CN109880902A - A kind of application of long-chain non-coding RP11-499F3.2 in head and neck cancer clinical detection and the treatment of reversing tumor Cetuximab drug resistance - Google Patents
A kind of application of long-chain non-coding RP11-499F3.2 in head and neck cancer clinical detection and the treatment of reversing tumor Cetuximab drug resistance Download PDFInfo
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Abstract
The invention belongs to tumor cells diagnosis and targeted therapy fields, the significant highly expressed lncRNA RP11-499F3.2 in head and neck cancer is found particular by bioinformatic analysis TCGA data, clinical sample verifies expression of the RP11-499F3.2 in head and neck scale carcinoma and is significantly higher than cancer beside organism, and closely related with head and neck scale carcinoma patient clinical prognosis.The high expression RP11-499F3.2 of function assessment experiment discovery can promote head and neck scale carcinoma cell proliferation in vitro, migration and invasion.Meanwhile it has also been found that RP11-499F3.2 can promote head and neck scale carcinoma cell to the drug resistance of Cetuximab.LncRNA RP11-499F3.2 disclosed by the invention helps to disclose the new pathogenesis of head and neck cancer, provides new tumor markers for the Prognosis scoveillance of head and neck cancer, while new thinking is provided for the clinical treatment of head and neck cancer.
Description
Technical field
The invention belongs to diagnosing tumors and molecular targeted therapy field, more specifically to a kind of long-chain non-coding RNA
Application of the RP11-499F3.2 in clinical detection and reversing tumor Cetuximab the drug resistance treatment of head and neck cancer.
Background technique
Head and neck cancer is the cancer type of ranking the 7th in tumor disease, accounts for about Whole Body malignancy disease
7%, wherein more than 90% head and neck cancer belong to head and neck scale carcinoma (Head and neck squamous cell carcinoma,
HNSCC).It is diagnosed as head and neck scale carcinoma per year over 500000 patients, Chinese neopathy number of cases accounts for about therein 1/5, annual dead
Case load about 5.6 ten thousand.Head and neck scale carcinoma is divided into carcinoma of mouth, nasopharyngeal carcinoma, oropharyngeal cancer and laryngocarcinoma etc. in general sense.Simultaneously as
The head and neck scale carcinoma patient of about a quarter is related to In vivo infection papillomavirus (HPV) (especially oropharyngeal cancer patient), neck
Squamous carcinoma can also be divided into HPV feminine gender and positive tumor.The bad life habits such as smoke and be addicted to drink are generally considered head and neck scale carcinoma hair
Raw inducement.Simultaneously because patient, which diagnoses, realizes poor, patient's body far-end transfer or relapse indications (Recurrent or
Metastatic, R/M) more reasons such as generally, the clinical early diagnosis and complex treatment of head and neck scale carcinoma disease are still by very big
Limitation, the mean survival time (MST) of head and neck scale carcinoma patient is only 5 years.
Long-chain non-coding RNA (Long non-coding RNA, lncRNA) is the weight of current research tumor disease mechanism
Point, definition are that non-coding RNA molecule nucleotide (nt) length is greater than 200, and lacks entire open reading frame (Open
Reading frame, ORF) RNA molecule.LncRNA tissue with higher and cell-specific, a large amount of lncRNA are reported
Road has differential expression spectrum in tumor disease, it is considered to be potential proto-oncogene and tumor suppressor gene, and it is expected to conduct
Clinical diagnosis by stages and treatment ideal targets.It is generally accepted currently, lncRNA participates in tumor disease occurrence and development, but its
Specific molecular mechanism still needs to numerous studies as experimental basis.Therefore, the lncRNA difference that the present invention passes through analysis head and neck scale carcinoma
Express spectra studies the functional mechanism of correlation lncRNA, it is desirable to find that the biomarker of head and neck scale carcinoma provides foundation.
The traditional approach such as operation excision, chemotherapy and radiotherapy are the common clinical treatments of head and neck scale carcinoma patient, but due to
Incidence organ site is special, such treatment relatively easily causes patient organ's functional, influences life quality.Simultaneously for quilt
It is diagnosed as advanced stage or the head and neck scale carcinoma patient of far-end transfer, local recurrence symptom occurs, the therapeutic effect of traditional treatment is often not
People's will to the greatest extent.As the molecular pathology research of malignancy disease is gradually carried out, the therapeutic effect of many tumor types is demonstrate,proved
Bright related with signal path regulation and mutant target gene, head and neck scale carcinoma targeted therapy also opens clinical treatment new approaches.
Transmembrane glycoprotein EGF-R ELISA (Epidermal growth factor receptor, EGFR) can lead to
Cross activation downstream passages (such as PI3K and ERK-1/2) and then the life such as modulate tumor cell Proliferation, invasion and drug resistance generate
Activity, is target molecules important in head and neck scale carcinoma, and 90% head and neck scale carcinoma patient is overexpressed EGFR.Cetuximab
(Cetuximab, trade name Erbitux) becomes the targeted therapy that first FDA approval is used for head and neck scale carcinoma on November 7th, 2011
Drug, it was reported that the head and neck scale carcinoma patient of only 10%-20% obtains complete incidence graph in Cetuximab long-term treatment.But
Currently, its drug resistance generation mechanism is still indefinite, the present invention passes through to head and neck scale carcinoma Cetuximab drug resistance correlation lncRNA's
Mechanism of action is studied, it is desirable to provide reference for clinical treatment.
Summary of the invention
1. goal of the invention
In view of the above-mentioned problems, the purpose of the present invention is find a kind of long-chain non-coding RNA RP11- relevant to head and neck cancer
499F3.2.The lnc RP11-499F3.2 and head and neck scale carcinoma clinical diagnosis and prognosis are closely related, and head and neck scale carcinoma can be promoted thin
Born of the same parents' in-vitro multiplication and transfer can be used as the potential biomarker of head and neck scale carcinoma.West can be improved for the lock nucleic acid of its design
The appropriate drug resistant sensibility of former times monoclonal antibody.
2. technical solution
To achieve the goals above, technical solution is as follows:
It is by inventor's comprehensive analysis cancer gene the invention discloses a kind of long-chain non-coding RNA RP11-499F3.2
Head and neck cancer and the lncRNA chip of expression spectrum data of normal cancer beside organism are found in map (TCGA) database one is significantly high
Express the lncRNA in neck cancerous tissue.It is located at human chromosomal 15:81,660,482-81,871,125 antisense strands, DNA
Sequence is as shown in SEQ ID NO.1.
Clinical testing procedure for head and neck cancer is real-time quantitative PCR, and the method includes:
Test sample is obtained from the object with head and neck cancer；
Determine the expression in the test sample including long-chain non-coding RNA RP11-499F3.2；With
The expression is analyzed to generate risk score, wherein the risk score can be used for providing the prognosis of object.
