Liver cancer cell line with LAPTM5 gene stable overexpression as well as construction method and application of liver cancer cell line

Abstract

The invention discloses a liver cancer cell line with LAPTM5 gene stable overexpression as well as a construction method and application of the liver cancer cell line. According to the invention, a human liver cancer cell line Huh7-LAPTM5 overexpression cell line is successfully constructed through a lentivirus transfection technology. Protein expression is detected through Western blot, and it is proved that overexpression cells are successfully constructed. Meanwhile, experiments prove that under the normal culture condition, LAPTM5 overexpression has no obvious influence on the processes of cell proliferation, apoptosis and the like. The Huh7-LAPTM5 overexpression cell line constructed by the invention plays a biological role by promoting a cell autophagy process, and the cell line is suitable for basic clinical cross correlation researches based on autophagy, drug resistance and the like.

Description

Technical Field

[0001] The present invention relates to a hepatocellular carcinoma cell line with stable overexpression of LAPTM5 gene, a construction method thereof and applications, belonging to the field of bioengineering technology. Background Art

[0002] LAPTM5 (Lysosomal-associated transmembrane protein 5), also known as CD40-linked activator-specific transcript 6 (CLAST6), is a transmembrane protein present in late endosomes and lysosomes, and its encoding gene is located on chromosome 1p34. LAPTM5 belongs to the late endosome / lysosome transmembrane protein family and was initially considered a protein homology regulator and an inflammatory signal regulator when it was first discovered. LAPTM5 plays a key role in the regulation of autophagy. Autophagy comes from the Greek word meaning "self-eating" and was first proposed by Christian de Duve. There are three forms of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy, and all these types promote the degradation of cytoplasmic components in lysosomes. Autophagy has an important regulatory role in the occurrence and development of tumors.

[0003] LAPTM5 plays a role in the process of protein transport from the Golgi apparatus to lysosomes and regulates the sorting process of lysosomes and cell membranes by interacting with the E3 ubiquitin ligase Nedd4. In the study of liver cancer, LAPTM5 affects drug resistance by regulating the autophagy pathway. The Huh7 cell line is a human-derived hepatocellular carcinoma cell line derived from the hepatocellular carcinoma tissue of a 57-year-old male patient. This cell line is widely used in basic and clinical research on the pathogenesis of human liver cancer and is also commonly used in the screening of anti-tumor drugs. However, the existing liver cancer models still have deficiencies in the study of autophagy. Summary of the Invention

[0004] The object of the present invention is to provide a hepatocellular carcinoma cell line with stable overexpression of LAPTM5 gene, a construction method thereof and applications. The Huh7-LAPTM5 overexpression cell line constructed by the present invention exerts biological effects by regulating the autophagy process of cells, and this cell line is suitable for research related to basic clinical intersections such as autophagy and drug resistance; through functional verification, the present invention shows that under normal culture conditions, overexpression of LAPTM5 has no significant effect on the proliferation and apoptosis of the cell line.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided the use of a biomaterial in the preparation of a kit for constructing a hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene, and the use in constructing a hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene. The biomaterial is a primer pair shown in SEQ ID NO: 1-2, or a target gene fragment obtained by PCR amplification using the primer pair, or an expression vector containing the target gene fragment.

[0007] Preferably, the expression vector is selected from viral, fungal or bacterial expression vectors.

[0008] In the second aspect of the present invention, there is provided a method for constructing a hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene, including: transfecting the hepatocellular carcinoma cell line Huh7 with a lentivirus carrying the target gene LAPTM5, and screening to obtain a hepatocellular carcinoma cell line with overexpression of LAPTM5, that is, obtaining a Huh7-LAPTM5 overexpressing hepatocellular carcinoma cell line.

[0009] In some embodiments of the present invention, the method specifically includes the following steps:

[0010] Step 1: Design primers for PCR amplification fragments, and introduce homologous sequences at the 5' end of the primers to the ends of the linearized cloning vector, so that the 5' and 3' terminal sequences of the amplification product are exactly the same as the two terminal sequences of the linearized cloning vector; the primer sequences are as shown in SEQ ID NO: 1-2;

[0011] Step 2: Clone the target gene fragment into the linearized cloning vector PGMLV-CMV-MCS-3xFlag-PGK-Puro after enzyme digestion to obtain a recombinant lentiviral vector for overexpression of LAPTM5;

[0012] Step 3: Transform the recombinant lentiviral vector assembled in Step 2 into DH5α competent cells; screen the transformants by colony PCR, send the positive clones for sequencing, and determine the positive clones by sequence alignment; amplify the positive clones and extract the plasmids;

[0013] Step 4: Combine the plasmid extracted in Step 3 with the viral packaging auxiliary plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system, and co-transfect Expi293F suspension cells using a transfection reagent PEI for virus packaging to obtain a virus solution;

[0014] Step 5: Infect the hepatocellular carcinoma cell line Huh7 with the virus solution obtained in Step 4, and screen to obtain a hepatocellular carcinoma cell line with overexpression of LAPTM5.

