CN113201540B - Non-coding RNA, RNA sequence containing non-coding RNA and application thereof - Google Patents

Non-coding RNA, RNA sequence containing non-coding RNA and application thereof Download PDF

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CN113201540B
CN113201540B CN202110414316.5A CN202110414316A CN113201540B CN 113201540 B CN113201540 B CN 113201540B CN 202110414316 A CN202110414316 A CN 202110414316A CN 113201540 B CN113201540 B CN 113201540B
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stem cells
pla2g16
breast cancer
coding rna
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CN113201540A (en
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柳满然
侯懿烜
刘水清
孙艳
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Chongqing Medical University
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Abstract

The invention belongs to the technical field of tumor stem cell research, and particularly relates to non-coding RNA and coding RNA, an RNA sequence containing the non-coding RNA and application thereof. The Sequence of the non-coding RNA is shown as Sequence No.1, the non-coding RNA is combined with the 3' -UTR-1 region of mRNA of the PLA2G16 gene to enhance the expression of the PLA2G16 gene, thereby promoting the stem characteristic of cells, and the non-coding RNA can be knocked out to inhibit the expression of the PLA2G16 gene in the tumor stem cells, thereby inhibiting the occurrence/development of the tumor stem cells. The invention can effectively inhibit the occurrence/development of breast cancer stem cells/tumors by effectively knocking out the expression of the non-coding RNA to inhibit PLA2G16 genes.

Description

Non-coding RNA, RNA sequence containing non-coding RNA and application thereof
Technical Field
The invention belongs to the technical field of tumor stem cell research, and particularly relates to non-coding RNA and coding RNA, an RNA sequence containing the non-coding RNA and application thereof.
Background
Tumor stem cells are thought to be the primary cause of poor prognosis in patients due to tumorigenesis, recurrence, metastasis, drug resistance, however, the regulatory mechanisms involved in maintaining tumor stem cell characteristics and biological behavior remain unknown. And the existing medicines for preventing or treating breast cancer have medicine resistance, so that the medicines have an obstructing effect for eliminating or inhibiting breast cancer cells/tumors. Long non-coding RNAs are a class of transcripts that are more than 200 nucleotides in length and do not encode proteins. More and more studies have shown that long-chain non-coding RNAs are key regulatory factors in various biological processes, including DNA damage repair, inflammation, metabolism, cell signaling, cell differentiation, proliferation, apoptosis, migration, invasion, and the like. Although some studies have shown that long non-coding RNAs are aberrantly expressed in tumor stem cells, only a small fraction has been identified as having important functions.
Disclosure of Invention
In view of this, the present invention aims to provide a non-coding RNA which is a long-chain non-coding RNA identified by analysis of lncRNA chip and which is highly expressed in breast cancer stem cells, and the present invention designates it as lncROPM. Meanwhile, lncROPM is highly expressed in clinical breast tumors and other solid tumors and is positively correlated with malignancy of disease, histological grading, and poor prognosis in patients. In vivo and in vitro functional gain-loss experiments indicate that lncrpm is necessary to maintain the stem cell stem characteristics of breast cancer. Mechanically, lncROPM regulates expression of the target gene PLA2G16 by directly binding to the 3' utr region of PLA2G16mRNA, maintaining its mRNA stability. Increased PLA2G16 promotes phospholipid metabolism and the production of free fatty acids, in particular arachidonic acid. Subsequently, increased arachidonic acid activates PI3K/AKT, wnt/β -catenin and Hippo/YAP signaling pathway, thereby participating in maintaining the stem characteristics of breast cancer stem cells. Importantly, lncrop and PLA2G16 also significantly promote breast cancer stem cell resistance. The combination of a clinically therapeutic agent (such as doxorubicin, cisplatin, or tamoxifen) with gellan (cytosolic phospholipase A2 inhibitor) is effective in eliminating breast cancer stem cells and tumorigenesis. In conclusion, lncROPM and its target gene PLA2G16 play a vital role in promoting the stem cell stem characteristics of breast cancer, and can be used as a marker of breast cancer stem cells or other tumor stem cells.