Further, the test sample is the tumor tissues and serum of Liquid nitrogen storage.
Further, the reagent for detecting biomarker expression level is real time quantitative PCR detecting reagent kit.
Detection primer sequence for real-time quantitative PCR is as shown in SEQ ID NO.2 and SEQ ID NO.3.
For assessing influence of the lnc RP11-499F3.2 to head and neck cancer cell function, specific experimental procedure is as follows:
It is overexpressed using lnc RP11-499F3.2 and silencing slow virus infects SCC4 cell respectively, filter out steady turn carefully
Then born of the same parents do cell Proliferation, migration invasion and scratch experiment detection respectively
The invention also discloses a kind of methods for establishing stable Cetuximab medicine-resistant cell line: being screened using concentration gradient
Method establishes SCC4/CTX drug-resistant cell strain.
In order to probe into effect of the lnc RP11-499F3.2 in Cetuximab drug resistance, we pass through design lock nucleic acid
The expression of specific regulatory control RP11-499F3.2.Specifically, this is specifically locked nucleotide sequence and sees SEQ ID NO.4.
Further, using the sequence of non-specific sequences NC as negative control, sequence is shown in SEQ ID NO.5, respectively
SCC4/CTX cell strain is transfected, is changed by detection mdr cell in-vitro multiplication and transfer ability, lnc RP11- is investigated
499F3.2 to the Susceptible change of Cetuximab mdr cell.
Meanwhile we investigate specificity lock nucleic acid to the drug resistant influence of Cetuximab in head and neck scale carcinoma body.Specifically
It says, by the way that repeatedly head and neck scale carcinoma Cetuximab drug resistance PDX model is established in passage in vivo, intratumor injection gives doses
The lock nucleic acid of lnc RP11-499F3.2 monitors tumor volume change.
3. beneficial effect
(1) present invention discover that lnc RP11-499F3.2 significantly high expression in head and neck scale carcinoma, and it is closely related with prognosis,
It can be used as the potential biomarker of head and neck scale carcinoma；
(2) present invention discover that expression of the lnc RP11-499F3.2 in head & neck cancer cell is significantly higher than oral epithelium
Cell can obviously inhibit proliferation, migration, invasion and the clone's shape of head & neck cancer cell after lnc RP11-499F3.2 loss of expression
At prompting its importance to growth and metastasis of tumours, provide reference for head and neck cancer targeted therapy；
(3) present invention establishes western appropriate former times drug resistance PDX model in vivo, finds by establishing western appropriate former times drug-resistant cell strain in vitro
Lowering lnc RP11-499F3.2 expression helps to restore head & neck cancer cell to the sensibility of Cetuximab, further proves
Lnc RP11-499F3.2 is the key that head and neck scale carcinoma Cetuximab drug-resistant phenotype generates lncRNA；
(4) present invention also attempts specificity interference RP11-499F3.2 in the other indications of Cetuximab, including colon
Cancer, the cancer of the esophagus, the effect in non-small cell lung cancer mdr cell model are that the clinical treatment of Cetuximab expands direction；
(5) experimental science that the present invention designs is reasonable, feasible effective, grinds to long-chain non-coding RNA RP11-499F3.2
Study carefully and gos deep into system；Based on the above discovery, lnc RP11-499F3.2 expression can be used as a new biomarker auxiliary
The reaction that Cetuximab is treated in the diagnosis of head and neck cancer and the prediction of grade malignancy, especially reversing drug resistance head and neck cancer patient
Property, therapeutic effect is improved, there is good translational medicine prospect.
Detailed description of the invention
Fig. 1 is the LncRNA chip dendrogram of differential expression in neck cancerous tissue and cancer beside organism, and data come from TCGA number
According to library；
Fig. 2 is lnc RP11-499F3.2 expression and the relationship that HNSCC patients overall survival leads, and data come from TCGA
Database；
Fig. 3 is that expression quantity of the lnc RP11-499F3.2 in 46 pairs of head and neck cancer tissues and cancer beside organism compares, and data are come
From TCGA database；
Fig. 4 is qPCR testing result (2 of the lnc RP11-499F3.2 in 50 pairs of HNSCC clinical samples-ΔΔctValue ratio
Compared with * P < 0.05, * * P < 0.01)；
Fig. 5 is that lnc RP11-499F3.2 expression and HNSCC patient in 50 HNSCC clinical tissue samples are total
The relational graph of survival rate；
Fig. 6 is that lnc RP11-499F3.2 expression and HNSCC patient in 30 HNSCC clinical serum samples are total
The relational graph of survival rate；
Fig. 7 is expression quantity comparison result figure of the lnc RP11-499F3.2 in HNSCC cell and mouth epithelial cells；
Fig. 8 is that lnc RP11-499F3.2 is overexpressed and silencing slow virus carrier plasmid map；
Fig. 9 is that lnc RP11-499F3.2 is overexpressed and silencing slow virus carrier transfects SCC4 cell and qPCR testing result
Figure；
Figure 10 is to be overexpressed and influence result figure of the silencing lnc RP11-499F3.2 to SCC4 ability of cell proliferation；
Figure 11 is the influence result figure being overexpressed with silencing lnc RP11-499F3.2 to SCC4 cell migration ability；
Figure 12 is the influence result figure being overexpressed with silencing lnc RP11-499F3.2 to SCC4 cell invasion ability；
Figure 13 is the IC50 value that mtt assay detects that parental generation SCC4 responds cetuximab；
Figure 14 is influence result figure of the cetuximab to SCC4 and SCC4/CTX ability of cell proliferation；
Figure 15 is the result figure that cetuximab influences SCC4 and SCC4/CTX cell cycle distribution；
Figure 16 is the result figure that cetuximab influences SCC4 and SCC4/CTX Apoptosis；
Figure 17 is the result figure that cetuximab influences SCC4 and SCC4/CTX cell clonal formation；
Figure 18 is the result figure that cetuximab influences SCC4 and SCC4/CTX cell tumor growth in vivo；
Figure 19 is the detection of lnc RP11-499F3.2 expression in SCC4 and SCC4/CTX cell strain；
Figure 20 is the tumour dynamic growth figure for establishing cetuximab medicaments insensitive PDX Transplanted tumor model；
Figure 21 is head and neck scale carcinoma cetuximab drug resistance PDX Transplanted tumor model tumour dynamic growth figure；
Figure 22 is lnc RP11-499F3.2 table in the HNSCC-PDX Model Tumor tissue of cetuximab sensitivity and tolerance
It is detected up to level；
It is swollen after lnc RP11-499F3.2 targeted therapy that Figure 23 is that the drug resistant HNSCC PDX model of cetuximab is given
Tumor dynamic growth figure；
It is swollen after lnc RP11-499F3.2 targeted therapy that Figure 24 is that the drug resistant HNSCC PDX model of cetuximab is given
Tumor internal anatomy；
Figure 25 is that the drug resistant HNSCC PDX model of cetuximab gives the master after lnc RP11-499F3.2 targeted therapy
Want the HE coloration result figure of internal organs.
