阅读说明：本技术 荜茇酰胺促进自噬及治疗口腔鳞状细胞癌的用途 (Application of piperlongumine in promoting autophagy and treating oral squamous cell carcinoma ) 是由 许小鸿 陈树伟 黄帅 郅程 苏涛 于 2020-08-04 设计创作，主要内容包括：本发明意外发现荜茇酰胺具有上调自噬启动基因Beclin-1和LC3表达的作用,并且发挥促进自噬,抑制口腔鳞状细胞癌增殖和迁移的作用,具有治疗口腔鳞状细胞癌的效果。(The invention unexpectedly discovers that the piperlongumine has the effect of up-regulating the expression of autophagy initiating genes Beclin-1 and LC3, plays the roles of promoting autophagy and inhibiting the proliferation and migration of oral squamous cell carcinoma, and has the effect of treating the oral squamous cell carcinoma.)

1. Use of piperlongumine in preparing autophagy promoter is provided.

2. Use according to claim 1, characterized in that: piperlongumine acts as an autophagy promoter by up-regulating Beclin-1 and/or LC 3.

3. Use according to claim 1, characterized in that: LC3 includes LC 3-I and LC 3-II.

4. Application of piperlongumine in preparing oral squamous cell carcinoma medicines is provided.

5. Use according to claim 4, characterized in that: piperlongumine inhibits proliferation and migration of oral squamous cell carcinoma cells.

6. Use according to claim 4, characterized in that: piperlongumine promotes autophagy of oral squamous cell carcinoma cells.

7. Use according to claim 4, characterized in that: the piperlongumine is used for up-regulating Beclin-1 and/or LC 3.

Use of Beclin-1 as a diagnostic marker for oral squamous cell carcinoma.

Use of LC3 as a diagnostic marker for oral squamous cell carcinoma.

10. Use according to claim 9, characterized in that: LC3 includes LC 3-I and LC 3-II.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of piperlongumine in promoting autophagy and treating oral squamous cell carcinoma.

Background

Oral Squamous Cell Carcinoma (OSCC) is a common head and neck malignancy, accounting for 3% of the total malignancy, 90% of the Oral malignancy, and has the characteristics of invasive growth, local metastasis, high recurrence rate, poor prognosis, and the like, with the malignancy ranking 6 th. OSCC is one of the common malignant tumors endangering human health and life, and seriously affects the quality of life and mental state of patients. With the continuous development of OSCC treatment, the surgical resection, combined radiotherapy, new adjuvant chemotherapy, targeted biological treatment and the like make great progress in diagnosis and clinical treatment, the survival rate of total OSCC patients is slightly improved, the 3-year survival rate reaches 50-75%, the 5-year survival rate is about 50%, but more than 60% of patients cannot be effectively controlled, and the postoperative survival rate is lower than 50%. Therefore, in order to improve the prognosis and survival rate of OSCC patients, the search for early diagnosis and targeted inhibition of the development and metastasis of OSCC patients is the key to preventing and treating OSCC.

Piperlongumine (CAS #:20069-09-4) alkaloid compounds are bioactive components of Piperaceae plant, and have various pharmacological effects of resisting blood platelet aggregation, resisting anxiety, and resisting depression. Researches show that the piperlongumine has a remarkable inhibiting effect on malignant tumors such as prostate cancer, colon cancer, lung adenocarcinoma, liver cancer, melanoma and the like, and no report is available so far about the inhibition of oral squamous cell carcinoma by the piperlongumine through regulating and controlling the autophagy of the oral squamous cell carcinoma.

