of medicines for inhibiting tumor cells in the prior art, the invention provides shRNA for an HSPA8 gene, namely shHSPA8, and application thereof in colorectal cancer treatment, and the purpose of inhibiting malignant tumor cell growth is realized by taking cytoplasmic chaperonin HSPA8 as a target and designing specific shRNA.
The invention firstly provides a shHSPA8 sequence, which takes cytoplasmic chaperone protein HSPA8 as a target, designs specific shRNA, can obviously reduce the expression level of mRNA and protein of the HSPA8 in colorectal cancer cells so as to inhibit the growth rate of the colorectal cancer cells, and the sequence of the shHSPA8 is shown as SEQ ID NO. 1.
The shHSPA8 of the present invention is useful for silencing HSPA8 genes.
Further, the target sequence of the HSPA8 is GGAGGTGTCTTCTATGGTTCT.
Still further, the oligonucleotide sequence for cloning the shHSPA8 sequence comprises a sense strand and an antisense strand, the oligonucleotide sequence for the sense strand being:
5’-CCGGGGAGGTGTCTTCTATGGTTCTCTCGAGAGAACCATAGAAGACACCTCCTTTTTG-3’；
the oligonucleotide sequences of the antisense strand are:
5’-AATTCAAAAAGGAGGTGTCTTCTATGGTTCTCTCGAGAGAACCATAGAAGACACCTCC-3’。
the invention also provides an application of the shHSPA8 in treating colorectal cancer, aiming at the HSPA8 as a treatment target.
The shHSPA8 sequence designed by the invention can obviously reduce the expression level of mRNA and protein of HSPA8 in colorectal cancer cells, thereby obviously inhibiting the growth rate of colorectal cancer cells and having very important significance for treating colorectal cancer. If the shRNA sequence is further developed into a clinical therapeutic drug, the shRNA sequence has wide market application prospect, and potential social and economic benefits are huge.
Drawings
Fig. 1 is a graph showing the effect of detecting shHSPA8 silencing HSPA8 gene expression by a fluorescent quantitative PCR method.
Fig. 2 shows the effect of immunoblotting to detect shHSPA8 silencing HSPA8 gene expression.
Fig. 3 is a comparison graph of cell count assays evaluating the inhibition of shHSPA8 on HCT116 colorectal cancer cell proliferation.
Detailed Description
The technical scheme of the invention is described in detail below with reference to specific embodiments.
Sources of materials involved in the examples:
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.
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 volume of 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.
The McCoy's 5A medium formulation is as follows: the McCoy's 5A powder 11.9 g and sodium bicarbonate 2.2 g are weighed, 800 mL of Milli Q ultrapure water is added, magnetic stirring is carried out, the mixture is fully dissolved, 10 mL of 100 Xpenicillin-streptomycin stock solution (penicillin concentration is 10000U/mL, streptomycin concentration is 10000 mug/mL) is added, pH is regulated to 7.2-7.4 by concentrated hydrochloric acid, milli Q water is fixed to 1L, filtration and sterilization are carried out by a microporous filter membrane of 0.22 mu m, 10% fetal bovine serum is added, and the mixture is preserved in a refrigerator at 4 ℃ for standby.
The remaining reagents and materials involved in this example are all commercially available and are not listed here.
The specific implementation steps of this embodiment are as follows:
(1) Design of shRNA for HSPA8 (i.e., shHSPA 8)
Nucleotide sequence information (NM-006597.6) for the human HSPA8 gene was obtained by means of the GenBank database (http:// www.ncbi.nlm.nih.gov/GenBank). Then, according to the design principle of shRNA, a shRNA sequence aiming at the human HSPA8 gene is designed and is marked as shHSPA8, and the sequence is shown as EQ ID NO. 1 and is used for silencing the human HSPA8 gene. The target sequence of shHSPA8 is: GGAGGTGTCTTCTATGGTTCT two oligonucleotide sequences were designed from the target sequence for cloning the shHSPA8 coding sequence into lentiviral vector pLKO.1. The sequence of the synthesized oligonucleotide is shown below:
the sense strand sequence is: 5'-CCGGGGAGGTGTCTTCTATGGTTCTCTCGAGAGAACCATAGAAGACACCTCCTTTTTG-3';
the antisense strand sequence is: 5'-AATTCAAAAAGGAGGTGTCTTCTATGGTTCTCTCGAGAGAACCATAGAAGACACCTCC-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, the sequence is as EQ ID NO:2, and the target sequence is as follows: CGCTGAGTACTTCGAAATGTC. Two oligonucleotide sequences were designed for the target sequence for cloning the shLuc coding sequence 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'.
