CN115417920A - Construction of targeted DHX38 polypeptide and application of targeted DHX38 polypeptide in anti-hepatocellular carcinoma stem cell activity - Google Patents

Abstract

The invention discloses the construction of a targeting DHX38 polypeptide and the anti-hepatocellular carcinoma activity thereof; the sequence for constructing the targeted DHX38 polypeptide comprises the targeted DHX38 polypeptide, wherein the nucleotide sequence of the targeted DHX38 polypeptide is shown as SEQ ID NO. 1; a construction method of targeting DHX38 polypeptide comprises the steps of synthesizing and connecting a nucleic acid sequence of the targeting DHX38 polypeptide with a pcDNA3.1 (+) eukaryotic expression vector under the action of T4 ligase to obtain a recombinant plasmid; the recombinant plasmid is transfected into hepatocellular carcinoma cells using liposomes. The present invention utilizes several functional measures to analyze the antagonism of hepatocellular carcinoma malignancy and stem cell property. The targeting DHX38 polypeptide is proved to have the potential of inhibiting hepatocellular carcinoma stem cell performance and enhancing cis-platinum mediated chemotherapy killing activity on the aspect of cell biology.

Description

Construction of targeted DHX38 polypeptide and application of targeted DHX38 polypeptide in anti-hepatocellular carcinoma stem cell activity

Technical Field

The invention relates to the technical field of bioengineering, relates to construction of a targeted DHX38 polypeptide and activity thereof in resisting liver cell cancer, and particularly discloses construction of the targeted DHX38 polypeptide and application thereof in targeted therapy of liver cell cancer.

Background

Hepatocellular carcinoma (hepatocellular carcinoma for short) is the main primary liver cancer type, and accounts for about 90% of the total incidence of liver cancer. Hepatocellular carcinoma is one of the major malignancies worldwide, with mortality rates third in all tumors. Hepatocellular carcinoma is particularly high in China, the incidence rate is 5 th in all tumors, and the mortality rate is second. Hepatocellular carcinoma is still mainly treated by combining surgical treatment with chemotherapy and radiotherapy at present, and most of patients in advanced stage have extremely poor prognosis due to lack of effective treatment means. The mechanism of hepatocellular carcinoma is complex, and the clinical value of some novel therapeutic modes such as targeted therapy on liver cancer is still not completely clear, so that the clinical application of the therapy is limited. Therefore, new tumor target treatment approaches are urgently needed for hepatocellular carcinoma to improve the clinical prognosis of patients. The occurrence of hepatocellular carcinoma is influenced by various factors, wherein tumor stem cells play a crucial role in the process of hepatocellular carcinoma, and therefore, the development of targeted therapeutic means for targeting hepatocellular carcinoma stem cells has a very positive significance in improving the prognosis of hepatocellular carcinoma.

In the process of hepatocellular carcinoma, tumor stem cells play a key role in the progression of liver cancer and in treatment tolerance. The previous literature indicates that tumor stem cells are mainly distributed at the front of hepatocellular carcinoma invasion and are key cell populations for mediating tumor metastasis, chemotherapy tolerance and immune escape, and if the tumor stem cells cannot be eliminated from hepatocellular carcinoma tissues, the tumor cannot be completely eliminated and rapidly recurs later, thus threatening the survival of patients. Targeting hepatocellular carcinoma stem cells is therefore a key mechanism to address tumor metastasis, drug resistance, and immune escape. We have recently discovered through research that DHX38, a protein that is involved in the transcriptional regulation of various oncogenes, plays a key role in the maintenance of hepatocellular carcinoma stem cells. The deletion of DHX38 can obviously reduce the expression of various cancer-related genes, including OCT4, CD44, LGR5 and the like, thereby inhibiting the formation and the proliferation of liver cell cancer stem cells and enhancing the sensitivity of hepatocellular carcinoma to cisplatin. Based on the design, a target polypeptide sequence of DHX38 is designed, and the target DHX38 polypeptide can obviously inhibit the expression of DHX38, so that the stem cell property and cisplatin resistance of hepatocellular carcinoma are inhibited, and the target polypeptide sequence has potential value of improving the prognosis of a patient when being applied to hepatocellular carcinoma clinical targeted therapy and has good prospect of being applied to hepatocellular carcinoma clinical therapy.

