HSPA1A gene, molecular marker and application

Abstract

The invention relates to the technical field of biological medicine, in particular to an HSPA1A gene, a molecular marker and application of the HSPA1A gene, the molecular marker and the application of the HSPA1A gene. The RAP1 cancer promoting pathway, the AMPK cancer promoting pathway, the Hippo cancer promoting pathway, the mTOR cancer promoting pathway and the Hedgehog cancer promoting pathway can be up-regulated by the expression of the RAP1 cancer promoting pathway, the AMPK cancer promoting pathway, the Hippo cancer promoting pathway and the mTOR cancer promoting pathway; and down-regulating a p53 cancer suppression pathway and apoptosis-related gene expression. The invention finds that the HSPA1A gene can simultaneously up-regulate a plurality of cancer promoting pathways related to drug-resistant transfer, such as RAP1, Hippo, Hedgehog, mTOR and the like. Cellular and animal level experiments prove that the nucleic acid and chemical drugs targeting the HSPA1A as a main switch have significant improvement and inhibition effects on drug resistance and metastasis of lung cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an HSPA1A gene and a molecular marker and an application thereof. Background Art

[0002] Lung cancer is divided into small cell lung cancer and non-small cell lung cancer, of which non-small cell lung cancer accounts for 85%. Cisplatin is the first-line drug in the clinical chemotherapy regimen for non-small cell lung cancer. The treatment cost is low, but most patients develop primary or secondary drug resistance. Previous studies have revealed the role of various molecular signaling pathways such as Hippo and Hedgehog in lung cancer resistance and metastasis. However, targeting one of the above pathways is still not enough to completely overcome the drug resistance and metastasis of cancer cells. Cancer cells can develop primary or stimulated resistance to treatment through bypass pathways, leading to tumor progression. Therefore, finding the upstream master switch that regulates the above signaling pathways and simultaneously targeting and inhibiting multiple downstream cancer-promoting pathways by turning off the master switch is of great value to the treatment of lung cancer. Summary of the invention

[0003] Based on this, the present invention proposes an HSPA1A gene and an inhibitor comprising the HSPA1A gene and an application thereof, which can be used as a diagnostic target for lung cancer and has good practical application value.

[0004] According to a first aspect of the present invention, there is provided an HSPA1A gene, comprising: a gene sequence in SEQ ID No. 1;

[0005] Used to upregulate the expression of RAP1 cancer-promoting pathway, AMPK cancer-promoting pathway, Hippo cancer-promoting pathway, mTOR cancer-promoting pathway and Hedgehog cancer-promoting pathway;

[0006] As well as downregulating the expression of p53 tumor suppressor pathway and apoptosis-related genes.

[0007] According to an embodiment of the present invention, the RAP1 cancer-promoting pathway includes the gene sequence in SEQ ID No. 2;

[0008] The AMPK cancer-promoting pathway includes the gene sequence in SEQ ID No. 3;

[0009] The Hippo cancer-promoting pathway includes the gene sequence in SEQ ID No. 4;

[0010] The mTOR cancer-promoting pathway includes the gene sequence in SEQ ID No.5

[0011] The Hedgehog cancer-promoting pathway includes PTCH1 key factors and SHH key factors;

[0012] The PTCH1 key factor includes the gene sequence in SEQ ID No.6;

[0013] The SHH key factor includes the gene sequence in SEQ ID No.7

[0014] According to an embodiment of the present invention, the p53 tumor suppressor pathway includes the gene sequence in SEQ ID No.8.

[0015] According to an embodiment of the present invention, the apoptosis-related genes include:

[0016] The CTSD gene is the gene sequence in SEQ ID No. 9;

[0017] The CASP7 gene is the gene sequence in SEQ ID No. 10;

[0018] The FAS gene is a gene sequence including SEQ ID No.11.

[0019] According to a second aspect of the present invention, there is provided a molecular marker formed by the HSPA1A gene, comprising: HSPA1A-F: 5'-CTGCGACAGTCCACTACCTT-3';

[0020] HSPA1A-R:5'-CGGCTCCGCTCTGAGATT-3'.

