阅读说明：本技术 Loc90024 rna的应用及诊断、预后评估和***的产品 (Application of LOC90024RNA and product for diagnosis, prognosis evaluation and treatment of tumors ) 是由 晏光荣 孟楠 陈敏 于 2019-12-04 设计创作，主要内容包括：本发明提供了一种LOC90024RNA的应用及诊断、预后评估和治疗肿瘤的产品,涉及生物技术领域。临床研究证实,LOC90024RNA在肿瘤组织中高表达,与肿瘤患者预后差呈正相关。癌组织中LOC90024RNA高水平的患者具有更短的生存期和更高的死亡率。抑制LOC90024的表达,抑制了肿瘤细胞的增殖、克隆形成和迁移侵袭。据此,LOC90024RNA作为生物标志物有效的提高了临床中使用试剂盒进行肿瘤预后判断的阳性率。例如研发新的检测LOC90024RNA的试剂盒以诊断肿瘤,或以LOC90024RNA为靶点的药物以治疗肿瘤等等,对于肿瘤的诊断和治疗具有重要的影响。(The invention provides an application of LOC90024RNA and a product for diagnosis, prognosis evaluation and tumor treatment, and relates to the technical field of biology. Clinical studies have confirmed that LOC90024RNA is highly expressed in tumor tissues and positively correlated with poor prognosis of tumor patients. Patients with high levels of LOC90024RNA in cancer tissues have shorter survival and higher mortality. Inhibition of the expression of LOC90024 inhibits the proliferation, clonogenic and migratory invasion of tumor cells. Therefore, the LOC90024RNA as the biomarker effectively improves the positive rate of tumor prognosis judgment by using the kit in clinic. For example, developing a new kit for detecting LOC90024RNA to diagnose tumor, or developing a drug targeting LOC90024RNA to treat tumor, etc., has important effects on the diagnosis and treatment of tumor.)

Use of LOC90024RNA for the preparation of a product for tumor diagnosis and/or prognosis evaluation.

2. The use of claim 1, wherein the tumor is a solid tumor;

preferably, the solid tumor comprises one or more of pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer or soft tissue sarcoma.

3. Use according to claim 1 or 2, wherein the product comprises an agent, kit or medicament.

4. A reagent for tumor diagnosis and/or prognosis evaluation, wherein the reagent comprises a marker for detecting LOC90024 RNA.

5. The reagent according to claim 4, wherein the label comprises a primer that binds to LOC90024 RNA;

preferably, the primers comprise Q-PCR primers for LOC90024RNA or RT-PCR primers for LOC90024 RNA.

6. The reagent according to claim 5, wherein the upstream primer of the Q-PCR primer of LOC90024RNA is shown as SEQ ID No.1, and the downstream primer of the Q-PCR primer of LOC90024RNA is shown as SEQ ID No. 2;

preferably, the upstream primer of the RT-PCR primer of LOC90024RNA is shown as SEQ ID NO.3, and the downstream primer of the RT-PCR primer of LOC90024RNA is shown as SEQ ID NO. 4.

7. A kit for tumor diagnosis and/or prognosis evaluation, comprising the reagent according to any one of claims 4 to 6.

8. The kit of claim 7, wherein the kit further comprises one or more of a reverse transcription reagent, an internal reference primer, a fluorescent substance for indicating the amount of DNA synthesis, a reaction buffer, dNTPs, a DNA polymerase, or water;

preferably, the reaction buffer solution is Q-PCR reaction buffer solution or RT-PCR reaction buffer solution;

preferably, the reference primer comprises a primer of GAPDH;

preferably, when the reaction buffer is a Q-PCR reaction buffer, the upstream primer of GAPDH is shown as SEQ ID NO.5, and the downstream primer of GAPDH is shown as SEQ ID NO. 6;

preferably, when the reaction buffer is an RT-PCR reaction buffer, the upstream primer of GAPDH is shown as SEQ ID NO.7, and the downstream primer of GAPDH is shown as SEQ ID NO. 8.

9. A medicament for the treatment of a tumour, characterised in that the medicament comprises an inhibitor of LOC90024 RNA.

10. The medicament of claim 9, wherein the inhibitor comprises siRNA of LOC90024 RNA;

preferably, the siRNA has a nucleotide sequence shown as SEQ ID NO.9 and SEQ ID NO.10, or SEQ ID NO.11 and SEQ ID NO. 12.

