CN108295261A - The function and purposes of PHF14 - Google Patents

Abstract

The invention belongs to chemical biology research fields in life science, and in particular to the function and purposes of PHF14.The present invention is after extensive and in-depth study, it finds for the first time, PHF14 combines individually or with KIF4A and can be used as lung cancer therapy target spot, inhibits the expression of PHF14 and/or KIF4A that can significantly inhibit growth and proliferation, the deterioration for inhibiting lung carcinoma cell, the inhibition lung carcinoma cell one-tenth knurl ability of lung carcinoma cell.Therefore, the present invention provides strong scientific evidence from clinical patient sample levels, cell function level and molecular level to the pathogenesis of lung cancer and the clinical treatment of lung cancer.

Description

The function and purposes of PHF14

Technical field

The invention belongs to chemical biology research fields in life science, and in particular to the function and purposes of PHF14.

Background technology

PHD finger albumen is that one kind is widely present in eucaryote, is had in gene transcription regulation and disease occur The PHD finger albumen of important function.In research in recent years, PHD finger albumen is classified as to identify histone methyl Change in " reader " albuminoid of modification (Lan et al., 2007；Org et al.,2008；Shi et al.,2006； Wysocka et al., 2006), this kind of protein family plays an important role in terms of adjusting cell epigenetic modification.No With PHD finger albumen can specificity different " the histone mark " of identification, by adjust activity inherently or The transcriptional expression (Baker et al., 2008) of gene is adjusted by adjusting the activity of its interaction protein.It is more and more Studies have shown that when point mutation, missing occur for the PHD finger structures of many genes coding or when chromosome translocation, these are different The past generation for causing human diseases of frequentation, including：Tumour, baryencephalia, immune deficiency etc. (Musselman and Kutateladze, 2009), this just more highlights PHD finger albumen as " reader " of an epigenetic in disease Important function (Baker et al., 2008) played in sick occurrence and development process.The generation of cancer is considered for a long time It is science of heredity and the change process that epigenetics is combined, finally promotes occurrence and development (the Jones and of cancer Baylin,2007).The relevant mutation of cancer and mistake adjusting are found to appear in various relevant with histone posttranslational modification In enzyme (Wang et al., 2007a, b).The result of study of our laboratories early periods finds that PHF14 can pass through its PHD1 It is combined with histone H 3 with two structural domains of PHD3, and self dimerization (Huang et al., 2013) can occur.From structure On, PHF14 contains multiple finger and nuclear localization sequence, thus, it is presumed that PHF14 is likely to as " histone a mark The reader of will ", by itself adjusting or by its adjusting to histone, the transcription table of gene is influenced in nucleus It reaches, and then influences the important cellular activities such as cell Proliferation, division, participate in tumour.At the same time, it has been found that many PHD finger albumen is reported not only influences the occurrence and development of tumour by epigenetic regulation, also participates in albumen drop Promote in various regulation and control such as solution, signal transduction and cell migration tumour generation (Akazawa et al., 2013； Bankovic et al.,2010；Chitalia et al.,2008；Kitagawa et al.,2012；Li et al., 2013；Zhou et al.,2005).Therefore, we conduct extensive research the PHF14 mechanism for participating in lung cancer generation.

In tumour correlation fatal disease worldwide, lung cancer is one of the most important cause of death (Jemal et al.,2011).Lung cancer is a worldwide public health problem, has had become since 1985 and sends out in the world Sick rate and the highest malignant tumour of the death rate, just having more than a million people died of the disease every year since 1993.Only 13% Lung cancer patient survive more than 5 years, the estimated number for infecting lung cancer still will gradually increase in several years of future.In all causes In the cause of the death, lung cancer ranking the tenth, in developing country because the increased reason of incidence will rise to the 5th.More and more grind Study carefully and tend to the screening of clinical sample level and study tumorigenic molecular marked compound, from molecular biology and science of heredity angle point Molecular mechanism of these important molecules in tumour generation is analysed, the expression of these genes is found from epidemiology angle and tumour is sent out The correlation of the processes such as raw, transfer, differentiation, to promote early screening and the detection of lung cancer.Due to the five year survival rate of lung cancer Extremely low, early stage of lung cancer detection, which can change disease outcome, seems even more important, however, current present situation is：The early stage of lung cancer is examined Section of cutting off the hands is either prohibitively expensive or sensitivity not enough (Henschke et al., 2006；Markowitz et al.,2007). On the other hand, currently, lung cancer therapy depends on the treatment mode of such as tumor grade and smoking history Clinical symptoms to passing through The characteristic on the molecular level of each case is analyzed so that it is determined that therapeutic scheme.Cause to improve therapeutic effect and reduce treatment Toxicity, researchers are dedicated to developing the individualized therapeutic scheme based on tumor cells characteristic.By finding on molecular level Biomarker come predicted treatment reaction and prognosis existence.Predictive marker object can be used to determine therapeutic scheme, and prognosis mark Note object can then predict independent for the treatment of mode treatment results (Belinsky et al., 1998；Gessner et al., 2004；Kettunen et al., 2004), the use of these molecular markers, which can become, overcomes chemotherapeutic bottleneck and drop The important means of toxicity caused by low chemotherapy.Some developed molecular targeted drug and are shown in treatment of cancer Effect, however to these drugs can have treatment reflection patient's ratio it is also extremely limited, further develop tumor cells target It is extremely urgent to drug.It is evident that：The screening of molecular labeling will be in the prevention of lung cancer, detection, clinical diagnosis and treatment It plays an increasingly important role in the process.It is found in the research of last decade, the abnormal sudden change of many genes may take part in The generation of lung cancer：The mutability of oncogene activates, such as：KRas, EGFR and MYC；The inactivation of tumor suppressor gene, such as：P53, p16 and RB；And a large amount of recurrent chromosome mutation (Sekido et al., 2003).In addition to gene alteration genetically, apparent something lost The abnormal sudden change caught may also cause tumour, these mutation may cause some in the generation, development of tumour with important Effect gene mutation activate or inhibit, to cause tumour (Esteller, 2008；Kwon and Shin,2011). Up to the present, a large amount of research is dedicated to relationship (the Shames et that the methylation state of lung cancer patient sample occurs with lung cancer al.,2006；Vaissiere et al., 2009), and it was found that many genes, such as APC (Virmani et al., 2001) and CDH13 (Toyooka et al., 2001), is reported in primary non-small cell lung cancer (non-small-cell lung Carcinomas, NSCLCs) in supermethylation has occurred.

Invention content

In order to overcome the problems of in the prior art, the work(that the purpose of the present invention is to provide PHF14 in lung cancer Energy and purposes.

To achieve the goals above and other related purposes, the present invention adopt the following technical scheme that：

The first aspect of the present invention provides the purposes that PHF14 inhibitor is used to prepare lung cancer therapy drug.

The lung cancer therapy drug at least has one of following function：The growth and increasing of lung carcinoma cell can be significantly inhibited The deterioration of lung carcinoma cell is grown, inhibited, inhibits lung carcinoma cell one-tenth knurl ability, destroys lung carcinoma cell mitosis process, extends lung cancer The cell cycle of cell.

The mitosis process of destroying includes but not limited to destroy chromosome condensation, influence the dynamic change of micro-pipe in turn Influence form and positioning, the influence cytokinesis of microtubule spindle.

The cell cycle for extending lung carcinoma cell includes but not limited to the M phases for extending the cell cycle.

Preferably, the PHF14 inhibitor refers to the molecule for having inhibition for PHF14.

Include but not limited to inhibition for PHF14：Inhibit PHF14 activity, or inhibits PHF14 genetic transcriptions Or expression.

The PHF14 inhibitor can be siRNA, shRNA, antibody, micromolecular compound.

As the embodiment of the present invention is enumerated, the PHF14 inhibitor can be siRNA, sequence such as SEQ ID NO.2 or Shown in SEQ ID NO.3.

The lung cancer therapy drug includes necessarily PHF14 inhibitor, and using PHF14 inhibitor as the effective of aforementioned function Ingredient.

In the lung cancer therapy drug, the active ingredient for playing aforementioned function can be only PHF14 inhibitor, also may include it He can play the molecule of similar function.

The lung cancer therapy drug can be single composition substance, also can be multi-component compound.

The form of the lung cancer therapy drug can be that solid, liquid, gel, semi-fluid, aerosol etc. are each without specifically limited Kind material form.

The targeted lung cancer of the lung cancer therapy drug can be non-small cell lung cancer.

The Remedies for lung cancer object mainly for object be mammal, such as such as rodent, primate.

The second aspect of the present invention provides a kind of method for treating lung cancer, to apply PHF14 inhibitor to object.

The object is the lung carcinoma cell of mammal or the mammal.The mammal is preferably Rodentia Animal, artiodactylous animals, Perissodactyla animal, Lagomorph, primate etc..The primate is preferably monkey, ape Or homo sapiens.The lung carcinoma cell can be in vitro lung carcinoma cell, including but not limited to A549, H2126, CRL-5803, CRL-5807, CRL-5810, CRL-5844, CRL-5872, CRL-5883, CRL5889, CRL-5908, CRL5928.

The object can suffer from the patient of lung cancer or the individual of Waiting treatment lung cancer or the object for lung cancer to suffer from The in vitro lung carcinoma cell of the individual of person or Waiting treatment lung cancer.

The PHF14 inhibitor can be applied before, during and after receiving lung cancer therapy to object.

The third aspect of the present invention, provides a kind of lung cancer therapy drug, including a effective amount of PHF14 inhibitor and medicinal Carrier.

The fourth aspect of the present invention provides a kind of lung cancer combination therapy pharmaceutical composition, including a effective amount of PHF14 inhibits Agent and other at least one lung cancer therapy drugs.

Other described lung cancer therapy drugs refer to the lung cancer therapy drug other than PHF14 inhibitor.

The combination therapy pharmaceutical composition can be any one in following form：

One) independent preparation is respectively prepared in PHF14 inhibitor and other lung cancer therapy drugs, the dosage form of preparation can be identical Or it is different, administration route also may be the same or different.

When other lung cancer therapy drugs are anti-tumour antibody, parenteral type is generally used.When other lung cancer therapies When drug is chemotherapeutics, form of medication can be relatively abundant, can be gastrointestinal administration can also be parenteral administration.One As recommend for each chemotherapeutics known administration route be administered.

Two) PHF14 inhibitor and other lung cancer therapy drugs are configured to compound preparation.By PHF14 inhibitor and its When his lung cancer therapy drug is simultaneously applied simultaneously using the administration of identical administration route, the shape that the two is configured to compound preparation can be used Formula.

Fifth aspect present invention provides a kind of lung cancer therapy method, to inhibit to object using a effective amount of PHF14 Agent, and apply other a effective amount of lung cancer therapy drugs to object and/or implement other lung cancer therapy means to object.