[0021] According to a third aspect of the present invention, there is provided a use of the above molecular marker in the diagnosis of lung cancer, wherein the above HSPA1A-F and / or HSPA1A-R are used to detect the expression level of HSPA1A in lung cancer lesions.

[0022] According to a fourth aspect of the present invention, there is provided a use of the above-mentioned HSPA1A gene as a target for lung cancer treatment.

[0023] It can be seen from the above technical solutions that the HSPA1A gene and molecular markers and applications provided by the present invention have the following beneficial effects:

[0024] The present invention found that the HSPA1A gene can simultaneously upregulate multiple cancer-promoting pathways related to drug resistance and metastasis, such as RAP1, Hippo, Hedgehog, and mTOR. Experiments at the cell and animal levels have confirmed that nucleic acids and chemical drugs targeting the master switch HSPA1A have a significant improvement and inhibitory effect on drug resistance and metastasis of lung cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a result diagram of detecting the expression level of HSPA1A protein in cancer patients according to an embodiment of the present invention; wherein, Figure 1 (A) is an immunohistochemical staining image of a cancer patient according to an embodiment of the present invention; Figure 1(B) is a statistical result diagram of the expression level of HSPA1A in cancer patients according to an embodiment of the present invention; Figure 1 (C) is a graph showing the relationship between the expression level of HSPA1A and the patient's prognosis in an embodiment of the present invention; Figure 1 (D) is a graph showing changes in the transcription level of HSPA1A in A549 drug-resistant cells detected by RT-qPCR in an embodiment of the present invention; Figure 1 (E) is a graph showing changes in protein levels in A549 drug-resistant cells detected by western blot method according to an embodiment of the present invention;

[0026] Figure 2 This is a diagram showing the expression effect of the HSPA1A gene in H520 cells in Example 1 of the present invention; wherein, Figure 2 (A) is a western blot experiment to confirm the protein level expression of HSPA1A in H520 cells transfected with knockdown or overexpression vectors of HSPA1A in the present invention; Figure 2 (B) RT-qPCR experiment to detect the expression level of the tag molecule in the knockdown vector (HSPA1A#6#7#8) of the embodiment of the present invention; Figure 2 (C) is the RT-qPCR experiment of the present invention to detect the expression level of the tag molecule in the knockdown vector (HSPA1A OE);

[0027] Figure 3 The results of the cell scratch test of A549 and H520 cells in the present invention are shown in the figure below. Figure 3 (A) is the result of the A549 cell scratch test in the present invention; Figure 3 (B) is the result of the H520 cell scratch test in the present invention;

[0028] Figure 4 The effects of overexpression of IGFL2-AS1 on cell invasion ability, stemness and drug sensitivity in the embodiments of the present invention are shown; wherein, Figure 4 (A) is the experimental results of cell invasion ability of A549 cells and H520 cells in the present invention; Figure 4 (B) is the experimental results of stemness of A549 cells and H520 cells in the present invention; Figure 4 (C) is the result of the drug sensitivity experiment of A549 cells and H520 cells in the present invention;

[0029] Figure 5 This is the effect of knocking down IGFL2-AS1 on cell invasion ability, stemness and drug sensitivity in the embodiment of the present invention; wherein, Figure 5 (A) is the experimental results of cell invasion ability of A549 cells and H520 cells in the present invention; Figure 5 (B) is the experimental results of stemness of A549 cells and H520 cells in the present invention; Figure 5 (C) is the result of the drug sensitivity experiment of A549 cells and H520 cells in the present invention;

[0030] Figure 6 RNA-seq experimental analysis was performed on A549 cells knocked down by the vector in the embodiment of the present invention, and the cancer-promoting signaling pathway diagram regulated by HSPA1A was screened; wherein, Figure 6 (A) is the KEGG analysis result of A549 cells knocked down by the vector of the present invention; Figure 6 (B) is the GSEA analysis result of A549 cells knocked down by the vector in the embodiment of the present invention;

