A biomarker for predicting liver cancer invasion and metastasis and its application

Abstract

The present invention relates to the field of genetic engineering, specifically to a biomarker oxoglutarate dehydrogenase analog (Oxoglutarate Dehydrogenase L; OGDHL) for predicting liver cancer metastasis and prognosis. The present invention also provides the use of a reagent for detecting the expression amount of OGDHL in liver cancer cells in the preparation of a kit for predicting liver cancer invasion and metastasis, and the use of OGDHL in the preparation of a drug for inhibiting liver cancer invasion and metastasis. The present invention provides a biomarker OGDHL for predicting liver cancer metastasis and prognosis, and reveals that increasing the expression level of OGDHL in cancer cells will effectively inhibit the invasion and metastasis of liver cancer. The conclusion of the present invention explains why low expression of OGDHL is widely prevalent in liver cancer cells, and provides a theoretical basis for evaluating the value of OGDHL in clinical samples as a new target for liver cancer diagnosis, prognosis, and treatment.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to the application of OGDHL protein in diagnosing liver cancer invasion and metastasis and developing targeted drugs. Background Art

[0002] Primary hepatocellular carcinoma is a malignant tumor that seriously threatens human health. The main reason for its poor prognosis is its high incidence of recurrence and metastasis. Surgical resection is still the most common and effective intervention method. The strong invasion and metastasis ability of malignant cells leads to a recurrence rate of 50-80% in 5 years after hepatocellular carcinoma surgery, and most recurrences occur within two years. The 5-year survival rate is only 5-6% worldwide.

[0003] The current important means of monitoring the recurrence of hepatocellular carcinoma in clinical practice are monitoring of serum alpha-fetoprotein (AFP), regular abdominal ultrasound and CT, etc. These methods each have certain shortcomings. Due to the low sensitivity and specificity caused by the technical level and unclear diagnosis, some patients with early recurrence of hepatocellular carcinoma lose the best time for surgery. Some preventive treatment measures are also taken clinically to prevent the recurrence and metastasis of hepatocellular carcinoma, but these preventive treatments are not targeted and are prone to overtreatment, increasing the economic burden and pain of patients. The above situation fully demonstrates that the metastasis risk, prognostic risk stratification, and tumor recurrence risk warning of postoperative HCC patients need to be supplemented by more accurate indicators. The use of molecular biological methods to study and identify effective molecular markers is one of the key means to assist existing clinical diagnosis, guide clinical intervention and early warning of cancer.

[0004] Previous studies have found that low expression of oxoglutarate dehydrogenase L (OGDHL) is common in liver tumors, and its expression level is negatively correlated with liver cancer histopathological stage, tumor size and patient survival time (Weiqi Dai, Ling Xu, Xiangnan Yu, Guangcong Zhang, Hongying Guo, Hailin Liu, Guangqi Song, Shuqiang Weng, Ling Dong, Jimin Zhu, Taotao Liu, Chuanyong Guo, Xizhong Shen. OGDHL silencing promotes hepatocellular carcinoma by reprogramming glutamine metabolism. J Hepatol 2020; 72(5): 909-923.).

[0005] However, there are no reports on the correlation between OGHDL and the invasion and metastasis of liver cancer cells. Summary of the invention

[0006] The purpose of the present invention is to provide a biomarker OGDHL for predicting liver cancer metastasis and prognosis, and reveals that increasing the expression level of OGDHL protein in cancer cells will effectively inhibit the invasion and metastasis of liver cancer.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] ①To explore the regulatory mechanism and significance of stable expression of HIF-1α protein in liver cancer cells under normoxic environment;

[0009] ②It is used to explain why low expression of OGDHL is widely prevalent in liver cancer cells;

[0010] ③ To provide a theoretical basis for evaluating the value of OGDHL in clinical samples as a new target for diagnosis, prognosis and treatment of liver cancer.

[0011] The present invention found that the loss of the gene OGDHL can increase the stability of HIF-1α protein in liver cancer cells under normoxic conditions, thereby promoting its epithelial-mesenchymal transition (EMT), further leading to the invasion and metastasis of liver cancer cells. Patients with low expression of OGDHL in primary tumors are more likely to have invasion and metastasis inside and outside the liver. Therefore, the present invention provides a biomarker OGDHL for predicting liver cancer metastasis and prognosis, and reveals that increasing the expression level of OGDHL in cancer cells will effectively inhibit the invasion and metastasis of liver cancer.

[0012] In a first aspect of the present invention, a biomarker for predicting liver cancer metastasis and prognosis is provided, wherein the biomarker is OGDHL.