A effective amount of PHF14 inhibitor can concurrently or sequentially be given and other at least one a effective amount of lung cancer are controlled Treat drug.

Be the newfound for the first time lung cancer therapy target spot of the present invention based on PHF14, with PHF14 inhibitor other than other lungs In cancer medicine drug combination, the effect of curative effect addition can be at least played, the inhibition for lung cancer is further enhanced.

Other described lung cancer therapy drugs include but not limited to：Anti-tumour antibody, chemotherapeutics or target medicinal etc..

The PHF14 inhibitor can be gastrointestinal administration or parenteral.Other described lung cancer therapy drugs can To be gastrointestinal administration or parenteral.For anti-tumour antibody or chemotherapeutics, parenteral is generally used.

The sixth aspect of the present invention, provides PHF14 inhibitor and KIF4A inhibitor combines and is provided commonly for preparing lung cancer and controls Treat the purposes of drug.

The lung cancer therapy drug at least has one of following function：The growth and increasing of lung carcinoma cell can be significantly inhibited The deterioration of lung carcinoma cell is grown, inhibited, inhibits lung carcinoma cell one-tenth knurl ability, destroys lung carcinoma cell mitosis process, extends lung cancer The cell cycle of cell.

The mitosis process of destroying includes but not limited to destroy chromosome condensation, influence the dynamic change of micro-pipe in turn Influence form and positioning, the influence cytokinesis of microtubule spindle.

The cell cycle for extending lung carcinoma cell includes but not limited to the M phases for extending the cell cycle.

Preferably, the PHF14 inhibitor refers to the molecule for having inhibition for PHF14；The KIF4A inhibitor It refer to the molecule that there is inhibition for KIF4A.

Include but not limited to inhibition for PHF14：Inhibit PHF14 activity, or inhibits PHF14 genetic transcriptions Or expression.Include but not limited to inhibition for KIF4A：Inhibit KIF4A activity, or inhibits KIF4A genetic transcriptions Or expression.

The PHF14 inhibitor can be siRNA, shRNA, antibody, micromolecular compound.The KIF4A inhibitor can Think siRNA, shRNA, antibody, micromolecular compound.

As the embodiment of the present invention is enumerated, the PHF14 inhibitor can be siRNA, sequence such as SEQ ID NO.2 or Shown in SEQ ID NO.3.The KIF4A inhibitor can be its sequence of siRNA such as SEQ ID NO.5 or SEQ ID NO.6 institutes Show.

The lung cancer therapy drug includes necessarily PHF14 inhibitor and KIF4A inhibitor, and with PHF14 inhibitor and Active ingredient of the KIF4A inhibitor as aforementioned function.

In the lung cancer therapy drug, the active ingredient for playing aforementioned function can be only PHF14 inhibitor and KIF4A inhibits Agent also may include that other can play the molecule of similar function.

The lung cancer therapy drug can be bi-component substances, also can be multi-component compound.

The form of the lung cancer therapy drug can be that solid, liquid, gel, semi-fluid, aerosol etc. are each without specifically limited Kind material form.

The targeted lung cancer of the lung cancer therapy drug can be non-small cell lung cancer.

The Remedies for lung cancer object mainly for object be mammal, such as such as rodent, primate.

The seventh aspect of the present invention provides a kind of method for treating lung cancer, for object apply PHF14 inhibitor and KIF4A inhibitor.

The object is the lung carcinoma cell of mammal or the mammal.The mammal is preferably Rodentia Animal, artiodactylous animals, Perissodactyla animal, Lagomorph, primate etc..The primate is preferably monkey, ape Or homo sapiens.The lung carcinoma cell can be in vitro lung carcinoma cell, including but not limited to A549, H2126, CRL-5803, CRL-5807, CRL-5810, CRL-5844, CRL-5872, CRL-5883, CRL5889, CRL-5908, CRL5928.

The object can suffer from the patient of lung cancer or the individual of Waiting treatment lung cancer or the object for lung cancer to suffer from The in vitro lung carcinoma cell of the individual of person or Waiting treatment lung cancer.

The PHF14 inhibitor and KIF4A inhibitor can be applied before, during and after receiving lung cancer therapy to object.

The eighth aspect of the present invention provides a kind of lung cancer therapy drug, including a effective amount of PHF14 inhibitor and KIF4A Inhibitor and pharmaceutical carrier.

The ninth aspect of the present invention provides a kind of lung cancer combination therapy pharmaceutical composition, including a effective amount of PHF14 inhibitor With KIF4A inhibitor and other at least one lung cancer therapy drugs.

Other described lung cancer therapy drugs refer to the Remedies for lung cancer other than PHF14 inhibitor and KIF4A inhibitor Object.

The combination therapy pharmaceutical composition can be any one in following form：

One) independent preparation is respectively prepared in PHF14 inhibitor and KIF4A inhibitor and other Remedies for lung cancer objects, made The dosage form of agent may be the same or different, and administration route also may be the same or different.

When other lung cancer therapy drugs are anti-tumour antibody, parenteral type is generally used.When other lung cancer therapies When drug is chemotherapeutics, form of medication can be relatively abundant, can be gastrointestinal administration can also be parenteral administration.One As recommend for each chemotherapeutics known administration route be administered.

Two) PHF14 inhibitor and KIF4A inhibitor and other Remedies for lung cancer objects are configured to compound preparation.It is inciting somebody to action PHF14 inhibitor and KIF4A inhibitor and other Remedies for lung cancer objects, can using the administration of identical administration route and when applying simultaneously Using the form that the two is configured to compound preparation.

Tenth aspect present invention provides a kind of lung cancer therapy method, to apply a effective amount of PHF14 inhibitor to object With KIF4A inhibitor, and applies other a effective amount of lung cancer therapy drugs to object and/or implement other lung cancer to object and control Treatment means.

A effective amount of PHF14 inhibitor and KIF4A inhibitor and at least one can concurrently or sequentially be given effectively Other lung cancer therapy drugs of amount.

Be the newfound for the first time joint lung cancer therapy target spot of the present invention based on PHF14 and KIF4A, with PHF14 inhibitor In other lung cancer therapy combination therapies other than KIF4A inhibitor, the effect of curative effect addition can be at least played, into one Inhibition of the step enhancing for lung cancer.

The eleventh aspect of the present invention provides：PHF14 is used to screen the purposes of lung cancer therapy drug.

Preferably, the lung cancer is non-small cell lung cancer.

Preferably, PHF14 for screen lung cancer therapy drug specifically refer to PHF14 as drug or preparation and be directed to lung cancer it is thin The action target of born of the same parents is applied to screening lung cancer therapy drug or preparation.

Further, PHF14 is applied to screening lung cancer therapy as drug or preparation for the action target of lung carcinoma cell Drug or preparation specifically refer to：Drug or preparation are screened using PHF14 as suppression target, to find, to reduce lung cancer thin The PHF14 inhibitor of the expression of intracellular PHF14, as lung cancer therapy drug candidate.

It is listed in some embodiments of the present invention by the target that PHF14 genes are RNA interference effects, has screened energy Enough significantly inhibit the siRNA of PHF14 gene expressions, the results showed that the siRNA can obviously weaken the proliferation energy of lung carcinoma cell Power, inhibits lung carcinoma cell one-tenth knurl ability at the deterioration for inhibiting lung carcinoma cell, can be used as with the medicine for inhibiting proliferation of lung cancer cells effect Object.In addition to this, such as antibody drug, small-molecule drug etc. also can be using PHF14 genes and its albumen as effective object.

Further, the twelveth aspect of the present invention, provide PHF14 and KIF4A combine be provided commonly for screening lung cancer control Treat the purposes of drug.

Preferably, the lung cancer is non-small cell lung cancer.

Preferably, PHF14 and KIF4A combine be provided commonly for screening lung cancer therapy drug specifically refer to PHF14 and KIF4A connection It closes and is applied to screening lung cancer therapy drug or preparation for the action target of lung carcinoma cell collectively as drug or preparation.

Further, PHF14 and KIF4A combines the action target application that lung carcinoma cell is directed to collectively as drug or preparation It is specifically referred in screening lung cancer therapy drug or preparation：PHF14 and KIF4A are combined collectively as suppression target, to drug or Preparation is screened, to find the inhibition that can simultaneously or separately reduce the expression of PHF14 and KIF4A in lung carcinoma cell Agent, as lung cancer therapy drug candidate.

It is RNA interference effects with PHF14 genes and KIF4A genes to be listed in some embodiments of the present invention respectively Target has screened the different siRNA that can significantly inhibit PHF14 genes and KIF4A gene expressions respectively as inhibitor, knot Fruit shows that the inhibitor can obviously weaken the proliferative capacity of lung carcinoma cell, the deterioration for inhibiting lung carcinoma cell, inhibit lung cancer thin Born of the same parents' one-tenth knurl ability can be used as with the drug for inhibiting proliferation of lung cancer cells effect.In addition to this, such as antibody drug, small molecule Drug etc. also can be using PHF14 genes and its albumen as effective object.

The thirteenth aspect of the present invention provides：PHF14 is used to prepare or screens the purposes of pulmonary cancer diagnosis reagent.

Preferably, it is non-small cell lung cancer that the lung cancer, which is the non-small lung cancer,.

Preferably, PHF14 is used to prepare or screens pulmonary cancer diagnosis reagent, including both sides content：

First, PHF14 is used to prepare pulmonary cancer diagnosis reagent, refer to examining as lung cancer using PHF14 genes or expression product Severed finger mark is applied to the preparation of pulmonary cancer diagnosis reagent.For example, can be right as standard items or the positive using PHF14 genes or expression product According to the detection for lung cancer.

Second, PHF14 is for screening pulmonary cancer diagnosis reagent, refer to using PHF14 genes or expression product as the knowledge of lung cancer The reagent of other target sieving specific recognition PHF14 genes or expression product, to be used as pulmonary cancer diagnosis reagent, to detect lung Cancer.

The fourteenth aspect of the present invention provides：PHF14 and KIF4A, which combines, is provided commonly for preparing or screen pulmonary cancer diagnosis examination The purposes of agent.

Preferably, the lung cancer is non-small cell lung cancer.

Preferably, PHF14 and KIF4A, which combines, is provided commonly for preparing or screens pulmonary cancer diagnosis reagent, including in both sides Hold：

First, PHF14 and KIF4A, which combine, is provided commonly for preparing pulmonary cancer diagnosis reagent, refer to by PHF14 genes or expression production Object and KIF4A genes or expression product combine the preparation for being applied to pulmonary cancer diagnosis reagent collectively as the diagnosis index of lung cancer.Example Such as, lung cancer can be used for using PHF14 genes or expression product and KIF4A genes or expression product as standard items or positive control Detection.