Technical Field

The invention relates to the technical field of biology, in particular to application of LOC90024RNA and a product for diagnosis, prognosis evaluation and tumor treatment.

Background

Cancer is a major disease threatening human health and life, and has become the second largest disease in our country in terms of morbidity and mortality. The incidence of cancer in China is increasing year by year and the cancer tends to be younger. Every year, 429 ten thousands of new cancer cases in China account for 20 percent of new cases worldwide and 281 ten thousands of deaths occur. The solid malignant tumor has hidden onset and no obvious specific symptoms, once the specific symptoms appear, the disease usually belongs to an advanced stage, about more than 80 percent of patients with the solid malignant tumor have the intermediate and advanced stages in the first diagnosis and treatment, and the optimal treatment opportunity is lost.

The occurrence of solid malignant tumors is a complex process of multiple factors and multiple steps, and the etiology of the malignant tumors is not completely understood at present. Despite the continuous understanding of the pathogenesis of malignant tumors and the continuous improvement of medical technical means, the prognosis of patients with advanced malignant tumors is still poor, and the lack of specific prognostic markers is a very important reason. Therefore, there is an urgent need for more effective and sensitive prognostic markers to facilitate diagnosis, predict prognosis of patients with malignant tumors and facilitate rational treatment protocols, thereby improving patient survival.

Long non-coding RNAs (lnc RNA) are a class of RNAs whose transcripts are longer than 200 nucleotides in length. lnc RNA plays an important role in regulation of protein activity, transcription and translation, RNA variable shearing, chromosome modification and other processes. The abnormally expressed lnc RNA can directly or indirectly regulate and control a target gene, and participate in invasion and metastasis of tumors to generate and develop. With the continuous research of lnc RNA by people, lnc RNA is expected to become a novel tumor marker and an anti-cancer drug intervention target, and has good clinical application prospect in the aspects of tumor diagnosis, treatment and prognosis.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

A first object of the present invention is to provide the use of LOC90024RNA for the preparation of a product for tumor diagnosis and/or prognosis evaluation, to alleviate at least one of the technical problems of the prior art.

The second object of the present invention is to provide a reagent for diagnosing tumor, and the third object of the present invention is to provide a kit for diagnosing tumor, so as to realize effective diagnosis of tumor.

The fourth purpose of the invention is to provide a medicine for treating tumors, so as to realize targeted treatment of tumors.

The invention provides an application of LOC90024RNA in preparing a product for tumor diagnosis and/or prognosis evaluation.

Further, the tumor is a solid tumor;

preferably, the solid tumor comprises one or more of pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer or soft tissue sarcoma.

Further, the product comprises a reagent, a kit or a medicament.

The present invention also provides a reagent for tumor diagnosis and/or prognosis evaluation, which comprises a marker for detecting LOC90024 RNA.

Further, the label comprises a primer that binds to LOC90024 RNA;

preferably, the primers comprise Q-PCR primers for LOC90024RNA or RT-PCR primers for LOC90024 RNA.

Further, an upstream primer of the Q-PCR primer of the LOC90024RNA is shown as SEQ ID NO.1, and a downstream primer of the Q-PCR primer of the LOC90024RNA is shown as SEQ ID NO. 2;

preferably, the upstream primer of the RT-PCR primer of LOC90024RNA is shown as SEQ ID NO.3, and the downstream primer of the RT-PCR primer of LOC90024RNA is shown as SEQ ID NO. 4.

The invention also provides a kit for tumor diagnosis and/or prognosis evaluation, which comprises the reagent.

Further, the kit also comprises one or more of a reverse transcription reagent, an internal reference primer, a fluorescent substance for indicating the DNA synthesis amount, a reaction buffer solution, dNTPs, DNA polymerase or water;

preferably, the reaction buffer solution is Q-PCR reaction buffer solution or RT-PCR reaction buffer solution;

preferably, the reference primer comprises a primer of GAPDH;

preferably, when the reaction buffer is a Q-PCR reaction buffer, the upstream primer of GAPDH is shown as SEQ ID NO.5, and the downstream primer of GAPDH is shown as SEQ ID NO. 6;

preferably, when the reaction buffer is RT-PCR reaction buffer, the upstream primer of GAPDH is shown as SEQ ID NO.7, and the downstream primer of GAPDH is shown as SEQ ID NO. 8.

In addition, the invention also provides a medicament for treating tumors, which comprises an inhibitor of LOC90024 RNA.

