CN114875065A - Design and application of eIF4E/c-Myc gene positive feedback loop self-control shRNA silencing vector - Google Patents
Design and application of eIF4E/c-Myc gene positive feedback loop self-control shRNA silencing vector Download PDFInfo
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Abstract
The invention discloses an eIF4E/c-Myc gene positive feedback loop self-controlled shRNA silencing vector, which comprises: an interfering nucleic acid gene, a promoter, a Kozak gene, an EGFP green fluorescent protein gene, a miR-30E gene, an SV40late pA gene, a gene driving the universal expression of a downstream marker gene, a resistance gene, a gene promoting the transcription termination of an upstream ORF, and a gene of a replication origin. After the vector is transfected into cancer cells, the c-Myc highly expresses in the cells, the c-Myc starts the plasmid to transcribe shRNA aiming at eIF4E by combining with a starting region of the plasmid, so that the expression of eIF4E is reduced, and the expression of the c-Myc is reduced, thereby inhibiting the functions of the cells.
Description
Technical Field
The invention relates to biotechnology, in particular to a shRNA vector with self-control property and application of the vector in tumor treatment.
Background
Eukaryotic translation initiation factor 4E (eukaryotic translation initiation factor, eIF4E) and c-Myc are both expressed at high levels in many tumor tissue cells. c-Myc is a transcription factor, eIF4E is a protein translation regulation factor, and the expression of the two factors is enhanced in the links of transcription and translation respectively to form an eIF4E/c-Myc positive feedback loop. In the past, the RNA interference technology is used for inhibiting eIF4E and c-Myc genes in human laryngeal squamous cell carcinoma Hep-2, but the siRNA or shRNA used in the research lacks specificity, namely the siRNA or shRNA can generate interference effect on all tissue cells (including tumor cells and normal cells). In fact, in normal tissue cells, eIF4E and c-Myc are expressed in small amounts and play a role in maintaining normal vital activities of cells. If the nonspecific siRNA or shRNA is used for gene therapy of tumors, damage can be caused to normal tissue cells which express c-Myc at a low level, and serious side effects can be caused to cause treatment failure. Therefore, designing and constructing a shRNA plasmid capable of realizing specific self-regulation is one of the methods for solving the problem.
Disclosure of Invention
The invention discloses an eIF4E/c-Myc gene positive feedback loop self-control shRNA silencing vector, which is transfected to a cancer cell with high concentration of c-Myc, and the c-Myc starts plasmid transcription to generate shRNA aiming at eIF4E by combining with a starting region of the plasmid, so that the expression of eIF4E is reduced, and further, the expression of c-Myc is reduced, and the malignant behavior of the tumor cell is inhibited.
In order to achieve the purpose, the invention adopts the following technical scheme:
an eIF4E/c-Myc gene positive feedback loop self-controlling shRNA silencing vector comprises: an interfering nucleic acid gene, a promoter, a Kozak gene, an EGFP green fluorescent protein gene, a miR-30E gene, an SV40late pA gene, a gene driving the universal expression of a downstream marker gene, a resistance gene, a gene promoting the transcription termination of an upstream ORF, and a gene of a replication origin.
Further, the interfering nucleic acid sequence comprises Seq NO.1, Seq NO.2, Seq NO. 3.
Further, the promoter comprises 4xc-Myc binding site/miniCMV.
Further, genes driving universal expression of downstream marker genes include CMV promoter;
further, the resistance gene comprises one or more genes of Puro and Ampicillin;
further, genes promoting the termination of transcription of the upstream ORF include BGH pA;
further, the origin of replication gene includes pUC ori.
