CN103495181A - Application of miR-375 - Google Patents

Abstract

The invention relates to the technical field of medicine and biology, and particularly relates to an application of miR-375. In-vitro experiments are done to research the influence of miR-375 on proliferation of adrenal cortex cancer cells. Overexpression of miR-375 is realized on human H295R cell lines by transient transfection, and results show that the overexpression of miR-375 can inhibit proliferation of adrenal cortex cancer cell line H295R. Therefore, miR-375 has regulation and control effects on proliferation of adrenal cortex cancer cells, and can be used for preparation of drugs for adrenocortical carcinoma or adrenal tumor.

Description

The application of miR-375

Technical field

The present invention relates to medicine and biological technical field, be specially medicine and diagnostic reagent with miRNA preparation and screening treatment adrenal gland neoplasms.

Background technology

In recent years, rise along with epigenetics, increasing research starts to pay close attention to non-encoding ribonucleic acid (noncoding RNA, ncRNA), and microRNA wherein (miRNA) more becomes the emerging study hotspot of the every field such as growth, metabolism, tumor.MicroRNAs (miRNAs) is the very conservative endogenous non-coding RNA on evolving that a class is about 19-25 nucleotide size, and the expression by post-transcriptional control to target gene exerts an influence; MicroRNAs extensively exists in animals and plants and microorganism, transcribed and generate precursor miRNA (pri-miRNA) through rna plymerase ii or III by DNA, pri-miRNA is cut and forms that length is about 70-100 base, the pre-miRNA tool hairpin structure by the Drosha enzyme action afterwards.The RNA of these hairpin structures is by core output albumen exportin5 transporte to cells matter, cut and form ripe miRNAs product by the Dicer enzyme action in endochylema again, the albumen such as same Ago2, TRBP form the silencing complex (RNA-induced silencing complex, RISC) that RNA induces together.Their bases by 3 ' the end untranslated region (3 ' untranslated region, 3 ' UTR) with target gene mRNA fully or incomplete pairing, cause the degraded of target gene mRNA and/or suppress the translation of this mRNA, thus the directly expression of Profilin matter.By research methoies such as high-flux sequences, find the gene of thousands of kinds of codified miRNA in recent years, and be subject to the regulation and control of these miRNA over 30% encoding proteins plasmagene.

From people's reported first such as Calin in 2002 have expression deletion and the downward of miR-15 and miR-16 in chronic lymphocytic leukemia, the effect of miRNA in tumorigenesis and the impact of oncotherapy curative effect, prognosis judgement is familiar with by people gradually.Now existing lot of documents has been reported the abnormal and regulatory mechanism that miRNA expresses in the kinds of tumors diseases such as breast carcinoma, gastric cancer, pulmonary carcinoma, carcinoma of prostate, hepatocarcinoma, itself or played the part of the role of proto-oncogene or antioncogene.And the function of miRNA in endocrine associated metabolic and tumor generating process also become study hotspot, as at cancer of pancreas, equal relevant report of visible miRNAs abnormal expression in thyroid, hypophysis, ovarian tumor.In adrenal gland diseases, for gene expression difference and with the dependency of disease and prognosis, also there are some research reports to studies have reported that in the recent period the differential expression of miRNA in the adrenal tumors such as pheochromocytoma, hypercortisolism, adrenocortical carcinoma, and for primary aldosteronism, there is not yet expression and the function of any report research non-coding RNA, wherein the Mechanism Study for the adrenal cortical cell hypertrophy lacks more.Compare the research situation of other tumors, this has restricted the understanding of people to primary aldosteronism to a great extent, has limited the development of diagnosis and treatment technology means.More and more evidences demonstrations, miRNA has participated in a plurality of links of oncobiology behavior.

Summary of the invention

The present invention is intended to miR-375 is applied to the medicine of preparation or screening treatment adrenal gland neoplasms.