Second, PHF14 and KIF4A, which combine, is provided commonly for screening pulmonary cancer diagnosis reagent, refer to by PHF14 genes or expression production Object and KIF4A genes or expression product combine the identification target sieving simultaneously or separately specific recognition collectively as lung cancer The reagent of PHF14 genes or expression product and KIF4A genes or expression product, to be used as pulmonary cancer diagnosis reagent, to detect lung Cancer.

The present invention lung cancer therapy drug or pulmonary cancer diagnosis drug include but not limited to：Nucleic acid molecules, carbohydrate, Lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or interference slow virus.

The nucleic acid includes but not limited to：NcRNA, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozyme, ribonucleic acid SiRNA (esiRNA) or short hairpin RNA (shRNA) prepared by restriction endonuclease III.

The fifteenth aspect of the present invention provides：PHF14 inhibitor is used to prepare lung carcinoma cell mitosis and upsets agent Purposes.

Agent is upset in the mitosis at least has one of following function：It destroys lung carcinoma cell mitosis process, extend The cell cycle of lung carcinoma cell.

The mitosis process of destroying includes but not limited to destroy chromosome condensation, influence the dynamic change of micro-pipe in turn Influence form and positioning, the influence cytokinesis of microtubule spindle.

The cell cycle for extending lung carcinoma cell includes but not limited to the M phases for extending the cell cycle.

The sixteenth aspect of the present invention provides：PHF14 inhibitor KIF4A inhibitor, which is combined, to be provided commonly for preparing lung cancer Cell mitogen upsets the purposes of agent.

Agent is upset in the mitosis at least has one of following function：It destroys lung carcinoma cell mitosis process, extend The cell cycle of lung carcinoma cell.

The mitosis process of destroying includes but not limited to destroy chromosome condensation, influence the dynamic change of micro-pipe in turn Influence form and positioning, the influence cytokinesis of microtubule spindle.

The cell cycle for extending lung carcinoma cell includes but not limited to the M phases for extending the cell cycle.

Compared with prior art, the present invention has the advantages that：

The present invention after extensive and in-depth study, has found for the first time, and PHF14 combines individually or with KIF4A and can be used as lung cancer Therapy target, inhibit PHF14 and/or KIF4A expression can significantly inhibit lung carcinoma cell growth and proliferation, inhibition lung cancer it is thin The deterioration of born of the same parents inhibits lung carcinoma cell one-tenth knurl ability.Therefore, the present invention from clinical patient sample levels, cell function level and divides Sub- level provides strong scientific evidence to the pathogenesis of lung cancer and the clinical treatment of lung cancer.

Description of the drawings

Figure 1A：PHF14 expressions in Non-Small Cell Lung Carcinoma (24).

Figure 1B：PHF14 immunohistochemical stainings are imaged in different cancerous lung tissues and cancer beside organism's chip, abbreviation：ADC, Adenocarcinoma (gland cancer)；SCC, Squamous Cell Carcinoma (squamous carcinoma)；LCC, Large Cell Carcinoma (large cell carcinoma)；Normal, normal structure；× 8, scale 1.0mm.；× 400, scale is 100 μm.

Fig. 1 C：PHF14 mrna expressions in adenocarcinoma of lung patient's sample.

Fig. 1 D：PHF14 expresses the correlation with gene copy number.

Fig. 1 E：Based on NCBI Geo Database (GSE3141, GSE19188) and Cancer Genome Atlas (TCGA) the PHF14 expressions of patient's sample carry out KM-plot survival analysis in.

Fig. 2A：It transiently transfects siRNA and knocks out PHF14 expression inhibiting A549 cell Proliferations.

Fig. 2 B：It transiently transfects siRNA and knocks out PHF14 expression inhibiting CRL-5810 cell Proliferations.

Fig. 2 C：Transfection PHF14 rescue plasmids can save part and be proliferated slack-off phenotype, left figure：Pass through the side of MTT Method detection control and the proliferation for transfecting cell after PHF14 siRNA, middle figure：Pass through in the cell of PHF14 siRNA transfections BrdU mixes the proliferation of method detection cell, for the statistical result of independent experiment three times, right figure：Immune-blotting method PHF14 The expression of albumen after transient transfection siRNA, β-actin are used as internal reference, data to be indicated with independent experiment average value ± SD three times, * P <0.05,**P<0.01。

Fig. 3 A：Inhibit PHF14 to express vicious transformation capable of inhibiting cell and its one-tenth knurl ability in Mice Body, stablizes The A549 cell strains of knockdown PHF14 are proliferated slack-off, left figure：Control cell strain and stabilization are detected by the method for MTT The proliferation of the A549 cell strains of knockdown PHF14, middle figure：Method, which is mixed, by BrdU detects control cell strain and stabilization The proliferation of the A549 cell strains of knockdown PHF14, for the statistical result of independent experiment three times, right figure：Immunoblotting is examined The expression of albumen after stablizing knockdown PHF14 is surveyed, β-actin are used as internal reference.

Fig. 3 B：Colony formation experiment in soft agar, cell observe the growth feelings of cell after being grown in soft agar 15 days Condition, the cell mass more than or equal to 50 cells are considered as the colony to be formed, left figure：The generation of cell growing state in soft agar Table photo, right figure：To the statistical analysis of growth ability of the shown cell in soft agar.Data average value ± SD tables Show, * * P<0.01.

Fig. 3 C：Tumor formation is tested in nude mouse, left figure：The cell as shown in the figure of equivalent amount is subcutaneously injected into 4 week old hero Property mouse omoplate at, the volume for the tumour that observation per week generates, the calculating of gross tumor volume uses following formula：Volume=1/ 2 (maximum gauge) x (minimum diameter) 2, every group of 4 mouse, data average value ± SD expressions, * * P<0.01, right figure：A549 pairs Photo cell and PHF14 stablize knockdown cells tumor formation situation representativeness picture in nude mouse, * P<0.05, * * P<0.01.

Fig. 4 A：The interaction of PHF14 and KIF4A, fast protein liquid chromatography experiment, PHF14, KIF4A, PARP-1 It is enriched in Ku70 in identical several components.

Fig. 4 B：Co-immunoprecipitation detects endogenous PHF14 and KIF4A and interacts, left figure：In the full lysate of A549 cells It is middle to immunoprecipitate PHF14, immune-blotting method PHF14 or KIF4A with preimmune serum or PHF14 antiserums, add in lysate Enter the combination of EB closings DNA, right figure：It is precipitated with preimmune serum or anti-KIF4A sero-immunities in A549 cell pyrolysis liquids The combination that EB closings DNA is mediated is added in lysate by KIF4A, immune-blotting method KIFA4 and PHF14.

Fig. 4 C：The measurement of PHF14 and KIF4A binding sites, upper figure：Co-immunoprecipitation GFP-KIF4A WT or deletion mutation Body and PHF14, plasmid shown in 293T cell transfectings prepare the full cell pyrolysis liquids of 293T, with the anti-blood of anti-PHF14 after 48 hours Clear or preimmune serum NS immunoprecipitates PHF14, and anti-PHF14, the antibody of anti-GFP is then used to carry out immunoblotting, under Figure：KIF4A mutant schematic diagrames, in conjunction with affinity from being classified as to weak by force：* *, by force；*, it is medium；*, weak.

Fig. 4 D：The measurement of PHF14 and KIF4A binding sites, upper figure：Co-immunoprecipitation PHF14 WT or deletion mutant and GFP-KIF4A, figure below：PHF14 mutant schematic diagrames.

Fig. 4 E：Immunofluorescence test endogenous PHF14 and KIF4A A549 cells common location, it is fixed after cell climbing sheet, Immunofluorescence test is done with anti-PHF14 (green) and anti-KIF4A (red), DAPI (blue) dyeing shows nucleus, Scale is 5 μm.

Fig. 5 A：PHF14 and KIF4A common height in lung cancer cell line and lung cancer sample, which is expressed and cooperateed with, promotes cell to increase, Expression of the PHF14 and KIF4A in non-small cell lung cancer clinical sample, left figure：Immunoblot results show PHF14 and KIF4A Common high expression, β-actin are used as internal reference, right figure in non-small cell lung cancer clinical sample：Correlation analysis result is shown The high expression of PHF14 and KIF4A is significantly correlated in non-small cell lung cancer clinical sample.

Fig. 5 B：QPCR shows PHF14 and KIF4A mRNA level in-sites in non-small cell lung cancer sample.

Fig. 5 C：PHF14 and KIF4A is in the expression of Lines, left figure：Immunoblot results are shown PHF14 and KIF4A common high expression, β-actin in Lines are used as internal reference, right figure：Correlation analysis As a result show that the high expression of PHF14 and KIF4A is significantly correlated in Lines.

Fig. 5 D：Knockdown PHF14 and/or transfection siRNA knock out KIF4A expression inhibiting A549 cell Proliferations, left figure： Stablize the proliferation of A549 cell strains after knockdown PHF14 and/or instantaneous knockout KIF4A by the method detection of MTT, it is right Figure：Immune-blotting method stablizes the expression of albumen after knockdown PHF14 and/or instantaneous knockout KIF4A, β-actin conducts Internal reference.

Fig. 5 E：It is overexpressed the total length expressed promotion HCT-116 cell Proliferations of PHF14 or KIF4A, but PHF14 NT 1-160aa (s) deletion mutant but cannot, left figure：Pass through method detection control and overexpression PHF14 WT of MTT, KIF4A and PHF14 The proliferation of HCT-116 cell strains, right figure after NT 1-160aa (s) deletion mutants：Immune-blotting method PHF14 WT, KIF4A With PHF14 NT 1-160aa (s) deletion mutant heterogenous expression efficiency, β-actin are used as internal reference.

Fig. 6 A：PHF14 and/or KIF4A missings lead to Mitotic abnomality and influence mitotic index, PHF14 missings Lead to mitotic cell micro-pipe and the form and abnormal distribution of chromosome, after turning siRNA in wink in Hela cells, immunofluorescence α-tubulin and chromosome are detected, is fixed after cell climbing sheet, is dyed with anti-α-tubulin (green) and show micro-pipe, DAPI (blue) dyeing shows nucleus.

Fig. 6 B：Stabilization checking PHF14 leads to Mitotic abnomality, left figure in A549 cell strains：It is fixed after cell climbing sheet, It is dyed with anti-α-tubulin (green) and shows micro-pipe, DAPI (blue) dyeing shows nucleus, right figure：WB detections are stablized The expression of albumen after knockdown PHF14, β-actin are used as internal reference.

Fig. 6 C：Living imaging experimental record HelaGFP-H₂When clockwise siRNA inhibits PHF14 and/or KIF4A in B cell The influence of cell cycle.

