CN113908279B - Application of MALT1 gene as marker in preparation of drug for treating colorectal cancer - Google Patents

Abstract

The invention discloses an application of MALT1 gene as a marker in preparing a medicine for treating colorectal cancer. The nucleotide sequence of the MALT1 gene is shown in SEQ ID NO. 1. The invention provides a novel colorectal cancer diagnosis marker MALT1 gene, which can effectively improve the sensitivity of colorectal cancer cells to MEK inhibitors and synergistically promote the inhibition effect of the MEK inhibitors on colorectal cancer by knocking down or inhibiting the MALT1 gene.

Description

Application of MALT1 gene as marker in preparation of drug for treating colorectal cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a MALT1 gene as a marker in preparation of a medicine for treating colorectal cancer.

Background

Colorectal Cancer (CRC) is a common malignancy worldwide. The results of the global cancer statistics report in 2018 show that the incidence and mortality of colorectal cancer are in the top three in common tumors. Colorectal cancer occurs in addition to genetic factors, to a large extent in connection with the dietary habits and lifestyles of people.

The early colorectal cancer patient is usually treated by surgical resection and is treated by some auxiliary medicines, so that the early colorectal cancer patient has a better treatment effect; patients with advanced disease or diagnosed unresectable disease are usually treated with chemotherapy, molecular targeted therapy, etc., but during treatment, patients often develop resistance problems, so the clinical prognosis of colorectal cancer is still not satisfactory, especially when CRC patients develop lymph node metastasis, the prognosis is worse.

In targeted therapy of colorectal cancer, the EGFR mab drug Cetuximab was first approved for the treatment of metastatic colorectal cancer. It can be used as first-line clinical therapeutic drug for KRAS wild type colorectal cancer patients. However, in colorectal cancer patients, KRAS mutant patients account for approximately 50%, and clinical studies have shown that patients carrying this mutation have a very low response rate to treatment with EGFR-related inhibitors. The low response rate caused by drug resistance of targeted therapy of colorectal cancer seriously restricts the clinical effect of targeted therapy of colorectal cancer, and becomes a problem which needs to be solved urgently in clinical treatment. The clinical benefit of targeted MAPK therapy for colorectal cancer patients is not high due to the activation of various compensatory transduction pathways that induce cell proliferation and survival, and the complexity of the signaling network, caused by gene mutations. The drug resistance problem of colorectal cancer needs to be solved urgently.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an application of a MALT1 gene as a marker in the preparation of a medicine for treating colorectal cancer, provides a novel colorectal cancer diagnosis marker MALT1 gene which can be used as a colorectal cancer treatment target and used for preparing a colorectal cancer treatment medicine and improving the sensitivity of colorectal cancer cells to MEK inhibitors.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problem is as follows:

the MALT1 is used as a marker in the application of preparing drugs for treating colorectal cancer, and the nucleotide sequence of the MALT1 gene is shown in SEQ ID No. 1.

An application of MALT1 gene expression inhibitor or knockout reagent in preparing medicine for treating colorectal cancer can inhibit proliferation of colorectal cancer cells and promote apoptosis.

Further, the concentration of the MALT1 gene expression inhibitor or knock-out agent is not less than 0.8. Mu.M.

An application of MALT1 gene expression inhibitor or knockout reagent in preparing adjuvant medicine for improving the therapeutic effect of colorectal cancer, the adjuvant medicine can inhibit the proliferation of colorectal cancer cells and promote apoptosis.

Further, the colorectal cancer is KRAS mutated colorectal cancer.

A kit for colorectal cancer diagnosis or prognosis detection comprises a reagent for detecting the expression quantity of a MALT1 gene.

Further, the kit comprises a reagent that specifically recognizes the MALT1 gene.

Further, the agent specifically recognizing MALT1 gene is selected from: primers for specifically amplifying MALT1 gene; or a probe that specifically recognizes MALT1 gene.

A combination comprising an inhibitor or knock-out agent of MALT1 gene expression and an inhibitor of colorectal cancer for the treatment of colorectal cancer.

Further, the colorectal cancer inhibitor is a MEK inhibitor or a MAPK inhibitor.

Further, inhibition of MALT1 gene expression can reduce the tolerance of cells to MEK inhibitors.