[0013] The second aspect of the present invention provides the use of a reagent for detecting the expression level of OGDHL in liver cancer cells in the preparation of a kit for predicting the invasion and metastasis of liver cancer.

[0014] Furthermore, in the above application, patients with low OGDHL expression are more likely to have intrahepatic and extrahepatic invasion and metastasis.

[0015] The third aspect of the present invention provides the use of a reagent for detecting the expression level of OGDHL in preparing a kit for predicting the prognosis of liver cancer.

[0016] The fourth aspect of the present invention provides the use of OGDHL in the preparation of a drug for inhibiting the invasion and metastasis of liver cancer.

[0017] Furthermore, OGDHL and HIF-1a expression showed a significant negative correlation; at the mRNA level, OGDHL was positively correlated with CDH1, an inhibitory epithelial marker for liver cancer metastasis; HIF-1a was negatively correlated with CDH1, an inhibitory epithelial marker for liver cancer metastasis, and positively correlated with Vimentin, a mesothelial marker that promotes liver cancer metastasis. Therefore, increasing the expression level of OGDHL in cancer cells can inhibit the expression of HIF-1a and effectively inhibit the invasion and metastasis of liver cancer.

[0018] A fifth aspect of the present invention provides use of a reagent for increasing the expression level of OGDHL in liver cancer cells in the preparation of a drug for inhibiting the invasion and metastasis of liver cancer.

[0019] Furthermore, the reagent for increasing the expression level of OGDHL in liver cancer cells is an OGDHL lentiviral overexpression vector.

[0020] In a sixth aspect of the present invention, there is provided the use of OGDHL as a target in screening drugs for inhibiting the invasion and metastasis of liver cancer, wherein the drug is a drug capable of increasing the expression level of OGDHL in liver cancer cells.

[0021] The advantages of the present invention are:

[0022] 1. The present invention explores the regulatory mechanism and significance of the stable expression of HIF-1α protein in liver cancer cells under normoxic environment, and finds that the loss of gene OGDHL can increase the stability of HIF-1α protein in liver cancer cells under normoxic environment, thereby promoting its epithelial-mesenchymal transition (EMT), further leading to the invasion and metastasis of liver cancer cells. Patients with low expression of OGDHL in primary tumors are more likely to have invasion and metastasis inside and outside the liver.

[0023] 2. The present invention provides a biomarker OGDHL for predicting liver cancer metastasis and prognosis, and reveals that increasing the expression level of OGDHL in cancer cells will effectively inhibit the invasion and metastasis of liver cancer.

[0024] 3. The conclusion of the present invention explains why low expression of OGDHL is widely prevalent in liver cancer cells, and provides a theoretical basis for evaluating the value of OGDHL in clinical samples as a new target for diagnosis, prognosis and treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1.OGDHL has the effect of inhibiting the invasion and metastasis of liver cancer cells. Figure A shows the expression of OGDHL at the protein level in various liver cancer cell lines; Figure B shows the overexpression / interference efficiency of the lentiviral vector after the OGDHL overexpression / RNA interference lentiviral vector was infected with HCC-LM3 or Bel-7402 respectively; Figures CF show the regulatory effect of OGDHL on HCC-LM3 cell metastasis (cell scratch assay; Figures C and D) and invasion (Transwell chamber assay; Figures E and F); Figures G and H show the regulatory effect of OGDHL on Bel-7402 cell metastasis (cell scratch assay; Figure G) and invasion (Transwell chamber assay; Figure H); ( ** P < 0.01, vs negative control group; n=3). The results showed that OGDHL could inhibit the invasion and metastasis of liver cancer cells.

[0026] Figure 2 .The regulatory mechanism of OGDHL on the stable expression of HIF-1α protein. Figures A and B show the expression of HIF-1α protein by OGDHL in HCC-LM3 and Bel-7402 cells ( * P<0.05, ** P < 0.01, vs negative control group; n=3). The results showed that under normoxic conditions, low-expressed OGDHL in liver cancer cells could enhance HIF-1α protein expression.