[0031] Figure 7 RNA-seq experimental analysis was performed on A549 cells knocked down by the vector in the embodiment of the present invention, and the tumor suppressor signaling pathway diagram regulated by HSPA1A was screened; wherein, Figure 7 (A) is the KEGG analysis result of A549 cells knocked down by the vector of the present invention; Figure 7 (B) is the gene enrichment analysis result of A549 cells knocked down by the vector of the present invention; Figure 7 (C) is the GSEA analysis result of A549 cells knocked down by the vector in the embodiment of the present invention;

[0032] Figure 8 The effects of HSPA1A inhibitor VER155008 alone and in combination with chemotherapy drugs on the metastasis of lung cancer cells in vivo in the lung cancer metastasis model of the present invention; wherein, Figure 8 (A) is a subcutaneous tumor model established by tail vein injection of A549 cells in the present invention; Figure 8 (B) is the in vivo imaging result of the metastasis of lung cancer cells in vivo according to an embodiment of the present invention; Figure 8 (C) is the HE staining result of the metastasis of lung cancer cells in vivo according to the embodiment of the present invention;

[0033] Fig. 9 The effects of HSPA1A inhibitor VER155008 alone and in combination with chemotherapy drugs on the proliferation of lung cancer cells in vivo in a subcutaneous tumor-bearing model in the present invention; wherein, Fig. 9 (A) is the in vivo imaging result of the proliferation of lung cancer cells in vivo according to an embodiment of the present invention; Fig. 9 (B) is the tumor growth curve result of the proliferation of lung cancer cells in vivo according to the embodiment of the present invention; Fig. 9 (C) is a comparison chart of lung cancer tumor sizes in an embodiment of the present invention; Fig. 9 (D) is a diagram showing the effect of the present invention on inhibiting the proliferation of lung cancer cells in vivo;

[0034] Fig.10The effects of HSPA1A inhibitor VER155008 alone and in combination with chemotherapy drugs on Ki67 nuclear protein in the subcutaneous tumor model of the present invention; wherein, Fig.10 (A) is the expression change of Ki67 nuclear protein in subcutaneous tumor tissue detected by immunohistochemical experiment in the embodiment of the present invention; Fig.10 (B) is an immunohistochemical experiment to detect the changes in HSPA1A expression in subcutaneous tumor tissues in an example of the present invention. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0036] According to a first aspect of the present invention, there is provided an HSPA1A gene, comprising: a gene sequence in SEQ ID No. 1;

[0037] Used to upregulate the expression of RAP1 cancer-promoting pathway, AMPK cancer-promoting pathway, Hippo cancer-promoting pathway, mTOR cancer-promoting pathway and Hedgehog cancer-promoting pathway;

[0038] As well as downregulating the expression of p53 tumor suppressor pathway and apoptosis-related genes.

[0039] According to an embodiment of the present invention, in the sequencing results of A549 and H520 cells overexpressing lincRNA IGFL2AS1 associated with lung cancer proliferation, migration, stemness and drug resistance, it was found that the HSPA1A gene was significantly upregulated.

[0040] According to an embodiment of the present invention, the RAP1 cancer-promoting pathway includes the gene sequence in SEQ ID No. 2;

[0041] The AMPK cancer-promoting pathway includes the gene sequence in SEQ ID No. 3;

[0042] The Hippo cancer-promoting pathway includes the gene sequence in SEQ ID No. 4;

[0043] The mTOR cancer-promoting pathway includes the gene sequence in SEQ ID No.5

[0044] The Hedgehog cancer-promoting pathway includes the key factors PTCH1 and SHH;

[0045] The PTCH1 key factor includes the gene sequence in SEQ ID No.6;

[0046] The SHH key factor includes the gene sequence in SEQ ID No.7

[0047] According to an embodiment of the present invention, the p53 tumor suppressor pathway includes the gene sequence in SEQ ID No.8.

[0048] According to an embodiment of the present invention, the apoptosis-related genes include: CTSD gene, CASP7 gene and FAS gene.

[0049] According to an embodiment of the present invention, the CTSD gene is the gene sequence in SEQ ID No.9.

[0050] According to an embodiment of the present invention, the CASP7 gene is the gene sequence in SEQ ID No.10.