The nucleic acid Sequence of the non-coding RNA is shown in Sequence No. 1.
Further, the non-coding RNA is located on the human 11q12.3-q13.1 chromosome, adjacent to the PLA2G16 gene.
Further, the non-coding RNA consists of two exons linked, with a length of 573bp.
Further, the non-coding RNA is located in the stem cell cytoplasm.
In certain embodiments, the stem cells comprise breast cancer stem cells, renal clear cell carcinoma stem cells, head and neck squamous cell carcinoma stem cells, ovarian serous cyst adenocarcinoma stem cells.
The present invention also provides an RNA sequence comprising: mRNA of PLA2G16 gene and the non-coding RNA described above.
Further, the RNA sequence is such that the non-coding RNA binds to the 3' -UTR-1 region of the mRNA of the PLA2G16 gene.
Further, the RNA Sequence is shown in Sequence No. 2.
The present invention also aims to provide a method for enhancing PLA2G16 gene expression, said method comprising: the aforementioned non-coding RNA was bound to the 3' -UTR-1 region of the mRNA of the PLA2G16 gene.
In certain embodiments, enhancing PLA2G16 gene expression is shown by binding the aforementioned non-coding RNAs to the 3' -UTR-1 region of the mature mRNA of the PLA2G16 gene mRNA, thereby extending the mRNA half-life of the PLA2G16 gene, increasing mRNA content, and PLA2G16 content.
The present invention also provides a method for prolonging the half-life of mRNA of PLA2G16 gene by binding the non-coding RNA to the 3' -UTR-1 region of mature mRNA of the PLA2G16 gene.
The present invention is also directed to a method of modulating transcription and/or translation of the PLA2G16 gene in a cell.
The method comprises the following steps: a) Knocking down the aforementioned non-coding RNA in the cell, thereby inhibiting transcription and/or translation of the PLA2G16 gene; or b) over-expressing the non-coding RNA as described above in the cell, thereby enhancing PLA2G16 gene transcription and/or translation.
In certain embodiments of the invention, the results of transcription and/or translation of the PLA2G16 gene are displayed by mRNA and protein levels of PLA2G 16.
The invention also provides a method for regulating the growth of stem cells in stem cells/non-stem cells in vitro.
The method comprises the following steps: a) Knocking out the non-coding RNA of any one of claims 1-4 in stem cells/non-stem cells, and inhibiting the growth of the stem cells; or b) overexpressing the non-coding RNA of claim 1 in stem cells/non-stem cells, promoting stem cell growth; or c) inhibiting PLA2G16 gene expression, inhibiting stem cell growth; or d) over-expressing PLA2G16 gene to promote stem cell growth.
Further, the cells/non-stem cells are cancer cells or normal cells.
Further, clear kidney cancer stem cells, head and neck squamous carcinoma stem cells, ovarian serous cystic adenocarcinoma stem cells, and breast cancer stem cells.
Further, the breast cancer stem cell includes: BT549 breast cancer stem cells, hs578T breast cancer stem cells, MDA231 breast cancer stem cells, MDA468 breast cancer stem cells, MDA436 breast cancer stem cells, MCF7 breast cancer stem cells, T47D breast cancer stem cells.
In certain embodiments of the invention, stem cell growth in stem cells/non-stem cells is modulated by modulating stem cell/non-stem cell characteristics.
The invention also provides a method of modulating the stem cell/non-stem cell stem characteristics.
The method comprises the following steps: a) Knocking out the aforementioned non-coding RNA or inhibiting expression of PLA2G16 gene in the stem cells/non-stem cells or attenuating the dry character using gellan pa; or b) overexpressing the aforementioned non-stem cells in stem cells/non-stem cellsCoding RNA or over-expressing PLA2G16 gene, facilitating dry character; the stem characteristic comprises expression of a stem related gene and/or stem cell balling rate and/or stem cell size and/or CD44 + /CD24 -/low A subpopulation of stem cells.
Further, the dryness gene includes: SOX2, nanog, OCT4, KLF4, beta-action.
Further, the stem/non-stem cells include: renal clear cancer stem cells/non-stem cells, head and neck squamous cancer stem cells/non-stem cells, ovarian serous cystic adenocarcinoma stem cells/non-stem cells, breast cancer stem cells/non-stem cells.
