WO2019174110A1 - Rhcg基因在制备治疗癌症药物及诊断性试剂盒中的应用 - Google Patents

Rhcg基因在制备治疗癌症药物及诊断性试剂盒中的应用 Download PDF

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WO2019174110A1
WO2019174110A1 PCT/CN2018/084505 CN2018084505W WO2019174110A1 WO 2019174110 A1 WO2019174110 A1 WO 2019174110A1 CN 2018084505 W CN2018084505 W CN 2018084505W WO 2019174110 A1 WO2019174110 A1 WO 2019174110A1
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rhcg
gastric cancer
cells
expression
gene
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黄剑飞
刘灿
王营静
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南通大学附属医院
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Definitions

  • the invention belongs to the technical field of cancer precise medical drugs, and particularly relates to the use of the RHCG gene as a target for diagnosis and treatment of gastric cancer.
  • Gastric cancer is one of the most common malignant tumors, and its occurrence is related to a variety of factors, such as genetic factors, Helicobacter pylori infection and eating habits. According to the latest GLOBOCAN data, gastric cancer is the fifth most common type of cancer in the world. In recent years, although the mortality rate of gastric cancer patients has decreased, it is still the third most common cause of cancer death, and the world is in 2012. About 951,000 newly diagnosed cases of gastric cancer caused about 723,000 deaths. More than 70% of gastric cancer cases occur in developing countries, and more than half are in East Asian countries. In China, according to epidemiological data, gastric cancer is the third most common cancer (about 29.24 per 100,000 people). And the third highest cancer with the highest mortality rate.
  • RHCG RHC glycoprotein
  • Rh Rhesus
  • Rh family proteins are divided into two groups according to their functions, namely RH transport aminoglycoprotein (RHAG, RHBG and RHCG) and non-transport (non-glycosylation) RH protein ( RHD and RHCE), the protein encoded by the RH gene family is a conserved protein, and members of this family share 12 transmembrane helically folded structures with 20-27% homology to MEP and Amt.
  • RHAG is expressed only in red blood cells and red blood cell precursor cells.
  • RHBG and RHCG are non-erythrocyte homologs, which are not expressed in red blood cells, but are expressed in important ammonia metabolism tissues, including kidney, liver, testis, and central nervous system. , gastrointestinal tract and skeletal muscle. Studies have shown that RHAG, RHBG and RHCG all have the function of transporting ammonia. Among them, RHCG is mainly responsible for the secretion of ammonia from the epithelial cell membrane of renal collecting duct. The mice knocking out the RHCG gene can cause blood due to the exclusion of NH 4 + in the urine. Abnormal acidification.
  • RHCG is located on human chromosome 15 (15q25), has 11 exons and a C/G-rich promoter, which can control epithelial expression extensively.
  • RHCG may carry complex N-glycans at the 48NLS 50 sequence, a novel multi-layered membrane glycoprotein that maintains normal cellular homeostasis in tissues as an epithelial transporter.
  • RHCG is expressed in the distal convoluted tubules and ligated cells of the kidney, type A sputum cells, and non-A non-B cells.
  • RHCG is not expressed in B-type sputum cells, primary cells and inner medullary collecting duct cells.
  • the expression of RHCG is closely related to tumors.
  • RHCG is highly expressed in renal chromophobe cell carcinoma and renal eosinophiloma, but is lowly expressed in renal clear cell carcinoma and renal papillary cell carcinoma. Because renal chromophobe and renal eosinophils appear to originate in type A sputum cells, the association of RHCG with tumors may be related to the origin of tumor cells.
  • RHCG is down-regulated in esophageal squamous cell carcinoma but expressed in multiple squamous epithelium.
  • the RHCG gene may have a dual role in tumor biology, namely, supporting tumorigenesis and anti-tumor.
  • RHCG is highly expressed in colon adenocarcinoma, hepatocellular carcinoma, but low in esophageal cancer and invasive breast cancer. It has been shown in the ATLAS database that high expression of RHCG leads to poor prognosis in lung adenocarcinoma, whereas in lung squamous cell carcinoma, high expression of RHCG leads to a good prognosis.
  • the relationship between RHCG and gastric cancer is still unclear, and its expression and specific regulation mechanism in gastric cancer are still unclear.
  • the object of the present invention is to provide an application of the RHCG gene in the preparation of a medicament for treating gastric cancer, and to meet the use requirements of the anticancer drug.
  • Another object of the present invention is to provide an application of the RHCG gene in the preparation of a kit for cancer diagnosis or prognosis.
  • the RHCG gene (National Gene Bank, Entrez ID: 51458) is used in the preparation of diagnostic kits for cancers with abnormal expression of RHCG.
  • the cancer is gastric cancer.
  • the cancer is gastric cancer.
  • the drug is designed with the RHCG gene as a target.
  • RHCG-sgRNA An inhibitor of the RHCG gene is RHCG-sgRNA, and its sequence is: 5'-CAGCGGGCGGTTCGGACGCT-3'.
  • the cancer is gastric cancer.
  • the present invention has been confirmed by immunohistochemistry that the expression in gastric cancer is significantly higher than that in adjacent normal tissues, and the patients with high expression of RHCG gene have poor prognosis; the specific sgRNA sequence is used to efficiently inhibit RHCG.
  • the expression of the gene in human gastric cancer cell lines by Western blot, cck8 test, E DU and other experimental methods, confirmed that reducing the expression of RHCG gene can inhibit tumor cell proliferation and increase the sensitivity of gastric cancer cells to oxaliplatin. It can be seen that the RHCG gene has a wide range of applications in the preparation of kits for the treatment of gastric cancer drugs, for cancer diagnosis or prognosis.
  • Figure 1 is a graph showing the expression of RHCG in gastric cancer and paracancerous tissues by IHC; a is a moderately and moderately differentiated gastric cancer tissue, b is a highly differentiated gastric cancer tissue, c is a severe atypical hyperplasia gastric tissue, and d is a normal gastric mucosal epithelial tissue. .
  • RHCG is negatively stained in normal tissue samples (green arrow). (a40 in a1-d1; x400 in a2-d2);
  • Figure 2 is a graph showing the relationship between the expression of RHCG and the prognosis of patients with gastric cancer;
  • A The overall survival rate of patients with high RHCG expression (blue line) is lower than that of patients with low RHCG expression (green line).
  • B Patients with high TNM stage (stage IV) (purple line) have a lower overall survival rate than patients with low TNM stage.
  • the probe number of C.RHCG is 219554_at.
  • the results were analyzed by Kaplan-Meier plotter database, the y-axis represents survival rate, the red line represents the patient whose RHCG expression level is higher than the median value, and the black line represents the patient whose RHCG expression level is lower than the median value;
  • Figure 3 is a graph showing the expression of RHCG and PCNA in gastric cancer and corresponding adjacent tissues by Western Blot;
  • Figure 4 is a graph showing the expression of RHCG in transfected and untransfected gastric cancer cell lines;
  • A Protein expression level of RHCG in undifferentiated gastric cancer cell line HGC27, gastric cancer cell lines MKN1, AGS, MKN45, SNU719 (*p ⁇ 0.05).
  • B Protein expression of RHCG in the control group, blank group, and successfully transfected Flag-RHCG group (*p ⁇ 0.05).
