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

Abstract

This invention discloses the construction of a DHX38-targeting peptide and its anti-hepatocellular carcinoma activity. Specifically, it provides a sequence for constructing the DHX38-targeting peptide, comprising a DHX38-targeting peptide with a nucleotide sequence as shown in SEQ ID NO:1; and a method for constructing the DHX38-targeting peptide, comprising synthesizing and ligating the nucleic acid sequence of the DHX38-targeting peptide to a pcDNA3.1(+) eukaryotic expression vector using T4 ligase to obtain a recombinant plasmid; and transfecting the recombinant plasmid into hepatocellular carcinoma cells using liposomes. This invention utilizes various functional methods to analyze its antagonistic effects on the malignancy and stem cell nature of hepatocellular carcinoma. At the cell biology level, it demonstrates that this DHX38-targeting peptide has the potential to inhibit the stem cell nature of hepatocellular carcinoma and enhance cisplatin-mediated chemotherapeutic killing activity.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and specifically to the construction of a DHX38-targeting peptide and its anti-hepatocellular carcinoma activity. In particular, this invention discloses the construction of a DHX38-targeting peptide and its application in the targeted therapy of hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the main type of primary liver cancer. Current treatment for HCC primarily involves surgery combined with chemotherapy and radiotherapy. Many patients with advanced-stage HCC suffer from extremely poor prognoses due to the lack of effective treatment options. The pathogenesis of HCC is complex, and the clinical value of some novel treatments, such as targeted therapy, for liver cancer remains unclear, limiting their clinical application. Therefore, there is an urgent need for new tumor-targeted therapies to improve the clinical prognosis of HCC patients. The development of HCC is influenced by multiple factors, among which tumor stem cells play a crucial role in its progression. Therefore, developing targeted therapies that target HCC stem cells is of great significance for improving the prognosis of HCC.

[0003] In the development of hepatocellular carcinoma (HCC), tumor stem cells play a crucial role in the progression and treatment resistance of the disease. Previous literature indicates that tumor stem cells are primarily distributed at the forefront of HCC invasion and are a key cell population mediating tumor metastasis, chemotherapy resistance, and immune escape. If tumor stem cells cannot be eliminated from HCC tissue, the tumor cannot be completely eradicated and will rapidly recur, threatening patient survival. Therefore, targeting HCC stem cells is a key mechanism for addressing tumor metastasis, drug resistance, and immune escape. Our recent research has revealed that DHX38, a transcriptional regulatory protein associated with multiple oncogenes, plays a crucial role in the maintenance of HCC stem cells. The absence of DHX38 significantly reduces the expression of various oncogenes, including OCT4, CD44, and LGR5, thereby inhibiting the formation and proliferation of HCC stem cells and enhancing the sensitivity of HCC to cisplatin. Based on this, we designed a DHX38-targeting peptide sequence and found that this DHX38-targeting peptide can significantly inhibit the expression of DHX38, thereby inhibiting the stem cell-like nature and cisplatin resistance of hepatocellular carcinoma. It has the potential value of being applied to the clinical targeted therapy of hepatocellular carcinoma to improve the prognosis of patients and has a good prospect for clinical application in the treatment of hepatocellular carcinoma. Summary of the Invention

[0004] As one aspect of the present invention, the present invention overcomes the deficiencies of the prior art and provides the construction of a DHX38-targeting peptide and its antagonistic value against hepatocellular carcinoma stem cells.