Further, the concentration of the MALT1 gene expression inhibitor in the combination drug is more than 0.2 μ M, and the concentration of the colorectal cancer inhibitor is not less than 0.25 μ M.

Further, the concentration of the MALT1 gene expression inhibitor in the combination drug is 0.4. Mu.M, and the concentration of the colorectal cancer inhibitor is 0.25. Mu.M.

Further, the concentration of the MALT1 gene expression inhibitor in the combination drug is 0.4. Mu.M, and the concentration of the colorectal cancer inhibitor is 0.5. Mu.M.

Furthermore, the combined medicine is a medicine capable of inhibiting the proliferation of colorectal cancer cells and promoting apoptosis.

The invention has the beneficial effects that:

the invention provides a novel colorectal cancer diagnosis marker MALT1 gene, which can effectively improve the sensitivity of colorectal cancer cells to MEK inhibitors and synergistically promote the inhibition effect of the MEK inhibitors on colorectal cancer by knocking down or inhibiting the MALT1 gene.

Drawings

FIG. 1 is a graph showing the effect of knockdown of MALT1 on proliferation of HCT116 cells. (A) detecting the knocking-down efficiency of shRNA on MALT1 by q-PCR; (B) MTS 72 hour assay control (plko.1) and MALT1 knockdown cells respond to MEKi; (C) Long-term (12-14 days) clonogenic assays detect the response of PLKO.1 and MALT 1-knockdown cells to MEKi;

FIG. 2 is a graph showing that MTS detects the response of HCT116 cells to MALT1 inhibitor-MI-2 at 72 hours;

FIG. 3 is a graph of the effect of MALT1 inhibitors and MEK inhibitors on the long-term proliferation of cellular HCT 116; long-term (12-14 days) clonogenic experiments examined the response of HCT116 to MI-2, AZD, MI-2+ AZD. Treating the cells with MI-2 of different concentrations (0, 0.2, 0.4, 0.8, 1, 2 μ M) and AZD6244 of different concentrations (0, 0.125, 0.25, 0.5 μ M) every other day for 12-14 days, and staining the cells with 0.05% crystal violet staining solution;

FIG. 4 shows a Western blot to detect the effect of MALT1 inhibitor and MEK inhibitor on NF- κ B expression in HCT 116; (A) The effect of different concentrations of MI-2 and 1. Mu.M AZD on total p-NF- κ B expression of HCT 116; (B) The effects of 2. Mu.M MI-2 and 1. Mu.M AZD on NF-. Kappa.B expression in the nucleus and cytoplasm of HCT 116;

FIG. 5 shows a Western blot to examine the effect of different concentrations of MALT1 inhibitor and MEK inhibitor on HCT116 pathway proteins;

FIG. 6 shows the effect of Western blot detection on proteins associated with PI3K pathway in HCT116 after knockdown of MALT 1;

FIG. 7 shows a Western blot to detect the effect of MALT1 inhibitor on RTKs expression of HCT116 and PI3K pathway protein;

FIG. 8 shows a Western blot to examine the effect of knocking down MALT1 on the RTKs expression of HCT116 and PI3K pathway protein.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

The colorectal cancer cell lines HCT116 and HEK293T were purchased from the American Type Culture Collection (ATCC).

Example 1 Effect of the amount of MALT1 expression and sensitivity of cells to MEKi

1. Knock-down of MALT1

The RNAi is used for targeted silencing of the gene, and the result of the knockdown efficiency of the shRNA to the MALT1 shows that the relative expression quantity of the MALT1 in a knockdown cell is lower than 30 percent and meets the basic requirement.

Next, to examine the effect of knocking down MALT1 on cell proliferation in HCT116, we treated the control group (plko.1) and the cell knocking down MALT1 HCT116 (shMALT 1-3) with different concentrations (0, 0.01, 0.1, 1, 10 μ M) of MEKi (MEK inhibitor, AZD 6244), respectively, and examined the effect of the drug on cell proliferation by MTS method 72 hours after drug treatment (fig. 1B).

