CN102961739A

CN102961739A - Application of KLOTHO protein

Info

Publication number: CN102961739A
Application number: CN2012105330757A
Authority: CN
Inventors: 黄曙; 汤小伟; 季国忠; 范志宁; 王敏
Original assignee: 2nd Affiliated Hospital of Nanjing Medical University
Current assignee: 2nd Affiliated Hospital of Nanjing Medical University
Priority date: 2012-12-12
Filing date: 2012-12-12
Publication date: 2013-03-13

Abstract

The invention provides application of a KLOTHO protein in preparation of medicines for treating liver cancer. An amino acid sequence of the KLOTHO protein is shown in SEQ NO.1; and the invention also provides application of the KLOTHO protein in preparation of formula for diagnosing the liver cancer, and the amino acid sequence of the KLOTHO protein is shown in SEQ NO.1. The application of the KLOTHO protein has the following technical effects that: (1) the prediction occurrence of the liver cancer is reduced by detecting expressions of individual liver tissues and (or) KLOTHO proteins in blood and related composition components because the KLOTHO protein expression in the cancer tissues of a liver cancer patient is lower than para-carcinoma tissues; (2) the liver cancer is treated by improving the expression level of the KLOTHO proteins and related composition components in the liver cancer tissues because the growth of the liver cancer is inhibited by the KLOTHO proteins; and (3) because the KLOTHO participates a pathological process related to cell activities of malignant proliferation, apoptosis, invasion and the like, experience and application base is provided to deeply discuss about the relation between KLOTHO and other diseases for the future.

Description

The purposes of KLOTHO albumen

Technical field

The invention provides the purposes of KLOTHO albumen, belong to the biological medicine technology field.

Background technology

Primary hepatocellular carcinoma (hcc) (is called for short hepatocarcinoma, HCC) be one of modal malignant tumor in the world wide, the data show of in December, 2007 ACS (ACS) issue: increase 66.7 ten thousand examples hepatocarcinoma year, dead 59.8 ten thousand examples, wherein about 50% new trouble case and death are in China, be that hepatocarcinoma is larger to China people's health threat, become China second tumor killer, the patient suffering be can't bear, survival rate is low, and brings serious economy and psychological burden for family, society.

Klotho is discovery in 1997 and old and feeble relevant gene, is positioned at human chromosomal 13q12, and length is about 50kb, is comprised of 5 exons and 4 introns.The cDNA of Koltho gene contains the different fragment of two-stage nitration, the two kinds of different albumen of encoding respectively: one is membranous type, is positioned on the cell membrane, and the protein sequence of its extracellular region is similar to beta-glucosidase; Two is secreting type, is present in the blood circulation, is considered to a kind of peptide hormone.The expression deletion of Klotho gene in mice causes its various phenotypes that the mankind aging occurs being similar to, such as arteriosclerosis, osteoporosis, emphysema, the lost of life, atrophoderma, infertility, movement disorder etc.In recent years, increasing research discovery klotho gene can be regulated IGF, the signal paths such as TGF-β 1, FGF, participate in the oxidative stress process, and relevant with the immunodeficiency of body, prompting klotho plays a significant role in the carcinogenesis of human of malignant tumor, and can further utilize klotho to be used for the antineoplastic treatment as the mark of tumor generation, classification and as target spot.

Domestic and foreign literature and patent to prior art are retrieved, and so far there are no, and any KLOTHO albumen and relevant components composition thereof are used for the research report that hepatocellular carcinoma is diagnosed and treated.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art part, the purposes of a kind of KLOTHO albumen in the preparation cancer treatment drug is provided, the aminoacid sequence of described KLOTHO albumen is shown in SEQ NO.1.

Another object of the present invention provides the purposes of a kind of KLOTHO albumen in preparation diagnosing cancer of liver preparation, and the aminoacid sequence of described KLOTHO albumen is shown in SEQ NO.1.

The present invention has following technique effect:

(1) the KLOTHO protein expression is lower than cancer beside organism in the liver cancer patient cancerous tissue, by detect individual hepatic tissue and (or) in the blood expression of KLOTHO albumen and relevant components composition thereof reduce the generation of predicting liver cancer;

(2) KLOTHO albumen is inhibited to liver cancer growth, by the expression Hepatoma therapy of KLOTHO albumen and relevant components composition thereof in the raising liver cancer tissue;

(3) because klotho has participated in the relevant pathological process of the cellular activities such as malignant proliferation, apoptosis, invasion and attack, so the present invention provides experience and application foundation to the relation of further investigated klotho and other diseases from now on.

