CN105749295B - Application of high expression of RecQL4 gene as a target in the preparation of anti-gastric cancer drugs - Google Patents

Abstract

Application of high expression of RecQL4 gene as a target in the preparation of anti-gastric cancer drugs. The invention relates to the application of RecQL4 gene as a novel molecular marker in predicting the sensitivity and prognosis of gastric cancer chemotherapy. The present invention finds for the first time that the expression level of RecQL4 is highly consistent with the resistance of gastric cancer cells to the first-line chemotherapy drug cisplatin, and the RecQL4 gene inhibitor can be used as an effective ingredient in the anti-gastric cancer drug, and the inhibitor can also be combined with platinum The drug combination is used for the treatment of gastric cancer, and the expression level of RecQL4 can be used as an independent molecular marker to predict the sensitivity and prognosis of gastric cancer to chemotherapy drugs. The invention provides guiding significance for predicting the drug resistance and prognosis of gastric cancer chemotherapy, and creates a new standard for clinical guidance of medication and prognosis evaluation.

Description

Application of high expression of RecQL4 gene as a target in the preparation of anti-gastric cancer drugs

technical field

The invention relates to the fields of biochemistry and molecular biology, in particular to the application of high expression of RecQL4 gene as a target in the preparation of anti-gastric cancer drugs.

Background technique

Gastric cancer ranks second among all cancers in terms of morbidity and mortality, and has an increasing trend year by year. Currently, the clinical treatment of gastric cancer is mainly based on platinum-based chemotherapy, but even for patients of the same type and disease period In terms of treatment effects, there will also be significant differences (Shah MA.J Clin Oncol. 2015). At the same time, in the course of clinical chemotherapy, gastric cancer patients often develop resistance to chemotherapeutic drugs, which eventually leads to a significant decrease in the therapeutic effect. Therefore, how to better predict the sensitivity of gastric cancer to chemotherapy drugs such as cisplatin is of great significance for the clinical treatment and diagnosis of gastric cancer.

As a member of the human RecQ helicase family, RecQL4's main function is to open DNA double strands and play an important role in DNA replication, transcription, damage repair and maintenance of genome stability (Croteau DL. TrendsGenet 2012; Dietschy T, Life Sci, 2007; Larizza L, Cancer Lett, 2006). Mutations in the RecQL4 gene lead to RTS syndrome (Rothmund-Thomson syndrome), RAPADILLINO syndrome (Siitonen HA, AmJ Hum Genet. 2003), and BGS syndrome (Baller-Gerold syndrome), which are characterized by premature aging and high tumor incidence ( Siitonen, Eur J Hum Genet 2009; Wang LL, Cancer Inst, 2003). Previous studies have shown that the RecQL4 gene is abnormally highly expressed in osteoblastoma (Maire G, Neoplasia. 2009), breast cancer (Fang HB, PLoSOne. 2013) and prostate cancer (Su Y, Cancer Res. 2010). Its expression in gastric cancer and its relationship with the sensitivity of gastric cancer to drug-resistant chemotherapy have not been reported.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of high expression of RecQL4 gene as a target in the preparation of anti-gastric cancer drugs.

In order to achieve the purpose of the present invention, the high expression of RecQL4 gene provided by the present invention is used as a target in the preparation of anti-gastric cancer drugs, and a RecQL4 gene inhibitor is used as an effective ingredient in the anti-gastric cancer drug.

The present invention also provides an anti-gastric cancer drug, the active ingredient of which is a RecQL4 gene inhibitor. The inhibitor is at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compound, peptide, antibody and the like.

Specifically, the RecQL4 gene inhibitor provided by the present invention is shRNA, which is a hairpin structure containing the following base sequence: 5'-GCTCAAGGCCAATCTGAAAGG-3'.

The present invention also provides an anti-gastric cancer combined medicine, the effective components of which are the above-mentioned anti-gastric cancer medicine and platinum-based medicine.

The present invention also provides the application of the RecQL4 gene as a marker in the preparation of a gastric cancer detection and/or therapeutic effect evaluation reagent. The curative effect refers to the curative effect of chemotherapy based on platinum drugs for the treatment of gastric cancer.