[0005] This invention provides the following technical solution: a construction sequence for a DHX38-targeting polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1:

[0006] 1 SMYQLWILGA LDNTGGLTST

[0007] 21 GRLMVEFPLD PALSKMLIVS

[0008] 41 CDMGCSSEIL LIVSMLSVPA

[0009] 61 IFYRPKGREE ESDQIREKFA

[0010] 81 VPESDHLTYL NVYLQWKNNN

[0011] 101 YSTIWCNDHF IHAKAMRKVR

[0012] 121 EVRAQLKDIM VQQRMSLASC

[0013] 141 GTDWDIVRKC ICAAYFHQAA

[0014] 161 KLKGIGEYVN IRTGMPCHLH

[0015] 181 PTSSLFGMGY TPDYIVYHEL

[0016] 201 VMTTKEYMQC VTAVDGEWLA

[0017] 221 ELGPMFYSVK QAGKSRQENR

[0018] 241 RRAKEEASAM EEEMALAEEQ

[0019] 261 LRARRQEQEK RSPLGSVRST

[0020] 281 KIYTPGRKEQ GEPMTPRRTP

[0021] 301 ARFGL

[0022] The encoding cDNA sequence is shown in SEQ ID NO:2:

[0023] 1 TCCATGTACC AACTTTGGAT CTTGGGGGCC TTGGATAACA CTGGGGGTCTTACATCTACA

[0024] 61 GGGAGACTGA TGGTGGAATT TCCCTTGGAT CCCGCACTGA GCAAGATGTTGATCGTCTCT

[0025] 121 TGTGACATGG GATGTTCATC CGAGATTCTT CTCATCGTAA GTATGCTCTCAGTTCCAGCC

[0026] 181 ATCTTCTATC QUESTIONAGGG CHANGE QUESTIONCC CHANGEQUAKATTTGCT

[0027] 241 GTCCCHATA TRANSACTION TACCTACCTT AATGTATATC TGCAGTGGAAGAATAATAAT

[0028] 301 TACTCCACTA TCTGGTGTAA CGATCACTTT ATACACGCCA AGGCAATGAGGAAGGTCCGC

[0029] 361 GAGGTTCGAG CTCAACTCAA GGACATTATG GTTCAACAGC GCATGTCCCTTGCATCCTGT

[0030] 421 GGCACGGATT GGGACATAGT AAGGAAGTGC ATTTGCGCTG CGTATTTCCACCAGGCAGCC

[0031] 481 AAACTTAAGG GGATTGGAGA ATACGTAAAC ATTCGCACCG GGATGCCTTGTCATCTCCAT

[0032] 541 CCAACGTCTT CTCTCTTCGG AATGGGTTAC ACGCCAGATT ACATTGTGTATCACGAATTG

[0033] 601 GTAATGACTA CGAAAGAATA CATGCAGTGC GTTACTGCGG TAGATGGGGAGTGGCTGGCC

[0034] 661 GAGTTGGGAC CAATGTTTTA TAGTGTGAAA CAAGCAGGTA AAAGCAGACAGGAAAATCGC

[0035] 721 AGACGGGCCA AGGAGGAGGC GAGCGCTATG GAAGAGGAGA TGGCACTGGCCGAAGAGCAA

[0036] 781 CTTAGGGCAC GGCGACAAGA ACAGGAAAAG CGGTCACCAC TCGGCTCTGTTAGATCCACC

[0037] 841 AAAATATACA CTCCGGGGCG GAAAGAGCAA GGCGAGCCCA TGACGCCACGACGCACTCCT

[0038] 901 GCTCGCTTCG GTCTG

[0039] Furthermore, this invention provides a method for constructing and expressing a DHX38-targeting polypeptide, comprising: artificially synthesizing the gene encoding the aforementioned polypeptide, treating it with HindIII and XhoI restriction endonucleases, mixing it with a linearized pcDNA3.1(+) expression vector digested with BamHI and HindIII restriction endonucleases at a molar concentration of 3:1, adding 10% volume of T4 DNA ligase buffer and 1 μl of T4 DNA ligase, incubating at room temperature for 2 h to obtain a recombinant vector, verifying the obtained recombinant vector by PCR and sequencing, introducing it into hepatocellular carcinoma cells using liposomes, and detecting the expression of the DHX38-targeting polypeptide and its antagonistic effects on downstream molecules promoting stem cell growth and hepatocellular carcinoma stem cells.

