CN107789624B

CN107789624B - Application of KIF15 inhibitor in preparation of lung cancer treatment drug

Info

Publication number: CN107789624B
Application number: CN201711167295.1A
Authority: CN
Inventors: 刘子玲; 姜艳; 丛晓凤; 陈晨; 李智
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2017-11-21
Filing date: 2017-11-21
Publication date: 2020-04-28
Anticipated expiration: 2037-11-21
Also published as: CN107789624A

Abstract

The invention belongs to the field of biomedical research, and particularly relates to an application of a KIF15 inhibitor. The invention is widely and deeply researched, and the first discovery shows that KIF15 can be used as a lung cancer treatment target. The KIF15 inhibitor can inhibit the proliferation rate of lung cancer cells, change the cell cycle distribution of lung cancer cells, promote the apoptosis of lung cancer cells and inhibit the growth of lung cancer tissues, thereby treating lung cancer and opening up a new direction for the treatment of lung cancer.

Description

Application of KIF15 inhibitor in preparation of lung cancer treatment drug

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a KIF15 inhibitor in preparation of a lung cancer treatment drug.

Background

Primary bronchogenic carcinoma (lung cancer for short) is the most common malignant tumor in recent years, and the rate of incidence is high at the top of various tumors. Despite the emerging therapeutic approaches to lung cancer, 5-year survival rates are only 14.1%, with 60% of patients dying within 1 year after diagnosis. The diagnosis and prognosis evaluation of lung cancer are related to the formulation of individual treatment schemes, and directly influence the treatment and survival of patients. In the era of functional genomics and proteomics, the development of molecular pathology, the study and diagnosis of diseases from the gene level and the gene product-protein level, has become the frontier of current medical biology. The discovery of the molecular genetic changes of lung cancer may become a new cancer diagnostic system and biomarker, which can be used for prognosis and monitoring of treatment response, and is beneficial to more personalized treatment and prevention of recurrence of lung cancer.

Disclosure of Invention

In order to overcome the problems of the prior art, the invention aims to provide a novel application of a KIF15 inhibitor.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the invention, there is provided the use of an inhibitor of KIF15 for the preparation of a medicament for the treatment of lung cancer.

Further, the lung cancer treatment drug has at least one of the following functions:

inhibiting proliferation rate of lung cancer cells, changing lung cancer cell cycle distribution, promoting lung cancer cell apoptosis, and inhibiting lung cancer tissue growth.

Further, the KIF15 inhibitor is a molecule having an inhibitory effect on KIF 15.

Inhibitory effects on KIF15 include, but are not limited to: inhibiting KIF15 activity, or inhibiting KIF15 gene transcription or expression.

The KIF15 inhibitor can be siRNA, shRNA, antibody, small molecule compound.

As exemplified in the examples herein, the KIF15 inhibitor can be an siRNA or shRNA. The target sequence of the siRNA or the target sequence of the shRNA is shown in SEQ ID NO. 1.

The lung cancer treatment medicine necessarily comprises a KIF15 inhibitor, and takes the KIF15 inhibitor as an effective component of the aforementioned functions.

In the lung cancer treatment drug, the effective component playing the roles can be only a KIF15 inhibitor, and other molecules playing similar roles can also be contained.

That is, the KIF15 inhibitor is the only active ingredient or one of the active ingredients of the lung cancer therapeutic drug.

The lung cancer treatment medicine can be a single-component substance or a multi-component substance.

The form of the lung cancer treatment drug is not particularly limited, and the lung cancer treatment drug can be in the forms of various substances such as solid, liquid, gel, semifluid, aerosol and the like.

The lung cancer treatment drug mainly aims at mammals such as rodents, primates and the like.

In a second aspect of the invention, a method is provided for treating lung cancer by administering to a subject an inhibitor of KIF 15.

The subject may be a mammal or a mammalian lung cancer cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

The subject may be a patient suffering from lung cancer or an individual for whom treatment of lung cancer is desired. Or the subject is an isolated lung cancer cell from a lung cancer patient or an individual expected to treat lung cancer.

The KIF15 inhibitor can be administered to a subject before, during, or after receiving treatment for lung cancer.

In a third aspect of the present invention, there is provided a medicament for treating lung cancer, comprising an effective amount of an inhibitor of KIF 15.

Further, the lung cancer treatment drug comprises an effective dose of KIF15 inhibitor and a medicinal carrier.

In a fourth aspect of the invention, there is provided a combination therapy for lung cancer comprising an effective amount of an inhibitor of KIF15 and at least one other therapeutic agent for lung cancer.

