TW201432051A - Gene markers for colorectal cancer, method for detecting colorectal cancer using the same and kit containing the same - Google Patents

Abstract

The current invention provides gene markers for colorectal cancer, these gene markers include CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1. These gene markers have high accuracy, sensibility, and specificity, and can be utilized as screen tool for colorectal cancer, or to predict response of certain course of the treatment or assess further course of the treatment for patients, before and after therapy.

Description

Gene marker for colorectal cancer, method for detecting colorectal cancer thereof, and kit containing the same

The present invention relates to a genetic marker associated with colorectal cancer, and more particularly to a genetic marker in peripheral blood, which can be used for screening colorectal cancer or evaluating the prognosis of colorectal cancer.

Colorectal cancer (CRC) is a common cancer worldwide. In the United States, it is estimated that in 2009, 146,970 patients with colorectal cancer were added, and 49,920 people died of the cancer. Screening for CRC can reduce the incidence of cancer and provide a better assessment of prognosis and survival. Traditional CRC screening methods include fecal occult blood test, sigmoid colonoscopy, barium enema or colonoscopy, etc. Although the above screening methods are commonly used, they all have their limitations, such as high denaturing sensitivity (from 37% to 80%). Or the interaction with the diet leads to errors in the screening results. In addition, for some of the above-mentioned highly invasive tests, the willingness to screen is also reduced, and the early diagnosis rate of colorectal cancer is low.

The spread or migration of malignant cells of a tumor from a primary tumor is a key cause of cancer progression. In many clinical cases, cancer cells have metastasized even before the primary cancer is diagnosed, and these cancer cells in the human circulatory system may be the earliest forms of cancer metastasis. Therefore, it has been reported that polymerase chain reaction (PCR)-based analysis can detect keratin, tumor embryonic antigen (CEA), and epidermal growth factor receptor in peripheral blood samples of patients with CRC. The mRNA of the (epidermal growth factor receptor, EGFR) gene is used as a molecular marker for early tumor molecular screening. However, these conventional genes also have the disadvantages of low sensitivity and low specificity, so that it is not possible to properly detect colorectal cancer (Sergeant G, Penninckx F, Topal B: Quantitative RT-PCR detection of colorectal tumor cells in peripheral blood- -a systematic review.J SuRg Res 2008, 150:144-152) , and the recently discovered molecular marker ProtM also has problems with poor sensitivity ( Schuster R, Max N, Mann B, Heufelder K, Thilo F, Grone J, Rokos F, Buhr HJ, Thiel E, Keilholz U: Quantitative real-time RT-PCR for detection of disseminated tumor cells in peripheral blood of patients with colorectal cancer using different mRNA markers. Int J Cancer 2004, 108: 219-227 ) .

Therefore, it is still necessary to find a genetic marker for colorectal cancer that is more sensitive and highly specific.

The present invention provides a genetic marker for detecting colorectal cancer and/or evaluating its prognosis, which comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1.

According to the genetic marker of the present invention, it has an increased performance in colorectal cancer cells. In a preferred embodiment, the colorectal cancer cell line is present in the peripheral blood of the individual.

Another aspect of the present invention provides a method for detecting whether an individual has colorectal cancer, the steps comprising: (a) obtaining a test sample comprising a nucleic acid from a body; and (b) measuring a gene marker expression in the sample. And the gene marker comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1; (c) normalizing the expression level of the gene marker by the housekeeping gene expression amount of the individual; d) comparing the normalized gene marker expression amount with the gene marker expression amount of a health control group, if the gene marker expression amount is increased, it indicates that the individual has a colorectal cancer risk.

Yet another aspect of the present invention provides a method of monitoring and/or evaluating a prognostic response to a treatment of a colorectal cancer patient, the steps comprising: (a) obtaining a patient from the patient prior to receiving treatment a pre-treatment sample comprising a nucleic acid; (b) obtaining a post-treatment sample comprising the nucleic acid from the patient after receiving the treatment; (c) measuring a gene marker expression of the pre-treatment sample and the post-treatment sample, wherein The gene marker comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1; (d) comparing the pre-treatment sample with the gene marker expression of the post-treatment sample, if the post-treatment gene marker expression amount Increased, representing the poor prognosis of the individual.

According to the method of the present invention, wherein the test sample is a body fluid, and in a preferred embodiment, the body fluid is peripheral blood. Further in accordance with the method of the present invention, wherein the gene expressed in step (b) is mRNA or protein.

In a further aspect of the invention, a kit for detecting colorectal cancer and/or assessing its prognosis is provided, the kit comprising: a specific primer pair of CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 genes.