  • C Protein expression of RHCG in the control group, blank group, and successfully transfected RHCG-sgRNA group (*p ⁇ 0.05);
  • Figure 5 is a graph showing the proliferation index of transfected and untransfected gastric cancer cells at the corresponding time points in the CCK-8 assay
  • Figure 6 is a diagram showing the number of clones of gastric cancer cells in the clone formation analysis control group and the transfection group;
  • Figure 7 is a graph showing the effect of EHC assay on the proliferation of gastric cancer cells by RHCG;
  • Figure 8 is a graph showing the cell viability curves of HGC27 and MKN1 cells treated with different concentrations of oxaliplatin
  • Figure 9 shows different concentrations of oxaliplatin in the treatment of RHCG-sgRNA-transfected HGC27 cells and Flag-RHCG-transfected MKN1 cells, and the CCK-8 assay was used 24 hours later;
  • Figure 10 is a diagram showing the IC50 of the corresponding group according to the numerical results of Figure 9;
  • Figure 11 is a graph showing the results of cell assays in which RHCG-sgRNA-transfected HGC27 cells and Flag-RHCG-transfected MKN1 cells were incubated with oxaliplatin (8 ⁇ mol/L), respectively, after 24 hours.
  • the main reagents used in the following examples are: two-step immunohistochemistry test kit: Beijing Zhongshan Jinqiao Company; mouse anti-human RHCG monoclonal antibody (for immunohistochemistry): novusbio; HRP-labeled goat anti-mouse/rabbit IgG polymer (for immunohistochemistry): Beijing Zhongshan Jinqiao Company; antibody dilution: Beijing Zhongshan Biotechnology Co., Ltd.; 0.01mol/L citrate buffer (pH6.0): Beijing Zhongshang Jinqiao Company; DAB: Dako Company Xylene, neutral gum, etc. are provided by the pathology department.
  • Cell cryopreservation solution RPMI-1640 complete medium, fetal bovine serum and DMSO were prepared at a ratio of 5:4:1 and stored at 4 °C.
  • 1 ⁇ TBST1L Take Tris 2.42g, NaCl 8.0g, Tween-200.5mL, mix and dissolve, dilute to 1L, and store at room temperature.
  • 1 ⁇ transfer Buffer1L glycine 14.4g, Tris 3.03g, add appropriate amount of double distilled water to stir and dissolve, add 200mL of anhydrous methanol, dilute to 1L, mix well (timely prepared).
  • 100 mL of blocking solution Take 5 g of skim milk powder, add 100 mL of 1 ⁇ TBST, and mix and dissolve (if necessary).
  • tissue chip maker Beecher Instruments, USA
  • automated immunohistochemical stainer (2D) LABVISION, USA.
  • Inverted phase contrast microscope Olympus, Japan
  • gel imaging system BIO-RAD, USA
  • multi-function microplate reader Thermo, USA
  • BD Accuri TM C6 flow cytometer Becton Dickinson, USA.
  • the gastric cancer tissue samples from 2007 to 2017 were collected from the Department of Pathology of the affiliated Hospital of Nantong University, including 179 fresh frozen gastric cancer tissues and 163 benign tissues. These tissue samples were fixed in formalin, embedded in paraffin, and graded according to the latest WHO diagnostic criteria. All cases were determined by histopathology by two pathologists. The patients did not receive immunotherapy, chemotherapy or radiotherapy before surgery, and the clinical case data were detailed and complete.
  • 163 cases of benign tissue included 44 cases of adjacent tissues and 179 benign cases (21 cases of chronic gastritis, 29 cases of intestinal metaplasia, 32 cases of low grade intraepithelial neoplasia, 37 cases of advanced intraepithelial neoplasia).
  • the tissue chip was frozen at 4 ° C for about 4 hours, and then the tissue array block was corrected with a fully automatic tissue microtome at a speed of 20 mm/rev, and all the tissue cores were completely exposed.
  • Slice the tissue array block with a microtome float the serial slices in cool water, let them expand naturally, and then transfer the slices to a 45 °C warm water for about 2 minutes. The slide-protected slides were allowed to dry.
  • the slice was baked in an environment of 60 ° C for 3 minutes, and the baked piece was continued at 58 ° C for 16 hours.
  • the prepared tissue chip is stored in a slice box and placed in a refrigerator at 4 ° C for use.
  • coverslips and slides firstly soak the coverslips and slides with concentrated acid for at least 24h, rinse in running water, ddH 2 O, then soak in 95% ethanol for 24h, and finally re-set Bake in an electric oven at 60 ° C for use.
  • Sectioning and roasting The gastric cancer tissue was embedded in paraffin, serially sliced, and the thickness was about 5 ⁇ m. It was attached to a clean glass slide and placed in a thermostat oven at 60 ° C for about 6-8 hours.
  • Blocking endogenous peroxidase Apply a layer of lipstick to the edge of the tissue, quickly drip 3% H 2 O 2 on the section , incubate for 15 min at room temperature, then rinse with PBS for 5 min ⁇ 3 times.
  • Incubation of the primary antibody gently remove the remaining PBS solution on the section, add a primary antibody to the tissue, and dilute the antibody to a concentration of 1:100, incubate for 1 hour at room temperature, and then rinse with PBS solution for 5 min ⁇ 3 times.
  • (10) lining add appropriate amount of hematoxylin on the gastric cancer tissue section for 10-20s, then buffer in tap water, then put it in hydrochloric acid-ethanol color separation solution for about 2-3s, and finally use running water slowly. Rinse for 5 minutes.
  • Dehydration Prepare ethanol solutions with different concentration gradients (70% ethanol, 80% ethanol, 95% ethanol, absolute ethanol), soak them in sequence for 3 min ⁇ 1 time, 3 min ⁇ 1 time, 5 min ⁇ 1 time, 5 min. ⁇ 1 time, the ethanol on the slice was slightly dried, and then immersed in a xylene solution for 8 min ⁇ 2 times.
  • (12) Covering A drop of neutral resin is placed in the center of the gastric cancer tissue section, covered with a cover glass and gently pressed. No bubbles are generated in this process, and it is air-dried in a fume hood.
  • the two pathologists used a double-blind method to observe a representative region of each slice under a 400-fold field of view, randomly selected five fields of view, and counted 500-1000 cells to calculate the positive rate of cells.
  • RHCG is mainly localized in the cell membrane, and is also partially distributed in the cytoplasm, and the positive part is brownish yellow or brown.
  • Tumor cell positive rate score 0 points (positive cells equal 0%); 1 point (positive cells 1-33%), 2 points (positive cells 34-66%), 3 points (positive cells 67-100%) ).
  • Cell staining intensity score 0 (no coloring), 1 minute (weak, light yellow), 2 points (medium, brownish yellow), 3 points (strong, tan).
  • the positive cell ratio and the staining intensity score were multiplied, and the results obtained were 0-2 for low expression and 3-6 for high expression.
  • the Kaplan-Meier survival curve showed that the overall survival rate of the RHCG high expression group was lower than that of the RHCG low expression group *P ⁇ 0.05.
  • Table 1 IHC staining of RHCG in gastric cancer tissues, adjacent tissues, benign sections of the stomach
  • SDS-PAGE gel electrophoresis (1) Clean the glass plate, install it on the electrophoresis tank as required, pour ddH 2 O for 1-2 min, prepare 10% polyacrylamide separation gel according to the formula, pipette Quickly blow until the mixture is evenly mixed. Quickly inject the separation gel into the interlayer of the glass plate to a suitable height. Finally, slowly inject a layer of isopropyl alcohol from the left to the right of the separation gel to about 1-2 mL to make the glue flat and cool at room temperature. Set the polymerization for about 30 minutes.
  • Transfer film (1) The PVDF membrane of the same size as the gel was immersed in methanol and double distilled water for 1 min, and then placed in the pre-cooled transfer solution for 10 min.