The present invention also is applied to miR-375 the medicine of preparation or screening treatment adrenocortical carcinoma.

The present invention also is applied to miR-375 the medicine of preparation or screening regulation and control or adrenal cortex cancer cell multiplication.

Study miRNA-375(miR-375 by experiment in vitro) on the impact of adrenal cortex cancer cell multiplication.On people H295R cell line, with the mimics transient transfection, make miR-375 cross expression, utilize CCK-8, EDU dyeing is observed miRNA and is crossed the cell proliferation variation that expression causes.

The result demonstration, miR-375 crosses expression can suppress adrenal cortex cancerous cell line H295R propagation.

Therefore, miR-375 has regulating and controlling effect for the propagation of adrenal cortex cancerous cell, can be used for the medicine of preparation screening adrenocortical carcinoma or adrenal gland neoplasms.

The present invention has determined the relation of miR-375 and adrenal cortex cancer cell multiplication, can be using miR-375 as the index of screening, by detecting the expression of mi-375, screen preparation treatment adrenal gland neoplasms and especially treat the medicine of adrenocortical carcinoma or the medicine of regulation and control adrenal cortex cancer cell multiplication.

The accompanying drawing explanation

The A-E of Fig. 1 is in embodiment 3, the differential expression of miRNA-375, miRNA-7, miRNA-29a, miRNA-29b and miRNA-29c in adenoma, nodal-like hyperplastic tissue and normal control tissue in primary aldosteronism.

Fig. 2 is in embodiment 5, miR-375 expression when H295R transient transfection mimic

Fig. 3 is in embodiment 5, suppresses the result of cell proliferation in H295R cell line during overexpression miR-375, and A is the CCK8 testing result, and B is EDU dyeing situation.

The specific embodiment

Embodiment 1

Be diagnosed as patient's 128 examples of the parallel operative treatment of primary aldosteronism

I. examination object choice standard:

(1) JNC(Joint National Committee on the Prevention, Evaluation, and Treetment of High BLood Pressure) hypertension grading 2 grades of (BP>160～179/100～109mmHg), 3 grades (BP>180/110mmHg);

(2) refractory hypertension (combine and use 3 kinds of antihypertensive drug therapies, wherein must comprise diuretic, and every kind of antihypertensive drugs all reaches conventional therapy dosage, blood pressure still is greater than 140/90mmHg);

(3) hypokalemia due to spontaneity or diuretic;

(4) Adrenal Incidentalomas;

(5) early onset family history of hypertension or early (being less than 40 years old) cerebrovascular accident family history;

(6) there is hypertensive first degree relative in former aldehyde patient.

II. the diagnosis of alternative

Aldosterone/feritin (ARR): all object of study are stopped using to the influential medicine of RASS system, comprise that diuretic, Radix Glycyrrhizae and preparation thereof are more than 4 weeks, stop using angiotensin converting enzyme inhibitor, angiotensin receptor antagonist, dihydropyridine calcium ion antagonist, beta-blocker, nonsteroidal antiinflammatory drug more than 2 weeks, and blood pressure lowering can use non-dihydropyridine calcium ion antagonist instead or the α receptor blocking agent is controlled blood pressure.

Get up early morning and keep non-clinostatism state 2h (can stand, walk about, seat), after rest 15min, in 10: 00, front seat extracted blood aldosterone and feritin, and calculated ARR.ARR > 300 further row normal saline tests.Improve other hormonal readinesses and biochemistry detection simultaneously, comprise blood, urine electrolyte, improve blood ACTH, blood hydrocortisone, UFC, blood plasma metanephrine (MN), blood plasma methoxyepinephrine (NMN) detection simultaneously.