[0027] Figure 3 . Low expression of OGDHL promotes EMT of liver cancer cells by enhancing the stability of HIF-1α protein expression. Figures A and B show the regulatory effects of OGDHL on the epithelial marker E-cadherin and mesothelial marker Vimentin proteins during epithelial-mesenchymal transition in four types of liver cancer cells (HCC-LM3, Bel-7402, SMMC-7721, and MHCC-97H); Figure C shows the protein expression levels of epithelial marker E-cadherin and mesothelial marker Vimentin after HCC-LM3 cells were co-infected with HIF-1α and OGDHL lentiviral overexpression vectors; Figures D and E show the effects of OGDHL on the cell morphology of Bel-7402 (Figure D) and HCC-LM3 (Figure E) ( n=3). The results showed that low expression of OGDHL promoted the EMT process of liver cancer cells, and this process depended on the regulation of OGDHL on HIF-1α protein.

[0028] Figure 4.The expression and correlation of OGDHL with HIF-1α and EMT-related markers were studied in human tissue specimens. Figures A and B show the protein expression of OGDHL in primary tumors and metastatic liver cancer in 24 human liver cancer specimens; Figures C and D show the correlation between OGDHL and HIF-1a protein expression in the above tumor specimens; Figures E, F and G show the expression information of each gene in liver cancer samples extracted from the GEO database (GSE14520, 225 human liver cancer tissues) DETAILED DESCRIPTION

[0029] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0030] Embodiment 1:

[0031] 1. Experimental Methods

[0032] 1. Explore the regulatory mechanism of OGDHL on the stable expression of HIF-1α protein

[0033] (1) Lentivirus construction: shOGDHL-A / B and Lenti-OGDHL lentivirus overexpression vectors were purchased from commercial companies, and the OGDHL high-expressing human liver cancer cell line Bel-7402 and the OGDHL low-expressing human liver cancer cell line HCC-LM3 were cultured in vitro. Bel-7402 (sh-OGDHL-A / B) and HCC-LM3 (Lenti-OGDHL) cell lines were established, and the regulatory effect of OGDHL on the expression of HIF-1α, E-cadherin and Vimentin proteins was subsequently analyzed by western blotting.

[0034] (2) Western blotting experiment:

[0035] ① Gel filling and sample loading after cell protein extraction:

[0036] After aligning the glass plate, put it into the clamp and clamp it; then clamp it vertically on the rack and prepare for glue filling;

[0037] Prepare the separation gel of appropriate concentration according to the previous method, add TEMED and shake well immediately before pouring the gel;

[0038] When pouring glue, you can use a 10mL gun to suck 5mL of glue and release it along the glass until the glue surface rises to the height of the middle line of the green belt; then add a layer of water on the glue, and the glue will solidify faster after liquid sealing; when there is a refraction line between the water and the glue, it means that the glue has solidified; wait for another 3 minutes for the glue to fully solidify, then pour out the water on the glue and absorb the water with absorbent paper;

[0039] Prepare 4% concentrated gel according to the previous method, add TEMED and shake well immediately before pouring the gel;

[0040] Fill the remaining space with concentrated glue and then insert the comb into the concentrated glue;

[0041] When pouring glue, the glue should also flow down along the glass plate to avoid bubbles in the glue;

[0042] Keep the comb level when inserting it;

[0043] Since the volume of the gel will shrink when it solidifies, thus reducing the loading volume of the sample well, it is necessary to frequently add gel on both sides during the solidification process of the concentrated gel;

[0044] After the concentrated gel solidifies, pinch both sides of the comb with both hands and gently pull it out vertically upwards;

[0045] Rinse the stacking gel with water and place it in the electrophoresis tank;

[0046] Based on the protein quantification results, calculate the volume of the solution containing 50 mg of protein as the loading amount;

[0047] The sample was taken out to a 0.5 mL centrifuge tube and 5× SDS loading buffer was added to a final concentration of 1×.

[0048] Before loading, the sample should be boiled in boiling water for 5 minutes to denature the protein;

[0049] Load the sample, and run the electrophoresis for 1 to 3 hours, preferably at a voltage of 80V to 120V;

[0050] The electrophoresis can be terminated when the bromophenol blue just runs out, and the membrane can be transferred.

[0051] ②Transfer:

[0052] The cut PVDF membrane was placed in methanol for 30 min for later use;

[0053] Place the transfer clips, two sponge pads, a glass rod, filter paper and the soaked membrane in the enamel dish with transfer solution;

[0054] Open the clamp so that the black side is level;

[0055] Place a sponge pad on top, pry off the glass plate before peeling off the glue, and after removing the small glass plate, gently scrape off the concentrated glue;

[0056] Carefully peel off the separation gel and cover it on the filter paper. Use your hands to adjust it to align with the filter paper and gently use a glass rod to remove bubbles.

