CN111420058A - A gene inhibitor for the treatment of prostate cancer - Google Patents

Abstract

The invention finds that long-chain non-coding RNA L INC01841 can be used as a drug target for treating the prostatic cancer through experiments, and also finds that the L INC01841 gene inhibitor can inhibit the proliferation rate of prostatic cancer cells, inhibit the migration of the prostatic cancer cells and inhibit the invasion of the prostatic cancer cells.

Description

A gene inhibitor for the treatment of prostate cancer

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a gene inhibitor for treating prostate cancer.

Background technique

Prostate cancer is one of the most common malignant tumors in the male genitourinary system, and it is one of the main malignant tumors that cause the death of males worldwide. Although the incidence of prostate cancer in my country is much lower than that in western developed countries, due to the continuous aging of the population in my country, the incidence of prostate cancer is increasing year by year, which seriously threatens the health of middle-aged and elderly men, and affects the lifestyle and life of Chinese men. The impact of quality is becoming more and more prominent. Early symptoms of prostate cancer are insidious, so many patients are usually diagnosed at a later stage. At present, traditional treatment methods such as local surgical resection, chemotherapy, radiotherapy and hormone blockade have their own shortcomings and deficiencies, such as large side effects and easy recurrence after treatment. Therefore, the choice of new treatment modalities and therapeutic drugs has become more and more urgent.

Long non-coding RNA is a kind of non-coding RNA with a length of more than 200nt. Although the number of non-coding RNAs is far more than the number of coding genes, for a long time in the past, non-coding genes have been regarded by scientists as Some meaningless transcripts. However, with the advent of high-throughput gene sequencing technology, people have gradually realized the importance of lncRNAs. Current studies have found that lncRNAs play an important role in many aspects of cell occurrence, development, proliferation, and differentiation. At the same time, studies have found that lncRNAs play an important role in the occurrence and development of tumors. At present, they have become important markers and drug targets for tumor diagnosis and treatment. The study of lncRNA in tumor pathogenesis provides new ideas for the selection of new tumor therapeutic targets and the development of new precision therapeutic drugs.

Currently, there is no relevant report on the role of LINC01841 in prostate cancer.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a gene inhibitor with inhibitory effect on LINC01841 prepared by taking LINC01841 as an action target and used for preparing a drug for the treatment of prostate cancer, aiming at the deficiencies of the prior art.

For achieving the above object, the present invention provides the following technical solutions:

The present invention provides the use of a LINC01841 gene inhibitor in the preparation of a medicament for treating prostate cancer. The transcript of the LINC01841 is NR_134908.1, and the sequence of the LINC01841 is shown in SEQ ID NO.1.

Preferably, the LINC01841 gene inhibitor is a molecular inhibitor prepared by targeting LINC01841 and having an inhibitory effect on LINC01841; the molecular inhibitor is one of a nucleic acid molecule, an antibody or a small molecule compound.

Preferably, the nucleic acid molecules include shRNA, siRNA, dsRNA and microRNA.

Preferably, the nucleic acid molecule is siRNA.

Preferably, the sequence of the sense strand of the siRNA is shown in SEQ ID NO.6, and the sequence of the antisense strand of the siRNA is shown in SEQ ID NO.7.

In addition, the present invention provides a gene inhibitor, the gene inhibitor is a LINC01841 gene inhibitor, and the LINC01841 gene inhibitor is a molecular inhibitor prepared by targeting LINC01841 with an inhibitory effect on LINC01841, the The LINC01841 gene inhibitor can be used to prepare a drug for treating prostate cancer.

Preferably, the LINC01841 gene inhibitor is siRNA, the sequence of the sense strand of the siRNA is shown in SEQ ID NO.6, and the sequence of the antisense strand of the siRNA is shown in SEQ ID NO.7.

In addition, the present invention provides a pharmaceutical composition for the treatment of prostate cancer, characterized in that the pharmaceutical composition comprises a LINC01841 gene inhibitor.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or dressing.

Preferably, the pharmaceutically acceptable carrier and/or dressing comprises: adhesive, diluent, lubricant, surfactant, adsorbent carrier.