  • Protein immunoassay (1) Configure the blocking solution, ie 5% skim milk with PBST. The transferred PVDF membrane was placed therein, and sealed at room temperature for 2 h; (2) PBST was washed for 5 min ⁇ 3 times; (3) The primary antibody was incubated at 4 ° C overnight. (4) The PVDF membrane was taken out from the refrigerator, first warmed for 1 h, and then the membrane was washed with PBST for 5 min ⁇ 3 times. (5) Configure the secondary antibody, incubate the PVDF membrane with the secondary antibody, incubate for 2 h at room temperature in the dark, and wash the membrane with PBST for 10 min ⁇ 3 times.
  • the PVDF film is blotted dry with filter paper and placed on the corresponding position of the developing device.
  • the ECL illuminating liquid is mixed with the A liquid and the B liquid in equal proportion before use, and uniformly added to the PVDF film, and the gel image is formed.
  • the system takes pictures and saves.
  • the protein expression level of RHCG in 8 pairs of gastric cancer and its adjacent normal tissues was examined by the WB method. Among the 7 pairs of tissues, the expression of RHCG in gastric cancer tissues was significantly higher than that of the corresponding adjacent tissues, and it was consistent with the expression of PCNA.
  • the strip gray scale of RHCG was compared with the strip gray scale of GAPDH, and the relative expression amount of the RHCG strip was calculated to obtain a quantified map. The results suggest that the expression levels of RHCG and PCNA are increased in gastric cancer tissues compared with adjacent tissues (p ⁇ 0.05), and the expression trends of CNA and RHCG are consistent (Fig. 2).
  • the RHCG-sgRNA sequence is: 5'-CAGCGGGCGGTTCGGACGCT-3'.
  • the gastric cancer cell line including: HGC27 cells, AGS cells, MKN45 cells, MKN1 cells, and SNU719 cells were cultured in RPMI1640 complete medium, maintained at a temperature of 37 ° C, 5% CO 2 saturated humidity in the incubator, and routinely subcultured in an incubator. The medium was changed once in 1-2 days, and the cells in the logarithmic growth phase were selected for the experiment.
  • the proteins of five gastric cancer cells were extracted as described above. Western blot was used to detect the expression of EphA8 in five breast cancer cells. High and low expression cells were screened.
  • RHCG The expression of RHCG in HGC27, MKN1, AGS, MKN45 and SNU719 gastric cancer cell lines was detected by Western blot. The results showed that RHCG was highly expressed in HGC27 and AGS gastric cancer cells, and the highest expression was found in undifferentiated gastric cancer cells HGC27; RHCG was in MKN45 and The expression of SNU719 gastric cancer cells is relatively low.
  • the gastric cancer cells were seeded in a six-well plate at a suitable density, and the density was increased to 70%, and then replaced with a complete medium without antibiotics for further 2 hours; (2) the transfection solution was configured according to the instructions of Lipofectamine 2000. (3) Sterilize 1.5 mL of EP tube, add basal medium + liposome (lip2000) to EP tube I, add basal medium + plasmid to EP tube II, and gently pipette to uniformity with pipette. Incubate for 5 min at room temperature. (4) Mix the contents of EP tube I and EP tube II, gently pipette until mixed, and let stand for 20 min at room temperature.
  • lip2000 basal medium + liposome
  • RHCG-sgRNA was transfected into HGC27 and AGS gastric cancer cells, and Flag-RHCG was transfected into MKN45 and SNU719 gastric cancer cells.
  • Western blot analysis showed that the expression of RHCG was significantly decreased in HGC27 and AGS cells transfected with RHCG-sgRNA, and the expression of RHCG was significantly increased in MKN1 and SNU719 cells transfected with Flag-RHCG (Fig. 4).
  • HGC27 cells RHCG-sgRNA transfected and untransfected groups
  • GES-1 cells RHCG-Flag transfected and untransfected groups
  • a total of four groups of cells each approximately 200 per culture dish
  • the density of the cells was diluted, and the cells were gently pipetted to uniformly disperse the cells, and cultured in a cell culture incubator for about 14 days.
  • HGC27 cells and AGS cells RHCG-sgRNA transfected and untransfected
  • MKN1 cells and SNU719 cells RHCG-Flag transfected and untransfected
  • a cell suspension (concentration: 5 ⁇ 10 4 /mL) was added to a well of a control group and a transfection group in a volume of 100 ⁇ L in each well of a 96-well culture plate, and cultured in an incubator, and maintained in the incubator 37 °C temperature, 5% CO 2 saturated humidity.
  • the cells were treated after 24 hours.
  • the plate was taken out at the corresponding time point, and 10 ⁇ L of CCK-8 reagent was added to each well in the dark, and gently mixed until mixed, and then incubated for 60 min in the dark.
  • the absorbance of each well at a wavelength of 450 mm was measured by a microplate reader.
  • HGC27 cells RHCG-sgRNA transfected and untransfected
  • MKN1 cells RHCG-Flag transfected and untransfected
  • a total of 4 groups of cells were made into cell suspensions ( The concentration was 8 ⁇ 10 4 /mL), and 100 ⁇ L of the control and transfected cells were added to each well of a 96-well culture plate, and cultured in an incubator. The temperature in the incubator was maintained at 37 ° C, 5%.
  • HGC27 cells RHCG-sgRNA transfected and untransfected groups
  • MKN1 cells RHCG-Flag transfected and untransfected
  • a total of 4 groups of cells were made into cell suspensions, 1 ⁇ 10 per well
  • the cell density of 4 was inoculated into a 96-well plate and cultured in an incubator for 24 hours.
  • the temperature in the incubator was maintained at 37 ° C and 5% CO 2 saturation humidity.
  • HGC27 cells and MKN1 cells were stimulated with oxaliplatin at a concentration of 8 concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 g/mL) for 24 h, respectively, and then determined by CCK-8 assay. The resulting cell viability.
  • HGC27 cells RHCG-sgRNA transfected and untransfected groups
  • MKN1 cells RHCG-Flag transfected and untransfected
  • the temperature in the incubator was maintained at 37 ° C and 5% CO 2 saturated humidity. After the cells were attached, the original medium was discarded, and the solution was changed.
  • the complete medium containing different concentrations of oxaliplatin (0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 4 ⁇ g/mL, 8 ⁇ g/mL, 16 ⁇ g/mL, 32 ⁇ g/mL) was further cultured for 24 h.
  • EDU labeling cell solidification, staining: (1) Dilute the EdU solution in a ratio of 1000:1 with complete cell culture medium, prepare an appropriate amount of 50 ⁇ M EdU medium, and (2) incubate 100 ⁇ L of 50 ⁇ M medium in each well. 2 hours, the amount of EdU medium is preferably no cells; (3) discard the culture solution, wash the cells 1 to 2 times for 5 minutes each time in PBS solution; the purpose of cleaning is to elute EdU without infiltration into DNA. (4) Add 100 ⁇ L of cell fixative (PBS containing 4% paraformaldehyde) to each well for 30 minutes at room temperature. Low concentration of paraformaldehyde is beneficial to the maintenance of cell structure.
  • TritonX-100 is required.
  • the cells are facilitated to facilitate entry of the antibody dye into the cells; (5) 2 mg/mL glycine is added to each well, and the bleaching shaker is incubated for 5 minutes to neutralize paraformaldehyde. (6) Add 100 ⁇ L of PBS to each well and wash for 5 minutes on a decolorizing shaker; then add 100 ⁇ L of penetrant (0.5% Triton X-100 in PBS) to each well for 10 minutes on a decolorization shaker; wash once with PBS for 5 minutes.
  • RHCG-sgRNA and Flag-RHCG were transfected into corresponding gastric cancer cells respectively.
  • CCK-8 assay, colony formation assay and EDU assay were used to analyze the regulation of RHCG in gastric cancer cells.