Disclosure of Invention

As one aspect of the invention, the invention overcomes the defects in the prior art and provides the construction of the target DHX38 polypeptide and the value of the target DHX38 polypeptide in antagonizing the stem cell property of the hepatocellular carcinoma.

The invention provides the following technical scheme: a construction sequence of a targeting DHX38 polypeptide, a cDNA sequence thereof and an expressed polypeptide amino acid sequence:

coding cDNA sequence:

GAAATTTGCT

GTGGCTGGCC

661 GAGTTGGGAC CAATGTTTTA TAGTGTGAAA CAAGCAGGTA AAAGCAGACA

GGAAAATCGC

721 AGACGGGCCA AGGAGGAGGC GAGCGCTATG GAAGAGGAGA TGGCACTGGC

CGAAGAGCAA

781 CTTAGGGCAC GGCGACAAGA ACAGGAAAAG CGGTCACCAC TCGGCTCTGT

TAGATCCACC

841 AAAATATACA CTCCGGGGCG GAAAGAGCAA GGCGAGCCCA TGACGCCACG

ACGCACTCCT

901 GCTCGCTTCG GTCTG

in addition, the invention provides a construction and expression method of a targeted DHX38 polypeptide, which comprises the steps of artificially synthesizing gene codes of the polypeptide, treating the gene codes by HindIII and XhoI restriction enzymes, mixing the gene codes with a BamHI and HindIII restriction enzyme digestion linearized pcDNA3.1 (+) expression vector at the molar concentration of 3.

The invention clones the target DHX38 polypeptide coding nucleic acid sequence into pcDNA3.1 (+) eukaryotic expression vector by utilizing the biological engineering technology. The successful construction of recombinant clone is determined by PCR identification and DNA sequencing, the eukaryotic expression vector is transfected into hepatocellular carcinoma cells, the expression of the target DHX38 polypeptide in the hepatocellular carcinoma cells is determined by immunoblotting, the expression change of the downstream stem cell related target gene is verified by real-time quantitative PCR, and the change of the stem cell cancer stem cell character is analyzed by a stem cell sphere formation experiment. The research proves that the targeted DHX38 polypeptide has the function of specifically inhibiting a stem cell pathway and a phenotype in hepatocellular carcinoma on a cell biology level, and can obviously inhibit key cells which induce a tumor malignant process in hepatocellular carcinoma tissues.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive labor. Wherein:

FIG. 1 shows the successful construction of a vector encoding a targeted DHX38 polypeptide;

FIG. 2 is a graph of targeting the expression of a DHX38 polypeptide in hepatocellular carcinoma cells;

FIG. 3 is a schematic representation of targeting a DHX38 polypeptide to downregulate the expression of DHX 38;

fig. 4 is a diagram showing that targeting DHX38 polypeptide expression down-regulates stem cell gene expression;

FIG. 5 is a graph of the effect of targeting a DHX38 polypeptide on hepatocellular carcinoma stem cell sphere formation;

FIG. 6 is a graph of targeting a DHX38 polypeptide to enhance sensitivity of hepatocellular carcinoma to cisplatin.

