阅读说明：本技术 LncRNA XIST及与其结合的miR-132作为抑制胃癌细胞增殖、侵袭与转移的用途 (LncRNA XIST and application of miR-132 combined with LncRNA XIST in inhibition of gastric cancer cell proliferation, invasion and metastasis ) 是由 李丽丽 刘佳利 黄娅 于 2020-04-15 设计创作，主要内容包括：本发明公开了一种长链非编码RNA XIST和与之结合的miR-132在抑制胃癌细胞增殖、侵袭与转移的用途。本发明的长链非编码RNA XIST的si-XIST,能够沉默LncRNA XIST,导致LncRNA XIST对miR-132的吸附能力下降,而miR-132能与CUL4B的基因结合,最终下调CUL4B的表达,从而抑制胃癌细胞的增殖、侵袭转移及成瘤能力,促进胃癌细胞的凋亡。本发明公开的LncRNA XIST和miR-132的应用可能有助于胃癌的靶向治疗和临床治疗。(The invention discloses application of a long-chain non-coding RNA XIST and miR-132 combined with the long-chain non-coding RNA XIST in inhibition of proliferation, invasion and transfer of gastric cancer cells. The si-XIST of the long-chain non-coding RNA XIST can silence LncRNA XIST, so that the adsorption capacity of the LncRNA XIST on miR-132 is reduced, the miR-132 can be combined with the gene of CUL4B, and the expression of CUL4B is finally reduced, so that the proliferation, invasion and metastasis and tumorigenicity of gastric cancer cells are inhibited, and the apoptosis of the gastric cancer cells is promoted. The application of LncRNA XIST and miR-132 disclosed by the invention is possibly beneficial to targeted therapy and clinical therapy of gastric cancer.)

1. The long-chain non-coding RNA is LncRNAXIST, and is characterized in that si-XIST of the LncRNAXIST is used for inhibiting proliferation, invasion and metastasis of gastric cancer cells.

2. The use as claimed in claim 1, wherein the lncrnaxaist has a nucleotide sequence as set forth in SEQ id no: 1 is shown.

3. The use according to claim 1, wherein the nucleotide sequence of si-XIST is as set forth in SEQ id no: 2, respectively.

4. The use according to claim 1, wherein the nucleotide sequence of si-XIST is as set forth in SEQ id no: 3, respectively.

5. Use of miR-132 capable of directly binding to lncrnaxaist of claim 1 for inhibiting gastric cancer cell proliferation, invasion and metastasis.

6. The use of claim 5, wherein the nucleotide sequence of miR-132 is as shown in SEQ ID NO: 4, respectively.

7. The use of claim 5, wherein the target gene of miR-132 is CUL 4B.

8. The use according to claim 5, wherein said miR-132 binds to the 3' UTR of the CUL4B gene in gastric cancer cells.

Technical Field

The invention relates to a long-chain non-coding RNA related to gastric cancer and application of microRNA combined with the long-chain non-coding RNA in inhibition of proliferation, invasion and transfer of gastric cancer cells, wherein the long-chain non-coding RNA is LncRNA XIST, and the microRNA is miR-132, belongs to the field of biomedicine, and particularly belongs to the technical field of molecular biology.

Background

Gastric Cancer (GC) is a common malignant tumor, and the incidence of various malignant tumors is the first in China. Since most of the gastric cancers have no obvious symptoms or specific symptoms at the early stage, the gastric cancers are easy to be overlooked, so that the early diagnosis rate of the gastric cancers is low at present. Once found, it has progressed to a middle to late stage, resulting in a high fatality rate, reaching 75%. The main causes of the development of the gastric cancer are 4, namely, regional environment and dietary life factors, helicobacter pylori (Hp) infection, precancerous lesion and genetic factors; among them, helicobacter pylori infection. There is a major risk factor for gastric cancer. Many advanced examination and diagnostic methods for gastric cancer have emerged. The diagnosis method comprises the following steps: x-ray barium meal examination, fibrogastroscopy, abdominal ultrasound, spiral CT and positron emission imaging examination; or detecting tumor markers (such as CEA, CA50, CA72-4, CA19-9, etc.); the treatment method comprises standard treatment, such as surgery, chemotherapy or targeted drug therapy. The chemotherapy drugs are irinotecan (irinotecan), taxane (taxanes), fluoropyrimidine (fluoropyrridine), platinum base (platinum base) and the like; the targeted therapeutic medicine comprises apoptosis promoter, epidermal growth factor receptor inhibitor, matrix metalloproteinase inhibitor, angiogenesis inhibitor, cell cycle inhibitor, etc. However, the mean survival of gastric cancer patients is still low, only about 8-10 months, and the prognosis is poor, and the main reasons for this phenomenon may be the difficulty of early detection of gastric cancer and the lack of effective therapeutic targets. Therefore, researchers have focused on the research of early diagnosis and effective therapeutic targets of gastric cancer.