[0051] According to an embodiment of the present invention, the FAS gene is a gene sequence including SEQ ID No.11.

[0052] According to a second aspect of the present invention, there is provided a molecular marker formed by the HSPA1A gene described above, for detecting the expression level of HSPA1A in lung cancer, the molecular marker comprising:

[0053] HSPA1A-F:5'-CTGCGACAGTCCACTACCTT-3';

[0054] HSPA1A-R:5'-CGGCTCCGCTCTGAGATT-3'.

[0055] According to a third aspect of the present invention, there is provided a use of the above molecular marker in the diagnosis of lung cancer, wherein the above HSPA1A-F and / or HSPA1A-R are used to detect the expression level of HSPA1A in lung cancer lesions.

[0056] According to a fourth aspect of the present invention, there is provided a use of the above-mentioned HSPA1A gene as a target for lung cancer treatment. HSPA1A inhibitors can be used to inhibit HSPA1A gene expression to treat lung cancer, and the inhibitors include: VER155008 (C 25 H 23 CL2N7O4, molecular weight: 556.40). Or the inhibitor of HSPA1A gene is used in combination with the chemotherapy drug DDP and the chemotherapy drug 5-FU. The administration method is intraperitoneal injection. VER155008 can inhibit the expression of HSPA1A in tumor cells, thereby downregulating the cancer-promoting pathway and upregulating the cancer-suppressing pathway, and ultimately achieve the effect of treating cancer.

[0057] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0058] Example 1

[0059] Virus transfection: A549 and H520 cells in the logarithmic growth phase were plated into 6-well plates at an appropriate density. When the cell density reached 60%-70%, the virus transfection reagent and the corresponding CTRL, IGFL2-AS1 OE and SHV, IGFL2-AS1#2 lentivirus and CTRL, HSPA1A OE and SHV, HSPA1A#6 / #7 / #8 plasmids were added at a ratio of 1:1000. After 48 hours of transfection, puromycin (1 mg / mL) was added to select stable cell lines, and A549 cells and H520 cells containing HSPA1A overexpression vector (HSPA1A OE), knockdown vector (HSPA1A#6#7#8) and corresponding control vector (CTRL) were collected as samples for subsequent experiments.

[0060] Experimental example

[0061] Experimental methods

[0062] The properties of A549 and H520 cells prepared in Example 1 after overexpression of IGFL2-AS1 and knockdown of HSPA1A were verified by the following method. The test method is as follows:

[0063] (1) Cell transfection

[0064] Virus transfection: A549 and H520 cells in the logarithmic growth phase were plated into 6-well plates at an appropriate density. When the cell density reached 60% to 70%, the transfection reagent and the corresponding CTRL, IGFL2-AS1 OE and SHV, IGFL2-AS1#2 lentivirus and CTRL, HSPA1A OE and SHV, HSPA1A#6 / #7 / #8 plasmids were added at a ratio of 1:1000 of virus transfection reagent: complete medium. After 48 hours of transfection, puromycin (1 mg / mL) was added to select stable transfected cell lines, and the cells were collected for subsequent experiments.

[0065] (2) Cell scratch

[0066] Take cells in the logarithmic growth phase and calculate 2×10 5 Each well was inoculated with cells / well in a 12-well plate, with 3 replicate wells in each group. After 24 hours, a "X" scratch was made in the center of each well with a 200 μL sterile pipette tip, the culture medium was discarded, and the culture medium was replaced with serum-free culture medium after washing with PBS. The scratch distance L0 at 0 hour after scratching was photographed under a microscope at 0 hour. After 48 hours of culture, the scratch distance L1 at the same position was photographed, and the migration rate was calculated as (L1-L0) / L0×100%.

[0067] (3) Transwell

[0068] The Transwell chamber was placed in a 24-well plate, 800 μL of culture medium containing 20% ​​fetal bovine serum was added to the lower chamber of the 24-well plate, and 200 μL (containing 8000 cells) of serum-free culture medium was added to the upper chamber. After incubation for 48 hours, the cells were washed with PBS, fixed with formaldehyde, and stained with crystal violet for 2 minutes. The floating crystal violet was washed off with PBS, and the cells were dried and photographed under an inverted fluorescence microscope to count the number of cells that passed through the membrane.