The 4xc-Myc binding site/miniCMV is a specific promoter of plasmid, which contains 4 CACGTG sequences (4 sequences are added to ensure that the promoter has higher sensitivity and high efficiency), the c-Myc protein can be combined with the CACGTG sequences, once the c-Myc protein in the cell is combined with one CACGTG sequence, the 4xc-Myc binding site/miniCMV is activated to transcribe each downstream gene (the arrow of the 4xc-Myc binding site/miniCMV points to the downstream gene direction). Kozak sequences can promote transcription of downstream genes. EGFP is a green fluorescent protein gene and is used for detecting the efficiency of the plasmid transfected into cells, namely the plasmid can be judged to be transferred into a target cell and express a fluorescent protein by recognizing green fluorescence through a fluorescent microscope. miR-30E can promote maturation and processing of shRNA. Three interference nucleic acid sequences shRNA are inserted into the plasmid to perform high-efficiency interference on the eIF4E gene so as to achieve a better silencing effect. The sequences of shRNA #1, shRNA # 2, and shRNA #4(shRNA #3 was discarded due to the cleavage site) were:
Seq NO1:(shRNA#1)：
CCCAAAGATAGTGATTGGTTATTAGTGAAGCCACAGATGTAATAACCAATCACTATCTTTGGA；
Seq NO2(shRNA#2)：
ACGGCTGATCTCCAAGTTTGATTAGTGAAGCCACAGATGTAATCAAACTTGGAGATCAGCCGC；
Seq NO3(shRNA#4)：
ACCGACTACAGAAGAGGAGAAATAGTGAAGCCACAGATGTATTTCTCCTCTTCTGTAGTCGGG。
SV40late pA: simian Virus 40 terminal polyadenylation signals facilitate the termination of transcription of upstream ORFs. CMV promoter: the human cytomegalovirus early promoter can drive the universal expression of downstream marker genes. Puro: puromycin (Puromycin) resistance gene, which makes the target cell resistant to Puromycin. BGH pA: the bovine growth hormone polyadenylation signal promotes the termination of transcription of upstream ORFs. pUC ori: the pUC origin of replication (the plasmid carrying this gene is present in high copy number in E.coli). Ampicillin: ampicillin resistance gene, which can screen the growth of colibacillus resisting ampicillin to ensure the quality of plasmid and avoid the contamination of mixed bacteria.
The eIF4E/c-Myc gene positive feedback loop self-controlling shRNA silencing vector is used for inhibiting the biological behavior of tumor cells.
Further, the cancer cell is a cancer cell that is a high c-Myc expression cancer cell.
Compared with the prior art, the invention has the following beneficial effects:
the prior art directly designs shRNA of c-Myc, silences the c-Myc gene and simultaneously reduces the expression of eIF4E protein; or the shRNA directly designed by eIF4E is studied to reduce the expression of eIF4E and c-Myc protein. The eIF4E/c-Myc positive feedback loop is further developed and utilized, so that shRNA interfering eIF4E has high selectivity, and in a cell highly expressing c-Myc, the c-Myc starts plasmid transcription to generate shRNA aiming at eIF4E by combining with a starting region of the plasmid, so that the expression of eIF4E is reduced, and the expression of c-Myc is reduced, so that the function of the cell is inhibited; in a cell with low expression of c-Myc, the combination probability of the c-Myc and the promoter region of the plasmid is low, the plasmid can not transcribe shRNA aiming at eIF4E, the function of the cell is not inhibited or the inhibition capacity is smaller than that of the cell, and the specificity, namely self-control, of the eIF4E/c-Myc positive feedback loop is realized.
Drawings
FIG. 1A plasmid map of the present invention;
FIG. 2 is a schematic diagram of the eIF4E/c-Myc gene positive feedback of the present invention;
FIG. 3 shows the fluorescence expression of the plasmid of the present invention after transfection of Hela cells;
FIG. 4 mRMA and protein Table of Hela cell empty plasmid control group eIF4E, c-MycThe result is achieved. mock group vs shRNA-nc group: # /P>0.05。
FIG. 5 shows the results of cell proliferation of mock group and shRNA-nc group of Hela cells; mock group vs shRNA-nc group: # /P>0.05；
FIG. 6 clone formation results of Hela cells. mock group vs shRNA-nc group: # /P>0.05；
FIG. 7 shows the results of the Hela cell invasion assay. A: mock group, B: shRNA-nc group; mock group vs shRNA-nc group: # /P>0.05；
FIG. 8HeLa cell migration assay. A: mock group, B: shRNA-nc group; mock group vs shRNA-nc group: # /P>0.05；
FIG. 9 results of mRMA and protein expression of eIF4E, c-Myc after transfection of Hela cells with the autocopulation plasmid. shRNA-nc group vs shRNA-eIF4E/c-Myc group: # /P>0.05；*/P<0.05；**/P<0.01；
FIG. 10 shows the cell proliferation results of the shRNA-nc group and the shRNA-eIF4E/c-Myc group of HeLa cells; shRNA-nc group vs shRNA-eIF4E/c-Myc group: p < 0.01;