After the 21 st century, with the rapid development of modern biotechnology, especially the maturation of gene sequencing technology and genome analysis technology, scientists found that most genes were transcribed into non-coding RNAs (ncRNAs). Non-coding RNA is classified into two types according to the difference in coding length of RNA. One is RNA with length less than 200 nucleotides, mainly micro RNA (miRNAs), small interfering RNA (siRNAs), etc. Among them, miRNAs are small (about 22 nucleotides) non-coding RNAs, which play important roles in various biological and pathological processes such as cell proliferation, differentiation, apoptosis, canceration, etc.; miRNAs participate in various stages of tumor development, and show that the abnormal expression of miRNA plays an important regulatory role in the expression of known oncogenes or cancer suppressor genes in the tumor development process. In the process of gastric cancer occurrence and development, miRNA can play a role in promoting tumor occurrence and development or inhibiting tumor occurrence and development, and has great research and application prospects in diagnosis, prognosis, detection and treatment of gastric cancer. Another non-coding RNA is called long non-coding RNA (LncRNA) because it is more than 200 nucleotides in length. The Long non-coding RNA is generally between 200 and 100000 nucleotides in length, is located in nucleus or cytoplasm, and can be divided into five types, namely antisense Long non-coding RNA (antisense LncRNA), intron non-coding RNA (intron tran), Long endogenous non-coding RNA (lincRNA), Promoter-associated LncRNA (Promoter-associated LncRNA), and untranslated region LncRNA (UTAnacardiatedLncRNA), according to the source. LncRNA has various functions and is closely related to the occurrence, development and prevention and treatment of human diseases. For example, LncRNA may be involved in regulating gene expression at the epigenetic, transcriptional and post-transcriptional levels; participate in a plurality of important regulation processes such as X chromosome silencing, genome imprinting, chromatin modification, transcription activation, transcription interference, intranuclear transportation and the like. In addition, the literature reports that long-chain non-coding RNA participates in invasion and migration of gastric cancer cells and lymph node metastasis, and the long-chain non-coding RNA has good research value and application prospect in diagnosis, prognosis, detection and treatment of gastric cancer. Therefore, screening and further studying miRNAs and/or lncrnas associated with the development of gastric cancer is a very promising direction for early diagnosis and/or treatment of gastric cancer.

The long non-coding RNA plays a role of a miRNA sponge and can regulate and control the expression of a target gene. LncRNA X-inactive transcript (LncRNA XIST) is a long non-coding RNA which plays an important role in human cell gene maintenance, proliferation and differentiation and can regulate the X chromosome at the transcription level. Scientists found that LncRNA XIST is up-regulated in non-small cell lung cancer (NSCLC), while miR-137 is abnormally down-regulated in non-small cell lung cancer. This indicates that LncRNA XIST is a competitive endogenous rna (cerna) of miR-137, and can promote survival and invasion of NSCLC cells; miR-137 targets the 3' UTR of Paxillin (PXN), so that the activity and invasion of non-small cell lung cancer cells can be inhibited; meanwhile, miR-137 is in negative correlation with PXN expression, namely LncRNA XIST is in positive correlation with PXN expression. Paxillin (pxn), a focal adhesion aptamer with multiple functions, plays an important role in focal adhesion, tumor progression and migration, endothelial cell barrier dysfunction, inflammatory response, oxidative stress, and the like. PXN is also frequently expressed abnormally in human cancer progression, and has been reported to correlate with the cell invasiveness of gastric cancer cell line AGS. Meanwhile, research shows that the PXN gene knockout or miR-137 abnormal expression can obviously inhibit the in-vitro proliferation, migration and invasion of colorectal cancer cells and inhibit the growth and metastasis of tumors. Therefore, the inhibition of miR-137-induced PXN overexpression promotes the metastasis and development of colorectal cancer tumors, and the inhibition can be used as an independent prognostic index of colorectal cancer patients. In addition, the expression level of miR-132in gastric cancer tissues is obviously higher than that of corresponding normal tissues, and the miR-132 is related to more lymph node metastasis, more lymph duct cancer emboli and more advanced gastric cancer, so that miR-132 can also be used as an effective prognostic factor of gastric cancer patients and becomes another promising diagnosis and/or treatment target of gastric cancer. In the occurrence and development of gastric cancer, miR-132 and/or LncRNA XIST possibly play an important role through PXN or other targets, and provides scientists with a new research direction in the diagnosis and/or treatment of gastric cancer.