The platinum drug involved in the present invention is cisplatin, a first-line chemotherapy drug.

The present invention further provides the application of the RecQL4 gene as a marker in the preparation of a reagent for evaluating the sensitivity and/or prognosis of gastric cancer to the chemotherapeutic drug cisplatin.

In the present invention, by detecting the expression level of RecQL4 protein in 7 gastric cancer cells (MGC-803, AGS, HGC-27, MKN45, NCI-N87, SUN-1, SNU-16), it is found that RecQL4 protein in all 7 gastric cancer cells The expression levels were significantly higher than the control samples (Figure 1). Subsequently, 11 pairs of gastric cancer clinical samples and adjacent controls were collected to detect the expression of RecQL4 protein. It was found that 8 pairs of gastric cancer samples of the 11 pairs of clinical samples had significantly higher RecQL4 expression levels than the adjacent samples (Figure 2). The above results indicated that the expression of RecQL4 gene was significantly increased in gastric cancer.

In order to verify the expression level of RecQL4 gene in gastric cancer and its relationship with the sensitivity of gastric cancer drug resistance to chemotherapy, the present invention detected the sensitivity of 7 gastric cancer cells to clinical first-line chemotherapy drug cisplatin, and the results showed that gastric cancer with relatively high expression of RecQL4 Cells (AGS, NCI-N87, SNU-16) were also relatively resistant to cisplatin, while gastric cancer cells with relatively low RecQL4 expression (SNU-1, MGC-803) were also relatively sensitive to cisplatin (Fig. 3). Although MKN45 cells with relatively low expression of RecQL4 and HGC-27 cells with relatively high expression of RecQL4 showed relatively moderate cisplatin resistance, the overall resistance trend still clearly indicates that the expression level of RecQL4 is related to the resistance of gastric cancer cells to cisplatin. Platinum resistance is closely related: cells with relatively high RecQL4 expression are more resistant to cisplatin, and cells with relatively low RecQL4 expression are more sensitive to cisplatin. Therefore, the expression level of RecQL4 can be used as an independent molecular marker to predict the sensitivity of gastric cancer to chemotherapy.

In order to further verify the relationship between RecQL4 and the drug resistance of gastric cancer cells, the present invention carried out a gastric cancer cell chemotherapy drug resistance detection (MTT method) experiment, as follows:

Three gastric cancer cells with relatively low RecQL4 expression (MKN45, SUN-1, MGC-803) were used to transfect the RecQL4 gene expression plasmid to significantly increase the expression level of RecQL4 in these three cells (upper Figure 4), and then detect Whether the resistance of cells to cisplatin changed after the expression of RecQL4 increased. The results showed that when the expression level of RecQL4 was increased, the resistance of cells to cisplatin was also significantly increased (Figure 4 bottom). Then, three gastric cancer cells with relatively high expression of RecQL4 (NCI-N87, AGS, HGC-27) were used to inhibit the expression of RecQL4 by adenovirus infection, and the resistance of gastric cancer cells to cisplatin was detected after the expression of RecQL4 was inhibited. The results showed that when the expression of RecQL4 was inhibited (the upper part of Fig. 5), the resistance of gastric cancer cells to cisplatin was also significantly reduced (the lower part of Fig. 5). It showed that the high expression of RecQL4 gene can significantly increase the resistance of gastric cancer cells to cisplatin. The above results show that the expression level of RecQL4 is highly consistent with the resistance of gastric cancer cells to cisplatin, that is, the level of RecQL4 expression can be used as a molecular marker to predict the resistance of gastric cancer cells to cisplatin.

The invention relates to the application of RecQL4 gene as a novel molecular marker in predicting the sensitivity and prognosis of gastric cancer chemotherapy. The present invention finds for the first time that the expression level of RecQL4 is highly consistent with the resistance of gastric cancer cells to the first-line chemotherapy drug cisplatin, and the RecQL4 gene inhibitor can be used as an effective ingredient in the anti-gastric cancer drug, and the inhibitor can also be combined with platinum The drug combination is used for the treatment of gastric cancer, and the expression level of RecQL4 can be used as an independent molecular marker to predict the sensitivity and prognosis of gastric cancer to chemotherapy drugs. The invention provides guiding significance for predicting the drug resistance and prognosis of gastric cancer chemotherapy, and creates a new standard for clinical guidance of medication and prognosis evaluation.