Fig. 6 D：Inhibit to significantly affect mitotic index, mitotic index calculation formula when PHF14 and/or KIF4A For：Average value ± variance, * * * P<0.0001, n for analysis number of cells, turn in wink in HeLa cells siRNA inhibit PHF14 and/ Or KIF4A, using 20 times of object lens high throughput shootings of high intension Image Acquisition analysis system 0peratta, every group of bat after 48 hours 9 visuals field (6,000 to 8,000 cell) are taken the photograph, mitotic cell shoots identification by the channel of 405 wavelength, and WB detects PHF14 With the knockout efficiency of KIF4A albumen, β-actin are used as internal reference.

Specific implementation mode

The present invention has found under study for action, and PHF14 combines individually or with KIF4A and can be used as lung cancer therapy target spot, inhibits PHF14 And/or the expression of KIF4A can significantly inhibit growth and proliferation, the deterioration for inhibiting lung carcinoma cell, the inhibition lung cancer of lung carcinoma cell Cell one-tenth knurl ability.

PHF14 inhibitor

Refer to the molecule that there is inhibition for PHF14.Include but not limited to inhibition for PHF14：Inhibit PHF14 activity, or inhibit PHF14 genetic transcriptions or expression.The PHF14 inhibitor include but not limited to siRNA, shRNA, Antibody, micromolecular compound.

It is that PHF14 vigor is instigated to decline to inhibit PHF14 activity.Preferably, it compares before inhibiting, PHF14 vigor declines at least 10%, at least 30%, then good reduction at least 50% are preferably reduced, more preferably reduces at least 70%, best reduction is at least 90%.

Inhibition PHF14 genetic transcriptions or expression refer to：So that the gene of PHF14 is not transcribed, or reduces turn of the gene of PHF14 Record activity, or the gene of PHF14 is made not express, or reduce the expression activity of the gene of PHF14.

Those skilled in the art can be adjusted the genetic transcription or expression of PHF14 using conventional method, such as gene It knocks out, homologous recombination, RNA interfering etc..

The inhibition of genetic transcription or the expression of PHF14 can detect expression quantity verification by PCR and Western Blot.

Preferably, compared with wild type, PHF14 genetic transcriptions or expression reduce at least 10%, preferably reduce at least 30%, then good reduction at least 50%, more preferably reduce at least 70%, and good reduction at least 90%, most preferably PHF14 genes It does not express completely.

KIF4A inhibitor

Refer to the molecule that there is inhibition for KIF4A.Include but not limited to inhibition for KIF4A：Inhibit KIF4A activity, or inhibit KIF4A genetic transcriptions or expression.The KIF4A inhibitor include but not limited to siRNA, shRNA, Antibody, micromolecular compound.

It is that KIF4A vigor is instigated to decline to inhibit KIF4A activity.Preferably, it compares before inhibiting, KIF4A vigor declines at least 10%, at least 30%, then good reduction at least 50% are preferably reduced, more preferably reduces at least 70%, best reduction is at least 90%.

Inhibition KIF4A genetic transcriptions or expression refer to：So that the gene of KIF4A is not transcribed, or reduces turn of the gene of KIF4A Record activity, or the gene of KIF4A is made not express, or reduce the expression activity of the gene of KIF4A.

Those skilled in the art can be adjusted the genetic transcription or expression of KIF4A using conventional method, such as gene It knocks out, homologous recombination, RNA interfering etc..

The inhibition of genetic transcription or the expression of PHF14 can detect expression quantity verification by PCR and Western Blot.

Preferably, compared with wild type, KIF4A genetic transcriptions or expression reduce at least 10%, preferably reduce at least 30%, then good reduction at least 50%, more preferably reduce at least 70%, and good reduction at least 90%, most preferably PHF14 genes It does not express completely.

Micromolecular compound

Middle finger of the present invention is made of several or tens atoms, and molecular mass is in 1000 compounds below.

PHF14 inhibitor (and KIF4A inhibitor) prepares drug

It is that one of main active or main active prepare drug with PHF14 inhibitor (and KIF4A inhibitor). In general, in drug other than active ingredient, according to the needs of different dosage forms, will also include one or more pharmaceutically acceptable Carrier or auxiliary material.

" pharmaceutically acceptable " refers to when biomolecule ontology and composition suitably give animal or people, they will not be produced Raw unfavorable, allergy or other adverse reactions.

" pharmaceutically acceptable carrier or auxiliary material " should be with PHF14 inhibitor (and KIF4A inhibitor) compatible, Ji Nengyu Its effect being blended without pharmaceutical composition is greatly lowered in general.Can be used as pharmaceutically acceptable carrier or The specific example of some substances of auxiliary material is carbohydrate, such as lactose, dextrose and saccharose；Starch, such as cornstarch and potato starch； Cellulose and its derivates, such as sodium carboxymethylcellulose pyce, ethyl cellulose and methylcellulose；Tragacanth powder；Malt；It is bright Glue；Talcum；Kollag, such as stearic acid and magnesium stearate；Calcium sulfate；Vegetable oil, such as peanut oil, cottonseed oil, sesame oil, olive Olive oil, corn oil and cupu oil；Polyalcohol, such as the third two liquor-saturated, glycerine, D-sorbite, mannitol and polyethylene glycol；Alginic acid； Emulsifier, such as Tween；Wetting agent, such as NaLS；Colorant；Flavoring agent；Tablet agent, stabilizer；Antioxidant；It is anti- Rotten agent；Apirogen water；Isotonic salting liquid；With phosphate buffer etc..These substances are used to help the stabilization of formula as needed Property or help to improve activity or it biological effectiveness or acceptable mouthfeel or smell are generated in the case of oral.

In the present invention, unless stated otherwise, pharmaceutical dosage form is not particularly limited, and can be made into injection, oral solution, piece The dosage forms such as agent, capsule, dripping pill, spray can be prepared by conventional method.The selection of pharmaceutical dosage form should be with administering mode phase Match.

Combination therapy pharmaceutical composition and method of administration

The combination therapy pharmaceutical composition can be any one in following form：

One) independent preparation is respectively prepared in PHF14 inhibitor (and KIF4A inhibitor) and other antitumor drugs, made The dosage form of agent may be the same or different, and administration route also may be the same or different.In use, can several medicines use simultaneously, also can be several Medicine successively uses.It, should be formerly with drug still to applying other drugs to body in body effective period when consecutive administration.

Two) PHF14 inhibitor (and KIF4A inhibitor) and other antitumor drugs will be configured to compound preparation.It is inciting somebody to action PHF14 inhibitor (and KIF4A inhibitor) and other antitumor drugs, can using the administration of identical administration route and when applying simultaneously Using the form that the two is configured to compound preparation.

The common administrated method of antibody is intravenous injection, intravenous drip or arterial perfusion.Its usage and dosage can refer to existing Technology.

The common administrated method of micromolecular compound can be gastrointestinal administration either parenteral.siRNA、 ShRNA, antibody then generally use parenteral.Can be local administration can also be Formulations for systemic administration.

It is can synchronizing or sequentially give a effective amount of PHF14 inhibitor (and KIF4A inhibitor) and it is a effective amount of its His lung-cancer medicament.In use, can two (three) plant medicine and use simultaneously, also can the successively uses of two kinds of (three) medicines.It, should when consecutive administration Formerly with drug still to applying other drugs to organism in organism effective period.

Chemotherapeutics include alkylating agent (such as Nimustine, Carmustine, lomustine, cyclophosphamide, ifosfamide and Glyciphosphoramide etc.), antimetabolite (such as deoxygenate fluorine guanosine, more west not bird pyridine, fluorouracil, mercaptopurine, methotrexate (MTX) nucleotide Analog), antitumor antibiotics (such as actinomycin D, adriamycin and daunorubicin antibiotic), antitumor animals and plants component drugs (such as vinorelbine, taxol, harringtonine, Irinotecan, taxotere and vincaleukoblastinum), antitumor hormone drug (such as Ah he Mei Tan, Anastrozole, aminoglutethimide, Letrozole, formestane and tamoxifen etc.) and such as cis-platinum, Dacarbazine, Ao Shali Platinum, Le Satin, can the Common Chemotherapies medicine such as platinum is difficult to understand husky, mitoxantrone and procarbazine.

Target medicinal includes that EGFR blocking agents such as Gefitinib (Gefitinib, Iressa and Iressa) and angstrom sieve replace Buddhist nun (Erlotinib, Tarceva), specific cells marker monoclonal antibody such as Cetuximab (Cetuximab, Erbitux) and anti-HER-2 monoclonal antibodies (Trastuzumab, Trastuzumab, Herceptin), tyrosine kinase receptor inhibitor as gram Azoles for Buddhist nun (Crizotinib, Xalkori), anti-tumor angiogenesis drug such as Bevacizumab, endostatin and Bevacizumab etc., Bcr-Abl tyrosine kinase inhibitors such as Imatinib and Dasatinib, anti-CD20 monoclonal antibody are such as Rituximab, IGFR-1 kinase inhibitor such as NVP-AEW541, mTOR kinase inhibitor such as CCI-779, Ubiquitin-proteasome Inhibitor such as Bortezomib etc..

Other oncotherapy means can be selected from operation excision, RF ablation, argon helium superconduction operative treatment, laser ablation are controlled It treats, high intensity focused ultrasound and radiotherapy include one or more in X- knives, R- knives, 3D-CRT and IMRT.

PHF14 is for screening lung cancer therapy drug, being used to prepare or screen tumour diagnostic reagent

PHF14 can be used as drug or preparation and be applied to screening lung cancer therapy drug or system for the action target of lung carcinoma cell Agent, specifically：Drug or preparation can be screened using PHF14 as suppression target, can be reduced in lung carcinoma cell with finding The PHF14 inhibitor of the expression of PHF14, as lung cancer therapy drug candidate.

PHF14 can be used for preparing pulmonary cancer diagnosis reagent, specifically, the examining as lung cancer using PHF14 genes or expression product Severed finger mark is applied to the preparation of pulmonary cancer diagnosis reagent.For example, can be right as standard items or the positive using PHF14 genes or expression product According to the detection for lung cancer.

PHF14 can be used for screening pulmonary cancer diagnosis reagent, specifically, using PHF14 genes or expression product as the knowledge of lung cancer The reagent of other target sieving specific recognition PHF14 genes or expression product, to be used as pulmonary cancer diagnosis reagent, to detect lung Cancer.