The experimental result shows that the proliferation of the cell shMALT1-3 is inhibited to a greater extent by the medicament, and the half inhibition concentration of the AZD6244 on the cell is reduced compared with PLKO.1, which indicates that the sensitivity of the knocked-down cell on MEKi is increased. In long-term colony formation experiments, when we treated the cells with different concentrations (0, 0.125, 0.25, 0.5 μ M) of MEKi every other day, the crystal violet staining results showed (FIG. 1C) that the cell shMALT1-3 proliferated more slowly during long-term proliferation compared to PLKO.1 and formed fewer colonies after AZD6244 treatment. These experimental results all indicate that knocking down MALT1 can cooperate with MEKi to inhibit cell proliferation in HCT 116.

2. Inhibition of MALT1 expression

Cells were treated with the MALT1 inhibitor MI-2 and we set up a panel of drug concentrations to test the response of HCT116 to MI-2 based on the IC50 (5.84. Mu.M) concentrations reported in the instructions for inhibitor MI-2. The effect of the drug on cell proliferation was examined 72 hours after drug treatment using the MTS method (FIG. 2). MTS assay results showed that half inhibitory concentration of MI-2 on HCT116 cells was between 2.5-3. Mu.M, whereas 5. Mu.M MI-2 completely inhibited proliferation of HCT116 cells.

Based on the short-term response of HCT116 to MI-2, we treated HCT116 with different concentrations (0, 0.2, 0.4, 0.8, 1, 2. Mu.M) of MI-2 and concentration gradients (0, 0.125, 0.25, 0.5) of AZD6244, alone or in combination, for the long-term (FIG. 3).

The results show that in long-term clonogenic, 0.8. Mu.M MI-2 alone acts on cells, and can effectively inhibit cell proliferation, and lower concentrations (0.2, 0.4. Mu.M) of MI-2 have little effect on cell proliferation; low concentrations (0.125, 0.25 μ M) of AZD6244 alone had no significant effect on HCT116 in inhibiting proliferation. In addition, probably because the cell density of the experiment is lower, 0.5 mu M AZD6244 has certain inhibition effect on cell proliferation. Meanwhile, we find that the combination of 0.4 mu M MI-2 and 0.25 mu M or 0.5 mu M AZD6244 can effectively inhibit the cell proliferation, and the inhibition effect is more obvious than that of the two medicines when the two medicines are used independently. This result indicates that MI-2 can synergize with MEKi to inhibit the proliferation of HCT 116.

The experimental results of RNA interference and target point inhibitor show that the inhibition of MALT1 expression can increase the sensitivity of HCT116 to MEKi, and the MEKi is cooperated to effectively inhibit the proliferation of HCT 116. To investigate the relationship between MALT1 and HCT116 for MEKi resistance changes, we further explored the function of MALT 1.

Example 2 investigation of the Effect of MALT1 on the tolerance of cellular MEKi

1. HCT11624 was treated with MI-2 of different concentrations (1, 2.5. Mu.M) and 1. Mu.M AZD6244, respectively or in combination, for 5363 hours (FIG. 4A), cellular proteins were extracted and then Western blot was used to detect protein expression. We found that MI-2 did not inhibit total intracellular p-NF- κ B and that MI-2 had some inhibitory effect on p-ERK expression. To further investigate the effect of MI-2 on NF-. Kappa.B, HCT was treated with 2. Mu.M MI-2 and 1. Mu.M MAZD6244 for another 24h period, and after cellular protein extraction, nucleoplasmic separation was performed on the protein (FIG. 4B) and protein expression was detected using Western blot. The results showed that NF-. Kappa.B was expressed more in cytoplasm, and MI-2 could not inhibit NF-. Kappa.B expression of HCT116 in cells.

The above results show that MI-2 is unable to inhibit NF-. Kappa.B expression in HCT116, indicating that the effect of MALT1 on the proliferation of cellular HCT116 is not caused by affecting NF-. Kappa.B. Therefore, the effect of the complex composed of MALT1 on the NF- κ B signal induced by AKT reported in the literature is not applicable in the present study. We speculate that MALT1 may have some relation to AKT.

