CN112877433B

CN112877433B - Colorectal cancer targeted therapy medicine

Info

Publication number: CN112877433B
Application number: CN202110184373.9A
Authority: CN
Inventors: 刘卡花; 姜顺顺; 刘玉敏
Original assignee: Suzhou Ruifeng Pharmaceutical R & D Co ltd
Current assignee: Suzhou Ruifeng Pharmaceutical R & D Co ltd
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2022-05-31
Anticipated expiration: 2041-02-08
Also published as: CN112877433A; CN114522179A; CN114522179B

Abstract

The invention provides a targeted therapeutic drug for colorectal cancer, belonging to the technical field of tumor cell biology. The invention provides a gene inhibitor of an AC092635.1 gene, which can effectively inhibit the expression of an AC092635.1 gene. Meanwhile, the targeted inhibition of the AC092635.1 gene is found to be capable of effectively inhibiting the proliferation, migration, invasion and EMT transformation of colorectal cancer cells by means of colony formation experiments, Transwell chamber experiments and Western Blot experiments, so that the targeted inhibition of the AC092635.1 gene can be used for preparing the medicine for treating colorectal cancer.

Description

Colorectal cancer targeted therapy medicine

Technical Field

The invention belongs to the technical field of tumor cell biology, and particularly relates to a targeted therapeutic drug for colorectal cancer.

Background

Colorectal cancer is one of the most common malignancies in the digestive system, and is characterized by high morbidity and mortality. Colorectal cancer has now become the third most malignant tumor in global incidence. The current main treatment means of colorectal cancer are endoscopic resection, surgical operation, new adjuvant chemotherapy, radiotherapy, targeted therapy and the like, and although many new drugs have remarkable curative effect in the treatment process of colorectal cancer, the five-year survival rate of patients is still poor due to relapse and metastasis of patients at the late stage and drug resistance of patients to drugs. Therefore, finding biomarkers closely related to the development of colorectal cancer is crucial to monitoring tumor progression and finding therapeutic targets.

Although many studies have found that various factors play an important role in the pathogenesis of colorectal cancer, the occurrence and evolution of colorectal cancer is a complicated regulatory process, and many problems still need further research.

Disclosure of Invention

The invention aims to provide a targeted therapeutic drug for colorectal cancer.

In order to achieve the purpose, the invention provides the following technical scheme:

in one aspect, the present invention provides a gene for colorectal cancer therapy, the gene being AC092635.1 gene; colorectal cancer can be effectively treated by specifically inhibiting the expression of the AC092635.1 gene.

Secondly, the invention provides an application of the AC092635.1 gene inhibitor in preparing a medicine for treating colorectal cancer.

Preferably, the AC092635.1 gene inhibitor is a molecule having an inhibitory effect on the AC092635.1 gene.

Preferably, the AC092635.1 gene inhibitor is one of siRNA, dsRNA and shRNA for reducing the expression level of AC092635.1 gene.

Preferably, the AC092635.1 gene inhibitor is an siRNA comprising the following nucleotide sequence: the sequence of the sense strand is shown as SEQ ID NO.6, and the sequence of the antisense strand is shown as SEQ ID NO. 7.

Thirdly, the invention provides a medicine for treating colorectal cancer, which comprises an effective dose of AC092635.1 gene inhibitor.

Fourthly, the invention provides siRNA for treating colorectal cancer, wherein the siRNA comprises the following nucleotide sequences: the sequence of the sense strand is shown as SEQ ID NO.6, and the sequence of the antisense strand is shown as SEQ ID NO. 7.

Fifthly, the invention provides application of an AC092635.1 gene inhibitor in preparing a colorectal cancer cell proliferation inhibitor.

Sixth, the invention provides an application of the AC092635.1 gene inhibitor in preparing colorectal cancer cell transfer inhibitors.