FIG. 11 shows the cloning formation results of the shRNA-nc group and the shRNA-eIF4E/c-Myc group of HeLa cells; shRNA-nc group vs shRNA-eIF4E/c-Myc group: p < 0.01;
FIG. 12 results of the invasion of the shRNA-nc group and the shRNA-eIF4E/c-Myc group of HeLa cells: a: shRNA-nc group; b: shRNA-eIF4E/c-Myc group; shRNA-nc group vs shRNA-eIF4E/c-Myc group: p < 0.01;
FIG. 13 migration results of the shRNA-nc group and shRNA-eIF4E/c-Myc group of HeLa cells: a: shRNA-nc group; b: shRNA-eIF4E/c-Myc group; shRNA-nc group vs shRNA-eIF4E/c-Myc group: p < 0.01;
FIG. 14 fluorescent expression of the plasmid of the present invention after transfection of C33A cells;
FIG. 15mock C33A Group and shRNA-nc C33A Group and shRNA-eIF4E/c-Myc C33A The mRMA and protein expression results of group cell eIF4E, c-Myc: mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05；
FIG. 16mock C33A Group, shRNA-nc C33A Group and shRNA-eIF4E/c-Myc C33A (ii) a group cell proliferation outcome; mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05；
FIG. 17mock C33A Group and shRNA-nc C33A Group and shRNA-eIF4E/c-Myc C33A Group cell clone formation results: mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05；
fig. 18C33A cell panel invasion results: a: mock C33A Group (d); b: shRNA-nc C33A Group (d); c: shRNA-eIF4E/c-Myc C33A Group (d); mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05；
fig. 19C33A cell migration results for each group: a: mock C33A Group (d); b: shRNA-nc C33A Group (d); c: shRNA-eIF4E/c-Myc C33A Group (d); mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05。
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
An eIF4E/c-Myc gene positive feedback loop self-controlling shRNA silencing vector comprises: an interfering nucleic acid gene, a promoter, a Kozak gene, an EGFP green fluorescent protein gene, a miR-30E gene, an SV40late pA gene, a gene driving the universal expression of a downstream marker gene, a resistance gene, a gene promoting the transcription termination of an upstream ORF, and a gene of a replication origin. The plasmid map is shown in FIG. 1.
Further, the interfering nucleic acid gene sequence comprises Seq NO.1, Seq NO.2 and Seq NO. 3.
Further, the promoter comprises 4xc-Myc binding site/miniCMV.
Further, genes driving universal expression of downstream marker genes include CMV promoter;
further, the resistance gene comprises one or more genes of Puro and Ampicillin;
further, genes promoting the termination of transcription of the upstream ORF include BGH pA;
further, the origin of replication gene includes pUC ori.
The 4xc-Myc binding site/miniCMV is a specific promoter of plasmid, which contains 4 CACGTG sequences (4 sequences are added to ensure that the promoter has higher sensitivity and high efficiency), the c-Myc protein can be combined with the CACGTG sequences, once the c-Myc protein in the cell is combined with one CACGTG sequence, the 4xc-Myc binding site/miniCMV is activated to transcribe each downstream gene (the arrow of the 4xc-Myc binding site/miniCMV points to the downstream gene direction). Kozak sequences can promote transcription of downstream genes. EGFP is a green fluorescent protein gene and is used for detecting the efficiency of the plasmid transfected into cells, namely the plasmid can be judged to be transferred into a target cell and express a fluorescent protein by recognizing green fluorescence through a fluorescent microscope. miR-30E can promote maturation and processing of shRNA. Three interference nucleic acid sequences shRNA are inserted into the plasmid to perform high-efficiency interference on the eIF4E gene so as to achieve a better silencing effect. The sequences of shRNA #1, shRNA # 2, and shRNA #4(shRNA #3 was discarded due to the cleavage site) were:
Seq NO1:(shRNA#1)：
CCCAAAGATAGTGATTGGTTATTAGTGAAGCCACAGATGTAATAACCAATCACTATCTTTGGA；
Seq NO2(shRNA#2)：
ACGGCTGATCTCCAAGTTTGATTAGTGAAGCCACAGATGTAATCAAACTTGGAGATCAGCCGC；
Seq NO3(shRNA#4)：
ACCGACTACAGAAGAGGAGAAATAGTGAAGCCACAGATGTATTTCTCCTCTTCTGTAGTCGGG。
SV40late pA: simian Virus 40 terminal polyadenylation signals facilitate the termination of transcription of upstream ORFs. CMV promoter: the human cytomegalovirus early promoter can drive the universal expression of downstream marker genes. Puro: puromycin (Puromycin) resistance gene, which makes the target cell resistant to Puromycin. BGH pA: the bovine growth hormone polyadenylation signal promotes the termination of transcription of upstream ORFs. pUC ori: the pUC origin of replication (the plasmid carrying this gene is present in high copy number in E.coli). Ampicillin: ampicillin resistance gene, which can screen the growth of colibacillus resisting ampicillin to ensure the quality of plasmid and avoid the contamination of mixed bacteria.