The combination therapy drug combination may be in any one of the following forms:

firstly), the KIF15 inhibitor and other lung cancer treatment drugs are respectively prepared into independent preparations, the preparation forms can be the same or different, and the administration routes can be the same or different.

When the other lung cancer-treating drug is an antibody, a parenteral administration type is generally employed. When other lung cancer treatment medicines are chemical medicines, the administration forms can be rich, and the administration can be carried out in the gastrointestinal tract or the parenteral tract. Known routes of administration for each chemical are generally recommended.

And secondly) the KIF15 inhibitor and other lung cancer therapeutic agents are prepared into a compound preparation, and when the KIF15 inhibitor and other lung cancer therapeutic agents are administered by the same administration route and are applied simultaneously, the two can be prepared into the form of the compound preparation.

In a fifth aspect of the invention, a method is provided for treating lung cancer by administering to a subject an effective amount of a KIF15 inhibitor and administering to the subject an effective amount of another lung cancer treatment agent and/or administering to the subject another means for treating lung cancer.

An effective amount of a KIF15 inhibitor and at least one effective amount of another lung cancer therapeutic agent may be administered simultaneously or sequentially.

Based on that KIF15 is the lung cancer treatment target discovered for the first time, the invention can at least play a role in adding curative effects when being used in combination with other lung cancer treatment medicines except for KIF15 inhibitors, thereby further enhancing the treatment effect on lung cancer.

Other lung cancer therapeutic agents include, but are not limited to: antibody drugs, chemical drugs or targeted drugs, etc.

The KIF15 inhibitor may be administered parenterally or parenterally. The other lung cancer therapeutic agent may be administered gastrointestinal or parenteral. For antibody drugs, parenteral administration is generally employed.

In a sixth aspect of the invention, there is provided the use of an inhibitor of KIF15 in the manufacture of a medicament having the effect of any one or more of: inhibiting proliferation rate of lung cancer cells, changing lung cancer cell cycle distribution, promoting lung cancer cell apoptosis, and inhibiting lung cancer tissue growth.

In a seventh aspect of the invention, the use of KIF15 for screening a drug for treating lung cancer is provided.

The application of KIF15 in screening lung cancer treatment medicines specifically refers to the application of KIF15 as an action target in screening lung cancer treatment medicines.

The application of KIF15 as an action target in screening lung cancer treatment medicines specifically refers to the application of KIF15 as an action object in screening candidate substances so as to find molecules with an inhibitory effect on KIF15 as candidate lung cancer treatment medicines.

In an eighth aspect of the present invention, there is provided a method for screening a therapeutic agent for lung cancer, the method comprising:

(1) treating a system expressing KIF15 with a candidate agent; and

(2) detecting expression of KIF15 in said system;

wherein, if the candidate substance can inhibit the expression of KIF15, the candidate substance is a potential substance of the lung cancer treatment drug.

Compared with the prior art, the invention has the following beneficial effects:

the invention is widely and deeply researched, and the first discovery shows that KIF15 can be used as a lung cancer treatment target. The KIF15 inhibitor can inhibit the proliferation rate of lung cancer cells, change the cell cycle distribution of lung cancer cells, promote the apoptosis of lung cancer cells and inhibit the growth of lung cancer tissues, thereby treating lung cancer and opening up a new direction for the treatment of lung cancer.

Drawings

FIG. 1: qPCR measures the efficiency of target gene depletion at the mRNA level.

FIG. 2: and (3) detecting the target point by Western Blot to reduce the protein level expression of the target gene.

FIG. 3: MTT experiment detects the proliferation rate change of the lung cancer cells after shRNA lentivirus infection.

FIG. 4: after shRNA lentivirus infection, the periodic distribution of lung cancer cells changes.

FIG. 5: after shRNA lentivirus infection, the number of lung cancer cells undergoing apoptosis in the experimental group was changed.

Detailed Description

The invention develops the lung cancer gene related information in the TCGA database and further refers to a large number of literatures. Meanwhile, functional gene screening methods such as expression profile difference analysis, high-throughput cell screening technology and the like are combined. Finally, a possible specific oncogene KIF15 promoting lung cancer transformation is successfully screened out, and the gene has not been further developed and researched in the field of lung cancer so far.

Subsequently, the present invention confirmed the role of KIF15 gene in lung cancer development from a cell functional point of view. Detecting the expression conditions of mRNA and protein level target genes in two groups of lung cancer cell lines by constructing a target gene shRNA lentivirus, transfecting lung cancer cells with the lentivirus and comparing the lung cancer cells with transfection control vector lentivirus; and then cell proliferation, apoptosis, cell cycle and other detection are carried out through cytofunctional experiments, and the results show that the lung cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group and the increase degree of the cell apoptosis rate of the shRNA group is higher than that of the control group compared with the control group.