[0040] This invention utilizes bioengineering technology to clone the DHX38-targeting peptide encoding nucleic acid sequence into a pcDNA3.1(+) eukaryotic expression vector. The successful construction of the recombinant clone was confirmed by PCR and DNA sequencing. This eukaryotic expression vector was transfected into hepatocellular carcinoma cells. Western blotting confirmed the expression of the DHX38-targeting peptide in hepatocellular carcinoma cells. Real-time quantitative PCR verified the expression changes of downstream stem cell-related target genes, and stem cell spheroidization assays analyzed changes in hepatocellular carcinoma stem cell sex. This study demonstrates at the cell biology level that this DHX38-targeting peptide specifically inhibits stem cell sex pathways and phenotypes in hepatocellular carcinoma, significantly suppressing key cells that induce malignant progression in hepatocellular carcinoma tissues. Attached Figure Description

[0041] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0042] Figure 1 Successful construction of the DHX38 peptide-encoding vector;

[0043] Figure 2 To target the expression of DHX38 peptide in hepatocellular carcinoma cells;

[0044] Figure 3 Targeting the DHX38 peptide can downregulate the expression of DHX38;

[0045] Figure 4 To target DHX38 peptide expression and downregulate stem cell gene expression;

[0046] Figure 5 To target the effect of DHX38 peptide on the formation of hepatocellular carcinoma stem cell spheres;

[0047] Figure 6 To target the DHX38 peptide to enhance the sensitivity of hepatocellular carcinoma to cisplatin. Detailed Implementation

[0048] Figure 1 This demonstrates the successful construction of a vector encoding the DHX38 peptide.

[0049] The DNA sequence encoding the DHX38-targeting peptide was synthesized in its entirety at Shanghai Sangon Biotech Co., Ltd. 4 μg of plasmid was added to sterile water to a final volume of 34 μl, followed by 4 μl of 10× restriction enzyme buffer and 1 μl each of BamHI and HindIII restriction endonucleases. The mixture was incubated overnight at 37°C. After incubation, the DHX38-targeting peptide-coding DNA was recovered using a PCR DNA recovery kit according to the manufacturer's instructions. The DHX38-targeting peptide-coding DNA was then mixed with the linearized pcDNA3.1(+) expression vector, and sterile water was added to a final volume of 17 μl. 2 μl of T4 DNA ligase buffer and 1 μl of T4 DNA ligase were also added. Ligation was performed at room temperature for 2 hours, and the ligation product was transformed into DH5α competent bacteria. Single colonies were picked and identified by colony PCR: a single colony was picked with a 20 μl pipette tip and added to 50 μl LB medium. Then, 2 μl of the LB medium containing bacteria was added to 2 μl of 5 μM primer mixture (primer 1: 5'-GGGGGCCTTGGATAACACTG-3'; primer 2: 5'-CTCCTCCTCTCTCCCTTTCGG-3'), 11 μl of sterile water, and 15 μl of 2*PCR mixture. PCR amplification was performed in a PCR instrument according to the following program: 95℃ pre-denaturation for 5 min, 30 cycles (95℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 30 s), and 72℃ extension for 10 min. After PCR, 10 μl of the sample was loaded into a 2% agarose gel for electrophoresis. Electrophoresis was performed at 120V for 30 min. After electrophoresis, gel imaging revealed that all obtained clones contained DNA encoding the DHX38-targeting polypeptide. Figure 1 ).

[0050] Figure 2-4

[0051] Application Example 1: Targeting the expression of DHX38 peptide in hepatocellular carcinoma cells

[0052] Hepatocellular carcinoma Huh7 cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin under the following conditions: 37°C cell culture incubator, 5% CO2, and seeded at 1.5 × 10⁶ cells / year. 6 Cells were transferred to 6cm culture dishes and transfected after 24 hours.

