CN108295261B - Function and use of PHF14 - Google Patents

Abstract

The invention belongs to the field of chemical and biological research in life science, and particularly relates to a function and application of PHF 14. The invention is widely and deeply researched, and firstly discovers that PHF14 alone or combined with KIF4A can be used as a lung cancer treatment target, and the inhibition of the expression of PHF14 and/or KIF4A can obviously inhibit the growth and proliferation of lung cancer cells, inhibit the deterioration of the lung cancer cells and inhibit the tumor forming capability of the lung cancer cells. Therefore, the invention provides strong scientific evidence for the pathogenesis of lung cancer and the clinical treatment of lung cancer from the level of clinical patient samples, the level of cell functions and the level of molecules.

Description

Function and use of PHF14

Technical Field

The invention belongs to the field of chemical and biological research in life science, and particularly relates to a function and application of PHF 14.

Background

PHD finger proteins are a class of PHD finger proteins that are widely present in eukaryotes and play important roles in gene transcription regulation and disease occurrence. In recent studies, PHD finger proteins have been classified as a class of proteins that recognize histone methylation modifications (Lan et al, 2007; Org et al, 2008; Shi et al, 2006; Wysocka et al, 2006), and this family of proteins plays an important role in regulating cell epigenetic modifications. Different PHD finger proteins specifically recognize different "histone marks", and regulate the transcriptional expression of genes by either modulating their intrinsic activity or by modulating the activity of their interacting proteins (Baker et al, 2008). An increasing number of studies have shown that when many genes encode PHD finger structures that undergo point mutations, deletions or chromosomal translocations, these abnormalities often trigger the development of human diseases, including: tumors, mental retardation, immunodeficiency, etc. (Musselman and Kutateladze,2009), which further underscores the important role played by PHD finger protein as an epigenetic "reader" in the development of disease (Baker et al, 2008). The development of cancer has long been recognized as a combined genetic and epigenetic process of alteration that ultimately contributes to the development and progression of cancer (Jones and Baylin, 2007). Cancer-related mutations and dysregulations are found to occur in various enzymes associated with post-translational modification of histones (Wang et al, 2007a, b). Our pre-laboratory findings found that PHF14 is able to bind histone H3 via its two domains, PHD1 and PHD3, and is able to undergo auto-dimerization (Huang et al, 2013). Structurally, PHF14 contains multiple finger and nuclear localization sequences, and therefore, we speculate that PHF14 is likely to be a "histone mark" reader, and by its own regulation or its regulation of histones, it affects the transcriptional expression of genes in the nucleus, and further affects important cellular activities such as cell proliferation and division, and is involved in tumorigenesis. Meanwhile, we found that many PHD finger proteins are reported to influence the development of tumors not only through epigenetic regulation, but also participate in the regulation of multiple aspects such as protein degradation, signal transduction and cell migration to promote the development of tumors (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 conducted extensive studies on the mechanism of PHF14 involved in lung cancer development.

Among tumor-related fatal diseases worldwide, lung cancer is one of the most prominent causes of death (Jemal et al, 2011). Lung cancer is a worldwide public health problem that has become the most prevalent malignancy of morbidity and mortality in the world since 1985, with over one million people dying from the disease annually since 1993. Only 13% of lung cancer patients survive more than 5 years, and the number of people infected with lung cancer is expected to increase in the coming years. Among all causes of death, lung cancer ranks tenth and will climb to fifth in developing countries due to increased incidence. More and more researches are inclined to screen and research molecular markers of tumorigenesis at the clinical sample level, analyze the molecular mechanism of important molecules in tumorigenesis from the aspects of molecular biology and genetics, and look for the correlation between the expression of genes and the processes of tumorigenesis, metastasis, differentiation and the like from the aspect of epidemiology, so that the early screening and detection of lung cancer are promoted. Since the five-year survival rate of lung cancer is extremely low, early lung cancer detection can change the disease outcome and is more important, however, the current situation is that: early lung cancer is either too expensive or not sensitive enough (Henschke et al, 2006; Markowitz et al, 2007). On the other hand, lung cancer treatment is currently mainly dependent on treatment patterns of clinical features such as tumor grade and smoking history to determine a treatment plan by analyzing characteristics at a molecular level of each case. In order to improve the therapeutic effect and reduce the toxicity caused by the therapy, researchers have been working on developing personalized therapeutic regimens based on the molecular properties of tumors. Therapeutic response and prognostic survival are predicted by the discovery of biomarkers at the molecular level. Predictive markers can be used to determine treatment regimens, while prognostic markers can predict treatment outcomes independent of treatment pattern (Belinsky et al, 1998; Gessner et al, 2004; Kettunen et al, 2004), and the use of these molecular markers can be an important means of overcoming the bottleneck of chemotherapy and reducing the toxicity associated with chemotherapy. Some developed molecular targeted drugs have shown efficacy in cancer treatment, however, the proportion of patients who can respond well to these drugs is still very limited, and further development of tumor molecular targeted drugs is urgent. It is apparent that: the screening of molecular markers will play an increasingly important role in the prevention, detection, clinical diagnosis and treatment of lung cancer. In the last decade of research, it has been found that abnormal mutations in many genes may be involved in the development of lung cancer: mutational activation of oncogenes, such as: kRas, EGFR and MYC; inactivation of cancer suppressor genes, such as: p53, p16 and RB; and a number of recurrent chromosomal mutations (Sekido et al, 2003). In addition to genetic changes, epigenetic mutations can also trigger tumors, and these mutations can trigger mutational activation or suppression of some genes that play an important role in tumor development and development, thus triggering tumors (Esteller, 2008; Kwon and Shin, 2011). To date, a great deal of research has been devoted to the correlation of methylation status of lung cancer patient samples with lung cancer development (Shames et al, 2006; Vaissiere et al, 2009), and many genes, such as APC (virani et al, 2001) and CDH13(Toyooka et al, 2001), have been reported to be hypermethylated in primary non-small cell lung cancer (NSCLCs).

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the function and the application of PHF14 in lung cancer.

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

in a first aspect of the invention, there is provided the use of a PHF14 inhibitor for the manufacture of a medicament for the treatment of lung cancer.

The lung cancer treatment drug has at least one of the following functions: can obviously inhibit the growth and proliferation of the lung cancer cells, inhibit the deterioration of the lung cancer cells, inhibit the tumorigenic capacity of the lung cancer cells, destroy the mitotic process of the lung cancer cells and prolong the cell cycle of the lung cancer cells.

Such disrupting mitotic processes include, but are not limited to, disrupting chromosome condensation, affecting microtubule dynamics, which in turn affects microtubule spindle morphology and localization, affecting cytokinesis.

Extending the cell cycle of the lung cancer cells includes, but is not limited to, extending the M phase of the cell cycle.

Preferably, the PHF14 inhibitor refers to a molecule having an inhibitory effect on PHF 14.

Having inhibitory effects on PHF14 include, but are not limited to: inhibiting PHF14 activity, or inhibiting PHF14 gene transcription or expression.

The PHF14 inhibitor can be siRNA, shRNA, antibody and small molecule compound.

As exemplified in the examples herein, the PHF14 inhibitor can be an siRNA, the sequence of which is shown in SEQ ID NO.2 or SEQ ID NO. 3.

The lung cancer treatment drug necessarily comprises a PHF14 inhibitor, and the PHF14 inhibitor is used as an effective component of the functions.

In the lung cancer treatment drug, the effective component exerting the functions can be only the PHF14 inhibitor, and other molecules playing similar functions can also be contained.

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

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

The lung cancer targeted by the lung cancer therapeutic drug may be non-small cell lung cancer.

The lung cancer therapeutic drug is mainly aimed at mammals such as rodents, primates and the like.

In a second aspect of the invention, a method of treating lung cancer is provided by administering to a subject an inhibitor of PHF 14.

The subject is a mammal or a lung cancer cell of the mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. Preferably, the primate is a monkey, ape or homo sapiens. The lung cancer cell can be an isolated lung cancer cell, including but not limited to A549, H2126, CRL-5803, CRL-5807, CRL-5810, CRL-5844, CRL-5872, CRL-5883, CRL5889, CRL-5908, and CRL 5928.

The subject may be a patient suffering from lung cancer or an individual in whom treatment of lung cancer is desired, or the subject is an ex vivo lung cancer cell of a lung cancer patient or an individual in whom treatment of lung cancer is desired.

The PHF14 inhibitor can be administered to a subject before, during, or after receiving treatment for lung cancer.

In a third aspect of the present invention, there is provided a medicament for treating lung cancer, comprising an effective amount of a PHF14 inhibitor and a pharmaceutically acceptable carrier.

In a fourth aspect of the invention, there is provided a combination therapy for lung cancer comprising an effective amount of a PHF14 inhibitor and at least one other therapeutic agent for lung cancer.

The other lung cancer treatment drug is a lung cancer treatment drug except for the PHF14 inhibitor.

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

firstly), the PHF14 inhibitor and other lung cancer therapeutic drugs are respectively prepared into independent preparations, the preparation forms can be the same or different, and the administration routes can be the same or different.

When the other lung cancer therapeutic agent is an antitumor antibody, a parenteral administration type is generally adopted. When the other lung cancer treatment medicines are chemotherapy medicines, the administration forms can be rich, and the administration can be carried out in a gastrointestinal tract or a parenteral tract. Known routes of administration for each chemotherapeutic agent are generally recommended.