Description of drawings

Fig. 1 is hepatocarcinoma and the KLOTHO of cancer beside organism protein immunization group dyeing (* 400) result: (A) KLOTHO albumen strong positive in renal cells is expressed, as the positive control of KLOTHO protein immunization group dyeing; (B) KLOTHO albumen high expressed in hepatocarcinoma cancer beside organism; (C) KLOTHO albumen low expression in liver cancer tissue.

Fig. 2 is the Kaplan-Meier survival curve.

Fig. 3 is that Klotho high-expression vector pCMV6-Entry-KL raises KLOTHO protein expression effect;

Fig. 4 is the propagation result that transfection Klotho high-expression vector pCMV6-Entry-KL suppresses hepatoma Hep G 2 cells;

Fig. 5 is the apoptosis result that transfection Klotho high-expression vector pCMV6-Entry-KL promotes hepatoma Hep G 2 cells;

Fig. 6 is that transfection klotho gene specific siRNA suppresses KLOTHO protein expression effect;

Fig. 7 is the propagation result that transfection klotho gene specific siRNA promotes hepatoma Hep G 2 cells;

Fig. 8 is the apoptosis result that transfection klotho gene specific siRNA suppresses hepatoma Hep G 2 cells;

Fig. 9 suppresses the propagation result of hepatoma Hep G 2 cells for adding exogenous secreting type KLOTHO albumen;

Figure 10 is that the exogenous secreting type KLOTHO of lumbar injection albumen is to tumor bearing nude mice liver cancer tissue affects on the growth result;

Figure 11 is that the exogenous secreting type KLOTHO of lumbar injection albumen is on the result that affects of KLOTHO protein expression in the tumor bearing nude mice liver cancer tissue.

The specific embodiment

Further specify the present invention below in conjunction with specific embodiment.Should be noted that following examples only are used for explanation the present invention, limit the scope of the invention and be not used in.

The KLOTHO albumen of mentioning in the following example is the KLOTHO albumen of aminoacid sequence shown in SEQ NO.1.

The KLOTHO protein expression is lower than the clinical meaning of cancer beside organism in the embodiment 1 liver cancer patient cancerous tissue

Adopt Immunohistochemical method, detect the expression of KLOTHO albumen in the organization chip that is formed by 52 routine hepatocarcinoma and 54 routine cancer beside organisms, and carry out statistical analysis in conjunction with clinical and pathological data.The result shows shown in Fig. 1, Fig. 2, table 1,2 KLOTHO protein expression positive rate is lower than cancer beside organism in the liver cancer tissue( P＜0.05); And the clinical stages of KLOTHO protein expression and hepatocarcinoma, is negative correlation, is proportionate with the case classification in the liver cancer tissue( P＜0.05), with the dependency not statistically significant of Gender, age, tumor size etc. ( P0.05).

The liver cancer patient of including research in is carried out survival analysis to be found The positive expression of KLOTHO albumen becomes positive correlation with patient's prognosis(mean survival time is 28.132 months to the life span of KLOTHO protein positive group, 95% credibility interval CI is 23.726-32.538) (mean survival time is 16.772 months than the negative group of KLOTHO albumen life span, 95% credibility interval CI is 11.550-21.993) long, the Log-rank check show two groups of differential survivals have statistical significance ( P=0.016).

COX risk ratio model analysis presentation of results KLOTHO can be used as independently predictor life cycle( P=0.043). KLOTHO albumen high expressed may delay the death of hepatocarcinoma patient, it is lower that the patient that KLOTHO expresses the positive carves dead relative risk at a time than the negative patient of klotho expression, is 0.397.

The expression of these results suggest KLOTHO albumen in hepatocarcinoma reduces, and may participate in generation and the progress of hepatocarcinoma, and judging for the diagnosis and prognosis of hepatocarcinoma provides reference frame.