In another aspect of the invention, a kit for detecting colorectal cancer and/or assessing its prognosis is provided, the kit comprising: a specific antibody against the anti-CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 gene proteins.

definition

The term "a" or "an", when used in conjunction with "including", is used in the scope of the application or the specification, which may be one, but also one or more or at least one.

The term "overexpression" or "rising performance" means that the amount of gene expression in a cell or organism is higher than that of a normal cell or organism.

The term "treatment" means the use of any means, such as surgery, chemotherapy or drug control, to reduce the frequency, extent, severity and/or duration of symptoms experienced by patients with colorectal cancer.

The term "alleviation", "alleviation" or "improvement" means reducing the symptoms of one or more diseases, disorders or conditions.

The term "gene marker" means a change (up or down) in the amount of expression of a DNA in an individual due to a particular disease, and can be used to guide the presence or absence of a particular disease or an increase in the risk of abnormality," Terms such as "gene marker" or "molecular marker" can be used interchangeably.

The term "prognosis" refers to the prediction of the possible development and outcome of a disease, especially the possibility of recovery.

The term "relapse" refers to the re-emergence of a cancer cell after the individual has been treated and returned to the pre-treatment state.

As used herein, "individual" refers to a mammal, including but not limited to humans, primates, rodents, cats, dogs, cattle, and the like.

The term "sample" as used herein refers to a biological sample, such as tissue or fluid isolated from an individual (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva, or tissue sections) or in vitro cell culture. Things.

Colorectal cancer gene marker of the present invention

The present invention provides a gene marker for detecting colorectal cancer and/or evaluating its prognosis, and the gene marker comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 (hereinafter referred to as the 7 gene of the present invention) mark).

In a preferred embodiment, the present invention utilizes a peripheral blood sample to detect genetic markers of colorectal cancer using an instant polymerase chain reaction reaction technique. In detail, after the collected blood samples are extracted from total RNA, reverse transcription is performed to generate cDNA, and the polymerase chain reaction is performed by using a pre-selected specific primer pair to compare the difference between the normal individual and the colorectal cancer patient. Gene expression, select the gene markers, and use the logistic regression statistical method to evaluate the gene marker prediction model, and obtain 12 sets of microarray data sets from the public network information, namely GSE 4107, 4183, 8671, 9438, 10961, 13067 13,294, 13471, 14333, 15960, 17538, and 18105, including 519 adenocarcinomas and 88 normal mucosal control groups, and the 12 published microarray data were used as external validation to detect candidate genes as cancer molecular markers. feasibility. The 5 gene model disclosed in EP201987A2 and the 7 gene model of Marshall et al. ( Marshall KW, et al.: A blooa-based biomarker panel for stratifying current risk for colorectal cancer. Int J Cancer 2010, 126:1177) -1186 ) and Han et al. 5 gene model ( Han et al.: Novel blood-basea, five-gene biomarker set for the detection of colorectal cancer. Clin Cancer Res 2008 , 14 : 455-460 ) The 12 published microarray datasets were externally validated and subjected to multivariate logistic regression analysis to obtain the 7 gene markers of the present invention with better accuracy, sensitivity and specificity: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 can be used as screening for CRC, before or after treatment, to predict response to a specific course of treatment, or to assess subsequent course of treatment for a patient.

Logistic regression is used to predict the probability of an event by fitting data to a logical curve. The logistic regression model yields the probability of the reaction occurring as an independent variable exponential function. The model is expressed in probability (P) and ranges from 0 to 1. Logistic regression is represented by a logical equation:

The input value is Y and the output value is f(Y), which is P. The output value is limited to between 0 and 1. The variable Y represents exposure to certain risk factors, while f(Y) represents the probability of a particular result derived from the risk factors. This variable Y is an overall measure of all the risk factors used in the model, also known as the pair Number of advantages and disadvantages (logit).

The variable Y is usually defined as Y = β ₀ + β ₁ x ₁ + β ₂ x ₂ + ... + β _k x _k , β ₀ is called intercept, β ₁ , β ₂ , β ₃ etc. They are called regression coefficients of x ₁ , x ₂ , and x ₃ . When all risk factors are zero (ie, the z value of an individual without any risk factor), the Y value is equal to the intercept. Each regression coefficient describes the contribution of the risk factor. A positive regression coefficient indicates that the risk factor increases the probability of the result, while a negative regression coefficient indicates that the risk factor reduces the probability of the result; a larger regression coefficient indicates that the risk factor significantly affects the probability of the result; The regression coefficient indicates that the risk factor will only slightly affect the probability of the outcome.