Secondly), the PHF14 inhibitor and other lung cancer treatment medicines are prepared into a compound preparation. When the PHF14 inhibitor and the other lung cancer therapeutic agent are administered by the same route of administration and administered simultaneously, they may be formulated into a combination preparation.

In a fifth aspect of the invention, a method of treating lung cancer is provided, comprising administering to a subject an effective amount of a PHF14 inhibitor, and administering to the subject an effective amount of an additional lung cancer treatment agent and/or administering to the subject an additional lung cancer treatment modality.

An effective amount of a PHF14 inhibitor and an effective amount of at least one other lung cancer therapeutic agent may be administered simultaneously or sequentially.

Based on that PHF14 is the first newly discovered lung cancer treatment target point, the compound can at least play a role in adding curative effects when being used together with other lung cancer treatment medicaments except a PHF14 inhibitor, and further enhance the inhibition on the lung cancer.

Such other lung cancer therapeutic agents include, but are not limited to: antitumor antibodies, chemotherapeutic drugs or targeted drugs, etc.

The PHF14 inhibitor may be administered parenterally or parenterally. The other lung cancer therapeutic agent may be administered gastrointestinal or parenteral. For antitumor antibodies or chemotherapeutic drugs, parenteral administration is generally employed.

In a sixth aspect of the invention, there is provided the use of a PHF14 inhibitor in combination with a KIF4A inhibitor for the manufacture of a medicament for the treatment of lung cancer.

The lung cancer treatment drug has at least one of the following functions: can obviously inhibit the growth and proliferation of the lung cancer cells, inhibit the deterioration of the lung cancer cells, inhibit the tumorigenic capacity of the lung cancer cells, destroy the mitotic process of the lung cancer cells and prolong the cell cycle of the lung cancer cells.

Such disrupting mitotic processes include, but are not limited to, disrupting chromosome condensation, affecting microtubule dynamics, which in turn affects microtubule spindle morphology and localization, affecting cytokinesis.

Extending the cell cycle of the lung cancer cells includes, but is not limited to, extending the M phase of the cell cycle.

Preferably, the PHF14 inhibitor refers to a molecule having an inhibitory effect on PHF 14; the KIF4A inhibitor is a molecule having an inhibitory effect on KIF 4A.

Having inhibitory effects on PHF14 include, but are not limited to: inhibiting PHF14 activity, or inhibiting PHF14 gene transcription or expression. Inhibitory effects on KIF4A include, but are not limited to: inhibiting KIF4A activity, or inhibiting KIF4A gene transcription or expression.

The PHF14 inhibitor can be siRNA, shRNA, antibody and small molecule compound. The KIF4A inhibitor can be siRNA, shRNA, antibody, small molecule compound.

As exemplified in the examples herein, the PHF14 inhibitor can be an siRNA, the sequence of which is shown in SEQ ID NO.2 or SEQ ID NO. 3. The KIF4A inhibitor can be siRNA, the sequence of which is shown in SEQ ID NO.5 or SEQ ID NO. 6.

The lung cancer treatment drug necessarily comprises a PHF14 inhibitor and a KIF4A inhibitor, and the PHF14 inhibitor and the KIF4A inhibitor are used as effective components of the aforementioned functions.

In the lung cancer treatment drug, the effective components playing the functions can be only a PHF14 inhibitor and a KIF4A inhibitor, and other molecules playing similar functions can also be contained.

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

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

The lung cancer targeted by the lung cancer therapeutic drug may be non-small cell lung cancer.

The lung cancer therapeutic drug is mainly aimed at mammals such as rodents, primates and the like.

In a seventh aspect of the invention, there is provided a method of treating lung cancer by administering to a subject an inhibitor of PHF14 and an inhibitor of KIF 4A.

The subject is a mammal or a lung cancer cell of the mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. Preferably, the primate is a monkey, ape or homo sapiens. The lung cancer cell can be an isolated lung cancer cell, including but not limited to A549, H2126, CRL-5803, CRL-5807, CRL-5810, CRL-5844, CRL-5872, CRL-5883, CRL5889, CRL-5908, and CRL 5928.

The subject may be a patient suffering from lung cancer or an individual in whom treatment of lung cancer is desired, or the subject is an ex vivo lung cancer cell of a lung cancer patient or an individual in whom treatment of lung cancer is desired.

The PHF14 inhibitor and KIF4A inhibitor can be administered to a subject before, during, or after receiving treatment for lung cancer.

In the eighth aspect of the invention, a medicament for treating lung cancer is provided, which comprises effective amounts of a PHF14 inhibitor, a KIF4A inhibitor and a medicinal carrier.

The ninth aspect of the invention provides a lung cancer combination therapy drug combination, which comprises effective dose of PHF14 inhibitor and KIF4A inhibitor and at least one other lung cancer treatment drug.

The other lung cancer treatment drug is a lung cancer treatment drug except for a PHF14 inhibitor and a KIF4A inhibitor.

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

firstly), the PHF14 inhibitor, the KIF4A inhibitor and other medicaments for treating the lung cancer are respectively prepared into independent preparations, the preparation forms can be the same or different, and the administration routes can be the same or different.

When the other lung cancer therapeutic agent is an antitumor antibody, a parenteral administration type is generally adopted. When the other lung cancer treatment medicines are chemotherapy medicines, the administration forms can be rich, and the administration can be carried out in a gastrointestinal tract or a parenteral tract. Known routes of administration for each chemotherapeutic agent are generally recommended.

Secondly), the PHF14 inhibitor, the KIF4A inhibitor and other lung cancer treatment medicines are prepared into a compound preparation. When the PHF14 inhibitor and the KIF4A inhibitor and other lung cancer therapeutic agents are administered by the same administration route and administered simultaneously, both may be formulated in the form of a combined preparation.

In a tenth aspect of the invention, a method of treating lung cancer is provided, comprising administering to a subject an effective amount of a PHF14 inhibitor and a KIF4A inhibitor, and administering to the subject an effective amount of another lung cancer treatment agent and/or administering to the subject another means of lung cancer treatment.

An effective amount of a PHF14 inhibitor and a KIF4A inhibitor and an effective amount of at least one other lung cancer therapeutic agent may be administered simultaneously or sequentially.

Based on PHF14 and KIF4A as combined lung cancer treatment targets newly found for the first time, the combination of the PHF14 and the KIF4A with other lung cancer treatment medicaments except a PHF14 inhibitor and a KIF4A inhibitor can at least play a role in adding curative effects, and further enhance the inhibition on the lung cancer.

In an eleventh aspect of the present invention, there is provided: the PHF14 is used for screening lung cancer treatment drugs.

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

Preferably, the PHF14 is used for screening lung cancer treatment medicines, and particularly, the PHF14 is used for screening lung cancer treatment medicines or preparations as an action target of the medicines or preparations for lung cancer cells.

Further, the application of PHF14 as a drug or a preparation for screening a lung cancer therapeutic drug or preparation against an action target of lung cancer cells specifically means: the PHF14 is used as an inhibition target to screen a drug or a preparation so as to find a PHF14 inhibitor capable of reducing the expression level of PHF14 in lung cancer cells, and the PHF14 inhibitor is used as a candidate drug for treating lung cancer.

In some embodiments of the invention, the PHF14 gene is listed as a target of RNA interference effect, and siRNA capable of obviously inhibiting PHF14 gene expression is screened, and the result shows that the siRNA can obviously weaken the proliferation capability of lung cancer cells, inhibit the deterioration of the lung cancer cells and inhibit the tumor formation capability of the lung cancer cells, and can be used as a medicament with the effect of inhibiting the proliferation of the lung cancer cells. In addition, PHF14 gene and its protein can be used as targets of action, such as antibody drugs, small molecule drugs, etc.

Further, according to a twelfth aspect of the present invention, there is provided a use of PHF14 and KIF4A in combination for screening a therapeutic agent for lung cancer.

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

Preferably, the PHF14 and the KIF4A are jointly and jointly used for screening the lung cancer treatment drugs, in particular the PHF14 and the KIF4A are jointly and jointly used as the drug or the preparation to be applied to screening the lung cancer treatment drugs or the preparation aiming at the action targets of the lung cancer cells.

Further, the PHF14 and the KIF4A are jointly used as the drug or the preparation for screening the lung cancer treatment drug or the preparation by being taken as the action target of the drug or the preparation aiming at the lung cancer cells, and specifically refer to the following steps: the PHF14 and the KIF4A are jointly used as inhibition targets, and the drug or preparation is screened to find the inhibitor capable of simultaneously or respectively reducing the expression levels of PHF14 and KIF4A in lung cancer cells, so that the inhibitor can be used as a candidate drug for treating lung cancer.

In some embodiments of the invention, different siRNAs capable of respectively and significantly inhibiting expressions of PHF14 gene and KIF4A gene are screened as inhibitors by respectively taking PHF14 gene and KIF4A gene as targets of RNA interference, and the results show that the inhibitors can significantly reduce proliferation capacity of lung cancer cells, inhibit deterioration of the lung cancer cells and inhibit tumorigenicity of the lung cancer cells, and can be used as drugs with the effect of inhibiting proliferation of the lung cancer cells. In addition, PHF14 gene and its protein can be used as targets of action, such as antibody drugs, small molecule drugs, etc.

A thirteenth aspect of the present invention provides: the PHF14 is used for preparing or screening lung cancer diagnostic reagents.