  • Inhibition of RHCG expression in HGC27 and AGS cells reduced the value-adding ability of gastric cancer cells compared to the control group; overexpression of RHCG in MKN1 and SNU719 cells increased the value-adding ability of gastric cancer (Fig. 5).
  • Example 3 the effect of RHCG expression level on chemosensitivity of gastric cancer cells
  • the inhibitory effect of oxaliplatin on gastric cancer cells was detected by CCK-8 assay. It was found that in the two gastric cancer cell lines, the cell viability decreased with the increase of drug dosage, and the high concentration of oxaliplatin on gastric cancer. Cell proliferation has a significant inhibitory effect (Figure 8). On this basis, RHCG-sgRNA-transfected HGC27 cells and Flag-RHCG-transfected MKN1 cells were stimulated with 7 concentrations (0.5, 1, 2, 4, 8, 16 and 32 ⁇ g/mL) of oxaliplatin. After 24h, the cell viability was also measured by CCK-8 assay, and the corresponding histogram was obtained by quantitative analysis.

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Abstract

本发明公开了RHCG基因在制备有关癌症诊断性试剂盒及靶向治疗药物中的应用。本发明利用组织水平验证及免疫组化证实，结果显示RHCG在胃癌组织中表达增高，且RHCG表达较高的患者预后较差；本发明通过体外细胞功能学实验，证实转染RHCG-sgRNA后的HGC27和AGS细胞，RHCG的基因表达明显降低、细胞增殖能力下降，而转染了Flag-RHCG后的MKN45和SNU719细胞，RHCG的基因表达明显增加、细胞增殖能力升高；本发明证实敲除RHCG胃癌细胞的对奥沙利铂的化疗敏感性提高；而过表达RHCG胃癌细胞的对奥沙利铂的化疗敏感性降低；RHCG在制备癌症诊断性试剂盒和癌症靶向治疗药物有很好的应用。

Description

RHCG基因在制备治疗癌症药物及诊断性试剂盒中的应用 技术领域
本发明属于癌症精准医疗药物技术领域，具体涉及RHCG基因作为胃癌诊疗靶点的用途。
背景技术
胃癌(gastric cancer，GC)是最常见的恶性肿瘤之一，它的发生与多种因素相关，如遗传因素、幽门螺杆菌感染和饮食习惯等。根据最新GLOBOCAN数据显示，胃癌是世界上第五大最常见的癌症类型，近年来尽管胃癌患者的死亡率有所下降，但是它仍是第三大最常见的癌症死亡相关原因，2012年全世界约有95.1万新诊断的胃癌病例，造成约72.3万人死亡。70％以上的胃癌病例发生在发展中国家，且一半以上在东亚国家，在中国，根据流行病学资料显示，胃癌是第三大发病率最高的癌症(每10万人中约有29.24人)和第三大死亡率最高的癌症。目前胃癌的治疗方法多种多样，其中最有效的治疗方法为根治性切除和化疗组合，但尽管如此，患者复发和转移的风险仍很高，5年生存率低于40％。我国胃癌患者的早期诊断率低，预后差，严重威胁居民的健康。因此，探索胃癌的发生发展机制、寻找新的敏感且特异的分子治疗靶点对胃癌诊断和治疗至关重要。
RHCG(RHC glycoprotein)属于Rhesus(Rh)家族，Rh家族蛋白根据功能的不同分为两个组，即RH转运氨糖蛋白(RHAG，RHBG和RHCG)和非转运(非糖基化)RH蛋白(RHD和RHCE)，RH基因家族编码的蛋白为保守蛋白，该家族的成员共享12个跨膜螺旋折叠结构，其与MEP和Amt具有20-27％的同源性。RHAG仅在红细胞和红细胞前体细胞中表达，RHBG和RHCG为非红细胞同系物，它们在红细胞中不表达，但在重要的氨代谢组织中都有表达，包括肾、肝、睾丸、中枢神经系统、胃肠道和骨骼肌。已有研究显示RHAG，RHBG和RHCG均具有运输氨的功能，其中RHCG主要负责肾集合管上皮细胞膜的氨分泌，敲除RHCG基因的小鼠由于其尿液中NH 4 +排除受损可导致血液的异常酸化。RHCG定位于人的15号染色体(15q25)，有11个外显子和富含C/G的启动子，可广泛控制上皮的表达。体外研究表明，RHCG可能在48NLS 50序列处携带有复杂的N-聚糖，RHCG是一种新型的多层膜糖蛋白，在组织中其可作为上皮转运蛋 白维持正常细胞稳态。根据与H +-ATP酶、水通道蛋白-2、钙结合蛋白-D28k共定位实验显示，RHCG在肾脏的远曲小管和连接段细胞、A型闰细胞和非A非B细胞中表达，而在B型闰细胞，主细胞和内髓集合管细胞中不表达。RHCG的表达与肿瘤密切相关，在肾脏肿瘤中，RHCG在肾嫌色细胞癌和肾嗜酸细胞瘤中高表达，但在肾透明细胞癌和肾乳头状细胞癌中低表达。因肾嫌色细胞癌和肾嗜酸细胞瘤似乎起源于A型闰细胞，故RHCG与肿瘤的相关性可能与肿瘤细胞的起源有关。在食管癌中，RHCG在食管鳞状细胞癌中表达下调，但在多个鳞状上皮中表达。因此RHCG基因在肿瘤生物学中可能具有双重作用，即支持肿瘤发生发展和抗肿瘤性。在TCGA数据库中显示，RHCG在结肠腺癌中、肝细胞肝癌中高表达，但在食管癌和浸润性乳腺癌中低表达。在ATLAS数据库中显示，高表达RHCG在肺腺癌中导致不良预后，而在肺鳞状细胞癌中，高表达RHCG导致好的预后。但是，RHCG与胃癌的关系目前还不明确，其在胃癌中的表达和具体调节机制还不清楚。
发明内容
发明目的：针对现有技术中存在的不足，本发明的目的是提供一种RHCG基因在制备用于治疗胃癌药物中的应用，满足抗癌药物的使用需求。本发明的另一目的是提供一种RHCG基因在制备用于癌症诊断或预后判断的试剂盒中的应用。
技术方案：为了实现上述发明目的，本发明采用的技术方案如下：
RHCG基因(国家基因库，Entrez ID：51458)在制备用于RHCG表达异常的癌症的诊断性试剂盒中的应用。所述的癌症为胃癌。
RHCG基因在制备用于治疗RHCG表达异常的癌症的药物中的应用。所述的癌症为胃癌。所述药物以RHCG基因为靶点设计而成。