According to the research results, a new method for diagnosing and treating the gene is further explored and developed, so that more choices can be provided for the diagnosis and treatment of the lung cancer patient.

KIF15 inhibitor

Refers to a molecule having inhibitory effect on KIF 15. Inhibitory effects on KIF15 include, but are not limited to: inhibiting KIF15 activity, or inhibiting KIF15 gene transcription or expression. The KIF15 inhibitor includes but is not limited to siRNA, shRNA, antibodies, small molecule compounds.

Inhibiting KIF15 activity refers to a decrease in KIF15 activity. Preferably, KIF15 activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to its activity prior to inhibition.

Inhibiting the transcription or expression of KIF15 gene refers to: the method comprises the steps of making a gene of KIF15 not be transcribed, or reducing the transcription activity of a gene of KIF15, or making a gene of KIF15 not be expressed, or reducing the expression activity of a gene of KIF 15.

One skilled in the art can use conventional methods to modulate gene transcription or expression of KIF15, such as gene knock-outs, homologous recombination, interfering RNA, and the like.

Inhibition of gene transcription or expression of KIF15 can be verified by PCR and Western Blot detection of expression level.

Preferably, KIF15 gene transcription or expression is reduced by at least 10%, preferably by at least 30%, even more preferably by at least 50%, even more preferably by at least 70%, even more preferably by at least 90%, most preferably by no expression of KIF15 gene as compared to wild type.

Small molecule compounds

The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.

KIF15 inhibitor preparation medicine

A KIF15 inhibitor is used as a main active ingredient or one of the main active ingredients to prepare a medicament for treating lung cancer. Generally, the medicament may comprise one or more pharmaceutically acceptable carriers or excipients in addition to the active ingredient, according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

A "pharmaceutically acceptable carrier or adjuvant" should be compatible with, i.e., capable of being blended with, an inhibitor of KIF15 without substantially reducing the effectiveness of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as glycerol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

Combination therapeutic drug combinations and methods of administration

firstly), the KIF15 inhibitor and other lung cancer treatment drugs are respectively prepared into independent preparations, the preparation forms can be the same or different, and the administration routes can be the same or different. When in use, several medicines can be used simultaneously or sequentially. When administered sequentially, the other drugs should be administered to the body during the period that the first drug is still effective in the body.

The antibody is usually administered by intravenous injection, intravenous drip or arterial infusion. The usage and the dosage can refer to the prior art.

The small molecule compounds are usually administered by either gastrointestinal or parenteral administration. The siRNA, shRNA and antibody are generally administered parenterally. Can be administered locally or systemically.

When in use, an effective amount of KIF15 inhibitor and an effective amount of other lung cancer treatment drugs can be used simultaneously, or the effective amount of KIF15 inhibitor and the effective amount of other lung cancer treatment drugs can be used sequentially. When administered sequentially, the other drug should be administered to the organism during the period that the first drug is still effective for the organism.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1

Materials (A) and (B)

Lentiviruses and vectors were purchased from Kjekay, Shanghai; the related antibody used for the immunohistochemistry of the cell functional science and clinical samples is a Proteintech product; b27 and CD133 were purchased from invitrogen; DMEM/F12, RPMI-1640, and fetal bovine serum were purchased from Gibco; matrigel is a product of BD company; hoechst33342 and Annexin v/PI apoptosis detection kits were purchased from Binyun biological products; FACS Calibur flow cytometer is BD, usa; the Model550 enzyme-linked immunoassay instrument is Bio-rad company; the inverted microscope was OlympusCK, japan; the laser confocal fluorescence microscope is Leica company of Germany; h1299 and A549 cell strains were purchased from the cell book Collection of Wuhan university.

(II) investigation method

2.1 screening of specific oncogenes for promoting transformation of Lung cancer cells

The gene screening platform for promoting lung cancer transformation is from a cancer and tumor gene map (TCGA) database constructed by the national cancer institute. Similar to the Human Genome Project (HGP), TCGA is another genome-based major scientific research project that studies genomic changes in cancer based on the results of the human genome project. It was initiated by the U.S. government and attempted to map genomic variations of all human cancers (recently targeted at 50 tumors including subtypes) by genomic analysis techniques, particularly by large-scale genomic sequencing, and to perform systematic analysis, aiming to find out the minor variations of all oncogenes and cancer suppressor genes, understand the mechanisms of carcinogenesis and development of cancer cells, and to obtain new diagnostic and therapeutic methods based thereon, and finally draw out a whole new "cancer prevention strategy". Over the course of several years of effort, TCGA has become the largest database of cancer genetic information at present, where much valuable information is embedded. The research is carried out by combining researchers of medical colleges of Maryland university, USA, the lung cancer gene related information in a TCGA database is developed, and a large amount of documents are further consulted, and functional gene screening methods such as expression spectrum difference analysis, high-throughput cell screening technology and the like are combined. Finally, a possible specific oncogene that promotes transformation of lung cancer cells is screened and has not been further developed and studied in the lung cancer field so far. Subsequently, the present study will demonstrate the role of this gene in lung cancer development from different perspectives, respectively.