Embodiment 2 confirms the effect of klotho in hepatocarcinoma occurs and makes progress in the cell model level

(1) transfection klotho high-expression vector suppresses the growth of hepatoma carcinoma cell and the apoptosis of promotion hepatoma carcinoma cell

As shown in Figure 3, its contrast empty carrier of transfection klotho high-expression vector pCMV6-Entry-KL(pCMV6-Entry in hepatoma carcinoma cell HepG2) after 48 hour, detect KLOTHO protein expression level checking high-expression vector effect by Western Blot, the result shows that the KLOTHO protein expression obviously raises among the hepatoma carcinoma cell HepG2 ^* P＜0.05, show that this klotho high-expression vector can effectively raise the KLOTHO protein expression.

Hepatoma carcinoma cell HepG2 is at its contrast of transfection klotho high-expression vector pCMV6-Entry-KL(empty carrier pCMV6-Entry) after 48 hours, detect cell proliferation and Apoptosis by Flow Cytometry situation by MTT.The result show its cell proliferation be suppressed ( ^* P＜0.05, as shown in Figure 4), increasing apoptosis many ( ^* P＜0.05, as shown in Figure 5), prompting KLOTHO protein expression increases the growth that has suppressed hepatoma carcinoma cell and the apoptosis that promotes hepatoma carcinoma cell.

(2) the Klotho gene RNA disturbs the growth that promotes hepatoma carcinoma cell and the apoptosis that suppresses hepatoma carcinoma cell

As shown in Figure 6, in hepatoma carcinoma cell HepG2, the siRNA(siRNA-kl of transfection klotho gene specific organizes respectively) and irrelevant sequence contrast siRNA (siRNAc group) 48h, the interference effect that the KLOTHO protein expression level is verified klotho gene specific siRNA detected by Western Blot.The result shows that the KLOTHO protein expression obviously reduces among the hepatoma carcinoma cell HepG2, ^* P＜0.05, prompting klotho gene specific siRNA can effectively reduce the KLOTHO protein expression.

Transfection klotho gene specific siRNA(siRNA-kl group among the hepatoma carcinoma cell HepG2) and the irrelevant sequence siRNA(siRNAc group of its contrast) after 48 hours, by MTT detection cell proliferation and Apoptosis by Flow Cytometry situation.The result show its cell proliferation accelerate ( ^* P＜0.05, as shown in Figure 7), apoptosis obviously reduce ( ^* P＜0.05, as shown in Figure 8), prompting KLOTHO albumen reduces the growth that has promoted hepatoma carcinoma cell.

(3) exogenous secreting type KLOTHO albumen suppresses the growth of hepatoma carcinoma cell

In hepatoma carcinoma cell HepG2, add respectively exogenous secreting type KLOTHO albumen 0.05 μ g, 0.1 μ g, 0.15 μ g, 0.2 μ g, 0.25 μ g and its solvent control and process after 48 hours, detect the multiplication capacity of hepatoma Hep G 2 cells by MTT.The result as shown in Figure 9, the result shows, along with increasing of exogenous secreting type KLOTHO albumen concentration for the treatment of, concentration dependent ground suppresses the growth of hepatoma carcinoma cell.

Embodiment 3 confirms that in the animal model level KLOTHO albumen is to the inhibitory action of liver cancer growth

The BALB/cASlac-nu type nude mice in 10 6 ~ 8 ages in week, male and female half and half (being purchased from Jiangsu Province Nanjing Medical University zoopery center).Be 2 * 10 at every nude mice back subcutaneous injection cell concentration ⁶/ 100 μ l HepG2 cells 200 μ l observed for 2 weeks, will become 8 nude mices of tumor success to be divided at random two groups, injecting normal saline in one group of abdominal cavity (Control group), the other one group of exogenous secreting type KLOTHO of lumbar injection protein solution.Every injection in 2 days once, altogether inject 3 times.Injected rear 5 weeks of Continuous Observation for the last time, measured respectively weekly the size (as shown in table 3) of tumor bearing nude mice liver cancer tissue; And when the 5th week was put to death nude mice, measure the weight (shown in Figure 10 and table 3) of liver cancer tissue, find that exogenous secreting type KLOTHO albumen is processed after, size and the weight of tumor bearing nude mice liver cancer tissue are significantly less than matched group, P＜0.05, show that KLOTHO albumen can suppress the growth of liver cancer tissue in the tumor bearing nude mice.

Simultaneously, the albumen of the tumor bearing nude mice liver cancer tissue after 5 weeks is observed in extraction, carries out Western Blot and detects.As shown in figure 11, after the result showed the exogenous secreting type KLOTHO of tumor bearing nude mice lumbar injection albumen, the KLOTHO protein expression in its liver cancer tissue increased.