Autophagy is a life phenomenon which is highly conserved in the biological evolution process and widely exists in eukaryotic cells, is a very important physiological process and an indispensable metabolic mode in the bodies of eukaryotes and mammals, participates in various physiological and pathological processes of organisms, and has important influence on the whole life cycle of cells including cell growth, proliferation, aging and apoptosis. The autophagy is mainly characterized in that a large number of autophagosomes and lysosomes are present in cytoplasm, and the autophagosomes formed by fusing the autophagosomes degrade redundant or damaged long-life proteins and organelles and other macromolecular substances in the cytoplasm and release saccharides, fatty acids, amino acids and other micromolecular substances for the organism to absorb and utilize again so as to complete the metabolism requirement of cells and the update of certain organelles; autophagy is also motile, and it self-inhibits or self-promotes through a variety of regulatory mechanisms to maintain cellular homeostasis when the external environment changes, such as under starvation, stress, injury, etc. Research shows that the change of autophagy activity is related to the occurrence and development of tumors, on one hand, autophagy maintains the intracellular environment stability by removing reactive active oxygen clusters, damaged organelles and proteins, thereby inhibiting the occurrence of tumors and playing a role in inhibiting cancers of the tumors, and the deficiency of autophagy activity can cause the intracellular environment disorder and DNA mutation of the tumors and promote the malignant progression of cancer cells. Among them, Beclin-1 and LC3 are autophagy promoter genes, and up-regulation of autophagy promoter genes will help to treat diseases associated with autophagy abnormalities.

In view of this, the present invention is proposed.

Disclosure of Invention

The invention aims to: 1. providing a new application of piperlongumine in preparing autophagy promoter; 2. provides a new application of piperlongumine in treating oral squamous cell carcinoma; use of Beclin-1 as a diagnostic marker for oral squamous cell carcinoma; use of LC3 as a diagnostic marker for oral squamous cell carcinoma.

The technical scheme of the invention comprises any one of the following items:

the invention provides application of piperlongumine in preparation of autophagy promoters.

Furthermore, the piperlongumine plays a role of autophagy promoter by up-regulating Beclin-1 and/or LC 3.

Further, LC3 includes LC 3-I and LC 3-II.

The invention provides application of piperlongumine in preparation of oral squamous cell carcinoma medicines.

Further, piperlongumine inhibits proliferation and migration of oral squamous cell carcinoma cells.

Further, piperlongumine promotes autophagy of oral squamous cell carcinoma cells.

Further, the piperlongumine is used for up-regulating Beclin-1 and/or LC 3.

The invention provides application of Beclin-1 as a diagnostic marker of oral squamous cell carcinoma.

The invention provides a use of LC3 as a diagnostic marker for oral squamous cell carcinoma.

Further, LC3 includes LC 3-I and LC 3-II.

The invention has the following technical effects:

the invention unexpectedly discovers that the piperlongumine has the effect of up-regulating the expression of autophagy initiating genes Beclin-1 and LC3, plays the roles of promoting autophagy and inhibiting the proliferation and migration of oral squamous cell carcinoma, and has the effect of treating the oral squamous cell carcinoma.

Drawings

FIG. 1: immunohistochemical detection of Beclin-1 and LC3 protein expression in oral squamous cell carcinoma tissue is carried out, wherein, the picture A shows that Beclin1 is strongly positively expressed in non-tumor tissue beside cancer, and the pictures B and C show that the expression intensity of Beclin1 in tongue squamous cell carcinoma tissue is reduced; panel D shows positive expression of LC3 in paraneoplastic non-neoplastic tissue, and panels E and F show down-regulation of LC3 expression in squamous cell carcinoma tissue;

FIG. 2: western test of the effect of PPLGM treatment on the expression of Beclin-1 and LC3 proteins on oral squamous cell carcinoma cells;

FIG. 3: effect of PPLGM treatment of oral squamous cell carcinoma cells on autophagy;

FIG. 4: the scratch test detects the influence of PPLGM on the migration when the oral squamous cell carcinoma cells are treated;

FIG. 5: the MTT method detects the cell proliferation of oral squamous cell carcinoma treated by PPLGM with different concentrations for different time periods.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The experimental methods and experimental conditions used in the following experimental examples were carried out by a conventional method or a manufacturer's manual unless otherwise specified, and the materials, reagents and the like used in the following experimental examples were commercially available unless otherwise specified.