The invention has the beneficial effects that:

the invention discovers that the AC092635.1 gene is highly expressed in the colorectal cancer tissue for the first time, and the expression difference of the AC092635.1 gene in the colorectal cancer and the tissue beside the cancer has excellent diagnostic value, so the AC092635.1 gene can be used for preparing a diagnostic kit for the auxiliary diagnosis of the colorectal cancer.

Meanwhile, the expression level of the AC092635.1 gene in the transferred colorectal cancer tissue is obviously higher than that of the non-transferred colorectal cancer tissue, and differential expression has excellent diagnostic value, so that the AC092635.1 gene can be used for preparing a diagnostic kit for diagnosing colorectal cancer transfer.

In addition, the AC092635.1 gene is highly expressed in colorectal cells, so that the method can be used for screening colorectal cancer medicines by detecting the expression level of the AC092635.1 gene in the colorectal cancer cells.

In addition, the targeted inhibition of the AC092635.1 gene is found to be capable of effectively inhibiting the proliferation, migration, invasion and EMT transformation of colorectal cancer cells by means of colony formation experiments, Transwell chamber experiments and Western Blot experiments, so that the targeted inhibition can be used for preparing the medicine for treating colorectal cancer.

Drawings

FIG. 1 the difference in expression of the AC092635.1 gene in colorectal tumor tissue and in paraneoplastic tissue;

FIG. 2 ROC curves for differences in gene AC092635.1 expression in colorectal cancer tissues and paracarcinoma tissues;

FIG. 3 difference in expression level of gene AC092635.1 in metastatic colorectal cancer tissue and non-metastatic colorectal cancer tissue;

FIG. 4 ROC curves for the difference in AC092635.1 expression in metastatic and non-metastatic cancer tissues;

figure 5 expression differences of AC092635.1 in colorectal cancer cells;

FIG. 6 detection of knockdown effect of siAC092635.1;

FIG. 7 Targeted inhibition of the regulation of SW480 cell clonogenic by AC 092635.1;

FIG. 8 Targeted inhibition of the modulation of cell migration ability of AC092635.1 on SW480 cells;

figure 9 targeted inhibition of the modulation of the cell invasive capacity of AC092635.1 on SW480 cells;

figure 10 modulation of EMT transformation-associated proteins by targeted inhibition of AC 092635.1.

Detailed Description

In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.

Tumor tissues and corresponding paracarcinoma tissues of 30 colorectal cancer patients in the central hospital of Qingdao city were collected, wherein 16 tumor tissues with metastasis and 14 tissues without metastasis were collected. All colorectal cancer patients underwent any chemoradiotherapy treatment before surgery, and the sample collection was requested and requested by the ethics committee of the compound hospital and the patient's consent was as follows:

number of	Sex	Age of diagnosis	Type of sample
				1	Female	67	Metastasis
2	Female	49	Metastasis
				3	Male	24	Metastasis
4	Female	39	Metastasis
				5	Male	64	Un-metastasis
6	Female	63	Metastasis
				7	Male	24	Un-metastasis
8	Female	22	Un-metastasis
				9	Female	43	Metastasis
10	Female	58	Un-metastasis
				11	Male	58	Metastasis
12	Female	68	Un-metastasis
				13	Female	43	Metastasis
14	Male	65	Metastasis
				15	Female	65	Un-metastasis
16	Male	54	Un-metastasis
				17	Male	82	Un-metastasis
18	Female	46	Metastasis
				19	Male	54	Un-metastasis
20	Male	41	Un-metastasis
				21	Female	51	Metastasis
22	Male	59	Metastasis
				23	Male	62	Metastasis
24	Male	47	Un-metastasis
				25	Female	46	Metastasis
26	Male	63	Metastasis
				27	Male	53	Un-metastasis
28	Female	50	Un-metastasis
				29	Male	63	Metastasis
30	Female	46	Un-metastasis

Example 1

Detecting the expression difference of the AC092635.1 gene in colorectal tumor tissue and paracarcinoma tissue

1. Extraction of RNA from tissue

(1) Putting 50mg of colorectal cancer tissues and tissues beside the cancer into a precooled mortar, and quickly grinding into powder;