Disclosure of Invention

In order to research the effect of miRNA related to gastric cancer and related long-chain non-coding RNA in gastric cancer, and therefore to screen appropriate related long-chain non-coding RNA or miRNA which can be used for inhibiting gastric cancer cell proliferation, invasion and metastasis. 65 cases of stomach cancer and tissues beside the cancer which are removed by operation are collected from hospitals, and LncRNA XIST which can be used for inhibiting proliferation, invasion and metastasis of stomach cancer cells and miR-132 which can be directly combined with the LncRNA XIST are found from the tissues by related technical means.

Therefore, the invention provides an LncRNA XIST, and the use of si-XIST of the LncRNA XIST for inhibiting the proliferation, invasion and metastasis of gastric cancer cells.

Preferably, the nucleotide sequence of lncrrna XIST of the present invention is as set forth in SEQ ID NO: 1 is shown.

Preferably, the nucleotide sequence of si-XIST described in the present invention is as shown in SEQ ID NO: 2, respectively.

Preferably, the nucleotide sequence of si-XIST described in the present invention is as shown in SEQ ID NO: 3, respectively.

On the other hand, the invention also provides application of miR-132 capable of being directly combined with LncRNAXIST in inhibiting proliferation, invasion and metastasis of gastric cancer cells.

Preferably, the nucleotide sequence of miR-132 provided by the invention is shown in SEQ ID NO: 4, respectively.

Preferably, the target gene of miR-132 described in the invention is CUL 4B.

Preferably, the miR-132 described in the invention binds to the 3' UTR of the CUL4B gene in gastric cancer cells.

The si-XIST disclosed by the invention can silence LncRNA XIST, so that the adsorption capacity of the LncRNA XIST on miR-132 is reduced, and the expression of CUL4B is finally reduced, so that the proliferation, invasion and metastasis and tumor forming capacity of gastric cancer cells are inhibited, and the apoptosis of the gastric cancer cells is promoted. In addition, the miR-132 can be combined with the gene of CUL4B to down-regulate the expression of CUL4B, so that the proliferation, invasion and metastasis and tumorigenicity capacity of gastric cancer cells are inhibited, and the apoptosis of the gastric cancer cells is promoted. The application of LncRNA XIST and miR-132 disclosed by the invention is possibly beneficial to targeted therapy and clinical therapy of gastric cancer.

Drawings

Wherein, indicates that P <0.05, indicating significant difference.

FIG. 1 expression and localization of LncRNA XIST in gastric cancer tissues and cell lines:

FIG. 1A: RT-PCR detects the relative expression level of LncRNA XIST in gastric cancer tissues and paracancer normal tissues, wherein Adjacent normal tissues represent paracancer normal tissues, and GC tissues represent gastric cancer tissues;

FIG. 1B: RT-PCR detects the relative expression level of LncRNA XIST in the stomach cancer tissue without metastasis and the stomach cancer tissue with metastasis, wherein without metastasis represents the stomach cancer tissue without metastasis, and with metastasis represents the stomach cancer tissue with metastasis;

FIG. 1C: RT-PCR detects the relative expression level of LncRNA XIST in different gastric cancer cell lines;

FIG. 1D: FISH experiments, LncRNA XIST indicates localization of long non-coding RNA XIST, merge with DAPI indicates co-localization of long non-coding RNA XIST with nuclei, red portion in the figure represents LncRNA XIST, blue portion represents nuclei,. times.200.