Further, the inhibitor includes siRNA of LOC90024 RNA;

preferably, the siRNA has a nucleotide sequence shown as SEQ ID NO.9 and SEQ ID NO.10, or SEQ ID NO.11 and SEQ ID NO. 12.

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

the RNA LOC90024 related to tumors provided by the invention can be used for preparing products for tumor diagnosis and/or prognosis evaluation. Prior to the present invention, there has been no public report concerning the use of the LOC90024RNA of the present invention for tumor prognosis evaluation and diagnosis. Clinical studies have confirmed that LOC90024RNA is highly expressed in tumor tissues and positively correlated with poor prognosis of tumor patients. Patients with high levels of LOC90024RNA in cancer tissues have shorter survival and higher mortality. Therefore, the LOC90024RNA as the biomarker effectively improves the positive rate of tumor prognosis judgment by using the kit in clinic. Inhibition of the expression of LOC90024 inhibits the proliferation, clonogenic and migratory invasion of tumor cells. Different products can be developed aiming at the LOC90024RNA, for example, a new kit for detecting the LOC90024RNA is developed to diagnose tumors, or a medicine taking the LOC90024RNA as a target is used for treating the tumors, and the like, and the kit has important influence on the diagnosis and treatment of the tumors.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a comparative graph showing RT-PCR results of LOC90024 expression in the RT-PCR detection of gastric cancer and liver cancer tissues in example 1 of the present invention;

FIG. 2 is a graph showing the comparison of the Q-PCR results of LOC90024 expression in the tissues of gastric cancer and liver cancer detected by Q-PCR in example 2 of the present invention;

FIG. 3 is a comparison graph of the RT-PCR results of LOC90024 expressed by colon cancer cell line SW-620 capable of silencing LOC90024 in example 3 of the present invention;

FIG. 4 is a graph comparing the Q-PCR results of LOC90024 expression of a colon cancer cell line SW-620 silencing LOC90024 in example 3 of the present invention;

FIG. 5 is a comparison graph of the cell proliferation results of LOC90024 expression in the silent colon cancer cell line HCT-116 in example 4 of the present invention;

FIG. 6 is a comparison graph of the results of cell cloning of LOC90024 expression in the silent colon cancer cell line HCT-116 in example 4 of the present invention;

FIG. 7 is a graph comparing the results of LOC90024 expression in the colon cancer cell line HCT-116 according to example 4 of the present invention in terms of cell migration ability and invasion ability.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

According to one aspect of the present invention, there is provided the use of LOC90024RNA for the preparation of a product for tumor diagnosis and/or prognosis evaluation.

Long Non-Coding RNA (Long Non-Protein Coding RNA, lncRNA) was originally considered as "noise" of genome transcription and had no biological function. However, it is generally accepted in the art that most lncrnas do not encode any protein, even though lncrnas actually regulate the expression level of genes in RNA form at various levels (epigenetic regulation, transcriptional regulation, and post-transcriptional regulation).

Long Intergenic Non-Protein Coding RNA (lincRNA) is one of lncRNA, LOC90024(Gene ID:90024) is a newly discovered lincRNA, and the function thereof has not been clarified. The LOC90024RNA is related to tumors, particularly solid tumors, and can express protein polypeptides.

Based on this, the present invention provides a novel marker for tumor prognosis evaluation judgment: LOC90024 RNA. Clinical research proves that the high expression of the polypeptide in tumor tissues is in obvious positive correlation with poor prognosis of tumor patients. Patients with high levels of LOC90024RNA in cancer tissues have shorter survival and higher mortality. Inhibition of the expression of LOC90024 inhibits the proliferation, clonogenic and migratory invasion of tumor cells. Therefore, the LOC90024RNA as the biomarker effectively improves the positive rate of tumor prognosis judgment by using the kit in clinic.

It should be noted that the increased expression level of LOC90024RNA is indicative of a tumor and/or indicative of poor prognosis of a tumor.

In some preferred embodiments, the tumor is a solid tumor;

preferably, the solid tumor comprises one or more of pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer or soft tissue sarcoma.

In some embodiments, the solid tumor is selected from advanced or metastatic malignant solid tumors.

The term "advanced or metastatic malignant solid tumor" refers to a histologically or cytologically confirmed diagnosis of advanced, unresectable and/or metastatic relapsed or refractory malignant solid tumor that is ineffective or absent treatment proven effective against standard therapy. According to the present invention, malignant solid tumors include, but are not limited to, carcinomas, sarcomas, melanomas, lymphomas, or the like.