Description of drawings

FIG. 1 is the experimental result of the protein expression level of RecQL4 in human gastric cancer cell line in Example 1 of the present invention. Among them, Normal is a human normal gastric tissue sample, and MGC-803, AGS, HGC-27, MKN45, NCI-N87, SUN-1, and SNU-16 are 7 gastric cancer cell lines. Figure 1 shows the use of Western blot method to extract the protein supernatant of each cell line, after separation by denaturing polyacrylamide electrophoresis, transfer to nitrocellulose membrane, then incubate with anti-RecQL4 primary antibody, and then with matching secondary antibody After incubation, chemiluminescence was used to observe the intensity of RecQL4 protein bands. GAPDH levels were used to calibrate the amount of protein spiked per cell line.

FIG. 2 is the experimental result of the protein expression level of RecQL4 in human gastric cancer clinical samples in Example 1 of the present invention. Among them, N represents normal paracancerous tissue, and T represents gastric cancer tissue. In this figure, the protein supernatant of each pair of gastric cancer tissues was extracted by Western blot method, separated by denaturing polyacrylamide electrophoresis, transferred to nitrocellulose membrane, incubated with anti-RecQL4 primary antibody, and then mixed with matching secondary antibody. After anti-incubation, the intensity of the RecQL4 protein band was observed by chemiluminescence. β-actin levels were used to calibrate the amount of protein spiked for each sample.

FIG. 3 is the experimental result of the resistance of seven human gastric cancer cells to cisplatin in Example 2 of the present invention. Among them, 5 concentration gradients (0, 0.5, 1, 1.5, 2, 2.5 μg/ml) were set for cisplatin, and 10 parallel repetitions were set for each concentration gradient. Each point is the mean ± standard deviation, and it is the result of three independent repeated experiments.

FIG. 4 is the experimental result of overexpression of RecQL4 and resistance to cisplatin in the gastric cancer cell line with low expression of RecQL4 in Example 3 of the present invention. Among them, the picture above shows that gastric cancer cells were transfected with the expression vector to increase the expression level of RecQL4, and then the cell protein supernatant was extracted, and the level of RecQL4 was detected by Western blot method. The following figure shows the resistance of cells to cisplatin detected by MTT method after the level of RecQL4 was changed. Five concentration gradients (0, 0.5, 1, 1.5, 2, 2.5 μg/ml) were set for cisplatin, and each concentration gradient was set. 10 parallel repetitions. Each point is the mean ± standard deviation, and it is the result of three independent repeated experiments.

FIG. 5 is the experimental result of virus inhibition of RecQL4 expression and resistance to cisplatin in a gastric cancer cell line with high RecQL4 expression in Example 3 of the present invention. Among them, the above picture shows that the expression of RecQL4 in gastric cancer cells was inhibited by adenovirus infection, and then the cell protein supernatant was extracted, and the level of RecQL4 was detected by Western blot method. The following figure shows the resistance of cells to cisplatin detected by MTT method after the level of RecQL4 was changed. Five concentration gradients (0, 0.5, 1, 1.5, 2, 2.5 μg/ml) were set for cisplatin, and each concentration gradient was set. 10 parallel repetitions. Each point is the mean ± standard deviation, and it is the result of three independent repeated experiments.

FIG. 6 is the experimental result of virus inhibition of RecQL4 expression and resistance to cisplatin in breast cancer cells in Example 4 of the present invention. Adenovirus infection was used to inhibit the expression of RecQL4 in breast cancer cells, and then the cell protein supernatant was extracted, and the level of RecQL4 was detected by Western blot. The following figure shows the resistance of cells to cisplatin detected by MTT method after the level of RecQL4 was changed. Five concentration gradients (0, 0.5, 1, 1.5, 2, 2.5 μg/ml) were set for cisplatin, and each concentration gradient was set. 10 parallel repetitions. Each point is the mean ± standard deviation, and it is the result of three independent repeated experiments.