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

Preferably, PHF14 is used for preparing or screening a lung cancer diagnostic reagent, comprising:

firstly, the application of PHF14 in preparing lung cancer diagnostic reagents refers to the application of PHF14 gene or expression products as lung cancer diagnostic indicators in preparing lung cancer diagnostic reagents. For example, the PHF14 gene or expression product can be used as a standard or positive control for the detection of lung cancer.

Secondly, the PHF14 is used for screening lung cancer diagnostic reagents, and is a reagent which takes a PHF14 gene or an expression product as a recognition target of lung cancer to screen and specifically recognize a PHF14 gene or the expression product, so that the reagent is used as a lung cancer diagnostic reagent to detect the lung cancer.

A fourteenth aspect of the present invention provides: the PHF14 and KIF4A are jointly and jointly used for preparing or screening lung cancer diagnostic reagents.

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

Preferably, PHF14 and KIF4A are used jointly in the preparation or screening of a lung cancer diagnostic reagent, including two aspects:

firstly, the PHF14 and the KIF4A are jointly used for preparing the lung cancer diagnostic reagent, and the PHF14 gene or the expression product and the KIF4A gene or the expression product are jointly used as the diagnostic index of the lung cancer to be applied to the preparation of the lung cancer diagnostic reagent. For example, the PHF14 gene or expression product and KIF4A gene or expression product can be used as standard or positive control for lung cancer detection.

Secondly, the PHF14 and the KIF4A are jointly and jointly used for screening the lung cancer diagnostic reagent, which means that the PHF14 gene or the expression product and the KIF4A gene or the expression product are jointly and jointly used as recognition targets of lung cancer to screen reagents which simultaneously or respectively specifically recognize the PHF14 gene or the expression product and the KIF4A gene or the expression product, so that the reagents are used as the lung cancer diagnostic reagent to detect the lung cancer.

The lung cancer therapeutic agent or lung cancer diagnostic agent of the present invention includes, but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

Such nucleic acids include, but are not limited to: ncRNA, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (esiRNA) produced by endoribonuclease III, or short hairpin RNA (shRNA).

In a fifteenth aspect of the present invention, there is provided: use of a PHF14 inhibitor for the preparation of a disrupting agent for the mitosis of lung cancer cells.

The mitotic disruptor has at least one of the following functions: the mitosis process of the lung cancer cells is destroyed, and the cell cycle of the lung cancer cells is prolonged.

Such disrupting mitotic processes include, but are not limited to, disrupting chromosome condensation, affecting microtubule dynamics, which in turn affects microtubule spindle morphology and localization, affecting cytokinesis.

Extending the cell cycle of the lung cancer cells includes, but is not limited to, extending the M phase of the cell cycle.

In a sixteenth aspect of the present invention, there is provided: use of a PHF14 inhibitor, KIF4A inhibitor, in combination for the preparation of a disrupting agent for the mitosis of lung cancer cells.

The mitotic disruptor has at least one of the following functions: the mitosis process of the lung cancer cells is destroyed, and the cell cycle of the lung cancer cells is prolonged.

Such disrupting mitotic processes include, but are not limited to, disrupting chromosome condensation, affecting microtubule dynamics, which in turn affects microtubule spindle morphology and localization, affecting cytokinesis.

Extending the cell cycle of the lung cancer cells includes, but is not limited to, extending the M phase of the cell cycle.

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

the invention is widely and deeply researched, and firstly discovers that PHF14 alone or combined with KIF4A can be used as a lung cancer treatment target, and the inhibition of the expression of PHF14 and/or KIF4A can obviously inhibit the growth and proliferation of lung cancer cells, inhibit the deterioration of the lung cancer cells and inhibit the tumor forming capability of the lung cancer cells. Therefore, the invention provides strong scientific evidence for the pathogenesis of lung cancer and the clinical treatment of lung cancer from the level of clinical patient samples, the level of cell functions and the level of molecules.

Drawings

FIG. 1A: PHF14 was expressed at levels in non-small cell lung cancer tissues (24 cases).

FIG. 1B: PHF14 immunohistochemical staining imaging in different lung cancer tissues and paracarcinoma tissue chips, abbreviation: ADC, adenocarinoma (Adenocarcinoma); SCC, Squamous Cell Carcinoma (Squamous Cell Carcinoma); LCC, Large Cell Carcinoma; normal, Normal tissue; x 8, scale 1.0 mm.; x 400, scale 100 μm.

FIG. 1C: PHF14 gene mRNA expression level in lung adenocarcinoma patient samples.

FIG. 1D: correlation of PHF14 expression with gene copy number.

FIG. 1E: KM-plot survival analyses were performed based on PHF14 expression levels in patient samples from NCBI Geo Database (GSE3141, GSE19188) and Cancer Genome Atlas (TCGA).

FIG. 2A: transient transfection of siRNA knockdown PHF14 expression inhibited a549 cell proliferation.

FIG. 2B: transient transfection of siRNA knockdown PHF14 expression inhibited CRL-5810 cell proliferation.

FIG. 2C: transfection of the PHF14 rescue plasmid was able to rescue a partially slowed proliferation phenotype, left panel: proliferation of cells after control and transfection of PHF14siRNA was detected by MTT method, middle panel: proliferation of cells was detected by the BrdU incorporation method in PHF14siRNA transfected cells, which is a statistical result of three independent experiments, right panel: immunoblotting examined protein expression after transient transfection of PHF14 with β -actin as an internal reference, and data were expressed as mean ± SD of three independent experiments with P <0.05 and P < 0.01.

FIG. 3A: inhibition of PHF14 expression inhibits cell malignant transformation and its tumorigenicity in mice, and a549 cell strain stabilizing knockdown PHF14 becomes slow in proliferation, left panel: proliferation of the control cell line and a549 cell line of stable knockdown PHF14 was detected by MTT method, in which: proliferation of the control cell strain and the a549 cell strain of stable knockdown PHF14 was detected by the BrdU incorporation method, which is a statistical result of three independent experiments, right panel: the expression of protein after stabilizing knock down PHF14 is detected by immunoblotting, and beta-actin is used as an internal reference.

FIG. 3B: colony formation experiment in soft agar, after cells were grown in soft agar for 15 days, the growth of the cells was observed, and a cell mass of 50 or more cells was considered as a formed colony, left panel: representative photograph of cells grown in soft agar, right panel: statistical analysis of the ability of the indicated cells to grow in soft agar. Data are expressed as mean ± SD,. P < 0.01.

FIG. 3C: in vivo tumor formation experiment in nude mice, left panel: an equal number of cells shown in the figure were injected subcutaneously at the scapular region of 4-week-old male mice, and the volume of the generated tumor was observed every week, and the tumor volume was calculated using the following formula: volume 1/2 (maximum diameter) x (minimum diameter) 2, 4 mice per group, data expressed as mean ± SD, × P <0.01, right panel: representative pictures of tumorigenesis of a549 control cells and PHF14 stable knockdown cells in nude mice, P <0.05, P < 0.01.

FIG. 4A: the interaction of PHF14 with KIF4A, a rapid protein liquid chromatography experiment, PHF14, KIF4A, PARP-1 and Ku70 were all enriched in the same several fractions.

FIG. 4B: co-immunoprecipitation assay endogenous PHF14 interacts with KIF4A, left panel: pre-immune serum or PHF14 antiserum is used for immunoprecipitating PHF14 in A549 cell total lysate, immunoblotting is used for detecting PHF14 or KIF4A, EB is added into the lysate for blocking the combination of DNA, and the right graph shows that: a549 cell lysate is subjected to immunoprecipitation of KIF4A by using preimmune serum or anti-KIF4A serum, and immunoblotting is carried out to detect KIFA4 and PHF14, wherein EB is added into the lysate to block DNA-mediated binding.

FIG. 4C: determination of PHF14 and KIF4A binding sites, upper panel: co-immunoprecipitation GFP-KIF4A WT or deletion mutant and PHF14, 293T cells were transfected with the indicated plasmids, 293T whole cell lysates were prepared 48 hours later, PHF14 was immunoprecipitated with either anti-PHF14 antiserum or preimmune serum NS, followed by immunoblotting with anti-PHF14, anti-GFP antibodies, as shown in the following figure: schematic representation of KIF4A mutants, the binding affinities were graded from strong to weak as: x, strong; x, medium; weak.

FIG. 4D: determination of PHF14 and KIF4A binding sites, upper panel: co-immunoprecipitation of PHF14WT or deletion mutant and GFP-KIF4A, panels below: schematic representation of PHF14 mutant.

FIG. 4E: the co-localization of endogenous PHF14 and KIF4A in A549 cells is detected by immunofluorescence, the cells are fixed after cell slide, the immunofluorescence detection is carried out by anti-PHF14 (green) and anti-KIF4A (red), cell nuclei are shown by DAPI (blue) staining, and the scale is 5 mu m.

FIG. 5A: PHF14 and KIF4A are co-highly expressed in lung cancer cell lines and lung cancer samples and synergistically promote cell proliferation, and PHF14 and KIF4A are expressed in non-small cell lung cancer clinical samples, and the left graph shows that: the immunoblotting result shows that PHF14 and KIF4A are commonly highly expressed in a non-small cell lung cancer clinical sample, beta-actin is used as an internal reference, and the right picture: the correlation analysis result shows that the high expression of PHF14 and KIF4A are obviously correlated in the non-small cell lung cancer clinical sample.

FIG. 5B: qPCR showed mRNA levels of PHF14 and KIF4A in non-small cell lung cancer samples.