Specific embodiment
The present invention is further described below combined with specific embodiments below.
Embodiment 1
Number of people neck cancer tissue is analyzed with the lncRNA sequencing result with normal tissue
Oncogenome map (TCGA) plan by U.S. National Cancer Institute (NCI) and
National Human Genome Research Institute (NHGRI) is in the project of combined launch in 2006, using big
Genome analysis technology based on scale sequencing carries out large scale experiment, TCGA genome analysis center for 36 kinds of cancers
(GCC) tumour and normal tissue are compared, gene mutation relevant to each cancer or hypotype, amplification or missing are found.For reason
The molecular mechanism of cancer is solved, people is improved and provides help to the scientific knowledge of pathogenesis of cancer molecular basis.
Into TCGA (https: //cancergenome.nih.gov/) the head and neck scale carcinoma option page, clinical is selected
And RNAseq option, click to enter the page.Rsem.genes.normalized_ is chosen in RNAseq option
Results.txt file, and All Files in METADATA and clinical catalogue are chosen, it is downloaded after obtaining address；Obtain head
After carcinoma of neck tissue gene expresses data, RNA standardized data is renamed according to METADATA the file information, to facilitate and base
Because of expression data file and clinical pathology information comparison；Statistics shares 500 head and neck scale carcinoma cancerous tissues and 46 necks after comparison
Squamous carcinoma cancer beside organism, corresponding rna expression data and clinical pathology information are complete.
It selects R lingware (3.1 version) analysis head and neck scale carcinoma tissue gene to express data, screens differential expression
LncRNA, R lingware are the gene chip expression difference algorithms being modified by chip expression abundance and noise height；
Using head and neck scale carcinoma tissue gene expression data as standardized data typing Excel software, the vacancy value of expression is taken out more than 80%
The lncRNA data of sample size；Differential expression lncRNA, the screening conditions of differential expression lncRNA are analyzed using R lingware
It is set as FDR < 0.05, Fold change > 2, i.e., screening is in head and neck scale carcinoma cancerous tissue and normal tissue expression level error
The lncRNA of different P value < 0.05 and 2 times of > of difference value (absolute value) use 3.0 software package of Cluster to draw clustering
Figure carries out visual analyzing to head and neck scale carcinoma tissue high throughput gene expression data.
563 differential expressions are obtained according to screening conditions herein from 13964 candidate lncRNA in the result is shown in Figure 1
LncRNA (fold change > 2, P < 0.05), wherein expressed in head and neck scale carcinoma tissue significantly raise have 254, lower
Have 309.
Embodiment 2
The relationship that the screening of lnc RP11-499F3.2 and its expression and HNSCC patients overall survival lead
All clinical sample indifferences are bisected into two groups (training set group and test set groups) at random, every group is faced for 250
Bed sample；Training set clinical sample is divided into high risk and low risk group to have screened lncRNA relative expression levels, with
The survival of patients time is independent variable, chooses variable using lasso method, draws Kaplan-Meier survivorship curve；Utilize test
Collect the lncRNA for screening training set group to verify, screening technique and condition are identical.
Table 1.Kaplan-Meier survivorship curve method is screened and HNSCC life cycle significant relevant lncRNA
For further verify and screen head and neck scale carcinoma diagnose correlation lncRNA, the present invention to the 8 kinds of lncRNA screened into
Row ROC curve is drawn, and is assessed above-mentioned lncRNA to the diagnosis prediction ability of head and neck scale carcinoma patient survival, is screened head and neck scale carcinoma
Ideal diagnosis molecular marker.
2 ROC curve method of table screens head and neck scale carcinoma patient survival correlation lncRNA
After lncRNA in conjunction with AUC < 0.6 in table 1, the rejecting ROC curve of table 2, qualified lncRNA is filtered out altogether
Totally 4: RP11-499F3.2, LINC00460, LINC00958 and ST3GAL4-AS1, and show its specificity and sensitivity compared with
It is high.Wherein, in the AUC area highest of the highly expressed lnc RP11-499F3.2 of head and neck scale carcinoma tissue specificity, training set and
The AUC of test set is respectively 0.714 and 0.748, and susceptibility and specificity are above 80%, can be used as head and neck scale carcinoma ideal
Diagnosis molecular marker.
The result is shown in Figure 1, the expression of lnc RP11-499F3.2 and HNSCC prognosis are aobvious in training set and test set
Work property is related.
Embodiment 3
Lnc RP11-499F3.2 is in HNSCC tumor tissues and with the expression analysis in normal tissue
According to the method for embodiment 1 obtain 43 pairs of HNSCC sample cancerous tissues and cancer beside organism clinical information and
Corresponding lnc RP11-499F3.2 expresses data.As a result see Fig. 2, compared with the normal cancer beside organism of pairing, lnc RP11-
Expression of the 499F3.2 in HNSCC significantly increases, and considers the index that can be used as the early diagnosis of head and neck cancer clinic.
Embodiment 4
Expression of the lnc RP11-499F3.2 in head and neck cancer patient and normal cancer beside organism.
(1) acquisition of sample
In the case where patient's informed consent, head and neck cancer and cancer beside organism's sample are acquired in art, after physiological saline cleaning,
It is stored in spare in liquid nitrogen or -80 DEG C of refrigerators.
(2) design of primers
All exon sequences of the gene are searched in ensemble database according to the information of lnc RP11-499F3.2
Information, and Primer Premier5.0 design primer is used according to the sequence information of acquisition, sequence is as follows:
Upstream primer (SEQ ID NO.2)
Downstream primer (SEQ ID NO.3)
(3) expression of the real-time quantitative PCR detection lnc RP11-499F3.2 in head and neck cancer patient and normal cancer beside organism.
The total serum IgE for collecting sample is extracted by the Trizol specification of life company, then with NanoDrop ND-1000 nucleic acid
Quantitative instrument quantifies the purity and concentration of extracted RNA, and agarose quality inspection ensures to extract the integrality of RNA.It is tried using TaKaRa
Agent box PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) is to extraction
Total serum IgE reverse transcription synthesizes cDNA.Using TaKaRa kitPremix Ex TaqTM II(Tli RNaseH Plus)
Carry out qPCR reaction.Reaction system is as follows:
3 PCR reaction system of table
Above-mentioned component is pressed into following procedure: 95 DEG C of 30s initial denaturations, 40 circulations after mixing；95 DEG C of 5s, 60 DEG C
30s。
According to the specificity of melting curve judgement reaction, the opposite of lnc RP11-499F3.2 is calculated by formula 2- Δ Δ Ct
Expression quantity.As a result see Fig. 4, in 75% or so head and neck cancer sample, the expression of lnc RP11-499F3.2 is significantly higher than
Normal cancer beside organism.
