CN111019944B

CN111019944B - SiRNA (small interfering RNA) for targeted silencing of cZNF292 gene and application thereof

Info

Publication number: CN111019944B
Application number: CN201911228958.5A
Authority: CN
Inventors: 屠志刚; 嵇梦晨; 谢旺旺; 刘晗青; 卢子文
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2023-08-22
Anticipated expiration: 2039-12-04
Also published as: CN111019944A

Abstract

The invention relates to the fields of molecular biology and biological medicine, in particular to siRNA for targeted silencing of a cZNF292 gene and application thereof. According to the structural characteristics of the circular RNA, an siRNA crossing a shear site is designed at a splice site of a cZNF292 sequence, and the siRNA is named as si-cZNF292. The cell proliferation experiment result proves that the siRNA can obviously interfere with the expression of cZNF292, obviously inhibit the proliferation activity of lung adenocarcinoma PC9 cells and HCC827 cells, and further inhibit the growth of lung adenocarcinoma. Can be used as a target drug to pertinently interfere the expression of the cZNF292, provides new thought and scientific basis for the research and development of novel anti-lung adenocarcinoma drugs, and has potential application value in the future lung adenocarcinoma treatment research.

Description

SiRNA (small interfering RNA) for targeted silencing of cZNF292 gene and application thereof

Technical Field

The invention relates to the fields of molecular biology and biological medicine, in particular to siRNA for targeted silencing of a cZNF292 gene and application thereof.

Background

Lung cancer is the most common tumor, with the first leading aspect of morbidity and mortality. The prediction results in 2018 show that 2100 ten thousand new lung cancer patients will develop each year, accounting for 11.6% of new tumors throughout the year. At the same time, 1800 ten thousand lung cancer patients are expected to die, and the number of the patients who die due to tumors in the whole year is 18.4 percent. About 88% of lung cancers are non-small cell lung cancers, and despite the great progress that has been made in the treatment of non-small cell lung cancers, the survival rate of patients remains poor, with only 15% of patients with advanced non-small cell lung cancers having a survival rate of 5 years. Among non-small cell lung cancers, lung adenocarcinoma is the most common. Better understanding of the molecular mechanism of lung adenocarcinoma is critical to diagnosis, prognosis and treatment of lung adenocarcinoma. This may also provide new biomarkers or therapeutic targets for diagnosis and treatment of non-small cell lung cancer.

The lung cancer is treated according to different pathological types, but the traditional treatment means generally uses surgical treatment and combined chemotherapy to carry out comprehensive treatment, but the treatment effect is not satisfactory, so that the lack of effective treatment methods and means, and the search for new methods has become an important subject to be solved urgently in the field. Numerous studies have found that the main cause of high morbidity and mortality of lung cancer is the unlimited proliferation of malignant tumors, so finding a method that can effectively inhibit lung adenocarcinoma cell proliferation is a key to improving survival rate of lung adenocarcinoma patients.

RNA interference (RNAi) refers to the phenomenon of highly conserved, double-stranded RNA-induced, highly efficient, specific degradation of homologous mRNA during evolution. Since the RNAi technology can specifically eliminate or shut down the expression of a specific gene, the technology has been widely used in recent years in the field of gene therapy for exploring gene functions and malignant tumors.

Circular RNAs are a novel class of non-coding RNAs, mostly produced by selective reverse cleavage of pre-mRNA. With the rapid development of RNA deep sequencing technology and bioinformatics, more and more circular RNAs have been identified. Among many tumors, including gastric, ovarian, colorectal and lung cancers, circular RNAs have been reported to be involved in many biological processes, such as proliferation, migration, invasion, apoptosis and autophagy. Recently, it was found that the cyclic RNA cZNF292 produced from exon 1A to exon 4 of ZNF292 gene was significantly up-regulated under hypoxic conditions, and that silencing of this cyclic RNA could reduce angiogenesis in vitro. However, the role of cZNF292 in NSCLC remains to be studied. At present, no RNA interference technology is applied, small molecular RNA is used as a targeting drug, and the expression of cZNF292 is interfered in a targeted manner, so that the effect of inhibiting the proliferation of lung adenocarcinoma cells is achieved.