FIG. 5C: expression of PHF14 and KIF4A in non-small cell lung cancer cell lines, left panel: immunoblot results showed that PHF14 and KIF4A were co-highly expressed in non-small cell lung cancer cell lines, β -actin as internal reference, right panel: correlation analysis results showed that high expression of PHF14 and KIF4A were significantly correlated in non-small cell lung cancer cell lines.

FIG. 5D: knockdown PHF14 and/or transfected siRNA knockdown KIF4A expression inhibits a549 cell proliferation, left panel: the proliferation of a549 cell line after stable knockdown PHF14 and/or transient knockout of KIF4A was detected by MTT method, right panel: and (3) carrying out immunoblot detection on the expression of the protein after stabilizing knock down PHF14 and/or instantaneously knocking out KIF4A, wherein beta-actin is used as an internal reference.

FIG. 5E: full-length expression of either over-expressed PHF14 or KIF4A promoted HCT-116 cell proliferation, but PHF14 NT 1-160aa(s) deletion mutant did not, left panel: detection of proliferation of HCT-116 cell lines by MTT method after control and overexpression of PHF14WT, KIF4A and PHF14 NT 1-160aa(s) deletion mutant, right panel: the foreign expression efficiency of the deletion mutants of PHF14WT, KIF4A and PHF14 NT 1-160aa(s) is detected by immunoblotting, and beta-actin is used as an internal reference.

FIG. 6A: PHF14 and/or KIF4A deletion leads to mitotic abnormalities and influences mitotic index, PHF14 deletion leads to abnormal shape and distribution of mitotic cell microtubules and chromosomes, alpha-tubulin and chromosomes are detected by immunofluorescence after transient siRNA transfer in Hela cells, cell climbing post-fixation, microtubules are shown by anti-alpha-tubulin (green) staining, and cell nucleus is shown by DAPI (blue) staining.

FIG. 6B: stable inhibition of PHF14 in a549 cell line resulted in mitotic abnormalities, left panel: cell climbing post-fixation, microtubules visualized by anti- α -tubulin (green) staining, nuclei visualized by DAPI (blue) staining, right panel: WB detects the expression of protein after stabilizing knock down PHF14, and beta-actin is used as an internal reference.

FIG. 6C: record HelaGFP-H in vivo imaging experiment₂Effect of anterograde siRNA in B cells on cell cycle when inhibiting PHF14 and/or KIF 4A.

FIG. 6D: inhibition of PHF14 and/or KIF4A significantly affects the mitotic index, which is calculated as: mean ± variance,. P <0.0001, n is the number of cells analyzed, siRNA inhibition by transient siRNA in HeLa cells PHF14 and/or KIF4A was performed 48 hours later, high throughput imaging was performed using 0 pertta 20 fold objective lens in high content image acquisition and analysis system, 9 fields of view (6,000 to 8,000 cells) were imaged per group, mitotic cells were identified by channel imaging at 405 wavelengths, WB assay for knock-out efficiency of PHF14 and KIF4A proteins, β -actin was used as an internal reference.

Detailed Description

The research of the invention discovers that PHF14 alone or combined with KIF4A can be used as a lung cancer treatment target, and the inhibition of the expression of PHF14 and/or KIF4A can obviously inhibit the growth and proliferation of lung cancer cells, inhibit the deterioration of the lung cancer cells and inhibit the tumor forming capability of the lung cancer cells.

PHF14 inhibitors

Refers to a molecule having inhibitory effect on PHF 14. Having inhibitory effects on PHF14 include, but are not limited to: inhibiting PHF14 activity, or inhibiting PHF14 gene transcription or expression. The PHF14 inhibitor includes but is not limited to siRNA, shRNA, antibody and small molecule compound.

Inhibiting PHF14 activity refers to a decrease in PHF14 activity. Preferably, the PHF14 activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to its activity prior to inhibition.

Inhibiting PHF14 gene transcription or expression refers to: the method comprises the steps of preventing the transcription of the gene PHF14, reducing the transcription activity of the gene PHF14, preventing the expression of the gene PHF14, and reducing the expression activity of the gene PHF 14.

The regulation of PHF14 gene transcription or expression can be accomplished by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

Inhibition of gene transcription or expression of PHF14 was confirmed by PCR and Western Blot detection of expression level.

Preferably, the PHF14 gene transcription or expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, still more preferably by at least 90%, most preferably the PHF14 gene is not expressed at all, compared to the wild type.

KIF4A inhibitors

Refers to a molecule having inhibitory effect on KIF 4A. Inhibitory effects on KIF4A include, but are not limited to: inhibiting KIF4A activity, or inhibiting KIF4A gene transcription or expression. The KIF4A inhibitor includes but is not limited to siRNA, shRNA, antibody, small molecule compound.

Inhibiting KIF4A activity refers to a decrease in KIF4A activity. Preferably, KIF4A activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to its activity prior to inhibition.

Inhibiting the transcription or expression of KIF4A gene refers to: the method comprises the steps of making the gene of KIF4A not be transcribed, or reducing the transcription activity of the gene of KIF4A, or making the gene of KIF4A not be expressed, or reducing the expression activity of the gene of KIF 4A.

One skilled in the art can use conventional methods to modulate gene transcription or expression of KIF4A, such as gene knock-outs, homologous recombination, interfering RNA, and the like.

Inhibition of gene transcription or expression of PHF14 was confirmed by PCR and Western Blot detection of expression level.

Preferably, KIF4A gene transcription or expression is reduced by at least 10%, preferably by at least 30%, even more preferably by at least 50%, even more preferably by at least 70%, even more preferably by at least 90%, most preferably the PHF14 gene is not expressed at all, compared to wild type.

Small molecule compounds

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

PHF14 inhibitor (and KIF4A inhibitor) for preparing medicine

A PHF14 inhibitor (and KIF4A inhibitor) is used as main active ingredient or one of the main active ingredients for preparing medicine. Generally, the medicament may comprise one or more pharmaceutically acceptable carriers or excipients in addition to the active ingredient, according to the requirements of different dosage forms.

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

A "pharmaceutically acceptable carrier or adjuvant" should be compatible with, i.e., capable of being blended with, the PHF14 inhibitor (and KIF4A inhibitor) without substantially reducing the effectiveness of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as glycerol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

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

Combination therapeutic drug combinations and methods of administration

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

firstly), the PHF14 inhibitor (and KIF4A inhibitor) and other antitumor drugs are respectively prepared into independent preparations, the preparation formulations can be the same or different, and the administration routes can be the same or different. When in use, several medicines can be used simultaneously or sequentially. When administered sequentially, the other drugs should be administered to the body during the period that the first drug is still effective in the body.

Secondly), the PHF14 inhibitor (and KIF4A inhibitor) and other antitumor drugs are prepared into a compound preparation. When the PHF14 inhibitor (and KIF4A inhibitor) and the other antitumor agent are administered by the same route of administration and simultaneously administered, both may be formulated in the form of a combination preparation.

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

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

An effective amount of PHF14 inhibitor (and KIF4A inhibitor) and an effective amount of other lung cancer drugs may be administered simultaneously or sequentially. When in use, two or three medicines can be used simultaneously, or two or three medicines can be used successively. When administered sequentially, the other drug should be administered to the organism during the period that the first drug is still effective for the organism.

Chemotherapeutic agents include alkylating agents (e.g., nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, and glyphosate), antimetabolites (e.g., nucleotide analogs such as doxifluridine, doxycycline, fluorouracil, mercaptopurine, methotrexate), antitumor antibiotics (e.g., antibiotics such as actinomycin D, doxorubicin, and daunorubicin), antitumor animal and plant components (e.g., vinorelbine, taxol, cephalotaxine, irinotecan, taxotere, and vinblastine), antitumor hormonal agents (e.g., atalmentane, anastrozole, aminoglutethimide, letrozole, formestane, and tamoxifen), and conventional chemotherapeutic agents such as cisplatin, dacarbazine, oxaliplatin, lesonidine, carboplatin, mitoxantrone, and procarbazine.

Targeted drugs include EGFR blockers such as Gefitinib (Gefitinib, Iressa and Iressa) and Erlotinib (Erlotinib, Tarceva), monoclonal antibodies to specific cell markers such as Cetuximab (Cetuximab, Erbitux) and anti-HER-2 mabs (Herceptin, Trastuzumab, Herceptin), tyrosine kinase receptor inhibitors such as Crizotinib (Crizotinib, Xalkori), anti-tumor angiogenesis drugs such as Bevacizumab, endostatin and Bevacizumab, etc., Bcr-Abl tyrosine kinase inhibitors such as Imatinib and Dasatinib, anti-CD 20 mabs such as Rituximab, IGFR-1 kinase inhibitors such as NVP-AEW541, mTOR kinase inhibitors such as CCI-779, ubiquitin-proteasome inhibitors such as Bortezomib, etc.

Other tumor treatment modalities may be selected from one or more of surgical resection, radio frequency ablation, argon helium superconducting surgical treatment, laser ablation therapy, high intensity focused ultrasound, and radiation therapy including X-ray, R-ray, 3D-CRT, and IMRT.

PHF14 used for screening lung cancer therapeutic drugs, and for preparing or screening tumor diagnostic reagent

The PHF14 can be used as a drug or a preparation for screening lung cancer treatment drugs or preparations aiming at the action target of lung cancer cells, and specifically comprises the following steps: the PHF14 can be used as an inhibition target to screen a drug or a preparation so as to find a PHF14 inhibitor capable of reducing the expression level of PHF14 in lung cancer cells, and the PHF14 inhibitor can be used as a candidate drug for treating lung cancer.