[0069] (4) Stem cell sphere formation

[0070] Cells in the logarithmic growth phase were taken, single-cell suspension was prepared with serum-free medium, cells were counted with a cell counter, 4000 cells / well were inoculated into low-adhesion 12-well plates, 3 replicate wells were set up for each group, and spheres were induced in serum-free medium containing 20ng / mL EGF, 20ng / mL FGF and 2% B27 for 2 weeks. Pictures were taken with an inverted microscope, and the number of spheres in each group was counted.

[0071] (5) IC50 assay

[0072] After counting the cells in each group in the logarithmic growth phase, 4000 cells / well were inoculated in a 96-well plate, and 3 replicates were set for each group. After culturing in the incubator for 24 hours, the cells were divided into DDP group, 5-FU group, gefitinib group and osimertinib group. Each group was added with drugs at corresponding concentrations according to the drug concentration gradient, DDP: 0, 0.5, 1, 2, 4, 8 μg / mL; 5-FU: 0, 2, 4, 8, 16, 32 μg / mL. After 48 hours of drug action, 5 μL of CCK-8 reagent was added to each well and incubated in the incubator for 2 hours. The cells without drug addition were used as the blank group, and the absorbance value at a wavelength of 450 nm was measured by an enzyme marker. The formula for calculating the proliferation inhibition rate = [(control group OD-experimental group OD) / control group OD] × 100% The half-maximal inhibitory concentration (IC50) of the drug was calculated using Graphpad Prism8.0 software and the drug dose-effect curve was drawn.

[0073] (6) RT-qPCR Total RNA was extracted using the Trizol method, and cDNA was synthesized using a reverse transcription kit for q-PCR detection. The concentration and purity of cDNA were detected by a spectrophotometer. GAPDH was used as the internal reference gene, and RT-qPCR was performed on the relevant genes in the study. The reaction system and conditions were as follows: 2×SYBER Super mix plus 10μL, template cDNA 100μg, upstream and downstream primers 1μL each, and ddH2O was added to 20μL. The three-step RT-qPCR reaction procedure was: 95℃4min, 95℃15s, 54℃20s, 72℃20s, 40 cycles. The data obtained were used for 2 -△△CT The primers used for statistical analysis are listed in Table 1.

[0074] (7) Western blotting

[0075] The cell pellet was collected by centrifugation, and the protein lysis buffer was added to lyse on ice for 30 minutes. The supernatant was centrifuged and the total protein concentration was determined using a BCA protein detection kit (China Sangon Biotechnology Co., Ltd.). 5× loading buffer was added and boiled at 95℃ for 10 minutes. 25μg of protein was loaded. After SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane, and then blocked with 5% milk at room temperature for 1 hour. The ABCG5 antibody (1:1000) was incubated at 4℃ overnight, and then the corresponding horseradish peroxidase (HRP)-labeled secondary antibody (1:2000) was added and incubated at room temperature for 1 hour before development. Finally, the gel imager was used to take pictures, with α-Tublin as the internal reference.

[0076] (8) RNA-seq

[0077] The A549 cells with HSPA1A knockdown and normal control were collected and sent to Novogene Biotech Co., Ltd. for subsequent mRNA sequencing, and the NovoCloud analysis cloud platform was used for data analysis. (9) Immunohistochemical staining

[0078] Embed tissue in paraffin, slice, and place slides in xylene-xylene-100% alcohol-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol for dewaxing, and place in each reagent for 10 minutes. After dewaxing, add 3% H2O2 and incubate for 10 minutes, wash in clean water, add citric acid buffer, cook in a microwave oven for 15 minutes (medium heat), and cool to room temperature. Pour out the citric acid buffer, wash twice with water, add serum and incubate at 37℃ for half an hour for blocking. Add primary antibody at 4℃ overnight, incubate secondary antibody at 37℃ for half an hour, wash with PBS, and then develop color with BCA. Counterstain with hematoxylin, dehydrate, and seal. When reading the slides, randomly select 10 fields of view for each group and analyze using Image J software.