In certain implementations of the invention, the method includes: a) Knocking out the non-coding RNA in breast cancer stem cells, and weakening the characteristic of dryness; or b) overexpressing non-coding RNA in breast cancer non-stem cells to promote a stem trait.
The present invention is directed to a method of identifying stem cells and/or non-stem cells.
The method comprises the following steps: detecting the expression level of the PLA2G16 gene in the cell, a) a stem cell if the expression level is high, or b) a non-stem cell if the expression level is low.
Further, the method comprises: detecting whether the non-coding RNA in the cell binds to the 3' -UTR-1 region of the mRNA of the PLA2G16 gene, and if so, determining that the cell is a stem cell; if not, it is a non-stem cell.
In certain embodiments of the invention, the method comprises: a) The stem cells and the non-stem cells are breast cancer stem cells/non-stem cells, and the high expression level of PLA2G16 is breast cancer stem cells; or b) the stem cells are embryonic stem cells and stem cells derived from the embryonic stem cells, and the high expression level of PLA2G16 is the embryonic stem cells; or c) the stem cells and the non-stem cells are liver stem cells and liver cells of differentiated cells, and the liver stem cells with high expression level of PLA2G16 are obtained.
The invention also provides a method for activating/inhibiting PI3K/AKT, wnt/beta-catenin and Hippo/YAP signal paths.
The method comprises the following steps: a) Overexpression of the non-coding RNA or the PLA2G16 gene activates PI3K/AKT, wnt/beta-catenin and Hippo/YAP signal channels; or b) knocking out the non-coding RNA or inhibiting PLA2G16 gene expression, and inhibiting PI3K/AKT, wnt/beta-catenin and Hippo/YAP signal paths.
The present invention also aims to provide a method for inhibiting/promoting the arachidonic acid content of cells, which comprises the following steps: a) Inhibiting the cellular arachidonic acid content by knocking out the non-coding RNA of claim 1 or inhibiting expression of the PLA2G16 gene; or overexpressing the non-coding RNA of claim 1 or overexpressing the PLA2G16 gene to promote the cellular arachidonic acid content.
In certain embodiments, the representative products Cer (ceramide) and FFA (free fatty acid) levels were significantly reduced after knockdown of lncrpm and the Cer and FFA levels were significantly increased after overexpression of lncrpm, indicating that Cer and FFA may be key metabolites of lncrpm-PLA 2G16 driving the stem cell stem characteristics of breast cancer. The more active PLA2G16 expression, the higher the AA (arachidonic acid) content; meanwhile, compared with non-stem cells, AA is obviously enriched in breast cancer stem cells; in addition, the embodiment of the invention also finds that AA is obviously enriched in advanced tumor tissues, recurrent tumor tissues and drug-resistant tissues. AA (arachidonic acid) is a major part of FFA (free fatty acid).
The present invention also provides a method of inhibiting tumorigenesis/progression comprising: knocking down the aforementioned non-coding RNA in the cells of the tumor.
Further, the tumor is a kidney transparent tumor, a head and neck squamous tumor, an ovarian serous cyst gland tumor, and a breast tumor.
In certain embodiments, knocking down lncrpm reduces the initiation frequency of the tumor in the tumor stem cells; or b) over-expressing lncROPM in tumor non-stem cells increases the initiation frequency of tumors.
The invention also provides a method for regulating the sensitivity of cancer stem cells/non-stem cells to drugs.
The method comprises the following steps: a) Knocking down the aforementioned non-coding RNAs and/or inhibiting PLA2G16 gene expression increases cancer stem cell/non-stem cell sensitivity to drugs; or b) over-expressing the non-coding RNA and/or over-expressing the PLA2G16 gene as described above reduces the drug sensitivity of the stem/non-stem cells.
Further, the cancer stem cells/non-stem cells are breast cancer stem cells/non-stem cells.