Therefore, statistical analysis of the amount of gene marker expression can be used to predict an individual's risk of colorectal cancer. The Odds Ratio (OR) estimates the relative risk of colorectal cancer. The odds ratio (OR) is a measure of the effect value, indicating the strength of the association or the non-independence between two binary data values, used as a descriptive statistic and as a logical regression. An important role. The odds ratio (OR) is defined as the success rate of one event occurring in one group relative to occurring in another group. Statistically, the success rate is the probability of an event occurring divided by the probability that the event will not occur. The odds ratio is a method for comparing the probability of occurrence of a particular event for two groups. When the odds ratio is equal to 1, it shows that the event has the same tendency to occur in the two groups. When the odds ratio is greater than 1, it shows that the event is more likely to occur in the first group. When the odds ratio is less than 1, it is shown that the event is less prone to occur in the first group. If the probability of an event occurring in each group is p ₁ (first group) and p ₂ (second group), then the odds ratio is:

According to the 7 gene marker of the present invention, CPEB4 binds to target mRNAs of cytoplasmic polyadenylation elements (CPE) and controls cytoplasmic polyadenylation and activation translation during growth and development ( Huang YS, Kan) MC, Lin CL, Richter JD: CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. EMBO J 2006, 25: 4865-4876 ); EIF2S3 is a eukaryotic transcription initiation factor 2 The largest subunit of eukaryotic translation initiation factor 2 (EIF2) may be involved in the inhibition of prostate cancer metastasis through the downstream regulatory gene 1 of the N-myc pathway ( Tu LC, Yan X, Hood L, Lin B: Proteomics analysis of the interactome of N-myc downstream regulated gene 1 and its interactions with the androgen response program in prostate cancer cells. Mol Cell Proteomics 2007 , 6 : 575-588 ). The first of the present invention found the correlation of these seven genes in colorectal cancer and was combined for use as a genetic marker for colorectal cancer. These gene markers have a specificity of 96.8% accuracy, sensitivity of 99.4%, and 96.6%, and can be used as a screening for CRC, before or after treatment, to predict a specific course of treatment, or to assess a patient's response. Follow-up treatment methods.

According to the genetic marker of the present invention, it has an increased performance in colorectal cancer cells. In a preferred embodiment, the colorectal cancer cell line is present in the peripheral blood of the individual.

Method for monitoring and/or evaluating colorectal cancer of the present invention

Another aspect of the present invention provides a method for detecting whether an individual has colorectal cancer, the steps comprising: (a) obtaining a test sample comprising a nucleic acid from a body; and (b) measuring a gene marker expression in the sample. And the gene marker comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1; (c) normalizing the expression amount of the gene marker by the housekeeping gene expression amount of the individual; And (d) comparing the normalized gene marker expression amount with the gene marker expression amount of a health control group, and if the gene marker expression amount is increased, it indicates that the individual has a colorectal cancer risk.

Yet another aspect of the present invention provides a method of monitoring and/or evaluating a prognostic response of a colorectal cancer patient to a treatment comprising the steps of: (a) obtaining a pre-treatment sample comprising nucleic acid from the patient prior to receiving the treatment; (b) after the patient is treated, obtaining a post-treatment sample containing the nucleic acid from the patient; (c) measuring the gene marker expression of the pre-treatment sample and the post-treatment sample, wherein the gene marker comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1; (d) Comparing the pre-treatment sample with the gene marker expression amount of the post-treatment sample, if the post-treatment gene marker expression amount is increased, it means that the individual has a poor prognosis.

According to the method of the present invention, wherein the test sample is a body fluid, and in a preferred embodiment, the body fluid is peripheral blood.

The expression levels of the aforementioned CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 gene markers may be mRNA expression levels, and the gene markers of the present invention are in cancer sample samples and non-cancer sample samples (or the same patient is being treated) Any change in the amount of mRNA expression of the sample obtained before and after, any skilled person can easily use any conventional method including, but not limited to, northern blotting, reverse transcription polymerase chain reaction (Reverse Transcription) Polymerase Chain Reaction (RT-PCR) or real-time polymerase chain reaction (Q-RT-PCR) and other known correlation mRNA analysis techniques.