[0079] (9) Animal experiments

[0080] 5-8 week old BALB / c-nu female mice were selected and 5×106 luciferase-labeled A549 cells were injected subcutaneously in the right axilla (to construct a subcutaneous tumor-bearing model) or injected into the tail vein (to construct a lung metastasis model).

[0081] Subcutaneous tumor model: After the tumor grows to a diameter of about 3-5mm, the mice are divided into Ctrl group (100μl saline), DDP or 5-Fu group (2mg / kg), VER-155008 group (HSPA1A inhibitor, 40mg / kg), VER-155008+DDP or 5-Fu group (40mg / kg+2mg / kg), 6 mice in each group, and the drugs are intraperitoneally injected twice a week. The tumor diameter and mouse weight are measured every other day; the tumor growth is detected once a week by in vivo imaging of small animals. After 5 weeks of treatment, the nude mice are killed and the following indicators are detected for the tumor: tumor weight, immunohistochemical detection of Ki-67 and HSPA1A expression levels.

[0082] Lung metastasis model: Two weeks after inoculation, the mice were given drugs in the same manner as above, with 6 mice in each group. The lung tissue metastasis was detected once a week by in vivo imaging of small animals. After 8 weeks, the nude mice were killed and lung tissue sections were taken for HE staining.

[0083] Experimental content

[0084] (1) Immunohistochemical staining was used to detect the expression of HSPA1A protein in lung cancer tissue and paracancerous tissue of 79 cancer patients, and the expression difference between lung cancer tissue and paracancerous tissue was analyzed, as well as the relationship between HSPA1A expression and patient prognosis. Figure 1 As shown in Figure 2, immunohistochemistry results showed that the expression of HSPA1A in lung cancer tissues was significantly higher than that in adjacent tissues. Figure 1 (A)- Figure 1 As shown in (C), the expression of HSPA1A in cancer tissues is upregulated, and the expression is negatively correlated with the patient's prognosis, that is, patients with high expression of HSPA1A in lung cancer have a short survival period and a poor prognosis. RT-qPCR and western blot methods were used to detect the changes in the transcription and protein levels of HSPA1A in drug-resistant cells. The results showed that the transcription and protein levels of HSPA1A were upregulated in the drug-resistant cell lines A549 / DDP and A549 / 5FU, and the mRNA transcription level of HSPA1A in A549 / DDP and A549 / 5FU resistant cell lines was significantly higher than that in A549 cells ( Figure 1 (D)), the protein level of HSPA1A in A549 / DDP and A549 / 5FU resistant cell lines was significantly higher than that in A549 cells ( Figure 1 (E)).

[0085] (2) The HSPA1A overexpression vector (HSPA1A OE), knockdown vector (HSPA1A#6#7#8) and corresponding control vector were transferred into H520 cells by cell transfection in Example 1. The knockdown and overexpression effects of HSPA1A were confirmed by western blot experiments. Figure 2 (A) As shown. RT-qPCR experiments detected the changes in the expression levels of metastasis-related marker molecules (E-cadherin, Twist, Snail) in H520 cells after HSPA1A knockdown and overexpression. The results showed that after HSPA1A knockdown and overexpression, the expression of E-cadherin, Twist, and Snail was also downregulated or increased, respectively. Figure 2 (B) and Figure 2 (C) shown.

[0086] (3) The scratch assay was used to detect the effects of overexpression of IGFL2-AS1 and knockdown of HSPA1A after overexpression of IGFL2-AS1 on the metastatic ability of A549 and H520 cells. Figure 3 As shown, Figure 3 (A) Overexpression of IGFL2-AS1 in A549 cells enhanced the cell metastasis ability. On this basis, knocking down HSPA1A could reverse the effect of IGFL2-AS1 upregulation on cell metastasis ability. Figure 3 (B) In H520 cells, overexpression of IGFL2-AS1 enhanced the cell metastasis ability. On this basis, knocking down HSPA1A can reverse the effect of IGFL2-AS1 upregulation on cell metastasis ability.