The eIF4E/c-Myc gene positive feedback loop self-controlling shRNA silencing vector is used for inhibiting the biological behavior of tumor cells.
Furthermore, the tumor cell is a tumor cell with high expression of c-Myc.
The prior art shows that: firstly, in different cells, an RNA interference technology (siRNA or shRNA) is utilized to interfere two genes of eIF4E or c-Myc, so that the effect of silencing the genes is achieved, and the genes are inhibited; ② another study proves that a positive feedback regulation loop exists between eIF4E and c-Myc gene as shown in figure 2.
The invention is different from the prior art: directly designing shRNA of c-Myc, silencing the c-Myc gene and simultaneously reducing eIF4E protein expression; or the shRNA directly designed by eIF4E is studied to reduce the expression of eIF4E and c-Myc protein. The invention further develops and utilizes the eIF4E/c-Myc positive feedback loop, and aims to enable shRNA interfering eIF4E to have high selectivity, namely: in a cell with high expression of c-Myc, c-Myc starts the plasmid to transcribe shRNA aiming at eIF4E by combining with the promoter region of the plasmid, so that the expression of eIF4E is reduced, and the expression of c-Myc is reduced, thereby inhibiting the function of the cell; in cells with low expression of c-Myc, the c-Myc level is low, the probability of combination with the promoter region of the plasmid is low, the plasmid can not transcribe shRNA aiming at eIF4E, the cell function is not inhibited or the inhibition capacity is smaller than that of the former, and the specificity of eIF4E/c-Myc positive feedback loop can be realized or the self-control property can be realized.
EXAMPLE 1 preparation of the support
And (3) selecting an externally purchased vector, and connecting the vector with the interference gene by double enzyme digestion. Such as the technology shown in patent CN 107988254A.
Example 2 vector transformation
The prepared plasmid vector is transformed into competent cells, as shown in the patent CN 107988254A.
Example 3 Effect of eIF4E/c-Myc Positive feedback Loop-controlled shRNA silencing plasmid on Hela cells
The cervical adenocarcinoma cell line Hela (HPV18+) was divided into three groups: mock (negative control group/Hela cell without plasmid transfection), shRNA-nc (positive control group/Hela cell transiently transfected with unloaded plasmid), shRNA-eIF4E/c-Myc (experimental group/Hela cell transiently transfected with the self-regulated plasmid disclosed by the invention).
Results of the experiment
Observation of the Effect of plasmid transfected Hela cells under fluorescent microscope
FIG. 3 shows that after the empty-load plasmid (shRNA-nc) carrying green fluorescence and the self-control plasmid (shRNA-eIF4E/c-Myc) are transferred into Hela cells for 24h by a liposome method, the fluorescence expression conditions of each group are observed under a fluorescence microscope.
In Hela cells, the difference between the molecular and cellular levels of the shRNA-nc group and the mock group is not statistically significant.