Embodiment 5
The relationship that lnc RP11-499F3.2 expression and 50 HNSCC patients overall survivals lead
50 HNSCC patients are divided into high risk and low risk group according to lncRNA relative expression levels, with patient
Life span is independent variable, chooses variable using lasso method, draws Kaplan-Meier survivorship curve.
As a result see Fig. 5, there are correlation, lnc for the survival rate of the expression of lnc RP11-499F3.2 and head and neck cancer patient
The patients overall survival of RP11-499F3.2 low expression, which leads, is significantly higher than the highly expressed patient of TMEM170B, further confirms lnc
A New Set of the RP11-499F3.2 as head and neck cancer prognosis.
Embodiment 6
Expression of the lnc RP11-499F3.2 in head and neck cancer patient and Healthy Human Serum
(1) acquisition of sample
In the case where patient's informed consent, in preoperative collection head and neck cancer patient and healthy volunteer's serum sample, save
It is spare in -80 DEG C of refrigerators.
(2) table of real-time quantitative PCR detection lnc RP11-499F3.2 in head and neck cancer patient and healthy volunteer's serum
It reaches
Design of primers and specific detection method are the same as embodiment 5.
As a result see Fig. 6, compared with healthy volunteer, expression of the lnc RP11-499F3.2 in HNSCC is significantly increased,
Consider to can be used as the serological index that head and neck cancer clinic early diagnoses.
Embodiment 7
Detect the lnc RP11-499F3.2 expression in HNSCC cell and normal oral epithelial cell
Extract head and neck cancer SCC4, SCC9, SCC13, the total serum IgE of CAL27 and normal oral epithelial cell HIOEC, qPCR inspection
Lnc RP11-499F3.2 is surveyed, specific detection method is the same as embodiment 5.As a result see Fig. 7, head and neck scale carcinoma cell line lnc RP11-
499F3.2 is expressed obviously higher than HIOEC cell.
Embodiment 8
Lnc RP11-499F3.2 is overexpressed preparation and virus transfection Efficiency testing with silent carrier
Synthesis imports for the full-length cDNA of lnc RP11-499F3.2 and is overexpressed slow virus carrier (Fig. 8), according to shRNA
Design rule, (sequence is respectively SEQ ID to small molecules interference RNA of the design three for lnc RP11-499F3.2 exon
NO.6, SEQ ID NO.7 and SEQ ID NO.8) silencing slow virus carrier (Fig. 8) is imported, by the same packaging plasmid of above-mentioned plasmid
DR8.9 and envelope plasmid VSVG.2 corotation enter in 293T cell to generate virus, after transfecting 48h, collect in the virus of cell
Clearly, SCC4 cell is infected.After infection for 24 hours, addition puromycin screening obtains the steady lnc RP11-499F3.2 that turns and is overexpressed and sinks
Silent cell strain.It collects the total serum IgE for surely turning cell, lnc RP11-499F3.2 is detected by qPCR (specific method is with embodiment 5)
Expression variation.
As a result see Fig. 9, transfect the transfection cell that cell lnc RP11-499F3.2 is overexpressed slow virus plasmid according to the observation
Apparent GFP protein expression can be observed under fluorescence, lnc RP11-499F3.2 expression generates significant compared with control group CTRL
It raises (P < 0.01).It is tested simultaneously according to GFP expression and qRT-PCR, sh-c interferes slow virus plasmid transfection effect as the result is shown
Rate highest.
Embodiment 9
It is overexpressed and influence of the silencing lnc RP11-499F3.2 to SCC4 ability of cell proliferation
Using mtt assay detection be overexpressed and silencing lnc RP11-499F3.2 after to the active shadow of SCC4 cell Proliferation
It rings.Head & neck cancer cell is collected when culture to 90% or more density with trypsin digestion in 37 DEG C, the incubator of 5%CO2, is used
Culture solution is resuspended cell and counts under the microscope, and cell concentration is adjusted to 3.0 × 104A/mL, cell suspension inoculation is arrived
In 96 orifice plates, every 100 μ L of hole, and cultivated in 37 DEG C, 5%CO2 incubator.Respectively in culture 0h, for 24 hours, after 48h and 72h to
The MTT of 20 μ L 5mg/mL is added in every hole in 96 orifice plates, continues to cultivate 4h.Culture medium is sucked, it is molten that 100 μ L DMSO are added in every hole
Solution.It in Detection wavelength is 570am with microplate reader, reference wavelength is the place 630nm measurement light absorption value, and calculates growth inhibition ratio
(proliferation inhibition, PI).
Test is independently repeated 3 times, and the result tested is indicated with mean ± SD, and is carried out statistics T and examined, * P < 0.05
For significant difference, * * P < 0.01 is extremely significant sex differernce.
The result is shown in Figure 10, the group for being overexpressed lnc RP11-499F3.2 then generate on extremely significant ability of cell proliferation
It adjusts, and silencing lnc RP11-499F3.2 declines head and neck scale carcinoma ability of cell proliferation.This display lnc RP11-499F3.2 tool
There is the external ability for promoting head and neck scale carcinoma cell Proliferation.
Embodiment 10
It is overexpressed and influence of the silencing lnc RP11-499F3.2 to SCC4 cell migration ability
Head & neck cancer cell SCC4 is inoculated into the cell transwell, every 100 μ L of hole, room is added at transwell
Complete medium of the 0.6mL containing 10%FBS stimulates cell migration, and in 5%CO2,37 DEG C of cultures are for 24 hours.Kong Zhongpei liquid is discarded, is used
90% alcohol room temperature fixes 30min, and 0.1% crystal violet room temperature dyes 10min, and clear water rinses, and dabs off upper layer not with cotton swab
Migrating cell, microscopically observation simultaneously select four visuals field to take pictures counting.According to formula computation migration inhibiting rate (migration
Inhibition rate, MIR):
Wherein Ntest is the cell migration number of test group, and Ncontrol is the cell migration number of blank control group.Test is only
Vertical to be repeated 3 times, the result tested calculates mean ± SD, and carries out statistics t and examine, and * P < 0.05 is significant difference, * *
P < 0.01 is extremely significant sex differernce.
The result is shown in Figure 11, SCC4 cell quantity of Successful migration after being overexpressed lnc RP11-499F3.2 significantly increase,
In contrast, the SCC4 cell for striking low lnc RP11-499F3.2 significantly reduces migrating cell quantity after cultivating 48h.This says
Bright lnc RP11-499F3.2 can promote cell migration in head and neck scale carcinoma cell line SCC4.
Embodiment 11
It is overexpressed and influence of the silencing lnc RP11-499F3.2 to SCC4 cell invasion ability
By 10mg/mL Matrigel culture medium with 1: 3 dilution, it is coated on the cell transwell film, is air-dried at room temperature.