[0015] In some embodiments of the present invention, the method for virus packaging in step 4 includes: co-transfecting Expi293F suspension cells for 72 h, collecting the cell culture supernatant, centrifuging (at 4 °C, 1000 - 2000×g, for 5 - 15 min) to remove cell debris, adding PEG-it virus precipitation reagent (to a final concentration of 5%) and precipitating overnight; centrifuging the next day (at 4 °C, 1000 - 2000×g, for 40 - 50 min), discarding the supernatant, and resuspending the virus particles with PBS to obtain a high-titer (titer ≥ 1×10^9 TU / mL) lentivirus concentrate overexpressing LAPTM5.

[0016] In the third aspect of the present invention, a liver cancer cell line with stable overexpression of the constructed LAPTM5 gene is provided.

[0017] In the fourth aspect of the present invention, the application of the liver cancer cell line with stable overexpression of the LAPTM5 gene described in the third aspect in constructing a research model for the function of the LAPTM5 gene is provided.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] After a large number of attempts, the present invention uses Expi293F suspension cells (Thermo Fisher Scientific) to replace traditional adherent 293T cells, and adopts an innovative virus packaging process for efficient virus packaging and concentration to obtain a high-titer lentivirus concentrate overexpressing LAPTM5; then, the liver cancer cell line Huh7 is transfected with the lentivirus to construct a cell line with stable overexpression of Huh7-LAPTM5, and the transfection efficiency is high; moreover, through experiments, it is verified that under normal culture conditions, overexpression of LAPTM5 has no significant effect on processes such as cell proliferation and apoptosis, and this model does not affect the cell state under normal culture conditions and can be used as an ideal model for studying autophagy. Description of the Drawings

[0020] Figure 1 Plasmid structure diagram with [vector name] as the vector.

[0021] Figure 2 For detecting the overexpression level of LAPTM5 and its effect on apoptosis of the cell line by Western blot.

[0022] Figure 3 For detecting the effect of LAPTM5 on the proliferation ability of Huh7 by CCK8 experiment. Detailed Embodiments

[0023] To make the present invention more obvious and understandable, preferred embodiments are provided below in conjunction with the accompanying drawings for detailed description as follows.

[0024] For the experimental methods without specific conditions in the following examples, they were carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used were all commercially available conventional products without special instructions.

[0025] Example

[0026] I. Construction of Huh7-LAPTM5 overexpression cell line:

[0027] 1. Design and synthesize primers

[0028] 1) Design primers for PCR amplification fragments and introduce homologous sequences at the 5' end of the primers to the ends of the linearized cloning vector, so that the 5' and 3' terminal sequences of the amplification product are exactly the same as the two terminal sequences of the linearized cloning vector respectively.

[0029] 2) Synthesize the designed primer sequences (as shown in Table 1)

[0030] Table 1 Primer sequences

[0031]

[0032]

[0033] 2. Double digestion of vector PGMLV-CMV-MCS-3xFlag-PGK-Puro (as Figure 1 shown)

[0034] 1) Incubate the bacterial solution containing the vector plasmid overnight, and take 3 - 5 ml of fresh bacterial solution to extract the plasmid. The specific method refers to the QIAGEN plasmid miniprep instruction manual.

[0035] 2) Take 1 μg of fresh plasmid and perform double digestion with the corresponding restriction endonucleases. The digestion system is as follows:

[0036] vector 1 μg green Buffer 3 μL XhoI 1.5 μL BamHI 1.5 μL <![CDATA[ddH2O]]> Make up to 30 μL

[0037] Digest at 37 °C for about 3 h.

[0038] 3) Perform agarose gel electrophoresis on the digestion products. After electrophoresis, perform gel extraction. The steps are as follows: Under ultraviolet light, cut the gel strip containing the target fragment. Weigh the total weight with a balance and subtract the weight of the empty tube to calculate the weight of the gel. Calculate the volume of the gel according to 100 mg = 100 μL, and add 1 times the gel volume of Binging Solution and place it in a 65 °C water bath to completely melt the gel. Shake the EP tube appropriately during this period to accelerate the dissolution of the gel.

[0039] 4) Transfer all of the above liquid into the filter column and centrifuge at 13,000 rpm for 30 s (this can be repeated once). Then discard the liquid in the tube, add 500 μL of WA Solution to the column, and centrifuge at 13,000 rpm for 30 s. Discard the liquid in the tube, add 500 μL of Wash Solution to the column, and centrifuge at 13,000 rpm for 30 s (this can be repeated once). Then centrifuge without liquid for 3 min. Place the filter column in a new 1.5 mL EP tube and let it dry at room temperature. Finally, add 35 μL of ddH2O to the column, let it stand for 5 min, and centrifuge at 13,000 rpm for 1.5 min. To improve the recovery rate, the dissolved DNA can be added to the column again and centrifuged for one minute. Discard the column, and the recovered vector fragment is obtained and its concentration is measured.

