CN116814700B

CN116814700B - Application of ACSM5-P425T in construction of drug detection model for treating Xuanwei lung cancer

Info

Publication number: CN116814700B
Application number: CN202310973886.7A
Authority: CN
Inventors: 何成禄; 李娅; 石洪琼; 段勇
Original assignee: First Affiliated Hospital of Kunming Medical University
Current assignee: First Affiliated Hospital of Kunming Medical University
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2024-01-30
Anticipated expiration: 2043-08-03
Also published as: CN116814700A

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of ACSM5-P425T in constructing a drug detection model for treating Xuanwei lung cancer, wherein the ACSM5-P425T sequence is formed by single nucleotide polymorphism caused by missense mutation of a C base of chr16:20442608 locus in a gene ACSM5 into an A base.

Description

Application of ACSM5-P425T in construction of drug detection model for treating Xuanwei lung cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of ACSM5-P425T in constructing a drug detection model for treating Xuanwei lung cancer.

Background

The Xuanwei lung cancer is a endemic disease in Yunnan province, has the characteristics of high incidence rate and mortality rate of lung adenocarcinoma of non-smoking females, younger incidence age, family aggregation, poor sensitivity of radiotherapy and chemotherapy, high drug resistance and the like, and is attractive worldwide because of clear incidence characteristics. In recent years, a variety of targeted therapeutic drugs such as TK inhibitors (Tyrosine Kinaseinhibitors, TKIs) gefitinib have been developed, which pace the precise treatment of lung cancer. However, only a few patients with epidermal growth factor receptor (Epidermal growth factorreceptor, EGFR) mutations (e.g., L858R, G719C, etc.) were effective for gefitinib treatment, and cases without EGFR mutations were almost ineffective for gefitinib-targeted treatment, suggesting that our premise of accurate treatment needs to be based on accurate diagnosis. Although many lung cancer related gene mutation sites have been identified at present for the targeted treatment of TKIs, the pathogenesis features of Xuanwei lung cancer are unique and TKIs treatment is poorly effective, and there may be a different driver mutation spectrum than non-Xuanwei lung cancer.

Intrinsic factors for the development of tumorigenesis include activation of body oncogenes and inactivation of oncogenes. Searching related oncogenes and cancer suppressor genes of the Xuanwei lung cancer, deeply constructing related cells and animal models, screening more effective medicaments for the accurate treatment of the Xuanwei lung cancer, and revealing the influence of the medicaments on the occurrence and the development of the Xuanwei lung cancer from the aspects of in vitro cell experiments and in vivo animal tumorigenesis experiments.

Disclosure of Invention

The invention mainly aims to provide a method for constructing a detection model of a medicament for treating Xuanwei lung cancer, and the prepared model can be used for accurately treating the Xuanwei lung cancer and screening more effective medicaments.

The invention provides the following technical scheme for realizing the purposes:

the application of ACSM5-P425T in constructing a drug detection model for treating Xuanwei lung cancer is that the ACSM5-P425T sequence is formed by single nucleotide polymorphism caused by missense mutation of C base at chr16:20442608 site in gene ACSM5 into A base.

Further, the model overexpresses the ACSM5-P425T gene sequence.

Further, the model is an Xuanwei lung cancer cell line model or an animal model.

Further, the Xuanwei lung cancer cell line model construction method comprises the following steps: cell culture, construction of a target gene plasmid containing ACSM5-P425T gene sequence, carrying out slow virus packaging on the target gene plasmid, carrying out cell infection and detecting the target gene plasmid titer of slow virus packaging.

Further, the method for constructing the animal model comprises the following steps: and injecting cells which over-express ACSM5-P425T gene sequence into mice, and culturing to obtain the mice model.

The invention also provides a recombinant plasmid, wherein the ACSM5-P425T sequence is formed by single nucleotide polymorphism caused by missense mutation of C base at chr16:20442608 locus in the gene ACSM5 into A base.

The invention also provides an application of knocking out or inhibiting the expression of ACSM5 genes and/or coded proteins thereof in constructing a drug detection model for treating Xuanwei lung cancer.

The invention achieves the technical effects that:

1. according to the invention, through early-stage experiments and screening, ACSM5-P425T is considered to be related oncogene of Xuanwei lung cancer, ACSM5 is considered to be an oncogene, and through deep and relevant cell and animal model construction, the method can be used for accurately treating the Xuanwei lung cancer, screening more effective medicines, and revealing the influence of the medicines on the occurrence and the development of the Xuanwei lung cancer from the aspects of in-vitro cell experiments and in-vivo animal tumorigenesis experiments

2. The stable expression wild ACSM5, A549 and JT cell proliferation, plate cloning capacity, migration and invasion which are successfully constructed by the method are obviously inhibited, the cell cycle is blocked in the G0/G1 phase, the early apoptosis cells are increased, and compared with the mutant ACSM5-P425T, the opposite result is obtained. Notably, the effects of ACSM5 wild type and ACSM5-P425T mutant on JT cell proliferation were greater than a549 cells, whereas the plate clonality was reversed, indicating that a549 cells were more malignant than JT cells; compared with ACSM5 wild type, the taxol has the drug concentration CC50 of 3.507 mug/ml and 75.95 mug/m in ACSM5-P425T mutant A549 cells and JT cells respectively, and the comparison difference between groups has statistical significance (P is less than 0.05), which indicates that the taxol is more resistant to JT cells, and further shows the application of ACSM5-P425T mutant cells in screening drugs for treating Xuanwei lung cancer.

3. The in vivo nude mice tumor-bearing experimental results show that compared with a wild type ACSM5 group with stable expression, the ACSM5-P425T mutant group has the advantages of increased growth speed, increased weight, increased ki-67 expression and reduced Tunel apoptosis body of transplanted tumors, and the comparison difference between groups has statistical significance (P is less than 0.05).

Drawings

FIG. 1A schematic diagram of ACSM5 wild type and mutant sequences (substitution of nucleotide at position 1273 of ORF region with A) and PReceiver-Lv201 vector;

FIG. 2 shows Western Blot analysis results of JT cells of lung carcinoma of ventilating lung and ACSM5 total protein expression of different NSCLC lines;

FIG. 3 shows the effect of Sanger reverse sequencing of plasmid (A) of ACSM5 wild-type strain and ACSM5-P425T mutant strain on forward sequencing of A549 cells and JT cells (B);

FIG. 4 shows a graph of qPCR detection results after lentivirus infection of 293T cells; (amplification of titer CT plot (A), standard plot (B) and titer (C))

FIG. 5A 549 and JT cells construct a control empty strain, ACSM5 wild strain, and ACSM5-1 mutant, and a graph of the results of the verification; ((A) A549 cells construct empty-load strain, ACSM5 wild strain and ACSM5-1 mutant stable vector and verification result, (B) JT cells construct empty-load strain, ACSM5 wild strain and ACSM5-1 mutant stable vector and verification result, (C) Western Blot verification of A549 and JT cells in empty-load strain, ACSM5 wild strain and ACSM5-1 mutant

FIG. 6 is a graph of the effect of NC, ACSM5-W wild type (ACSM 5) and ACSM5-T mutant (ACSM 5-1) on proliferation potency of A549 and JT cells;

FIG. 7 (40X) is a graph of the effect of NC, ACSM5-W wild type (ACSM 5) and ACSM5-T mutant (ACSM 5-1) on the clonogenic capacity of A549 and JT cells;

FIG. 8 is a graph showing the effects of wild-type ACSM5-W (ACSM 5) and mutant ACSM5-T (ACSM 5-1) on cycle of A549 cells (A) and JT cells (B);

FIG. 9 NC, graphs showing the effect of wild-type ACSM5-W (ACSM 5) and mutant ACSM5-T (ACSM 5-1) on apoptosis of A549 cells (A) and JT cells (B);

FIG. 10 is a graph showing the effect (4X) of scratch assay on migration ability of NC, ACSM5-W wild type (ACSM 5) and ACSM5-T mutant (ACSM 5-1) on A549 cells (A) and JT cells (B);

FIG. 11 is a graph of the effect (10X) of NC, ACSM5-W wild type (ACSM 5) and ACSM5-T mutant (ACSM 5-1) on migration ability of A549 cells (A) and JT cells (B) by Transwell experiments;