In addition, the expression levels of the aforementioned CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 gene markers may also be protein expression, whereas the CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 of the present invention. Gene labeling changes in the protein expression of a sample of a cancer sample and a sample of a non-cancer sample (or a sample sample obtained by the same patient before and after treatment), and any skilled person can easily use any conventional method. Including but not limited to enzyme-linked immunosorbent assay (ELISA), immunofluorescence, immunohistochemistry, enzyme immunoassay (EIA), radioimmunoassay ( Radioimmunoassay (RIA) or Western blotting (Western blotting) and other known protein analysis techniques.

Those skilled in the art can screen for drugs for the treatment of colorectal cancer by identifying whether the expression of the CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1 gene markers is promoted or inhibited. For example, a green fluorescent protein gene can be ligated using the promoter regions labeled with CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1 genes, and the fluorescence intensity of the tested drug is observed from the cells.

The kit for detecting colorectal cancer and/or evaluating its prognosis of the present invention

In a further aspect of the invention, a kit for detecting colorectal cancer and/or assessing its prognosis is provided, the kit comprising: a specific primer pair of CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 genes.

The term "primer" as used herein refers to a strand of DNA used in a PCR reaction as a starting point for replication of a particular gene, and another strand of DNA that replicates the starting point at the other end of the particular gene is collectively referred to as a primer pair. It can pinch out the specific gene to be amplified.

Using the primer pair in the kit of the present invention, the expression levels of the aforementioned CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1 gene markers can be detected, and a reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain) can be used. Reaction, RT-PCR or real-time PCR (Q-RT-PCR) and other known related mRNA analysis techniques determine whether an individual has colorectal cancer and/or whether the prognosis is good.

In another aspect of the invention, a kit for detecting colorectal cancer and/or assessing its prognosis is provided, the kit comprising: a specific antibody against the anti-CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1 gene proteins.

The specific antibody can further be provided with a chemical probe, a fluorescent protein, a radioactive substance or an enzyme, and the kit can be directly subjected to ELISA, immunofluorescence staining, immunohistochemistry, enzyme immunoassay or radioimmunoassay. Or the amount of expression of the genetic marker.

Example

Hereinafter, the present invention will be further described in detail with reference to the embodiments. However, it is to be understood that these examples are only intended to aid in the understanding of the invention and are not intended to limit the scope of the invention.

Example 1

The 5-gene model (model 1) disclosed in EP2019871A2 and the 7-gene model disclosed in the prior art (model 2, see Marshall KW, et al.: A blood-based biomarker panel for stratifying current risk for colorectal cancer. Int J Cancer 2010, 126: 1177-1186 , hereinafter referred to as Marshall et al. 7 gene model) and 5 gene model (Model 3, see Han M, et al.: Novel blood-based, five-gene biomarker set for the detection of colorectal cancer .Clin Cancer Res 2008 , 14 : 455-460 , hereinafter referred to as Han et al. 5 gene model), using a collection of 12 public microarray data sets for external validation, performing multivariate logistic regression analysis. The results are shown in Table 1 below.

From the above Table 1, it can be seen that the HL test results of the 7 gene model of Marshall et al. of Model 2 are significant (p value is 0.044), which represents that the model data is not fitted and the fitness is poor. Those with good fitness are models 1, 3 and 4. The model 4 is a 7-gene model obtained from the results of pairwise comparison in models 1, 2, and 3, and the model 4 exhibits the best logic compared to models 1, 2, and 3. The results of the regression analysis (HL p=1.000, R ² =0.951, AUC=0.999, accuracy=0.968, specificity=0.966 and sensitivity=0.994) met the excellent diagnostic test criteria.

As a result of the above examples, since cancer cells of any kind of cancer are likely to be scattered by tumor tissues, penetrate and invade blood vessels, and circulate in the peripheral blood, the circulating cancer cells can be used. Predicting clinical outcomes in cancer patients (Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF: Circulating tumor cells, disease progression, and survival in Metastatic breast cancer. N Engl J Med 2004, 351:781-791) , which differs in the performance of the health control group and CRC patients, further the 5 gene model of EP2019871A2 and the 7 gene of Marshall et al. A total of 17 genes were used in the model and Han et al., and 12 public microarray datasets were used as external validation. Multivariate logistic regression analysis was performed to find a set of 7 gene models (CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1), this group of 7 gene models have excellent accuracy, sensitivity and specificity, so they can also be used for early detection and diagnosis of colorectal cancer. , or can be used to predict the response of a specific course of treatment before or after treatment, or to assess the subsequent course of treatment of the patient.

The 7 gene model of the invention can adjust the number of detection genes according to cost, and the detection sequence is (1) detecting only MGC20553; (2) detecting (1), increasing EIF2S3; (3) detecting (2), increasing TNFAIP6; (4) In addition to the detection (3), increase the CPEB4; (5) increase the ANXA3 in addition to the detection (4); (6) increase the MAS4A1 in addition to the detection (5); or increase the IL2RB in addition to the (7) detection (6). Detection accuracy and calculation The calculation parameters are shown in Table 2 below.