Make a definite diagnosis: " primary aldosteronism diagnosis and treatment guide " (the Case Detection delivered with reference to International Society of Endocrinology in 2008, Diagnosis, the test method of making a definite diagnosis former aldehyde of and Treatment of Patients with Primary Aldosteronism:An Endocrine Society Clinical Practice Guideline) recommending, we have adopted normal saline inhibition test normal saline test wherein: 1h must lie up before test, start at 8: 00, at 4h angular vein instillation normal saline 2000ml (500ml/h, drip speed constant), need close Monitoring of blood pressure and changes in heart rate in whole process.Measure respectively blood aldosterone and blood potassium before quiet He after quiet.Aldosterone level after normal saline > the former aldehyde diagnosis of 100pg/mL establishment.

Classification diagnosis

(1) adrenal CT scanning: be the first-selected test mode of level diagnosis, the unenhanced Enhanced CT that adds of patient's conventional row bilateral adrenal glands, bed thickness 3.75mm.CT scan can point out adrenal gland's form normal, and one-sided adrenal gland's limb thickens, bilateral adrenal glands nodal-like hypertrophy, one-sided or bilateral adenoma.

(2) Adrenal venous sampling (AVS): be the goldstandard experiment of distinguishing one-sided and bilateral primary aldosteronism.Intubate 8:00-9:00 in the morning starts, and in whole process, the patient keeps clinostatism.Intubate is carried out under the DSA guiding, and after the right lateral thigh venipuncture, intubate is to the postcava and left and right adrenal veins of renal veins level, and the sampling censorship, analyze relatively bilateral adrenal glands vein and inferior caval aldosterone and cortisol levels.Bilateral adrenal glands vein hydrocortisone than postcava hydrocortisone equal>3 prompting intubate successes; A higher side aldosterone points out than offside aldosterone and Cortisol Ratio>2 secretion that has superiority with Cortisol Ratio.If the discovery advantage is capable operative treatment.

Judge adrenal gland's nodal-like hypertrophy or adenoma,adrenal by pathological diagnosis.

III. sample collection and preservation

The constitutional aldosterone patient who includes the underwent operative treatment of research in amounts to 128 examples, and Pathological diagnoses is adenoma person's 88 examples, and nodal-like increases survivor's 40 examples.All primary aldosteronism patients' specimens from pri is all excised by experienced doctor in operating room under aseptic condition from the pathological changes interrenal tissue taken out, and is put in immediately preservation in liquid nitrogen until use.Basic clinical data is as table 1.

The normal control specimen is: the capable frozen section of the other interrenal tissue's cut-out tissue of tumor of adrenal gland's nonfunctional adenoma corrective surgery excision, HE dyeing, after being judged as normal adrenal cortex by experienced pathologists, will remaining interrenal tissue and preserve (8 example) as the experiment Normal group.

The basic clinical data of table 1. patient

The continuous variable means with mean ± SD or median (quartile), quantity for classified variable (percentage ratio) expression

Male's 40 examples in primary aldosteronism adenoma patient, male's 18 examples in nodal-like hypertrophy patient, two groups of sex compositioies are than zero difference (p=0.964).Hypertensive disease, systolic pressure and diastolic pressure level no significant difference between two groups.Primary aldosteronism adenoma patient is similar to nodal-like hypertrophy patient's Clinical symptoms, but the former serum aldosterone level is apparently higher than the latter: the serum aldosterone level is 560.6 (329.1-901.7) pg/ml in adenoma group, nodal-like hypertrophy group is 436.5 (233.1-655.2) pg/ml, and both differences have statistical significance (p=0.026).The aldosterone renin activity obviously raises in than nodal-like hypertrophy group in the aldosteronoma group than ARR, and difference has statistical significance (p=0.005).And plasma renin activity, ratio, blood potassium, blood sodium, twenty-four-hour urine potassium and urine sodium equal not statistically significant of comparing difference between two groups.In adrenal CT scanning, adenoma patient's focus diameter is 1.50 (1.20-2.00) cm, and with nodal-like hypertrophy patient size of tumor 1.50 (1.00-2.00) cm relatively, both differences are not statistically significant also.