2.2 cell functional Angle-related Studies

2.2.1 cell inoculation and culture

(1) Respectively culturing H1299 and A549 cells to the density of about 90%, blowing and beating the cells in each position in a cell bottle by using A5 ml gun head, collecting the mixed solution to a 15ml test tube, and centrifuging for 3min at 800 rpm.

(2) The supernatant was removed, 1ml of complete medium was added, and cells were counted and seeded at 5X 105 per flask in T25 flasks for subsequent experiments.

2.2.2 cell infection lentivirus parameter determination

(1) One day before the experiment, 3-5X 104 cells of interest were seeded in a 96-well plate at a volume of 90. mu.l (slow lentiviral expression, fluorescence was observed after typically three to four days to ensure a degree of fusion of the cells at the time of viral infection of 30% -40%).

(2) Diluting polybrene to 50 μ g/ml with Complete Medium to obtain a final polybrene concentration of 5 μ g/ml, and labeling polybrene (M); polybrene was diluted to 50. mu.g/ml using ENi.S as a working solution to a final polybrene concentration of 5. mu.g/ml and labeled polybrene (E); virus was diluted to three titers using eni.s: group a 1x108(MOI ═ 100), group B1 x107(MOI ═ 10), and group C1 x106(MOI ═ 1) were subjected to cell infection by replacing the culture medium and adding the virus solution in groups.

(3) After mixing, the culture is continued, and after 8-12 hours, the culture medium is replaced by fresh medium.

(4) After 3-4 days of infection, fluorescence expression was observed.

(5) The infection condition of the target cells is determined by the infection effect of the cells as polybrene (M), and the infection parameter MOI is 10.

2.2.3 Each lung cancer cell strain is divided into two groups of experiment and control groups, and the two groups are respectively infected with target gene RNAi and control vector lentivirus

(1) Respectively culturing H1299 and A549 cells to the density of about 90%, blowing and beating the cells in the cell bottle by using A5 ml gun head, collecting the mixed solution to a 15ml test tube, and centrifuging for 3min at 800 rpm.

(2) The supernatant was removed, 1ml of complete medium was added, the cells were counted, and the cells were inoculated into 2.5ml of medium per flask, 4X 105 cells per flask according to the experimental contents, and cultured overnight in an incubator at 37 ℃ with 5% CO 2.

(3) Transfection can be carried out after 24h, but cell densities of 40-50% are generally preferred.

(4) The target gene RNAi diluted by Polybrene serum-free medium with 5. mu.g/ml and 500. mu.l of its control vector virus solution (50. mu.l of virus solution with 1X108 titer) were added to the medium at an MOI of 10 respectively to carry out virus infection for 10 hours.

(5) After changing 5ml of complete medium per flask and continuing the culture for 3 days, the medium was used for subsequent experiments.

2.2.4 reduction of protein level expression of target gene by Western blot detection target

1. Protein extraction

(1) The NP-40 lysate is thawed at room temperature in advance, the volume used for the experiment is estimated for subpackaging, and PMSF with the subpackaging volume of 1% is mixed for standby.

(2) Adding a corresponding volume of NP-40 lysate according to the mass and volume of each sample, placing on ice, blowing the lysate to a single cell suspension by using a pipette, and then continuing to place on the ice and standing for 5 min.

(3) Starting a low-temperature refrigerated centrifuge, centrifuging at 12000rpm and 4 ℃ for 10min, and separating supernatant to obtain the obtained protein extract.

2. Protein quantification

(1) Preparation of a standard curve: 0.5. mu.g/. mu.l BSA standard solution was dispensed to wells of the microplate in volumes of 0, 1, 2, 4, 8, 12, 16, and 20. mu.l, and the remaining volume was made up to 20. mu.l with PBS buffer.

(2) Protein solution preparation: mixing 1 μ l of the protein extract of the sample to be tested with 19 μ l of PBS buffer solution.

(3) BCA reaction: according to the formula of solution A: the volume ratio of the solution B is 50: 1, estimating the volume of the BCA working solution required by the experiment according to 200 mul per hole, adding the prepared working solution into each hole, repeatedly blowing and beating by using a pipette, fully and uniformly mixing, and reacting at 37 ℃ for 20min, wherein the solution is changed from green to purple.