FIG. 12 is a graph showing the effect (10X) of NC, ACSM5-W wild type (ACSM 5) and ACSM5-T mutant (ACSM 5-1) on the invasive capacity of A549 cells (A) and JT cells (B) by Transwell experiments;

FIG. 13 is a graph showing the expression level of EMT-related proteins in each experimental group in A549 cells and JT cells detected by Western blot experiments;

FIG. 14 is a graph showing the effect of CCK-8 on CC50 (in μg/ml) of paclitaxel in different treatment groups;

FIG. 15 is a graph showing the comparison of tumor growth rates of the nude mice tumor-bearing model establishment (A) and the transplantation tumor growth rates of the respective cell groups (B);

figure 16A tumor mass was removed 15 days after single-point inoculation of each group of cells in the subcutaneous anterior axillary limb, and the size of the cell transplantation tumor of each group was compared; B. statistically analyzing the weight map of each group of cell transplantation tumor;

FIGS. 17A and C are graphs of Ki-67 staining and statistical analysis of transplanted tumor tissue for each group of cells (10X); b and d. Engrafted tumor tissue TUNEL staining of each group of cells (10×);

in the above figures, inter-group comparisons are referred to: * : p is less than 0.05; * A.x; p is less than 0.01; * X; p is less than 0.005; * P < 0.001;

Detailed Description

The conception and technical effects of the present application will be clearly and completely described below with reference to examples and drawings to fully understand the objects, features and effects of the present application. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have meanings commonly understood by those of ordinary skill in the art, and the purchase of goods in the test methods, such as those not explicitly stated herein, is performed under conventional conditions or conditions suggested by the manufacturer, and reagents or equipment used, such as those not explicitly stated by the manufacturer, may be obtained by commercially available purchase of conventional products.

In the invention, ACSM5 gene is a public sequence, and NCBI sequence number is: NM-017888.2.

In the drawings and in the text of the description, the mutations ACSM5-T, ACSM5-1 and ACSM5-P425T refer to chr16 in the gene ACSM 5: the missense mutation of C base at 20442608 site is single nucleotide polymorphism caused by A base.

The early-stage study of the invention shows that ACSM5 plays an anti-cancer gene role in Xuanwei lung cancer patients, and when SNP locus chr16 of ACSM 5: when the 1273 locus C base missense mutation of 20442608 is changed into A base, the coded hydrophobic proline is changed into hydrophilic threonine, so that the mutant ACSM5-P425T can possibly play an oncogene role. qPCR, westernblot, immunohistochemistry and other experiments further prove that the high and low expression ACSM5 has correlation with clinical related information such as tumor size, stage and the like. In summary, it is believed that the ACSM5-P425T mutant may be involved in the development and progression of lung cancer, and thus, constructing a cell or animal model containing the ACSM5-P425T mutant, or a cell or animal model with low expression of ACSM5, can pertinently screen drugs for treating lung cancer.

As a specific embodiment, ACSM5-P425T can be applied to the construction of a drug detection model for treating Xuanwei lung cancer, and the sequence of the ACSM5-P425T is chr16 in the gene ACSM 5: the missense mutation of C base at 20442608 site is formed by single nucleotide polymorphism caused by A base, and the model overexpresses ACSM5-P425T gene sequence.

As some embodiments, the selectable model is an xuwei lung cancer cell line model or an animal model.

As a more specific embodiment, the method for constructing the Xuanwei lung cancer cell line model comprises the following steps: cell culture, construction of a target gene plasmid containing ACSM5-P425T gene sequence, carrying out slow virus packaging on the target gene plasmid, carrying out cell infection and detecting the target gene plasmid titer of slow virus packaging.

As another more specific embodiment, the method for constructing an animal model is as follows: and injecting cells which over-express ACSM5-P425T gene sequence into mice, and culturing to obtain the mice model.

As another embodiment, the invention also provides a recombinant plasmid, which contains ACSM5-P425T base sequence, wherein the ACSM5-P425T base sequence is chr16 in gene ACSM 5: the missense mutation of C base at 20442608 site is the formation of single nucleotide polymorphism caused by A base.

As another embodiment, knocking out or inhibiting expression of the ACSM5 gene and/or its encoded protein can be applied in constructing a drug detection model for treating xuway lung cancer.

As mentioned above, preferred embodiments are shown to facilitate understanding. The scope of the invention is not limited to the embodiments and examples specifically described herein, and is limited only by the scope of the claims. The examples of the present invention are specifically shown below in terms of test procedures.

Example 1

Protein expression condition of 1 ACSM5 in Xuanwei lung cancer JT cell line and NSCLC cell line

Compared with HBE cells, the ACSM5 protein expression level of A549 cells in NSCLC cell lines (A549, H460, H1299, H1975) is slightly lower than that of HBE, and the ACSM5 protein expression level of Xuanwei lung cancer JT cells is slightly higher than that of HBE, so that the difference is not statistically significant (P > 0.05). However, the expression of ACSM5 was significantly higher for both NSCLC lines H1299 and H1975 than for HBE, with H460 significantly lower than for HBE, with statistical differences (P < 0.05) in the group comparisons (fig. 2). Thus, subsequent in vitro and in vivo cell experiments will select for a549 cells and JT cells.

Plasmid construction and verification of 2 ACSM5 wild strain and ACSM5-P425T mutant strain

Plasmids of ACSM5 wild strain (ACSM 5-W) and ACSM5-P425T mutant strain (ACSM 5-T) constructed according to step 2.3 of the present invention were sequenced by Sanger to ensure successful construction of the corresponding plasmids (specific process delegated completion by Beijing Optimago Co., ltd.) prior to transfection and selection of cell lines stably expressing the gene of interest. Reverse sequencing of the plasmid showed: the 1273 base of ACSM5-W plasmid is C, the 1273 base of ACSM5-T plasmid is A, which shows that the construction of the plasmid of the target gene is successful (see FIG. 3-A).

Meanwhile, the candidate A549 cells and JT cells are sequenced through Sanger to ensure that the candidate A549 cells and JT cells do not have natural 1273 site C base mutation into base A before transfection of corresponding plasmids and screening of cell strains, and the sequencing result is consistent with the sequencing result of a wild type plasmid, which shows that the base at 1273 site of the A549 cells and JT cells does not have natural mutated A base (see figure 3-B).

The above results indicate that: the plasmids of the ACSM5 wild strain and the ACSM5-P425T mutant strain are successfully constructed, and meanwhile, the 1273 site of the ACSM5 in the A549 cells and JT cells has no natural mutation of the A base, so that the accuracy assurance is provided for the subsequent plasmid transfection and the cell strain screening of stably expressing the target gene.

3 lentivirus titre detection

According to the step 2.3.3 of the invention, after a series of processes of lentivirus packaging, 293T cell infection, concentration and purification, lentivirus RNA extraction in supernatant, reversion and the like, the titer of the target gene plasmid packaged by the lentivirus is detected by a qPCR method so as to ensure the success rate of subsequent infection and screening of cells A549 and JT stably expressing the target gene plasmid. Fig. 4 shows: the titre of the target gene ACSM5 (wild type) and ACSM5-1 (mutant) and the titre of the empty PRecceiver are both higher than the medium concentration standard value of 16.13. This result shows that: the target gene titer accords with the concentration value of the stable transfer A549 and JT cells of the subsequent infection and screening.

4 wild-type ACSM5-W and mutant ACSM5-T stable vector construction

The PReciver-ACSM 5-W wild slow virus vector, the PReciver-ACSM 5-T mutant slow virus vector and the control vector PReciver-GFP which are constructed by the invention express green fluorescence GFP, JT cells and A549 cells are infected, puromycin with the concentration of 1.5ug/ml is used for screening out infected positive cells until the infection positive rate reaches more than 80%, and the screened positive cells are subjected to passage expansion culture, so that the cells are used for subsequent experiments. And the construction effect of the stable cell line was verified by qPCR and WesternBlot experiments (fig. 5).

The above results indicate that: stable expression control empty, wild type ACSM5-W and mutant ACSM5-T a549 and JT cell lines were successfully constructed.