Figure 4 shows that at mRNA and protein expression levels: compared with the mock group, the mRNA and protein expression difference of the shRNA-nc group eIF4E and the c-Myc has no statistical significance (P)>0.05). mock group vs shRNA-nc group: # /P>0.05。
FIG. 5 shows that, in terms of proliferation potency: the OD values of mock groups of 0h, 24h, 48h and 72h are respectively 0.304 +/-0.005, 0.451 +/-0.014, 0.721 +/-0.030 and 1.321 +/-0.084. The OD values of 0h, 24h, 48h and 72h of the shRNA-nc group are respectively 0.317 +/-0.025, 0.418 +/-0.050, 0.672 +/-0.101 and 1.149 +/-0.166. The difference of OD values in the two groups of time periods has no statistical significance (P)>0.05). mock group vs shRNA-nc group: # /P>0.05。
FIG. 6 shows that, in terms of clonogenic capacity: the monoclonal formation rate of mock group cells is 0.490 +/-0.059 percent, and the monoclonal formation rate of shRNA-nc group cells is 0.492 +/-0.043 percent. Compared with the mock group, the difference of the cell monoclonal formation rate of the shRNA-nc group has no statistical significance (P)>0.05). mock group vs shRNA-nc group: # /P>0.05。
fig. 7, in terms of invasiveness: the difference in invasive potential between the shRNA-nc groups compared to the mock group was not statistically significant (P>0.05). A: mock group, B: shRNA-nc group. mock group vs shRNA-nc group: # /P>0.05。
as shown in fig. 8, in terms of migration capability: the difference in the migration ability of the shRNA-nc group compared to the mock group was not statistically significant (P)>0.05). A: mock group, B: shRNA-nc group, mock group vs shRNA-nc group: # /P>0.05
as shown in FIG. 9, in Hela cells, the mRNA and protein expression levels of eIF4E and c-Myc of the shRNA-eIF4E/c-Myc group are:
compared with the shRNA-nc group, the expression difference of c-Myc mRNA of the shRNA-eIF4E/c-Myc group has no statistical significance (P)>0.05), eIF4E mRNA expression decreased 28.333 + -4.868%, and the difference had significant statistical significance (P)<0.01). Compared with the shRNA-nc group, the expression of the eIF4E and the c-Myc protein of the shRNA-eIF4E/c-Myc group is respectively reduced by 64.167 +/-17.75 percent and 47.267 +/-16.614 percent, and the differences have obvious statistical significance (P)<0.01). shRNA-nc group vs shRNA-eIF4E/c-Myc group: # /P>0.05； ** /P<0.01
as shown in fig. 10, in terms of proliferation potency: the OD values of 0h, 24h, 48h and 72h of shRNA-nc group are 0.329 +/-0.011, 0.515 +/-0.014, 0.917 +/-0.016 and 1.559 +/-0.038 respectively.
The OD values of 0h, 24h, 48h and 72h of the shRNA-eIF4E/c-Myc groups are respectively 0.316 +/-0.009, 0.415 +/-0.026, 0.668 +/-0.053 and 1.076 +/-0.137. Compared with the shRNA-nc group, the cell proliferation inhibition rates of 24h, 48h and 72h of the shRNA-eIF4E/c-Myc group are 19.417%, 27.154% and 30.981% respectively, and the differences have obvious statistical significance (P)<0.01). shRNA-nc group vs shRNA-eIF4E/c-Myc group: ** /P<0.01
as shown in fig. 11, in terms of clonogenic ability: the monoclonality formation rate of shRNA-nc group cells is 0.348 +/-0.020%, and the monoclonality formation rate of shRNA-eIF4E/c-Myc group cells is 0.157 +/-0.018%. Compared with the shRNA-nc group, the monoclonal formation rate of the shRNA-eIF4E/c-Myc group cells is reduced by 58.89%, and the difference has obvious statistical significance (P)<0.01). shRNA-nc group vs shRNA-eIF4E/c-Myc group: ** /P<0.01
as shown in fig. 12, in terms of invasiveness: compared with the shRNA-nc group, the invasion rate of the shRNA-eIF4E/c-Myc group is reduced by 67.720 +/-14.771%, and the difference is remarkably uniformSignificance of design (P)<0.01). A: shRNA-nc group; b: shRNA-eIF4E/c-Myc group; shRNA-nc group vs shRNA-eIF4E/c-Myc group: ** /P<0.01。
as shown in fig. 13, in terms of migration capability: compared with the shRNA-nc group, the migration rate of the shRNA-eIF4E/c-Myc group is reduced by 70.698 +/-6.745%, and the difference has obvious statistical significance (P)<0.01). A: shRNA-nc group; b: shRNA-eIF4E/c-Myc group; shRNA-nc group vs shRNA-eIF4E/c-Myc group: ** /P<0.01。
and (4) conclusion:
hela cells are cervical adenocarcinoma cell lines, HPV18 type infection is positive, and c-Myc and eIF4E are expressed at high level in Hela cells. At the molecular level, both eIF4E mRNA and protein expression were reduced, but c-Myc was only protein level reduced, and mRNA did not show a reduction, indicating that c-Myc exerted effects at the eIF4E gene transcript level, while eIF4E exerted a role at the c-Myc mRNA translation level, consistent with the illustrated pattern of regulation between eIF4E and c-Myc. The c-Myc and eIF4E are two important genes for the survival and development of Hela cells, and the proliferation, clone formation, invasion and migration capabilities of the Hela cells are remarkably inhibited after the expression of the c-Myc and eIF4E is reduced.