PHF14 and KIF4A, which combines, to be provided commonly for screening screening lung cancer therapy drug, combines to be provided commonly for preparing or screen and swell Tumor diagnostic reagent

PHF14 and KIF4A can combine is applied to screening collectively as drug or preparation for the action target of lung carcinoma cell PHF14 and KIF4A are specifically combined collectively as suppression target, are carried out to drug or preparation by lung cancer therapy drug or preparation Screening, to find the inhibitor that can simultaneously or separately reduce the expression of PHF14 and KIF4A in lung carcinoma cell, as lung Cancer treats drug candidate.

PHF14 and KIF4A, which combines, is provided commonly for preparing pulmonary cancer diagnosis reagent, specifically, by PHF14 genes or expression product And KIF4A genes or expression product combine the preparation for being applied to pulmonary cancer diagnosis reagent collectively as the diagnosis index of lung cancer.For example, It can be used for the inspection of lung cancer using PHF14 genes or expression product and KIF4A genes or expression product as standard items or positive control It surveys.

PHF14 and KIF4A, which combines, is provided commonly for screening pulmonary cancer diagnosis reagent, specifically, by PHF14 genes or expression product And KIF4A genes or expression product combine the identification target sieving simultaneously or separately specific recognition PHF14 collectively as lung cancer The reagent of gene or expression product and KIF4A genes or expression product, to be used as pulmonary cancer diagnosis reagent, to detect lung cancer.

PHF14 inhibitor (and KIF4A inhibitor) is used to prepare lung carcinoma cell mitosis and upsets agent

Agent is upset in the mitosis at least has one of following function：It destroys lung carcinoma cell mitosis process, extend The cell cycle of lung carcinoma cell.The mitosis process of destroying includes but not limited to destroy chromosome condensation, influence micro-pipe Dynamic change and then form and positioning, the influence cytokinesis for influencing microtubule spindle.

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The test method of actual conditions is not specified in the following example, Usually according to normal condition, or according to the condition proposed by each manufacturer.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Embodiment 1

Embodiment 1, experiment material and experimental method

One, experiment material

1.1 bacterial strain：Bacillus coli DH 5 alpha：Structure and amplification of the bacterial strain for plasmid.

1.2 mammalian cell strain

(1)Hfl-1：Human embryonic lung fibroblasts derive from normal male fetus lungs, and form is fibroblast sample, Normal diploid structure, adhere-wall culture and passage, as normal lung cancer cell control group.

(2) people source lung cancer cell line：A549, H2126, CRL-5803, CRL-5807, CRL-5810, CRL-5844, CRL- 5872, CRL-5883, CRL5889, CRL-5908, CRL5928.

(3)HCT-116：Human colon cancer cell derives from adult male colon cancer, and form is epithelioid cell, adherent training It supports and passage, Tumor formation is strong, in the cell line, loss of expression in PHF14 (Ivanov et al., 2007), thus subsequent reality It tests middle selection cell strain and carries out functional experiment as natural PHF14 deletion cells models.

1.3 mouse types

Tumor-bearing mice strain is used by this experiment：Nude mouse BALB/cAnN-malm- (BALBlc Nude).

Origin：It is handed over by BALB/cABOIl-nu and BALB/cAnNCrj-nu in Charles River Japan (CRJ) Match and backcrossing obtains.Pregnancy BALB/CArlNCrj-nu female mices with stringent family record were in the CRJ that induces one in 1985, and same Year is bred.This mouse is inbreeding, and genetic monitoring is the result shows that be BALB/c nude mices.Hair color：It is hairless, albefaction.

Characteristics and uses：Retarded growth, reproductive capacity is low, and severe infections easily occur.Athymism, for the 11st pair of dye Colour solid recessive inheritance.Due to athymia, only thymus gland vestiges or Abnormal epithelial, T cell normal differentiation cannot be made, therefore T cell is exempted from Epidemic disease defect.Bone-marrow-derived lymphocyte is normal, but functional defect, and antibody is mainly 1gM, only a small amount of 1gG, contactless sensibility, no transplanting Rejection, therefore can be widely used for the research of rabbit epidemiology, oncology and disease genesis mechanism.

1.4 clinical lung cancer samples

All for the cancer sample tissue tested includes clear-cell carcinoma, liver cancer and non-small cell lung cancer sample standard deviation from patient It is obtained in the cancerous tissue cut off after operation.

All samples and clinical information acquisition are through patient or family members' informed consent, all sample collections, preservation and operation Meet relevant law and Ethics Committee's regulation.

The postoperative quilt of cancer and cancer beside organism detaches and quick-frozen in liquid nitrogen at once.Hereafter -80 DEG C of refrigerators are placed on to preserve.To carry The RNA and albumen sample, group for taking cancer and cancer beside organism are woven in liquid nitrogen environment and are smashed with mortar.About equal homogenous quantities The cancerous tissue of (50-100mg) and cancer beside organism are used to extraction RNA and albumen sample.

1.5 plasmid

(1) pRK5-Rs-PHF14WT-myc and pRKs-RS-GFP-PHF14 Α WT：The people source full length coding regions PHF14 α CDNA is from people source Hela cells (Huang et al., 2013).

(2) PHF14 deletion mutants：Using EcoRI/XbalI and XbalI/SalI restriction enzyme sites by its full-length cDNA and Deletion mutant is cloned into pRK5-Rs-3 ' myc and pRK5-Rs-5 ' GFP zero loads, PHF14 WT (full-length):1- 948aa；PHF14 Delta 1-160:161-948aa；PHF14 Delta 1-310:311-948aa；PHF14 Delta 1- 410:411-948aa；PHF14 Delta 1-500:501-948aa.

(3) pEGFP-C1-KIF4AWT and pGEX-5X1-KIF4A858-1232aa (s) uses BglII and SalI digestions Its deletion mutant is cloned into pEGFP-C1 zero loads, MS (motor and Stalk domain) by site:1-1018aa；ST (Stalk and tail domain):335-1232aa；M(motor domain):1-335aa；S(Stalk domain): 335-1018aa；T(tail domain):912-1232aa.

(4)pSIREN-RetroQ-NC/297i/1958：Respectively by Negative control siRNA of synthesis, The oligonucleotide sequence of PHF14siRNA and PHF14 RNAi plasmids is cloned into RNAi-ReadypSIREN-RetroQ carriers (Clontech,USA)。

(5)pcL10A1：Reverse transcriptase packaging virus carrier, is arrived with pSIREN-RetroQ-NC/297i/1958i corotation After HEK293T cells, prepared by the packaging that is efficiently easy to get for recombinant virus.

It is determined via DNA sequencing after the completion of all plasmid constructions.

1.6 antibody

Anti-KIF4A：The immunogene of mouse source antibody is GST-fused KIF4A WT 858-1232aa (s).

Commercial antibodies：anti-Ku70(Santa Cruz,sc-5309)；anti-KIF4A(Bethyl Laboratories, USA, Cat.#A301-074A).

1.7 oligonucleotide chains

Negative control siRNA, the PHF14siRNA that synthesized by Shanghai Ying Jun Bioisystech Co., Ltd and The oligonucleotide sequence of PHF14RNAi plasmids is：

NC:UUCUCCGAACGUGUCACGU(SEQ ID NO.1)

PHF14-297i:GGAAAAGAAGGAAGAAGAA(SEQ ID NO.2)

PHF14-1958i:AGCUUCAUGUAGAAUAUAA(SEQ ID NO.3)

By the oligonucleotide sequence of Negative control siRNA of Shanghai Ji agate biosynthesis, KIF4AsiRNA For：

NC:UUCUCCGAACGUGUCACGU(SEQ ID NO.4)

KIF4A-i-1:GCAAGAUCCUGAAAGAGAUTT(SEQ ID NO.5)

KIF4A-i-2:GCAGAUUGAAAGCCUAGAGTT(SEQ ID NO.6)

Two, experimental method

2.1 Biochemistry Experiment

1) extraction of animal and tissue full RNA and albumen

Tumor tissues RNA used is all extracted using Trizol (invitrogen, USA).Operating process in strict accordance with Trizol reagent operation instructions.Specific test procedure is referring to Trizol operation instructions.

After patient's sample tissue collecting, lysate with high salt (1%NP-40,0.5M NaCl, 10%sucrose, 40mM is added Tris-HCl pH 7.5 and protease inhibitor cocktail), it is ground using Multi-example tissue grinder, 65Hz, 1-5min, centrifugation, 13,000rpm, 4 DEG C, 30 minutes, take supernatant use BCA standard measures, be added sample-loading buffer sample preparation, 100 DEG C Heating 10 minutes, loading 50g protein electrophoresis.

2) lung cancer sample tissue immunohistochemistry scores

The scoring of lung cancer sample tissue immunohistochemistry is evaluated using double blind.It is divided into level Four according to positive cell percentage：1.1- 10% positive cell；2.11-50% positive cells；3.51-75% positive cells；4.>75% positive cell.Staining power is from low It is supreme to be divided into：1. negative；2. weakly positive；3. positive；4. strong positive.Appraisal result is integrated using immune response (immunoreactive score, IRS) standard.Immune response scoring=staining power (staining intensity, SI) × positive percentage (postivity percentage, PP), i.e. IRS=S I × PP.From down to high score be 4 grades：1-4, No positive dyeing；5-8, weakly positive dyeing；9-12, positive staining；13-16, strong positive dyeing.Patients clinical sample data is in disease It records, collects under people and family members' informed consent.

3) vitro binding assay (In vitro binding assay)

First determine amount that bacterial supernatant contains albumen with the method for small scale purification albumen, with determine test each time in albumen Dosage.Cell pyrolysis liquid is first non-to remove some in conjunction with 2 hours with 10 μ l Glutathione Sepharose 4B beads The albumen of specific binding.10 μ l Glutathione Sepharose 4B are added to the bacterial lysate containing gst fusion protein Beads, 4 DEG C be incubated 3-5 hour after with 1 × HTNG wash beads, centrifugation, 5,000rpm, 4 DEG C, 1 minute.Add into each sample Enter the cell pyrolysis liquid containing 0.5-1 milligrams of total proteins；4 DEG C are incubated 3-5 hours or stay overnight；1 × HTNG is washed 3 times.Precipitation loading Buffer solution elutes loading.

4) fast protein liquid chromatography purifying (Fast Protein Liquid Chromatography, FPLC)

After the 2ml nuclei lysis buffer cracking of sub- separating obtained Hela nuclear components：

A) pillar is rinsed：About 2 hours (column volumes of pillar are rinsed with ddH2O：1ml；Flow velocity：About 1ml/min；Temperature：16 ℃)。

B) pillar is balanced：It is balanced with balance buffer (50mM Tris-HCl pH8.0,0.3M NaCl, 1mM EDTA) Pillar (HiLoad 16/60Superdex 200) is steady to baseline.

C) loading：2ml Hela nuclear components sample inspiration syringes are turned down into bubble with sample loop loading, from into Sample valve is disposably pushed into sample introduction.