Example 2

Influence of biological behavior in vitro of wild-type ACSM5-W and mutant ACSM5-T cell strains on proliferation potency of 1 cells

NC cells (motivecontrol), wild type A549 cells and JT cells, mutant type A549 cells and JT cells were inoculated in 96-well plates, respectively, and the proliferation capacity of the cells was measured every 24 hours by CCK-8 method, and a growth curve was drawn with time as the horizontal axis and absorbance relative to the change of the cells as the vertical axis (FIG. 6). The results show that: (1) the proliferation capacity of JT cells of each group is stronger than that of A549 cells, and the JT cells are more obvious from 72 hours; (2) the ACSM5-W wild type A549 cells and JT cells are obviously inhibited in growth (P is less than 0.05) compared with the other two groups, which indicates that the ACSM5-P425T mutant can restore the proliferation capacity of the ACSM5-W wild type A549 cells and JT cells; (3) the NC group was not significantly different from the mutant A549 and JT groups (P < 0.05), suggesting that the lentiviral expression vector had no effect on cell proliferation.

The above results indicate that: the ACSM5 wild strain can inhibit cell proliferation (JT cells > A549 cells); the ACSM5-P425T mutant promotes the above biological processes (JT cells > A549 cells).

2 Effect of cell clone formation

Cell proliferation experiments suggest that the ACSM5-P425T mutant can restore the proliferation capacity of ACSM5-W wild type A549 cells and JT cells, and in order to further verify that the ACSM5-P425T mutant has higher clone forming capacity than the ACSM5-W wild type, NC cells, wild type A549 cells and JT cells, mutant A549 cells and JT cells are respectively verified by adopting a plate clone forming experiment (figure 7). The results show that: (1) in A549 cells and JT cells, the ACSM5-P425T mutant has stronger clone forming ability (P < 0.05) than the ACSM5-W wild type, but has weaker clone forming ability (P < 0.05) than the NC group; (2) although JT cells are more proliferative than A549 cells (5.1), A549 cells are more clonogenic than JT cells.

The above results indicate that: the ACSM5 wild strain can inhibit cell clone formation (malignant degree A549 cells > JT cells); the ACSM5-P425T mutant can promote the above biological processes.

3 Effect of cell cycle

In the A549 cell group, ACSM5-W wild-type A549 cells were stably transfected compared to the ACSM5-P425T mutant group and NC control group (FIG. 8-A): (1) the proportion of cells in the G0-G1 phase is increased (P < 0.05) and is obviously lower than that of JT cells (53.85% < 70.71%, A549 VSJT); (2) the ratio of the cells in the G2-M phase is between that of the ACSM5-P425T mutant group and that of the NC control group, the cells are different from that of the NC group (P is less than 0.05), and the cells in the G2-M phase are not different from that of the ACSM5-P425T mutant group (P is more than 0.05); (3) the proportion of cells in S phase was reduced (P < 0.05).

In the JT cell group, ACSM5-W wild-type JT cells were stably transfected compared to the ACSM5-P425T mutant group and NC control group (FIG. 8-B): (1) the proportion of cells in the G0-G1 phase is obviously increased (P is less than 0.05); (2) the proportion of cells in the G2-M phase is reduced, and the comparison among groups has no statistical difference (P is more than 0.05); (3) the proportion of S-phase cells was reduced (P < 0.05).

The above results indicate that: the ACSM5 wild strain can promote cells to arrest in the G0-G1 phase; the ACSM5-P425T mutant inhibits the above biological processes.

Influence of apoptosis in 4 cells

Annexin-V/7-ADD flow cytometry detected the proportion of early apoptotic cells in NC, ACSM5-W wild type and ACSM5-T mutant in A549 cells and JT cells, as shown in FIG. 9, by flow analysis of early apoptotic cells, i.e., Q4 population, and statistical analysis of early apoptotic cell ratios. The results show that: compared with ACSM5-P425T mutant group and NC control group, the ratio of early apoptosis cells of ACSM5-W wild type A549 and JT cells is obviously increased (P < 0.05), wherein the ratio of early apoptosis cells of ACSM5-T mutant JT cells is higher than that of corresponding A549 cells, and the difference is statistically significant (P < 0.05).

The above results indicate that: the ACSM5 wild strain can promote early apoptosis of cells; the ACSM5-P425T mutant can inhibit early apoptosis of A549 cells and JT cells.

5 Effect of cell migration ability

5.1 scratch test

In the A549 cell group (FIG. 10-A), the number of migrating cells under the scratch area was hardly affected by time-dependence in the ACSM5-W wild-type group. In contrast, the ACSM5-P425T mutant group and the NC control group showed a time-dependent increase in the number of migrating cells under the scratch area, the number of migrating cells under the scratch area of the NC control group was significantly higher than that of the ACSM5-P425T mutant group (P < 0.05), and the number of migrating cells under the scratch area of the NC control group was significantly higher than that of the ACSM5-P425T mutant group, and the comparison between groups had a statistical difference (P < 0.05).

In the JT cell group (FIG. 10-B), the tendency of the number of migrating cells under the scratch area is substantially consistent with that of A549 cells, however, the number of migrating cells is significantly smaller than that of the A549 cell group.

The above results indicate that: the ACSM5 wild strain has the capacity of inhibiting cell migration; the ACSM5-P425T mutant has the ability to promote cell migration (A549 cells > JT cells).

5.2 Transwell migration experiment

In the A549 cell group (FIG. 11-A) and the JT cell group (FIG. 11-B), the ACSM5-W wild-type group entered the lower chamber at about ((443/view) and (385/view)) less than the ACSM5-P425T mutant group at about ((583/view) and (523/view)) and the NC control group at about ((655/view) and (575/view)) (P < 0.05). The ACSM5-W wild strain is proved to obviously influence the migration capacity of A549 cells and JT cells, and the conclusion is consistent with a 5.5.1 scratch experiment.

The above 5.5.1+5.5.2 results indicate that: the ACSM5 wild strain has the capacity of inhibiting cell migration; the ACSM5-P425T mutant has the ability to promote cell migration.

6 Effect of cell invasiveness

In the A549 cell group (FIG. 12-A) and the JT cell group (FIG. 12-B), the ACSM5-W wild-type group penetrated Matrigel gel, the number of cells entering the lower chamber was about ((408/field) and (363/field)) less than that of the ACSM5-P425T mutant group ((487/field) and (408/field)) and the NC control group about ((624/field) and (558/field)) (P < 0.05). The ACSM5-W wild strain was demonstrated to significantly affect the invasive capacity of A549 cells and. JT cells.

7 epithelial mesenchymal transition

Epithelial cell mesenchymal transition (epigheli-MesenchymalTransition, EMT) is the process by which Epithelial cells lose the characteristics of Epithelial cells and acquire characteristics of mesenchymal cells, and is judged by relevant literature to be key to invasion and metastasis, and the reduction of the expression level of the relevant Epithelial protein E-Cadherin, and the increase of the expression levels of the relevant mesenchymal proteins N-Cadherin and Vimentin, and the trend of the change of the expression levels of the relevant proteins often indicate the occurrence of EMT.

Thus, the wild type ACSM5-W has the ability to inhibit A549 and JT cell invasion, whereas the mutant ACSM5-T, in contrast, promotes A549 and JT cell invasion. We verified the changes in expression levels of the three EMT-related proteins at the molecular level by Western-blot experiments to verify whether A549 and JT cells stably expressing ACSM5-W wild-type and ACSM5-T mutant inhibit or promote the EMT process.

The results of fig. 13 show that: (1) in A549 cells, the expression levels of the mesenchymal cell-related proteins N-Cadherin and Vimentin were increased to different extents in the NC group and the ACSM5-T mutant group compared with the ACSM5-W wild-type group, the group comparison had a statistical difference (P < 0.05), while the expression level of the epithelial cell-related protein E-Cadherin was significantly weaker (not counted); (2) in JT cells, only the expression levels of the Vimentin protein were compared among three groups, with statistical differences (P < 0.05).

The above results have not clearly demonstrated that the wild-type strain of ACSM5 and the mutant strain of ACSM5-P425T inhibit or promote the process of epithelial cell mesenchymal transition in cells.