Culture is arrived to the head & neck cancer cell trypsin digestion of logarithmic growth phase, collects, uses blank cultures after being washed twice with PBS
It is resuspended.Cell concentration is adjusted to 1 × 105A/mL.It seeds cells into the cell transwell, every 100 μ L of hole,
Complete medium of the 0.6mL containing 10%FBS, which is added, in room under transwell stimulates cell invasion, in 5%CO2,37 DEG C of cultures
24h.Kong Zhongpei liquid is discarded, fixes 30min with 90% alcohol room temperature, 0.1% crystal violet room temperature dyes 10min, and clear water rinses, and uses
Cotton swab dabs off the cell that upper layer is not invaded, and microscopically observation simultaneously selects four visuals field to take pictures counting.According to formula
Calculate invasion inhibiting rate (Invasion inhibition rate, IIR):
Wherein Ntest is the cell invasion number of test group, and Ncontrol is the cell invasion number of blank control group.Test is only
Vertical to be repeated 3 times, the result tested calculates mean ± SD, and carries out statistics t and examine, and * P < 0.05 is significant difference, * *
P < 0.01 is extremely significant sex differernce.
The result is shown in Figure 12, the SCC4 cell quantity that success is invaded after being overexpressed lnc RP11-499F3.2 significantly increase,
In contrast, the SCC4 cell for striking low lnc RP11-499F3.2 significantly reduces invasion cell quantity after cultivating 48h.This says
Bright lnc RP11-499F3.2 can promote cell invasion in head and neck scale carcinoma cell line SCC4.
Embodiment 12
The IC50 value that mtt assay detection parent's sensitivity SCC4 responds cetuximab
Cetuximab 10nM, 20nM, 40nM, 80nM, 160nM, 320nM drug concentration are set, with docetaxel (10 μ
It g/ml) is positive drug, MTT detects the zengzhi situation of SCC4 cell under different pharmaceutical concentration, and specific method is shown in embodiment 9.
The result is shown in Figure 13, Cetuximab significantly increase the proliferation inhibiting effect of SCC4 cell with drug concentration promotion,
For the cell strain to Cetuximab medicaments insensitive, IC50 value of the SCC4 cell under Cetuximab drug effect is 80nM, is made
For the initial concentration for screening Cetuximab drug-resistant cell strain.
Embodiment 13
Influence of the cetuximab to SCC4 and SCC4/CTX ability of cell proliferation
(1) it recovers and cultivates SCC4 cell, when SCC4 cell grows to 70% density, discard culture medium in culture bottle, more
It is changed to the DMEM complete medium of the final concentration of 80nM of drug, at 37 DEG C, 48h is cultivated in the incubator containing 5%C02, then use
No pharmaceutical culture medium is stablized passage 3 times or more；
(2) restore to stablize growth to cell, cell density reaches 70% density, starts Cetuximab in multiplication culture medium
Drug concentration repeats the above steps, and each drug dose is kept for cell culture 15-20 days；
(3) the SCC4 cell for periodically freezing each drug concentration induction, measures the IC50 value of inducing cell, is finally resistant to
The SCC4 cell of 1280 nM Cetuximabs, is named as SCC4/CTX for mdr cell.
(4) using mtt assay detection various concentration Cetuximab to the proliferative capacity of two kinds of cells of SCC4 and SCC4/CTX
Influence, specific method is shown in embodiment 9.
The result is shown in Figure 14, compared to parent's SCC4 drug sensitive cell, the mdr cell SCC4/CTX that the present invention establishes exists
Cell proliferation level in Cetuximab tolerance dose still significantly increases.
Embodiment 14
Influence of the cetuximab to SCC4 and SCC4/CTX cell cycle distribution
(1) when SCC4 and SCC4/CTX grow to logarithmic growth phase, addition 0.25% trypsin solution digestion goes to EP
1000rpm is centrifuged 5min after pipe, is resuspended and is laid with to six porocyte culture plates cultures, when cell density grows to 60%, discards
Culture medium, is separately added into the DMEM serum free medium containing 0nM, 40nM, 320nM Cetuximab drug concentration, and six orifice plates are set
In 37 DEG C, 48h is cultivated in the incubator containing 5%CO2；
(2) digestion of 0.25% trypsin solution is added, goes to 900rpm centrifugation 5min after centrifuge tube, then be resuspended with PBS
Cell is washed, calculates cell concentration using cell counter, concentration of cell suspension is adjusted to 1 × 106A/ml；
(3) it takes 1ml cell suspension, the 70% pre-cooled ethanol solution of 500 μ l is added, 2h is fixed at 4 DEG C to overnight,
It is discarded supernatant after 1000rpm centrifugation 3min, PBS washs cell twice, washes away remaining fixer；
(4) dyeing working fluid is prepared according to the volume ratio that RNase:PI working solution is 1: 9,500 μ l are added in each sample
Dyeing working fluid is protected from light at 4 DEG C and is incubated for 30min；
(5) it using the machine testing cell cycle on flow cytometer, chooses and records each cell sample at excitation wavelength 488nn
Red fluorescent；
(6) data analysis uses FlowJo v5.7.3 software, using Dean-Jett-Fox model of fit, with cell quantity
As ordinate, PI red fluorescent intensity is abscissa, distribution of the statistics sample cell in each cell cycle.
The result is shown in Figure 15, cell distribution ratio of the SCC4 cell in the G1 phase are promoted with drug concentration and dramatically increase (P <
0.05)；S phase cell distribution ratio is promoted with drug concentration and is remarkably decreased (P < 0.05), and Cetuximab grows cell
Blocked in the G1 phase, inhibits SCC4 cell Proliferation vigor.And under the effect of Cetuximab different pharmaceutical concentration, SCC4/CTX is thin
G1, S, G2/M phase cell distribution of born of the same parents is without significant difference.
Embodiment 15
Influence of the cetuximab to SCC4 and SCC4/CTX Apoptosis
(1) when SCC4 and SCC4/CTX grow to logarithmic growth phase, 0.25% trypsin solution digestion, after going to EP pipe
1000rpm is centrifuged 5min, is resuspended and is laid with to six porocyte culture plates cultures, when cell density grows to 60%, discards culture
Base, is separately added into the DMEM serum free medium containing 0nM, 40nM, 320nM Cetuximab drug concentration, and six orifice plates are placed in 37
DEG C, 48h is cultivated in the incubator containing 5%CO2；
(2) digestion of 0.25% trypsin solution is added, 900rpm centrifugation 5min after centrifuge tube is gone to, with the PBS of pre-cooling
Buffer is resuspended and rinses cell twice, removes remaining pancreatin in cell suspension, calculates cell concentration using cell counter, adds
Enter 400 μ l of Annexin V combination liquid, cell is resuspended and adjusts cell concentration to 1 × 106A/ml；
(3) 5 μ l of Annexin V-FITC dyeing liquor is added to cell suspension, mixes gently, be protected from light at 4 DEG C and be incubated for 15min,
The PI dyeing liquor for adding 10 μ l, mixes gently, and is protected from light at 4 DEG C and is incubated for 5min；
(4) it chooses using machine testing Apoptosis on flow cytometer at once and records excitation wavelength 488nn, transmitted wave
The fluorescence signal of cell sample at long 530 nm, while detecting the mono- positive pipe of the Annexin V-FITC without drug-treated cell
And single positive pipe of PI, determine the position of fluorescence compensation numerical value and cross quadrant door；
(5) data analysis uses FlowJo v5.7.3 software, using PI red fluorescent intensity as ordinate,
Annexin V-FITC green florescent signal intensity is abscissa.In experimental result, upper right Q2 quadrant belongs to non-viable apoptotic cell,
Bottom right Q3 quadrant belongs to viable apoptotic cell, and lower-left Q4 quadrant belongs to normal cell, uses Q2 quadrant when counting apoptosis rate
And Q3 quadrant.