[0057] Place the film on the glue to cover the entire glue (the film cannot be moved after it is covered) and remove bubbles;

[0058] Cover the membrane with 3 pieces of filter paper and remove air bubbles;

[0059] Finally, cover with another sponge pad and roll it a few times to close the clip;

[0060] The entire operation is carried out in transfer liquid;

[0061] Put the clip into the transfer slot, with the black side of the clip facing the black side of the slot, and the white side of the clip facing the red side of the slot;

[0062] Heat is generated during the electrical transfer, so place a piece of ice on one side of the tank to cool it down;

[0063] 20V transfer 30min;

[0064] After transfer, the membrane was stained with 1× Ponceau red dye for 5 min (shaking on a decolorizing shaker);

[0065] Then wash away the unstained dye with water to see the protein on the membrane;

[0066] Allow the membrane to dry for later use.

[0067] ③Immune response:

[0068] After soaking the membrane with TBS from bottom to top, move it to a plate containing blocking solution and shake it on a decolorizing shaker at room temperature for 1 hour;

[0069] Dilute the primary antibody to an appropriate concentration with TBST (in a 1.5 mL centrifuge tube);

[0070] Tear off a piece of plastic wrap of appropriate size and spread it on the laboratory table. Soak the four corners with water to keep the plastic wrap flat.

[0071] Add the antibody solution to the plastic wrap;

[0072] Take out the membrane from the blocking solution, absorb the residual liquid with filter paper, and place the membrane protein side down on the antibody liquid surface, and lift the four corners of the membrane to drive out the residual bubbles;

[0073] After incubation at room temperature for 1 to 2 h, wash twice with TBST on a decolorizing shaker at room temperature for 10 min each time;

[0074] Wash once more with TBS for 10 min;

[0075] Prepare the secondary antibody dilution solution as above and contact the membrane. After incubation at room temperature for 1 to 2 hours, wash with TBST.

[0076] Wash twice on a decolorizing shaker at room temperature for 10 min each time;

[0077] Wash once more with TBS for 10 min;

[0078] The membrane was scanned and the net optical density of the target band was analyzed using a correlation image processing system.

[0079] (3) Cell scratch assay to detect the effect of OGDHL on the migration of liver cancer cells

[0080] ①First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, using a ruler to measure, approximately every 0.5 to 1 cm, across the holes. At least 5 lines should be drawn across each hole;

[0081] ② Add about 5×10 6 The specific number of cells varies with the cell type, but it is important to ensure that the cells can be fully confluent overnight;

[0082] ③ On the second day, use the tip of the gun to measure the ruler and try to make it vertical to the horizontal scratch mark on the back. The tip of the gun should be vertical and not tilted.

[0083] ④ Wash the cells three times with PBS, remove the scratched cells, and add serum-free culture medium to the control group and OGDHL intervention group;

[0084] ⑤ Place in a 37°C 5% CO2 incubator for incubation. Take samples at 0 and 24 hours and take photos.

[0085] (4) In vitro Transwell chamber migration assay to detect the effect of OGDHL on the migration of hepatoma cell lines

[0086] ① Hydrate the basement membrane: add 70ul serum-free culture medium to each well and incubate at 37℃ for 30min, then remove the culture medium;

[0087] ② In the logarithmic growth phase, trypsinize the cells, suspend them in medium containing 0.2% serum, count them, and dilute them to a density of 1×10 5 / ml, add 200 μl of cell suspension to each well;

[0088] ③ Add 600 μl of culture medium containing 30% newborn calf serum to the lower chamber of the 24-well plate;

[0089] ④ After 24 hours, discard the culture medium in the wells, wash twice with PBS, fix with methanol for 20 minutes, stain with 0.1% crystal violet for 15-20 minutes, wash with water for more than 3 times, and air dry naturally;

[0090] ⑤ Observe the cells in five random fields under a 100x microscope and count them;

[0091] ⑥Elute with 33% acetic acid for 10 minutes and measure the OD value at 570nm on a microplate reader in a small chamber.