8 paclitaxel resistance index detection

Paclitaxel is one of the antitumor drugs commonly used in combination with cisplatin in advanced lung cancer chemotherapy, but after a period of use, it is easy to cause drug resistance to affect the chemotherapy effect. Thus, the study treated NC, ACSM5-W wild-type and ACSM5-T mutant A549 and JT cells with different concentrations of paclitaxel (0 μg/ml,5 μg/ml,10 μg/ml,20 μg/ml,40 μg/ml,80 μg/ml and 160 μg/ml) and the concentration of half cell death drug (50% cytoxicity concentration, CC50) was calculated using the CCK-8 method. The results of fig. 14 show that: the ACSM5-T mutant group had poorer sensitivity to paclitaxel (P < 0.05), i.e., higher resistance (JT cells > A549 cells), than the NC group and the ACSM5-W wild type group.

The above results indicate that: stable expression of ACSM5-P425T mutant strain is easier to generate drug resistance to paclitaxel than wild strain ACSM5 (JT cells > A549 cells).

Example 3

Influence of the in vivo tumorigenicity of wild-type ACSM5-W and mutant ACSM5-T cell strains

1 effects of tumor volume and mass in vivo:

animal models of non-Xuanwei lung cancer and Xuanwei lung cancer were established by NC, A549 cells stably expressing ACSM5-W wild type and ACSM5-T mutant, and JT cells (FIG. 15-A). Since the a549 cell nude mouse tumor-bearing model is more pronounced than JT cells (JT cell nude mouse tumor-bearing formation is less optimal and not shown herein), the a549 cell model was selected for subsequent in vivo nude mouse tumor-bearing experiments.

The growth curves of transplanted tumors of each group of cells (NC, A549 cells stably expressing ACSM5-W wild type and ACSM5-T mutant type) show (figures 15-B and C), the growth speed of the group stably expressing ACSM5-W wild type is obviously lower than that of the group NC and stably expressing ACSM5-T mutant type (P is less than 0.05), which indicates that after the group stably expressing ACSM5-W wild type, the growth speed of tumor cells is obviously inhibited, and the growth speed of tumor cells can be promoted by the ACSM5-T mutant type.

After 15 days, the peeled tumor bodies were weighed, and the weights of the tumor bodies in each group were compared. Results display (fig. 16): the weight of the transplanted tumor of the wild group with the stable expression of ACSM5-W is lighter than that of the NC group and the group with the stable expression of ACSM5-T mutant (P is less than 0.05). It is demonstrated that the wild type ACSM5-W can inhibit the tumorigenicity of tumor cells, while ACSM5-T mutant can promote tumorigenesis of tumor cells.

2 effects of proliferation and apoptosis of transplanted tumor cells:

nude mice were sacrificed 15 days later, and the cell proliferation activity of each group was observed by Ki-67 immunohistochemical staining after paraffin embedding, slicing, and the like of the transplanted tumor. As shown (fig. 17-a): compared with a wild type group of the ACSM5-W stably expressed, NC and tissue sections of the group of the ACSM5-T stably expressed mutant have significantly increased Ki-67 positive staining (green fluorescence, indicated by red arrows), and the difference between the groups has statistical significance (P < 0.05), which indicates that the wild type of the ACSM5-W can inhibit the proliferation of tumor cells, and the mutant type of the ACSM5-T can promote the proliferation of tumor cells. This is consistent with the results of the prior in vitro cell proliferation assay.

And observing early apoptosis of transplanted tumor tissue cells by using TUNEL apoptosis detection method. The results show (FIG. 17-B): compared to NC and the stable ACSM5-T mutant group, the stable ACSM5-W wild group tissue sections showed significantly more TUNEL stained (green fluorescence, red arrow) cells. The results suggest that the wild type ACSM5-W can significantly promote NSCLC apoptosis, while the mutant ACSM5-T can inhibit NSCLC apoptosis. This is consistent with the results of the early in vitro Annexin V/7-AAD flow assay.

The above results indicate that: the ACSM5 wild strain can inhibit NSCLC tumor cell proliferation and promote apoptosis, whereas the ACSM5-P425T mutant strain can promote NSCLC tumor cell proliferation and inhibit apoptosis.

In summary, the invention constructs A549 and JT cells which stably express ACSM5 wild type and ACSM5-P425T mutant, observes the proliferation, plate cloning, cell cycle and apoptosis, migration and invasion, epithelial cell interstitial transformation, taxol resistance and other capacity changes of the two cells at the in vitro cell level, and proves that ACSM5 can inhibit Xuanwei lung cancer cell proliferation, plate cloning, migration and invasion, block cells in G0/G1 phase, promote early apoptosis, play the role of an cancer suppressor gene, and the mutant ACSM5-P425T can promote the change of the biological behaviors of the cells, play the function of 'driver mutation', thereby affecting the prognosis of Xuanwei lung cancer patients; in vivo nude mice transplanted tumor level, also get the conclusion consistent with in vitro cell experiment; paclitaxel resistance index experiments also suggest that JT cells are more resistant than a549 cells.

Research materials and methods

1 Experimental materials

1.1 Experimental cell lines

The Xuanwei lung cancer cell line JT is preserved by a medical laboratory of a first affiliated hospital of Kunming medical university, and the cell properties are referred to (Ma Liju, wang hong Zhi, bian Li, etc.. The establishment of XLA-07 of Xuanwei lung adenocarcinoma cell line and the characteristics [ J ]. J.Chinese J pathology, 2012, 41 (5): 335-339. JT cells in the present invention are passage cells of the cell line XLA-07); 4 non-small cell lung carcinoma NSCLC cell lines (A549, H460, H1299, H1975) and normal human bronchial epithelial cell lines HBE were purchased from the Kunming animal institute cell bank of the national academy of sciences.

1.2 laboratory animals

BALB/C-nude female nude mice (N0.430727210100378365) 4 weeks old, from Stokes Levoda laboratories, inc. of Hunan province, had a weight of about 120mg when purchased. The living environment is maintained at proper temperature and humidity, and water and feed are added at regular time.

1.3 Main reagents and consumables

1.4 routine laboratory reagent preparation

(1) Cell culture PBS phosphate buffer (1L, used after high temperature and high pressure sterilization): naCl (8.0 g), KCl (0.2 g), na2HPO4.12H2O (2.86 g), KH2PO4 (0.27 g). (2) SDS running buffer (1 mL): glycine (14.4 g), tris (3 g), SDS (1 g).

(3) SDS transfer buffer (1 mL): glycine (14.4 g), tris (3 g), 200ML methanol.

(4)6×SDSloadingbuffer：4×Tris-HCl/SDS(pH6.8)(7mL)，Glycerol(3.0mL)，DTT(0.933g)，Bromophenolblue(1.2mg)。

2 Experimental methods

2.1 cell culture

JT and A549 cell culture medium is RPMI1640, 10% FBS; the remaining cell culture medium was DMEM,10% fbs. 100U/ml of enollin and 100. Mu.g/ml of treptomycin were added to both JT and A549 cell culture media for screening stably expressing the target gene.

2.2 Protein expression condition of ACSM5 in Xuanwei lung cancer and lung cancer cell lines and candidate cell line screening

(1) Collecting target proteins: the density in the 6-hole plate is 5 multiplied by 10 in advance ⁵ JT cells in logarithmic growth phase of cells/mL/well, cells of each cell line of NSCLC (A549, H460, H1299, H1975) and normal human bronchial epithelial cell line H BE is cultured for 24 hours by RPMI1640 or DMEM serum-free culture medium, the culture solution is removed, the cells are washed three times by PBS buffer, after the PBS buffer is removed, 200 mu L of RIPA lysate and 2 mu L of protease inhibitor and 2 mu L of phosphatase inhibitor are added into each hole of a 6-hole plate, the mixture is placed on ice for cracking for 30 minutes, and the mixture is centrifuged at 12000rpm for 5 minutes, and the supernatant is sucked to obtain the total protein of each cell line. (2) measurement of the target total protein by BCA assay. (3) SDS-PAGE was performed. (4) analysis results: gray scale values of each cell line were analyzed according to Image software, and differences in total protein expression amount of ACSM5 among each cell line were compared. (5) Selecting candidate cell lines for subsequent in vivo and in vitro experiments according to step (4).