(4) Data reading: and (3) starting the microplate reader for preheating 15min in advance, placing the microplate on the carrier, setting the reading wavelength to be 570nm for reading, and recording data.

(5) And drawing a standard curve according to the standard protein concentration and the corresponding light absorption value, calculating the protein concentration of the sample through a regression equation, and multiplying the protein concentration by the dilution multiple to obtain the protein concentration of the sample.

3、SDS-PAGE

(1) Assembling an electrophoresis device: and (3) sucking the surface moisture of the cleaned glass plate by using filter paper, then airing the glass plate at room temperature, and starting assembling after confirming that the surface has no water stain or dust. The long plate is arranged outside, the short plate is arranged inside, the bottom edges are aligned and fixed by a wedge, the bottom edges are sealed, and the glue filling can be started after the two surfaces are clamped.

(2) Preparation of polyacrylamide gel: selecting polyacrylamide gel with corresponding concentration according to the molecular weight of a target protein, wherein the concentration of concentrated gel is 5%, the concentration of separation gel is 8% and 12% in the experiment, preparing the separation gel from bottom to top in the sequence of gel filling, adding APS and TEMED before the gel filling, uniformly mixing, filling along one side of a glass plate, and sealing the upper layer with water to promote gel polymerization; pouring off the water layer after the separation gel is polymerized, pouring the prepared concentrated gel, adding APS and TEMED before the gel is poured, finally sealing with a comb, waiting for about 30min, pulling out the comb, and starting to sample.

(3) Protein loading solution preparation: the protein sample was diluted with 5 × Loading Buffer and PBS, boiled in boiling water bath for 5min, and made into a Loading solution for use. The loading volume for this experiment was 20. mu.l, containing 40. mu.g of protein.

(4) SDS-PAGE: after the comb is pulled out, respectively injecting electrophoresis liquid into the anode and the cathode of the electrophoresis tank, adding 20 mul electrophoresis sample liquid into each hole, finally adding 5 mul protein Marker, communicating the electrodes, adjusting the current to the maximum, adjusting the voltage to 80V, and performing constant voltage electrophoresis for 2.5 h.

4. Transfer printing

(1) Preparing: firstly, a transfer buffer solution is prepared and then is placed into a refrigerator for precooling at 4 ℃; secondly, dipping filter paper, sponge and the like for transfer printing into a transfer printing buffer solution for fully soaking when the electrophoresis is about to finish; finally, an appropriately sized PVDF membrane was cut out, labeled at the front corners of the membrane, soaked in anhydrous methanol, immersed in the transfer buffer as well, and shaken on a shaker.

(2) Preparation of a "sandwich": after electrophoresis is finished, taking out the glass plate, opening the glass plate by using a blade, cutting off the concentrated gel part of the gel and the bottom of the separation gel, peeling off the remaining whole gel into a container filled with a transfer buffer solution, and soaking for 10 min; firstly laying a layer of sponge on one side of a transfer printing clamp cathode, then laying 5 layers of filter paper, then laying gel, then laying a PVDF film, wherein the front surface of the film is in contact with the gel, and finally laying 5 layers of filter paper and a layer of sponge on the PVDF film; forming a "sandwich" shape of "sponge-filter-membrane-gel-filter-sponge".

(3) After the sandwich is laid, the transfer clamp is clamped and inserted into a transfer groove, sufficient transfer buffer solution is injected, an electrode is switched on, the current is adjusted to the maximum, and the voltage is 70V for transfer printing for 1.5 h.

5. Sealing of

(1) Preparing: 5% (M/V) skimmed milk powder is prepared by TTBS buffer solution for standby.

(2) And taking out the PVDF membrane after the transfer printing is finished, immersing the PVDF membrane into TTBS, and shaking the shaking table for 5 min.

(3) The TTBS was decanted off, the PVDF membrane was immersed in the skimmed milk powder solution and the shaker was shaken slowly for 1 h.

6. Incubation primary antibody

(1) Diluting the antibody with 5% (M/V) skimmed milk powder to obtain antibody working solution.

(2) Pouring the antibody working solution into a hybridization bag, putting the hybridization bag into a sealed PVDF membrane, sealing the hybridization bag by using a film pressing machine, and incubating the antibody.

7. Incubation secondary antibody

(1) The PVDF membrane after incubation of the primary antibody was removed from the hybridization bag, immersed in TTBS, shaken on a shaker for 5min, and the procedure was repeated 4 times.

(2) Diluting the antibody with 5% (M/V) skimmed milk powder to obtain a second antibody working solution.