In certain embodiments, the method comprises: a) Knocking down the non-coding RNA and/or inhibiting the PLA2G16 gene expression and/or adding arachidonic acid reduces the drug sensitivity of BT549 breast cancer stem cells/non-stem cells to doxorubicin; or b) overexpressing the non-coding RNA and/or the overexpressing PLA2G16 gene reduces the drug sensitivity of BT549 breast cancer stem cells/non-stem cells to doxorubicin; or c) knocking down the non-coding RNA or inhibiting the PLA2G16 gene and/or adding arachidonic expression to increase the drug sensitivity of BT549 breast cancer stem cells/non-stem cells to cisplatin; or d) overexpressing the non-coding RNA or the overexpressing PLA2G16 gene reduces drug sensitivity of BT549 breast cancer stem cells/non-stem cells to cisplatin; or e) knocking down the non-coding RNA or inhibiting the PLA2G16 gene and/or adding arachidonic acid to increase the sensitivity of BT549 breast cancer stem cells/non-stem cells to tamoxifen; or f) overexpression of the non-coding RNA or overexpression of PLA2G16 gene reduces sensitivity of BT549 breast cancer stem cells/non-stem cells to tamoxifen.
In certain embodiments, the method comprises: a) Knocking down the non-coding RNA or inhibiting PLA2G16 gene expression in the breast cancer stem cells or adding arachidonic acid to the BT549 breast cancer stem cells treated by Ji Lipa can restore the drug resistance of the breast cancer stem cells to doxorubicin; or b) knockdown of the non-coding RNA of claim 1 or inhibition of PLA2G16 gene expression or treatment with Ji Lipa of MCF7 breast cancer stem cells, the drug resistance of the stem cells to tamoxifen can be restored after arachidonic acid is added.
The invention also provides an in vitro method for inhibiting the occurrence/development of breast cancer stem cells/tumors.
The method comprises the following steps: the use of gelipenda in combination with doxorubicin, cisplatin, tamoxifen, respectively, inhibits the expression of the aforementioned non-coding RNAs and PLA2G16 genes, thereby inhibiting the occurrence/progression of breast cancer stem cells/tumors.
In certain embodiments, the method comprises: a) The combined use of gellan pa and doxorubicin inhibits the occurrence/development of BT549/Hs578T breast cancer stem cells/tumors; or b) inhibition of the occurrence/progression of BT549/Hs578T breast cancer stem cells/tumors in combination with cisplatin; or c) the use of gelipapide in combination with tamoxifen inhibits the occurrence/progression of MCF7 breast cancer stem cells/tumors.
The invention has the beneficial effects that:
the lncROPM provided by the invention can be directly combined with the 3' UTR region of PLA2G16 mRNA to maintain the stability of the mRNA so as to regulate the expression of the target gene PLA2G 16; increased PLA2G16 promotes phospholipid metabolism and the production of free fatty acids, in particular arachidonic acid. Subsequently, increased arachidonic acid activates PI3K/AKT, wnt/β -catenin and Hippo/YAP signaling pathway, thereby participating in maintaining the stem characteristics of breast cancer stem cells.
The lncROPM and PLA2G16 provided by the invention also significantly promote the drug resistance of breast cancer stem cells, and the combination of the drug (such as doxorubicin, cisplatin or tamoxifen) and gellanpatadine (cytosolic phospholipase A2 inhibitor) can effectively eliminate the breast cancer stem cells and tumorigenesis.
Drawings
FIG. 1 shows MCF7 breast cancer stem cells (CD 44 + CD24 -/low Microspheres) and non-stem cells (CD 44 -/low CD24 + ) lncRNAs map of differential expression in (a).
FIG. 2 is a graph of lncRNAs associated with metabolic pathways in breast cancer stem cells.
FIG. 3 shows the sequence of long non-coding RNA lncROPM.
FIG. 4 shows the expression of lncROPM in cancer stem cells of various breast cancer cell lines.
FIG. 5 is a RNA Fish assay to locate lncROPM.
FIG. 6 is a subcellular separation assay positioning lncROPM.
FIG. 7 is a TaNRIC database search for lncROPM expression in patients.
FIG. 8 is a graph showing that high expression of lncROPM is significantly correlated with poor prognosis in breast cancer patients.