2.3 construction of plasmids for ACSM5 wild-type strain and ACSM5-P425T mutant strain

The ORF sequence of ACSM5 gene was searched from NCBI website, wild type sequence and mutant sequence (nucleotide at 1273 of ORF region was replaced by A) were carried on PRECeiver-Lv201 empty vector, and a large number of objective vectors were obtained by E.coli amplification and ampicillin screening, which were carried out by Guangzhou complex gene Co., ltd (FIG. 1).

2.3.1 vector sequencing identification

2.3.1.1 vector amplification

(1) The lentiviral plasmids synthesized by 10. Mu.L of the complex were aspirated and added to 100. Mu.L of E.coli DH5a competent cells, respectively, and the walls of the flick tube were mixed and left on ice for 30min. It was placed in a circulating water bath preheated to 42 ℃ for about 90s and then quickly moved to an ice-water bath for incubation for 2min. Then, 500. Mu.LLB medium was added thereto, followed by shaking culture on a shaker at 37℃for 1 hour. And (3) uniformly coating a proper amount of bacterial liquid on a flat plate containing ampicillin, and placing the flat plate in a constant temperature incubator for inverted culture for 16 hours.

(2) Single colonies were inoculated into LB liquid medium containing ampicillin and cultured at 37℃for 12-16h. Plasmid extraction was then performed using the EndoFreemmidiiPrambkit.

(3) Collecting the overnight culture bacterial liquid in a 5mL centrifuge tube, centrifuging at 12000rpm for 2min, collecting bacterial liquid, discarding supernatant, adding 250 μL of cell heavy suspension, sufficiently oscillating to uniformly suspend the bacterial liquid, adding 250 μL of cell lysate, adding 10 μL of proteinase K, reversing for 6 times, mixing uniformly, and standing for 1-2min to crack the bacterial body until the bacterial body is clear. After 350. Mu.L of the neutralization solution was added, the mixture was again mixed upside down to precipitate the protein, the mixture was left in an ice bath for 5min, centrifuged at 10000rpm for 10min, the protein was discarded, the supernatant was collected into a sterile E tube, centrifuged at 12000rpm for 5min, the supernatant was transferred into a recovery column, and centrifuged again at 12000rpm for 1min, and the lower-layer waste liquid was discarded. Adding 600 mu L of the pre-prepared rinsing liquid, centrifuging at 12000rpm for 1min, discarding the lower layer waste liquid, centrifuging at 12000rpm for 2min, removing the residual rinsing liquid again, transferring the recovered column to a new 1.5mLEP tube on a super clean bench, standing for 10-20mmin, adding 95 mu LNuclease-freeWater into the recovered column after naturally airing, standing for 2min, centrifuging at 2000rpm for 2min, collecting samples, numbering, electrophoresis, measuring concentration and quality inspection.

2.3.1.2 electrophoretic identification

(1) And (3) glue preparation: the method comprises the steps of weighing 0.5g of agarose by an electronic balance, boiling for 1min in a microwave oven, dissolving in 50ml of TAE buffer solution (1×), cooling to 50-60 ℃, adding 5 mu L of nucleic acid dye, gently mixing, carefully pouring into a gel preparation groove in which a comb is inserted in advance, taking action to be gentle so as to prevent a large amount of bubbles, if the action is careless, puncturing the gel by a sterile gun head, standing for 40min, and naturally solidifying to obtain the 1% agarose gel.

(2) Loading: before loading, the prepared gel is put into an electrophoresis tank filled with TAE buffer solution, and bubbles in the gel holes are exhausted. When in loading, the sample is loaded according to the proportion of 2 mu L of mixed 1 mu LLoadingbuffer, and 15000bp DNAmarker with the same volume is loaded on the leftmost well or the rightmost well. (3) electrophoresis: the voltage is set to 150V during electrophoresis, and the electrophoresis time is 15-20min. (4) judging: and (3) turning off the power supply after electrophoresis, taking out the gel block, putting the gel block into a gel imager, turning on an ultraviolet transmission lamp to observe electrophoresis strips, judging the molecular length, and selecting a sample with bright strips, single and consistent molecular weight to send to sequencing.

2.3.1.3 sequencing

Sequencing and comparing the vicinity of the mutation site of the ORF region of the ACSM5 gene carried by the vector, and the specific process is entrusted to completion of Beijing qingke biotechnology company.

2.3.2 cell sequencing identification

The lentiviral vector is infected with 293T cells, DNA is extracted for sequencing, and whether ACSM5 is mutated is compared, and the specific sequencing process is completed by Beijing qing family biotechnology company.

2.3.3 lentiviral titer assay

2.3.3.1 lentiviral packaging

(1) Preparation of lentiviral packaging helper cells 293T

Recovering 293T cells, when the 293T cells grow to 90%, discarding old culture solution, adding PBS solution, gently shaking, washing the cell growth surface, discarding the PBS solution, adding a proper amount of EDTA-pancreatin, digesting for 1-2 min at 37 ℃, observing under an inverted microscope, when the cells are separated from each other and become round, pouring out pancreatin solution, adding fresh complete culture medium, uniformly mixing the cells, and inoculating into a 10cm cell culture plate. Lentiviral packaging was performed when the cells increased to 80%.

(2) The virus was packed in sterile centrifuge tubes, diluted 2.5ug of vector to be transfected and 5 ul of Lenti-PacHIV with 200 ul of Opti-MEM medium, and in another centrifuge tube, diluted 15 ul of endoFectinLenti with 200 ul of Opti-MEM medium and incubated for 5min at room temperature. The diluted endoFectinlenti was added dropwise to the diluted carrier, gently shaken in a centrifuge tube containing the mixed solution, and incubated at room temperature for 20min. Taking cells inoculated on a 10cm culture plate, discarding old culture solution when the cells grow to reach the fusion degree of 80%, washing for 2 times by PBS, and replacing the old culture medium in the culture plate with new culture medium (DMEM culture medium containing 10% of inactivated fetal calf serum and without double antibodies). Adding the incubated carrier-EndoFectinLenti complex into a 293T cell culture plate, uniformly mixing, and placing into a cell culture box for culturing for 8-14h. The old medium was replaced with DMEM medium containing 5% inactivated fetal bovine serum, 1% diabody, and 1/500 volume of titeboost was added. And (3) respectively collecting 293T cell culture solution after 48h and 72h of transfection, centrifuging for 10min at 500g, and collecting supernatant for concentrating or split charging and freezing at-80 ℃ for later use.

(3) Lentivirus concentration and purification

After completion of the lentiviral packaging operation, the supernatant, i.e., the supernatant containing the lentiviral particles, is collected from the tool cell culture plate or flask. The supernatant may be centrifuged at 2000g for 10 minutes at 4℃to remove cell debris, or alternatively, the cell debris may be filtered through a 0.45 μm filter; according to the volume of lentivirus liquid: the lentivirus supernatant and the concentrated reagent (the concentrated reagent is added directly to the stock solution of 6X) are mixed in a ratio of concentrated reagent volume=5:1, and incubated at a temperature of 0 to 4 ℃ for 2 hours or more (or overnight). After the incubation was completed, 3500g of the mixture was centrifuged at 4℃for 25 minutes; after centrifugation, the supernatant was carefully aspirated off and discarded; weighing 1/10-1/100 volume of DMEM or PBS of the stock solution, and re-blowing to suspend slow virus precipitation; the resuspended lentivirus liquid is concentrated, and can be stored at-80 ℃ after sub-packaging, and a small amount of concentrated lentivirus titer can be measured.

2.3.3.2 lentivirus titer assay

(1) Viral RNA extraction:

add 0.25ml RNAzol to 50 or 100. Mu.L of stock solution or 10. Mu.L of concentrate, dissolve virus 10 times upside down, incubate at room temperature for more than 15 minutes. Add 50. Mu.L of pure water to a 50. Mu.L virus stock tube or 10. Mu.L virus concentrate, and total virus solution volume to total water volume is 100. Mu.L. Centrifugation is carried out at 20℃and 18000g for 10min. The supernatant was transferred to a fresh 1.5ml centrifuge tube, and 1. Mu.L of 1.5mg/ml of linearlycramide was added to each 100. Mu.L of supernatant. Adding equal volume of isopropanol or three times volume of absolute ethanol, mixing, and standing in a refrigerator at-20deg.C for 4 hr or overnight. Centrifugation was performed at 10℃for 18000g for 20min, and the supernatant was discarded. RNA was washed twice with 0.5ml of 75% ethanol, each at 18000g, and centrifuged for 5min at 10 ℃. After drying in air for 3min, 50. Mu.L of water was added to dissolve RNA.