The PHF14 can be used for preparing lung cancer diagnostic reagents, and specifically, the PHF14 gene or expression product is used as a diagnostic index of lung cancer for preparing the lung cancer diagnostic reagents. For example, the PHF14 gene or expression product can be used as a standard or positive control for the detection of lung cancer.

The PHF14 can be used for screening lung cancer diagnostic reagents, in particular, the PHF14 gene or expression product is used as a recognition target of lung cancer to screen a reagent for specifically recognizing the PHF14 gene or expression product, so that the reagent is used as a lung cancer diagnostic reagent to detect the lung cancer.

PHF14 and KIF4A are jointly and jointly used for screening lung cancer treatment medicines and preparing or screening tumor diagnosis reagents

PHF14 and KIF4A can be jointly used as a medicine or a preparation and applied to screening of a lung cancer treatment medicine or a preparation aiming at an action target of a lung cancer cell, specifically, PHF14 and KIF4A are jointly used as an inhibition target to screen the medicine or the preparation so as to find an inhibitor capable of simultaneously or respectively reducing the expression levels of PHF14 and KIF4A in the lung cancer cell, and the inhibitor is used as a candidate medicine for treating the lung cancer.

The PHF14 and the KIF4A are jointly used for preparing the lung cancer diagnostic reagent, and particularly, the PHF14 gene or expression product and the KIF4A gene or expression product are jointly used as the diagnostic index of lung cancer to be applied to the preparation of the lung cancer diagnostic reagent. For example, the PHF14 gene or expression product and KIF4A gene or expression product can be used as standard or positive control for lung cancer detection.

PHF14 and KIF4A are jointly used for screening lung cancer diagnostic reagents, specifically, PHF14 gene or expression product and KIF4A gene or expression product are jointly and jointly used as recognition targets of lung cancer to screen reagents which can simultaneously or respectively specifically recognize PHF14 gene or expression product and KIF4A gene or expression product, and thus the reagents are used as lung cancer diagnostic reagents to detect lung cancer.

PHF14 inhibitor (and KIF4A inhibitor) for preparing lung cancer cell mitosis disturbing agent

The mitotic disruptor has at least one of the following functions: the mitosis process of the lung cancer cells is destroyed, and the cell cycle of the lung cancer cells is prolonged. Such disrupting mitotic processes include, but are not limited to, disrupting chromosome condensation, affecting microtubule dynamics, which in turn affects microtubule spindle morphology and localization, affecting cytokinesis.

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

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

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

Example 1

Example 1, test materials and test methods

First, experimental material

1.1 strains: e.coli DH5 α: the strain is used for constructing and amplifying plasmids.

1.2 mammalian cell lines

(1) Hfl-1: the human embryonic lung fibroblast is derived from the lung of a normal male fetus, is in the form of a fibroblast-like structure, has a normal diploid structure, and is cultured and subcultured in an adherent manner to be used as a normal lung cancer cell control group.

(2) Human lung cancer cell line: a549, H2126, CRL-5803, CRL-5807, CRL-5810, CRL-5844, CRL-5872, CRL-5883, CRL5889, CRL-5908 and CRL 5928.

(3) HCT-116: the human colon cancer cell is derived from adult male colon cancer, is in the form of an epithelial-like cell, is cultured and passaged adherently, has strong tumorigenicity, and is deleted and expressed in PHF14 (Ivanov et al, 2007) in the cell line, so that the cell line is selected as a natural PHF14 deleted cell model in subsequent experiments for functional experiments.

1.3 mouse types

The tumor-bearing mouse product adopted in the experiment is as follows: nude mice BALB/cAnN-malm- (BALLc Nude).

Origin: obtained by mating and backcrossing BALB/cABOIl-nu and BALB/cAnNCrj-nu in Charles River Japan (CRJ). Pregnant BALB/CArlNCrj-nu females with strict pedigree records received CRJ introductions in 1985 and bred in the same year. The mice were inbred and genetic monitoring indicated BALB/c nude mice. And (3) hair color: no hair, whitened.

Characteristics and uses: poor growth and development, low fertility and easy generation of serious infection. Thymus is deleted and is recessive inheritance of chromosome 11. T cell immunodeficiency is caused by the inability of the thymus, only thymic remnants or abnormal epithelia, to differentiate normally into T cells. B lymphocyte is normal but has function defect, the antibody is mainly 1gM, only a small amount of 1gG, and the antibody has no contact sensitivity and transplant rejection, so the antibody can be widely used for the research of rabbit epidemics, oncology and disease occurrence mechanism.

1.4 clinical Lung cancer samples

All cancer sample tissues used for the experiments, including renal cell carcinoma, liver cancer and non-small cell lung cancer samples, were obtained from cancer tissues excised after surgery of the patient.

All samples and clinical information were collected with informed consent from the patient or family members, and all sample collection, preservation and handling were in compliance with the relevant legal and ethical committee regulations.

Cancer and paracarcinoma tissues were immediately isolated after surgery and snap frozen in liquid nitrogen. And then stored in a refrigerator at-80 ℃. To extract RNA and protein samples from cancer and paracarcinoma tissues, the tissues were ground into powder in a liquid nitrogen environment using a mortar. Approximately equal masses (50-100mg) of cancerous tissue and paracancerous tissue are used to extract RNA and protein samples.

1.5 plasmids

(1) pRK5-Rs-PHF14WT-myc and pRKs-RS-GFP-PHF14 AMT: the human PHF14 α full-length coding region cDNA was derived from human Hela cells (Huang et al, 2013).

(2) PHF14 deletion mutant: cloning of full-length cDNA and deletion mutants thereof into pRK5-Rs-3 'myc and pRK 5-Rs-5' GFP-unloaded using EcoRI/XbalI and XbaI/SalI enzymatic cleavage sites, PHF14WT (full-length):1-948 aa; PHF14 Delta 1-160:161-948 aa; PHF14 Delta 1-310:311-948 aa; PHF14 Delta 1-410:411-948 aa; PHF14 Delta 1-500:501-948aa.

(3) pEGFP-C1-KIF4AWT and pGEX-5X1-KIF4A858-1232aa(s), and the deletion mutants thereof are cloned into pEGFP-C1 no-load using BglII and SalI enzyme cleavage sites, MS (motor and stage domain) 1-1018 aa; ST (staged and tail domain) 335-; m (motor domain) 1-335 aa; s (staged domain) 335 aa; t (tail domain) 912-1232aa.

(4) pSIREN-RetroQ-NC/297 i/1958: the synthetic oligonucleotide sequences of Negative control siRNA, PHF14siRNA and PHF14RNAi plasmid were cloned into RNAi-ReadypSIREN-RetroQ vector (Clontech, USA), respectively.

(5) pcL10a 1: the reverse transcriptase packaging virus vector is co-transferred to HEK293T cells together with pSIREN-RetroQ-NC/297i/1958i, and is used for packaging and preparing the recombinant virus with high efficiency and easy obtaining.

All plasmid constructs were determined by DNA sequencing after completion.

1.6 antibodies

Anti-KIF 4A: the immunogen of the murine antibody is GST-fused KIF4A WT 858-1232 aa(s).

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

1.7 oligonucleotide strands

Oligonucleotide sequences of Negative control siRNA, PHF14siRNA and PHF14RNAi plasmid synthesized by Shanghai Yingjun Biotechnology limited are as follows:

NC:UUCUCCGAACGUGUCACGU(SEQ ID NO.1)

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

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

the oligonucleotide sequences of Negative control siRNA and KIF4AsiRNA biosynthesized by Shanghai Jima are as follows:

NC:UUCUCCGAACGUGUCACGU(SEQ ID NO.4)

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

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

second, Experimental methods

2.1 Biochemical experiments

1) Extraction of total RNA and protein from animal and human tissue

All tumor tissue RNA used was extracted using Trizol (invitrogen, USA). The procedure was carried out exactly according to the instructions for Trizol reagent. See Trizol instructions for specific test procedures.

After the patient sample tissue was collected, a high-salt lysate (1% NP-40,0.5M NaCl, 10% sucrose,40mM Tris-HCl pH 7.5, and protease inhibitor cocktail) was added, ground using a multi-sample tissue grinder, 65Hz, 1-5min, centrifuged, 13,000rpm, 4 ℃, 30 min, the supernatant was quantitated by BCA method, a loading buffer was added, heated at 100 ℃ for 10 min, and 50g of protein was loaded for electrophoresis.

2) Lung cancer sample tissue immunohistochemical scoring

The lung cancer sample tissue immunohistochemical score was assessed by double-blind assessment. The classification was four-point according to the percentage of positive cells: 1.1-10% positive cells; 2.11-50% positive cells; 3.51-75% positive cells; 4.> 75% positive cells. The staining intensity was classified from low to high: 1. negative; 2. weak positive; 3. positive; 4. strong positive. Scoring results were measured using an Immune Response Score (IRS) standard. The immunoreaction score is Staining Intensity (SI) x positive cell percentage (PP), i.e., IRS is S I × PP. The low to high ratio is 4 grades: 1-4, no positive staining; 5-8, weak positive staining; 9-12, positive staining; 13-16, strong positive staining. The clinical sample data of the patients are recorded and collected under the informed consent of the patients and family members.