The result is shown in Figure 16, for SCC4 cell under 40nM and 320nM Cetuximab drug effect, apoptosis rate is significant
Increase；And SCC4/CTX only generates apoptosis rate under 320nM drug concentration pressure and dramatically increases, and identical drug concentration
Under, SCC4/CTX apoptosis rate is reduced compared with SCC4 cell conspicuousness.Thus, it can be said that under Cetuximab drug pressure,
SCC4/CTX has the function of that stronger apoptosis is resisted compared with SCC4.
Embodiment 16
Influence of the cetuximab to SCC4 and SCC4/CTX cell clonal formation
(1) 0.56g low melting-point agarose powder is weighed, 10ml distilled water is added, is formulated as 5.6% agarose gel mother
Liquid, high pressure sterilization 30min at 121 DEG C, puts the constant temperature into 50 DEG C of water-baths；
(2) 0.8% bottom agar carbohydrate gums are prepared: taking 5.6% agarose gel mother liquor 1.5ml, temperature maintains 40 DEG C of left sides
The right side prevents from solidifying, and 9ml DMEM complete medium is added, uniformly, the packing hole 0.5ml/ to 12 porocyte culture plates is spread altogether for piping and druming
If 18 holes, cooled and solidified 5min at 4 DEG C；
(3) complete medium of DMEM containing Cetuximab for preparing 45.7nM and 365.7nM respectively, digests and collects place
In SCC4 the and SCC4/CTX cell of logarithmic growth phase, two kinds of equal equivalent of cell are divided into three parts, using DMEM complete medium,
45.7nM dosing DMEM culture medium and 365.7nM dosing DMEM culture medium are resuspended, cell count, adjustment cell concentration to 1 × 104
A/ml；
(4) 0.7% top-layer agar carbohydrate gums prepare: draw 5.6% agarose gel mother liquor 1.2ml, respectively to 6 5ml from
In heart pipe, it is separately added into each 1.4ml of cell suspension of preparation of above-mentioned SCC4 and SCC4/CTX, slowly piping and druming uniformly, dispenses 0.5
Into 12 porocyte culture plates for being laid with lower layer's glue, the identical drug concentration of allogenic cell sets three multiple holes in the hole ml/, and every kind
Cell suspension is laid with 9 holes altogether, and it is cooled and solidified 5min at 5 × 103,4 DEG C that every hole, which is laid with cell concentration,；
(5) after agarose gel solidification, corresponding DMEM is added into hole respectively by colony formation group and cultivates completely
Base, 40nM dosing DMEM culture medium and 320nM dosing DMEM culture medium, every each 0.5ml in hole；
(6) 12 orifice plates are placed in 37 DEG C, the incubator culture containing 5%CO2 15-20 days, replace upper layer every three days and accordingly train
Base is supported, when there are macroscopic cell clone, stopping cell culture in culture plate；
(7) it draws upper layer culture medium and discards, 0.01% violet staining liquid of 1ml is added in every hole, be protected from light dyeing 1h,
PBS buffer decolourizes high-visible to cloning, and tissue culture plate is placed in inverted microscope, observes and count SCC4 and SCC4/
The cell clone amount of CTX.
The result is shown in Figure 17, as drug concentration increases, SCC4 cell clone formation number is remarkably decreased, and SCC4/
CTX is under various concentration drug pressure, and cell clonal formation amount, which does not generate, to be decreased obviously, and there was no significant difference, this shows
Cetuximab inhibits the ability of SCC4/CTX cell clonal formation weaker.
Embodiment 17
Cetuximab influences the tumour growth of Transplanted tumor model in SCC4 and SCC4/CTX cell body
(1) mass propgation SCC4 and SCC4/CTX cell are digested using 0.25% trypsin solution, and cell is outstanding after terminating digestion
Liquid 1000rpm is centrifuged 5min, counts after cell is resuspended with the DMEM culture medium of serum-free, adjustment cell concentration to 5 × 107It is a
/ml；
(2) every nude mice (orders 4-6 week old, weight at female BAl BIc/c nude mice totally 24 of 14-16g, moves at SPF grades
Object receptacle adaptive feeding 1 week) the corresponding other cell suspension of group of 200 μ l of left side oxter inoculation, injection cell concentration is 1 × 107
It is a；
(3) the tumour growth situation at close observation nude inoculation position after being inoculated with, the 7th day after inoculation, inoculation position occurs
White tubercle, can be in subcutaneous activity, with tumor tissue growth after touch, and inoculation position gradually forms hard tumor mass, and about 14 days swollen
Tumor tissue average external volume reaches 100mm3, and BALB/c nude mice is divided at random to four groups, every group 6, starts the weight of animals when administration
For 16-18g；
(4) transplantable tumor volume was measured and recorded every two days, calculation formula is such as gross tumor volume (Tumor volume, TV)
Under:
Gross tumor volume=0.5 × a × b^2
Wherein, a is transplantable tumor length, and b is transplantable tumor width.
The result is shown in Figure 18, in SCC4 Transplanted tumor model, Cetuximab group (20mgkg-1) gross tumor volume obviously subtract
It is few, have extremely significant sex differernce (P < 0.001)；In SCC4/CTX Transplanted tumor model, Cetuximab group (20mgkg-1)
Apparent reduced tumor growth is not generated under Cetuximab sustained drug pressure, tumor growth curve is close with feminine gender group.This
Show that SCC4/CTX cell transplantation tumor model generates drug-resistant phenotype to Cetuximab, SCC4/CTX cell has western appropriate in vivo
Former times monoclonal antibody drug resistance.