(2) Dnase I treatment: the reagents were added according to the following system: DEPCwater- -1.5. Mu. L, lentiviralRNA- -20. Mu. L, DNaseIbuffer (10X) - -2.5. Mu. L, DNaseI- -1.0. Mu.L, incubation at 37℃for 30-60 min followed by treatment at 75℃for 10min to inactivate DNaseI.

(3) Reverse transcription: the reagents were added according to the following system:

(DNaseI treated) RNA- -10. Mu.L, 4.0. Mu.L of DNAsynthesisprimer- -5. Mu.L at 70℃for 5min, after which the sample was cooled on ice.

The reagents were added according to the following system:

10 XReverseTranscriptionBuffer-2.0. Mu.L, 25mM DNTP-1.0. Mu. L, RNaseInhibitor-1.0. Mu. L, reverseTranscriptase-1.0. Mu.L, 60min at 37℃and 90℃for 10min, and the product was used directly for subsequent qPCR or stored in a refrigerator at-20 ℃.

(4) qPCR detection of viral titer: the reagents were added according to the following system: water- -6.0. Mu.L, 2Xall-in-Oneq PCRMIX- -10.0. Mu.L of qPCRcrimermix (2.5 uMeach) - -2.0. Mu. L, DNAstrandardorcDNAsampleorwater- -2.0. Mu.L.

The reaction was carried out according to the following procedure

Temperature	Interval	Duration	Read
				72--95℃	0.5℃	6sec/each	On
30℃		30sec	off

(5) Data analysis

CT value corresponds to standard curve copy number = CT value corresponds to titer x PCR system standard (sample) volume, virus titer = CT value corresponds to standard curve copy number x dilution (copies/ml)

Dilution fold = (RNA volume/virus sample volume) × (total DNaseI treated volume/RNA volume for DNaseI treated) × (total reverse transcription volume/RNA volume for reverse transcription) × (1000 μl/ml/volume for qPCR).

2.4 construction of stably transfected cell lines

2.4.1 cell culture

(1) Cell resuscitation: the temperature of the water bath was adjusted to 37 ℃. Frozen A549 and JT cells were removed from the liquid nitrogen tank according to the record, rapidly shaken in a 37℃water bath, rapidly transferred to a 15ml centrifuge tube containing PBS after dissolution, and centrifuged at 1000r/min for 5min. The supernatant was removed, the cells were resuspended in medium, and after air-blow mixing, the cell suspension was transferred to a T25 cell flask and incubated in a 5% co2 incubator at 37 ℃.

(2) Cell passage: when the cells grow to 80%, the culture solution in the cell culture flask is sucked and removed, the cells are washed by PBS for 2 times, a proper amount of 0.25% pancreatin is added for digestion, when most cells are rounded and separated from each other, a proper amount of RPMI1640 complete culture medium containing fetal bovine serum is added for stopping digestion, the cells are gently blown to prepare single cell suspension, and the single cell suspension is centrifuged for 5 minutes at 1000r/min and passaged for 1/3.

(3) Sequencing and identification: and extracting DNA from the JT and A549 cells after passage, sequencing, and comparing whether the ACSM5 has natural mutation or not, wherein the specific sequencing process is completed by Beijing qing biotechnology company.

2.4.2 infection of A549 and JT cells with wild-type and mutant ACSM5 lentiviruses

A549 cells and JT cells (106) with the growth density of 75% are infected by the slow virus and the culture medium according to the ratio of 1:1, and Polybrene with the final concentration of 8ug/ml is added to be beneficial to virus infection. 8h after infection, the old medium was replaced with fresh medium containing 5% FBS (inactivated) and 1% diabody and continued to be placed in C02 incubator. After 72h fresh medium containing puromycin at a concentration of 1.5ug/ml was added and infection positive cell selection was performed. The selection of infection positive cells was performed with the optimal concentration of puromycin at every 2 days. And (3) until the infection positive rate reaches more than 80%, carrying out passage expansion culture on the screened positive cells, and harvesting the cells for subsequent experiments. And respectively taking wild ACSM5 and mutant ACSM51 cells of the two strains of A549 and JT, and respectively carrying out qPCR, proliferation, migration, invasion, cycle, apoptosis and other detection by taking the normally cultured A549 and JT cells as blank strains and taking the A549 and JT cells PReceiver-Lv201 as empty strains.

2.4.3 detection of mRNA level Change of ACSM5 Gene associated with stably transfected cell lines

2.4.3.1 extraction of Total RNA from cells

(1) After the adherent cells to be tested are cultured, carefully pouring out the culture solution, lightly adding PBS prepared in advance, washing twice, centrifuging at 4000rpm for 10min, and discarding the supernatant for later use. (2) 1ml of Trizol reagent was added to the flask, the flask was shaken left and right to allow sufficient contact with the cells, and left to stand on ice for 10 minutes to allow the cells to lyse sufficiently. (3) After cell lysis, it was transferred to labeled and chilled 1.5ml RNaesEP-free tubes. (4) Chloroform was added in an amount of 200. Mu.L of chloroform added to 1ml of Trizol reagent, and the mixture was vigorously shaken manually for 15 seconds and allowed to stand at room temperature for 2 to 3 minutes. (5) 12000g, centrifuged at 4℃for 15 minutes, the visible liquid separated into three layers, the lower pink phenol and chloroform, the middle white, and the RNA remained in the colorless upper aqueous phase. (6) The upper aqueous phase was carefully aspirated into another EP tube, taking care not to aspirate the middle and lower layers (if aspirated carelessly, it was necessary to gently squeeze them out), followed by the addition of 0.5ml of 100% isopropyl alcohol, mixing upside down, and standing for 10 minutes (which can be placed in a-20℃refrigerator overnight when the sample volume is small to increase RNA extraction). (7) After centrifugation at 12000g for 10min at 4℃white RNA precipitate was formed at the bottom of the tube (no visible precipitate was observed with the naked eye at small sample volumes, nor did it affect normal operation). (8) The supernatant was discarded by gently tilting the nozzle, taking care not to pour out the pellet, sucking the nozzle with absorbent paper, adding 1ml of 75% ethanol to the pellet, floating the pellet by inverting the pellet to a few minutes, and standing for 2 minutes. (9) 7500g, 4℃for 5 minutes, and again the precipitate was attached to the bottom of the tube, this step was repeated twice as necessary to clean the impurities. (10) Discarding the supernatant, reversely buckling the centrifuge tube on absorbent paper, naturally drying for 10 minutes or drying in an ultra-clean workbench to volatilize ethanol and water as much as possible but not too dry, otherwise, influencing the subsequent dissolution of RNA. (11) Adding 30-50 mu L of RNase water into the dried RNA precipitate, standing for 15 minutes to thoroughly dissolve the RNA, taking 2 mu L for electrophoresis detection, taking 3 mu LRNA, diluting to 3ml with TAE buffer, detecting OD260 and OD280 by a spectrophotometer, and calculating the purity/concentration of the RNA and the loading amount of the following steps. (12) The remaining RNA was immediately reverse transcribed or frozen in a-80℃freezer.

2.4.3.2 electrophoresis detection

(1) And (3) glue preparation: the method comprises the steps of weighing 0.5g of agarose by an electronic balance, boiling for 1min in a microwave oven, dissolving in 50ml of TAE buffer solution (1×), cooling to 50-60 ℃, adding 5 mu L of nucleic acid dye, gently mixing, carefully pouring into a gel preparation groove in which a comb is inserted in advance, taking action to be gentle so as to prevent a large amount of bubbles, if the action is careless, puncturing the gel by a sterile gun head, standing for 40min, and naturally solidifying to obtain the 1% agarose gel. (2) sample loading: before loading, the prepared gel is put into an electrophoresis tank filled with TAE buffer solution, and bubbles in the gel holes are exhausted. The sample was applied in a ratio of 2. Mu.L of 1. Mu.L of the mixture to 1. Mu.LLoadingbuffer, with DNAmaroker of the same volume on the leftmost well or the rightmost well. (3) electrophoresis: the voltage is set to 150V during electrophoresis, and the electrophoresis time is 15-20 min. (4) judging: and (5) turning off the power supply after electrophoresis, taking out the gel block, putting the gel block into a gel imager, turning on an ultraviolet transmission lamp to observe an electrophoresis strip, judging the quality of RNA (18 s strip, 28s strip, brightness and the like), photographing and storing.