2. HCT116 cells were treated with MI-2 and 1. Mu.M AZD6244 at different concentrations (2, 5. Mu.M) for 1h and 24h, respectively or simultaneously, and protein expression was detected by Western blot (FIG. 5). From the experimental results, it can be seen that the cells treated with different concentrations of MI-2 can significantly inhibit the expression of MALT1 after 24h, the expression level of MALT1 is not changed after 1h treatment, and the inhibition effects of 2. Mu.M and 5. Mu.M of MI-2 on MALT1 are not significantly different. MI-2 has obvious inhibition on the expression of P-AKT (S473) when used alone for 24h, and has no influence on P-P70S6K and P-ERK; when MI-2 and AZD are combined to treat the cells for 24h, the expression of P-AKT (S473) is also obviously reduced, the expression of P-P70S6K is also inhibited to a certain extent, and the P-ERK is almost completely inhibited. The results of the above experiments indicate that the combined use of MI-2 and MEKi can inhibit MAPK pathway of HCT116 cells, and simultaneously influence the activity of PI3K pathway through the inhibition of P-AKT and P-P70S 6K.

To rule out the effect of non-specific targeted inhibition of the inhibitor, we next continued validation in cells where shRNA targets knockdown MALT 1. We treated the control (PLKO.1) and cells knocked down for 1h, 24h with 1. Mu.M AZD6244, and after extracting cellular proteins, western blot was used to detect protein expression (FIG. 6). We found that in PLKO.1 group, MEKi had no effect on the expression of P-P70S6K, P-AKT, but in cells knocked down MALT1, the expression level of P-AKT (S473) was significantly reduced compared to the control at MEKi concentration of 0, and that P-AKT (S473) was also lower after MEKi treatment, indicating that knocking down MALT1 was able to inhibit the expression of P-AKT (S473) in cells. Meanwhile, the cells with the MALT1 knockdown function have P-P70S6K expression reduced after AZD treatment for 24 hours, which indicates that MEKi can influence the expression of P-P70S6K in the cells with the MALT1 knocked down function. In addition, MEKi still significantly inhibited the expression of the p-ERK protein downstream of the MAPK pathway in several groups of cells. The above experimental results show that after cellular knockdown of MALT1, the activity of PI3K pathway is inhibited, and after combined use of MEKi, MAPK pathway is blocked.

Example 3 investigation of the Effect of MALT1 on PI3K pathway Activity

The above results indicate that, when expression of MALT1 is inhibited in HCT116, activation of p-AKT (S473) and activity of downstream proteins in cells are affected, and thus the activity of PI3K pathway is also affected. Therefore, we hypothesize that the reason why HCT116 is resistant to MEKi may be that cell proliferation is maintained by the PI3K pathway after inhibition of the MAPK pathway, whereas inhibition of MALT1 affects the activity of the PI3K pathway, so inhibition of MALT1 can cooperate with MEKi to inhibit cell proliferation.

Therefore, we need to further explore the effect of MALT1 on PI3K pathway activity. The effect of MALT1 on PI3K pathway-associated proteins was observed by modulating the expression of MALT1 with small molecule inhibitors and mimicking the AKT activation process with growth factors in vitro (figure 7).

Cells HCT116 were starved for 24 hours under serum-free conditions, and then stimulated with 20ng/mL EGF and 20ng/mL IGF-1 for 15min and 30min, respectively. We found that the expression level of p-EGFR (Tyr 1068) of the cells after EGF stimulation is obviously increased, while the expression levels of p-AKT (S473) and p-ERK are obviously increased only when the cells are stimulated for 15min, and the expression level is basically restored to a normal level when the cells are stimulated for 30min, which is probably a degradation phenomenon caused by over-activation. After IGF-1 stimulation, the expression level of p-IGF1-R (Tyrr 1135/1136) of the cells is increased to a certain extent, the expression of p-AKT (S473) is obviously increased at 15min and 30min of stimulation, and the expression level of p-ERK is not obviously influenced. The above experimental phenomena indicate that the growth factor can effectively activate the receptor and the downstream protein expression.

Next, the effect of MALT1 on the activation of the signaling pathway was observed. After treating the cells with 2 μ M MI-2 for 24 hours while starving and then stimulating the cells with growth factors, we found that the expression level of p-EGFR of the cells was significantly increased when EGF was stimulated for 15min, the expression level was significantly decreased when EGF was stimulated for 30min compared to 15min, and the expression level of downstream p-AKT (S473) was not substantially increased when compared to no growth factor; under the same conditions, after IGF-1 stimulates cells, the results showed that the expression levels of p-IGF1-R and p-AKT were significantly increased and were not inhibited by the addition of MI-2.