FIG. 9 shows qRT-PCR assay to detect expression of lncROPM in breast cancer tissue and paired adjacent normal breast tissue.
FIG. 10 is a representative image of the expression of lncROPM as a function of tumor size, TNM stage, tissue grade, lymph node metastasis, tumor recurrence.
FIG. 11 shows the expression relationship of lncROPM with the dry marker SOX2 in breast cancer tissue.
Fig. 12 is the relationship of lncROPM to dry marker OCT4 expression in breast cancer tissue.
FIG. 13 shows the expression of lncROPM in breast cancer stem cells and non-stem cells.
FIG. 14 shows the expression of lncROPM in breast cancer tissues that are/are not resistant to chemotherapeutic agents.
Fig. 15 is a relationship of lncap expression in tissue grade increase in Head and Neck Squamous Cell Carcinoma (HNSCC).
FIG. 16 shows the relationship of increasing lncROPM expression in tissue grade in ovarian serous cystic adenocarcinoma (OV).
FIG. 17 shows the expression of lncROPM in stem cells and non-stem cells of renal clear cell carcinoma (KIRC).
Fig. 18 shows lncrpm expression in stem cells and non-stem cells of Head and Neck Squamous Cell Carcinoma (HNSCC).
Fig. 19 shows lncrpm expression in stem cells and non-stem cells of ovarian serous cystic adenocarcinoma (OV).
FIG. 20 is a graph showing the case of short hairpin RNAs (shRNAs) silencing lncROPM in different breast cancer stem cells.
FIG. 21 is a graph showing the overexpression of lncROPM by lentiviral vectors in different breast cancer stem cells.
FIG. 22 shows expression of the genes related to dryness (SOX 2, nanog, OCT4, KLF 4) after efficient knockdown of lncROPM in different breast cancer stem cells.
Fig. 23 shows the condition of stem cell pelleting and size physical after effective knockout of lncrpm in different breast cancer stem cells.
Fig. 24 is a graph showing the spheroidization rate/stem cell size of stem cells after effective knockdown of lncrpm in different breast cancer stem cells.
FIG. 25 shows the expression of lncROPM and a stem related gene (SOX 2, nanog, OCT4, KLF 4) in non-stem cells.
FIG. 26 shows the condition of stem cells forming pellets and size objects after overexpression of lncROPM in non-stem cells.
FIG. 27 shows the cell pelleting rate/stem cell size after overexpression of lncROPM in non-stem cells.
FIG. 28 is a plot of the initial frequency of lncROPM deficiency in breast cancer stem cells versus breast tumor in vivo experiments.
FIG. 29 is a plot of the initial frequency of lncROPM deficiency in breast cancer stem cells versus breast tumor in vitro.
FIG. 30 is a plot of lncROPM overexpression loss in breast cancer non-stem cells versus initiation frequency of breast tumors in vivo.
FIG. 31 is a plot of lncROPM overexpression loss in breast cancer non-stem cells versus initiation frequency of breast tumors in an in vitro experiment.
FIG. 32 shows the expression of the target gene PLA2G16 after knockdown of lncROPM in breast cancer stem cells.
FIG. 33 shows the expression of PLA2G16 protein as target gene after knockdown of lncROPM in breast cancer stem cells.
FIG. 34 shows the expression of PLA2G16 as a target gene after overexpression of lncROPM in non-stem cells of breast cancer.
FIG. 35 shows the expression of PLA2G16 protein as a target gene after overexpression of lncROPM in non-stem cells of breast cancer.
FIG. 36 shows the relationship between the expression of lncROPM and the expression of PLA2G16 in breast cancer tissue samples.
FIG. 37 is a representative image of highly expressed PLA2G16 with advanced TNM stage, high tissue grade, lymph node metastasis, tumor recurrence.
FIG. 38 is a graph showing the expression of PLA2G16 in drug resistant/sensitive breast cancer tissues.
FIG. 39 is a graph showing poor prognosis and drug resistance associated with PLA2G16 in breast cancer patients.
FIG. 40 shows the expression levels of PLA2G16 pre-mRNA and mature mRNA after knockdown of lncROPM in MCF7 breast cancer stem cells.