2.4.3.3 concentration determination

Starting a spectrophotometer in advance, taking 3 mu L of extracted RNA sample, diluting to the volume of 3ml by using TAE, zeroing by using TAE buffer solution, replacing the sample into a cuvette, respectively measuring OD260 and OD280, and calculating the ratio of the two, wherein if the ratio is between 1.8 and 2.0, the RNA purity is better. The concentration of RNA was then calculated and,

The formula is RNA concentration (ug/ml) =od 260 x dilution x 0.04.

2.4.3.4 mRNA expression level detection of AMSC5 gene

(1) Reverse transcription of mRNA to cDNA

Using Genecopoeia, guangzhou Co

SureScript-First-strand-cDNA-synthesis-kit, operated according to the instructions, was specified as follows: taking out the components of the reverse transcription kit, melting at room temperature, centrifuging briefly, placing on ice, and sequentially adding the reagents and solutions in the following table on the ice:

after brief centrifugation, reverse transcription was performed on a conventional PCR instrument according to the conditions in the following table:

note that, since the loading amounts of the surescript rtasemix (20×) and surescript reactionbuffer (5×) are extremely small, they can be mixed with a proper amount of ddH2O (RNase/dnafebre) in advance and then mixed with RNA, respectively, so that the loading accuracy can be improved, unnecessary errors can be reduced, and all the loading operations can refer to this method.

(2) RT-PCR (real-time fluorescent quantitative PCR) detection

The reverse transcription product cDNA is diluted 5-20 times (all samples must be diluted according to the same multiple, the concentration is too high or too low to influence the detection result, gradient verification can be performed first to determine the optimal dilution), and then the following Q-PCR reaction is performed. RT-PCR detection of mRNA BlazeTaq from Genecopoeia, guangzhou ^TM GreenqPCRMix2.0 kit, reagents in the following tables were added sequentially:

5X BlazeTaqqPCRMix, PCRforwardprimer (2. Mu.M), PCRreverse primer (2. Mu.M) and ddH2O (RNase/DNaserree) may be mixed in advance in the calculated ratio, and then mixed with cDNA in the total volume thereof, so that the loading accuracy can be improved.

When the sample loading operation is performed, whether bubbles exist in the gun head or not should be checked after the liquid is blown into the hole once, whether the liquid remains in the gun head or not should be checked after the liquid is blown into the hole once, if the liquid remains, the residual liquid instrument should be blown into the corresponding hole, and the sample loading operation must be performed at one time, other experimental operations can not be performed at the same time or things can not be done, so that the consistency of repeated holes is ensured.

After brief centrifugation, 40 cycles of reaction were performed on a CFX96 real-time quantitative fluorescent PCR instrument under the following conditions:

collecting and recording fluorescence, preparing an amplification curve and a dissolution curve, reading Ct values, and analyzing the relative expression quantity of ACSM 5. And (3) injection: the ACSM5 primer sequences are as follows:

the upstream primer is as follows: 5'-TTGTGGATGATGAGGGCAACG-3'

The downstream primer is: 5'-AGAAACAGAAGGGCCGAGTGG-3'

2.4.3.5 AMSC5 protein expression level detection: this step is the same as invention 2.2.

2.5 stable cell line function experiment

2.5.1 cell proliferation assay

A549 and JT cells (empty control cells, ACSM5 transfected wild-type or mutant cells) in logarithmic growth phase were separately digested and resuspended and then separately inoculated into 96-well plates at 100. Mu.L (2000 cells) per well, and after culturing for 24h, 48h, 72h, 96h and 120h, 10. Mu.LCCK 8 solution was added to each well, and the blank control was supplemented with the corresponding amounts of cell culture medium and CCK8 solution but no cells. The plates were incubated in the incubator for 4 hours. The absorbance at 450nm was measured with a microplate reader and the cell proliferation differences of each group were analyzed.

2.5.2 apoptosis detection

(1) After digestion of A549, JT cells (empty control cells, cells transfected with ACSM5 wild-type or mutant), the cell fluid was transferred to an EP tube, centrifuged at 1000rpm for 5min, and the supernatant was discarded. (2) The supernatant was rinsed twice with 1ml of pre-chilled PBS and the cells were resuspended to 1X 106 cells/ml with 1 Xbindingbuffer (10X, double distilled water dilution). (3) Transfer 100. Mu.L of cell suspension (105) to centrifuge tube, add 5. Mu.L of Annexin V-PE and 5. Mu.L of 7-AAD, mix and incubate at room temperature in the dark for 15min. (4) Then, 300. Mu.L of 1 Xbindingbuffer was added thereto, and the mixture was detected by an upflow meter within one hour. (5) The changes in apoptosis after transfection of lentiviruses were compared by taking the sum of the second quadrant (late apoptosis) and the fourth quadrant (early onset apoptosis).

2.5.3 cell cycle detection

(1) Preparation of the staining solution: and preparing corresponding PI staining solution according to the number of the samples.

(2) Collecting sample cells: the cell culture broth was carefully aspirated, and the cells were digested with pancreatin to prepare a single cell suspension. 1000g centrifugation for 3-5min, cell precipitation, supernatant removal, 1ml ice-bath precooled PBS rinse cell once. Cells were collected by centrifugation, resuspended in approximately 1ml of pre-chilled PBS, pelleted by centrifugation, the supernatant carefully aspirated, and resuspended in 1ml of ice-cooled PBS. The number of cells was 1X 10 ⁶ And each ml. (3) cell immobilization: 4ml of pre-chilled 95% ethanol was taken and vortexed at low speed while 1ml of cell suspension was added dropwise. The final concentration of ethanol is maintained between 70 and 75 percent. Immediately after mixing, the mixture was placed on ice. Cells were fixed at 4℃for 2 hours or longer, and the effect of fixation for 12-24 hours was better. The fixed cells can be stored at-20deg.C for 1 week at 4deg.C for 2 days. (4) cell resuspension: 1000g centrifugation for 3-5min, precipitation of cells, if insufficient precipitation for a particular cell, may be performed for a suitable prolonged period of centrifugation or slightly increased. The supernatant was carefully aspirated and about 50. Mu.L of ethanol could be left to avoid aspiration of the cells. About 5m of addition L ice-bath pre-chilled PBS, re-suspending the cells, collecting the cells by centrifugation, carefully pipetting the supernatant, and 50. Mu.L of PBS can be reserved to avoid pipetting the cells. The bottom of the centrifugal tube is lightly flicked to disperse cells properly and avoid cell agglomeration. (5) staining of cells: 500. Mu.L of the prepared staining solution was added to each tube of sample, and the cells were slowly and thoroughly resuspended. Incubate at 37℃for 30min in the absence of light. After the completion, the sample can be stored for a short time at 4 ℃ in a dark place, but the sample needs to be detected within 24 hours, and the flow detection is completed on the same day. (6) flow detection and analysis: detection was performed by flow cytometry at an excitation wavelength of 488nm, while detecting light scattering. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software modfit-1.

2.5.4 cell invasion assay

(1) The split-packed matrigel is diluted to 300ug/ml with culture medium in advance, 200 mu L is taken and added on the bottom poly carbonic acid membrane of a Transwell cell with the aperture of 8 mu m, so that all the apertures on the membrane can be covered by matrigel. Drying overnight at 37 ℃. (2) mu.L of five serum medium was added to the 24-well plate to just touch the bottom of the Transwell chamber. (3) A549, JT cells (no-load control cells, cells transfected with ACSM5 wild type or mutant type) were each suspended in 200 μl (in 10% fbs-containing complete medium), inoculated in a Transwell upper chamber, incubated in an incubator for 5h, medium was changed after cell attachment, the upper chamber was washed with PBS, serum-free medium was added, the lower chamber was removed after 24h incubation with 10% fbs-containing complete medium, fixed with methanol for 10min, methanol was discarded, dried (5) staining: staining with 0.5% crystal violet solution (formaldehyde) for 20min, cotton swab was used to wipe out PET membrane upper cells and again rinsed with PBS to background clarity (6) acetic acid decolorization to read OD values (7) step 4 (8) sealing and cell counting were repeated.