3) In vitro binding assay (In vitro binding assay)

The amount of protein in the bacterial supernatant was determined by first using a small amount of purified protein to determine the amount of protein used in each experiment. Cell lysates were first bound for 2 hours with 10. mu.l of Glutathione Sepharose 4B beads to remove some non-specifically bound proteins. Mu.l of Glutathione Sepharose 4B beads was added to the bacterial lysate containing the GST fusion protein, incubated at 4 ℃ for 3-5 hours, washed with 1 XHTNG, centrifuged at 5,000rpm, 4 ℃ for 1 minute. Adding to each sample a cell lysate containing 0.5-1 mg total protein; incubating at 4 ℃ for 3-5 hours or overnight; 1 × HTNG 3 times. The pellet was eluted with loading buffer.

4) Rapid Protein Liquid Chromatography purification (FPLC)

After sub-separation of the Hela nuclear fraction obtained after lysis with 2ml of nucleoleis lysis buffer:

a) and (3) washing the column: the column was flushed with ddH2O for about 2 hours (column volume: 1 ml; flow rate: about 1 ml/min; temperature: 16 ℃ C.).

b) Column equilibration: the column (HiLoad 16/60Superdex 200) was equilibrated to baseline with equilibration buffer (50mM Tris-HCl pH8.0,0.3M NaCl,1mM EDTA).

c) Loading: and (3) loading the sample by using a sample ring, sucking 2ml of Hela cell nucleus component sample into a syringe, pushing bubbles away, and pushing the sample into a sample injection valve for one time.

d) Collecting: buffer was used to try to wash the breakthrough peak back to baseline. Collecting components from the peak, wherein each 200l component is collected until the peak disappears, collecting 344 components, wherein the last 10 components are multi-washing components, and storing the collected sample in a refrigerator at-80 ℃ or detecting.

e) Column washing and preservation: after the end of the collection, the column was washed with ddH2O and finally stored with 20% ethanol.

5) Dot mark (Dot Blotting)

Spotting the sample on a cellulose acetate membrane, after the sample is completely dried and adsorbed on the membrane, sealing the membrane for 1 hour by using 1 xNET-gelatine after the completion, eliminating non-specific binding sites of the antibody, and then performing the following operation steps of immunoblotting, primary antibody incubation, secondary antibody incubation and ECL substrate reaction development.

6) Identification by mass spectrometry

2.2 cell biology experiments

1) Culture of cells

The mammalian cells used in the experiment were all cultured in a 5% CO 237 ℃ incubator with saturated water vapor. All cell culture work was performed on a sterile clean bench. 293T, Hela and Hela GFP-H2B cells were cultured in DMEM and all lung cancer cell lines were cultured in RPMI Medium-1640.

2) Establishment of PHF14RNAi Stable cell Strain

Firstly, according to the result of instantaneous siRNA knockdown, selecting fragments with good RNAi effect, annealing the synthesized nucleotide according to the RNAi-Ready pSIREN-RetroQ plasmid construction method, and connecting the annealed nucleotide into an empty vector. And carrying out enzyme digestion identification to construct a PHF14RNAi plasmid.

The pcL10A1 plasmid was co-transferred with pSIREN-RetroQ-NC/297i/1958i to HEK293T cells of 60% confluence, the supernatant was harvested after 48 hours, stored at 4 ℃ and supplemented with 5ml of culture medium for further culture, and after another 24 hours the supernatant was harvested, combined twice and filtered through HEPES and poly-brene filters at 0.22 μm. Subpackaging and storing at-80 deg.C.

2ml of the virus solution was added to a 6-well plate plated with A549 cells, centrifuged at 2500rpm at 30 ℃ for 30 minutes, the virus solution was removed, cultured at 37 ℃ and screened by adding 500ng/ml puromycin after 24 hours. Control cells all died after two days. After screening, the positive cells were cultured in pool cells, which were part of the stably transfected cells, and 1000 other cells stably expressing PHF14RNAi were seeded in a 10cm dish, and after they formed colonies, monoclonal cells were picked. The PHF14 expression quantity of each cell and pool cell is detected by an immunoblotting method, and clones with good RNAi effect are selected for seed conservation and amplification to carry out subsequent functional experiments.

3) Cell proliferation assay

MTT assay and BrdU incorporation assay were used to measure cell proliferation.

MTT test: after transfection, the cells were seeded in 96-well plates, each containing 1000 cells and 5000 cells, one set of the cells was taken after the cells were attached to the wall, 20l of 5mg/ml MTT was added to the culture solution, the culture solution was cultured for 4 hours, the culture solution was removed, 100. mu.l of DMSO was added, the purple crystals formed were dissolved by standing in the dark for 30 minutes, the light absorption at a wavelength of 570nm was measured using a spectrophotometer, each set of three wells was repeated, and the measurement was performed every 24 hours thereafter, and the experiment was continued for 5 to 7 days. Results of three replicates were statistically analyzed.

BrdU incorporation experiments: transiently transfected cells were transfected 48 hours before BrdU incorporation experiments. Specific experimental procedures were performed according to BrdU incorporation assay kit instructions (Roche, Pensberg, Germany). The brief record is as follows: after the cell density reached 50-70%, BrdU was added to the culture medium to a final concentration of 10. mu.M and the culture was continued for 30 minutes, and the cell nuclei of the fixed cells, Brdu positive cells and all cells were stained with BrdU antibody and DAPI, respectively. The percentage of BrdU and DAPI positivity was recorded. At least 1000 cells were counted per cell. Results of three replicates were statistically analyzed.

4) Soft agar colony formation assay

Dissolving agar in deionized water to obtain 0.6% and 1.2% solutions, sterilizing at high temperature, and storing at 4 deg.C. Before the experiment, soft agar was melted in a microwave oven and placed in a water bath at 40 ℃. 1.2% soft agar and preheated 2 XDMEM at 40 ℃ are mixed in equal proportion, 1/10 volume of fetal calf serum is added, 2ml of fetal calf serum is uniformly mixed and then evenly spread in a 6-hole culture dish, and the mixture is kept stand for 15 minutes at room temperature until the agar is solidified. The laid 6-hole plate can be put in a refrigerator at 4 ℃ for standby. Mixing 0.6% soft agar with 2 × DMEM at 40 deg.C at equal ratio, adding 1/10 volume fetal calf serum, mixing, inoculating 2500 cells per ml, spreading 2ml in 6-well plate with bottom agar, standing at room temperature for 15 min to allow the upper gel to coagulate. Culturing in 37 deg.C incubator. After two weeks of culture, colonies with a cell number of more than 50 were counted under a microscope.

5) In vivo tumor formation experiment in mice

Each cell was cultured in a petri dish to about 90% confluence. Trypsinized and resuspended in incomplete medium. The cell suspension was inoculated subcutaneously into the right scapula of nude mice using a 1ml syringe. Each cell was injected 4X 10 per mouse⁶And (4) cells. The maximum and minimum diameters of the formed tumors were measured with a vernier caliper every other week. The volume of tumor formed was calculated according to the following formula: volume 1/2 x (maximum diameter) x (minimum diameter)². At least 4 mice were inoculated per cell. The volume of the formed tumors is expressed as mean ± SD (standard deviation).

6) HeLa Living body imaging recording (Live-cell imaging)

Transfecting various groups of Hela GFP-H2B stable-transformed cell strains by using siRNA, then shooting by using a living cell high-speed laser confocal and total internal reflection multi-dimensional image workstation, selecting 3-5 visual fields for each group, shooting once every 5 minutes for 8-12 hours, and synthesizing a continuous image file.

7) Determination of cell division index (Mitotic index)

Hela cells are paved on a 24-well plate, are subjected to siRNA transfection treatment for 48 hours, are fixed and are subjected to DAPI staining, the cells in the 24-well plate are subjected to full-automatic high-flux bright field and fluorescence microscopic imaging by an Operetta high content cell analysis system of Perkinelmer company in a turntable confocal imaging mode, DAPI channel full-well shooting is carried out, and the microscopic images are rapidly subjected to fluorescence intensity analysis and cell morphology analysis and data statistics treatment by using powerful Harmony software. Cell mitosis index is mitotic cell number/total cell number x 100%.

Data statistics and analysis

1) Data statistics

All immunoblots were quantitated using Quantity One software (Bio-Rad laboratories Inc., Hercules, Calif.). All data were statistically analyzed using Excel 2003(Microsoft corp., Redmond, Wash.) software. The statistical data are expressed using mean ± SD (standard deviation). Paired, two-tailed student t-test analysis to account for statistical differences between the two groups. P <0.05 was considered significantly different; p <0.01 is considered to be very significantly different.

2) Clinical case data analysis

GEO Database (GSE19188) was derived from NCBI, and expression levels of unpaired tumor tissue and paracancerous tissue were detected and analyzed by Welch's correction using PHF14(reporter:229085_ at) as a probe. Calibration Analysis and Kaplan-Meier plot of subvalval Analysis were analyzed using the software SPSS 16.0software (SPSS Inc., 1989-2007). Fisher's Exact Test according tohttp:// www.langsrud.com/fisher.htm。

Example 2 detection of expression levels of PHF14 in tumor samples

To examine the relationship of PHF14 to cancer, five types of clinical tumor samples and their corresponding paracancerous samples (renal, gastric, colon, liver and non-small cell lung cancer) were collected. We found that PHF14 was significantly upregulated in lung cancer, whether at gene copy number, mRNA, or protein level, expression of PHF14 (fig. 1A-D). WB and immunohistochemistry results suggest that more than 80% of lung cancer tissue samples show high expression levels of PHF14 protein relative to paracarcinoma tissues. qPCR also showed that 71% of patients had PHF14 increased at the mRNA level. Analysis of the correlation between PHF14 expression level and clinical data (Table 1) shows that high expression (score >12) is correlated with adenocarcinoma, and is significantly high expression (score ≧ 9) in early stage lung cancer (TNM I, or tumor diameter ≦ 3 cm). We further carried out bioinformatics calculation by searching published lung cancer related microarray dataset, analyzed PHF14 expression in more lung cancer specimens and correlation with clinical indexes, and searched universal rule of PHF14 expression in lung cancer. The results show that PHF14 is in multiple numbers

The databases (Cancer Genome Atlas, GSE74095and GSE74116) all present a high expression profile in lung Cancer samples, and the expression level is inversely proportional to the survival time of the patients (fig. 1E).