The beneficial effects of the present invention are:

The present invention discovers for the first time that LINC01841 can be used as a target for prostate cancer treatment. Moreover, the siRNA provided by the present invention can inhibit the proliferation, migration and invasion of prostate cancer cells. Therefore, the gene inhibitor provided by the present invention can be used to prepare drugs for the treatment of prostate cancer and provide a new direction for the development of drugs for prostate cancer. .

Description of drawings

Figure 1 Expression of LINC01841 in normal prostate cancer epithelial cells RWPE-1 and prostate cancer cells DU-145, PC3 and LNCaP.

Figure 2 The effect of transfection of si-LINC01841 on the cell proliferation ability of PC3 cells

Figure 3 The effect of transfection of si-LINC01841 on the cell proliferation-related proteins c-myc and cyclinD1 of PC3 cells.

Figure 4 Effects of transfection of si-LINC01841 on cell migration and cell invasion of PC3 cells.

Figure 5. Effects of transfection of si-LINC01841 on EMT-related proteins E-cadherin and Vimentin in PC3 cells.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be noted that the protection scope of the present invention is not limited to the following specific embodiments, and it should be understood that the terms used in the embodiments of the present invention are used to describe specific The specific embodiments are not intended to limit the scope of protection of the present invention.

In addition, in the following examples, the test methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by various manufacturers. The reagents and medicines involved in the examples are common commercial products unless otherwise specified.

Example 1

Expression of LINC01841 in normal prostate cancer epithelial cells RWPE-1 and prostate cancer cells DU-145, PC3, and LNCaP.

1. RNA extraction

(1) Inoculate RWPE-1 cells, DU-145 cells, PC3 cells, and LNCaP cells in a six-well plate and culture for 48 hours. When the cells are confluent to 90%, add 500 μl Trizol, mix well, and transfer the mixture Transfer to a 2ml centrifuge tube and place at room temperature for 5min;

(2) Place the centrifuge tube in a low-temperature high-speed centrifuge, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant;

(3) Add 100 μl of chloroform, invert and mix, and let stand for 15 minutes at room temperature;

(4) Place the centrifuge tube in a low-temperature high-speed centrifuge and centrifuge at 12,000 rpm for 5 minutes;

(5) Transfer the supernatant to a new 2ml RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, invert and mix, and let stand on ice for 10 minutes;

(6) Place the centrifuge tube in a low-temperature high-speed centrifuge, 12000rpm, centrifuge for 10min, carefully remove the supernatant with a pipette, and retain the white precipitate;

(7) Add 500 μl of 75% ethanol, invert and mix, place the centrifuge tube in a low-temperature high-speed centrifuge, 4°C, 8000 rpm, and centrifuge for 5 minutes;

(8) Carefully remove the supernatant and dry the centrifuge tube at room temperature. After drying, add DEPC water to dissolve the precipitate completely, and measure the RNA concentration with a Nanodrop 2000 spectrophotometer.

2. Fluorescence quantitative PCR to detect the expression of LINC01841

(1) Reverse transcription reaction

Refer to takara reverse transcription kit

A: Removal of genomic DNA

Reagents added amount 5×gDNA Eraser Buffer 2.0μl gDNA Eraser 1μl Total RNA 1μg RNase Free dH2O up to 10μl

Reaction conditions: 42°C for 120s, 4°C.

B: reverse transcription reaction

Reagents added amount The reaction solution of step (A) 10μl PrimeScript RT Enzyme Mix I 1.0μl RT Primer Mix 1.0μl 5 x PrimeScript Buffer 2 4.0μl RNase Free dH2O 4.0μl

Reaction conditions: 37°C for 15 min; 85°C for 5s, 4°C.

(2) Fluorescent PCR detection

Primer sequence

LINC01841

Forward 5'- CCGCTAAGCACACAGAGGAA-3' (SEQ ID NO. 2)

Reverse 5'- AACAGCAAGGTGTCAGCAGA-3' (SEQ ID NO. 3)

GAPDH

Forward 5'- GGTGTGAACCATGAGAAGTATGA-3' (SEQ ID NO. 4)

Reverse 5'-GAGTCCTTCCACGATACCAAAG-3' (SEQ ID NO. 5)

Reagents

Reagents added amount SYBR Green Premix Ex Taq (2×) 10μl Forward primer 0.4μl Reverse primers 0.4μl cDNA template 2μl ddH2O 7.2μl

Reaction conditions: 95°C for 10 min; 95°C for 12s, 59°C for 40s, 40 cycles; 60°C for 5min.