FIG. 2LncRNA XIST involved in regulating biological function of gastric cancer cells [ si-NC group (without any treatment), si-XIST-1 group (transfection interfering XIST-1 plasmid), si-XIST-2 (transfection interfering XIST-2 plasmid) ]:

FIG. 2A: qRT-PCR detects the expression of LncRNA XIST;

FIG. 2B: EdU measures proliferation capacity (x 400) of each group of cells, where EdU indicates proliferating cells, DAPI stained nuclei indicating whole viable cells, and Merge indicates cells where EdU and DAPI coincide;

FIG. 2C: cell scratch assay test migration capacity of each group of cells (x 40);

FIG. 2D: transwell measures the invasion capacity (x 200) of each group of cells;

FIG. 2E: detecting the apoptosis capacity of each group of cells by flow cytometry;

FIG. 2F: in vivo tumor formation experiments in mice examined the effect of LncRNA XIST on tumor formation growth.

FIG. 3 prediction of the binding relationship between XIST and miR-132 and its effect on the biological function of gastric cancer cells [ mic-NC group (without any treatment), miR-132 mic group (miR-132 micid plasmid transfection), inhibitor-NC group (without any treatment), miR-132inhibitor group (miR-132 inhibitor plasmid transfection), miR-132 micid + si-XIST group (miR-132 micid and si-LncRNA XIST plasmids are transfected at the same time) ]:

FIG. 3A: miR-132 and LncRNA XIST binding site, wherein Lnc RNA XIST-WT represents wild type, Lnc RNA XIST-MUT represents mutant type;

FIG. 3B: the result of detecting the fluorescence activity of miR-132 and LncRNA XIST;

FIG. 3C: detecting the binding of LncRNA XIST, miR-132 and Ago2 by RIP;

FIG. 3D: RNA-pull down analysis of the binding of LncRNA XIST to miR-132, wherein NC-miR-132 represents the untreated control group, WT-miR-132 represents the wild-type group, and MUT-miR-132 represents the mutation group;

FIG. 3E: EdU measures proliferation capacity (x 400) of each group of cells, where EdU indicates proliferating cells, DAPI stained nuclei indicating whole viable cells, and Merge indicates cells where EdU and DAPI coincide;

FIG. 3F: cell scratch assay test migration capacity of each group of cells (x 40);

FIG. 3G: transwell measures the invasion capacity (x 200) of each group of cells;

FIG. 3H: detecting the apoptosis capacity of each group of cells by flow cytometry;

FIG. 3I: the in vivo tumor formation experiment of the mice detects the influence of miR-132 on the tumor formation and growth.

FIG. 4 Targeted prediction of miR-132 and CUL4B (Cullin-4B) and statistics of expression associated with XIST, miR-132 and CUL4B [ mic-NC group (without any treatment), miR-132 mic group (transfection of miR-132 mic plasmid), inhibitor-NC group (without any treatment), miR-132inhibitor group (transfection of miR-132inhibitor plasmid), miR-132 mic + si-XIST group (transfection of miR-132 and si-LncRNA XIST plasmids) ]:

FIG. 4A: the binding site of miR-132 to CUL 4B;

FIG. 4B: detecting the fluorescence activity of miR-132 and CUL 4B;

FIG. 4C: WB detects the expression gray development pattern of CUL4B protein;

FIG. 4D: and (3) carrying out column statistics on expression of CUL4B protein by WB detection.