The results show that MI-2 has certain effect on the activation of p-EGFR and has obvious inhibition effect on the activation of p-AKT (S473) downstream of EGFR, so that the activity of a PI3K pathway is influenced by the addition of MI-2. From the above experimental results, it is clear that MI-2 only inhibits p-AKT activated by p-EGFR, and p-AKT activated by p-IGF1-R is not inhibited by MI-2, indicating that MALT1 plays a role in the regulation of p-AKT by p-EGFR and thus affects the activity of signaling pathway.

Under the same conditions of the experiment, after the cells are stimulated by 10% serum for 30min, only the expression level of the p-ERK is obviously increased, and the activation of other proteins is not influenced by the serum, so that the influence of the serum in a culture medium on the experiment result can be eliminated.

Example 4

To rule out the effect of non-specific inhibition by small molecule inhibitors, we performed similar validation in cells that knockdown MALT1, along with the experiments with MALT1 inhibitors described above (fig. 8). Cells from control (PLKO.1) and knockdown MALT1 cells were first starved for 24h in serum-free medium, then stimulated with 20ng/mL EGF and 20ng/mL IGF-1 for 15min and 30min, respectively, and each group of cells was treated with 10% serum for 30min as a control. After extracting cell protein, western blot detects the protein expression condition.

The experimental results show that in the MALT 1-knocked-down HCT116, the activation expression level of p-EGFR and p-AKT (S473) after EGF stimulation is lower than that of the p-EGFR and p-AKT (S473) of the control group (PLKO.1) after stimulation. After IGF1 stimulation, the expression of p-IGF1-R, p-AKT (S473) was increased in both PLKO.1 and MALT 1-knockdown cells, and there was no significant difference in expression levels between the two groups of cells.

The results of this experiment indicate that knocking down MALT1 inhibits activation of p-EGFR and p-AKT downstream of p-EGFR (S473). In addition, we also found that p-EGFR activation of p-AKT (S473) was inhibited in cells that knockdown MALT1, whereas p-IGF1-R activation of p-AKT was not affected, again demonstrating that MALT1 plays a role in EGFR regulation of AKT, thereby affecting PI3K pathway activity.

In conclusion, the sensitivity of the colorectal cancer cells to the MEK inhibitor can be effectively improved by knocking down or inhibiting the MALT1 gene, and the inhibition effect of the MEK inhibitor on the colorectal cancer cell proliferation can be synergistically promoted.

Sequence listing

<110> Sichuan university

Application of <120> MALT1 gene as marker in preparation of medicine for treating colorectal cancer

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Claims (8)

1. The application of a MALT1 gene expression inhibitor MI-2 in the preparation of a medicine for treating colorectal cancer is characterized in that the medicine can inhibit the proliferation of colorectal cancer cells and promote apoptosis.

2. The application of a MALT1 gene expression inhibitor MI-2 and a MEKI inhibitor AZD624 in the combined preparation of medicaments for treating colorectal cancer is characterized in that the medicaments can inhibit the proliferation of colorectal cancer cells and promote the apoptosis.

3. The use according to claim 1 or 2, wherein the concentration of MALT1 gene expression inhibitor MI-2 is not less than 0.6 μ Μ.

4. Use according to claim 3, wherein the colorectal cancer is KRAS mutated colorectal cancer.

5. A combination for the treatment of colorectal cancer comprising the MALT1 gene expression inhibitor MI-2, and the MEKI inhibitor AZD624.

6. Combination according to claim 5, wherein the concentration of the inhibitor of MALT1 gene expression, MI-2, is not less than 0.2 μ M and the concentration of the MEKI inhibitor AZD624 is not less than 0.25 μ M.

7. Combination according to claim 6, wherein the concentration of the MALT1 gene expression inhibitor MI-2 and the concentration of the MEKI inhibitor AZD624 in the combination is 0.4 μ M or 0.5 μ M.

8. The combination of claim 5~7 wherein the combination is a drug capable of inhibiting proliferation and promoting apoptosis in colorectal cancer cells.

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