In sum, the KLOTHO protein expression is reduced in the genesis of hepatocellular carcinoma and plays an important role, can by detect individual hepatic tissue and (or) in the blood expression of KLOTHO albumen and relevant components composition thereof reduce the generation of predicting liver cancer, improve individual and (or) expression of KLOTHO albumen and relevant components composition thereof is used for the treatment of hepatocarcinoma in the liver cancer tissue, KLOTHO albumen and relevant components composition thereof judge for diagnosis, the prognosis of hepatocarcinoma and treatment biology provides new reference frame.

Organization Applicant

----------------------

Street :

City :

State :

Country :

PostalCode :

PhoneNumber :

FaxNumber :

EmailAddress :

＜110〉OrganizationName: The Second Affiliated Hospital of Nanjing Medical University

Individual Applicant

--------------------

Street :

City :

State :

Country :

PostalCode :

PhoneNumber :

FaxNumber :

EmailAddress :

＜110〉LastName: Huang

＜110〉FirstName: daybreak

<110> MiddleInitial :

<110> Suffix :

Application Project

-------------------

＜120〉Title: the purposes of KLOTHO albumen

<130> AppFileReference :

<140> CurrentAppNumber :

<141> CurrentFilingDate : ____-__-__

Sequence

--------

<213> OrganismName :

<400> PreSequenceString :

MPASAPPRRP RPPPPSLSLL LVLLGLGGRR LRAEPGDGAQ TWARVSRPPA PEAAGLFQGT 60

FPDGFLWAVG SAAYQTEGGW QQHGKGASIW DTFTHHPLAP PGDSRNASLP LGAPSPLQPA 120

TGDVASDSYN NVFRDTEALR ELGVTHYRFS ISWARVLPNG SAGVPNREGL RYYRRLLERL 180

RELGVQPVVT LYHWDLPQRL QDAYGGWANR ALADHFRDYA ELCFRHFGGQ VKYWITIDNP 240

YVVAWHGYAT GRLAPGIRGS PRLGYLVAHN LLLAHAKVWH LYNTSFRPTQ GGQVSIALSS 300

HWINPRRMTD HSIKECQKSL DFVLGWFAKP VFIDGDYPES MKNNLSSILP DFTESEKKFI 360

KGTADFFALC FGPTLSFQLL DPHMKFRQLE SPNLRQLLSW IDLEFNHPQI FIVENGWFVS 420

GTTKRDDAKY MYYLKKFIME TLKAIKLDGV DVIGYTAWSL MDGFEWHRGY SIRRGLFYVD 480

FLSQDKMLLP KSSALFYQKL IEKNGFPPLP ENQPLEGTFP CDFAWGVVDN YIQVDTTLSQ 540

FTDLNVYLWD VHHSKRLIKV DGVVTKKRKS YCVDFAAIQP QIALLQEMHV THFRFSLDWA 600

LILPLGNQSQ VNHTILQYYR CMASELVRVN ITPVVALWQP MAPNQGLPRL LARQGAWENP 660

YTALAFAEYA RLCFQELGHH VKLWITMNEP YTRNMTYSAG HNLLKAHALA WHVYNEKFRH 720

AQNGKISIAL QADWIEPACP FSQKDKEVAE RVLEFDIGWL AEPIFGSGDY PWVMRDWLNQ 780

RNNFLLPYFT EDEKKLIQGT FDFLALSHYT TILVDSEKED PIKYNDYLEV QEMTDITWLN 840

SPSQVAVVPW GLRKVLNWLK FKYGDLPMYI ISNGIDDGLH AEDDQLRVYY MQNYINEALK 900

AHILDGINLC GYFAYSFNDR TAPRFGLYRY AADQFEPKAS MKHYRKIIDS NGFPGPETLE 960

RFCPEEFTVC TECSFFHTRK SLLAFIAFLF FASIISLSLI FYYSKKGRRS YK 1012

<212> Type : PRT

<211> Length : 1012

SequenceName : 1

SequenceDescription :

Claims

1. the KLOTHO albumen purposes in the preparation cancer treatment drug, the aminoacid sequence of described KLOTHO albumen is shown in SEQ NO.1.

2. the KLOTHO albumen purposes in preparation diagnosing cancer of liver preparation, the aminoacid sequence of described KLOTHO albumen is shown in SEQ NO.1.