Example 4 Effect of eIF4E/C-Myc Positive feedback Loop automatic shRNA silencing plasmid on C33A cells
Dividing the cervical adenocarcinoma cell line C33A (HPV-) into three groups of mock C33A Group (negative control group/plasmid-free transfected C33A cells), shRNA-nc C33A Group (positive control group/C33A cells transiently transfected with empty plasmid), shRNA-eIF4E/C-Myc C33A Panel (experimental panel/C33A cells transiently transfected with the self-controlling plasmid disclosed herein).
Effect of plasmid transfection on C33A cells under fluorescent microscope
As shown in FIG. 14, after the empty plasmid (shRNA-nc) carrying green fluorescence and the autonomous plasmid (shRNA-eIF4E/C-Myc) were transferred into C33A cells for 24h, the fluorescence expression of each group was observed under a fluorescence microscope. Fluorescence expression after transfection of plasmid C33A cells (10X 10)
As shown in FIG. 15, in C33A cells, the mRNA and protein expression levels of eIF4E and C-Myc of shRNA-eIF4E/C-Myc group
Phase comparison mock C33A Group, shRNA-nc C33A The group c-Myc, eIF4E mRNA and protein expression differences were not statistically significant (P)>0.05). Comparison of shRNA-nc C33A Group, shRNA-eIF4E/c-Myc C33A The group c-Myc mRNA level was elevated 29.967 + -12.788%, the differences were statistically significant (P)<0.05); eIF4E mRNA level was reduced by 5.433. + -. 0.351%, with significant statistical significance for the difference (P)<0.01). Comparison of shRNA-nc C33A Group, shRNA-eIF4E/c-Myc C33A Group c-Myc protein differences were not statistically significant (P)>0.05), the level of eIF4E protein was reduced by 24.933 + -5.012%, and the difference was statistically significant (P)<0.01)。mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): (xi)/P<0.05， ** /P<0.01
As shown in fig. 16, in terms of proliferation potency: mock C33A The OD values of the groups 0h, 24h, 48h and 72h are respectively 0.178 +/-0.030, 0.213 +/-0.071, 0.285 +/-0.105 and 0.387 +/-0.137. shRNA-nc C33A The OD values of the groups 0h, 24h, 48h and 72h are respectively 0.180 +/-0.030, 0.214 +/-0.067, 0.274 +/-0.101 and 0.359 +/-0.121. shRNA-eIF4E/c-Myc C33A The OD values of the groups 0h, 24h, 48h and 72h are respectively 0.180 +/-0.025, 0.207 +/-0.038, 0.267 +/-0.048 and 0.344 +/-0.037. No statistical significance was observed for the group differences at each time point (P)>0.05)。mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05。
as shown in fig. 17, in terms of clonogenic ability: mock C33A The formation rate of the group clone is 36.562 + -3.171%. shRNA-nc C33A The formation rate of the group clone was 33.468. + -. 4.491%. shRNA-eIF4E/c-Myc C33A The formation rate of the group clone is 32.308 + -3.173%. Phase comparison mock C33A Group, shRNA-nc C33A Group differences were not statistically significant (P)>0.05); comparison of shRNA-nc C33A Group, shRNA-eIF4E/c-Myc C33A Group differences were not statistically significant (P)>0.05)。mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vsshRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05。
as shown in fig. 18, in invasive ability: phase comparison mock C33A Group, shRNA-nc C33A The difference in group invasive potential was not statistically significant (P)>0.05). Comparison of shRNA-nc C33A Group, shRNA-eIF4E/c-Myc C33A The difference in group invasive potential was not statistically significant (P)>0.05)。
A：mock C33A Group (d); b: shRNA-nc C33A Group (d); c: shRNA-eIF4E/c-Myc C33A Group (d); mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05。
as shown in fig. 19, in terms of migration capability: phase comparison mock C33A Group, shRNA-nc C33A The difference in group migration capacity was not statistically significant (P)>0.05). Comparison of shRNA-nc C33A Group, shRNA-eIF4E/c-Myc C33A The difference in group migration capacity was not statistically significant (P)>0.05)。
A：mock C33A Group (d); b: shRNA-nc C33A Group (d); c: shRNA-eIF4E/c-Myc C33A Group (d); mock C33A Group vs shRNA-nc C33A Group (2): # /P>0.05；shRNA-nc C33A group vs shRNA-eIF4E/c-Myc C33A Group (2): # /P>0.05。