Example 1 expression of the autophagy-promoting genes Beclin-1 and LC3 in oral squamous cell carcinoma

1.1 Beclin-1 and LC3 protein assays

Firstly, thermally treating a tissue slice at 65 ℃ for 1h, and then dewaxing the tissue slice in xylene for 2 times and 5min each time; hydrating the slices with 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and pure water for 1min each time; taking out, adding 3% hydrogen peroxide dropwise, incubating at room temperature for 10min, and inactivating endogenous peroxidase; placing the slices in a container containing EDTA pH9.0 repairing solution, and repairing under high pressure for 10 min; taking out the slices, surrounding the tissues by an immunohistochemical pen, rinsing the tissues for 3min by using PBS buffer solution, dripping goat serum confining liquid (reagent A) after spin-drying, and incubating for 20min at room temperature; removing the reagent A, dropwise adding a primary antibody working solution diluted by an antibody diluent, and incubating overnight at 4 ℃; removing primary antibody, rinsing with PBST buffer solution for 3 times and 5min each time, spin-drying, dripping biotin-labeled secondary antibody working solution (reagent B), and incubating at room temperature for 20 min; discarding the reagent B, rinsing with PBST buffer solution for 3 times and 5min each time, dripping horse radish peroxidase-labeled streptavidin working solution (reagent C) after spin-drying, and incubating at room temperature for 10 min; removing the reagent C, rinsing the mixture for 3 times with PBST buffer solution for 5min each time, dripping DAB developing solution after spin-drying, and incubating the mixture for 1-5 min at room temperature; immersing the slices in tap water to stop color development, re-staining with hematoxylin staining solution for 1min, washing with tap water, and differentiating with hydrochloric acid alcohol for 5 s; dehydrating the slices with 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 3min each time, and drying in a drying oven at 37 deg.C for 30min to obtain environmentally friendly resin sealed tablet.

The tissue to be detected is from paraffin specimen of retained oral squamous cell carcinoma and paracarcinoma non-tumor tissue of department of pathology of Guangzhou medical science and institute of technology

1.2 results of the assay

As shown in fig. 1, Beclin1 was strongly positively expressed in paracancerous non-tumor tissues (fig. 1A) and was less strongly expressed in tongue squamous carcinoma tissues (fig. 1B and 1C); LC3 was positively expressed in paraneoplastic non-neoplastic tissue (fig. 1D), but down-regulated in tongue squamous carcinoma tissue (fig. 1D and 1E). As can be seen, the expression levels of the autophagy promoting genes Beclin-1 and LC3 in oral squamous carcinoma tissues are obviously lower than those in paracancerous non-tumor tissues. Therefore, the reagent for detecting Beclin-1 and LC3 can be independently used as an oral squamous cell carcinoma diagnostic reagent.

Example 2 Piperlongumine promotes the expression of Beclin-1 and LC3 proteins in oral squamous cell carcinoma

2.1 Piperlongumine treatment

Oral squamous cell carcinoma cells, Cal27 and UM1, were treated with 3 μ M and 6 μ M of piperlongumine (abbreviated as PPLGM), respectively, as piperlongumine treated groups and DMSO as normal controls.

