CN108949827B

CN108949827B - shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression and construction method and application thereof

Info

Publication number: CN108949827B
Application number: CN201810801240.XA
Authority: CN
Inventors: 袁建辉; 张洪; 谢妮; 左然; 任晓虎; 吴德生; 李萍; 毛吉炎
Original assignee: SHENZHEN NANSHAN CENTER FOR DISEASE CONTROL AND PREVENTION
Current assignee: SHENZHEN NANSHAN CENTER FOR DISEASE CONTROL AND PREVENTION
Priority date: 2018-07-20
Filing date: 2018-07-20
Publication date: 2022-03-25
Anticipated expiration: 2038-07-20
Also published as: CN108949827A

Abstract

The invention discloses a shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression and a construction method and application thereof, belonging to the technical field of genetic engineering. The shRNA lentiviral expression vector constructed by inserting the PPARd-targeting shRNA oligonucleotide sequence into the pLVX-shRNA2-Puro expression vector has the advantages of high transfection efficiency, sustainability, high efficiency, stability and specificity in the expression of the PPARd gene of the lung cancer, good inhibition effect and the like, and can be used for the research of the lung cancer, the drug screening of the lung cancer, the gene therapy of the lung cancer and the like.

Description

shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression, and a construction method and application thereof.

Background

PPARD is an important member of peroxisome proliferator-activated receptor family, and has high expression level in lipid metabolism related tissues such as small intestine, heart and fat tissue in human body, and also has high expression level in macrophage and skin. PPARDs may be involved in glycolipid metabolism, muscle energy metabolism, embryonic cell development, wound healing, and inflammatory responses, among others. In addition, the expression of PPARD exists in various tumors, including breast cancer, lung cancer, glioma, colon cancer, liver cancer, pancreatic cancer, prostate cancer, thyroid cancer and the like, and the PPARD expression difference is considered to be related to the stage and prognosis of the tumors.

Most studies have shown that PPARD is a risk factor for lung cancer. The PPARD has the effects of promoting the growth of cancer cells, promoting the generation of new vessels and resisting apoptosis in lung adenocarcinoma.

The RNA interference technology can efficiently and specifically silence the expression of target genes and is widely applied to the expression of specific suppressor genes and the function research thereof. The PPARD gene silent lung cancer cell established by the technology is used for research on lung cancer occurrence, lung cancer drug screening, lung cancer gene therapy and the like. At present, researches on PPARD and lung cancer and related products are very few, and no report is reported for establishing PPARD gene silencing lung cancer cell strains and applying the PPARD gene silencing lung cancer cell strains to lung cancer researches by using an RNAi technology.

Disclosure of Invention

The invention aims to solve the problems of lung cancer research, lung cancer drug screening and lung cancer gene therapy, and provides a shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression, and a construction method and application thereof.

The invention inserts shRNA oligonucleotide sequence containing BamH I enzyme cutting site and EcoR I enzyme cutting site into multiple cloning site of pLVX-shRNA2-Puro expression vector to successfully construct shRNA slow virus expression vector of the target PPARd gene, uses slow virus to infect lung cancer cell, and integrates shRNA of the target PPARd gene into genome of the lung cancer cell, so that the shRNA of the target PPARd gene can be expressed stably for a long time, and the effect of inhibiting PPARd gene expression efficiently, durably and specifically is generated, thereby completing the invention.

The purpose of the invention is realized by the following technical scheme: an shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression specifically comprises:

pLVX-shRNA2-Puro expression vector and shRNA oligonucleotide sequence of target PPARd gene;

the shRNA oligonucleotide sequence of the targeted PPARd gene comprises a target nucleotide sequence GTGGAAGCAGTTGGTGAAT and a target nucleotide sequence complementary sequence ATTCACCAACTGCTTCCAC.

The shRNA oligonucleotide sequence of the targeted PPARd gene is positively inserted into the multiple cloning site of the pLVX-shRNA2-Puro expression vector.