FIG. 5 Effect of CUL4B expression on gastric cancer cell biological function [ oe-NC group (without any treatment), oe-CUL4B group (transfection of CUL4B overexpression plasmid), oe-CUL4B + si-XIST group (transfection of oe-CUL4B and si-LncRNAXIST plasmid at the same time) ]:

FIG. 5A: qRT-PCR detects the expression of LncRNA XIST;

FIG. 5B: EdU measures proliferation capacity (x 400) of each group of cells, where EdU indicates proliferating cells, DAPI stained nuclei indicating whole viable cells, and Merge indicates cells where EdU and DAPI coincide;

FIG. 5C: cell scratch assay test migration capacity of each group of cells (x 40);

FIG. 5D: transwell measures the invasion capacity (x 200) of each group of cells;

FIG. 5E: detecting the apoptosis capacity of each group of cells by flow cytometry;

FIG. 5F: in vivo mouse tumorigenesis experiments examined the effect of CUL4B on tumor formation growth.

Detailed Description

The invention is further illustrated below with reference to specific examples. The various starting materials mentioned in the following examples are all commercially available unless otherwise specified.

The experimental method comprises the following steps:

1.1 sample Collection

65 specimens of gastric cancer and tissues beside the cancer (more than 5cm away from a canceration part and no cancer tissues in pathological examination) are collected from the collection of surgical excision from 2 month and 1 day to 2017 year and 2 month and 1 day in the hospital, and 42 and 23 males and 23 females in specimen source patients are aged 29-82 years and are aged (57.35 +/-17.74) on average. All patients did not receive any radiotherapy or chemotherapy before surgery. There were 13 cases of no regional lymph node metastasis (N0), 16 cases of 1 to 6 regional lymph node metastasis (N1), 17 cases of 6 to 15 regional lymph node metastasis (N2), and 19 cases of more than 15 regional lymph node metastasis (N3). The collected sample tissues are divided into two types, wherein one part of tissues are collected and immediately stored in a liquid nitrogen tank, and the other part of tissues used for subsequent experiments are fixed by paraformaldehyde and then are embedded by paraffin. The study was approved by the ethical committee of my hospital and all patients signed informed consent.

1.2 cell culture

Culturing human gastric cancer cell strains BGC-823, SGC7901, MKN45, MKN28, HGC-27, AGS and human normal gastric mucosal epithelial cell strain GES-1. The cells except for the AGS cell line were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 100U/ml streptomycin solution, and the AGS cell line was cultured in F12 medium containing 10% fetal bovine serum and 100U/ml streptomycin solution. All cells were cultured in a 5% CO2 cell culture chamber at 37 ℃. When the cell density reached about 90%, the cells were passaged, the medium in the cell flask was aspirated away, 1ml of sterile PBS solution was added and washed once, and 1ml of 0.25% pancreatin was added for digestion. When the cells appear oval, the digestion is stopped by adding 4ml of the culture medium, the cells are gently pipetted and mixed evenly, the cells are counted and then passaged according to a certain proportion, and the final screening is carried out by qRT-PCR. The cell lines were purchased from Wuhan Punuoist Life technologies, Inc.

1.3 cell grouping and transfection

The gastric cancer cells SGC7901 obtained by screening are collected and divided into 11 groups: si-NC group (without any treatment), si-XIST-1 group (transfection interfering XIST-1 plasmid), si-XIST-2 (transfection interfering XIST-2 plasmid), mimic-NC group (without any treatment), miR-132 mimic group (transfection miR-132 mimic plasmid), inhibitor-NC group (without any treatment), miR-132inhibitor group (transfection miR-132inhibitor plasmid), miR-132 mimic + si-XIST group (simultaneous transfection of miR-132 mimic and si-LncRNAXIST plasmids), oe-NC group (without any treatment), oe-CUL4B group (transfection CUL4B overexpression plasmid), oe-CUL4B + si-XIST group (simultaneous transfection of oe-CUL4B and si-LncXIST plasmids). si-XIST, miR-132 mimic, miR-132inhibitor, oe-CUL4B were all purchased from Ruibo, Guangzhou. Cell transfection procedure was as follows: appropriate amounts of cells were seeded into 24-well plates and cultured, and when the cell density was 50% -60%, gastric cancer cells were transfected according to the procedure of Lipofectamine (TM) (Invitrogen, USA). Lipofectamine 2000 and RNA to be transferred were prepared in sterile EP tubes, two respectively: 1 μ L lipofectamine 2000+50 μ L serum-free medium, left at room temperature for 5min until RNA transfer (20pmlo) +50 μ L serum-free medium; mixing the liquids in the two EP tubes to enable the RNA and the liposome to form a complex, and standing for 20min at room temperature; finally, adding the mixture into the culture dish cells to be transfected; placing the mixture in a 37 ℃ and 5% CO2 incubator for continuous culture, and adding the complete culture medium after 6-8 h.