(2) Construction of lentiviral recombinant plasmid pLKO.1-shHSPA8
The sequence encoding shHSPA8 is cloned to a lentiviral expression vector pLKO.1 by using a synthesized oligonucleotide sequence, and a lentiviral recombinant plasmid pLKO.1-shHSPA8 is constructed. 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; then the diluted reaction product is connected with a double enzyme cutting carrier pLKO.1 by using T4 DNA ligase according to the conventional methodAnd transforming the connection product into competent escherichia coli DH5 alpha, selecting a monoclonal for overnight culture, and extracting plasmids to obtain recombinant plasmids. 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-shHSPA8.
Packaging lentiviruses with recombinant plasmids: the lentivirus recombinant plasmids pLKO.1-shHSPA8 and pLKO.1-shLuc with accurate identification are taken, and the lentivirus is packaged by referring to the operation instruction book of 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 is prepared by respectively adding three plasmids (the total amount of the plasmids is 2.34 mug) into a centrifuge tube according to the mass ratio of the plasmids pLKO.1-shHSPA8 to pCMV-VVsVg to pCMV-delta-R8.2=3:1:2, uniformly mixing, and standing at room temperature for later use;
(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.
(4) Infection of HCT116 cells with shRNA lentiviruses and evaluation of silencing Effect
And detecting the effect of shHSPA8 in silencing the expression of the HSPA8 gene by adopting a fluorescent quantitative PCR method and an immunoblotting method. Packaging lentivirus with recombinant plasmid pLKO.1-shHSPA8, and infecting human colorectal cancer cells HCT116 in logarithmic growth phase; then, mRNA and protein levels of intracellular HSPA8 were detected by fluorescent quantitative PCR and immunoblotting, respectively, to evaluate the effect of shHSPA8 in inhibiting HSPA8 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-shHSPA8 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 HSPA8 mRNA in cells is detected by a fluorescent quantitative PCR method; the other was used to extract total protein and relative levels of HSPA8 protein in cells were detected by immunoblotting. Finally, the gene silencing effect of shHSPA8 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).
(5) Cell counting method for detecting inhibition of shHSPA8 on HCT116 cell proliferation
Inoculating HCT116 cells with HSPA8 gene silencing into a 6-hole cell culture plate, and placing CO 2 Culturing was continued in the incubator and cells were counted at different time pointsThe effect of shHSPA8 on cell proliferation was evaluated. 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 HSPA8 gene inhibited proliferation of HCT116 cells. The results are shown in figure 3, where shHSPA8 experimental group cells were significantly inhibited compared to shLuc control group cells. Therefore, the shHSPA8 sequence designed by the invention can 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. shRNA for HSPA8 gene, namely shHSPA8, is characterized in that the sequence is shown as SEQ ID NO. 1.
2. shHSPA8 according to claim 1, characterized in that the shHSPA8 is used to silence HSPA8 genes.
3. shHSPA8 according to claim 2, characterized in that the target sequence of shHSPA8 is GGAGGTGTCTTCTATGGTTCT.
4. A shHSPA8 according to claim 3, characterized in that the shHSPA8 sequence comprises an oligonucleotide sequence comprising a sense strand and an antisense strand, the oligonucleotide sequence of the sense strand being:
5’-CCGGGGAGGTGTCTTCTATGGTTCTCTCGAGAGAACCATAGAAGACACCTCCTTTTTG-3’；
the oligonucleotide sequences of the antisense strand are:
5’-AATTCAAAAAGGAGGTGTCTTCTATGGTTCTCTCGAGAGAACCATAGAAGACACCTCC-3’。
5. shHSPA8 according to any one of claims 1 to 4 for use as a therapeutic target for HSPA8 in the treatment of colorectal cancer.
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