Disclosure of Invention

In view of the above, the present invention aims to provide an siRNA targeting silencing cZNF292 gene, and the targeting gene transfected with the siRNA finds positive correlation with proliferation activity of human lung adenocarcinoma cells PC9, HCC827, and can be used for preparing anti-lung cancer drugs, especially anti-lung adenocarcinoma drugs.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

the invention provides a siRNA for targeted silencing of cZNF292 gene, which designs an siRNA crossing a cutting site at a splicing site of the cZNF292 according to the structural characteristics of annular RNA, and the sense strand sequence of the siRNA is shown as SEQ ID NO.1, namely: 5'-GGATAAAGGCTTGCCCCAGTT-3' the antisense strand sequence is shown in SEQ ID NO.2, namely: 5'-CUGGGGCAAGCCUUUAUCCTT-3'.

In some embodiments, the present invention transfects the above siRNA into human lung adenocarcinoma cells PC9 and HCC827, and uses qRT-PCR technique to detect the expression level of cZNF292 with siGL as reference; the upstream primer cZNF292-F of the qRT-PCR primer is shown as SEQ ID NO.3, namely: 5'-GCTCAAGAGACTGGGGTGTG-3', the downstream primer cZNF292-R is shown as SEQ ID NO.4, namely: 5'-AGTGTGTGTTCTGGGGCAAG-3'. The test result shows that the siRNA provided by the invention can obviously inhibit the expression quantity of the cZNF292 in the PC9 and HCC827 human lung adenocarcinoma cells, and can effectively silence the cZNF292 in the lung adenocarcinoma cells, so that the expression of the annular RNA is obviously reduced, thereby achieving the purpose of treating lung adenocarcinoma.

In some embodiments, the invention further provides methods of detecting the effect on cell proliferation activity following transfection of human lung adenocarcinoma cells with the above-described siRNA by MTT staining. The test result shows that the siRNA transfected si-cZNF292 provided by the invention has stronger cell killing power on human lung adenocarcinoma cells PC9 and HCC827, the relative cell survival rate is obviously reduced, and the proliferation activity of lung adenocarcinoma cells can be effectively inhibited.

The invention also provides an application of the siRNA for targeted silencing of cZNF292, wherein the siRNA is used for preparing biological agents for inhibiting the expression of the cZNF292 gene, medicines for treating lung cancer, medicines for treating lung adenocarcinoma and the like.

The invention also provides a medicine for treating lung adenocarcinoma, which comprises the siRNA targeting silencing cZNF292 gene and other pharmaceutically acceptable auxiliary materials.

The invention also provides a medicine for treating lung cancer, which comprises the siRNA for targeted silencing cZNF292 and other pharmaceutically acceptable auxiliary materials.

The invention also provides a biological agent for inhibiting MYU expression, which comprises the siRNA for targeted silencing cZNF292 and further comprises other pharmaceutically acceptable carriers.

Further, the invention also provides a method for inhibiting the expression of cZNF292, wherein the method is to interfere with the expression of the cZNF292 after the siRNA is transfected.

The beneficial effects of the invention are as follows:

according to the structural characteristics of the circular RNA, an siRNA crossing a shear site is designed at a splice site of a cZNF292 sequence, and the siRNA is named as si-cZNF292. The cell proliferation experiment result proves that the siRNA can obviously interfere with the expression of cZNF292, obviously inhibit the proliferation activity of lung adenocarcinoma PC9 cells and HCC827 cells, and further inhibit the growth of lung adenocarcinoma. In view of the fact that cZNF292 is used as a potential lung cancer treatment target, the siRNA and the related biological reagent thereof provided by the invention can be used for researching and preparing medicaments for treating related diseases of lung cancer. Meanwhile, the invention proves that the siRNA can inhibit proliferation of human lung adenocarcinoma cells PC9 and HCC827, can be used as target medicaments to pertinently interfere cZNF292 expression, provides new thought and scientific basis for research and development of novel medicaments for resisting lung adenocarcinoma, and has potential application value in future lung adenocarcinoma treatment research.

Drawings

FIG. 1 is a graph comparing the expression levels of si-cZNF292 and siGL2 on PC9 cells;

FIG. 2 is a graph comparing the expression levels of si-cZNF292 and siGL2 on HCC827 cells;

FIG. 3 is a graph comparing the relative survival rates of si-cZNF292 and siGL2 versus PC9 cells;

FIG. 4 is a graph comparing the relative survival rates of si-cZNF292 and siGL2 versus HCC827 cells.

Detailed Description

The invention is further illustrated by the following examples, which are provided to illustrate the invention and are not intended to limit the scope of the invention. Those skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. The experimental materials used in the examples described below are all commercially available unless otherwise specified. The present invention uses all techniques known in the art, except as noted specifically.