7 is the experimental result of virus inhibition of RecQL4 expression and resistance to cisplatin in intestinal cancer cells in Example 4 of the present invention. Adenovirus infection was used to inhibit the expression of RecQL4 in colorectal cancer cells, and then the cell protein supernatant was extracted, and the level of RecQL4 was detected by Western blot. The following figure shows the resistance of cells to cisplatin detected by MTT method after the level of RecQL4 was changed. Five concentration gradients (0, 0.5, 1, 1.5, 2, 2.5 μg/ml) were set for cisplatin, and each concentration gradient was set. 10 parallel repetitions. Each point is the mean ± standard deviation, and it is the result of three independent repeated experiments.

FIG. 8 is the experimental result of the protein expression level of RecQL4 in human gastric cancer cell line in Example 5 of the present invention. Among them, Normal is a human normal gastric tissue sample, and MGC-803, BGC-823, AGS, HGC-27, MKN45, NCI-N87, and SGC-7901 are 7 gastric cancer cell lines. The protein supernatant of each cell line was extracted by Western blot method, separated by denaturing polyacrylamide electrophoresis, transferred to nitrocellulose membrane, incubated with anti-RecQL4 primary antibody, and then incubated with matching secondary antibody. The intensity of RecQL4 protein bands was observed by chemiluminescence. GAPDH levels were used to calibrate the amount of protein spiked per cell line.

FIG. 9 is the experimental result of drug resistance of three human gastric cancer cell lines MGC-803, BGC-823 and SGC7901 to cisplatin in Example 5 of the present invention.

FIG. 10 is the experimental result of the drug resistance of 6 human gastric cancer cells to 4-NQO in Example 5 of the present invention. Five concentration gradients (0, 0.1, 0.2, 0.3, 0.4, 0.5 μg/ml) were set for 4-NQO drug, and 10 parallel replicates were set for each concentration gradient. The results are the results of three independent replicate experiments.

FIG. 11 is the experimental result of the drug resistance of 6 human gastric cancer cells to 5-FU in Example 5 of the present invention. Sensitivity curve of 6 gastric cancer cells to 5-FU treatment. Five concentration gradients (0, 10, 50, 100, 150, 200 μg/ml) were set for 5-FU drug, and 10 parallel replicates were set for each concentration gradient. The results are the results of three independent replicate experiments.

FIG. 12 is the experimental result of the resistance of 6 human gastric cancer cells to doxorubicin in Example 5 of the present invention. Sensitivity curve of 6 gastric cancer cells to doxorubicin treatment. Five concentration gradients (0, 0.2, 0.4, 0.6, 0.8, 1 μg/ml) were set for doxorubicin, and 10 parallel replicates were set for each concentration gradient. The results are the results of three independent replicate experiments.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the examples are in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Sources of cell lines involved in the following examples: AGS, NCI-N87, SNU-1, SUN-16 were purchased from ATCC, MKN45 was purchased from European ECACC cell bank, HGC-27 cells were purchased from Japan JCRB cell bank, MGC-803, BGC-823 and SGC7901 cells were purchased from the Basic Medical Cell Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences.

The methods for detecting the expression of RecQL4 gene in gastric cancer and the methods for detecting drug resistance of RecQL4 and gastric cancer cells involved in the following examples are as follows:

1. Identification of RecQL4 mRNA levels in gastric cancer clinical tissues and gastric cancer cell lines

The total RNA of tumor cells was extracted by guanidine lysis reagent, and then the RNA was reverse transcribed into template cDNA by reverse transcriptase. The RecQL4 mRNA level was then quantitatively analyzed by real-time quantitative PCR (RT-qPCR).

2. Western Blot identification of RecQL4 protein levels in tumor cells and clinical gastric cancer samples

The harvested human tissue or cells are homogenized in lysis buffer and centrifuged to obtain the protein supernatant. Proteins were then separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with RecQL4 antibody (SDIX Cat. #25470002). It was then incubated with a secondary antibody (Vector LaboratoriesCat. #PI-1000) matched to the RecQL4 antibody, and the protein expression level of RecQL4 in human tissues or cells was detected by chemiluminescence.