The products described in the present invention preferably comprise reagents, kits or medicaments.

According to a second aspect of the present invention, there is provided a reagent for tumor diagnosis and/or prognosis evaluation, which comprises a marker for detecting LOC90024 RNA.

As the agent for detecting RNA, a known agent known to those skilled in the art can be used, for example, a nucleic acid capable of hybridizing with the RNA and labeled with a fluorescent label.

In some preferred embodiments, the label comprises a primer that binds to LOC90024 RNA.

Preferably, the primers comprise Q-PCR primers for LOC90024RNA or RT-PCR primers for LOC90024 RNA.

Both Q-PCR primers and RT-PCR primers can be conveniently used to detect LOC90024RNA to measure the transcription level of LOC90024 RNA.

In some preferred embodiments, the upstream primer of the Q-PCR primer of LOC90024RNA is shown as SEQ ID NO.1, and the downstream primer of the Q-PCR primer of LOC90024RNA is shown as SEQ ID NO. 2;

preferably, the upstream primer of the RT-PCR primer of LOC90024RNA is shown as SEQ ID NO.3, and the downstream primer of the RT-PCR primer of LOC90024RNA is shown as SEQ ID NO. 4.

When the Q-PCR primer and the RT-PCR primer with the specific sequences are selected to detect the LOC90024RNA, the expression level of the LOC90024RNA in the tumor cells can be quantitatively detected, the specificity is stronger, the sensitivity is higher, and the obtained detection result is more accurate.

According to a third aspect of the present invention, there is provided a kit for tumor diagnosis and/or prognosis evaluation, the kit comprising the above-described reagents.

The kit can amplify cDNA reverse transcribed from LOC90024 RNA.

In some preferred embodiments, the kit further comprises one or more of a reverse transcription reagent, an internal reference primer, a fluorescent substance for indicating the amount of DNA synthesis, a reaction buffer, dNTPs, a DNA polymerase, or water;

it should be noted that, the corresponding reaction buffer can be selected according to different detection techniques, for example, when the detection is performed by using Q-PCR means, the kit can contain Q-PCR reaction buffer; when RT-PCR is used for detection, RT-PCR reaction buffer may be included in the kit.

Preferably, the reference primer comprises a primer of GAPDH. By setting internal reference contrast, the validity of the detection result can be judged more accurately.

Preferably, when the reaction buffer is a Q-PCR reaction buffer, the upstream primer of GAPDH is shown as SEQ ID NO.5, and the downstream primer of GAPDH is shown as SEQ ID NO. 6;

preferably, when the reaction buffer is RT-PCR reaction buffer, the upstream primer of GAPDH is shown as SEQ ID NO.7, and the downstream primer of GAPDH is shown as SEQ ID NO. 8.

Alternatively, the DNA polymerase is selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, Klenow fragment.

Alternatively, the reagent or kit provided by the invention is used for detecting cell lysate of tumor tissue or tissues beside cancer of a subject.

As used herein, "tissue lysate," "cell lysate," "lysed sample," "tissue extract," or "cell extract" refers to a sample and/or biological sample material comprising lysed tissue or cells, i.e., wherein the structural integrity of the tissue or cells has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining a lysate. This process is encompassed by the term "lysis".

In some specific embodiments, the present invention also relates to a method of assessing a tumor, for example assessing a solid tumor, the method comprising:

(i) measuring the expression amount of LOC90024RNA in a sample using the above-described reagent or kit for specifically detecting LOC90024 RNA;

(ii) optionally, measuring the concentration of one or more other tumor markers in the sample;

(iii) (iii) assessing the tumor using the measurement of step (i) and optionally the measurement of step (ii), wherein an increased expression level of LOC90024RNA is indicative for the tumor.

According to a fourth aspect of the present invention, there is provided a medicament for the treatment of a tumour, said medicament comprising an inhibitor of LOC90024 RNA. By inhibiting the expression of LOC90024RNA, a potent inhibitory effect on tumor cells can be achieved.

In some preferred embodiments, the inhibitor comprises an siRNA of LOC90024 RNA;

preferably, the siRNA has a nucleotide sequence shown in SEQ ID NO.9 and SEQ ID NO.10 or SEQ ID NO.11 and SEQ ID NO. 12.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.