Experimental materials and reagents: du Erba modified eagle's medium, distilled water without DNase/RNase, reduced serum medium, liposomes 2000 were all purchased from America Life technologies, inc., life Technologies; cell incubator was purchased from sameimers; trypsin was purchased from sigma aldrich trade limited; the nvitrogen reverse transcription kit was purchased from english strapdesk trade limited; thiazole blue was purchased from BIOSHARP company. Instrument: ABI7300 type fluorescent quantitative PCR instrument, burley, USA.

The human lung adenocarcinoma cells PC9 and HCC827 used in the invention are purchased from American type culture Collection (American type culture Collection), and American type culture collection; human lung adenocarcinoma cells PC9 and HCC827 were treated with Du Erba family modified eagle medium (DMEM) containing 10% fetal calf serum, 10U/ml penicillin, 0.1mg/ml streptomycin, and treated with 5% CO ₂ Culturing in a cell culture box at 37 ℃. Observing the growth state of cells once a day, and carrying out passage in time when the cells grow to 70% or more of the surface area of the culture flask, and keeping the cells in a good state and in a logarithmic growth phase for later use.

The invention is based on the structural characteristics of cyclic cZNF292 (with the sequence shown as SEQ ID NO. 5) and passes through BLOCK-iT of Thermo Fisher Scientific ^TM RNAi Designer designs an siRNA sequence crossing a cleavage site at a splice site of the cZNF292 sequence, and the sense strand sequence of the siRNA sequence is shown as SEQ ID NO.1, namely: 5'-GGATAAAGGCTTGCCCCAGTT-3' the antisense strand sequence is shown in SEQ ID NO.2, namely: 5'-CUGGGGCAAGCCUUUAUCCTT-3'. The si-cZNF292 powder was synthesized by Shanghai Ji Ma pharmaceutical technologies Co., ltd according to the primer sequence, and the siGL2 powder in the comparative example was also purchased from Shanghai Ji Ma pharmaceutical technologies Co., ltd; the si-cZNF292 powder and the si GL2 powder were prepared into 20. Mu.M si-cZNF292 and si-GL2 powder with distilled water (DNase/RNase-Free Distilled Water) free of DNase/RNase, respectively, as required in the specification, and stored in a refrigerator at-20℃for use.

Example 1 qRT-PCR experiments to detect silencing efficacy of si-cZNF292

Dilute si-cZNF292 and si-GL2 at 20. Mu.M to 2. Mu.M working concentration with DNase/RNase-Free Distilled Water, and then take 2 EP tubes and dilute si-cZNF292 and si-GL2 with serum-reduced medium (OPTI-MEM) to 25nM final concentration, respectively, and incubate 100. Mu.L of the mixture with 2. Mu.L of liposome 2000 (lipofectamine 2000) at room temperature for 15min.

The T25 flask for culturing PC9 and HCC827 cells was removed, and 10. Mu.l of the cell suspension was taken out to a cell counting plate for counting after cell passage treatment of the resuspended cells by a conventional method. The cell suspension volume per well was 900. Mu.l, 4.5X10 were seeded ⁵ Cell by cell, cell suspension was calculated and formulated.

A six-well plate was taken, 100. Mu.l of the incubated mixture was added to each of the 4 wells, and then 900. Mu.l of the cell suspension with the adjusted cell density was added thereto, and the mixture was gently shaken and placed in a cell incubator for culturing for 72 hours. After 72h, six-well plates were removed, cells in the wells were digested with trypsin in a general passaging step, transferred to EP tubes for labeling, centrifuged at 400g for 5min, supernatant was aspirated off, resuspended in 1ml PBS in each EP tube, and the supernatant was centrifuged off once again for subsequent qRT-PCR experiments.

The relative expression level of cZNF292 in cells is detected by a real-time fluorescent quantitative RT-PCR technology, the used qRT-PCR primer sequence is synthesized by a biological engineering (Shanghai) Co., ltd, and the upstream primer cZNF292-F is shown as SEQ ID NO.3, namely: 5'-GCTCAAGAGACTGGGGTGTG-3', the downstream primer cZNF292-R is shown as SEQ ID NO.4, namely: 5'-AGTGTGTGTTCTGGGGCAAG-3'.