2.5.5 cell migration assay

(1) 500. Mu.L of serum-free medium was added to the 24-well plate to just touch the bottom of the Transwell chamber.

(2) A549, JT cells (empty control cells, cells transfected with ACSM5 wild-type or mutant), each group taken 3.5×10 each ⁴ The cells were suspended at 200. Mu.L/well (in complete medium with 10% FBS, inoculated)Culturing in a Transwell upper chamber for 5h in an incubator, replacing the culture medium after cell attachment, cleaning the upper chamber with PBS, adding serum-free culture medium, and culturing in a lower chamber with 10% FBS-containing complete culture medium for 24h. (3) Taking out after 24 hours, fixing with methanol for 10 minutes, discarding the methanol, and airing. (4) dyeing: dyeing with 0.5% crystal violet solution (prepared by formaldehyde) for 20min, wiping off cells on the upper part of the PET film by using a cotton swab, and rinsing again with PBS until the background is clear.

2.5.6 scratch test

(1) Cell preparation: a549, JT cells (empty control cells, cells transfected with ACSM5 wild-type or mutant) in the logarithmic growth phase were removed, the original culture broth was aspirated, and the cells were washed with sterile PBS. Cells were digested by adding 1m10.25% pancreatin digest, all cells were observed under a microscope to complete shrinkage and rounding, and complete medium was added to terminate digestion. The cell suspension was collected in a 15ml centrifuge tube and centrifuged at 800rpm/min for 5min at room temperature. Cell counting was performed with a high-precision flow image counter FILPLUS, the supernatant was discarded, and the cells were resuspended in medium. At a rate of 3.5 to 4 multiplied by 10 per hole ⁴ Cell density was seeded onto 6-well plates and cultured for one day in RPMI1640 medium with 10% fetal bovine serum. Preferably, the material can be spread overnight. (2) straight line scratch: cells confluent with six well plates were removed and the wells were scored from one end to the other with a 200. Mu.L gun head perpendicular to the cell surface. The transverse line scratch which is vertical to the back surface as much as possible is formed, and the gun head is vertical and cannot incline. At this time, the cell surface can be clearly seen to be a scratch in the shape of a # on the surface of the culture dish. (3) PBS washing cells and culturing: the old medium was aspirated, the cell surfaces of each group were washed 3 times with sterile PBS, the scraped cells were removed, and medium containing 1% fetal bovine serum was added. The six-well plate was gently shaken to cover the whole 6-well plate with the cell culture solution, and the whole was placed in a 5% C02 incubator at 37℃for culture. (4) observation of results: the scratch widths of the scratch tracks of different treatment groups are observed after the scratches are respectively taken for 0 hour, 24 hours and 48 hours. The results can be seen: the control group showed a recovery of about 90% in cell scratch width 48h after cell scratch, and the experimental group showed a recovery of about 60%, indicating that the cell migration of the experimental group was inhibited, while the control group maintained the original migration ability, and the scratch was masked by migration after 48 h.

2.5.7 plate clone formation experiments

(1) Cell preparation: the cells of each experimental group A549, JT (empty control cells, cells transfected with ACSM5 wild-type or mutant) in logarithmic growth phase were pancreatin digested, resuspended in complete medium, made into cell suspension, and counted. (2) cell seeding: about 100 cells/well (the normal proliferation speed is 1:5-1:10 passage, 3 days full cells can be inoculated with 50-200 cells, the rest proliferation is slow cells can be inoculated with 800-1000 cells) of each experimental group are inoculated in a 6-well culture plate, the gradient dilution of the cell suspension is noted during the inoculation, the cell density is observed so as not to cause the deviation of experimental results due to inaccurate counting, 3 multiple wells are arranged in each experimental group, and the culture medium is a complete culture medium containing 10% FBS. (3) Shaking the inoculated cells, then, putting the cells in an incubator for continuous culture, changing the liquid every 3 days, observing the cell state, and observing the clone size under a microscope. (4) About 10-14 days of culture, the culture was terminated when the number of cells in most of the individual clones in the wells was about 50, the supernatant was discarded, and the cells were washed 1 time with PBS.

(5) 1mL of 4% paraformaldehyde (toxic, note operating in a safety cabinet) was added to each well, cells were fixed in a refrigerator at 4℃for 60min, and the cells were washed 1 time with PBS. (6) 1000 mu L of clean and impurity-free crystal violet dye solution is added into each hole to dye cells for 2min. (0.5% to 0.1% in methanol in solvent, PBS in dilution), (50 mg 10ml methanol in, and then 5-fold dilution with PBS). (7) The cells were washed several times with ddH20, air-dried, photographed in a digital camera, and counted in clones.

2.5.8 ACSM5 and ACSM5-P425T drug resistance to paclitaxel

(1) Paclitaxel was diluted as follows: 0. 5, 10, 20, 40, 80 and 160. Mu.g/ml.

(2) After the cells of each experimental group are passaged and attached, paclitaxel with the concentration gradient diluted is added to continuously culture the cells, and the rest steps are the same as invention 2.5.1.

2.6 in vitro animal experiments

(1) Experimental grouping: BALB/c nude mice are randomly divided into three groups, namely A549-NC groups,

Groups A549-ACSM5-1 (mutant) and A549-ACSM5 (wild type), 6 in total, 18. (2) Before the experiment, three groups of cells, namely an A549-NC group, an A549-ACSM5-1 (mutant type) and an A549-ACSM5 group (wild type) in the logarithmic growth phase, are resuspended to 1X 106, and then are configured and fully mixed with matrigel according to the proportion of 1:1. (3) The three groups of cell suspensions were aseptically injected into the forelimb axilla of each nude mouse from 100 μl to the random group of step (1). (4) parameter recording: from the beginning of tumor formation, the tumor size (long diameter and short diameter) is measured once every other day, nude mice are sacrificed after two weeks, the complete tumor is taken, and after measurement and photographing, the nude mice are divided into two parts, part of the nude mice are fixed in paraformaldehyde (the subsequent pathological section is made), and part of the nude mice are frozen in a refrigerator at-80 ℃ for relevant detection such as PCR (polymerase chain reaction).

2.6 statistical analysis

Each experiment was repeated at least three times. Analysis was performed using SPSS21.0 software. The normalization test is performed before the data is analyzed. Normal data are represented by x±sd, and bias data are represented by median (P25-P75). The comparison between two groups of normal distribution data adopts t-test of two groups of independent samples, and the comparison between multiple groups adopts ANOVA single-factor variance analysis; the bias distribution data were compared between two groups using the Mann-WhitneyU test and between groups using the Kruskal-Wallis rank sum test. The count data is expressed as a rate (%), and the comparison between groups is performed using chi-square test. P < 0.05 is statistically significant for the differences.

Claims

The application of ACSM5-P425T in constructing a drug detection model for treating non-small cell lung cancer is characterized in that the ACSM5-P425T sequence is characterized in that the missense mutation of C base at 1237 locus in the functional sequence of the gene ACSM5 is A base, and the NCBI sequence number of the ACSM5 sequence is NM_017888.2;

the model overexpresses the ACSM5-P425T gene sequence.
2. The use according to claim 1, wherein the model is a cell line model or an animal model.
3. The use according to claim 2, wherein the cell line model construction method is: cell culture, construction of a target gene plasmid containing ACSM5-P425T gene sequence, carrying out slow virus packaging on the target gene plasmid, carrying out cell infection and detecting the target gene plasmid titer of slow virus packaging.
4. The use according to claim 2, wherein the animal model is constructed by the following method: and injecting cells which over-express ACSM5-P425T gene sequence into mice, and culturing to obtain the mice model.
5. The application of the recombinant plasmid in constructing a drug detection model for treating non-small cell lung cancer is characterized in that the recombinant plasmid contains ACSM5-P425T base sequence, the ACSM5-P425T sequence is C base missense mutation of 1237 locus in gene ACSM5 functional sequence to A base, and NCBI sequence number of the ACSM5 sequence is NM_017888.2.