(3) Pouring the secondary antibody working solution into a hybridization bag, putting the rinsed PVDF membrane into the hybridization bag, sealing the hybridization bag by using a film pressing machine, and incubating the antibody.

8. Substrate luminescence

(1) The PVDF membrane after incubation of the secondary antibody was removed from the hybridization bag, immersed in TTBS, shaken on a shaker for 5min, and this step was repeated 6 times.

(2) ECL chemiluminescent reagent A, B was mixed in equal volumes for use.

(3) Spreading a preservative film on the table top, sucking water on the back of the PVDF by using filter paper, spreading the PVDF on the preservative film, uniformly spraying ECL luminous liquid, and standing for 5min for reaction.

(4) Covering it with another preservative film, removing excess liquid with a glass rod, transferring into a dark box, and exposing in a dark room.

9. Antibody deprivation

(1) And taking the exposed PVDF film out of the preservative film, immersing the PVDF film into distilled water, shaking the table for 5min, and repeating the steps for 2 times.

(2) The container was decanted, an appropriate amount of stripping solution was added to completely cover the PVDF membrane, and the shaker was shaken for 15 min.

(3) The stripping solution was decanted from the container, briefly rinsed with distilled water, replaced with fresh distilled water, shaken on a shaker for 10min, and repeated 2 times.

10. Sealing of

(2) The PVDF membrane was removed, immersed in TTBS, and shaken on a shaker for 5 min.

11. Incubation internal reference antibody

12. Incubation secondary antibody

(1) The incubated PVDF membrane after internal control was removed from the hybridization bag, immersed in TTBS, shaken on a shaker for 5min and this step was repeated 4 times.

13. ECL substrate luminescence

(2) ECL chemiluminescent reagent A, B was mixed in equal volumes for use.

14. Analysis of results

The film was scanned and the optical density values of the target bands were analyzed using a Gel image processing system (Gel-Pro-Analyzer software).

3.2.5 qPCR detection of target point to reduce target gene mRNA level expression

1. Extraction of Total RNA

The total RNA of the sample is extracted by using a total RNA extraction kit (Tiangen), and the method comprises the following steps:

(1) 1ml of RZ lysate is added to the sample, mixed well and left at room temperature for 5 min.

(2) Add 200. mu.l chloroform, cover the tube, vortex the tube sufficiently for 15s, and let stand at room temperature for 3 min.

(3) Centrifugation is carried out at 10005g for 10min at 4 ℃, and the sample is divided into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 50% of the used lysis solution RZ reagent. The aqueous phase was transferred to a new tube and subjected to the next step.

(4) Slowly adding 0.5 times volume of absolute ethyl alcohol, and uniformly mixing. The resulting solution and precipitate were transferred together into adsorption column CR3, centrifuged at 10005g at 4 ℃ for 30s and the waste liquid in the collection tube was discarded.

(5) Mu.l of deproteinized solution RD was added to the adsorption column CR3, and the mixture was centrifuged at 10005g at 4 ℃ for 30 seconds, and the waste solution was discarded.

(6) To the adsorption column CR3 was added 500. mu.l of the rinsing solution RW, and the mixture was allowed to stand at room temperature for 2min, and centrifuged at 10005g at 4 ℃ for 30s to remove the residual liquid.

(7) The adsorption column was placed in a 1.5ml collection tube and centrifuged at 10005g at 4 ℃ for 2min to remove the residual liquid.

(8) Transferring the adsorption column CR3 into a new centrifuge tube, adding 40. mu.l of RNase-free ddH₂And O, standing at room temperature for 2min, and centrifuging at the temperature of 4 ℃ for 2min at 10005g to obtain the total RNA of the sample.

2. Reverse transcription

The RNA samples obtained from the previous experiment were reverse transcribed to obtain the corresponding cDNA.

(1) The following reaction mixture was added to an ice-bath nuclease-free centrifuge tube:

adding an RNA sample according to the concentration of the extracted RNA sample to ensure the consistency of the RNA sample concentration;

oligo(dT)₁₅1μl；

random 1μl；

dNTP(2.5mM each) 2μl；

add ddH₂O to the total reaction volume 14.5. mu.l.

(2) Heating at 70 deg.C for 5min 2 min. After the reaction solution is collected by brief centrifugation, the following components are added:

5×Buffer 4μl；

RNasin 0.5μl。

add 1. mu.l (200U) M-MLV and mix gently with a pipette.

(3) Bathing at 25 deg.C for 10min, and at 42 deg.C for 50 min.

(4) Heating at 95 deg.C for 5min to terminate reaction, and performing subsequent experiment or freezing storage on ice.