The 2- ^△△Ct method was used to process the experimental data, and the results are shown in Figure 1.

Experimental results:

As can be seen from Figure 1, the expression of LINC01841 in prostate cancer cells was higher than that in normal cells, and the difference was statistically significant (P < 0.05). Therefore, PC3 cells were selected for subsequent cell experiments.

Example 2

The effect of transfection of si-LINC01841 on the cell proliferation ability of PC3 cells detected by CCK-8

(1) Inoculate 5×10 ³ PC3 cells transfected with si-NC or si-LINC01841 into a 96-well plate, with 90 μl per well, set up 3 duplicate wells in each group, and culture in a constant temperature cell incubator;

(2) The cells were detected at 0h, 24h, 48h, and 72h respectively. During the detection, 10 μl of CCK-8 detection solution was added to each well, and then the cells were incubated in a constant temperature cell incubator for 4 hours;

(3) Place the cell culture plate in a microplate reader, detect the absorbance at 450 nm, and draw a growth curve. The results are shown in Table 1 and Figure 2.

The sequence of si-LINC01841 is

Sense strand 5'-GGUCAAGAGUUCAAGACCAAU-3' (SEQ ID NO. 6)

Antisense strand 5'-UGGUCUUGAACUCUUGACCUU-3' (SEQ ID NO. 7)

si-LINC01841 was synthesized by Shanghai Zima Gene, and si-NC was provided by the company.

Table 1 Proliferation changes of PC3 cells after transfection of si-LINC01841 and si-NC

time si-NC si-LINC01841 0h 0.096±0.007 0.097±0.009 24h 0.325±0.046 0.186±0.042 48h 0.675±0.049 0.394±0.041 72h 0.986±0.057 0.612±0.046

Experimental results

It can be seen from Table 1 and Figure 2 that after transfection of si-LINC01841, the proliferation rate of PC3 cells was significantly inhibited compared with transfection of si-NC, indicating that inhibiting LINC01841 can inhibit the proliferation of prostate cancer cells.

Example 3

Western blot detection of the effect of transfection of si-LINC01841 on proliferation-related proteins

(1) 2×10 ⁵ PC3 cells were seeded on 6-well culture plates, and after 24 hours of culture, the cells were transfected according to Lipofectin 2000, and the transfection group was divided into si-NC group and si-LINC01841 group;

(2) 48 hours after transfection, wash cells with PBS, add 150µl RIPA cell lysate, and place on ice for 30 minutes;

(3) After fully lysing, transfer the cells to a centrifuge tube, centrifuge at 10,000g/min and 4°C for 15 minutes;

(4) Aspirate the supernatant with a pipette and transfer it to a new centrifuge tube;

(5) Pipette 2µl, measure the protein concentration according to the BCA instructions, add 5×SDS loading buffer, and cook in a boiling water bath for 5 minutes to obtain a protein sample.

(6) Assemble the electrophoresis tank, configure 5% upper layer glue and 10% lower layer glue;

(7) Add 20µg of protein sample and protein marker indicator to each well, add the newly configured electrophoresis buffer, 80V for 20min, 120V for 120min;

(8) Assemble the film transfer clip, transfer it into the electroporation tank, add electrotransfer fluid, 200mA for 1.5h;

(9) After transferring the membrane, take out the NC membrane, transfer it to 5% nonfat milk powder, and seal it with a shaker at room temperature for 1 hour;

(10) Take out the PVDF membrane, wash the membrane with TBST, 5 min each time, a total of 3 times, incubate with C-myc, CyclinD1 and β-actin primary antibody dilutions at 4°C and incubate overnight on a shaker.

(11) Aspirate the primary antibody, wash the membrane with TBST, 5 min each time, a total of 3 times, incubate the secondary antibody at room temperature, and incubate on a shaker for 1 h;

(12) In the dark room, quickly add luminescent droplets to the PVDF membrane for imaging.