3. Detection of chemotherapy drug resistance in gastric cancer cells (MTT method)

Collect log-phase cells, adjust the concentration of cell suspension, add 5000 cells per well, and the volume is 100 μl. After culturing in the incubator for 24 hours, add the concentration gradient drug, continue to culture for 72 hours, blot the medium, add 100 microliters of fresh medium and 10 microliters of 5 mg/ml MTT solution to each well, continue to culture for 4 hours, and then drain the medium , add 100 microliters of dimethyl sulfoxide to each well, shake for 10 minutes, and measure the absorbance on a microplate reader. Data were recorded and cell viability curves were drawn.

Example 1 Abnormally high expression of RecQL4 gene in gastric cancer cells and clinical tissues

In this example, the expression level of RecQL4 protein in 7 gastric cancer cells (MGC-803, AGS, HGC-27, MKN45, NCI-N87, SUN-1, SNU-16) was detected, and it was found that RecQL4 in all 7 gastric cancer cells The protein expression levels were significantly higher than those in the control samples (Figure 1). Subsequently, 11 pairs of gastric cancer clinical samples and adjacent controls were collected to detect the expression of RecQL4 protein. It was found that 8 pairs of gastric cancer samples of the 11 pairs of clinical samples had significantly higher RecQL4 expression levels than the adjacent samples (Figure 2). The above results indicated that the expression of RecQL4 gene was significantly increased in gastric cancer.

Example 2 The expression level of RecQL4 is closely related to the drug resistance of gastric cancer cells

In order to detect the correlation between RecQL4 and the drug resistance of gastric cancer cells, the sensitivity of 7 gastric cancer cells to the clinical first-line chemotherapy drug cisplatin was tested in this example, and the results showed that gastric cancer cells (AGS, NCI-N87, SNU- 16) The resistance to cisplatin was also relatively high, while gastric cancer cells with relatively low RecQL4 expression (SNU-1, MGC-803) were also relatively sensitive to cisplatin (Figure 3). Although MKN45 cells with relatively low RecQL4 expression and HGC-27 cells with relatively high RecQL4 expression showed relatively moderate cisplatin resistance, the overall drug resistance trend still clearly shows that the level of RecQL4 expression is related to the resistance of gastric cancer cells to cisplatin Platinum resistance is closely related: cells with relatively high RecQL4 expression are more resistant to cisplatin, and cells with relatively low RecQL4 expression are more sensitive to cisplatin. Therefore, the expression level of RecQL4 can be used as an independent molecular marker to predict the sensitivity of gastric cancer to chemotherapy.

Example 3 RecQL4 enhances the resistance of gastric cancer cells to cisplatin

In order to further verify the relationship between RecQL4 and the drug resistance of gastric cancer cells, in this example, three gastric cancer cells (MKN45, SUN-1, MGC-803) with relatively low expression of RecQL4 were used to transfect the expression plasmid containing the RecQL4 gene to make the 3 gastric cancer cells. The expression level of RecQL4 in the cell line was significantly increased (Figure 4 upper), and then whether the resistance of the cells to cisplatin changed after the increased expression of RecQL4 was detected. The results showed that when the expression level of RecQL4 was increased, the resistance of cells to cisplatin was also significantly increased (Figure 4 bottom). Then, three gastric cancer cells with relatively high expression of RecQL4 (NCI-N87, AGS, HGC-27) were used to inhibit the expression of RecQL4 by adenovirus infection, and the resistance of gastric cancer cells to cisplatin was detected after the expression of RecQL4 was inhibited. The results showed that when the expression of RecQL4 was inhibited (the upper part of Fig. 5), the sensitivity of gastric cancer cells to cisplatin was also significantly increased (the lower part of Fig. 5). It showed that the high expression of RecQL4 gene can significantly increase the resistance of gastric cancer cells to cisplatin. These results indicate that the expression level of RecQL4 is highly consistent with the resistance of gastric cancer cells to cisplatin, that is, the level of RecQL4 expression can be used as a molecular marker for predicting the resistance of gastric cancer cells to cisplatin.