D) it collects：Peak will be penetrated as possible with buffer solution and wash back baseline.It is collected since appearance, is a component per 200l, Until peak vanishes, collect 344 each components, wherein last 10 groups are divided into the component more washed, sample is placed in -80 DEG C after acquisition altogether Refrigerator preserves, or for detecting.

E) pillar is rinsed and is preserved：After collection, pillar is washed with ddH2O, is finally preserved with 20% ethyl alcohol.

5) the point marking (Dot Blotting)

By sample spot on cellulose acetate film, after sample is completely dried and is adsorbed onto on film, after the completion with 1 × NET- Gelatine is closed 1 hour, after the nonspecific binding site for eliminating antibody, the next same immunoblotting of operating procedure, and one Anti-, secondary antibody is incubated, ECL substrate reactions develop.

6) Mass Spectrometric Identification

2.2 Cell Biology Experiment

1) culture of cell

Experiment mammalian cell used is cultivated in 37 DEG C of incubators of 5%CO2 and has the vapor of saturation.Institute Some cell culture work is carried out in sterile super-clean bench.293T, Hela and Hela GFP-H2B cells are cultivated with DMEM, are owned Lung cancer cell line is cultivated with RPMI Medium-1640.

2) foundation of PHF14 RNAi stable cell lines

First, according to the siRNA knockdown turned in wink as a result, the good segment of selection RNAi effects, according to RNAi- Ready pSIREN-RetroQ plasmid construction methods, by the annealing oligonucleotide of synthesis, and are connected into empty carrier.Structure is identified in digestion Build PHF14 RNAi plasmids.

PcL10A1 plasmids and the HEK293T of pSIREN-RetroQ-NC/297i/1958i corotation to 60% degree of converging is thin Born of the same parents receive supernatant after 48 hours, 4 DEG C of preservations separately fill 5ml cultures and continue to cultivate, then receive supernatant after 24 hours, and supernatant closes twice And add HEPES and poly-brene and 0.22 μm of filter filtering.Packing, -80 DEG C of preservations.

2ml virus liquids are added to the 6 orifice plates for being covered with A549 cells, 2500rpm, 30 DEG C centrifuge 30 minutes, remove virus liquid, 37 DEG C of cultures, after 24 hours plus 500ng/ml puromycin are screened.Control cell is all dead two days later.It is positive after screening Cell continues to cultivate pool cell of the part as stable transfected cells, another 1000 cells for stablizing expression PHF14 RNAi Kind waits for that its forms collection and falls behind picking monoclonal cell in 10cm culture dishes.The PHF14 expression quantity of each cell and pool cells It is detected by the method for immunoblotting, chooses the good clone's conservation of RNAi effects, amplification, carries out follow-up function experiment.

3) cell proliferation experiment

MTT experiment and BrdU incorporation experiments are used for measuring the proliferation of cell.

MTT experiment：It is planted after cell transfecting in 96 orifice plate kinds, each 1000-5000 cell of hole kind waits for that cell is adherent Afterwards, one of which cell is taken, the MTT of 20l5mg/ml is added in culture solution, is cultivated 4 hours, culture solution is removed, 100 μ l are added DMSO places 30 minutes lysigenous purple crystals in dark, is inhaled using light of the spectrophotometer measurement at wavelength 570nm It receives, every group of three holes repeat, and measure primary, Therapy lasted 5-7 days every 24 hours later.The result of three repeated experiments is united Count credit analysis.

BrdU incorporation experiments：The cell of transient transfection carries out BrdU incorporation experiments after 48 hours to be transfected.Specific experiment is grasped Make according to BrdU incorporation experiment reagent box specifications (Roche, Pensberg, Germany).Summary record is such as Under：It waits for that cell density grows to 50-70%, final concentration of 10 μM of BrdU is added in culture solution and continues culture 30 minutes, Gu Determine cell, the nucleus of Brdu positive cells and all cells uses BrdU antibody and DAPI to dye respectively.Record BrdU and DAPI Positive percentage.Each cell at least counts 1000.The result of three repeated experiments carries out statistical analysis.

4) soft agarose generates experiment

Agar is dissolved in deionized water, 0.6% and 1.2% solution, high-temperature sterilization, 4 DEG C of preservations are made into.Test it Before, melt soft agar with micro-wave oven, is placed in 40 DEG C of water-baths.1.2% soft agar and 40 DEG C of 2 × DMEM equal proportions preheated is mixed It closes, and the fetal calf serum of 1/10 volume is added, take 2ml to be uniformly layered in 6 hole culture dishes after mixing, be stored at room temperature 15 minutes and wait for fine jade Fat solidifies.It is for use that the 6 orifice plates completed can be placed on 4 DEG C of refrigerators.0.6% soft agar and 40 DEG C of 2 × DMEM equal proportions is mixed It closes, and the fetal calf serum of 1/10 volume is added, the cell detected needed for the concentration inoculation after mixing by every milliliter of 2500 cells, It takes 2ml to be uniformly layered in the 6 orifice plates of bottom-layer agar, is stored at room temperature and waits within 15 minutes upper layer gel condensation.It is put into 37 DEG C of incubators Middle culture.Culture counts the colony number that cell number is more than 50 under the microscope after two weeks or so.

5) tumor formation is tested in Mice Body

Each cell is cultivated in culture dish to about 90% degree of converging.Pancreatin is digested and is resuspended in incomplete culture solution. The right omoplate that cell suspension is seeded in nude mice with 1ml syringes is subcutaneous.Each cells per mouse injection 4 × 10⁶A cell.Often Every the maximum gauge and minimum diameter of the tumour that one week is formed with vernier caliper measurement.The gross tumor volume of formation is counted as follows It calculates：Volume=1/2 × (maximum gauge) × (minimum diameter)².Each cell is at least inoculated with 4 mouse.The body of the tumour of formation Long-pending average value ± SD (standard variance) indicates.

6) HeLa living imagings record (Live-cell imaging)

Each group Hela GFP-H2B stable cell strains are transfected with siRNA, then, are copolymerized with living cells high-rate laser burnt and complete Internal reflection multidimensional image work station is shot, 3-5 visual field of every group of selection, and shooting in every 5 minutes is primary, and it is small to shoot 8-12 altogether When, synthesize continuous image file.

7) cell division index measures (Mitotic index)

After Hela cells are laid on 24 orifice plates, after siRNA transfections processing 48 hours, fixed, DAPI dyeing is used The Operetta high intensions cell analysis system of PerkinElmer companies is by turntable co-focusing imaging mode, in 24 orifice plates Cell carry out full-automatic high-throughput light field and fluorescent microscopic imaging, the channels DAPI complete opening shoot and utilize powerful Harmony softwares quickly carry out fluorescence intensity to micro-image, cellular morphology carries out analysis and data statistics processing.Cell mitogenic Splitting index=mitotic cell number/total cell number × 100%.

Three, data statistics and analysis

1) data statistics

The result of all immunoblottings with Quantity One softwares (Bio-Rad laboratories Inc., Hercules, CA) carry out quantitative analysis.All data using Excel 2003 (Microsoft Corp., Redmond, Wash.) software statistics are analyzed.Statistical data is indicated using average value ± SD (standard variance).The student t- of pairing, double tail Test analyzes to count the significant difference between two groups.P<0.05 is considered significant difference；P<0.01 is considered having pole Its significant difference.

2) clinical case analysis

GEO Database (GSE19188) derive from NCBI, with PHF14 (reporter:229085_at) it is used as probe The expression of non-matching tumor tissues and cancer beside organism is detected and Welch's correction are analyzed. Correlation Analysis and Kaplan-Meier plot of survival analysis use SPSS 16.0software (SPSS Inc., 1989-2007) software is analyzed.Fisher's Exact Test according tohttp:// www.langsrud.com/fisher.htm。

The detection of embodiment 2, the PHF14 expression in tumor sample

In order to detect the relationship of PHF14 and cancer, we have collected five type clinical tumor samples and its corresponding cancer Other sample (kidney, gastric cancer, colon cancer, liver cancer and non-small cell lung cancer).It was found that PHF14 either genes in lung cancer The expression of PHF14 all significantly raises (Figure 1A-D) on copy number, mRNA or protein level.WB and ImmunohistochemistryResults Results prompt are super The cancerous lung tissue sample for crossing 80% or more shows the high expression level of PHF14 albumen with respect to cancer beside organism.QPCR is also shown 71% patient P HF14 has in mRNA level in-site to be increased.For PHF14 expressions and clinical data correlation analysis (table 1), show Show its height expression (score>12) related to gland cancer, and (TNM I or diameter of tumor≤3cm) is just presented in the early stage of lung cancer Apparent high expression (score >=9).The relevant microarray dataset of lung cancer that we have further been delivered by searching, Bioinformatics calculating is carried out, analyzes expressions of the PHF14 in more lung cancer specimens, and its related to clinical indices Property, universal laws of the searching PHF14 in Expressions in Lung Cancer.The results show that PHF14 is in multiple numbers

It is presented in lung cancer sample according to (Cancer Genome Atlas, GSE74095and GSE74116) in library high The rule of expression, and the life span of its expression and patient are inversely proportional (Fig. 1 E).

Table 1.PHF14 lung cancer clinical sample immunohistochemistry scores and clinical and pathological data correlation analysis

Embodiment 3, downward PHF14 expression can significantly inhibit proliferation of lung cancer cells

Based on the discovery of tumour patient clinical sample level, we choose people source lung cancer cell line and carry out function assessment experiment. Pass through the expression of PHF14 in two kinds of different people source lung carcinoma cells of method knockdown A549 and CRL-5810 of RNAi, observation The influence to lung cancer proliferation and tumor formation is lowered in PHF14 expression.MTT results significantly suppress after showing PHF14 knockdown The growth of A549 and CRL-5810 lung carcinoma cells and proliferation (Fig. 2A, 2B)；BrdU incorporations experiment (speed of detection DNA synthesis) Being also demonstrated that, which reduces PHF14 expression, can inhibit the DNA of cell to synthesize and then inhibit the proliferation of cell.In addition, we build The PHF14 expression plasmids of RNAi resistant carry out the slack-off table of cell growth after rescue PHF14 knockdown

As a result type is shown：The slack-off phenomenon of seen cell Proliferation (figure can be saved by expressing rescue plasmids 2C).The result shows that：Proliferation of lung cancer cells can be significantly inhibited by lowering PHF14 expression.