1.4FISH(Fluorescence in situ hybridization,FISH)

Expression of LncRNA XIST was detected in Situ in gastric cancer cells using the Floriscent in Situ Hybridization Kit (C10910, Boehringer, Guangzhou). Placing the cell slide at the bottom of a 24-well plate, taking the cells growing in the logarithmic phase to digest on the slide (about 6 × 104/well), and fixing for 10min at room temperature by using 4% paraformaldehyde; after PBS cleaning, 1mL of precooled liquid permeable is added into each hole, and the mixture is kept stand for 5min at 4 ℃; discarding the permeation solution, adding 1 × PBS to wash the cells for 3 times, adding 200 μ L of prehybridization solution into each well, and sealing at 37 deg.C for 30 min; adding 2.5 μ L of 20 μm FISH Probe Mix stock solution into the hybridization solution under dark condition; discarding the prehybridization solution in each hole of cells, adding a proper amount of probe hybridization solution containing the probe, keeping out of the sun, and hybridizing at 37 ℃ overnight; washing the cells in each well at 42 ℃ in the dark to reduce the background signal, and washing the cells at 42 ℃ in the dark for 1 time by using a washing solution II; washing the cells for 1 time at 42 ℃ in a dark place; cells were washed with 1 × PBS in the dark. DNA staining: shading, dyeing with DAPI staining solution for 10 min; light protected, cells were washed 3 times with 1 × PBS. Sealing: under the condition of keeping out of the light, the cell slide is carefully taken out from the hole and fixed on a glass slide by using a sealing agent for fluorescence detection. Specific probes for LncRNA XIST were synthesized by Ribo Bio Inc.

1.5 Dual luciferase reporter assays

And (3) carrying out binding site analysis on the LncRNA XIST, the miR-132 and the CUL4B by using a biological prediction website, and obtaining a fragment sequence containing an action site. The full length of LncRNA XIST and the 3' UTR region of CUL4B were cloned into pmirGLO (E1330, Promega, USA) Luciferase vector, named pLncRNA XIST-Wt and pCUL4B-Wt, respectively. Respectively constructing pLncRNA XIST-Mut and pCUL4B-Mut vectors, wherein the internal references are pRL-TK vector (E2241, Promega, USA) for expressing renilla Luciferase, the miR-132 mimic group and the mimic-NC group are co-transfected with a Luciferase report vector for SGC7901 cells (CRL-1415, ATCC, USA), Dual Luciferase Reporter Gene Assay Kit (GM-040502A, promastigotes, China) at 560nm (firefly RLU) and 465nm (renilla RLU), and the ratio of firefly RLU/renilla RLU is used for judging the binding strength.

1.6RNA-pull down assay

Gastric cancer cells SGC7901 were digested with 0.25% trypsin (Gibco, USA), the pellet was centrifuged and resuspended in RIP lysate, lysed on ice for 5min, the lysate was aliquoted into aliquots (one of which was input) and placed at-80 ℃. Using a Magnetic RNA-Protein Pull-Down kit (Pierce, USA), 1. mu.g of biotin-labeled RNA was taken out and put into an EP tube, 500. mu.L of Structure Buffer was added thereto, and the mixture was subjected to a water bath at 95 ℃ for 2min and then to an ice bath for 3 min. Resuspend the beads well and take 50. mu.L of the bead suspension in an EP tube overnight at 4 ℃. Centrifuge at 3000rpm for 3min, and discard the supernatant. Add 500. mu.L RIP Wash Buffer and Wash 3 times, add 10. mu.L cell lysate, and let stand at room temperature for 1 h. And eluting the protein in the incubated magnetic bead-RNA-protein mixture. And (3) detecting protein expression by Western-blotting after the protein concentration is detected by a BCA method.