Embodiment 2

The total RNA that gets embodiment 1 acquisition carries out the microRNA chip analysis

(1) the RNA quality control detects

The primary aldosteronism adenoma of having extracted, nodal-like hyperplastic tissue and normal interrenal tissue send LC Sciences company to carry out the detection of RNA quality.Up-to-standard RNA carries out next step microarray gene expression detection.

(2) μ Paraflo ^tMthe experiment of microRNA microarray gene expression

Send the sample of surveying chip to comprise that adenoma organizes the RNA6 example, the RNA6 of nodal-like hyperplastic tissue example and the normal RNA3 of interrenal tissue example.The microarray gene expression test experience is completed by LC Sciences company.Concrete experiment flow is as follows: carrying out Microarray Experiments needs the total RNA sample of 10 μ g.Total RNA separates and obtains the little RNA that length is less than 300nt by the micro-centrifugal filtration post of YM-100 (Millipore).Little RNA3 ' the end obtained in separation uses Poly (A) polymerase to add poly (A) tail, then connects 1 oligonucleotide labelling with the fluorescent labeling for follow-up after this poly (A) tail.If two sample experiments, select two little RNA samples of two different label labellings.Utilize the Circulation of micro circulation pump (Atactic Technologies) on μ ParafloTM micro-fluid chip, spend the night and carry out hybridization.On μ ParafloTM micro-fluid chip, every detector probe be all by one with the chemically modified nucleoside acid encoding section (deriving from miRBase, http://microrna.sanger.ac.uk/sequences/) of target microRNA complementation or with other with Quality Control or the RNA of customization sequence complementation and expansion coding section that one is comprised of Polyethylene Glycol with

The spacer of the spacing of substrate forms.All detector probe use PGR(photogenerated reagent) the chemical method original position is synthetic.The detector probe of chemical modification is for balance hybridization melting temperature.With the 100 μ L6xSSPE(0.90M NaCl that contain 25% Methanamide, 60mM Na2HPO4,6mM EDTA, pH6.8) as hybridization buffer, hybridization temperature is 34 ℃.Cy3 and the Cy5 fluorescent dye of detection specificity after hybridization.After hybridization finishes, utilize laser scanner (GenePix4000B, Molecular Device) gather image and use Array-Pro image analysis software (Media Cybernetics) to carry out the image digitazation conversion to result.

(3) at first data analysis is the subduction background value, then uses LOWESS to filter (Locally-Weighted Regression) and carries out signal normalization process.For two mark experiments, calculate the ratio (log2 conversion, balance) of two groups of detection signals, carry out t-test, analyze p-values; When p-value<0.01, be considered to the significance difference opposite sex and express.

In carrying out the chip detection process, we have selected the primary aldosteronism adenoma to organize 6 examples, nodal-like hyperplastic tissue 6 examples, and normal interrenal tissue 3 examples, total RNA of each sample has carried out respectively μ Paraflo ^tMmicroRNA microarray gene expression analysis of spectrum.Result shows, in the chip average signal strength is greater than 500 miRNAs, the miRNAs that Analysis of variance obtains differential expression p<0.01 in normal interrenal tissue, nodal-like hyperplastic tissue and adenoma tissue has 30,8 of miRNA of up-regulated in former aldehyde tissue wherein, 22 of down-regulated expression; The miRNAs of the p of tool differential expression<0.05 has 65, and wherein in former aldehyde tissue 39 of up-regulated, 26 of down-regulated expression.Can see the rise of a plurality of miRNA expressions simultaneously or lower presenting increasing or decreasing gradually to adenoma tissue again from normal adrenal cortex to hypertrophy and sexually revising, the formation reason of hypertrophy or adenoma tissue may be expressed the degree difference changed with miRNA and be correlated with.