The pLVX-shRNA2-Puro expression vector comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence and a promoter sequence of a PLVX-shRNA2-Puro expression vector.

The multiple cloning sites comprise BamH I enzyme cutting sites and EcoR I enzyme cutting sites.

The shRNA oligonucleotide sequence of the targeted PPARd gene consists of a BamH I enzyme cutting site, a target nucleotide sequence, a stem-loop structure sequence, a target nucleotide sequence complementary sequence, a termination site sequence and an EcoR I enzyme cutting site.

The preferred sequence of the stem-loop structure sequence is TTCAAGAGAGA;

the termination site sequence is preferably RNA PolyIII polymerase transcription termination site TTTTTT;

the shRNA oligonucleotide sequence targeting the PPARd gene is preferably a forward sequence PPARD-T1: gatccGTGGAAGCAGTTGGTGAATTTCAAGAGAATTCACCAACTGCTTCCACTTTTTT and reverse complement sequence PPARD-D1: aattAAAAAAGTGGAAGCAGTTGGTGAATTCTCTTGAAATTCACCAACTGCTTCCACg are provided.

The construction method of the shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression comprises the following steps:

(1) shRNA design and Synthesis

The PPARD shRNA is designed to have the basic structure as follows: six regions of BamH I enzyme cutting site + target nucleotide sequence + stem-loop structure (TTCAAGAGA) + target sequence complementary sequence + RNA PolyIII polymerase transcription termination site (TTTTTT) + EcoR I enzyme cutting site are respectively named as PPARD-T1 and PPARD-D1; the sequence is shown as follows:

PPARD-T1：

gatccGTGGAAGCAGTTGGTGAATTTCAAGAGAATTCACCAACTGCTTCCACTTTTTT；

PPARD-D1：

aattAAAAAAGTGGAAGCAGTTGGTGAATTCTCTTGAAATTCACCAACTGCTTCCACg；

(2) shRNA interference vector construction

Carrying out double digestion and recovery on the pLVX-shRNA2-Puro vector by using EcoR1 and BamH1, and then annealing the PPARD shRNA;

(3) constructing a recombinant interference vector:

connecting the annealed PPARD shRNA with PLVX-shRNA2-Puro, and taking a connecting product to transform competent cells for culture;

(4) extracting and identifying positive clone plasmid:

taking cultured connecting product competent cells, and extracting recombinant plasmids; and obtaining the target shRNA lentiviral expression vector by adopting double enzyme digestion and sequencing confirmation.

The competent cell in the step (3) is preferably a JM107 competent cell.

The double enzyme digestion in the step (4) is preferably one of EcoR1 and Kpn1 double enzyme digestion or BamH1 and Kpn1 double enzyme digestion.

The shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression is applied to the screening of lung cancer drugs.

The shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression is applied to the preparation of lung cancer gene medicines.

Compared with the prior art, the invention has the following advantages and effects:

by adopting the technical scheme, the shRNA lentiviral expression vector constructed by inserting the PPARd-targeting shRNA oligonucleotide sequence into the pLVX-shRNA2-Puro expression vector has the advantages of high transfection efficiency, sustainability, high efficiency, stability and specificity in lung cancer PPARd gene expression, good inhibition effect and the like, and can be used for lung cancer research, lung cancer drug screening, lung cancer gene therapy and the like.

Drawings

FIG. 1 shows that EcoR1 and Kpn1, BamH1 and Kpn1 were subjected to double digestion to identify (1, 3, 5: PPARD-1, 2, 3 plasmid KpnI, EcoRI double digestion; 2, 4, 6: PPARD-1, 2, 3 plasmid KpnI, BamHI double digestion), respectively);

FIG. 2 shows the relative expression level of PPARD detected by QPCR;

FIG. 3 shows that QPCR detects PPARd gene expression changes in lung cancer cells;

FIG. 4 shows the expression of the WB detection PPARd protein in lung cancer cells.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1: construction of target PPARd gene lentivirus shRNA interference vector