FIG. 41 shows the expression levels of PLA2G16 pre-mRNA and mature mRNA after knockdown of lncROPM in BT549 breast cancer stem cells.
FIG. 42 shows the expression of PLA2G16 pre-mRNA and mature mRNA after the lncROPM has been knocked down.
FIG. 43 shows the interaction of lncROPM with the 3' -UTR region of PLA2G16 mRNA.
FIG. 44 shows the binding of lncROPM to the 3' -UTR region of PLA2G16 mRNA.
FIG. 45 shows the luciferase activity of overexpressing lncROPM and constructs containing PLA2G 16' -UTR regions.
FIG. 46 is a half-life of PLA2G16 mRNA in breast cancer stem cells deleted/overexpressed lncROPM.
FIG. 47 shows the expression of PLA2G16 in breast cancer stem/non-stem cells.
FIG. 48 shows the relationship between the expression of PLA2G16 and the expression of the dryness-related gene SOX2 in breast cancer tissue samples.
FIG. 49 shows the relationship between PLA2G16 expression and dry related gene OCT4 expression in breast cancer tissue samples.
FIG. 50 shows the expression of PLA2G16 gene in breast cancer stem cells knocked down with PLA2G 16.
FIG. 51 shows the expression of PLA2G16 protein in breast cancer stem cells knocked down with PLA2G 16.
FIG. 52 shows PLA2G16 gene expression in breast cancer non-stem cells that overexpress PLA2G 16.
FIG. 53 shows PLA2G16 protein expression in breast cancer non-stem cells that overexpress PLA2G 16.
FIG. 54 is a graph of the inhibition of PLA2G16 enzyme activity by Gilipapide.
FIG. 55 shows the expression of endogenous PLA2G16 knockdown or post-use gellan Pade in various breast cancer stem cells (SOX 2, OCT4, KLF 4).
FIG. 56 is a graph showing stem cell balling after knockdown of endogenous PLA2G16 in various breast cancer stem cells or use of Gilipadine.
FIG. 57 is a graph showing the spheroidization rate of stem cells after knockdown of endogenous PLA2G16 in various breast cancer stem cells or using Gilipapid.
FIG. 58 is a graph showing stem cell size following knockdown of endogenous PLA2G16 in various breast cancer stem cells or use of Gillepada.
FIG. 59 is a graph showing stem cell balling after overexpression of PLA2G16 in different breast cancer non-stem cells.
FIG. 60 is a graph showing the spheroidization rate of stem cells after overexpression of PLA2G16 in various breast cancer non-stem cells.
FIG. 61 shows the expression of the genes related to dryness (SOX 2, OCT4, KLF4, beta-action) after overexpression of PLA2G16 in different breast cancer non-stem cells.
FIG. 62 is a graph showing the spheroid size of stem cells after overexpression of PLA2G16 in various breast cancer non-stem cells.
FIG. 63 shows high/low expression of PLA2G16 and CD44 + /CD24 -/low Stem cell subpopulation expression relationship.
FIG. 64 shows the expression relationship of high/low expression PLA2G16 and dryness-associated genes.
FIG. 65 shows the expression of PLA2G16 in embryonic stem cells and embryoid bodies.
FIG. 66 shows PLA2G16 expression in embryonic stem cells and stem cells derived therefrom.
FIG. 67 shows PLA2G16 expression in cells of differentiated cells and stem cells in the liver.
FIG. 68 shows PLA2G16 expression in mast cells via stem cells.
FIG. 69 shows the arachidonic acid content of MCF7 breast cancer stem cells after knockdown of PLA2G16 or inhibition of PLA2G16 enzymatic activity.
FIG. 70 shows the content of arachidonic acid after overexpression of PLA2G16 in MCF7 breast cancer non-stem cells.
FIG. 71 shows the enrichment of arachidonic acid in breast cancer stem cells/non-stem cells.
FIG. 72 shows the enrichment of arachidonic acid in late stage tumor tissue, recurrent tumor tissue, and drug resistant tissue.
FIG. 73 shows the expression of the suppressed arachidonic acid-added stem-related genes (SOX 2, OCT4 and KLF 4) in stem cells after knocking down lncROPM or PLA2G 16.