2.2 Beclin-1 and LC3 protein assays

Taking about 100mg of cells to be detected, grinding the cells in a liquid nitrogen by using a mortar, adding RIPA lysate to the crushed cells on ice for cracking for 30min, collecting the lysate, centrifuging the lysate for 15min at 4 ℃ under the condition of 12,000g, collecting supernatant, carrying out protein quantification according to the BCA protein quantification kit instruction, adding 5 multiplied by sample loading buffer solution with the volume of 1/4 into the supernatant, carrying out boiling water bath for 10min, and carrying out ice bath for 5 min. Using SDS PAGE protein electrophoresis system, the loading amount is 30 μ g total protein, the electrophoresis parameters are 10% separation gel, 5% concentration gel, 100V electrophoresis 90min until the trypan blue indicator migrates to the bottom, then electrotransfer is performed, and the PVDF membrane with 0.45 μm is used, and 300mAl ice bath is used for 90 min. After the electrotransfer is finished, sealing the membrane by using 5% skimmed milk powder, rinsing by TBST for 3 times and 5min, adding a primary antibody (1:1000), incubating overnight at 4 ℃, rinsing by TBST for 3 times and 5min, adding a secondary antibody diluted by a sealing solution, incubating at room temperature for 1h, rinsing by TBST for 3 times and 5min, adding an ECL luminous solution, placing the membrane in a pressing clamp, sensitizing by an X-ray negative film for 1min to 20min, and carrying out development and fixation treatment: the immunoblot detection was performed with GAPDH as a control.

2.3 results of detection

As shown in fig. 2, the levels of Beclin-1, LC3 and expression in the piperlongumine-treated group were significantly increased compared to the normal control group. Therefore, the piperlongumine can be used as an autophagy promoter because the piperlongumine promotes the expression of Beclin-1 and LC3 proteins in oral squamous cell carcinoma.

Example 3 Piperlongumine promotion of oral squamous cell carcinoma autophagy

3.1 Piperlongumine treatment

Oral squamous cell carcinoma cells, Cal27, were treated with 10. mu.M Piperlongumine (PPLGM) as a Piperlongumine treatment group and DMSO as a normal control group.

3.2 autophagy assay

Pancreatin digestion of cells, centrifugation, fixation of the cell mass in 2.5% glutaraldehyde and 0.1% osmium tetroxide in PBS at 4 ℃ overnight; the plates were dehydrated with a gradient of ethanol, soaked with epoxy resin and cut into thin slices. After the fixed sections were stained, the formation of autophagosome was observed by electron microscopy.

3.3 scratch test

Cells were seeded into six-well plates with approximately 5 × 10 additions⁵Individual cell, 5% at 37%CO₂Culturing until the fusion degree is 100%, marking a trace in the middle of each hole by using a gun head or a sterile toothpick, washing the cells for 3 times by using sterile PBS after marking is finished, washing the cells which do not adhere to the wall, replacing a fresh culture medium to continue culturing, taking out the cells at 0, 24 and 48 hours, and observing and taking pictures by using a microscope line.

3.4 results of the experiment

As shown in fig. 3 and 4, the piperlongumine-treated group showed autophagosome and decreased cell migration compared to the normal control group. Therefore, the piperlongumine can be used as an autophagy promoter and can inhibit oral squamous cell carcinoma migration.

Example 4 Piperlongumine inhibits proliferation of oral squamous cell carcinoma cells

4.1 MTT assay for cell proliferation

The oral squamous cell carcinoma cells Cal27 and UM1 were digested with 0.25% trypsin at a cell concentration of 3 × 10⁴Uniformly inoculating each cell/mL of the cell suspension into a 96-well culture plate, wherein each well is 100 mu L, adding 6, 12 and 24 mu M of piperlongumine (PPLGM for short) after the cell is attached to the wall, establishing a DMSO control group, each group is provided with 6 multiple wells which are respectively 24h, 48h and 72h, adding 10 mu L of MTT reagent, incubating for 4h in a 37 ℃ incubator, adding 150ul of dimethyl sulfoxide into each well, and detecting the light absorption value A (490nm) of each well by using a multifunctional microplate reader to observe the influence of the piperlongumine on cell proliferation along with the change of time.

4.2 results of detection

As shown in fig. 5, PPLGM was able to significantly inhibit the proliferation of oral squamous cell carcinoma cells. Therefore, the piperlongumine can be used as an oral squamous cell carcinoma medicament.