(1) ShRNA design and Synthesis

Aiming at a ppard gene sequence, three pairs of RNA interference target sequences are designed by using an RNA interference sequence design principle provided in a public website, evaluation and determination are carried out according to years of design experience and design software of a company, an optimal kinetic parameter target is selected to enter a subsequent experiment process, and besides a shRNA target sequence which is designed by self, some sequences (shRNAc-T and shRNAc-D) recognized in an RNA interference experiment are also used. The PPARD shRNA has the basic structure as follows: six regions of BamH I enzyme cutting site + target nucleotide sequence + stem-loop structure (TTCAAGAGA) + target sequence complementary sequence + RNA PolyIII polymerase transcription termination site (TTTTTT) + EcoR I enzyme cutting site are named PPARD-T1 and PPARD-D1, PPARD-T2 and PPARD-D2, PPARD-T3 and PPARD-D3, shRNAc-T and shRNAc-D, respectively. The specific sequence is shown in the following table 1:

TABLE 1 PPARD Gene shRNA sequence Listing

(2) shRNA interference vector construction

Carrying out double digestion on pLVX-shRNA2-Puro by using EcoR1 and BamH1, carrying out digestion for 30min at 37 ℃, carrying out agarose gel electrophoresis, and recycling by using a DNA gel recycling kit; the digestion reaction system is shown in the following table 2:

TABLE 2 enzymatic digestion Components Table

Annealing of PPARD shRNA: each set of shRNA was dissolved with ultrapure water to a final concentration of 10. mu.M, and 10. mu.l of each shRNA was mixed well.

Placing in boiled water for slow cooling, adding 1mL ddH after annealing₂O。

(3) Constructing a recombinant interference vector:

the annealed PPARD shRNA is connected with pLVX-shRNA2-Puro, and the reaction system is shown in the following table 3:

TABLE 3 connected Components Table

Carrying out thermostatic water bath for 2h at the temperature of 16 ℃.

6ul of each overnight ligation was transformed into 100ul of JM107 competent cells: uniformly mixing the ligation product and competent cells, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 70s, immediately placing on ice for 2min, adding 150ul of TB medium preheated to room temperature, carrying out shake culture at constant temperature of 250rpm and 37 ℃ for 1h, taking 100ul of TB medium by a pipette, uniformly coating the TB medium on a TB plate containing 100ug/ml Ampicillin resistance, inverting, and carrying out culture in a constant temperature incubator at 37 ℃ overnight.

(4) Extracting and identifying positive clone plasmid:

selecting monoclonal on TB plate, adding TB culture medium, culturing, and extracting recombinant plasmid with plasmid miniextract kit.

The PPARD gene interference vector is identified by double enzyme digestion:

the extracted plasmids were digested simultaneously with EcoR1 and Kpn1, BamH1 and Kpn1, respectively, and the digestion reaction systems are shown in Table 4. Carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis result is shown in figure 1; the BamH1 and Kpn1 enzyme digestion vectors generate two bands, and the EcoR1 and Kpn1 double enzyme digestion vectors only have one band, which indicates that the exogenous shRNA is successfully inserted, and the EcoR1 enzyme digestion site formed by the recombinant vector is damaged and can not be digested by enzyme

TABLE 4 composition of enzyme digestion reaction

Sequencing identification of PPARD gene interference vector:

and (3) sending the plasmid with the correct enzyme digestion identification fragment to Shanghai workers for sequencing identification, wherein the sequencing result is correct.

Example 2: targeted PPARd gene shRNA interference vector screening

(1) Plasmid vector transfected HEK293 cell

a. Culturing HEK293 cells, spreading HEK293 cells in six-well plates (10 per well)⁶Single cell, 37 ℃, 5% CO₂Culturing for 20 h;

b. respectively adding 0.25ml of opti-MEM culture medium into two EP tubes, respectively adding 6ul of lipo2000 and 3 ug of plasmid (PLVX-PPARDshRNA1, PLVX-PPARDshRNA2, PLVX-PPARDshRNA3 and PLVX-shRNAC), mixing, and standing for 5 min;

c. plasmid and lipo2000 complexes were added to six well plates and vortexed gently to disperse the plasmid and lipo2000 homogeneously in cell culture medium at 37 ℃ with 5% CO₂Culturing for 7h, and replacing fresh DMEM complete culture medium.