Embodiment 18
The detection of lnc RP11-499F3.2 expression after SCC4 and SCC4/CTX cell in-vivo tumour is administered；
In SCC4 and SCC4/CTX cell body after Transplanted tumor model administration, cuts open tumor tissue and carry out qRT-PCR monitoring, it is real
The result is shown in Figure 19 is tested, in Cetuximab drug pressure (20mgkg-1) under, the lnc RP11- of SCC4 cell transplantation tumor tissue
499F3.2 expression shows as being remarkably decreased (P < 0.05), and the expression of SCC4/CTX cell line does not generate obvious change
Change, expression lnc RP11-499F3.2 still has extremely significant sex differernce (P < 0.01) compared with SCC4 cell.lnc RP11-
499F3.2 expression is positively correlated with Cetuximab drug-resistant phenotype, may be the reverse drug resistant target of head and neck scale carcinoma Cetuximab
Point molecule.
Embodiment 19
Establish the tumour dynamic growth situation of cetuximab medicaments insensitive PDX Transplanted tumor model
(1) head and neck scale carcinoma flesh tissue sample is directly obtained by postoperative, is put into sterile vessel after sample is in vitro rapidly, is transported
To aseptic operating platform；
(2) fresh tumor tissue uses the physiological saline rapid cleanup containing antibiotic, is immediately transferred into DMEM containing antibiotic
The 10cm Tissue Culture Dish of culture medium cuts off necrotic tumor tissue, fibr tissue and adipose tissue in tumor, cleans again；
(3) residual activity tumor tissues are cut into the tumor tissue of 2 × 2 × 2mm3 size.Take 2-3 BALB/c naked
Mouse, 75% ethanol disinfection forelimb carry on the back skin of abdomen, and it is broken to cut 2mm skin in mouse forelimb underarm region for isoflurane anesthetized mice
Mouthful, tumor tissue is filled into No. 18 canula puncture needles, and puncture is subcutaneous to mouse forelimb shoulder back, forms subcutaneous 10mm long and migrates
Sinus pushes canula puncture needle needle core that tumor tissue is made to be inoculated in shoulder back position subcutaneous, at every mouse inoculation two, every sample
2-3 mouse of this inoculation (determines) that sample is no more than 4h, head and neck scale carcinoma to tumor tissues migration process in vitro depending on tumor sample amount
PDX Transplanted tumor model is named as G1 generation；
(4) 150mm is grown to PDX Transplanted tumor model mean tumour volume in each experimental group3, and identical algebra PDX mould
Type mouse quantity meets grouping and requires (no less than 8), starts internal Cetuximab administration, verifies PDX parental generation model mice
To the sensibility of Cetuximab drug；
(5) each experimental group PDX model mice is divided at random to two groups, and every group of 4 mouse, group setting is respectively that G1 feminine gender is right
According to (physiological saline), G2 Cetuximab (20mgkg-1), dosage period is 21 days.
As a result see Figure 20, it is that drug is quick that nude mouse tumor volume, which is reduced after drug-treated more than 50% PDX definition,
Sense group, it is drug resistance group that gross tumor volume, which increases above 35% definition, after drug-treated, remaining definition is drug substance stable
Group.2 medicaments insensitive group PDX models are obtained altogether, and the present invention chooses the PDX model (C2 group, Patients With Oral Squamous Cell Carcinoma) of medium sensitivity
As the parental generation model for establishing Cetuximab drug resistance head and neck scale carcinoma PDX Transplanted tumor model, vivo medicine-feeding experiment is carried out.
Embodiment 20
Head and neck scale carcinoma cetuximab drug resistance PDX Transplanted tumor model tumour dynamic growth situation
After the head and neck scale carcinoma medicaments insensitive PDX transplantable tumor of screening is passed on, to tumor volume growth to 150mm3, into
Row Cetuximab vivo medicine-feeding, the generation of induced drug sensitivity PDX Transplanted tumor model drug-resistant phenotype.It is single by three-wheel western appropriate former times
There is significant drug-resistant phenotype in anti-vivo medicine-feeding and tumor tissues passage, C2 group PDX model, and gross tumor volume is in Cetuximab drug
It still generates and rises appreciably under pressure.As a result see that Figure 21, Cetuximab sensitivity oral squamous cell carcinomas PDX model (OSCC) are single in western appropriate former times
After anti-administration, gross tumor volume is generated compared with negative control group (physiological saline group) and is remarkably decreased, and the multiple body of Cetuximab
The oral squamous cell carcinomas PDX model (OSCC-CR) that interior administration induction generates, negative control group tumour growth volume is compared with medicaments insensitive mould
Type (OSCC) is significantly higher (P < 0.01), occurs apparent tumour growth, gross tumor volume under Cetuximab drug pressure
35% is increased above, significant drug resistance phenotype is showed, meets the definition of drug resistance group, that is, it is western appropriate to be successfully established head and neck scale carcinoma
Former times monoclonal antibody drug resistance PDX Transplanted tumor model.
Embodiment 21
Lnc RP11-499F3.2 expression in cetuximab sensitivity and the HNSCC-PDX Model Tumor tissue of tolerance
Detection
After cetuximab sensitivity and the HNSCC-PDX model vivo medicine-feeding of tolerance, cuts open tumor tissue and carry out qRT-PCR
Monitoring, experimental result is shown in Figure 22, the lnc RP11-499F3.2 of Cetuximab drug resistance OSCC-CR PDX model expresses water
It is flat to have extremely significant sex differernce (P < 0.01) compared with medicaments insensitive model.Meanwhile under Cetuximab drug pressure, medicaments insensitive
The lnc RP11-499F3.2 relative expression of the tumor tissues of model shows as being remarkably decreased (P < 0.05), and OSCC-CR model
The expression of tumor tissues does not generate significant change, and lnc RP11-499F3.2 expression is still more compared with medicaments insensitive model
Height, and there is extremely significant sex differernce (P < 0.01).The above results and lnc RP11-499F3.2 expression in SCC4/CTX
Unanimously (embodiment 18) proves that lnc RP11-499F3.2 expression and Cetuximab are resistance to again by clinical tumor tissue
Pharmacological property is positively correlated, while verifying Cetuximab tolerance head and neck scale carcinoma PDX model foundation success in the present invention.
Embodiment 22
The drug resistant HNSCC PDX model of cetuximab gives the tumour dynamic after lnc RP11-499F3.2 targeted therapy
Growth monitoring
To verify lnc RP11-499F3.2 in vivo in forming head and neck scale carcinoma Cetuximab drug resistance PDX Transplanted tumor model
Effect, the present invention design targeting lnc RP11-499F3.2 LNA carry out intratumor injection.