Experimental results

The experimental results are shown in Figure 3. It can be seen that the protein expressions of C-myc and Cyclin-D1 in cells transfected with si-LINC01841 were significantly lower than those in cells transfected with si-NC, indicating that inhibiting LINC01841 can inhibit C- The protein expression of myc and Cyclin-D1, the above results further verified that si-LINC01841 can inhibit the proliferation of prostate cancer cells.

Example 4

Effects of transfection of si-LINC01841 on cell migration of PC3 cells

(1) 2×10 ⁵ PC3 cells were seeded on 6-well culture plates, and after 24 hours of culture, the cells were transfected according to Lipofectin 2000, and the transfection group was divided into si-NC group and si-LINC01841 group;

(2) 24 hours after transfection, cells were digested with trypsin, collected into centrifuge tubes, washed with PBS and serum-free medium, and then suspended in serum-free medium for counting;

(3) 600 μL of medium containing 10% serum was added to the lower chamber of the Transwell chamber, 100 μL of 5×10 ⁴ cell suspension was added to the upper chamber, and the cells were cultured in a constant temperature incubator for 24 hours;

(4) Carefully take out the chamber with tweezers, blot the liquid in the upper chamber dry, then transfer the chamber to a culture plate with 800 μL of methanol, and fix it at room temperature for 30 minutes;

(5) Carefully take out the chamber with tweezers, dry the upper chamber fixative, transfer the chamber to a culture plate with 800 μL Giemsa staining solution, and stain at room temperature for 20 minutes;

(6) Take out the chamber, rinse and soak with water for 3 times, absorb the liquid in the upper chamber, carefully wipe off the cells on the membrane surface of the bottom of the upper chamber with a cotton swab, and place it under a microscope to take pictures.

Experimental results

The results are shown in the cell migration in Figure 4. It can be seen that compared with PC3 cells transfected with si-NC, PC3 cells transfected with si-LINC01841 significantly reduced the number of cells passing through the Transwell chamber, indicating that si-LINC01841 gene inhibitor Can inhibit the cell migration of prostate cancer cells PC3.

Example 5

Effects of transfection of si-LINC01841 on cell invasion of PC3 cells

(1) 2×10 ⁵ PC3 cells were seeded on 6-well culture plates, and after 24 hours of culture, the cells were transfected according to Lipofectin 2000, and the transfection group was divided into si-NC group and si-LINC01841 group;

(2) 24 hours after transfection, cells were digested with trypsin, collected into centrifuge tubes, washed with PBS and serum-free medium, and then suspended in serum-free medium for counting;

(3) Store the BD Matrigel gel frozen at -80°C at 4°C overnight to make it completely liquid;

(4) Pipette 200 μL of serum-free medium into a sterile centrifuge tube, add 40 μL of Matrigel glue, mix gently on ice, add 100 μL of Matrigel glue to the upper chamber of the Transwell chamber, and put the chamber into a constant temperature cell incubator , incubate until the Matrigel gel completely becomes solid;

(5) 600 μL of medium containing 10% serum was added to the lower chamber of the Transwell chamber, and 100 μL of 5×10 ⁴ cell suspension was added to the upper chamber, and the cells were cultured in a cell constant temperature incubator for 24 hours;

(6) Carefully take out the Transwell chamber with tweezers, suck up the liquid in the upper chamber, and then transfer the chamber to a cell culture plate with 800 μL of methanol, and fix it at room temperature for 30 minutes;

(7) After fixing, take out the chamber, dry the liquid in the upper chamber, then transfer the chamber to a cell culture plate with 800 μL Giemsa staining solution, and stain at room temperature for 20 minutes;

(8) Rinse the Transwell chamber with PBS, aspirate the liquid in the upper chamber, carefully wipe off the cells in the upper chamber of the Transwell chamber, and place it under a microscope to take pictures.

Experimental results

The experimental results are shown in Figure 4. The cell invasion is shown in Figure 4. It can be seen that, compared with PC3 cells transfected with si-NC, the number of cells transfected with si-LINC01841 through Matrigel and entering the lower part of the chamber is significantly reduced, indicating that si-LINC01841 LINC01841 gene inhibitor can inhibit the cell migration of prostate cancer cell PC3.