(2) adding 1ml of Trizol into a mortar, fully and uniformly mixing, transferring to an enzyme-free centrifuge tube, and standing for 5min at room temperature;

(3) setting the rotating speed of a centrifugal machine to be 12000rpm, and putting the centrifugal machine into a centrifugal tube for centrifugation for 5 min;

(4) adding 200 μ l chloroform into the centrifuge tube, reversing, mixing, and standing at room temperature for 10 min;

(5) setting the rotating speed of a centrifugal machine to be 12000rpm, and putting the centrifugal machine into a centrifugal tube for centrifugation for 10 min;

(6) dividing the liquid into three layers, transferring the supernatant of the upper layer into a new enzyme-free centrifuge tube, adding precooled isopropanol with the same volume, mixing uniformly, and standing on ice for 10 min;

(7) setting the rotating speed of a centrifugal machine to be 12000rpm, and putting the centrifugal machine into a centrifugal tube for centrifugation for 10 min;

(8) discarding the supernatant, adding 1ml of 75% ethanol prepared from DEPC water into the precipitate, mixing, placing in a centrifuge, and centrifuging at 7500rpm for 10 min;

(9) removing supernatant, placing in an ultraclean workbench, drying RNA white precipitate, adding 50 μ l DEPC water to dissolve precipitate, and detecting RNA purity and concentration with a trace quantitative detector.

Reverse transcription of cDNA

The method was performed according to TAKARA reverse transcription kit

(1) Removal of genomic DNA

A10. mu.l reaction system was prepared: 5 XgDNA Eraser Buffer 2.0. mu.l, gDNA Eraser 1.0. mu.l, Total RNA 1.0. mu.g, RNase Free dH2O up to 10. mu.l.

Reaction conditions of the PCR apparatus: 42 ℃ for 2 minutes, 4 ℃.

(2) Reverse transcription reaction

Prepare 20. mu.l reaction system: 10. mu.l of the reaction solution of step (1), 1.0. mu.l of PrimeScript RT Enzyme Mix I, 1.0. mu.l of RT Primer Mix, 24.0. mu.l of 5 XPimeScript Buffer, and 4.0. mu.l of RNase FreedH 2O 4.0.

Reaction conditions of the PCR apparatus: 15 minutes at 37 ℃, 5 seconds at 85 ℃ and 4 ℃.

3. Fluorescent quantitative PCR reaction

(1) Design of primers based on the sequence ENST00000434509.1 (SEQ ID NO. 1) of the AC092635.1 Gene

The primer sequences of AC092635.1 are as follows:

forward primer 5'-ACACACTAAAACTCCCACAGAA-3' (SEQ ID NO.2)

Reverse primer 5'-TACCTGTTTGTGACACCCGC-3' (SEQ ID NO. 3)

With GAPDH as an internal reference, the primer sequences of GAPDH are as follows:

forward primer 5'-TTCACCACCATGGAGAAGGC-3' (SEQ ID NO.4)

Reverse primer 5'-CCACCTGGTGCTCAGTGTAG-3' (SEQ ID NO.5)

(2) Reaction reagents are prepared according to a TAKARA fluorescent quantitative PCR kit:

reaction reagents, SYBR Green Premix Ex Taq (2X) 10. mu.l, forward primer 0.4. mu.l, reverse primer 0.4. mu.l, cDNA template 2. mu.l, ddH2O 7.2.2. mu.l were prepared.

The parameters of the fluorescent quantitative PCR instrument are set as follows: 5min at 95 ℃; 38 cycles of 95 ℃ for 15s and 60 ℃ for 40s

(3) GAPDH as reference gene, 2^-△△CtThe method calculates the relative expression level of the gene AC 092635.1.

The experimental results are as follows:

the difference of the expression level of the gene AC092635.1 in the paracarcinoma tissue and the colorectal cancer tissue is shown in figure 1. The relative expression level of the gene AC092635.1 in the colorectal cancer tissue is (162.6 +/-55.32)%, and it can be seen that the relative expression level of the gene AC092635.1 in the colorectal cancer tissue is obviously higher than that of the tissue beside cancer, and the difference has statistical significance.