[0053] ① Add 100 μl of opti-mum to a sterile EP tube, and add 4 μg of pcDNA3.1(+)-DHX38 polypeptide or empty vector plasmid;

[0054] ② In another sterile EP tube, add 100 μl opti-mum and 12 μl Lipofectamine 2000 (Thermofisher), mix well and let stand for 5 min;

[0055] ③ Mix the plasmid and Lipofectamine 2000 sample thoroughly, pipette 7-8 times, and let stand at room temperature for 15 minutes;

[0056] ④ Add the above mixture evenly to the cells, culture for 36 hours, and then collect the cells for immunoblotting to detect the expression of the DHX38-targeting peptide.

[0057] Cells were digested with 0.25% Trypsin solution at room temperature for 4 min. DMEM medium was then added, and the cell count was determined. Two × 10⁶ cells were collected. 6 Cells were centrifuged at 800g for 5 min, and the supernatant culture medium was removed. 100 μl of SDS loading buffer was added, and the mixture was boiled for 5 min. The sample was then loaded into 12% SDS-PAGE and electrophoresed at 100V for 40-60 min. Protein samples were then transferred to a PVDF membrane. After transfer, the membrane was blocked with TBST solution containing 5% skim milk for 1 h, followed by overnight incubation with Flag antibody (1:2000). The next day, the antibody was discarded, and the PVDF membrane was washed three times with TBST solution for 5 min each time. HRP-labeled goat anti-mouse secondary antibody (1:10000) was added and the membrane was incubated for 2 h. The DHX38-targeting peptide was visualized using ECL, and the results were obtained using a chemiluminescence immunoassay scanner. The results indicate that the DHX38-targeting peptide can be expressed in hepatocellular carcinoma. Figure 2 ).

[0058] Application Example 2: Targeting the DHX38 peptide to downregulate DHX38 expression

[0059] After the immunoblotting results were developed, the membrane was blocked with TBST solution containing 5% skim milk for 1 hour and incubated overnight with anti-DHX38 antibody (1:1000). The next day, the PVDF membrane was washed three times with TBST solution for 5 minutes each time, and HRP-labeled goat anti-rabbit secondary antibody (1:10000) was added and incubated for 2 hours. DHX38 protein expression was detected by ECL, and the DHX38 band was obtained by scanning. The results showed that the DHX38-targeting peptide could inhibit the expression of DHX38 in hepatocellular carcinoma. Figure 3 ).

[0060] Application Example 3: Downregulating Stem Cell Gene Expression by Targeting DHX38 Peptide Expression

[0061] Huh7 cells transfected with the control empty vector and the DHX38-targeting peptide were collected. Total RNA was extracted and lysed using Trizol. 1 μg of total RNA was reverse transcribed into cDNA using the SuperScript III reverse transcription kit. Real-time quantitative PCR was performed to detect the expression of stem cell-related genes OCT4, CD44, and LGR5. The specific methods are as follows:

[0062] RT-PCR primers

[0063] OCT4: Forward as shown in SEQ ID NO:3, reverse as shown in SEQ ID NO:4 (5'-GGG AGA TTG ATAACT GGT GTG TT-3' and 5'-GTG TAT ATC CCA GGG TGA TCC TC-3')

[0064] CD44: 5'-ATC ACC GAC AGC ACA GAC AGA AT-3' and 5'-AAC CAT GAA AAC CAA TCCCAG G-3'

[0065] LGR5: 5'-AGT CTT AAA GTT CTT ATG CTG C-3' and 5'-TAG CTG ATG TGG TTA GCATC-3'.

[0066] PCR reaction system: 10 μl 2*SYBR master mix (Takara), 0.5 μl cDNA, 1 μl primer mix (5 μM), 8.5 μl dH2O, total 20 μl.