[0092] (5) In vitro Transwell chamber invasion assay to detect the effect of OGDHL on the invasion and metastasis of liver cancer cell lines

[0093] ① The day before the experiment, place the Matrigel stock solution (BD) in a 4°C refrigerator to melt overnight;

[0094] ② Precool the pipette tip on ice for half an hour, dilute Matrigel with precooled serum-free medium at a ratio of 1:7, add 70 μl of the diluted Matrigel to each well, place in a 37°C incubator, and incubate for 5 hours;

[0095] ③ Hydrate the basement membrane: aspirate the residual liquid in the chamber, add 70ul serum-free culture medium to each well and incubate at 37℃ for 30min, then aspirate the culture medium;

[0096] ④ For cells in the logarithmic growth phase, trypsinize the cells, suspend them in medium containing 0.2% serum, count them, dilute them to a density of 1×105 / ml, and add 200 μl of cell suspension to each well;

[0097] ⑤ Add 600 μl of culture medium containing 30% newborn calf serum to the lower chamber of the 24-well plate;

[0098] ⑥ After 36 hours, discard the culture medium in the wells, wash twice with PBS, fix with methanol for 20 minutes, stain with 0.1% crystal violet for 15-20 minutes, wash with water for more than 3 times, gently wipe off the upper layer of non-migrated cells with a cotton swab, and air dry;

[0099] ⑦ Observe the cells in five fields of view at random under a 100x microscope and count them;

[0100] ⑧Elute with 33% acetic acid for 10 minutes and measure the OD value at 570nm on a microplate reader.

[0101] 2. Tissue specimen processing, tissue sectioning, tissue RNA and protein extraction

[0102] For patients with primary liver cancer and intrahepatic and extrahepatic metastasis who have surgical indications (24 cases), cancer tissue and adjacent tissue were obtained after surgery. One part was quickly stored in liquid nitrogen, and total RNA and total protein were extracted. After quantification, they were stored at -20°C for Real time RT-PCR and Western Blotting detection, respectively. The other part was fixed with 4% paraformaldehyde, and then ultramicro paraffin tissue sections were made for pathological tissue hematoxylin-eosin staining (HE staining) and immunohistochemical staining analysis. The above two parts of the experiment were used to detect the expression levels of OGDHL, EMT epithelial marker (E-cadherin) and EMT mesothelial marker (Vimentin) and HIF-1α molecules, and to analyze the expression and correlation of OGDHL and HIF-1α and EMT-related markers between tissues.

[0103] 2. Experimental Results

[0104] 1. OGDHL has the effect of inhibiting the invasion and metastasis of liver cancer cells

[0105] ① In vitro, different liver cancer cell lines (HCC-LM3 and Bel-7402 liver cancer cells) were cultured and the results of protein immunoblotting, cell scratch and invasion and metastasis Transwell chamber experiments showed that OGDHL has the effect of inhibiting the invasion and metastasis of liver cancer cells ( Figure 1 ).

[0106] ②Through protein immunoblotting and cell morphology observation under optical microscope, it was found that under normoxic environment, low-expression OGDHL in liver cancer cells can enhance HIF-1α protein expression ( Figure 2 ), low expression of OGDHL promoted the EMT process of liver cancer cells, and this process depended on the regulation of OGDHL on HIF-1α protein ( Figure 3 ).

[0107] The above experimental results show that the OGDHL silencing effect can enhance the stability of HIF-1α protein expression, thereby affecting the invasion and metastasis ability of liver cancer cells by regulating the EMT of liver cancer cells.

[0108] 2. Expression and correlation of OGDHL with HIF-1α and EMT-related markers

[0109] ① We collected 24 human liver cancer surgical resection specimens (12 of which had metastasis) from our hospital. We used the standard paraffin embedding method to prepare ultramicrotissue sections, and used immunohistochemical staining techniques to find that OGDHL protein was generally low-expressed in metastatic liver cancer samples compared with primary liver cancer and normal liver tissue samples ( Figure 4 .A and B).

[0110] ② The above 24 human liver cancer surgical resection specimens were collected in our hospital. Ultramicrotissue sections were prepared by paraffin embedding using standard methods. Immunohistochemical staining techniques were used to find that OGDHL and HIF-1a expression in the above 24 pairs of human liver cancer tissues showed a significant negative correlation in metastatic liver cancer samples ( Figure 4 .C and D).

[0111] ③ The expression information of each gene in liver cancer samples was extracted from the GEO database (GSE14520, 225 human liver cancer tissues). At the mRNA level, it was found that OGDHL was positively correlated with CDH1, an epithelial marker that inhibits liver cancer metastasis. In addition, HIF-1a was negatively correlated with CDH1, an epithelial marker that inhibits liver cancer metastasis, and positively correlated with Vimentin, a mesothelial marker that promotes liver cancer metastasis ( Figure 4 .E, F and G).

[0112] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. The use of a reagent for detecting the expression of OGDHL in liver cancer cells in the preparation of a kit for predicting liver cancer invasion and metastasis; patients with low OGDHL expression are more likely to have intrahepatic and extrahepatic invasion and metastasis.

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