Meanwhile, the present invention plots ROC curves for the difference in gene AC092635.1 between colorectal cancer tissues and paracarcinoma tissues, as shown in FIG. 2. Among them, the ROC curve has an AUC value of 0.8011, an std. error of 0.0559, a 95% confidence interval of 0.6916 to 0.9107, and a P of less than 0.0001, and shows excellent diagnostic value, so that whether colorectal cancer is caused or not can be judged by detecting the expression level of the gene AC092635.1 in colorectal cancer tissues and tissues adjacent to the cancer.

In addition, the present invention counted the difference in the expression level of gene AC092635.1 in the metastatic cancer tissue and the non-metastatic cancer tissue in the colorectal cancer tissue, and the results are shown in fig. 3. Wherein, compared with the non-metastatic cancer tissue, the relative expression level of the gene AC092635.1 in the metastatic cancer tissue is (129.1 +/-10.54)%, and it can be seen that the relative expression level of the gene AC092635.1 in the metastatic cancer tissue is obviously higher than that in the non-metastatic cancer tissue, and the difference has statistical significance.

The ROC curve of the difference in the expression level of AC092635.1 between metastatic and non-metastatic cancer tissues is shown in fig. 4. Among them, the ROC curve has an AUC value of 0.9107, an std. error of 0.0514, a 95% confidence interval of 0.8099 to 1.011, and P of less than 0.001, and shows excellent diagnostic value, so that whether colorectal cancer is metastasized can be judged by detecting the expression level of AC 092635.1.

Example 2

Detection of expression differences of AC092635.1 in colorectal cancer cells by fluorescent quantitative PCR

(1) Human normal colon epithelial cells in logarithmic growth phase NCM460 and HT29, SW620, SW480 and LOVO were seeded in cell culture plates;

(2) when the cell density reached 90%, Trizol was added to extract RNA, RNA extraction, reverse transcription reaction and fluorescent quantitative PCR reaction were performed as in example 1.

The difference in the expression level of AC092635.1 in different colorectal cancer cells is shown in fig. 5. It can be seen that the expression level of AC092635.1 in the colorectal cancer cells is significantly higher than that of NCM460 in normal human colon epithelial cells, and the result is identical with the result of expression level in tissues.

Example 3

Design interfering RNA of AC092635.1 and verify the interfering effect

(1) siRNA was designed according to the sequence ENST00000434509.1 of the AC092635.1 gene, and the sequence of si AC092635.1 was as follows:

sense strand: AAUUUUCCCCCUUUCUGUGGG, SEQ ID NO.6

Antisense strand: CACAGAAAGGGGGAAAAUUAG, SEQ ID NO. 7;

(2) SW480 cells were seeded in 6-well plates, when 80% of the cells grew, siNC and siAC092635.1 were transfected according to LipoFiter 3.0 instructions, and after 48h of transfection, RNA was extracted for fluorescent quantitative PCR detection, see example 1 for specific steps.

The knockdown effect of siac092635.1 is shown in fig. 6, where the relative expression level of AC092635.1 in the cells of the siac092635.1 group was (27.30 ± 6.02)%, and it can be seen that siac092635.1 can effectively inhibit the expression of AC 092635.1.

Example 4

Targeted inhibition of modulation of SW480 colorectal cancer cell proliferation by AC092635.1

(1) Digesting the SW480 cells of the transfected sinC and siAC092635.1 in the logarithmic growth phase by using pancreatin, and blowing, beating and uniformly mixing to prepare single cell suspension;

(2) counting cells, adjusting cell density to 1 × 10⁴Cell/ml, then adding 100 mul of cell suspension into a culture dish, adding 2ml of complete culture solution, placing in a cell culture box for culture, and setting 3 repeats per group;

(3) when cloning occurs in the culture dish, removing the culture medium, washing by using PBS (phosphate buffer solution), and then adding 4% paraformaldehyde for fixing for 15 min;

(4) discarding the fixing solution, adding crystal violet staining solution for staining, removing the staining solution after 20min, washing with PBS, and counting the number of clones in the culture dish.