(2) Extraction of RNA from each group of cells

The experimental procedures were described in RNeasy Mini Kit (QIAgene).

(3) Reverse transcription of RNA into cDNA

a. Reverse transcription of RNA into cDNA Using reverse transcription kit (PrimeScript)^TMRT reagent Kit) the specific experimental procedures were performed by reverse transcription of cDNA according to the reverse transcription Kit instructions, and a reverse transcription system (10. mu.l) was prepared on ice, as shown in Table 5;

b. reverse transcription setting: 15min at 37 ℃; 5s at 85 ℃; 4 ℃ for 10min

c. After the reverse transcription was complete, 90. mu.l of RNase Free dH was added₂The cDNA was diluted O and stored at-20 ℃.

TABLE 5 reverse transcription System Components Table

(4) Design of synthetic QPCR primers

(5) Detection of relative expression amount of PPARD Gene

a. The relative expression level of PPARD gene was quantitatively determined by fluorescence using cDNA as template, and the system is shown in Table 6.

TABLE 6 fluorescent quantitative detection system

b. Setting reaction conditions: performing a melting experiment in the range of 55-95 ℃; pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s at 1 cycle, annealing at 55 ℃ for 30s, annealing at 40 cycle, 5s at 95 ℃, 1min at 60 ℃ and 15s at 95 ℃;

c. after the reaction is finished, a standard curve, a melting curve and a relative quantitative value are obtained. And analyzing the relative expression quantity of the PPARD gene of each group of cells according to the detected data.

The experimental results show that:

(1) the relative expression level of the shRNA-interfering PPARD gene detected by QPCR is shown in table 7 below.

TABLE 7 QPCR detection of relative shRNA interference PPARD Gene expression

(2) And (3) taking beta-act as an internal reference, taking the sample shRNAC as a reference, calculating the relative expression quantity of the PPARD gene by a 2-delta Ct method, as shown in figure 2, and according to the QPCR detection result, finding out that the interference effect of the recombinant vector pLVX-PPARD shRNA1 is the best, wherein the recombinant vector is used for packaging the virus.

Example 3: PPARd gene silencing lentiviral packaging

24h before transfection, 293T cells in logarithmic growth phase were trypsinized and adjusted to a cell density of 2X 10 with 10% serum-containing medium⁷The cells/20 ml were re-inoculated in a 15cm cell culture dish at 37 ℃ with 5% CO₂Culturing in an incubator; the cell can be used for transfection after 24 hours when the cell density reaches 70-80%; the cell state is crucial for virus packaging, so it is necessary to ensure good cell state and less passage times;

replacing the cell culture medium with a serum-free medium 2h before transfection;

adding 20 mu g of lentivirus packaging vector, 10 mu g of PLVX-PPARDshRNA1 vector, 10 mu g of PLVX-PPARDshRNA2 vector and 10 mu g of PLVX-PPARDshRNA3 vector into a sterilized centrifuge tube, uniformly mixing with 2ml of Opti-MEM, adjusting the total volume to be 2.5ml, and incubating for 5 minutes at room temperature;

gently shaking Lipofectamine 2000, mixing 100. mu.l Lipofectamine 2000 with 2.4ml Opti-MEM in another tube, and incubating at room temperature for 5 min;

the diluted DNA was mixed with the diluted Lipofectamine 2000, and the mixture was gently inverted and mixed without shaking. Mixing must be done within 5 minutes;

after mixing, incubation for 20 minutes at room temperature to form a transfection complex of DNA with Lipofectamine 2000 dilution;

transferring the mixture of DNA and Lipofectamine 2000 to 293T cell culture medium, mixing, and culturing at 37 deg.C with 5% CO₂Culturing in a cell culture box;

after culturing for 8h, pouring out the culture medium containing the transfection mixture, adding 20ml of PBS (phosphate buffer solution) into each bottle of cells, slightly shaking the culture bottle left and right to wash the residual transfection mixture, and then pouring out;

cell culture medium 2 containing 10% serum was added to each flask of cells5ml, 5% CO at 37 ℃₂The incubator was allowed to incubate for 48 hours.