The above steps can be completed by a PCR instrument. Finally, 20. mu.l of cDNA sample is obtained, which can be stored at-20 ℃ or used in the next experiment.

3. PCR detection

(1) Establishing a PCR reaction system

The following reaction systems were added to the PCR tubes:

cDNA template 1. mu.l

Mu.l of each of the upstream and downstream primers

2×Taq PCR Master-mix 10μl

By ddH₂Make up to 20. mu.l of O

(2) The PCR reaction program was designed as follows:

4. electrophoretic analysis

(1) Preparing agarose gel: the PCR product was electrophoresed on a 1.5% gel.

And (3) placing the mixed gel liquid in a microwave oven to be heated to boiling so as to completely melt the gel, and placing at room temperature for cooling.

(2) Cooling to 50-60 ℃, adding Gold View dye, mixing uniformly, pouring the obtained gel liquid into a gel making template, removing bubbles, immediately inserting a comb, and cooling at room temperature.

Placing the gel and the gel-making plate into an electrophoresis tank, and allowing the electrophoresis buffer solution to just soak the gel surface, thereby carrying out sample application.

(3) And switching on an electrophoresis apparatus for electrophoresis and carrying out electrophoresis.

(4) And after electrophoresis, photographing the obtained gel by using a gel imaging system.

3.2.6MTT detection of Effect of target Gene knockdown on cell proliferation

1. Culturing each group of cells until the density is about 90%, collecting the cells, and centrifuging at 800rpm for 3 min.

2. Removing supernatant, adding 1ml complete medium, counting cells, and adjusting cell density to 1 × 10⁵Cells/ml were plated in 96-well plates in experimental groups of 5 replicates per well of 100l each, and plates were incubated at 37 ℃ in 5% CO₂IncubatorInternal culture for 30 min.

3. After the specified time had been reached, MTT was added to the wells at a concentration of 0.2mg/ml and incubated for 5 h.

4. Centrifugation was carried out for 10min at 1000rpm, the supernatant was carefully aspirated, 200. mu.l DMSO was added to dissolve the crystals formed by the cells, the OD at 490nm was measured on a microplate reader, data analysis was carried out, and the inhibition rate was calculated: the cell inhibition ratio (control group-experimental group)/control group × 100.

3.2.7 flow cytometry detection of Effect of target Gene knockdown on apoptosis

1. Lentiviral infection of cells as described above

2. Flow cytometry detection of apoptosis

(1) Each group of cells was collected after centrifugation at 1500rpm for 5min, the supernatant was carefully aspirated, the cells were washed twice with PBS, the cells were collected by centrifugation at 1500rpm for 5min, the supernatant was carefully aspirated, about 50. mu.l of PBS remained, and 500. mu.l of Binding Buffer was added to each tube of cell sample to gently resuspend the cells.

(2) Add 5. mu.l Annexin V-FITC and mix well, add 5. mu.l Propidium Iodid and mix well.

(3) Incubate 15min at room temperature in the dark.

(4) Then, the flow detection is carried out.

3.2.8 flow cytometry for detecting influence of target gene knockdown on cell cycle

1. Cell transfection

(1) Culturing the cells until the density is about 90%, collecting the cells, and centrifuging at 1000rpm for 5 min.

(2) Removing supernatant, adding 1ml complete medium, counting cells, adjusting cell density to 2X 10⁵Cells were seeded at 2ml per well in 6-well plates and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 30 min.

(3) Transfection reagents were prepared, and one well of a 6-well plate was used as an example below.

(4) 100l of 1640 basic medium was added to a centrifuge tube, 4.5l of transfection reagent and 2g of plasmid were added and mixed well.

(5) Standing at room temperature for 20min, and mixing the two solutions as much as possible.

The mixture was added dropwise to a 6-well plate, mixed well, and then cultured in an incubator containing 5% CO2 at 37 ℃ for 48 hours.

2. Flow cytometer for detecting cell cycle

(1) Cells were collected by centrifugation at 1500rpm for 5min, washed twice with PBS, collected by centrifugation at 1500rpm for 5min, and the supernatant carefully aspirated.

(2) Adding pre-cooled 70% ethanol, and fixing at 4 deg.C for 2 hr.

(3) The fixed cells are centrifuged at 1500rpm for 5min to collect the cells, the supernatant is discarded, the cells are washed with PBS for 2 times, centrifuged at 1500rpm for 5min, the supernatant is discarded, 500. mu.l of staining buffer is added into each tube of cell samples, and the cells are slowly and fully resuspended.

(4) After adding 25. mu.l of propidium iodide staining solution and mixing, 10. mu.l of RNase A was added and mixed.