Detailed Description

FIG. 1 successful construction of targeting DHX38 polypeptide encoding vectors

The DNA sequence encoding the targeted DHX38 polypeptide was synthesized from the Shanghai Biotech company in its entirety and 4. Mu.g of the plasmid was added to 34. Mu.l of sterile water, 4. Mu.l of 10 Xdigestion buffer and 1. Mu.l each of BamHI and HindIII restriction enzymes were added and incubated overnight at 37 ℃. And after the incubation is finished, recovering the target DHX38 polypeptide coding DNA after enzyme digestion by using a PCR DNA recovery kit according to the instruction. After mixing the coding DNA of the targeted DHX38 polypeptide after enzyme digestion with the pcDNA3.1 (+) expression vector which is also subjected to linearization treatment, adding sterile water until the total volume is 17 mu l, and adding 2 mu l of T4 DNA ligase buffer solution and 1 mu l of T4 DNA ligase. Ligation was carried out at room temperature for 2h, and the ligation products were transformed into DH 5. Alpha. Competent bacteria, from which single clones were picked and subjected to colony PCR identification: a single colony was picked with a 20. Mu.l pipette tip and added to 50. Mu.l of LB medium, and from 2. Mu.l of the strain-containing LB medium, 2. Mu.l of 5. Mu.M primer mixture (primer 1 '-GGGGGCCTTGGATAACACTG-3'; primer 2 '-CTCCTCTCTCTCCTGG-3'), 11. Mu.l of sterile water and 15. Mu.l of 2X PCR mix were added. PCR amplification was performed in a PCR apparatus according to the following procedure, with pre-denaturation at 95 ℃ for 5min, cycling 30 times (denaturation at 95 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 30 s), and extension at 72 ℃ for 10min. After the PCR is finished, 10 μ l of the sample is loaded into 2% agarose gel electrophoresis, electrophoresis is carried out for 30min under the condition of 120V, and after the electrophoresis is finished, the obtained clones are identified by a gel imager to contain the targeted DHX38 polypeptide coding DNA (figure 1).

FIGS. 2-4

Application example 1: targeting DHX38 polypeptide expression in hepatocellular carcinoma cells

Using DMEM medium (Gibco)Adding 10% fetal bovine serum (Hyclone) and 1% echinomycin to culture hepatocyte liver cancer Huh7 cell at 37 deg.C in cell culture box at 5% CO2, inoculating 1.5 × 10 ⁶ Cells were plated in 6cm dishes and cell transfection was performed after 24 h:

(1) add 100. Mu.l of opti-mum to the sterile EP tube and add 4. Mu.g of pcDNA3.1 (+) -DHX38 polypeptide or empty plasmid;

(2) adding 100 μ l of opti-mum and 12 μ l of Lipofectamine 2000 (Thermo fisher) into another sterile EP tube, mixing, and standing for 5min;

(3) mixing the plasmid and Lipofectamine 2000 sample, blowing and beating 7-8 times by a pipette, standing for 15min at room temperature;

(4) and uniformly adding the mixture into cells, culturing for 36h, collecting the cells, and performing immunoblotting to detect the expression of the target DHX38 polypeptide.

Digesting the cells with 0.25% Trypsin solution, digesting at room temperature for 4min, adding DMEM medium and counting the number of cells, and collecting 2X 10 cells ⁶ Cells were centrifuged at 800g for 5min, the supernatant medium was removed, 100 μ l of SDS loading buffer was added and boiled for 5min, the samples were loaded to 12% SDS-PAGE, then electrophoresed at 100V for 40-60min, and the protein samples were further transmembrane-coated into PVDF membranes, and after the end of transmembrane-coating, the membranes were blocked with TBST solution containing 5% skim milk for 1h, and further incubated with Flag antibody (1. The next day the antibodies were discarded and the PVDF membrane was washed 3 times with TBST solution for 5min each, HRP-labeled goat anti-mouse secondary antibody (1. The results indicate that the targeted DHX38 polypeptide can be expressed in hepatocellular carcinoma (figure 2).

Application example 2: targeting DHX38 polypeptides to down-regulate expression of DHX38

After the above immunoblot results were developed, the membrane was continuously blocked with TBST solution containing 5% skim milk for 1h, and the antibody against DHX38 (1. The results indicate that targeting DHX38 polypeptides can inhibit DHX38 expression in hepatocellular carcinomas (fig. 3).