RHCG基因在制备用于胃癌预后判断的诊断试剂盒中的应用。
RHCG基因的抑制剂，为RHCG-sgRNA，其序列为：5'-CAGCGGGCGGTTCGGACGCT-3'。
RHCG基因的抑制剂在制备用于治疗RHCG表达异常的癌症的药物中的应用。所述的癌症为胃癌。
有益效果：与现有的技术相比，本发明经免疫组化证实，在胃癌中表达明显高于癌旁正常组织，且RHCG基因高表达的患者预后较差；应用特异性sgRNA序列高效抑制RHCG基因在人胃癌细胞株的表达，经western blot、cck8实验、E DU等实验方法，证实降低RHCG基因的表达可以抑制肿瘤细胞增殖、升高胃癌细胞对奥沙利铂的化疗敏感性。可见，RHCG基因在制备用于治疗胃癌药物、用于癌症诊断或预后判断的试剂盒中的具有广泛的应用。
附图说明
图1是IHC检测RHCG在胃癌及癌旁组织中的表达结果图；a为中低度分化胃癌组织，b为高度分化胃癌组织，c为重度不典型增生胃组织，d为正常胃粘膜上皮组织。(a，b)RHCG在胃癌组织的胞膜和胞质中的呈强阳性染色(红色箭头)；(c)RHCG在重度不典型增生胃组织的胞膜和胞质中的呈弱阳性染色(红色箭头)；(d)RHCG在正常组织样本中呈阴性染色(绿色箭头)。(a1-d1中×40；a2-d2中×400)；
图2是RHCG的表达与胃癌患者预后的关系图；A.RHCG高表达(蓝线)的患者总体存活率低于RHCG低表达(绿线)的患者的。B.具有高TNM分期(IV期)(紫线)的患者的总生存率低于具有低TNM分期的患者。C.RHCG的探头编号为219554_at。通过Kaplan-Meier plotter数据库分析结果，y轴代表存活率，红线代表RHCG的表达水平高于中值的患者，黑线代表RHCG的表达水平低于中值的患者；
图3是Western Blot检测RHCG、PCNA在胃癌及相应癌旁组织中的表达结果图；
图4是RHCG在转染和未转染胃癌细胞系中的表达情况图；A.在未分化的胃癌细胞HGC27，胃癌细胞系MKN1、AGS、MKN45、SNU719中RHCG的蛋白表达水平(*p<0.05)。B.对照组、空白组、成功转染Flag-RHCG组中RHCG的蛋白表达(*p<0.05)。C.对照组、空白组、成功转染RHCG-sgRNA组中RHCG的蛋白表达(*p<0.05)；
图5是CCK-8实验在相应的时间点检测转染和未转染胃癌细胞的增殖指数图；
图6是克隆形成分析对照组和转染组胃癌细胞的克隆数目图；
图7是EDU实验检测RHCG对胃癌细胞的增殖能力的影响图；
图8是不同浓度的奥沙利铂处理HGC27和MKN1细胞，用CCK-8实验检测得细胞活力曲线图；
图9是不同浓度的奥沙利铂分别处理RHCG-sgRNA转染的HGC27细胞和Flag-RHCG转染的MKN1细胞，24小时后用CCK-8实验检测图；
图10是根据图9数值结果得出相应组别的IC50图；
图11是用奥沙利铂(8μmol/L)分别孵育RHCG-sgRNA转染的HGC27细胞和Flag-RHCG转染的MKN1细胞，24小时后用EDU测定细胞情况结果图。
具体实施方式
下面结合实施例和附图对本发明做更进一步地解释。下列实施例仅用于说明本发明，但并不用来限定本发明的实施范围。
以下实施例中使用的主要试剂为：二步法免疫组化检测试剂盒：北京中杉金桥公司；鼠抗人RHCG单克隆抗体(免疫组化试验用)：novusbio；HRP标记的羊抗鼠/兔IgG聚合物(免疫组化试验用)：北京中杉金桥公司；抗体稀释液：北京中山生物技术有限公司；0.01mol/L柠檬酸缓冲液(pH6.0)：北京中杉金桥公司；DAB：Dako公司；二甲苯、中性树胶等由病理科提供。DAB工作液：试剂C:试剂B＝1:50；胃癌细胞株购自南京科佰生物科技有限公司；1640培养基、胎牛血清：美国gibco公司；BCA蛋白测定试剂盒：Biosharp；PVDP膜(Westernblot试验用)：Bio-Rad公司；鼠抗人RHCG单克隆抗体(Westernblot试验用)：novusbio；PCNA抗体和cyclin D1抗体(Westernblot试验用)：Santa Cruz公司；GAPDH抗体(Westernblot试验用)：Reprotech公司；β-actin抗体(Westernblot试验用)：Reprotech公司；cyclin A抗体(Westernblot试验用)：Sigma公司；辣根过氧化物酶标记山羊抗兔/鼠IgG(Westernblot试验用)：abcam公司；ECL显影液(苏州新赛美公司)；RPMI-1640完全培养液：分别加入RPMI-1640与胎牛血清混匀，使其终浓度分别为90％，10％，1×，4℃保存。细胞冻存液：将RPMI-1640完全培养液、胎牛血清和DMSO按5∶4∶1比例配制，4℃保存。1×TBST1L：取Tris2.42g、NaCl8.0g、Tween-200.5mL，混合溶解，定容至1L，常温保存。1×转膜Buffer1L：甘氨酸14.4g、Tris3.03g，加适量双蒸水搅拌溶解，再加200mL无水甲醇，定容至1L，混合均匀(用时配制)。封闭液100mL：取脱脂奶粉5g， 加入100mL1×TBST，混合溶解即可(需用时配制)。
以下实施例中使用的主要仪器如下：组织芯片制作仪：美国Beecher Instruments公司；自动免疫组化染色仪(2D)：美国LABVISION公司。倒置相差显微镜：日本Olympus公司；凝胶成像系统：美国BIO-RAD公司；多功能酶标仪：美国Thermo公司；BD Accuri TM C6流式细胞仪：美国Becton Dickinson公司。
实施例1、RHCG在胃癌组织中的表达研究
从南通大学附属医院病理科收集2007-2017年间胃癌组织样本，其中新鲜冰冻胃癌组织179例，良性组织163例。这些组织样本均是由福尔马林固定，石蜡包埋，并根据最新的WHO诊断标准分级。所有的病例均是由两名病理学专家进行病理组织学确定，患者术前没有接受过免疫治疗、化疗或放疗，临床病例资料详细完整。
1.1免疫组织化学法标本：
163例良性组织中包含44例癌旁组织和179例良性病例(21例慢性胃炎、29例肠化生、32例低级上皮内瘤变、37例高级上皮内瘤变)。179例胃癌组织中，性别女41例，男138例；年龄低于60岁的61例，超过60岁的118例；组织学类型：管状腺癌141例，混合腺癌和粘液腺癌16例，低黏连性腺癌18例；肿瘤分级：高、中分化的胃癌组织为58例，低分化的胃癌组织为103例；TNM分期：0-Ⅰ期26例，Ⅱ期64例，Ⅲ期73例，Ⅳ期16例。委托生物样本库制作成组织芯片。
1)组织芯片的制作：
(1)根据HE染色切片的镜检结果，在蜡块上有代表性的癌巢区域作标记。
(2)1:1混合石蜡与蜂蜡，制作空白受体蜡块。在蜡块上设计10×7孔，共350点组织阵列，然后用组织芯片仪制成TMA空白蜡块。(3)将供体蜡块在标记的点上选取最有代表性的癌巢区域，取直径2mm的组织块，每例各取1个芯。(4)将取好的组织芯转移到受体蜡块的孔中，并取相应癌旁组织做对照。(5)组织阵列块在55℃的恒温烤箱中加热融合10分钟，在快融化之前放至室温冷却，使受体蜡块与供体组织融为一体。(6)将组织芯片置于4℃条件下冷冻4小时左右，随后用全自动组织切片机对组织阵列块进行修正，速度为20mm/转，等修到所有 组织芯完全曝露。(7)用切片机对组织阵列块进行切片，将连续切片分别漂在凉水中，使其自然展开，再将切片转移至45℃温水中展片2分钟左右，待展开后将其贴在经过防脱片处理的载玻片上晾干。(8)将切片置于60℃的环境下烤片3分钟，58℃继续烤片16h。(9)将做好的组织芯片保存于切片盒，置于冰箱4℃冷藏室备用。