[0067] The above reaction system was analyzed and detected using a Roche LightCycler 480 real-time quantitative PCR instrument. △△ The expression changes of the above genes after overexpression of the DHX38-targeted peptide were analyzed using the CT method. The results showed that targeting the DHX38-targeted peptide significantly inhibited the expression of stem cell-related genes OCT4, CD44, and LGR5. Figure 4 ).

[0068] Figure 5 Effects of DHX38-targeted peptide on hepatocellular carcinoma stem cell spheroid formation

[0069] Huh7 cells transfected with control empty vector and DHX38-targeted peptide were collected and seeded at a ratio of 500 cells / ml in stem cell spheroid culture medium (DMEM / F12, B27 and N2 additives, 20 ng / ml EGF, 20 ng / ml bFGF). Cells were cultured in very low adhesion dishes for 14 days. After culture, the number and size of stem cell spheroids formed in different groups were observed. The results showed that targeting DHX38 peptide could inhibit the formation of hepatocellular carcinoma stem cell spheroids. Figure 5 ).

[0070] Figure 6 Targeting the DHX38 peptide enhances the sensitivity of hepatocellular carcinoma to cisplatin.

[0071] In Huh7 cells transfected with control empty vector and DHX38-targeted peptide, cisplatin at 10 µg / ml was used for 24 h. After treatment, apoptosis was detected using the Annexin-V / PI apoptosis detection kit according to the product instructions. The results were plotted and statistically analyzed. The results showed that targeting DHX38 peptide can enhance the sensitivity of hepatocellular carcinoma to cisplatin. Figure 6 ).

[0072] This invention is based on the fact that tumor stem cells are key cell types mediating the occurrence and progression of hepatocellular carcinoma (HCC), playing a crucial role in tumor growth, drug resistance, and metastasis. A novel bioactive polypeptide has been developed that can specifically inhibit the expression of stem cell-related genes and suppress HCC stem cells. Molecular-level studies show that targeting the DHX38 polypeptide can inhibit the protein expression of DHX38 in HCC, thereby inhibiting the expression of downstream stem cell-related genes, thus suppressing the stem cell nature of HCC and enhancing the killing effect of cisplatin on HCC. This invention provides a preclinical exploration of targeted therapy for liver cancer using the DHX38 polypeptide, which has significant application value for clinical targeted treatment of HCC and improving patient prognosis.