The specific method is as follows:

1. Design a hairpin structure containing 21 bases according to the RecQL4 mRNA sequence. After being synthesized by a DNA synthesizer, it was ligated into the pShuttle vector to obtain a recombinant plasmid.

shRecQL4: 5'-GCTCAAGGCCAATCTGAAAGG-3' (SEQ ID NO: 1)

shControl: 5'-GAAGAGGACACGCCTTAGACT-3' (SEQ ID NO: 2)

2. Packaging and infection of adenovirus

1) Take 100ng of the recombinant plasmid to linearize with PmeI single enzyme digestion.

2) Transfer to BJ5183 competent cells containing pAdEasy, spread on LB plates containing kana antibiotics, and pick smaller single colonies.

3) The extracted plasmid is identified by PacI single enzyme digestion. If agarose gel electrophoresis shows a large fragment (about 30kb) and a small fragment (about 3.0 or 4.5kb), it is a positive recombinant adenovirus plasmid, and other Enzyme cleavage identification.

4) The recombinant adenovirus plasmid is re-transferred into DH5α, or stored in other recombinant-deficient strains.

5) 5 μg of recombinant adenovirus plasmid was linearized by single enzyme digestion with Pad, and purified with phenol-chloroform.

6) The day before transfection, HEK293 cells were inoculated in a 35mm petri dish, and cultured in high glucose DMEM containing 10% FBS, and the cells were grown to about 80% of the confluent petri dish on the day of transfection.

7) Transfect 2 μg of the linearized recombinant adenovirus plasmid according to the instructions of the transfection reagent.

8) Cultivate for 10-14 days until about 80% of the cells come off the wall, during which it can be observed whether plaques are formed. The medium cannot be changed during cultivation, and 1 ml of medium can be added.

9) Directly blow up the adherent cells, transfer the cell suspension to a 1.5ml EP tube, and freeze and thaw three times in liquid nitrogen/37°C.

10) Centrifuge at 12,000g at 4°C for 5 min, take the supernatant, quick-freeze in liquid nitrogen for amplification or aliquot and store at -80°C, marked as the first generation.

11) Inoculate HEK293 cells in a cell culture flask with a bottom area of 25 cm ² one day before an expansion, and grow to about 80% of the culture dish on the day of transfection. Take 500 μl of virus solution for one generation and add it to T25, and culture for 3-5 days, until about 70% of cells are detached.

12) Directly blow up the adherent cells, transfer the cell suspension to a 1.5ml EP tube, and freeze and thaw three times in liquid nitrogen/37°C.

13) Centrifuge at 12,000g at 4°C for 5min, take the supernatant, quick-freeze in liquid nitrogen for amplification or aliquot and store at -80°C, and mark it as the second generation.

14) Inoculate HEK293 cells in a cell culture flask with a bottom area of 75 cm ² one day before the secondary expansion, and grow to about 80% of the culture dish on the day of transfection. 500 μl of the second generation virus solution was added to T75 and cultured for 3 to 5 days until about 70% of the cells were detached.

15) Blow up the adherent cells directly, transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 4,000rpm for 10min, remove 11ml of cell culture medium, leave 4ml of culture medium for resuspension, and freeze and thaw 4 times in liquid nitrogen/-37°C. .

16) Centrifuge at 12,000g at 4°C for 5 min, take the supernatant, quick-freeze in liquid nitrogen and store at -80°C, mark it as the third passage, and use it to infect target cells.

17) Directly add an appropriate amount of virus liquid to the cells, and after 6-24 hours of infection (depending on the cell type), change to fresh medium to continue culturing.

Example 4 The overexpression of RecQL4 in breast and intestinal cancer cells does not affect the resistance of cells to cisplatin

In order to detect whether the expression level of RecQL4 is significantly correlated with the drug resistance of other types of tumors, in this example, two breast cancer cells (MCF-7, MDA-MB436) and one intestinal cancer cell (SW-620) were used for adenovirus Infection inhibits the expression of RecQL4 and then detects whether the resistance of cells to cisplatin changes. The results showed that when the expression of RecQL4 was significantly inhibited, the resistance of breast cancer cells (Fig. 6) and intestinal cancer cells (Fig. 7) to cisplatin did not change significantly. It showed that RecQL4 had no significant correlation with the drug resistance of breast cancer and intestinal cancer cells.