The effect of targeted inhibition of AC092635.1 on SW480 cell clonogenic is shown in fig. 7, and it can be seen that the number of SW480 cell clonogenic decreases significantly after transfection of siac092635.1, indicating that siac092635.1 is able to impair the in vitro proliferative capacity of SW480 cells, i.e. targeted inhibition of AC092635.1 is able to inhibit the in vitro proliferation of colorectal cancer cells.

Example 5

Modulation of SW480 colorectal cancer cell migration ability by targeted inhibition of AC092635.1

(2) counting cells, adjusting cell density to 5 × 10⁵Cells/ml;

(3) placing the Transwell chamber into a 24-well plate, adding 750 μ l of complete medium containing 10% FBS into the 24-well plate, adding 200 μ l of diluted cell suspension into the Transwell chamber, and culturing in a cell culture box;

(4) after culturing for 48h, taking out the chamber, gently cleaning the chamber by using PBS, and wiping off cells on the inner membrane of the chamber by using a cotton swab;

(5) adding 4% paraformaldehyde into a new hole of a 24-hole plate, and placing the small chamber into the paraformaldehyde for fixing for 15 min;

(6) the Transwell chamber was removed and washed with PBS for 5min, followed by staining with crystal violet for 10 min;

(7) the staining solution was removed, the Transwll cell was washed with clear water, placed under an inverted microscope for photography and 5 different fields of view were selected for counting.

Control of SW480 cell migration ability by targeted inhibition AC092635.1 as shown in fig. 8, it can be seen that after transfection of siac092635.1, the number of SW480 cells passing through the Transwell chamber was significantly reduced, indicating that siac092635.1 can impair the cell migration ability of SW480 cells, i.e., targeted inhibition AC092635.1 can inhibit the migration ability of colorectal cancer cells.

Example 6

Modulation of SW480 colorectal cancer cell invasion capacity by targeted inhibition of AC092635.1

(1) Placing the Matrigel in a refrigerator at 4 ℃ overnight to change the Matrigel into a liquid state;

(2) matrigel was diluted 1:8 using serum-free medium before placing the Transwell chamber in 24 plates;

(3) adding 100 μ l of diluted Matrigel into a Transwell chamber, and placing in a cell culture box to solidify;

(4) digesting the SW480 cells of the transfection siNC and siAC092635.1 in the logarithmic growth phase by using pancreatin, and blowing, beating and uniformly mixing to prepare a single cell suspension;

(5) counting cells, adjusting cell density to 5 × 10⁵Cells/ml;

(6) placing the Transwell chamber into a 24-well plate, adding 750 μ l of complete medium containing 10% FBS into the 24-well plate, adding 200 μ l of diluted cell suspension into the Transwell chamber, and culturing in a cell culture box;

(7) after culturing for 48h, taking out the chamber, gently cleaning the chamber by using PBS, and wiping off cells on the inner membrane of the chamber by using a cotton swab;

(8) adding 4% paraformaldehyde into a new hole of the 24-hole plate, and placing the small chamber into the paraformaldehyde for fixing for 15 min;

(9) the Transwell chamber was removed and washed with PBS for 5min, followed by staining with crystal violet for 10 min;

(10) the staining solution was removed, the Transwll cell was washed with clear water, placed under an inverted microscope for photography and 5 different fields of view were selected for counting.

Control of SW480 cell invasion capacity by targeted inhibition AC092635.1 as shown in fig. 9, it can be seen that after transfection of siac092635.1, the number of SW480 cells passing through the Transwell chamber was significantly reduced, indicating that siac092635.1 can impair the cell invasion capacity of SW480 cells, i.e., targeted inhibition of AC092635.1 can inhibit the invasion capacity of colorectal cancer cells.