[0073] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention. <110> Taizhou People's Hospital <120> Construction of DHX38-targeted peptide and its application in anti-hepatocellular carcinoma stem cell activity <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 305 <212> PRT <213> Artificial Sequence <400> 1 Ser Met Tyr Gln Leu Trp Ile Leu Gly Ala Leu Asp Asn Thr Gly Gly 1 5 10 15 Leu Thr Ser Thr Gly Arg Leu Met Val Glu Phe Pro Leu Asp Pro Ala 20 25 30 Leu Ser Lys Met Leu Ile Val Ser Cys Asp Met Gly Cys Ser Ser Glu 35 40 45 Ile Leu Leu Ile Val Ser Met Leu Ser Val Pro Ala Ile Phe Tyr Arg 50 55 60 Pro Lys Gly Arg Glu Glu Glu Ser Asp Gln Ile Arg Glu Lys Phe Ala 65 70 75 80 Val Pro Glu Ser Asp His Leu Thr Tyr Leu Asn Val Tyr Leu Gln Trp 85 90 95 Lys Asn Asn Asn Tyr Ser Thr Ile Trp Cys Asn Asp His Phe Ile His 100 105 110 Ala Lys Ala Met Arg Lys Val Arg Glu Val Arg Ala Gln Leu Lys Asp 115 120 125 Ile Met Val Gln Gln Arg Met Ser Leu Ala Ser Cys Gly Thr Asp Trp 130 135 140 Asp Ile Val Arg Lys Cys Ile Cys Ala Ala Tyr Phe His Gln Ala Ala 145 150 155 160 Lys Leu Lys Gly Ile Gly Glu Tyr Val Asn Ile Arg Thr Gly Met Pro 165 170 175 Cys His Leu His Pro Thr Ser Ser Leu Phe Gly Met Gly Tyr Thr Pro 180 185 190 Asp Tyr Ile Val Tyr His Glu Leu Val Met Thr Thr Lys Glu Tyr Met 195 200 205 Gln Cys Val Thr Ala Val Asp Gly Glu Trp Leu Ala Glu Leu Gly Pro 210 215 220 Met Phe Tyr Ser Val Lys Gln Ala Gly Lys Ser Arg Gln Glu Asn Arg 225 230 235 240 Arg Arg Ala Lys Glu Glu Ala Ser Ala Met Glu Glu Glu Met Ala Leu 245 250 255 Ala Glu Glu Gln Leu Arg Ala Arg Arg Gln Glu Gln Glu Lys Arg Ser 260 265 270 Pro Leu Gly Ser Val Arg Ser Thr Lys Ile Tyr Thr Pro Gly Arg Lys 275 280 285 Glu Gln Gly Glu Pro Met Thr Pro Arg Arg Thr Pro Ala Arg Phe Gly 290 295 300 Leu 305 <210> 2 <211> 915 <212> DNA <213> Artificial Sequence <400> 2 tccatgtacc aactttggat cttgggggcc ttggataaca ctgggggtct tacatctaca 60 gggagactga tggtggaatt tcccttggat cccgcactga gcaagatgtt gatcgtctct 120 tgtgacatgg gatgttcatc cgagattctt ccatcgtaa gtatgctctc agttccagcc 180 atcttctatc gaccgaaagg gagagaggag gagtcagacc agattagaga gaaatttgct 240 gtcccagaat cagaccacct tacctacctt aatgtatatc tgcagtggaa gaataataat 300 tactccacta tctggtgtaa cgatcacttt attacacgcca aggcaatgag gaaggtccgc 360 gaggttcgag ctcaactcaa ggacattatg gttcaacagc gcatgtccct tgcatcctgt 420 ggcacggatt gggacatagt aaggaagtgc atttgcgctg cgtatttcca ccaggcagcc 480 aaacttaagg ggattggaga atacgtaaac attcgcaccg ggatgccttg tcatctccat 540 ccaacgtctt ctctcttcgg aatgggttac acgccagatt acattgtgta tcacgaattg 600 gtaatgacta cgaaagaata catgcagtgc gttactgcgg taggggga gtggctggcc gagttgggac caatgtttta tagtgtgaaa caagcaggta aaagcagaca ggaaaatcgc agacgggcca aggaggc gagcgctatg tggcactggc cgaagagcaa 780 cttagggcac ggcgacaaga acaggaaaag cggtcaccac tcggctctgt tagatccacc aaatataca ctccggggcg gaaagagcaa ggcgagccca tgacgccacg acgcactcct gctcgcttcg gtctg 915 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <400> 3 gggagattga taactggtgt gtt <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 gtgtatatcc cagggtgatc ctc

Claims

1. A polypeptide targeting DHX38, characterized in that, Its polypeptide amino acid sequence is shown in SEQ ID NO:

1.

2. The DHX38-targeting polypeptide according to claim 1, characterized in that, Its encoding cDNA sequence is shown in SEQ ID NO:

2.

3. A method for constructing and expressing the DHX38-targeting polypeptide as described in claim 1, characterized in that, The gene encoding the artificially synthesized polypeptide, the gene encoding after treatment with the first restriction endonuclease, is mixed with the linearized expression vector pcDNA3.1(+) digested with the second restriction endonuclease at a molar concentration of 3:

1. Ligase buffer and ligase are added, and the mixture is ligated after being placed at room temperature to obtain the recombinant vector. The first restriction endonuclease includes HindIII and XhoI; The second restriction endonuclease includes BamHI and HindIII; The ligase buffer and ligase are 10% T4 DNA ligase buffer and 1 μl T4 DNA ligase, respectively.

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