Example 5 There is no significant correlation between the expression level of RecQL4 and the sensitivity of gastric cancer cells to other chemotherapeutic drugs

In order to detect the level of RecQL4 expression and the sensitivity of gastric cancer cells to other chemotherapeutic drugs, such as 4-nitroquinoline-1-oxide (4-NQO), 5-fluorouracil (5-FU), and doxrubincin To determine whether sex is highly correlated with cisplatin, 7 gastric cancer cells (MGC-803, BGC-823, AGS, HGC-27, MKN45, NCI-N87, SGC7901) were further selected and the expression level of RecQL4 was detected. The results are shown in Figure 8. The expression of RecQL4 in gastric cancer cells MGC-803, BGC-823 and MKN45 was relatively low, the expression of SGC-7901 was relatively moderate, and the expression of AGS, HGC-27 and NCI-N87 was relatively high. Subsequently, the resistance of three gastric cancer cell lines (MGC-803, BGC-823, SGC7901) to cisplatin was detected. The results are shown in Figure 9. The cells with relatively low RecQL4 expression (MGC-803, BGC-823) It is more sensitive to cisplatin, and SGC7901 cells with relatively moderate RecQL4 expression are much more resistant to cisplatin than the other two cell lines with relatively low RecQL4 expression, indicating that gastric cancer cell lines MGC-803, BGC-823 and SGC7901 The expression level of RecQL4 was highly correlated with cisplatin resistance. Three cell lines with relatively low expression of RecQL4 (MGC-803, BGC-823, MKN450), one cell line with relatively moderate expression of RecQL4 (SGC-7901) and two cell lines with relatively high expression of RecQL4 (AGS, HGC-27) to detect the sensitivity of these 6 cells to the chemotherapeutic drugs 4-NQO, 5-FU and doxrubincin, respectively. The results are shown in Figure 10. SGC7901 cells have high resistance to 4-NQO but their RecQL4 level is relatively moderate, MKN45 cells have relatively low RecQL4 expression levels but their resistance to 4-NQO is at a moderate level, MGC- 803, BGC-823, AGS and HGC-27 cells all had high sensitivity to 4-NQO, but MGC-803 and BGC-823 cells had relatively low levels of RecQL4 expression, while AGS and HGC-27 cells had RecLQ4 expression levels Relatively high. The above results indicated that the expression level of RecQL4 was not significantly correlated with the drug resistance of gastric cancer cells to 4-NQO. In addition, the resistance of these 6 cell lines to the chemotherapeutic drugs 5-FU (Fig. 11) and doxrubincin (Fig. 12) was also detected. It was also found that the expression level of RecQL4 was not significantly related to the sensitivity of gastric cancer cells to the two drugs. related. In conclusion, the expression level of RecQL4 is only highly correlated with the resistance of gastric cancer cells to cisplatin, and has no significant relationship with the resistance of gastric cancer cells to other chemotherapeutic drugs. A new approach to cisplatin drug sensitivity prediction.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (7)

1. Application of the 1.RecQL4 gene inhibitor in preparation anti-gastric cancer medicament.
2. 2. application according to claim 1, which is characterized in that the RecQL4 gene inhibitor be shRNA, siRNA, At least one of dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compound, peptide, antibody etc..
3. 3. application according to claim 2, which is characterized in that the RecQL4 gene inhibitor be shRNA, for containing The hair pin type structure of following base sequence: 5 '-GCTCAAGGCCAATCTGAAAGG-3 '.
4. Application of the 4.RecQL4 gene as marker in the detection of preparation gastric cancer and/or therapeutic evaluation reagent.
5. 5. application according to claim 4, which is characterized in that the curative effect refers to for curing gastric cancer with platinum medicine Based on chemotherapeutic efficacy.
6. 6. application according to claim 5, which is characterized in that the platinum medicine is cis-platinum.
7. 7.RecQL4 gene is as marker in preparing sensibility and/or prognostic evaluation reagent of the gastric cancer to chemotherapeutic drugs Cisplatin Application.