[0040] 3. Amplification of the target fragment

[0041] 1) Dilute the synthesized primers to a stock solution with a final concentration of 10 μmol / L.

[0042] 2) Use the diluted primers and template for PCR amplification. The reaction system is as follows:

[0043]

[0044]

[0045] Add the above components to a thin-walled tube, mix well and centrifuge briefly, then place it in a PCR instrument. Select the appropriate annealing temperature and extension temperature, and then start PCR amplification.

[0046] 3) After PCR, perform agarose gel electrophoresis and recover the target gene. The recovery method is the same as above.

[0047] 4. Ligation of the overexpression vector and the target fragment (seamless cloning)

[0048] 1) Measure the concentrations of the recovered vector and the target fragment;

[0049] 2) The optimal usage amount of the Hieff CloneTM recombination reaction system for the cloning vector is 0.03 pmol; the optimal molar ratio of the cloning vector to the inserted fragment is 1:2, that is, the optimal usage amount of the inserted fragment is 0.06 pmol. The DNA masses corresponding to these molar amounts can be calculated using the following formula:

[0050] Optimal usage amount of the cloning vector = [0.02 × base pairs of the cloning vector] ng (0.03 pmol)

[0051] Optimal usage amount of the inserted fragment = [0.04 × base pairs of the inserted fragment] ng (0.06 pmol)

[0052] 3) Ligation of the overexpression vector and the target fragment. The ligation system is as follows:

[0053] <![CDATA[ddH2O]]> Up to 20 μl 5×CE Buffer 4 μl linearized cloning vector 50 - 200 ng insert fragment amplification product 20 - 200 ng Exnase 2 μl

[0054] Ligate at 50 °C for 20 min.

[0055] 5. Transformation

[0056] 1) Place the DH5α competent cells on ice (4 °C). After natural thawing, take 10 μl of the ligation product and add it to the competent cells. Incubate on ice (4 °C) for 30 min.

[0057] 2) Then perform heat shock in a 42 °C water bath for 90 s. Immediately place it on ice (4 °C) for 2 - 3 min.

[0058] 3) Add 500 μL of SOC medium without antibiotics and incubate with shaking at 37 °C, 225 rpm for 45 min.

[0059] 4) Centrifuge at 3000 rpm for 2 min, discard 900 μL of the supernatant, resuspend the bacterial solution at the bottom of the tube, add it to the culture plate containing the corresponding resistance (ampicillin or kanamycin, etc.) on the vector, and spread it evenly with a sterilized spreader (the temperature of the spreader should not be too high to avoid killing the bacteria). Invert and incubate overnight in a 37 °C constant temperature incubator.

[0060] 6. Submission for Sequencing

[0061] Select two colonies from each clone and send them to a sequencing company for sequencing identification.

[0062] 7. High - efficiency Virus Packaging and Concentration

[0063] 1) Serum - free packaging system for suspension cells: Use Expi293F suspension cells (Thermo Fisher Scientific) to replace traditional adherent 293T cells. The seeding density is 2×10^6 cells / mL, and culture in a shaker at 37 °C and 8% CO2 (rotation speed 120 rpm).

[0064] 2) Optimization of transfection reagent: Use linear polyethyleneimine (PEI, Polysciences), mix the recombinant lentiviral vector, pSPAX2 and pMD2G plasmids (total DNA 25 μg) at a ratio of PEI:DNA = 3:1 (w / w), vortex and then let stand at room temperature for 15 min to form a complex.

[0065] 3) Innovation in virus concentration process: Collect the cell culture supernatant 72 h after transfection, centrifuge at 4 °C, 5000×g for 10 min to remove cell debris, and add PEG - it TMAdd virus precipitation reagent (System Biosciences (SBI), catalog number SBI-LV825A-1) to a final concentration of 5%, and precipitate overnight at 4°C. The next day, centrifuge at 4°C and 1500×g for 45 min, discard the supernatant, and resuspend the virus particles with PBS at 1 / 50 of the original volume to obtain a high-titer lentivirus concentrate overexpressing LAPTM5 (titer ≥ 1×10^9 TU / mL).

[0066] 4) After aliquoting, store in an ultra-low temperature freezer at -80°C.

[0067] II. Overexpression of LAPTM5 gene in Huh7 cells by lentivirus transfection

[0068] 1) Transfect the above-mentioned LAPTM5 lentivirus into the Huh7 cell line (serum-free, antibiotic-free, add 2 μg / μL polybrene);

[0069] 2) Replace with complete medium after 8 h;

[0070] 3) Add Puro to a final concentration of 2 μg / μL after 48 h and screen for 5 - 7 days;

[0071] 4) The screened cell line is used for subsequent verification.