2)免疫组化染色(EnVision两步法)
(1)盖玻片和载玻片的处理：首先用浓酸把盖玻片和载玻片浸泡至少24h，在流水、ddH 2O下冲洗，后于95％乙醇中浸泡24h，最后再置于60℃电热恒温干燥箱烘烤以备用。(2)切片和烤片：将胃癌组织用石蜡包埋，连续切片，厚度约为5μm，将其贴于干净的载玻片上，置于60℃电热恒温干燥箱烘烤约6-8h。(3)脱蜡、水化：备两份干净的二甲苯溶液，将胃癌组织切片依次浸于其中各约15分钟，而后再浸置于不同浓度梯度的乙醇溶液中，依次为：无水乙醇10min×2次→95％乙醇5min×2次→80％乙醇5min×1→70％乙醇5min×1次，最后用PBS溶液冲洗5min×3次。(4)抗原修复：将胃癌组织切片插入金属染色架上并置于瓷缸内，用0.01M枸橼酸钠缓冲溶液(pH6.0)填满，放置于煮沸的高压锅中，盖上锅盖，加热至小阀门升起喷气时开始计时，3min后去除热源，取出瓷缸自然冷却，用PBS冲洗切片上残留的枸橼酸钠缓冲溶液。(5)阻断内源性过氧化物酶：在贴近组织边缘处涂一层唇膏，迅速在切片上滴3％H 2O 2，室温下孵育15min，后用PBS冲洗5min×3次。(6)孵育一抗：轻轻甩去切片上残留的PBS溶液，在组织上滴加一抗，抗体的稀释浓度为1:100，于室温孵育1h，后用PBS溶液冲洗5min×3次。(7)轻轻甩去多余的PBS溶液，滴加试剂1(黄色)，用于改变细胞通透性，以便大分子更好与检测的一抗分子结合，室温下反应20分钟，后用PBS冲洗5min×3次。(8)轻轻甩去多余的PBS溶液，滴加试剂2(红色)，于室温下反应约20分钟，后用PBS溶液冲洗5min×3次。(9)显色：将DAB浓缩液和稀释液以1:20的比例配制成DAB工作液，后滴加适量的DAB工作液于胃癌组织切片上，反应约5min，在显微镜下观察反应程度，最后将切片置于自来水中缓慢地冲洗约5分钟。(10)衬染：于胃癌组织切片上滴加适量的苏木素复染10-20s，后在自来水中缓缓冲洗，然后再放于盐酸－乙醇分色液中约2-3s，最后用流水缓慢冲洗5分钟。(11)脱水：准备不同浓度梯度的乙醇溶液 (70％乙醇、80％乙醇、95％乙醇、无水乙醇)，按顺序依次浸泡3min×1次、3min×1次、5min×1次、5min×1次，后稍微吹干切片上的乙醇，再浸于二甲苯溶液中8min×2次。(12)封片：在胃癌组织切片中央处滴一滴中性树脂，盖上盖玻片并轻轻按压，此过程不要产生气泡，置于通风橱中风干。
3)免疫组化结果判断
两位病理学家采用双盲法使用光学显微镜，在400倍视野下，观测每张切片中具有代表性的区域，随机选取5个视野，计数500-1000个细胞，统计计算细胞的阳性率。
(1)RHCG主要定位于细胞膜，在细胞质中也有部分分布，阳性部分呈棕黄色或褐色。(2)肿瘤细胞阳性率评分：0分(阳性细胞等于0％)；1分(阳性细胞1-33％)，2分(阳性细胞34-66％)，3分(阳性细胞67-100％)。(3)细胞染色强度评分：0分(无着色)，1分(弱，淡黄色)，2分(中等，棕黄色)，3分(强，棕褐色)。(4)将阳性细胞比例和染色强度评分相乘，所得结果0-2为低表达，3-6为高表达。Kaplan-Meier生存曲线显示RHCG高表达组比RHCG低表达组总体生存率低*P<0.05。
免疫组化法检测了胃癌患者样本中RHCG的表达情况(179个胃癌样本，44个匹配的癌旁样本和116个良性组织样本)，RHCG在细胞膜和细胞质中均有阳性染色，但主要定位于细胞膜上。组化结果表明，在胃癌中RHCG的表达显著升高，但在癌旁或良性胃组织中RHCG低表达或呈阴性(图1)。胃癌组织中，60.89％(109/179)的胃癌样本发现RHCG高表达；良性组织中，慢性胃炎为19.05％(4/21)，肠上皮化生为55.17％(16/26)，低级上皮内瘤变43.75％(14/32)，高级上皮内瘤变51.35％(19/37)；癌旁组织为27.27％(12/44)(χ2＝27.395，p<0.001)(表1)。
表1:RHCG在胃癌组织，癌旁组织，胃良性组织切片中的IHC染色
Figure PCTCN2018084505-appb-000001
Figure PCTCN2018084505-appb-000002
所有数据均用统计软件SPSS V.22.0和STATA V.9.0处理，计量资料以均数±标准差表示，组间比较采用单因素方差分析，RHCG表达与胃癌患者的预后关系分析用Kaplan-Meier生存分析(图2)，所有检验结果P<0.05为差异有统计学意义。
1.2蛋白免疫印迹法标本：
8对新鲜胃癌组织及其对应的癌旁组织由南通大学附属肿瘤医院提供，上述组织手术离体后立即去除出血、坏死组织，用生理盐水冲洗血污，在30分钟内给以液氮冷冻处理，之后置于-80℃冰箱保存。
1)组织蛋白提取：
(1)从-80℃冰箱中取出8对新鲜的胃癌及癌旁正常组织，置于冰水混合物复温约30min，后将组织放于干净的1.5mL EP管中，在电子天平称上称重，最后按100mg：1mL的比例加入适量的蛋白裂解液。(2)用酒精消毒后镊子和剪刀，剪碎组织，放在冰上裂解10min，后将EP管中的组织转移至匀浆器，冰浴下顺时针匀浆，充分研磨。(3)匀浆结束后静置于冰盆中30min，后将组织转移到EP管中，4℃离心(12000r×15min)，最后取上清留用。(4)按比例配置成蛋白上样液，即：4×上样缓冲液：蛋白裂解液：DTT＝5:4:1，移液器吹打至均匀，置水浴锅中煮沸10-15min，后离心机4℃(2000r×15min)，取上清，用紫外分光亮度仪测浓度，记录，保存在-80℃冰箱备用，上样前取出煮沸3min。
2)western blot
SDS-PAGE凝胶电泳：(1)将清洗干净的玻璃平板，按要求安装在电泳槽子上，倒入ddH 2O验漏1-2min，按配方配制10％的聚丙烯酰胺分离胶，移液器快速吹打至混和均匀，将分离胶快速注入玻璃平板的夹层中至合适的高度，最后在分离胶上面缓慢地从左向右注入一层异丙醇约1-2mL，使胶平整，常温静置聚合30min左右。(2)倒去分离胶上的异丙醇，用ddH 2O缓慢冲洗干净，滤纸吸干残留的水，后制备5％的聚丙烯酰胺浓缩胶，向分离胶上加入浓缩胶到玻璃平板夹层的顶部，迅速插入配套的梳子，此过程注意不要产生气泡，常温静置聚合30min左右。(3)按比例配置上样缓冲液：4×上样缓冲液：蛋白裂解液：DTT＝5:4:1， 沸水中煮沸15min，随后4℃下离心(12000g×10min)。(4)按要求将凝胶板装入电泳槽中，缓缓加入1×SDS电泳缓冲液，垂直拔出Telfon梳子，用1×SDS电泳缓冲液轻轻冲洗加样孔，等量加入待测蛋白样品，对照孔中加入蛋白分子量标准样品。(5)以80V恒压分离浓缩胶中的蛋白质，后再增大恒压至120V，等到溴酚蓝的染料跑至凝胶板的末端时关闭电源。(6)取下电泳槽中的凝胶板，ddH 2O冲洗干净，置于预冷的转膜液中。