Example 4: construction of PPARd gene silencing lung cancer cell strain

Respectively culturing A549, NCI-H1975 and NCI-H1395 lung cancer cells, inoculating well-grown cells into six-well plate, 10 per well⁶After 12 hours, the cell density is about 50 percent, virus liquid is respectively taken, 10 times of DMEM complete culture medium is used for diluting the virus, and polybrene (polybrene) is added until the final concentration is 5 mug/mL; removing the culture medium in the six-well plate, adding a DMEM complete culture medium (containing 10% fetal calf serum) containing the virus, removing the DMEM complete culture medium containing the virus after 24 hours, replacing a fresh DMEM complete culture medium, and screening cells by puromycin with the concentration of 1 mu g/ml after 24 hours; screening for 10d, and replacing the culture medium once every 3d to finally obtain cell strains with silent A549 genes, NCI-H1975 genes and NCI-H1395 genes.

Example 5 fluorescent quantitative PCR detection of PPARd Gene expression level

According to the PPARd and beta-acting gene mRNA sequences, Primer design software Primer 5.O and Oligo 7.0 are utilized to design primers, and specific Primer sequences are shown in the following table 8.

TABLE 8 fluorescent quantitative PCR detection primers

A549, NCI-H1975 and NCI-H1395 lung cancer cells and A549, NCI-H1975 and NCI-H1395PPARd gene silencing cell strains are inoculated respectively. When the cell density reaches 80% -90%, extracting the total RNA of each group of cells by using RNeasy Mini Kit, performing reverse transcription on the mRNA into cDNA by using PrimeScript RT reagent Kit, and performing reverse transcription under the conditions that: 15min at 37 ℃; 5s at 85 ℃; 4 ℃, 10min, after the reverse transcription is finished, 90 mul of RNase Free dH is added₂The cDNA was diluted O and stored at-20 ℃ for later use in assays. Taking 1 mu l of cDNA of each group of cells as a template, taking beta-acting as an internal reference, detecting the relative expression quantity of PPARd by real-time fluorescence Quantitative PCR (QPCR), and setting reaction conditions: 95 ℃ for 30s, 95 ℃ for 5s, 55 ℃ for 30s, 40 cycles, 95 ℃ for 5s, 60 ℃ for 1min, 95 ℃ for 15s, detecting the relative expression of the PPARd gene of each group of cells by using SYBR Primescript RT-PCR Kit, and the result is shown in FIG. 3. The results show that when the PPARd gene-carrying interference fragment shRNA virus transduces A549, NCI-H1975 and NCI-H1395 lung cancer cells, the PPARd gene expression is obviously inhibited, and the inhibition efficiency is 82.6% + -0.15%, 87.4% + -0.16% and 86.5% + -0.12% respectively, so that the PLVX-shRNA2-Puro vector used in the experiment can specifically inhibit the PPARd gene expression by carrying shRNA, and the inhibition effect is very obvious.