TABLE 1 analysis of correlation between immunohistochemical score and clinical pathological data of clinical specimen of PHF14 lung cancer

Example 3 Down-regulation of PHF14 expression significantly inhibited lung cancer cell proliferation

Based on the discovery of clinical sample level of tumor patients, a humanized lung cancer cell strain is selected for functional experiments. The influence of the expression down-regulation of PHF14 on the lung cancer proliferation and the tumor formation is observed through the expression of PHF14 in two different human lung cancer cells, namely a knock down A549 cell and a CRL-5810 cell by an RNAi method. The MTT result shows that PHF14 knockdown significantly inhibits the growth and proliferation of A549 and CRL-5810 lung cancer cells (fig. 2A, 2B); BrdU incorporation experiments (measuring the rate of DNA synthesis) also demonstrated that decreasing PHF14 expression inhibited DNA synthesis and thus cell proliferation. In addition, we constructed the PHF14 expression plasmid for RNAi resistant to show that cell growth is slowed down after resee PHF14 knock down

Type, the results show: the observed slowing of cell proliferation can be rescued by expression of the rescue plasmid (fig. 2C). The results show that: the reduction of PHF14 expression can obviously inhibit the proliferation of lung cancer cells.

Example 4 Down-regulation of PHF14 expression significantly affected lung cancer cell proliferation, malignant transformation, and tumorigenicity in mice

We then selected 1958i this RNAi fragment to prepare PHF14 stable RNAi A549 cell line, and the established PHF14 stable knock down A549 cell line also showed slow cell proliferation (FIG. 3A). To examine whether expression of PHF14 correlates with malignant transformation of lung cancer, we performed colony formation in soft agar and in vivo tumor formation in nude mice. The stable cell strains A549/NC and A549/Cln2, A549/Cln9 cells are respectively planted in soft agar, and the A549/NC cells grow into a cell mass consisting of a plurality of cells in the soft agar after two weeks. Whereas the a549/Cln2 and a549/Cln9 cells failed to divide in soft agar, or only divided into cell clumps of less than ten cells. The results show that: expression of PHF14 was associated with anchorage-independent growth of lung cancer cells, and thus likely with malignant transformation of lung cancer (fig. 3B). In a nude mouse tumorigenicity experiment, the nude mouse tumorigenicity ability of A549 was remarkably inhibited after PHF14 expression was down-regulated (FIG. 3C). The results show that down-regulation of PHF14 expression significantly affects lung cancer cell proliferation, malignant transformation, and tumorigenic capacity in mice.

Example 5 interaction of PHF14 with KIF4A

In order to deeply research the action mechanism of PHF14 biological functions, we combined with SILAC (Stable Isotope Labeling with Amino Acids in Cell cultures), co-immunoprecipitation and MALDI-TOF/TOF mass spectrometry identification methods, found many proteins that may interact with PHF14 in HeLa cells, and the proteins mainly focused on DNA damage repair, transcriptional regulation, Cell division and proliferation and other aspects to exert biological effects. Of interest to us is chromatin motor protein KIF4A (also highly expressed specifically in lung cancer). Separating Hela cell nucleus components by Fast Protein Liquid Chromatography (FPLC) column, and detecting and identifying PHF14 possible interacting protein complex by dot blot, immunoblot, etc. As a result, it was found that: in the fast protein liquid chromatography experiments, PHF14, KIF4A, PARP-1 and Ku70 were all enriched in the same several components, suggesting that these proteins may be co-located in one large protein complex (fig. 4A). Immunoprecipitated PHF14 was able to detect KIF4A co-immunoprecipitated in a549 cells, and the same results were obtained after the addition of EB to exclude DNA-mediated protein binding, as well as the reverse co-immunoprecipitation results (fig. 4B). Immunofluorescence and laser confocal results show that: PHF14 and KIF4A co-localized in the nucleus during interphase, on chromatin, and on midbody during cytokinesis, both had good co-localization during the cell cycle in a549 cells (fig. 4E). Subsequently, we wanted to further define by which domains KIF4A and PHF14 interact. Through a series of constructions of mutant protein expression plasmids of PHF14 and KIF4A and co-immunoprecipitation experiments, we found that KIF4A interacted with PHF14N at 1-160aa(s) through its Stalk region (fig. 4C & D). In vitro binding of GST pull down assay also confirmed that binding of both was necessary at end 1-160aa(s) of PHF 14N. The combination of the above experimental results shows that: PHF14 binds directly to KIF4A in the region of 1-160aa(s) at the N-terminus of PHF14 and the Stalk domain of KIF 4A.

Interestingly, both were highly expressed and significantly positively correlated in both lung cancer cell lines and lung cancer samples (fig. 5A-C), not only at the mRNA level, but also at the protein level. Inhibition of PHF14 and/or inhibition of KIF4A both significantly inhibited a549 cell proliferation (fig. 5D), and overexpression of PHF14, full-length, promoted HCT-116 cell proliferation, but overexpression of PHF14 NT 1-160aa(s) mutant that did not bind to KIF4A, did not promote cell proliferation (fig. 5E), suggesting that PHF14 and KIF4A may play a synergistic role in promoting cell proliferation.

KIF4A is a chromatin motor protein that plays an important role in cell division. The literature reports that when cells enter mitosis, a deletion of KIF4A disrupts chromosome condensation; the dynamic change of the microtubules is influenced, and the shape and the positioning of microtubule spindles are further influenced; affecting cytokinesis, etc. (Bastos et al, 2014; Bastos et al, 2013; Kurasawa et al, 2004 a; 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). We speculate whether PHF14 might play a role in mitosis through interaction with KIF 4A. We down-regulate PHF14 expression by transient siRNA in Hela cells, and the immunofluorescence results show that: after inhibition of PHF14 expression, the mitotic cells showed abnormalities in both microtubule and chromosomal morphology and distribution (fig. 6A). Similar phenomena of chromosome aggregation, abnormal arrangement, aneuploidy, and abnormal microtubules were also observed in cell lines in which a549 stably inhibited PHF14 (fig. 6B). In vivo imaging experiments recorded that in the absence of PHF14, mitosis was affected once cells entered M phase, M phase was extended, cell cycle was extended, and in the absence of KIF4A we seen a similar phenotype with significant cell cycle retardation when both PHF14 and KIF4A were knocked out (fig. 6C). Transient sirnas inhibited expression of PHF14 and KIF4A, respectively, or both, significantly affecting mitotic index, suggesting a high probability of causing cell cycle prolongation (fig. 6D). Our study found that: RNAi PHF14 and RNAi KIF4A act similarly, and both disrupt chromosome aggregation and alignment, affect microtubule spindle morphology and localization, and cause prolongation of M phase. Deletion of PHF14 had a more pronounced effect on chromosomes than did KIF4A, whereas inhibition of KIF4A expression appeared to have a stronger effect on microtubules than did PHF14, with both expression being inhibited with the greatest effect on cell mitosis and cell cycle.

Example 6, summary and discussion

First, summarize the study that PHF14 and KIF4A are combined to participate in lung cancer development

1. The expression level of PHF14 in kidney cancer, colorectal cancer, gastric cancer and liver cancer has no significant change; and the high expression is obviously high in lung cancer tissue samples with over 80 percent of non-small cell lung cancer, and the high expression is obviously related to adenocarcinoma and poor clinical prognosis of lung cancer patients.

2. At the cellular level, down-regulation of PHF14 expression significantly inhibited lung cancer cell proliferation, malignant transformation, and tumorigenic capacity in mice.

3. PHF14 binds directly to KIF4A in the region of from 1 to 160aa(s) at the end of PHF14N to the staged domain of KIF 4A. Both have good co-localization during the cell cycle.

4. Over-expression of PHF14 or KIF4A in full length can remarkably promote cell proliferation, inhibition of PHF14 and/or KIF4A expression can remarkably inhibit cell proliferation, and combination of PHF14 and KIF4A can play an important role in the cell proliferation process.

5. Similarly to down-regulation of KIF4A, down-regulation of PHF14 disrupts chromosome aggregation and alignment, affects microtubule spindle morphology and localization, and results in an increase in the M phase of the cell cycle. And simultaneously, the expression of the two is inhibited, and the influence on cell mitosis and cell cycle is the largest.

6. PHF14 and KIF4A are highly expressed in non-small cell lung cancer clinical samples and cell lines, and the increase of the expression level is obviously related.