转膜：(1)将与胶差不多大小的PVDF膜依次在甲醇和双蒸水中各浸泡1min，然后再放在预冷的转膜液中10min。(2)组装电转膜夹：打开电转印夹，从正极到负极(即黑→白)按次序依次放置已浸透过转膜液的绵垫、滤纸、胶条、PVDF膜、滤纸和海绵垫(此过程中注意：滤纸和胶，胶和PVDF膜之间不要有气泡)，后夹好转印夹。(3)将组装好的电转印夹正确插入转膜槽内，按对应的极性方向将转膜槽放入电转仪中，加满已预冷的转膜液，转膜槽周围用大量的冰围住，连接电源。(4)调节电流为300mA，恒流转膜约90min(可根据相应的分子量调整转膜的时间)。结束后关闭电源，轻打开电转印夹，用镊子取出转印好的膜，已备用来进行蛋白质免疫检测。
蛋白质免疫检测：(1)配置封闭液，即用PBST配置5％脱脂牛奶。把转印好的PVDF膜放置其中，室温下封闭2h；(2)PBST洗膜5min×3次；(3)孵育一抗，4℃冰箱过夜。(4)从冰箱取出PVDF膜，先复温1h，然后用PBST液洗膜5min×3次。(5)配置二抗，用二抗孵育PVDF膜，室温下避光孵育2h，PBST液洗膜10min×3次。(6))洗膜结束后将PVDF膜用滤纸吸干，平铺于显影仪相应位置，ECL发光液在使用前等比例混合A液和B液，均匀滴加于PVDF膜上，凝胶成像系统拍照、保存。
通过WB方法，检测了在8对胃癌和其癌旁正常组织中RHCG的蛋白质表达水平。其中在7对组织中，RHCG在胃癌组织中表达明显高于对应的癌旁组织，且与PCNA的表达一致。将RHCG的条带灰度与GAPDH的条带灰度对比，算出RHCG条带的相对表达量，得出量化图。结果提示，相比于癌旁组织，在胃癌组织中RHCG和PCNA的表达量均升高(p<0.05)，且CNA和RHCG的表达趋势是一致的(图2)。
实例2、RHCG对胃癌细胞作用的功能研究
2.1sgRNA设计：
针对RHCG基因序列，委托广州复能基因有限公司制成sgRNA：
RHCG-sgRNA序列为：5'-CAGCGGGCGGTTCGGACGCT-3'。
2.2胃癌细胞系的培养
胃癌细胞系，包含：HGC27细胞、AGS细胞、MKN45细胞、MKN1细胞、SNU719细胞均用RPMI1640完全培养基培养，培养箱内保持温度37℃、5％的CO 2饱和湿度，培养箱内常规传代培养，1-2天换液一次，选取处于对数生长期的细胞进行实验。
2.3细胞蛋白质提取及蛋白量的测定
(1)各种胃癌细胞均培养于37℃培养箱，CO 2饱和湿度保持在5％，用RPMI1640完全培养基培养，1-2天换液，根据相应的密度传代。(2)按实验的需求将RHCG-Flag和RHCG-sgRNA转染至相应的胃癌细胞中，收集正常或转染后的胃癌细胞，弃培养基，用预冷的0.01MPBS洗涤细胞2次，弃PBS，用移液器将残留的PBS溶液吸取干净，以免稀释细胞蛋白。(3)根据细胞培养瓶的大小和细胞的生长密度，加入不同的1×SDS细胞裂解液，后用细胞刮子刮净细胞，转移至干净的EP管中。(4)将刮下的细胞蛋白在沸水中煮沸10-15min，结束后取出立刻放于冰上冷却。(5)4℃离心(12000r×10min)。(6)留上清，用紫外分光亮度仪测细胞蛋白的浓度，后保存在-80℃冰箱以备用。
按上述方法分别提取5种胃癌细胞的蛋白。用Westernblot检测五种乳腺癌细胞中EphA8的表达情况。筛选出高低表达细胞。
Westernblot检测HGC27、MKN1、AGS、MKN45、SNU719胃癌细胞株中RHCG的表达情况，结果表明RHCG在HGC27和AGS胃癌细胞中表达相对较高，其中在未分化胃癌细胞HGC27中表达最高；RHCG在MKN45和SNU719胃癌细胞中表达相对较低。
2.4细胞转染
(1)将胃癌细胞以合适的密度种在六孔板，待其密度长到70％后换成无抗生素的完全培养基继续培养2h；(2)按Lipofectamine2000说明书配置转染溶液。(3)将1.5mL的EP管灭菌，按比例EP管Ⅰ中加入基础培养基+脂质体(lip2000)，EP管Ⅱ中加入基础培养基+质粒，用移液器轻吹至均匀，室温静置孵育5min。(4) 混合EP管Ⅰ和EP管Ⅱ中的内容物，用移液器轻吹至混匀，常温静置反应20min。(5)用移液器吸去六孔板中的培养基，D-Hank’s洗一次，然后每孔中加入基础培养基2mL，分别对应地在每孔中加入配置好的转染溶液，移液器轻吹至混合均匀。(6)培养箱中培养6-8h后，弃原来的培养基，D-Hank’s洗涤细胞一次，后加入完全培养基继续培养。(7)再置于培养箱中培养48-72小时，后收集细胞提取蛋白或做其他实验。
为了进一步探究RHCG对胃癌细胞生物学行为的作用，本实施例分别在HGC27和AGS胃癌细胞中转染RHCG-sgRNA，在MKN45和SNU719胃癌细胞中转染Flag-RHCG。Western blot结果显示，转染了RHCG-sgRNA后的HGC27和AGS细胞，RHCG的基因表达明显降低，而转染了Flag-RHCG后的MKN1和SNU719细胞，RHCG的基因表达明显增加(图4)。
2.5克隆形成实验
(1)HGC27细胞(RHCG-sgRNA转染和未转染组)、GES-1细胞(RHCG-Flag转染和未转染组)，共四组细胞，均按照大约每个培养皿约200个细胞的密度进行稀释，轻轻吹打细胞使细胞分散均匀，置于细胞培养箱中培养约14天。
(2)当出现肉眼可见的小克隆细胞团后，取出培养皿，弃培养基，用细胞PBS洗涤2次，移液器吸净残留的液体，后用配置好的结晶紫染色30分钟，流水轻轻洗去染色液，空气中倒扣放置使之干燥。(3)在低倍镜下进行计数及统计分析。
2.6CCK-8实
2.6.1增值相关：(1)HGC27细胞和AGS细胞(RHCG-sgRNA转染和未转染的)、MKN1细胞和SNU719细胞(RHCG-Flag转染和未转染的)，共8组细胞制成细胞悬液(浓度为5×10 4个/mL)，在96孔培养板的每孔中分别加入100μL体积的对照组及转染组的细胞，放在培养箱培养，培养箱内维持37℃温度、5％的CO 2饱和湿度。(2)24h后开始处理细胞。分别于对应的时间点取出培养板换液，避光条件下向每孔中加入10μL CCK-8试剂，轻轻吹打至混匀，然后避光培养60min。(3)用酶标仪测定各孔在波长450mm处的吸光度。
2.6.2耐药相关：(1)HGC27细胞(RHCG-sgRNA转染和未转染的)、MKN1细胞(RHCG-Flag转染和未转染的)，共4组细胞制成细胞悬液(浓度为8×10 4个/mL)，在96孔培养板的每孔中分别加入100μL体积的对照组及转染组的细胞， 放在培养箱培养，培养箱内维持37℃温度、5％的CO 2饱和湿度；(2)等细胞贴壁后，换成含有不同浓度奥沙利铂(oxaliplatin)(0.5μg/mL，1μg/mL，2μg/mL，4μg/mL，8μg/mL，16μg/mL，32μg/mL)的完全培养基继续培养；(3)24h后开始处理细胞，避光条件下向每孔中加入10μL CCK-8试剂，轻轻吹打至混匀，然后避光培养60min。(4)用酶标仪测定各孔在波长450mm处的吸光度。(5)使用下述公式计算细胞抑制率：细胞抑制率(％)＝[1-(OD试验-OD空白)/(OD-空白对照)]×100％；半数抑制浓度(IC50)是由SPSS软件计算所得。