Example 6 detection of PPARd protein expression amount by Western blot

Respectively culturing A549, NCI-H1975 and NCI-H1395 lung cancer cells and A549, NCI-H1975 and NCI-H1395PPARd gene silencing cell strains in a large scale, removing a culture medium, washing 3 times by using cold PBS, adding 200 mu l of cell lysate, rapidly scraping the lysed cells from the bottle wall by using a cell scraper, collecting proteins into a 500 mu l EP tube, continuously lysing for 30min at 4 ℃, centrifuging for 20min at 12000rpm and 4 ℃, finally adding A5 xSDS-PAGE Sample Loading Buffer, denaturing for 5min at 100 ℃, carrying out 12% SDS-polyacrylamide gel electrophoresis, electrically transferring the proteins onto a PVDF membrane, and sealing by using 5% skimmed milk powder for 1H; respectively adding a PPARd antibody and a beta-activating antibody, incubating at 4 ℃ at room temperature overnight, washing the membrane for 3 times by TBST (tert-butyl-N-transferase) once every 10min, adding a secondary antibody, incubating at room temperature for 1h, washing the membrane for 3 times by TBST once every 10min, adding a Western blot chemiluminescence reagent, and performing imaging analysis. The GAPDH is used as an internal reference, the result shows that the lung cancer cells carrying PPARd gene interference fragment shRNA viruses A549, NCI-H1975 and NCI-H1395 can obviously inhibit the protein expression of the PPARd gene, and the highest inhibition effect of shRNA1 on the PPARd gene expression in various cells is shown in FIG. 4. And the PPARd gene expression of the cell of the control interference fragment shRNAc group is almost unchanged, and the result is consistent with the result of QPCR detection, which shows that the PLVX-shRNA2-Puro vector used in the experiment can specifically inhibit the PPARd gene expression by carrying shRNA.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. An shRNA lentiviral expression vector for specifically inhibiting the expression of a PPARd gene of lung cancer, which is characterized in that: the method specifically comprises the following steps:

the shRNA oligonucleotide sequence of the targeted PPARD gene is shown as a forward sequence PPARD-T1 shown as SEQ ID NO 3 and a reverse complementary sequence PPARD-D1 shown as SEQ ID NO 4;

the shRNA oligonucleotide sequence of the targeted PPARd gene consists of a BamH I enzyme cutting site, a target nucleotide sequence, a stem-loop structure sequence, a target nucleotide sequence complementary sequence, a termination site sequence and an EcoR I enzyme cutting site;

the stem-loop structure sequence is TTCAAGAGAGA.

2. The shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression according to claim 1, wherein the shRNA lentiviral expression vector comprises:

3. The shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression according to claim 1, wherein the shRNA lentiviral expression vector comprises:

the pLVX-shRNA2-Puro expression vector comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence and a promoter sequence of a PLVX-shRNA2-Puro expression vector; the multiple cloning sites comprise a BamHI enzyme cutting site and an EcoRI enzyme cutting site.

4. The shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression according to claim 1, wherein the shRNA lentiviral expression vector comprises:

the termination site sequence is RNA PolyIII polymerase transcription termination site TTTTTT.

5. The method for constructing the shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression according to any one of claims 1 to 4, which is characterized in that: the method comprises the following steps:

(1) shRNA design and Synthesis

The PPARD shRNA is designed to have the basic structure as follows: six regions of BamH I enzyme cutting site + target nucleotide sequence + stem-loop structure + target sequence complementary sequence + RNA PolyIII polymerase transcription termination site + EcoR I enzyme cutting site, which are respectively named as PPARD-T1 and PPARD-D1; the sequence is shown as follows:

PPARD-T1 is shown as SEQ ID NO 3;

PPARD-D1 is shown as SEQ ID NO 4;

the nucleotide sequence of the stem-loop structure is TTCAAGAGAGA;

the nucleotide sequence of the transcription termination site of the RNAPolyIII polymerase is TTTTTT;

(2) shRNA interference vector construction

(3) constructing a recombinant interference vector:

(4) extracting and identifying positive clone plasmid:

taking cultured connecting product competent cells, and extracting recombinant plasmids; obtaining a target shRNA lentiviral expression vector by adopting double enzyme digestion and sequencing confirmation;

the competent cell in the step (3) is a JM107 competent cell;

the double enzyme digestion in the step (4) is one of EcoR1 and Kpn1 double enzyme digestion or BamH1 and Kpn1 double enzyme digestion.

6. The application of the shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression in the screening of lung cancer drugs according to any one of claims 1 to 4.

7. The application of the shRNA lentiviral expression vector for specifically inhibiting the lung cancer PPARd gene expression in the preparation of lung cancer gene medicines according to any one of claims 1 to 4.