1.7 RIP (RNA Immunoprecipitation) assay

Gastric cancer cells were lysed with lysis buffer (25mM Tris-HCl pH7.4, 150mM NaCl, 0.5% NP-40, 2mM EDTA, 1mM NaF and 0.5mM dithiothreitol) containing RNase (Takara) and protease inhibitor cocktail (B14001a, Roche, USA). The lysate was centrifuged at 12000g for 30min and the supernatant was taken, then anti-human Ago-2 beads (BMFA-1, bemaiden bio, china) were added, and anti-IgG beads were added to the control group. After 4h incubation at 4 ℃ the beads were washed 3 times with wash buffer (50mM Tris-HCl, 300mM NaCl pH7.4,1mM MgCl2, 0.1% NP-40). RNA was extracted from the magnetic beads using TRIzol, and LncRNA XIST was detected by RT-PCR.

1.8 RT-PCR

Gastric cancer cells SGC7901SGC7901 growing in the logarithmic phase are transfected, and after 24 hours, the cells are collected and subjected to total RNA extraction by the Trizol (15596026, Invitrogen, Car, Cal, USA) method. RNA was reverse transcribed into cDNA according to the PrimeScript RT reagentKit (RR047A, Takara, Japan) reverse transcription kit instructions. The reaction conditions are as follows: 15min at 37 ℃; 20. mu.l of the system at 85 ℃ for 5 s. Primers for LncRNA XIST, miR-132, CUL4B, U6 and GADPH were designed, and were synthesized by Shanghai's engineering (see Table 1). The reverse transcription experiment system was performed in 20. mu.l, according to the EasyScript First-Strand cDNAsynthesis SuperMix (catalog number AE301-02, Beijing Quanyu, China) instructions. Taking the reaction solution to perform real-time fluorescent quantitative PCR operation, wherein the reaction system is 20 mul: 10. mu.l SYBR Premix, 2. mu.l cDNA template, 0.6. mu.l each of upstream and downstream primers and 6.8. mu.l DEPC water. RT-PCR experiments were performed using 7500 type fluorescent quantitative PCR from ABI, USA, with the reaction set-up conditions: pre-denaturation at 95 ℃ for 30s, annealing at 20s, extension at 72 ℃ for 30s, and 40 cycles. 2-delta-delta Ct represents the fold ratio relation of target gene expression of the experimental group and the control group, and the formula is as follows: the Δ Ct is Ct (target gene) -Ct (internal reference), Δ Ct is Δ Ct experimental group- Δ Ct control group. Ct is the number of diffusion cycles that elapsed when the real-time fluorescence intensity of the reaction reached a set threshold, where diffusion was increasing in log phase [ PMID: 16531770]. (the experiment was repeated 3 times) the expression level of LncRNAXIST, miR-132 and CUL4B in the cells was calculated.

TABLE 1 RT-PCR primer sequences

1.9 Western blotting

And (3) taking gastric cancer cells SGC7901 in a logarithmic growth phase to perform transfection according to cell grouping conditions, removing cell culture solution in a cell bottle after 48 hours, washing for three times by using precooled PBS (phosphate buffer solution), adding prepared RIPA (Ribose nucleic acid) lysate in advance, cracking for 30min on ice, centrifuging for 10min at 14000g, taking supernatant, measuring the protein concentration by using a BCA (burst amplification) method, and storing at-20 ℃ for later use. Then, SDS-PAGE gel kit is used for preparing 10% separation gel and 5% lamination gel, protein is separated on polyacrylamide gel by electrophoresis, then the protein is transferred to an NC membrane by adopting a wet transfer method, and 5% BSA is sealed for 1h at room temperature. The diluted primary anti-mouse anti-human CUL4B (ab32084, 1:1000) was added dropwise, overnight at 4 ℃, the membrane was washed with PBST 3 times the next day for 10min each time, the secondary anti-goat anti-mouse polyclonal antibody (ab6785,1:2000) was diluted with 5% skim milk, then the membrane was shaken on a shaker at room temperature for 1h, and washed with PBST again for 3 times for 15min each time. The developer was added and developed by bio-Rad gel imaging system (MG8600, Beijing Tomo Bio-technologies, Inc., China) for quantitative analysis using IPP7.0 software (Media Cybernetics, Singapore). The ratio of the CUL4B band to the internal reference GAPDH intensity value represents the respective content. All of the above antibodies were from Abcam, Cambridge, MA, USA.