Example 2

Further, according to the 12 sets of microarray data sets obtained from the disclosed network information, the 5 genomes CA7, SPIB, GUCA2B, IL6R and SPP1 are further analyzed to cooperate with the 7 gene models of the present invention, MGC20553, EIF2S3, TNFAIP6, CPEB4, ANXA3, MAS4A1. And IL2RB, the number of detection genes can be adjusted according to the cost, the detection sequence is (1) detection only CA7, (2) detection (1), increase IL6R; (3) detection (2), increase IL2RB; (4) detection (3) In addition, increase TNFAIP6; or (5) test (4), increase CPEB4. The accuracy and calculation parameters are shown in Table 3 below.

Although the embodiments are disclosed herein, it is to be understood that the scope of the present invention is not to be construed as a For the scope of the patent application below.

Claims (14)

A genetic marker for detecting colorectal cancer and/or assessing its prognosis, the gene marker comprising the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1. The gene marker of claim 1, wherein the gene marker has an increased amount of expression in colorectal cancer cells. The gene marker of claim 2, wherein the colorectal cancer cell line is present in the peripheral blood of the individual. A method for detecting whether an individual has colorectal cancer comprises the steps of: (a) obtaining a test sample containing nucleic acid from a body; and (b) measuring a gene marker expression amount in the sample, wherein the gene marker system The following genes are included: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1; (c) normalizing the expression level of the gene marker with the housekeeping gene expression amount of the individual; and (d) normalizing the gene The gene marker expression amount is compared with the gene marker expression amount of a health control group, and if the gene marker expression amount is increased, it indicates that the individual has a colorectal cancer risk. A method of monitoring and/or evaluating a prognostic response to a treatment of a colorectal cancer patient, the steps comprising: (a) obtaining a pre-treatment sample comprising nucleic acid from the patient prior to receiving the treatment; (b) administering to the patient After receiving the treatment, a post-treatment sample containing the nucleic acid is obtained from the patient; (c) measuring the gene marker expression amount of the pre-treatment sample and the post-treatment sample, wherein the gene marker comprises the following genes: CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1; (d) Comparing the pre-treatment sample with the gene marker expression amount of the post-treatment sample, if the post-treatment gene marker expression amount is increased, it means that the individual has a poor prognosis. The method of claim 4 or 5, wherein the sample is a body fluid. The method of claim 6, wherein the body fluid is peripheral blood. The method of claim 4 or 5, wherein measuring the gene expression amount according to requirements, the detection order is: (1) detecting only MGC 20553; (2) detecting (1), adding EIF2S3; (3) detecting (2) In addition, increase TNFAIP6; (4) test (3), increase CPEB4; (5) test (4), increase ANXA3; (6) test (5), increase MAS4A1; or (7) test (6) In addition, increase IL2RB. The method of claim 4 or 5, wherein the gene expresses an amount of mRNA or protein. The method of claim 9, wherein the mRNA is measured by Northern blotting, reverse transcriptase polymerase chain reaction, and real-time polymerase chain reaction. The method of claim 9, wherein the protein is measured by immunofluorescence staining, enzyme-linked immunosorbent assay, enzyme immunoassay, radioimmunoassay or Western blotting. A kit for detecting colorectal cancer and/or assessing its prognosis. The kit includes: specific primer pairs for CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553, and MAS4A1 genes. A kit for detecting colorectal cancer and/or assessing its prognosis, the kit includes: Specific antibodies against the proteins of CPEB4, EIF2S3, ANXA3, TNFAIP6, IL2RB, MGC20553 and MAS4A1. A method for detecting whether an individual has colorectal cancer, the steps comprising: (a) obtaining a test sample containing nucleic acid from a body; (b) measuring a gene marker expression amount in the sample; (c) The amount of housekeeping gene expression of the individual normalizes the amount of expression of the gene marker; and (d) comparing the normalized gene marker expression amount with the gene marker expression amount of a health control group, if the gene marker expression amount The increase indicates that the individual has a colorectal cancer risk; wherein the amount of the gene measured in the step (b) can be determined according to requirements, and the detection order is: (1) detecting only CA7; (2) detecting (1) outside , increase IL6R; (3) detect (2), increase IL2RB; (4) detect (3), increase TNFAIP6; or (5) detect (4), increase CPEB4.