(5) Incubate at 37 ℃ for 30min in the dark.

(6) Storing in ice bath and dark place, and immediately carrying out flow detection.

(III) results of the experiment

It should be noted that the sequence of the siRNA corresponding to the shRNA used is shown in SEQ ID No.2, which specifically includes: 5'-UGAAGUGAAGAGGCUCAAA-3' are provided. The sequence of the shRNA is shown as SEQ ID NO.3, and specifically comprises the following steps: 5'-CCGGGCTGAAGTGAAGAGGCTCAAACTCGAGTTTGAGCCTCTTCACTTCAGCTTTTTG-3' are provided. The sequence of the control shRNA is shown as SEQ ID NO.4, and specifically comprises the following steps: 5'-TTCTCCGAACGTGTCACGT-3' are provided.

1. As shown in FIG. 1, after 3 days of shRNA lentivirus infection, the expression level of the target gene in the lung cancer cells in the experimental group at the mRNA level is inhibited, and the inhibition rate is 81.2%. The target sequence of the shRNA is shown as SEQ ID NO.1, and specifically comprises the following steps: 5'-TGAAGTGAAGAGGCTCAAA-3' are provided.

2. From the Western Blot results, as shown in FIG. 2, the foreign expression of the target gene in the relevant cell line has a significant knock-down effect at the protein level.

3. In the MTT experiment, cells were plated in 96-well plates at 1000 plates 3 days after shRNA lentivirus infection. After continuous detection for 5 days, as shown in fig. 3, the proliferation rate of the lung cancer cells in the experimental group is remarkably inhibited, and the inhibition rate is 78.2%. Suggesting that the target gene is obviously related to the proliferation capacity of the lung cancer cells.

4. After 5 days of shRNA lentivirus infection, as shown in FIG. 4, the cells in the G1 phase of the experimental group are increased, the cells in the S phase are decreased, and the cells in the G2 phase are not obviously changed, which indicates that the target gene is obviously related to the cycle distribution of the lung cancer cells.

5. After 5 days of shRNA lentivirus infection, as shown in FIG. 5, the lung cancer cells undergoing apoptosis in the experimental group are significantly increased, suggesting that the target gene is significantly related to the apoptosis of the lung cancer cells.

In conclusion, the invention successfully screens out a possible specific cancer gene KIF15 which promotes the transformation of the lung cancer cells, and the function of the specific cancer gene KIF15 is proved through cell functional experiments.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

<110> Jilin university

Application of <120> KIF15 inhibitor in preparation of lung cancer treatment drug

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

tgaagtgaag aggctcaaa 19

<210>2

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ugaagugaag aggcucaaa 19

<210>3

<211>58

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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ccgggctgaa gtgaagaggc tcaaactcga gtttgagcct cttcacttca gctttttg 58

<210>4

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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ttctccgaac gtgtcacgt 19

Claims

The application of a KIF15 inhibitor in preparing a lung cancer treatment drug, wherein the KIF15 inhibitor is siRNA or shRNA, and the target sequence of the siRNA or the target sequence of the shRNA is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the lung cancer therapeutic agent has at least one of the following functions: inhibiting proliferation rate of lung cancer cells, changing lung cancer cell cycle distribution, promoting lung cancer cell apoptosis, and inhibiting lung cancer tissue growth.
3. The use of claim 1, wherein the inhibitor of KIF15 is the sole active ingredient or one of the active ingredients of the medicament for the treatment of lung cancer.
4. A lung cancer treatment drug comprises an effective dose of KIF15 inhibitor, wherein the KIF15 inhibitor is siRNA or shRNA, and the target sequence of the siRNA or the target sequence of the shRNA is shown in SEQ ID NO. 1.
5. The drug for the treatment of lung cancer according to claim 4, wherein the inhibitor of KIF15 is the only active ingredient or one of the active ingredients of the drug for the treatment of lung cancer.
6. A lung cancer combination therapy drug comprises an effective amount of KIF15 inhibitor and at least one other lung cancer treatment drug, wherein the KIF15 inhibitor is siRNA or shRNA, and the target sequence of the siRNA or the target sequence of the shRNA is shown in SEQ ID NO. 1.
Use of an inhibitor of KIF15 in the manufacture of a medicament having any one or more of the following effects: the kit comprises a KIF15 inhibitor, a siRNA or shRNA inhibitor, a target sequence of the siRNA or the shRNA inhibitor is shown in SEQ ID NO.1, and the kit can inhibit the proliferation rate of lung cancer cells, change the periodic distribution of the lung cancer cells, promote the apoptosis of the lung cancer cells and inhibit the growth of lung cancer tissues.