Embodiment 4, lower PHF14 expression significantly affect proliferation of lung cancer cells, one-tenth knurl ability in vicious transformation and Mice Body

We then have chosen 1958i this RNAi segment and are prepared for the A549 cell strains that PHF14 stablizes RNAi, foundation The A549 cell strains that PHF14 stablizes knockdown also show that the slack-off phenomenon of cell Proliferation (Fig. 3 A).For detection PHF14's Whether expression is related to the vicious transformation of lung cancer, and We conducted the colony formation experiment in soft agar and tumor formation in nude mouse are real It tests.Stable cell line A549/NC and A549/Cln2, A549/Cln9 cell are planted in soft agar respectively, after two weeks A549/NC Cell has grown into the cell mass of multiple cell compositions in soft agar.And A549/Cln2 and A549/Cln9 cells are in soft agar It is interior to divide, or only split into the cell mass less than ten cells.The result is shown：The expression of PHF14 and lung carcinoma cell Adherent dependent/non-dependent growth is related, and then may be related to the vicious transformation of lung cancer (Fig. 3 B).In tumor formation in nude mice, PHF14 After expression is lowered, the nude mice one-tenth knurl ability of A549 inhibits (Fig. 3 C) by highly significant.The results show that it is aobvious to lower PHF14 expression One-tenth knurl ability in work influence proliferation of lung cancer cells, vicious transformation and Mice Body.

The interaction of embodiment 5, PHF14 and KIF4A

In order in depth study the mechanism of action of PHF14 biological functions, we combine SILAC (Stable Isotope Labeling with Amino Acids in Cell Cultures), co-immunoprecipitation and MALDI-TOF/TOF Mass Spectrometric Identifications Method, be found that many albumen that may be interacted with PHF14, albumen are concentrated mainly on DNA damage in HeLa cells Reparation, transcriptional regulatory, cell division and proliferation etc. play biological effect.Us are wherein caused to be concerned with chromatin horse Up to dynein KIF4A (also in lung cancer specificity overexpression).Using fast protein liquid chromatography (Fast protein Liquid chromatography, FPLC) post separation Hela nuclear components, the detection such as some markings, immunoblotting, mirror is used in combination Determine the possible interaction protein compounds of PHF14.As a result, it has been found that：In fast protein liquid chromatography experiment, PHF14, KIF4A, PARP-1 and Ku70 are enriched in identical several components, prompt these albumen that may be in a big egg together In white compound (Fig. 4 A).In A549 cells, immunoprecipitates PHF14 and be able to detect that KIF4A is got off by co-immunoprecipitation, It is added after EB excludes the protein binding that DNA is mediated and also obtains similarly as a result, reverse co-immunoprecipition result is (Fig. 4 B) as the same.Exempt from Epidemic disease fluorescence and laser co-focusing result are shown：PHF14 and KIF4A A549 cells interkinesis common location in nucleus, Division stage common location is on chromatin, and also common location is on middle body when the cytokinesis phase, and in the cell cycle, the two has good Common location (Fig. 4 E).Then, we think further clear KIF4A and PHF14 is by which respective structural domain phase interaction With.Pass through the structure and co-immunoprecipitation experiment of the mutant protein expression plasmid of PHF14 and KIF4A a series of, it has been found that KIF4A is to interact (Fig. 4 C＆D) by its region Stalk and PHF14N end 1-160aa (s).GST pull are combined in vitro Down detections also demonstrate that PHF14N end 1-160aa (s) are necessary to the combination of the two.In summary experimental result explanation： PHF14 is bound directly with KIF4A, and calmodulin binding domain CaM is the Stalk domain of PHF14 N-terminal 1-160aa (s) and KIF4A.

Ironically, the two common high expression and notable positive correlation in more plants of lung cancer cell lines and lung cancer sample (Fig. 5 A-C), this is embodied in protein level not only in mRNA level in-site.Inhibit PHF14 and/or inhibits KIF4A expression A549 cell Proliferations (Fig. 5 D) are all significantly inhibited, HCT-116 cell Proliferations can be promoted by being overexpressed PHF14 overall lengths, but is overexpressed not PHF14 NT 1-160aa (s) mutant combined with KIF4A cannot but promote cell Proliferation (Fig. 5 E), prompt PHF14 and KIF4A may play synergistic effect in promoting proliferation process.

KIF4A is a kind of chromatin dynein played an important roll in fission process.Document report, when thin Born of the same parents enter mitosis, and KIF4A missings can destroy chromosome condensation；It influences the dynamic change of micro-pipe and then influences microtubule spindle Form and positioning；Influence cytokinesis etc. (Bastos et al., 2014；Bastos et al.,2013；Kurasawa et al.,2004a；Lee et al.,2001；Mazumdar et al.,2004；Samejima et al.,2012；Shrestha et al.,2012；Singh et al.,2014；Stumpff et al.,2012；Takemoto et al.,2009；Wandke et al.,2012；Wu and Chen,2008；Zhu and Jiang,2005；Zhu et al.,2005).It is presumed that Whether PHF14 may be played a role by the interaction with KIF4A during mitosis.Our in the winks in Hela cells Turn the expression that siRNA lowers PHF14, immunofluorescence results are shown：After inhibiting PHF14 expression, mitotic cell micro-pipe and dye All there is abnormal (Fig. 6 A) in the form of colour solid and distribution.Class is can also be observed that in the cell strain of A549 stabilization checkings PHF14 As chromosome condensation, arrangement be abnormal, phenomenon (Fig. 6 B) of aneuploid and micro-pipe exception.Living imaging experimental record When lacking PHF14, cell is once enter the M phases, and mitosis is affected, and the M phases are extended, and the cell cycle is extended, and are lacking When KIF4A, it is seen that similar phenotype, when knocking out PHF14 and KIF4A simultaneously, the cell cycle is by notable retardation (figure 6C).Turn in wink siRNA inhibit PHF14 and KIF4A respectively or both inhibit simultaneously expression when, all have to mitotic index aobvious The influence of work prompts the extension (Fig. 6 D) for being likely to cause the cell cycle.Our research is found：RNAi PHF14 and RNAi The effect of KIF4A is much like, can all destroy the agglutination and arrangement of chromosome, influences form and the positioning of microtubule spindle, and Cause the extension of M phases.It lacks influences of the PHF14 for chromosome to become apparent from compared with the KIF4A effects lacked, and inhibits KIF4A tables Seem stronger compared with the effect of inhibition PHF14 up to for the effect of micro-pipe, while inhibiting the expression of the two, for cell mitogen Influence with the cell cycle is maximum.

Embodiment 6 is summarized and is discussed

One, it summarizes PHF14 and is combined the research for participating in lung cancer generation with KIF4A

1, PHF14 in kidney, colorectal cancer, gastric cancer and liver cancer expression quantity without significant changes；And in non-small cell lung cancer Significantly high expression in cancerous lung tissue sample more than 80% or more, height expression and gland cancer and lung cancer patient it is poor clinic it is pre- It is significantly correlated afterwards.

2, in cellular level, lower PHF14 expression can significantly inhibit in proliferation of lung cancer cells, vicious transformation and Mice Body at Tumor ability.

3, PHF14 and KIF4A are bound directly, and calmodulin binding domain CaM is the Stalk of PHF14N end 1-160aa (s) and KIF4A domain.In the cell cycle, the two has good common location.

4, PHF14 or KIF4A overall lengths are overexpressed, cell Proliferation can be remarkably promoted, inhibit PHF14 and/or KIF4A expression Cell Proliferation is all significantly inhibited, the combination of PHF14 and KIF4A may play an important role in proliferation process.

5, similar to KIF4A is lowered, the agglutination and arrangement of chromosome can be destroyed by lowering PHF14, influence microtubule spindle Form and positioning, and cause the extension of phase cell cycle M.The expression for inhibiting the two simultaneously, for cell mitogen and carefully The influence in born of the same parents' period is maximum.

6, the common height of PHF14 and KIF4A is expressed in non-small cell lung cancer clinical sample and cell line, the rising of expression quantity It is significantly correlated.

Two, discussion of results

Lung cancer is that the change by gene in genetic level and epigenetics level generation a series of complex causes lung Cell irreversible infinite multiplication in portion's causes.Our research is found that the biomarker-PHD that a new lung cancer occurs Finger protein 14s (PHF14).This nucleoprotein is in upper highly conserved and wide expression of evolving, the height in lung cancer patient sample It expresses and related to the clinical prognosis that patient is poor, proliferation of lung cancer cells, vicious transformation and one-tenth knurl ability is had a significant impact；It is right The cell activities such as cell Proliferation, mitosis and cell cycle have a significant impact.Our work discloses for the first time PHF14 and potential tumor markers-chromatin motor power albumen KIF4A is bound directly and common location, in pulmonary carcinosis Common high expression and significantly correlated both in people's sample, interaction depend on PHF14N end 1-160aa (s) with The combination of the combination of KIF4A Stalk domain, the two may play an important role in proliferation process.We Result prompt：PHF14 may be a new potential pulmonary cancer diagnosis marker, by participating in lung with KIF4A collective effects Carcinogenesis.It is expected that our work can from clinical patient sample levels, the morbidity that cell function is horizontal and molecular level is to lung cancer The clinical treatment of mechanism and lung cancer provides strong scientific evidence.

PHD finger albumen is that one kind is widely present in eucaryote, is had in gene transcription regulation and disease occur The zinc finger protein of important function.In research in recent years, PHD finger albumen is classified as to identify histone methylated modification " reader " albuminoid in (Lan et al., 2007；Org et al.,2008；Shi et al.,2006；Wysocka Et al., 2006), this kind of protein family plays an important role in terms of adjusting cell epigenetic modification.Different PHD Finger albumen energy specificity identifies different " histone mark ", by adjusting activity inherently or passing through adjusting The activity of its interaction protein adjusts the transcriptional expression (Baker et al., 2008) of gene.More and more research tables Bright, when point mutation, missing or chromosome translocation occur for the PHD finger structures of many genes coding, these often draw extremely The generation of human diseases is sent out, including：Tumour, baryencephalia, immune deficiency etc., this just more highlights PHD finger albumen " reader " as an epigenetic played in disease development process important function (Baker et al., 2008).The generation of cancer is considered as science of heredity and the change process that epigenetics is combined for a long time, is finally promoted The occurrence and development (Jones and Baylin, 2007) of cancer.The relevant mutation of cancer and mistake adjusting are found to appear in In the various and relevant enzyme of histone posttranslational modification (Wang et al., 2007a, b).The research of our laboratories early periods As a result, it has been found that PHF14 can be combined by two structural domains of its PHD1 and PHD3 with histone H 3, and self dimerization can occur (Huang et al.,2013).From structure, PHF14 contains multiple finger and nuclear localization sequence, thus, it is presumed that PHF14 is likely to the reader as one " histone mark ", by itself adjusting or pass through its tune to histone Section influences the transcriptional expression of gene in nucleus, and then influences the important cellular activities such as cell Proliferation, division, participates in swollen Tumor occurs.At the same time, it has been found that many PHD finger albumen are reported not only to be influenced to swell by epigenetic regulation The occurrence and development of tumor also participate in various regulation and control such as protein degradation, signal transduction and cell migration and promote the hair of tumour Raw (Akazawa et al., 2013；Bankovic et al.,2010；Chitalia et al.,2008；Kitagawa et al.,2012；Li et al.,2013；Zhou et al.,2005).Therefore, we participate in the machine that lung cancer occurs for PHF14 System conducts extensive research.