FIG. 74 shows the pelleting of stem cells after addition of arachidonic acid after knockdown of lncROPM in stem cells.
FIG. 75 shows the pelleting of stem cells after the addition of arachidonic acid after knockdown of PLA2G16 in the stem cells.
FIG. 76 shows the relationship between the addition of arachidonic acid after lncROPM knockdown and PI3K/AKT, wnt/beta-catenin and Hippo/YAP signaling pathways.
FIG. 77 is a graph showing the relationship between the addition of arachidonic acid after PLA2G16 knockdown and PI3K/AKT, wnt/beta-catenin and Hippo/YAP signaling pathways.
FIG. 78 is a graph showing drug sensitivity of BT549 breast cancer stem cells to Doxorubicin (Doxorubicin) after lncROPM or PLA2G16 knockdown.
FIG. 79 shows drug sensitivity of BT549 breast cancer stem cells to Cisplatin (Cisplatin) after lncROPM or PLA2G16 knockdown.
FIG. 80 shows drug sensitivity of BT549 breast cancer stem cells to Tamoxifen (Tamoxifen) after knockdown of lncROPM or PLA2G 16.
FIG. 81 shows the drug sensitivity of stem cells to Doxorubicin (Doxorubicin) after the addition of arachidonic acid to BT549 breast cancer stem cells treated with the deletion of lncROPM or PLA2G16 and Ji Lipa.
FIG. 82 shows drug sensitivity of stem cells to Cisplatin (Cisplatin) after addition of arachidonic acid to BT549 breast cancer stem cells treated with the deletion of lncROPM or PLA2G16 and Ji Lipa.
FIG. 83 is a graph showing drug sensitivity of stem cells treated with deletion of lncROPM or PLA2G16 and Ji Lipa to Tamoxifen (Tamoxifen) after addition of arachidonic acid to MCF7 breast cancer stem cells.
FIG. 84 is a graph showing drug sensitivity to Doxorubicin (Doxorubicin) after overexpression of lncROPM or PLA2G16 in BT549 breast cancer non-stem cells.
FIG. 85 is a graph showing drug sensitivity to Cisplatin (Cisplatin) after overexpression of lncROPM or PLA2G16 in BT549 breast cancer non-stem cells.
FIG. 86 is a graph showing drug sensitivity to Tamoxifen (Tamoxifen) after overexpression of lncROPM or PLA2G16 in MCF7 breast cancer non-stem cells.
FIG. 87 shows the expression of lncROPM and PLA2G16 in stem cells of different breast cancer cells and stem cells of their parent cells.
FIG. 88 shows the expression of the susceptibility trait genes (OCT 4, SOX 2) in stem cells of various breast cancer cells and stem cells of their parent cells.
FIG. 89 is a graphical representation of the effect of Gilipadib (Gilipladib) in combination with Doxorubicin (Doxorubicin) or alone on BT549/Hs578T breast cancer stem cell balling.
FIG. 90 is an effect of Gilipadib (Gilipldib) in combination with Doxorubicin (Doxorubicin) or alone on the spheroidization rate of BT549/Hs578T breast cancer stem cells.
FIG. 91 is a graphical representation of the effect of Gilipadib (Gilipadib) in combination with Cisplatin (Cisplatin) or alone on the balling of BT549/Hs578T breast cancer stem cells.
FIG. 92 shows the effect of Gilipadib (Gilipadib) in combination with Cisplatin (Cisplatin) or alone on the spheroidization rate of BT549/Hs578T breast cancer stem cells.
Fig. 93 is a graphical representation of the effect of gilipendib (gilipendib) in combination with Tamoxifen (Tamoxifen) or alone on MCF7 breast cancer stem cell balling.
FIG. 94 is an effect of Gilipadib (Gilipadib) in combination with Tamoxifen (Tamoxifen) or alone on the rate of balling MCF7 breast cancer stem cells.
Figure 95 is a graph showing the case of combining gemfibrozil with doxorubicin and tumor volume.
Figure 96 shows the combination of geepadine with doxorubicin and tumorigenesis and growth.