1.10 EdU (5-ethyl-2' -deoxyuridine) assay

The EdU detection kit was purchased from Ruibo Bio Inc., China Triton X was purchased from Solebao, Inc., China, and the logarithmic phase of the cells was sampled and 5 × 10⁴The wells were plated in 96-well plates and after normal growth of the adherent cells, 500. mu.l of 50. mu. mol/l EdU medium was added to each well and incubated at 37 ℃ for 2 h. After fixation with 40g/l paraformaldehyde for 20min, 2mg/ml glycine was incubated for 10min and washed twice with PBS. Then 500. mu.l of 0.5% Triton X permeabilized cells were added, Apollo staining reaction solution was added and incubated in the dark for 30min, PBS was washed twice, Hoechst33342 reaction solution was added and incubated in the dark for 30min, 0.5% Triton X was washed twice, and observed under an inverted fluorescence microscope. Image-Pro Plus6.0 professional Image analysis software (IPP) cell numbers were counted.

1.11 scratch test

Stomach cancer cells SGC7901 were plated in 6-well plates at 5 × 10 per well⁵After 24 hours, marking a straight line on the cell surface by using a 200 mu L sterile gun head compared with a ruler, washing the cell surface for 3 times by PBS, then taking a picture under an inverted microscope (Olympus CX23) (× 100), taking out the six-well plate again after 48 hours, taking a picture and measuring the healing rate of the scratch.

1.12Transwell experiment

Diluting Matrigel (BD, USA) with precooled serum-free DMEM medium at a ratio of 1:10, sucking the well-mixed Matrigel, adding 100 mu L of diluted Matrigel into each upper chamber, standing at room temperature for 2h, adding 200 mu L of serum-free 1640 medium before use for washing, digesting the stem cells transfected for 24h, then re-suspending with the serum-free DMEM medium, counting and diluting to 3 × 10⁵Per mL, 100. mu.L of each was added to the upper chamber of a Transwell (Corning, USA) while 600. mu.L of 10% serum in DMEM medium (serum as a chemotactic factor) was added to the lower chamber, following the Transwell chamber instructions, crystal violet staining, three fields were randomly selected and the number of cells that crossed the membrane was counted.

1.13 flow cytometry detection

The apoptosis condition of the SGC7901 stem cell cultured and transfected for 48 hours is detected by an annexin V-FITC/PI double staining kit (556547, Shanghai Shuojia Biotech Co., Ltd., China), and the experimental steps are as follows: diluting the mixture into 1 × Binding Buffer by using deionized water 10 × Binding Buffer, centrifuging each group of cells at 2000rpm for 5min in a greenhouse, and collecting the cells. The cells were then washed by resuspension in precooled 1 × PBS, centrifugation at 200rpm for 5-10 min. Then 300. mu.L of 1 XBinding Buffer suspension cells were added. Add 5. mu. Lannexin V-FITC (fluorescein isothiocyanate) and mix well, keep out of the sun and incubate for 15min at room temperature. 5 μ L of PI (propidium iodide) was added 5min before the flow cytometer (Cube6, Partec, Germany), the ice bath was protected from light for 5min, the excitation wavelength was 480nm, FITC was detected at 530nm and PI was detected at > 575 nm.

1.14 nude mice tumorigenesis

BALB/C nude mice, 4 weeks old, 18-22g weight, unlimited male and female, 66 total, purchased from Shanghai Si Laike laboratory animals, GmbH, and raised under SPF environment, 6 mice per group 1 × 10⁶The stomach cancer cell SGC7901 is inoculated to the subcutaneous part of a nude mouse, the tumor volume is monitored 1 time every week, the tumor volume is calculated by using a V pi/6 (height ×, length × width) formula, the nude mouse is killed at the end of the third week, the tumor is collected after being taken out, the tumor mass is measured, and all experimental operations follow the international experimental animal ethical convention and meet the relevant national regulations.