Analysis chip express spectra data, the visible miRNA that gene expression abundance is higher in aldosteronoma comprises miR-1260b, miR-29 family, and express the miRNA obviously reduced, miR-375 and miR-7 are arranged, wherein again with miR-375 the differential expression in aldosteronoma and normal adrenal tissues the most obvious.And in nodal-like hyperplastic tissue, the gene expression abundance of these a few class miRNA, between aldosteronoma and normal control, is the relation of going forward one by one, as shown in table 2.

Table 2 adenoma, hypertrophy and normal adrenal tissues miRNA differential expression are analyzed

Embodiment 3

Analysis according to 2 kinds of chip results miRNA differential expressions of embodiment, to adenoma, nodal-like hyperplastic tissue and normal adrenocortical miR-375 in primary aldosteronism, miR-7 and miR-29a/b/c expression carry out the enlarged sample checking (expanded sample comprised the former adenoma of chip detection of sending organize 6 examples, nodal-like hyperplastic tissue 6 example and normal interrenal tissue 3 examples) of real-time PCR.Result is as Fig. 1-Fig. 3.Step is:

I. reverse transcription is cDNA

The reverse transcription process operation is undertaken by Exiqon Universal cDNA synthesis kit description.This reverse transcription mode can disposablely out be used all microRNA reverse transcriptions in order to follow-up real-time PCR.

II.Real-time?PCR

(1) design of primers and synthetic: complete by Exiqon company.Primer comprises: hsa-miR-375 (204362), hsa-miR-7 (204592), hsa-miR-29a (204698): hsa-miR-29b (204679), hsa-miR-29c (204729), U6snRNA (203907).

(2) after primer is delivered to, with 4 ℃, centrifuge, with the quick centrifugal Eppendorf pipe of putting primer of 17000g rotating speed, primer is collected in to pipe at the end, add respectively the tri-distilled water 110 μ l that do not contain nuclease to dissolve LNA Fwd Primer and LNA Rev Primer, vortex mixes, centrifugal collection, after dissolving 15min on ice, mix two pipe primers, fully mix, and be placed in after packing-20 ℃ of Refrigerator stores standby.

(3) take out and reverse the cDNA recorded, dissolving on ice, in the ratio of 1:80, with the tri-distilled water that does not contain nuclease, dilute cDNA, vortex mixes, centrifugal collection.

(4) take out Real-time pcr amplification reagent (SYBR Green master mix, Exiqon), dissolve 15min on ice, lucifuge.Turn upside down and mix reagent, then with using after the slight centrifugal collection of centrifuge.

(5) join Real-time PCR reactant liquor mixed liquor (the reactant liquor preparation will be carried out on ice), and packing joins in 384 orifice plates; Real-time PCR reaction system SYBR green master mix(2 *) 5 μ l, primer 1 μ l, add the cDNA template and complement to 10 μ l; After having added each component, with the plastic packaging film shrouding.

(6), with 3000 rev/mins of rotating speeds, centrifugal 3min, make reactant liquor be collected in the pipe end.

(7) use Roche LightCycler480Real-time pcr amplification instrument to be increased: Real-timePCR reaction condition: 95 ℃ of denaturations, 10min; 45 circulations, 95 ℃, 10s, 60 ℃, 1min; 1.6 ℃/s of alternating temperature speed

(8) analytical calculation of testing result: the comparison of microRNA expression between being organized according to the Ct value after the Ct value normalized of real-time PCR (Normalized CT value).The Ct value of the genes of interest that △ Ct is same sample and reference gene poor, i.e. △ Ct=Ctgene-CtU6.△ Ct is the poor of same group of sample genes of interest Ct value average and same group of sample reference gene Ct value average, i.e. △ Ct=Ctgene-CtU6.Ct markization=△ Ct+ △ Ct.Between each group, above-mentioned statistic is used in the variation analysis of microRNA expression, and in this sample of the larger explanation of Ct markization value, the microRNA expression is lower.