The tumorigenic research of the participation of PHF14 is rarely reported, initially speculates that PHF14 is in the research of cellular level Possible colon cancer tumor suppressor gene, but its exact function does not know (Ivanov et al., 2007).Nearest research table Bright, PHF14 unconventionality expressions in biliary tract cancer cell promote proliferation after inhibiting expression, prompt to participate in biliary tract carcinogenesis, but act on machine It makes indefinite (Akazawa et al., 2013).Since this two researchs all only carry out in individual cells system, however not excluded that It is the special phenomenon of cell line, or effects of the prompt PHF14 in different cell/tumours is different, while in view of them without phase The clinical data of pass and research, we do not know the clinical meaning of these results.Our result show that：PHF14 is in kidney, liver Expression quantity has no significant changes in cancer, colorectal cancer, gastric cancer；And it is more than 80% or more cancerous lung tissue sample in non-small cell lung cancer Significantly high expression in this.In clinical level, we have screened multiple tumor sample databases, and KM-plot survival analysis results are aobvious Show that the lung cancer patient clinical prognosis of PHF14 high expression is poor, further prompts PHF14 that there is important work to the occurrence and development of lung cancer With.Analysis is carried out to our obtained clinical case data and finds PHF14 gene high expressions and cancerous lung tissue pathological phase It closes related to gene copy number；KIF4A gene high expressions are related to cancerous lung tissue pathological, with the differentiation of lung cancer pathology and base Because copy number is significantly correlated.Generally speaking, from clinical tissue specimen samples level, PHF14 specificity, conspicuousness in lung cancer patient is high It expresses and there is poor clinical prognosis, thus speculate that it necessarily has in the occurrence and development of lung cancer and play important promotion Effect.

The mechanism study of PHF14 participation lung cancer occurrence and development is found, PHF14 and the KIF4A all high expression in lung cancer, And expression quantity is proportionate.The biological function of KIF4A, which is mainly reported, participates in cell mitogen and cytokinesis (Bastos et al.,2014；Bastos et al.,2013；Kurasawa et al.,2004a；Lee et al.,2001；Mazumdar et al.,2004；Samejima et al.,2012；Shrestha et al.,2012；Singh et al.,2014； Stumpff et al.,2012；Takemoto et al.,2009；Wandke et al.,2012；Wu and Chen,2008； Zhu and Jiang,2005；Zhu et al., 2005), DNA damage and reparation (Lee and Kim, 2003；Wu et al., 2008b) and tumour occur (Gao et al., 2011；Mazumdar et al.,2006；Minakawa et al., 2013；Taniwaki et al.,2007a).Tumour is more easy to that (Mazumdar et occur in the ES cells for knocking out KIF4 al.,2006).KIF4A high expression and, cell proliferation and tumour related to poor patients clinical prognosis in lung cancer patient Invasion, which have, to be significantly affected (Taniwaki et al., 2007b).In Patients with Gastric Cancer, KIF4A low expressions are right after overexpression Cell Proliferation and mouse nude mice one-tenth knurl ability are inhibited (Gao et al., 2011).KIF4A high tables in oral squamous cell carcinomas It reaches, by activating spindle assembly checkpoint (spindle assembly checkpoint, SAC) to promote cell Proliferation and mouth Chamber squamous carcinoma occurs.Up to the present, the research report to PHF14 and KIF4A in tumour is relatively limited.Our research first With such a possible lung cancer of KIF4A marker interaction can occur for the secondary PHF14 that demonstrates, the common high expression in lung cancer And it is significantly correlated, in conjunction with cell proliferation have significantly affect, this match with report before (Taniwaki et al., 2007b), thus it is presumed that the two plays important biological action in lung cancer generating process.Before studies have shown that KIF4A high expression in lung cancer, cell proliferation, tumor invasion have great influence (Taniwaki et al., 2007b), knot Fruit shows that KIF4 may be used as a clinical lung cancer predicting marker to play a significant role during lung cancer occurrence and development, may It is used to research and develop antitumor drug and tumor vaccine.Our research discovery PHF14 and KIF4A is in clinical lung cancer sample and carefully Common high up-regulated expression and significantly correlated, PHF14 cell proliferations, mitosis, tumor invasion and one-tenth knurl ability mistake in born of the same parents system It plays a significant role in journey, thus it is presumed that, PHF14 may be that a potential lung cancer marker influence lung cancer is sent out Exhibition, PHF14 very likely become a good medicine target protein.

The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation, It should be pointed out that for those skilled in the art, under the premise of not departing from the method for the present invention, can also make Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical scheme of the present invention It is interior.

SEQUENCE LISTING

<110>Shanghai Inst. of Life Science, CAS

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Claims (22)

1.PHF14 inhibitor is used to prepare the purposes of lung cancer therapy drug.

2. purposes according to claim 1, which is characterized in that the lung cancer therapy drug at least have following function it One：Can significantly inhibit lung carcinoma cell growth and proliferation, inhibit lung carcinoma cell deterioration, inhibit lung carcinoma cell one-tenth knurl ability, The cell cycle destroyed lung carcinoma cell mitosis process, extend lung carcinoma cell.

3. purposes according to claim 1, which is characterized in that the PHF14 inhibitor inhibits PHF14 activity, or suppression PHF14 genetic transcriptions or expression processed.

4. purposes according to claim 1, which is characterized in that the PHF14 inhibitor be siRNA, shRNA, antibody or Micromolecular compound.

5. a kind of method for treating lung cancer, to apply PHF14 inhibitor to object.

6. a kind of lung cancer combination therapy pharmaceutical composition, including a effective amount of PHF14 inhibitor and other at least one lung cancer therapies Drug.

7.PHF14 inhibitor and KIF4A inhibitor combine the purposes for being provided commonly for preparing lung cancer therapy drug.

8. purposes according to claim 7, which is characterized in that the lung cancer therapy drug at least have following function it One：Can significantly inhibit lung carcinoma cell growth and proliferation, inhibit lung carcinoma cell deterioration, inhibit lung carcinoma cell one-tenth knurl ability, The cell cycle destroyed lung carcinoma cell mitosis process, extend lung carcinoma cell.

9. purposes according to claim 7, which is characterized in that the PHF14 inhibitor inhibits PHF14 activity, or suppression PHF14 genetic transcriptions or expression processed；The KIF4A inhibitor inhibits KIF4A activity, or inhibits KIF4A genetic transcriptions or table It reaches.

10. purposes according to claim 7, which is characterized in that the PHF14 inhibitor be siRNA, shRNA, antibody or Micromolecular compound；The KIF4A inhibitor can be siRNA, shRNA, antibody or micromolecular compound.

11. a kind of method for treating lung cancer, to apply PHF14 inhibitor and KIF4A inhibitor to object.

12. a kind of lung cancer combination therapy pharmaceutical composition, including a effective amount of PHF14 inhibitor and KIF4A inhibitor and at least one Other lung cancer therapy drugs of kind.

13.PHF14 the purposes for screening lung cancer therapy drug.

14. purposes according to claim 13, which is characterized in that PHF14 is specifically referred to for screening lung cancer therapy drug PHF14 is applied to screening lung cancer therapy drug or preparation as drug or preparation for the action target of lung carcinoma cell；PHF14 makees For drug or preparation is applied to screening lung cancer therapy drug for the action target of lung carcinoma cell or preparation specifically refers to：It will PHF14 screens drug or preparation as suppression target, and the expression of PHF14 in lung carcinoma cell can be reduced to find PHF14 inhibitor, as lung cancer therapy drug candidate.

15.PHF14 and KIF4A combines the purposes for being provided commonly for screening lung cancer therapy drug.

16. purposes according to claim 15, which is characterized in that PHF14 and KIF4A combine be provided commonly for screening lung cancer control It treats drug and specifically refers to the action target that PHF14 and KIF4A combines collectively as drug or preparation for lung carcinoma cell and be applied to Screen lung cancer therapy drug or preparation；PHF14 and KIF4A combines the effect target that lung carcinoma cell is directed to collectively as drug or preparation Mark is applied to screening lung cancer therapy drug or preparation specifically refers to：PHF14 and KIF4A are combined collectively as suppression target, it is right Drug or preparation are screened, and the expression of PHF14 and KIF4A in lung carcinoma cell can be simultaneously or separately reduced to find Inhibitor, as lung cancer therapy drug candidate.

17.PHF14 is used to prepare or screens the purposes of pulmonary cancer diagnosis reagent.

18. purposes according to claim 17, which is characterized in that PHF14 is used to prepare or screens pulmonary cancer diagnosis reagent, packet Content of both including：First, PHF14 is used to prepare pulmonary cancer diagnosis reagent, refer to using PHF14 genes or expression product as lung The diagnosis index of cancer is applied to the preparation of pulmonary cancer diagnosis reagent；Second, PHF14 for screening pulmonary cancer diagnosis reagent, refer to by The reagent for identifying target sieving specific recognition PHF14 genes or expression product of PHF14 genes or expression product as lung cancer, To be used as pulmonary cancer diagnosis reagent, to detect lung cancer.

19.PHF14 and KIF4A combines the purposes for being provided commonly for preparing or screen pulmonary cancer diagnosis reagent.

20. purposes according to claim 19, which is characterized in that PHF14 and KIF4A, which combines, to be provided commonly for preparing or screen Pulmonary cancer diagnosis reagent, including both sides content：First, PHF14 and KIF4A, which combine, is provided commonly for preparing pulmonary cancer diagnosis reagent, It refer to the diagnosis index application combined PHF14 genes or expression product and KIF4A genes or expression product collectively as lung cancer In the preparation of pulmonary cancer diagnosis reagent；Second, PHF14 and KIF4A combine be provided commonly for screening pulmonary cancer diagnosis reagent, refer to by PHF14 genes or expression product and KIF4A genes or expression product combine collectively as lung cancer identification target sieving simultaneously or The reagent of specific recognition PHF14 genes or expression product and KIF4A genes or expression product respectively, to be used as pulmonary cancer diagnosis Reagent, to detect lung cancer.

21.PHF14 inhibitor is used to prepare the purposes that agent is upset in lung carcinoma cell mitosis.

22.PHF14 inhibitor KIF4A inhibitor, which is combined, is provided commonly for preparing the purposes that agent is upset in lung carcinoma cell mitosis.