[0072] III. Verification by Western blot

[0073] 1) Collect the above cell lines and lyse the cells by incubating with RIPA protein lysis buffer on ice for 15 min;

[0074] 2) Collect the cell lysate, centrifuge at 4°C and 12000 rpm for 15 min, and collect the supernatant;

[0075] 3) Add loading protein sample buffer in proportion and heat at 100°C or in a boiling water bath for 10 min to fully denature the protein;

[0076] 4) Detect the expression of protein LAPTM5 by Western blot. The results are as Figure 2 shown, indicating the successful construction of the Huh7-LAPTM5 overexpressing cell line.

[0077] IV. Functional verification:

[0078] 1) Cleaved-Caspase9 protein is a protein formed after the cleavage of Caspase9 protein. Caspase9 can be activated by self-cleavage and by the apoptosome (a protein complex composed of cytochrome c and apoptotic peptidase activating factor 1). Therefore, in this example, the expression of Cleaved-Caspase9 protein was detected by Western blot to detect the effect of overexpressing LAPTM5 on apoptosis in Huh7, as Figure 2 shown. Under normal culture conditions, overexpressing LAPTM5 in Huh7 had no significant effect on cell apoptosis, as evidenced by no significant difference in Cleaved-Caspase9 protein between the two groups. Therefore, under normal culture conditions, overexpressing LAPTM5 had no significant effect on the apoptosis of the hepatocellular carcinoma cell line Huh7, which can be used as a reliable model for studying autophagy.

[0079] 2) The effect of LAPTM5 on the proliferation ability of Huh7 was detected by CCK8 assay, as Figure 3 shown. Under normal culture conditions, overexpressing LAPTM5 in Huh7 had no significant effect on cell proliferation ability.

[0080] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form or substance. It should be noted that for those of ordinary skill in the art, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a biological material in the preparation of a kit for constructing a hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene, and use in constructing a hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene, characterized in that, The biomaterial is the primer pair shown in SEQ ID NO: 1-2, or the target gene fragment obtained by PCR amplification using the primer pair, or the expression vector containing the target gene fragment.

2. The application according to claim 1, characterized in that, The expression vector is selected from viral, fungal or bacterial expression vectors.

3. A method for constructing a liver cancer cell line with overexpression of the LAPTM5 gene, characterized in that, Including: Transfect the hepatocellular carcinoma cell line Huh7 with the lentivirus carrying the target gene LAPTM5, and screen to obtain the hepatocellular carcinoma cell line with overexpression of LAPTM5, that is, obtain the Huh7-LAPTM5 overexpressing hepatocellular carcinoma cell line.

4. The construction method according to claim 3, characterized in that, The method specifically includes the following steps: Step 1: Design primers for PCR amplification fragments, and introduce homologous sequences at the 5' end of the primers to the ends of the linearized cloning vector, so that the 5' and 3' terminal sequences of the amplification product are exactly the same as the two terminal sequences of the linearized cloning vector; the primer sequences are shown in SEQ ID NO: 1-2; Step 2: Clone the target gene fragment into the linearized cloning vector PGMLV-CMV-MCS-3xFlag-PGK-Puro after enzyme digestion to obtain the LAPTM5 overexpression recombinant lentiviral vector; Step 3: Transform the recombinant lentiviral vector assembled in Step 2 into DH5α competent cells; screen the transformants by colony PCR, send the positive clones for sequencing, and determine the positive clones by sequence alignment; amplify the positive clones and extract the plasmids; Step 4: Combine the plasmid extracted in Step 3 with the viral packaging helper plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system, and co-transfect Expi293F suspension cells using the transfection reagent PEI for virus packaging to obtain the virus solution; Step 5: Infect the hepatocellular carcinoma cell line Huh7 with the virus solution obtained in Step 4, and screen to obtain the hepatocellular carcinoma cell line with overexpression of LAPTM5.

5. The construction method according to claim 4, wherein The method for virus packaging in Step 4 includes: collecting the cell culture supernatant 72 h after co-transfecting Expi293F suspension cells, centrifuging to remove cell debris, adding PEG-it virus precipitation reagent for overnight precipitation; centrifuging the next day, discarding the supernatant, and resuspending the virus particles with PBS to obtain the LAPTM5 overexpressing lentivirus concentrate with a titer ≥ 1×10^9 TU / mL.

6. The hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene constructed by the construction method described in any one of claims 3-5.

7. Use of the hepatocellular carcinoma cell line with overexpression of the LAPTM5 gene described in claim 6 in constructing a research model for the function of the LAPTM5 gene.

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