Visible while comparing between the Ct value with after markization is organized, same Normal group (17.88 ± 0.9096) is compared, the Ct value that in the aldosterone producing adenoma tissue, miR-375 expresses is the highest by (27.97 ± 0.2681, p<0.0001), the miR-375 of nodal-like hyperplastic tissue expresses Ct value (25.37 ± 0.5249) between adenoma and Normal group, prompting miR-375 expresses all and reduces in primary aldosteronism patient pathological changes adrenal tissue, with more obvious in the adenoma tissue.Similar with the miR-375 expression, miR-7 expresses same (APA:27.65 ± 0.2409 that reduces in primary aldosteronism patient pathological changes adrenal tissue; Hyperplasia:25.65 ± 0.5257), in the adenoma tissue, express lower (p<0.0001), there is dependency (r=0.9667, p<0.0001) preferably in miR-375 with the miR-7 gene expression abundance.

In chip results, the expression compared with normal matched group of miR-29a in the adenoma tissue raise 3.51 times, yet in the enlarged sample proof procedure, only see that its expression in the adenoma tissue has rising trend (8.875 ± 0.1275), but same normal control (9.078 ± 0.1912) and nodal-like hyperplastic tissue (9.044 ± 0.1700) are relatively, the difference not statistically significant.MiR-29b and miR-29c expression difference in three groups is similar, i.e. relative Normal group, equal obvious rising (miR-29b:9.778 ± 0.1248vs11.48 ± 0.3191, p<0.001 of both expression in adenoma tissue; MiR-29c:6.160 ± 0.1187vs7.361 ± 0.2140, p<0.001), in nodal-like hyperplastic tissue, express rising (miR-29b:10.05 ± 0.1513vs11.48 ± 0.3191, p<0.001 also arranged; MiR-29c:6.419 ± 0.1491vs7.361 ± 0.2140, p<0.01), and with the differential expression of adenoma tissue, statistical significance is arranged.Above result and miRNA expression chip result are basically identical.

The continuous variable of each clinical indices parameter normal distribution is with mean ± standard deviation (means ± SD) expression, and the continuous variable of nonnormal distribution is with median (quartile) expression, and classified variable means with quantity (percent).MicroRNAs real-time PCR result is with Ct value representation after markization, and numerical variable means with mean ± standard error (means ± SEM).More all adopt the statistical method of two tail Student t checks in this research between two groups of the continuous variables of normal distribution.Between two groups of the continuous variable of nonnormal distribution and classified variables relatively by the statistical method of Mann-Whitney U check.When p<0.05, think and there is significant difference between two groups.

Embodiment 4

According to the result of embodiment 3, to the miR-375 expression, with primary aldosteronism patient clinical data correlation analysis, the miR-375 expression is relevant with tumor size in aldosterone producing adenoma, and result is as table 3.

Table 3miR-375 expression is with primary aldosteronism patient clinical phenotypic correlation

In all participating in 128 routine primary aldosteronism patients of research, it is visible when the Ct value representation miR-375 expression with after markization carries out correlation analysis, its Ct value is with patients serum's aldosterone level correlation (r=-0.222, p=0.018), along with the miR-375 expression reduces, patients serum's aldosterone level raises; The Ct value that miR-375 expresses is obvious positive correlation (r=0.350, p<0.0001) with adrenal gland's focus size, and, along with the reduction of miR-375 expression, adrenal gland's lesion volume increases.Under the impact of having proofreaied and correct above-mentioned size of tumor, in PA patient, the serum aldosterone level only is certain relevant trend with the miR-375 expression.And no matter whether the course of disease, systolic pressure, diastolic pressure, plasma renin activity, ARR, blood potassium, blood sodium level all proofreading and correct size of tumor all with miR-375 expression non-correlation in the primary aldosteronism patients.