(1) 4-6 week old, weight are ordered at female BAl BIc/c nude mice totally 20 of 14-16g, in SPF grades of animal feeding rooms
It adaptive feeding 1 week, recovers and is inoculated with OSCC-CR Cetuximab drug resistance oral squamous cell carcinomas model tissue block, close observation nude mice
The growth of transplanted human situation of inoculation position；
(2) when tumor tissues average external volume reaches 150mm3, BALB/c nude mice is divided at random to four groups, every group 5, group
Setting is respectively G1 negative control group (0.2ml/20g), G2 Cetuximab group (20mgkg-1), G3LNA group (5 mg
Kg-1), G4 Cetuximab+LNA group (Cetuximab: 20mgkg-1；LNA:5mgkg-1)；
(3) dosage period is 21 days, observes PDX model mice one week after dosage period, measures and records PDX model
Tumor volume change；
(4) mouse is put to death in dislocation after testing, and removes tumor mass, is calculated gross tumor volume and is taken pictures, each PDX model group is swollen
Tumor tissue block, which keeps sample, to carry out liquid nitrogen flash freezer and freezes.
As a result see that Figure 23, C2 group Cetuximab are resistant to the physiological saline group of oral squamous cell carcinomas PDX model (OSCC-CR), west
Appropriate former times monoclonal antibody group, LNA group, Cetuximab group+LNA group dosage period after, gross tumor volume be respectively (1959.08 ±
79.09)mm3、(832.08±92.08)mm3、(419.03±73.38)mm3And (97.05 ± 35.04) mm3(see Figure 24).
OSCC-CR model gross tumor volume under Cetuximab pressure increases above 35%, shows significant drug resistance phenotype, it was demonstrated that
OSCC-CR model has Cetuximab drug resistance；Individually applying LNA is remarkably decreased tumour growth volume compared with control group
(P < 0.001), it was demonstrated that lnc RP11-499F3.2 is lowered in LNA targeting can inhibit Cetuximab resistant models tumour growth；West
After appropriate former times monoclonal antibody group+LNA group dosage period, there is negative growth in gross tumor volume, i.e., OSCC-CR model is in Cetuximab medicine
Under object pressure, while targeting lnc RP11-499F3.2 expression in downward tumour can make script drug resistance symptom Phenotype transformation significant
Medication effect, this show LNA targeting lower lnc RP11-499F3.2 can make the drug resistant neck squama of Cetuximab
Cancer is multiple quick.
Embodiment 23
The drug resistant HNSCC PDX model of cetuximab gives the main organs after lnc RP11-499F3.2 targeted therapy
HE coloration result
After the drug resistant HNSCC PDX model of cetuximab gives lnc RP11-499F3.2 targeted therapy, modulus type
Rat heart, liver, spleen, lung, renal tissue, 10% formalin are fixed, are sliced after paraffin embedding, HE dyeing observation histopathology
Situation.Each experimental group tissue of head and neck scale carcinoma OSCC-CR PDX model lnc RP11-499F3.2 targeted therapy is collected, HE is carried out
Dyeing, observes each tissue pathologic condition.
As a result see that Figure 25, each group heart, liver,spleen,kidney internal organs do not occur obvious pathological symptom, G3 Cetuximab is given
There is lung's infiltration in medicine group, this shows that the group has metastases phenomenon, the i.e. drug resistant PDX transplanting of head and neck scale carcinoma Cetuximab
Occurs a degree of lung's transfer during tumor model foundation.
Embodiment 24
Lnc RP11-499F3.2 is to colon cancer parent and mdr cell HT29 (CTX-R) for targeting interference, and esophageal squamous cell carcinoma is thin
The influence of born of the same parents TE13 (CTX-R) and non-small cell lung cancer A549 (CTX-R) proliferative capacity
HT-29, TE13 and A549 drug-resistant cell strain are established using the method for cetuximab concentration gradient, specific method is shown in
Embodiment 13, using mtt assay detection lnc RP11-499F3.2 lock nucleic acid respectively to HT29, TE13 and A549 sensitivity and drug resistance
The influence of the proliferative capacity of cell, specific method are shown in embodiment 9.
It the results are shown in Table 4, compared to control group, the lnc RP11-499F3.2 that the present invention designs locks nucleic acid independent role energy
The proliferative capacity of HT-29, TE13 and A549 resistant cells significantly reduces, and proliferative capacity has extremely significant drop after combining cetuximab
It is low, illustrate that targeting specific reduces the expression of lnc RP11-499F3.2, Cetuximab drug resistance colon cancer, esophageal squamous cell can be made
Cancer and non-small cell lung cancer are multiple quick.
Influence of the targeting interference of the table 4 lnc RP11-499F3.2 to mdr cell
Test is independently repeated 3 times, and the result tested is indicated with mean ± SD, and is carried out statistics T and examined, * P < 0.05
For significant difference, * * P < 0.01 is extremely significant sex differernce.
Claims (10)
1. a kind of long-chain non-coding RP11-499F3.2 is treated in head and neck cancer clinical detection and reversing tumor Cetuximab drug resistance
In application.
2. a kind of long-chain non-coding RP11-499F3.2 according to claim 1, it is characterised in that the long-chain non-coding
RNA sequence is as shown in SEQ ID NO.1 in sequence table.
3. application according to claim 1, it is characterised in that the head and neck cancer is including oral squamous cell carcinomas, nasopharyngeal carcinoma
Head and neck squamous cell carcinoma.
4. application according to claim 1, which is characterized in that the clinical detection reagent for head and neck cancer is real-time
Quantitative PCR detecting reagent.
5. the application in reversing tumor Cetuximab drug resistance treatment according to claim 1, which is characterized in that pass through conjunction
At the expression of specificity lock nucleic acid (Locked Nucleic Acid, LNA) regulation lnc RP11-499F3.2.
6. the application in reversing tumor Cetuximab drug resistance treatment according to claim 1, which is characterized in that described
Tumour includes colon cancer, the cancer of the esophagus, non-small cell lung cancer.
7. the real time quantitative PCR detecting reagent kit according to claim 4 for applying a kind of clinical detection for head and neck cancer,
It is characterised in that it includes designing and synthesizing out specificity use according to the long-chain non-coding RNA that nucleotides sequence is classified as SEQ ID NO.1
In the detection primer of real-time quantitative PCR, primer sequence is as shown in SEQ ID NO.2 in sequence table and SEQ ID NO.3.
8. application according to claim 5, characterized in that it is SEQ ID NO.4 in sequence table that it, which locks nucleotide sequence,.
9. the method according to claim 7 for reversing the treatment of Cetuximab drug resistance, which is characterized in that the lock core is sweet
Sour synthesis mode is chemical synthesis, can be used as gene therapy medicament.
10. the administered in vivo of the lock nucleic acid of lnc RP11-499F3.2 according to claim 8 be in tumor it is in situ,
Tail vein is subcutaneously or intramuscularly injected.
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| CN202210931906.XA CN116397023A (en) | 2018-10-11 | 2018-10-11 | Application of long-chain non-coding RP11-499F3.2 in clinical detection of oral squamous cell carcinoma |
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| WO2017142484A1 (en) * | 2016-02-16 | 2017-08-24 | Agency For Science, Technology And Research | Epigenomic profiling reveals the somatic promoter landscape of primary gastric adenocarcinoma |
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