Example 7

Regulation of EMT conversion-related protein by targeted inhibition of AC092635.1

(1) SW480 cells were seeded in 6-well plates and when 80% of the cells grew, siNC and siAC092635.1 were transfected as per LipoFiter 3.0 instructions;

(2) after transfection for 48h, removing the culture medium and adding a proper amount of protein lysate containing protease inhibitor and phosphatase inhibitor;

(3) placing on a shaking bed, standing for 30min, transferring the lysate to 1.5ml EP tube, placing in a 4 deg.C precooled centrifuge, and centrifuging at 1200rpm for 10 min;

(4) after the centrifugation is finished, taking the supernatant into a new 1.5ml EP tube, determining the protein concentration by using a BCA method, adding a Loading Buffer, and boiling for 5min at 95 ℃ to obtain a protein sample;

(5) preparing 12% of separation glue and 5% of concentrated glue, and solidifying for later use;

(6) installing an electrophoresis frame, carrying out sample loading, setting the voltage to be 80V after sample loading, and adjusting the voltage to be 120V after 30min until bromophenol blue runs out;

(7) after electrophoresis is finished, an electric rotating clamp is installed according to a sandwich model, and 250mA electricity is rotated for 90 min;

(8) after the electrotransformation is finished, putting the membrane into 5% skimmed milk powder, and sealing for 1h at room temperature;

(10) diluting E-cadherin, N-cadherin, Vimentin, GAPDH primary antibody with BSA according to the antibody instructions and incubating the corresponding primary antibodies according to protein size, overnight incubation at 4 ℃;

(11) washing the membrane with TBST buffer for 3 times for 30min, incubating corresponding secondary antibody, and incubating for 1h at normal temperature in a shaking table;

(12) the secondary antibody was removed, and the PVDF membrane was washed 3 times with TBST for 30min and placed on a developing instrument for development.

The regulation of the protein related to EMT transformation by targeting inhibition AC092635.1 is shown in FIG. 10, and it can be seen that after the transfection of the siAC092635.1, the expression level of E-cadherin is obviously up-regulated, and the expression levels of N-cadherin and Vimentin are obviously down-regulated, which indicates that the siAC092635.1 can weaken the EMT transformation of SW480 cells, namely, targeting inhibition AC092635.1 can inhibit the EMT transformation of colorectal cancer cells.

Sequence listing

<110> Qingdao city central hospital

<120> colorectal cancer targeted therapy medicine

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 455

<212> DNA

<213> Human source (Human)

<400> 1

acacactaaa actcccacag aaagggggaa aattagaaaa ggggtgatat aatttgactc 60

tgtgtcacca cccaaatctc atcttgaatt gtaatccccg taatttctca cctggcaaga 120

gtgggaccag gtggagacaa ttggatcatg ggggcagttt tccccatgct cttctcgtga 180

taatgattga gtctcatggg atctgatgat tctataagtg tctggctttt ctcctacttg 240

cactcactcc atcctgccac cctgcgagga aggtgcctgc ttcttctttg ccttccgccg 300

tgagtgtgag tctcctgagg cctccccagc aatgtggaac tggatacgtc atggaacaga 360

gggcaacctg cctcctggaa atgtggagtg tctagggagt cacagctgag atcttccttg 420

gattaaaggc tgcgggtgtc acaaacaggt aggaa 455

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acacactaaa actcccacag aa 22

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tacctgtttg tgacacccgc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttcaccacca tggagaaggc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccacctggtg ctcagtgtag 20

<210> 6

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aauuuucccc cuuucugugg g 21

<210> 7

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cacagaaagg gggaaaauua g 21

Claims

Use of an AC092635.1 gene inhibitor in the manufacture of a medicament for the treatment of colorectal cancer, wherein the AC092635.1 gene inhibitor is an siRNA comprising the nucleotide sequence: the sequence of the sense strand is shown as SEQ ID NO.6, and the sequence of the antisense strand is shown as SEQ ID NO. 7.