[0087] (4) Transwell, stemness spheroid and IC50 assays were used to detect the effects of overexpression of IGFL2-AS1 and knockdown of HSPA1A after overexpression of IGFL2-AS1 on cell invasion, stemness and drug sensitivity in A549 and H520 cells. Figure 4 As shown in (A), overexpression of IGFL2-AS1 significantly enhanced the cell invasion ability in A549 and H520 cells. On this basis, knocking down HSPA1A can reverse the effect of IGFL2-AS1 on cell invasion ability. Figure 4 As shown in (B), overexpression of IGFL2-AS1 significantly enhanced the stemness of A549 and H520 cells. On this basis, knocking down HSPA1A can reverse the effect of IGFL2-AS1 on cell stemness. Figure 4 As shown in (C), in A549 and H520 cells, overexpression of IGFL2-AS1 reduced the sensitivity of cells to chemotherapeutic drugs. On this basis, knockdown of HSPA1A enhanced the sensitivity of cells to chemotherapeutic drugs.

[0088] (5) Transwell, stemness spheroid and IC50 assays were used to examine the effects of knocking down IGFL2-AS1 and overexpressing HSPA1A after knocking down IGFL2-AS1 on cell invasion, stemness and drug sensitivity in A549 and H520 cells. Figure 5 As shown in (A), in A549 and H520 cells, knockdown of IGFL2-AS1 significantly weakened the cell invasion ability. On this basis, overexpression of HSPA1A can reverse the effect of IGFL2-AS1 on cell invasion ability. Figure 5 As shown in (B), in A549 and H520 cells, knocking down IGFL2-AS1 significantly weakened the cell metastasis ability. On this basis, knocking down HSPA1A can reverse the effect of IGFL2-AS1 on cell metastasis ability. Figure 5 As shown in (C), in A549 and H520 cells, knockdown of IGFL2-AS1 enhanced the sensitivity of cells to chemotherapeutic drugs. On this basis, knockdown of HSPA1A decreased the sensitivity of cells to chemotherapeutic drugs.

[0089] (6) RNA-seq experiments were performed on A549 cells expressing knockdown vectors (HSPA1A#6#7#8) and corresponding control vectors (CTRL), and data were analyzed to screen for cancer-promoting signaling pathways regulated by HSPA1A. Figure 6 As shown in (A), after RNA-seq experiments, KEGG analysis results showed that after knocking down HSPA1A, the cancer-promoting RAP1 pathway, AMPK pathway, Hippo pathway, Hedghog pathway, cAMP pathway, and mTOR pathway were inhibited. Figure 6 As shown in (B), after RNA-seq experiments, GSEA analysis results showed that after knocking down HSPA1A, the cancer-promoting RAP1 pathway, AMPK pathway, Hippo pathway, Hedghog pathway, cAMP pathway, and mTOR pathway were inhibited.

[0090] (7) RNA-seq experiments were performed on A549 cells containing knockdown vectors (HSPA1A#6#7#8) and corresponding control vectors (CTRL). Data analysis and screening of tumor suppressor signaling pathways regulated by HSPA1A showed that after knockdown of HSPA1A, the tumor suppressor p53 pathway and genes related to apoptosis were activated, such as Figure 7 As shown in (A), after RNA-seq experiments, KEGG analysis results showed that after knocking down HSPA1A, the tumor suppressor p53 pathway and genes related to apoptosis were activated. Figure 7 As shown in (B), after RNA-seq experiment, gene enrichment analysis results showed that after knocking down HSPA1A, the expression of genes related to p53 pathway and apoptosis was upregulated. Figure 7As shown in (C), after RNA-seq experiments, GSEA analysis results showed that after knocking down HSPA1A, the tumor suppressor p53 pathway and genes related to apoptosis were activated.