2.7EDU实验
2.7.1细胞培养
增值相关：HGC27细胞(RHCG-sgRNA转染和未转染组的)、MKN1细胞(RHCG-Flag转染和未转染的)，共4组细胞制成细胞悬液，以每孔1×10 4的细胞密度接种于96孔板中，置于培养箱中培养24h，培养箱内维持37℃温度、5％的CO 2饱和湿度。
耐药相关：首先分别采用8种浓度(0，0.5，1，2，4，8，16和32g/mL)梯度的奥沙利铂刺激HGC27细胞和MKN1细胞24h，后用CCK-8实验测定所得细胞活力。其次制备HGC27细胞(RHCG-sgRNA转染和未转染组的)、MKN1细胞(RHCG-Flag转染和未转染的)，共4组细胞制成细胞悬液，以每孔1×10 4的细胞密度接种于96孔板中，置于培养箱中培养24h，培养箱内维持37℃温度、5％的CO 2饱和湿度；待细胞贴壁后，弃原培养基，换液，换成含有不同浓度奥沙利铂(oxaliplatin)(0.5μg/mL，1μg/mL，2μg/mL，4μg/mL，8μg/mL，16μg/mL，32μg/mL)的完全培养基继续培养24h。
2.7.2 EDU标记、细胞固化、染色：(1)用完全细胞培养基按1000：1的比例稀释EdU溶液，制备适量的50μM EdU培养基，(2)在每孔中加入100μL 50μMEdU培养基孵育2小时，EdU培养基用量以没过细胞为宜；(3)弃培养液，PBS溶液清洗细胞1～2次，每次5分钟；清洗目的是将未渗入DNA的EdU洗脱。(4)每孔加入100μL细胞固定液(含4％多聚甲醛的PBS)室温孵育30分钟，低浓度的多聚甲醛有利于细胞结构的保持，当需要抗体染色时，需要采用TritonX-100透化细胞以利于抗体染料进入细胞内；(5)每孔加入2mg/mL甘氨酸，脱色摇床孵育5分钟，其作用是中和多聚甲醛。(6)每孔加入100μL PBS，脱色摇床清洗 5分钟；后每孔再加入100μL渗透剂(0.5％TritonX-100的PBS)脱色摇床孵育10分钟；PBS清洗1次，5分钟。(7)每孔加入100μL的
Figure PCTCN2018084505-appb-000003
染色反应液，避光、室温、脱色摇床孵育30分钟；(8)除去染色反应液，加入100μL渗透剂(0.5％TritonX-100的PBS)脱色摇床清洗2～3次，每次10分钟；后每孔每次再加入100μL甲醇清洗1～2次，每次5分钟；PBS清洗1次，每次5分钟；
2.7.3 DNA染色：(1)用去离子水按100：1的比例稀释试剂F，制备适量1×Hoechst33342反应液，避光保存；(2)每孔加入100μL 1X Hoechst 33342反应液，避光、室温、脱色摇床孵育30分钟；(3)去除染色反应液后，每孔每次加入100μLPBS清洗1～3次，洗脱Hoechst 33342反应液。
2.7.4图像获取及分析：(1)染色完成后，最好立即进行观测；如果条件限制，可避光4℃湿润保存待测，但也不应超过3天。用荧光显微镜观测图像，每个孔随机选取五个视野。Edu阳性的细胞用绿色染料染色，根据五个视野中的平均细胞计数计算相对增殖阳性率。(2)本实验中所得出的数据均采用SPSS15.0统计分析软件，采用Pearsonχ2检验分析胃癌中RHCG的表达情况与临床病理参数的关联，Kaplan-Meier分析RHCG和胃癌患者的预后不良相关，Cox回归分析RHCG可作为胃癌的独立预后指标。检验水准α设为0.05，所有的数据均以
Figure PCTCN2018084505-appb-000004
±s表示，P<0.05表示差异具有统计学意义。
为了进一步探究RHCG是否影响胃癌细胞的增值。将RHCG-sgRNA和Flag-RHCG分别转染至对应胃癌细胞中，采用CCK-8实验、克隆形成实验、EDU实验分析RHCG在胃癌细胞中对其增殖能力的调节。与对照组相比，在HGC27和AGS细胞中抑制RHCG表达使胃癌细胞的增值能力下降；在MKN1和SNU719细胞中过表达RHCG使胃癌的增值能力升高(图5)。同时，克隆形成实验显示，降低RHCG的表达可抑制细胞克隆的形成；而提高RHCG的表达可促进细胞克隆的形成，且定量分析结果有显著差异(图6)。此外，我们通过EDU增值实验研究了RHCG对细胞增值活性的影响，如图所示，细胞荧光图像清楚地表明了敲除RHCG胃癌细胞的增殖明显减少；而过表达RHCG胃癌细胞的增殖明显增多。定量分析表明结果有显著差异(图7)。
实例3、RHCG的表达水平对胃癌细胞化疗敏感性的影响
应用CCK-8实验检测了奥沙利铂对胃癌细胞的抑制作用，发现在两种胃癌 细胞系中，随着药物剂量的增加细胞的活力随之降低，且高浓度的奥沙利铂对胃癌细胞的增值有明显的抑制作用(图8)。在此基础上，分别用7种浓度(0.5，1，2，4，8，16和32μg/mL)梯度的奥沙利铂刺激RHCG-sgRNA转染的HGC27细胞和Flag-RHCG转染MKN1细胞，24h后同样用CCK-8实验测定所得细胞活力，量化分析得出相应的柱状图；结果显示，敲除RHCG胃癌细胞的化疗敏感性提高；而过表达RHCG胃癌细胞的化疗敏感性降低(图9)。随后我们根据以上实验结果计算得出两种胃癌细胞系中奥沙利铂的半数抑制浓度(IC50)，如图所示，相对于空白组和对照组，敲除RHCG胃癌细胞奥沙利铂的IC50降低；而过表达RHCG胃癌细胞奥沙利铂的IC50升高(图10)。随后，在奥沙利铂孵育(8μg/mL)24h后，我们应用EDU细胞增值实验分别检测了RHCG-sgRNA转染的HGC27细胞和Flag-RHCG转染MKN1细胞；如图，细胞荧光结果细胞直观地显示了RHCG过表达的细胞其增殖活性高于对照组(*p<0.05；图11)；相反，RHCG敲除的细胞相对于对照组其增殖活性明显降低(*p<0.05)。

Claims (9)

  1. RHCG基因在制备用于RHCG表达异常的癌症的诊断性试剂盒中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的癌症为胃癌。
  3. RHCG基因在制备用于治疗RHCG表达异常的癌症的药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述的癌症为胃癌。
  5. 根据权利要求3所述的应用，其特征在于，所述药物以RHCG基因为靶点设计而成。
  6. RHCG基因在制备用于胃癌预后判断的诊断试剂盒中的应用。
  7. RHCG基因的抑制剂，其特征在于，所述抑制剂为RHCG-sgRNA，其序列为：5'-CAGCGGGCGGTTCGGACGCT-3'。
  8. RHCG基因的抑制剂在制备用于治疗RHCG表达异常的癌症的药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述的癌症为胃癌。
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