qRT-PCR experiments: the total RNA in the isolated cells was extracted by Trizol method, and then reverse transcribed by Invitrogen reverse transcription kit to synthesize first strand DNA, then qRT-PCR reaction system (volume 20. Mu.l, ultrapure water containing DNase RNase, 10 XPCR buffer, dNTP, SYBR Green I dye, ROX dye, pfu DNA polymerase, first strand DNA template obtained by reverse transcription of total RNA of the cells, front primer cZNF292-F, back primer cZNF 292-R) was prepared, and detection was performed by ABI7300 type fluorescent quantitative PCR instrument to transfect si-cZNF292 as experimental group, and transfected si GL2 as control group to compare the relative expression amount of cZNF292 in experimental group and control group. The PCR reaction procedure was: pre-denaturation at 95 ℃ for 2 min; denaturation at 95℃for 10 seconds, annealing at 60℃for 30 seconds, 40 cycles, and fluorescence signals were collected during cyclic extension of the PCR reaction. Finally use 2 ^-△△CT The relative expression level difference of cZNF292 between the experimental group and the control group was calculated by the method.

FIG. 1 is a graph comparing the expression levels of PC9 cells by experimental group si-cZNF292 and control group siGL 2; FIG. 2 is a graph comparing the expression levels of HCC827 cells by experimental group si-cZNF292 and control group siGL 2; as shown in fig. 1 and 2, the real-time fluorescent quantitative RT-PCR experiment result shows that compared with the control group siGL2, si-cZNF292 significantly inhibits the expression level of cZNF292 in PC9 cells and HCC827 cells (P <0.0001 is shown in the expression level, and the experiment frequency is equal to or greater than 3); silencing efficiency of the si-cZNF292 experimental group reached 83.77% ± 4.76% and 85.52% ± 3.31% in the two cell lines, respectively.

Example 2 influence of si-cZNF292 on cell proliferation Activity

In this example, the relative viability of the cells after the action of si-cZNF292 was examined by MTT staining to determine the effect of si-cZNF292 on the proliferation potency of PC9 and HCC827 cells.

In this example, the cell suspension was prepared in the same manner as in example 1, except that 2X 10 cells were used in the amount of 90. Mu.l per well of the cell suspension ³ Cell by cell, cell suspension was calculated and formulated. For each condition, 3 wells were multiplexed, 10. Mu.l of the incubated mixture was added to each well, and 90. Mu.l of the cells had a density of 2.3X10 ⁴ The cell suspension of each ml is gently shaken and placed into a cell culture box for culture. After 96h, the 96-well plate was removed and 10. Mu.l of 5mg/ml thiazole blue (MTT) was added to each well. After further incubation in an incubator for 1.5h, the broth was aspirated, 100 μl of dimethyl sulfoxide (DMSO) was added to each well, and blue-violet formazan crystals were dissolved by shaking in the dark for 15min on a shaker, and finally their absorbance was measured with an enzyme-linked immunosorbent assay at 550nm wavelength to measure the relative viability of the cells. The relative viability of PC9 cells after si-cZNF292 was examined 96h later by MTT assay.

FIG. 3 is a graph comparing the relative survival rates of si-cZNF292 and siGL2 versus PC9 cells; FIG. 4 is a graph comparing the relative survival rates of si-cZNF292 and siGL2 versus HCC827 cells; (the test times in the figure are more than or equal to 3). As shown in fig. 3 and 4, si-cZNF292 was more killing on PC9 and HCC827 cells compared to control reverse-transfected siGL2, and the relative cell viability was significantly reduced. After 96h, the cell viability of PC9 and HCC827 were 23.33% plus or minus 1.17% and 15.03% ± 4.03%, respectively.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims

1. The siRNA targeting silencing cZNF292 gene is characterized in that the sense strand sequence of the siRNA is shown as SEQ ID NO.1, and the antisense strand sequence is shown as SEQ ID NO. 2.

2. Use of the siRNA targeted to silence the cZNF292 gene of claim 1 in the preparation of a biologic that inhibits expression of the cZNF292 gene; the biological agent is used for treating lung adenocarcinoma.

3. Use of the siRNA targeted to silence cZNF292 gene of claim 1 in the manufacture of a medicament for treating lung adenocarcinoma.

4. A medicament for treating lung adenocarcinoma, comprising the siRNA of claim 1 targeted to silence the cZNF292 gene.

5. A biologic for inhibiting expression of cZNF292 comprising the siRNA of claim 1 targeted to silence the cZNF292 gene.

6. A medicament for treating lung adenocarcinoma, comprising the siRNA of claim 1 targeted to silence the cZNF292 gene.