Application example 3: targeted DHX38 polypeptide expression down-regulating stem cell gene expression

Collecting Huh7 cells transfected with control no-load and targeting DHX38 polypeptide, cracking and extracting total RNA by using Trizol, performing reverse transcription on 1 mu g of total RNA into cDNA by using a SuperScript III reverse transcription kit, and performing real-time fluorescent quantitative PCR (polymerase chain reaction) to detect the expression of stem cell related genes OCT4, CD44 and LGR5, wherein the specific method comprises the following steps:

RT-PCR primer

OCT4:5' GGG AGA TTG ATA ACT GGT GTG TT-3' and 5' GTG TAT ATC CCA GGG TGA TCC TC-3

CD44:5' ATC ACC GAC AGC ACA GAC AGA AT-doped 3' and 5' AAC CAT GAA AAC CAA TCC CAG G-doped 3

LGR5:5 'AGT CTT AAA GTT CTT ATG CTG C-3' and 5 'TAG CTG ATG TGG TTA GCA TC-3'.

And (3) PCR reaction system: mu.l 2 SYBR master mix (takara), 0.5. Mu.l cDNA, 1. Mu.l primer mix (5. Mu.M), 8.5. Mu.l dH2O, in total 20. Mu.l.

The reaction system is analyzed and detected in a LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche, utilizing ^△△ The ct method analyzes the expression change of the genes after the target DHX38 polypeptide is over-expressed. The results indicate that targeting DHX38 polypeptide can significantly inhibit expression of stem cell associated genes OCT4, CD44 and LGR5 (fig. 4).

FIG. 5 Effect of targeting DHX38 polypeptide on hepatocellular carcinoma Stem cell sphere formation

Huh7 cells transfected with control unloaded and DHX 38-targeted polypeptides were collected, inoculated into a stem cell pellet medium (DMEM/F12, B27 and N2 additives, 20ng/ml EGF and 20ng/ml bFGF) at a ratio of 500 cells/ml, cultured in an extremely low adhesion culture dish for 14 days, and differences in the number and size of stem cell pellets formed in different groups were observed after the culture was completed, indicating that DHX 38-targeted polypeptides can inhibit hepatocellular carcinoma stem cell pellet formation (FIG. 5).

FIG. 6 targeting DHX38 polypeptide enhances sensitivity of hepatocellular carcinoma to cisplatin

Treating the control unloaded and DHX38 polypeptide-targeted Huh7 cells with 10 mu g/ml cis-platinum for 24 hours, performing apoptosis detection by using an Annexin-V/PI apoptosis detection kit according to a product instruction after the treatment is finished, drawing a detection result after the detection is finished, and performing statistical analysis. The results indicate that targeting DHX38 polypeptides can enhance the sensitivity of hepatocellular carcinoma to cisplatin (fig. 6).

The invention discloses a novel bioactive polypeptide which can specifically inhibit expression of stem cell related genes and inhibit liver cell cancer stem cells based on the characteristic that tumor stem cells are key cell types mediating the occurrence and the process of hepatocellular carcinoma and play a key role in the growth, the drug resistance and the metastasis processes of hepatocellular carcinoma tumors. The research on a molecular level shows that the targeted DHX38 polypeptide can inhibit the protein expression of DHX38 in hepatocellular carcinoma, further inhibit the expression of related genes of downstream stem cells, further inhibit the stem cell property of the hepatocellular carcinoma, and enhance the killing capacity of cisplatin on the hepatocellular carcinoma. The invention provides the preclinical targeted DHX38 polypeptide for targeted therapy exploration of liver cancer, and has obvious application values for clinical targeted therapy of hepatocellular carcinoma and improvement of prognosis of patients.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (9)

1. A targeted DHX38 polypeptide, which is characterized by a cDNA sequence and an expressed polypeptide amino acid sequence:

1 SMYQLWILGA LDNTGGLTST

21 GRLMVEFPLD PALSKMLIVS

41 CDMGCSSEIL LIVSMLSVPA

61 IFYRPKGREE ESDQIREKFA

81 VPESDHLTYL NVYLQWKNNN

101 YSTIWCNDHF IHAKAMRKVR

121 EVRAQLKDIM VQQRMSLASC

141 GTDWDIVRKC ICAAYFHQAA

161 KLKGIGEYVN IRTGMPCHLH

181 PTSSLFGMGY TPDYIVYHEL

201 VMTTKEYMQC VTAVDGEWLA

221 ELGPMFYSVK QAGKSRQENR

241 RRAKEEASAM EEEMALAEEQ

261 LRARRQEQEK RSPLGSVRST

281 KIYTPGRKEQ GEPMTPRRTP

301 ARFGL。

2. the targeted DHX38 polypeptide of claim 1, wherein the cDNA sequence encodes:

1 TCCATGTACC AACTTTGGAT CTTGGGGGCC TTGGATAACA CTGGGGGTCT TACATCTACA

61 GGGAGACTGA TGGTGGAATT TCCCTTGGAT CCCGCACTGA GCAAGATGTT GATCGTCTCT

121 TGTGACATGG GATGTTCATC CGAGATTCTT CTCATCGTAA GTATGCTCTC AGTTCCAGCC

181 ATCTTCTATC GACCGAAAGG GAGAGAGGAG GAGTCAGACC AGATTAGAGAGAAATTTGCT

241 GTCCCAGAAT CAGACCACCT TACCTACCTT AATGTATATC TGCAGTGGAA GAATAATAAT

301 TACTCCACTA TCTGGTGTAA CGATCACTTT ATACACGCCA AGGCAATGAG GAAGGTCCGC

361 GAGGTTCGAG CTCAACTCAA GGACATTATG GTTCAACAGC GCATGTCCCT TGCATCCTGT

421 GGCACGGATT GGGACATAGT AAGGAAGTGC ATTTGCGCTG CGTATTTCCA CCAGGCAGCC

481 AAACTTAAGG GGATTGGAGA ATACGTAAAC ATTCGCACCG GGATGCCTTG TCATCTCCAT

541 CCAACGTCTT CTCTCTTCGG AATGGGTTAC ACGCCAGATT ACATTGTGTA TCACGAATTG

601 GTAATGACTA CGAAAGAATA CATGCAGTGC GTTACTGCGG TAGATGGGGAGTGGCTGGCC

661 GAGTTGGGAC CAATGTTTTA TAGTGTGAAA CAAGCAGGTA AAAGCAGACAGGAAAATCGC

721 AGACGGGCCA AGGAGGAGGC GAGCGCTATG GAAGAGGAGA TGGCACTGGCCGAAGAGCAA

781 CTTAGGGCAC GGCGACAAGA ACAGGAAAAG CGGTCACCAC TCGGCTCTGTTAGATCCACC

841 AAAATATACA CTCCGGGGCG GAAAGAGCAA GGCGAGCCCA TGACGCCACGACGCACTCCT

901 GCTCGCTTCG GTCTG。

3. a construction and expression method of the targeted DHX38 polypeptide as claimed in claim 1, which comprises artificially synthesizing the gene code of the polypeptide, treating with the first restriction enzyme, mixing with the linearized expression vector digested with the second restriction enzyme, adding ligase buffer and ligase, standing at room temperature, and ligating to obtain the recombinant vector.

4. The method of claim 3, wherein the first restriction enzyme comprises HindIII and XhoI.

5. The method for constructing and expressing as claimed in claim 3, wherein the second restriction enzyme includes BamHI and HindIII.

6. The method for constructing and expressing a polypeptide of claim 3, wherein the gene code processed by the first restriction enzyme is mixed with the linearized expression vector digested by the second restriction enzyme at a molar concentration of 3.

7. The construction and expression method according to claim 3, wherein the expression vector is pcDNA3.1 (+).

8. The method for constructing and expressing according to claim 3, wherein the ligase buffer and the ligase are 10% volume of T4 DNA ligase buffer and 1 μ l of T4 DNA ligase, respectively.

9. The use of the targeted DHX38 polypeptide of claim 1 in the targeted treatment of hepatocellular carcinoma.

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