[0091] (8) Figure 8 As shown in (A), subcutaneous injection and tail vein injection of A549 cells were used to construct subcutaneous tumor-bearing and tumor metastasis models, and the HSPA1A inhibitor VER155008 was used alone or in combination with chemotherapeutic drugs (DDP / 5FU). Figure 8 (B) and Figure 8 (C) The results showed that HSPA1A inhibitor VER155008 alone or in combination with chemotherapy drugs (DDP / 5FU) could inhibit the metastasis of lung cancer cells in vivo, and the combined treatment effect was more significant.

[0092] (9) Adoption Figure 8 (A) Subcutaneous tumor model shown Figure 8 A), and treated with HSPA1A inhibitor VER155008 alone and in combination with chemotherapy drugs (DDP / 5FU). Fig. 9 In vivo imaging results in (A) show that HSPA1A inhibitor VER155008 alone or in combination with chemotherapy drugs (DDP / 5FU) can inhibit the proliferation of lung cancer cells in vivo, and the combined treatment effect is more obvious. Fig. 9 The tumor growth curve results in (B) show that HSPA1A inhibitor VER155008 alone and in combination with chemotherapy drugs (DDP / 5FU) can inhibit the proliferation of lung cancer cells in vivo, and the combined treatment effect is more obvious. Fig. 9 (C) and Fig. 9 The statistical results of tumor size in (D) show that SPA1A inhibitor VER155008 alone and in combination with chemotherapy drugs (DDP / 5FU) can inhibit the proliferation of lung cancer cells in vivo, and the combined treatment effect is more obvious.

[0093] (10) Immunohistochemistry experiments were used to detect the changes in the expression levels of Ki67 and HSPA1A in subcutaneous tumor tissues. Fig.10 (A) shows that the expression of Ki67 nuclear protein was downregulated in both HSPA1A inhibitor VER155008 monotherapy and combined chemotherapy (DDP / 5FU) therapy, and the downregulation was more obvious in the combined treatment. Fig.10 (B) Immunohistochemistry results showed that HSPA1A expression was downregulated in both HSPA1A inhibitor VER155008 monotherapy and combined chemotherapy (DDP / 5FU) therapy, and the downregulation was more obvious in the combined treatment.

[0094] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A HSPA1A gene, characterized in that: include: The gene sequence in SEQ ID No. 1; Used to upregulate the expression of RAP1 cancer-promoting pathway, AMPK cancer-promoting pathway, Hippo cancer-promoting pathway, mTOR cancer-promoting pathway and Hedgehog cancer-promoting pathway; As well as downregulating the expression of p53 tumor suppressor pathway and apoptosis-related genes.

2. The HSPA1A gene according to claim 1, characterized in that The RAP1 cancer-promoting pathway includes the gene sequence in SEQ ID No. 2; The AMPK cancer-promoting pathway includes the gene sequence in SEQ ID No. 3; The Hippo cancer-promoting pathway includes the gene sequence in SEQ ID No. 4; The mTOR cancer-promoting pathway includes the gene sequence in SEQ ID No. 5; The Hedgehog cancer-promoting pathway includes PTCH1 key factors and SHH key factors; The PTCH1 key factor includes the gene sequence in SEQ ID No.6; The SHH key factor includes the gene sequence in SEQ ID No.

7.

3. The HSPA1A gene according to claim 1, characterized in that The p53 tumor suppressor pathway includes the gene sequence in SEQ ID No.

8.

4. The HSPA1A gene according to claim 1, characterized in that The apoptosis-related genes include: The CTSD gene is the gene sequence in SEQ ID No. 9; The CASP7 gene is the gene sequence in SEQ ID No. 10; The FAS gene is a gene sequence including SEQ ID No.

11.

5. A molecular marker for HSPA1A gene formation according to any one of claims 1 to 4, characterized in that: include: HSPA1A-F:5'-CTGCGACAGTCCACTACCTT-3'; HSPA1A-R:5'-CGGCTCCGCTCTGAGATT-3'.

6. Use of the molecular marker according to claim 5 in the diagnosis of lung cancer, characterized in that: The HSPA1A-F and / or HSPA1A-R described in claim 5 is used to detect the expression level of HSPA1A in lung cancer lesions.

7. Use of the HSPA1A gene according to any one of claims 1 to 4 as a target for lung cancer treatment.

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