All patients is carried out to grouped comparison, in 40 routine nodal-like hypertrophy patients, find that size of tumor only has relevant trend (p=0.066) with the miR-375 expression, and serum aldosterone, ARR are with miR-375 markization be proportionate (ALDO:r=-0.394, p=0.019 of Ct value afterwards; ARR:r=0.408, p=0.023),, along with the miR-375 expression reduces, patients serum's aldosterone and ARR level all raise, and after proofreading and correct the nodular hyperplasia size of tumor, this dependency disappears.Remaining systolic pressure, diastolic pressure, renin of blood activity, blood potassium, blood sodium level all with miR-375 expression non-correlation.

In patients with aldosterone-producing adenoma, only find that tumor size is proportionate (r=0.312, p=0.004) with Ct value after the miR-375 markization,, along with the miR-375 expression reduces, diameter of tumor progressively increases.There is certain relevant trend (p=0.073) in serum potassium with the miR-375 expression, but not statistically significant, and after the impact of considering tumor size, this relevant trend also disappears.Remaining systolic pressure, diastolic pressure, blood aldosterone, plasma renin activity, ARR, blood sodium index all with miR-375 expression non-correlation.

As can be seen here, the miR-375 expression is relevant to tumor size in aldosterone producing adenoma.

Embodiment 5

With miRNA-375 transient transfection adrenal cortex cancerous cell line H295R, make miR-375 cross expression, study the impact of miR-375 on the adrenal cortex cancer cell multiplication by experiment in vitro.

Adrenal cortex cancerous cell line H295R buys from national experimental cell resources shared platform (Beijing).

H295R cell transient transfection:

(1) H295R cell culture fluid preparation: DMEM/F12 culture medium 94.5ml, add Nu-Serum I serum substitute 2.5ml, 2mM glutamine 1ml, each 100IU/ml of penicillin/streptomycin and ITS premix 1ml, mix culture fluid, and 4 ℃ of preservations are stand-by.

(2) condition of culture and method: put in incubator and cultivate, the incubator condition is gas concentration lwevel 5%, humidity 90%, 37 ℃ of temperature.Cell changes liquid every other day, within every 6～7 days, by 1:3～1:4, goes down to posterity.

(3) do not use instead or not containing antibiotic complete culture solution, the H295R cell is seeded to 6 orifice plates, every porocyte density is about 3 * 10 ⁵, adhere-wall culture transfection after 72 hours.

Adopt liposome to carry out transient transfection, concrete operation step is with reference to Lipofectamine2000 test kit description.

As shown in Figure 2, transient transfection microRNA precursor sequence mimics on adrenal cortex cancerous cell line H295R, can obtain the high expressed of miR-375 to result, its expression compared with normal matched group 10000 times of left and right of can rising.

CCK-8 experimental result prompting, transient transfection contrast (NC) and miR-375mimics in cell line H295R, can make cell viability reduce approximately 20%, and difference has statistical significance (p<0.001).

With reference to Invitrogen Click-iT ^tMdescription operation in EdU Imaging Kits, in cell line H295R, transient transfection contrasts (NC) and miR-375mimics equally, in the fixing EDU that adds in first 6 hours, mix cell DNA, results suggest, after crossing expression miR-375, cell EDU dyeing percentage rate drops to 25.5% by former 34.9%, and difference has statistical significance (p<0.001).

Above result all illustrates that miR-375 crosses expression and can suppress adrenal cortex cancerous cell line H295R propagation.

Therefore, miR-375 has regulating and controlling effect for the propagation of adrenal cortex cancerous cell, can be used for the especially medicine of adrenocortical carcinoma of preparation screening adrenal gland neoplasms.

Claims (3)

1.MiR-375 for the preparation of or the medicine of screening treatment adrenal gland neoplasms.

2.MiR-375 for the preparation of or the medicine of screening treatment adrenocortical carcinoma.

3.MiR-375 for the preparation of or the medicine of screening regulation and control adrenal cortex cancer cell multiplication.

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