and (4) conclusion:
the C33A cell is a cervical cancer cell line, HPV infection is negative, and C-Myc and eIF4E are expressed at a low level in C33A compared with Hela cells. After the essence granule is transferred into a C33A cell, the low-level C-Myc has reduced binding capacity with a CACGTG sequence on the plasmid and cannot effectively start the transcription of a downstream gene out of shRNA so as to interfere the expression of eIF 4E. At the molecular level, the levels of eIF4E mRNA and protein in the plasmid interference group were slightly reduced, but the reduction was significantly smaller than that of Hela cells highly expressing c-Myc. The c-Myc mRNA level is increased, and the protein level is not obviously changed, which indicates that a feedback control mechanism exists in the cell gene expression, namely the c-Myc mRNA is increased probably due to cell compensation. C33A cells were independent of eIF4E and C-Myc, and showed no significant changes in proliferation, clonogenic, invasive and migratory capacity following transfection of an autonomous plasmid into the cells. This shows that eIF4E/c-Myc positive feedback loop automatic shRNA silencing plasmid only exerts significant inhibition effect on cells with high expression of c-Myc, but has limited effect on cells with low expression of c-Myc. Thus, normal tissue cells that express little or no c-Myc may not be affected, greatly facilitating the specificity of tumor gene therapy.
Sequence listing
<110> Guangdong university of medical science
<120> design and application of eIF4E/c-Myc gene positive feedback loop self-control shRNA silencing vector
<160> 3
<170> SIPOSequenceListing 1.0
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acggcgaccc aaggaaggaa gccacagaga acaaacggag acagccgc 48
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Claims (9)
1. An eIF4E/c-Myc gene positive feedback loop self-controlling shRNA silencing vector is characterized in that: the method comprises the following steps: an interfering nucleic acid gene, a promoter, a Kozak gene, an EGFP green fluorescent protein gene, a miR-30E gene, an SV40late pA gene, a gene driving the universal expression of a downstream marker gene, a resistance gene, a gene promoting the transcription termination of an upstream ORF, and a gene of a replication origin.
2. The eIF4E/c-Myc gene positive feedback loop automatic shRNA silencing vector of claim 1, wherein: the sequence of the interfering nucleic acid gene comprises: seq NO.1, Seq NO.2, Seq NO. 3.
3. The eIF4E/c-Myc gene positive feedback loop automatic shRNA silencing vector of claim 2, wherein: the promoter comprises 4xc-Myc binding site/miniCMV.
4. The eIF4E/c-Myc gene positive feedback loop automatic shRNA silencing vector of claim 3, wherein: genes driving universal expression of downstream marker genes include CMV promoter.
5. The eIF4E/c-Myc gene positive feedback loop automatic shRNA silencing vector of claim 4, wherein: the resistance gene comprises one or more gene sequences of Puro and Ampicillin.
6. The eIF4E/c-Myc gene positive feedback loop automatic shRNA silencing vector of claim 5, wherein: genes that promote the termination of transcription of the upstream ORF include BGH pA.
7. The eIF4E/c-Myc gene positive feedback loop automatic shRNA silencing vector of claim 6, wherein: the origin of replication gene includes pUC ori.
8. The use of the eIF4E/c-Myc gene positive feedback loop self-controlling shRNA silencing vector of claims 1-7, wherein: the eIF4E/c-Myc gene positive feedback loop is suppressed, and the malignant biological behavior of the cancer cell is further suppressed.
9. Use according to claim 8, characterized in that: the tumor cell is a tumor cell with high expression of c-Myc.
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Inventor after: Hu Xinrong Inventor after: Mu Chao Inventor after: Yao Yunhong Inventor after: Pang Tianyun Inventor after: Cai Wenjie Inventor before: Hu Xinrong Inventor before: Mu Chao Inventor before: Yao Yunhong Inventor before: Pang Tianyun Inventor before: Cai Wenjie |