Second, discussion of results

Lung cancer is caused by irreversible, unlimited proliferation of lung cells resulting from a complex series of genetic and epigenetic changes in the genes. Our study found a new biomarker for lung cancer development, PHD finger protein 14(PHF 14). The nucleoprotein is highly conserved and widely expressed in evolution, is highly expressed in a lung cancer patient sample and is related to poor clinical prognosis of the patient, and has obvious influence on lung cancer cell proliferation, malignant transformation and tumor forming capability; has obvious effect on cell life activities such as cell proliferation, mitosis and cell cycle. Our work revealed for the first time that PHF14 directly binds and co-localizes with a potential tumor marker, chromatin motor protein KIF4A, both are highly expressed and significantly associated in lung cancer patient specimens, and the interaction thereof mainly depends on the binding of PHF14N terminal 1-160aa(s) with KIF4A Stalk domain, and the binding of both may play an important role in the cell proliferation process. Our results suggest that: PHF14 may be a new potential lung cancer diagnostic marker involved in lung cancer development by interacting with KIF 4A. Our work is expected to provide strong scientific evidence from clinical patient sample levels, cellular functional levels, and molecular levels for the pathogenesis of and clinical treatment of lung cancer.

The PHD finger protein is a zinc finger protein which is widely existed in eukaryote and plays an important role in gene transcription regulation and disease occurrence. In recent studies, PHD finger proteins have been classified as a class of proteins that recognize histone methylation modifications (Lan et al, 2007; Org et al, 2008; Shi et al, 2006; Wysocka et al, 2006), and this family of proteins plays an important role in regulating cell epigenetic modifications. Different PHD finger proteins specifically recognize different "histone marks", and regulate the transcriptional expression of genes by either modulating their intrinsic activity or by modulating the activity of their interacting proteins (Baker et al, 2008). An increasing number of studies have shown that when many genes encode PHD finger structures that undergo point mutations, deletions or chromosomal translocations, these abnormalities often trigger the development of human diseases, including: tumors, mental retardation, immunodeficiency, etc., which further underscore the important role that PHD finger protein plays as an epigenetic "reader" in the development of disease (Baker et al, 2008). The development of cancer has long been recognized as a combined genetic and epigenetic process of alteration that ultimately contributes to the development and progression of cancer (Jones and Baylin, 2007). Cancer-related mutations and dysregulations are found to occur in various enzymes associated with post-translational modification of histones (Wang et al, 2007a, b). Our pre-laboratory findings found that PHF14 is able to bind histone H3 via its two domains, PHD1 and PHD3, and is able to undergo auto-dimerization (Huang et al, 2013). Structurally, PHF14 contains multiple finger and nuclear localization sequences, and therefore, we speculate that PHF14 is likely to be a "histone mark" reader, and by its own regulation or its regulation of histones, it affects the transcriptional expression of genes in the nucleus, and further affects important cellular activities such as cell proliferation and division, and is involved in tumorigenesis. Meanwhile, we found that many PHD finger proteins are reported to influence the development of tumors not only through epigenetic regulation, but also participate in the regulation of multiple aspects such as protein degradation, signal transduction and cell migration to promote the development of tumors (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 conducted extensive studies on the mechanism of PHF14 involved in lung cancer development.

There are few reports on the study of PHF14 in the involvement of tumorigenesis, and initial studies at the cellular level speculate that PHF14 is a possible colon cancer suppressor gene, but its exact function is unclear (Ivanov et al, 2007). Recent studies have shown that PHF14 is abnormally expressed in biliary tract cancer cells, and inhibits expression to promote proliferation, suggesting involvement in biliary tract carcinogenesis, but the mechanism of action is not clear (Akazawa et al, 2013). Since both studies were performed on single cell lines, it was not excluded that they were cell line specific or suggested that PHF14 might function differently in different cells/tumors, and the clinical significance of these results was unclear given that they had no relevant clinical data and studies. Our results show that: the expression level of PHF14 in kidney cancer, liver cancer, colorectal cancer and stomach cancer has no obvious change; and the expression is obviously high in lung cancer tissue samples with over 80 percent of non-small cell lung cancer. In clinical level, a plurality of tumor sample databases are screened, KM-plot survival analysis results show that the clinical prognosis of lung cancer patients with high PHF14 expression is poor, and further, the PHF14 is suggested to play an important role in the occurrence and development of lung cancer. The obtained clinical case data are analyzed to find that the high expression of the PHF14 gene is related to the pathological typing of the lung cancer tissue and the gene copy number; the high expression of the KIF4A gene is related to the pathological typing of lung cancer tissues, and is obviously related to the pathological differentiation and gene copy number of the lung cancer. In general, from the level of clinical tissue samples, PHF14 is specifically and significantly highly expressed in lung cancer patients and has a poor clinical prognosis, and thus it is presumed to play an important role in promoting the development of lung cancer.

The mechanism research of PHF14 participating in the occurrence and development of lung cancer shows that PHF14 and KIF4A are both highly expressed in the lung cancer, and the expression amounts are in positive correlation. The biological functions of KIF4A are primarily reported to be involved in cellular mitosis and cytokinesis (Bastos et al, 2014; Bastos et al, 2013; Kurasawa et al, 2004 a; Lee et al, 2001; Mazumdar et al, 2004; Samejima et al, 2012; shreshatha et al, 2012; Singh et al, 2014; Stumpff et al, 2012; Takemoto et al, 2009; wandet al, 2012; Wu and Chen, 2008; Zhu and Jiang, 2005; Zhu et al, 2005), DNA damage and repair (Lee and Kim, 2003; Wu et al, 2008b), and tumorigenesis (Gao et al, 2011; mamdar et al, 2014 et al, 2013; taniaal, 2007). Tumors were more likely to develop in mice ES cells that knock out KIF4 (Mazumdar et al, 2006). KIF4A is highly expressed in lung cancer patients and is associated with poor patient clinical prognosis, with significant effects on cell proliferation and tumor invasion (Taniwaki et al, 2007 b). In gastric cancer patients, KIF4A was low expressed and had inhibitory effects on cell proliferation and tumor-forming ability in mouse nude mice after overexpression (Gao et al, 2011). KIF4A is highly expressed in oral squamous cell carcinoma, and promotes cell proliferation and oral squamous cell carcinogenesis by activating Spindle Assembly Checkpoint (SAC). To date, PHF14 and KIF4A have been reported in limited studies in tumors. Our studies demonstrated for the first time that PHF14 interacts with KIF4A, a possible lung cancer occurrence marker that is both highly expressed and significantly associated in lung cancer, and that its binding has a significant effect on cell proliferation, consistent with previous reports (Taniwaki et al, 2007b), and therefore we speculate that both play important biological roles in lung cancer development. Previous studies show that KIF4A is highly expressed in lung cancer and has important influence on cell proliferation and tumor invasion (Taniwaki et al, 2007b), and the results show that KIF4 may play an important role in the development process of lung cancer as a clinical lung cancer prediction marker, and may be used for developing antitumor drugs and tumor vaccines. The research finds that PHF14 and KIF4A are up-regulated and significantly related in high expression in clinical lung cancer samples and cell lines, and PHF14 plays an important role in the processes of cell proliferation, mitosis, tumor invasion and tumor forming capacity, so that the inventor speculates that PHF14 is a potential lung cancer marker to influence the occurrence and development of lung cancer, and PHF14 is very likely to become a good drug target protein.

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

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Claims (9)

1. Use of a PHF14 inhibitor for the preparation of a medicament for the treatment of lung cancer, said PHF14 inhibitor being an siRNA or shRNA.
2. 2. The use according to claim 1, wherein the lung cancer therapeutic agent has at least one of the following functions: can obviously inhibit the growth and proliferation of the lung cancer cells, inhibit the deterioration of the lung cancer cells, inhibit the tumorigenic capacity of the lung cancer cells, destroy the mitotic process of the lung cancer cells and prolong the cell cycle of the lung cancer cells.
3. 3. The use of claim 1, wherein the PHF14 inhibitor inhibits PHF14 activity, or inhibits PHF14 gene transcription or expression.
4. 4. A drug combination product for combined treatment of lung cancer comprises effective amounts of a PHF14 inhibitor and a KIF4A inhibitor, wherein the PHF14 inhibitor is siRNA or shRNA, and the KIF4A inhibitor is siRNA or shRNA.
5. The use of a combination of a PHF14 inhibitor and a KIF4A inhibitor, said PHF14 inhibitor being an siRNA or shRNA, for the manufacture of a medicament for the treatment of lung cancer; the KIF4A inhibitor is siRNA or shRNA.
6. 6. The use according to claim 5, wherein the lung cancer therapeutic agent has at least one of the following functions: can obviously inhibit the growth and proliferation of the lung cancer cells, inhibit the deterioration of the lung cancer cells, inhibit the tumorigenic capacity of the lung cancer cells, destroy the mitotic process of the lung cancer cells and prolong the cell cycle of the lung cancer cells.
7. 7. A lung cancer combined treatment drug combination product comprises effective dose of PHF14 inhibitor, KIF4A inhibitor and at least one other lung cancer treatment drug, wherein the PHF14 inhibitor is siRNA or shRNA; the KIF4A inhibitor is siRNA or shRNA.
8. Use of a PHF14 inhibitor for the preparation of a lung cancer cell mitotic disruptor, said PHF14 inhibitor being an siRNA or shRNA.
9. Use of a PHF14 inhibitor and a KIF4A inhibitor in combination for the preparation of a lung cancer cell mitotic disruptor, the PHF14 inhibitor being an siRNA or shRNA and the KIF4A inhibitor being an siRNA or shRNA.

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