Example 6

Effects of transfection of si-LINC01841 on EMT-related proteins E-cadherin and Vimentin in PC3 cells.

(1) 2×10 ⁵ PC3 cells were seeded on 6-well culture plates, and after 24 hours of culture, the cells were transfected according to Lipofectin 2000, and the transfection group was divided into si-NC group and si-LINC01841 group;

(2) 48 hours after transfection, wash cells with PBS, add 150µl RIPA cell lysate, and place on ice for 30 minutes;

(3) After fully lysing, transfer the cells to a centrifuge tube, centrifuge at 10,000g/min and 4°C for 15 minutes;

(4) Aspirate the supernatant with a pipette and transfer it to a new centrifuge tube;

(5) Pipette 2µl, measure the protein concentration according to the BCA instructions, add 5×SDS loading buffer, and cook in a boiling water bath for 5 minutes to obtain a protein sample.

(6) Assemble the electrophoresis tank, configure 5% upper layer glue and 12% lower layer glue;

(7) Add 20µg of protein sample and protein marker indicator to each well, add the newly configured electrophoresis buffer, 80V for 20min, 120V for 120min;

(8) Assemble the film transfer clip, transfer it into the electroporation tank, add electrotransfer fluid, 200mA for 1.5h;

(9) After the membrane transfer, take out the PVDF membrane, transfer it to 5% nonfat milk powder, and seal it with a shaker at room temperature for 1 hour;

(10) Take out the PVDF membrane, wash the membrane with TBST, 5 min each time, a total of 3 times, incubate with E-cadherin, Vimentin and GAPDH primary antibody dilutions at 4°C and incubate overnight on a shaker.

(11) Aspirate the primary antibody, wash the membrane with TBST, 5 min each time, a total of 3 times, incubate the secondary antibody at room temperature, and incubate on a shaker for 1 h;

(12) In the dark room, quickly add luminescent droplets to the PVDF membrane for imaging.

Experimental results:

It can be seen from Figure 5 that in the cells transfected with si-LINC01841, the expression of E-cadherin is up-regulated and the expression of Vimentin is down-regulated, indicating that inhibiting LINC01841 can inhibit the EMT of prostate cancer cell PC3. Inhibits the migration and invasion of prostate cancer cells.

To sum up, the gene inhibitor si-LINC01841 provided by the present invention can inhibit the proliferation, migration and invasion of prostate cancer cells. Therefore, si-LINC01841 can be used to prepare a drug for the treatment of prostate cancer.

sequence listing

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Claims (10)

1. L INC01841 gene inhibitor is used for preparing medicine for treating prostatic cancer.

2. The use as claimed in claim 1, wherein the L INC01841 gene inhibitor is a molecular inhibitor prepared from L INC01841 as an action target and having an inhibiting effect on L INC01841, wherein the molecular inhibitor is one of a nucleic acid molecule, an antibody or a small molecular compound.

3. The use according to claim 2, wherein the nucleic acid molecule comprises shRNA, siRNA, dsRNA and microrna.

4. The use according to claim 3, wherein the nucleic acid molecule is an siRNA.

5. The use according to claim 4, wherein the sense strand sequence of said siRNA is shown as SEQ ID No.6 and the antisense strand sequence of said siRNA is shown as SEQ ID No. 7.

6. A gene inhibitor is L INC01841 gene inhibitor, wherein the L INC01841 gene inhibitor is a molecular inhibitor which is prepared by using L INC01841 as an action target and has an inhibiting effect on L INC01841, and the L INC01841 gene inhibitor can be used for preparing a medicament for treating prostatic cancer.

7. The L INC01841 gene inhibitor according to claim 6, wherein the L INC01841 gene inhibitor is siRNA, the sequence of the sense strand of the siRNA is shown as SEQ ID No.6, and the sequence of the antisense strand of the siRNA is shown as SEQ ID No. 7.

8. A pharmaceutical composition for treating prostate cancer, comprising the L INC01841 gene inhibitor of claim 6 or 7.

9. The pharmaceutical composition of claim 8, further comprising a pharmaceutically acceptable carrier and/or dressing.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutically acceptable carrier and/or dressing comprises: adhesive, diluent, lubricant, surfactant and adsorption carrier.

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