FIG. 97 shows the expression of the stem cell related transcription factor KLF4 in tumors after combination therapy.
In the figures, CSCs are stem cells, and non-CSCs are non-stem cells.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
In the examples of the present invention, human breast cancer cell lines (BT 549, hs578T, MDA, MDA468, MDA436, MCF7 and T47D) were from american type culture collection (ATCC, USA); all cells were at 37℃and 5% CO 2 Cultured in medium (Gibco, USA) of 10% fetal bovine serum (Gibco, USA) and 1% streptomycin/penicillin (Beyotime, shanghai, china).
In the examples of the present invention, in order to establish an MDA231 cell line (MDA 231/DDP) against Cisplatin (Cisplatin or DDP), a BT549 cell line (BT 549/DOX) against Doxorubicin (Doxorubicin or DOX), and an MCF7 cell line (MCF 7/TAM) against Tamoxifen (Tamoxifen or TAM), MDA231, BT549, and MCF7 cells were repeatedly exposed to increasing concentrations of the drug over a period of 6 months.
In the examples of the present invention, MDA231/DDP, BT549/DOX, MCF7/TAM cells were cultured in medium containing 2. Mu.M cisplatin (Med-ChemExpress, NJ, USA), 2. Mu.M doxorubicin (Med-ChemExpress, NJ, USA), 1. Mu.M tamoxifen (Med-ChemExpress, NJ, USA), respectively, in order to maintain the resistant phenotype.
In the examples of the present invention, arachidonic acid was purchased from Sigma-Aldrich (St.Louis, MO, USA); gilipadib, a cytoplasmic phospholipase A2 inhibitor, purchased from USBiological Life Sciences (Swampscott, mass., USA).
In an embodiment of the invention, human breast tumor tissue and its corresponding normal tissue (at least 5cm from tumor tissue) are obtained from a breast cancer patient who has undergone a primary surgery and has not undergone any pre-operative radiation or chemotherapy by an affiliated first hospital at Chongqing medical university, and then human breast cancer primary cells are isolated from the fresh tumor tissue. At the same time, breast tumor tissue sensitive and resistant to chemotherapeutic drugs is also obtained from patients whose breast was resected after neoadjuvant chemotherapy. The present invention is to evaluate the response of tumor tissue to neoadjuvant chemotherapy according to the guidelines for the assessment of solid tumor response criteria (RECIST). And obtaining clinical and pathological characteristic information of the breast cancer patient from the medical records according to institutional approved protocols. The excised specimens were fixed in 10% formalin, followed by paraffin embedding and cutting into 4 μm sections for subsequent analysis.
In the embodiment of the invention, the method for separating breast cancer stem cells by using the magnetic cell separation technology comprises the following steps: breast cancer stem cells and non-stem cells were sorted from breast cancer cells using magnetic beads coupled with CD44/CD24 antibodies (Miltenyi Biotec, bergisch Gladbach, germany), the sorting procedure following the manufacturer's instructions.
In the embodiment of the invention, the method for preparing RNA and quantifying reverse transcription PCR (qRT-PCR) comprises the following steps: total RNA was isolated from tissue specimens and cells by TRIzol reagent (Takara, japan) according to the manufacturer's instructions, and reverse transcription was performed using PrimeScript RT kit (Takara, japan). Gene expression was then detected by qRT-PCR experiments using the SYBR Premix Ex Taq II kit (Takara, japan). Relative mRNA levels were calculated by comparison CT values and normalized to the expression of β -actin, all experiments were performed at least 3 times.
In the embodiment of the invention, the method for measuring the formation and self-renewal capacity of mammary gland microspheres comprises the following steps: breast microspheres were cultured according to literature report methods, cells were grown at 1×10 in subsequent passages, respectively 4 cells/mL、5×10 3 Density inoculation of cells/mL was subcultured in six well plates with 2% Poly-2-hydroxyethyl methacrylate (Poly-HEMA, sigma-Aldrich, USA) gel; to culture breast microspheres, serum free DMEM/F12 (Gibco, USA) medium was supplemented with B27 (Gibco, USA), 20ng/mL epidermal growth factor (EGF, invitrogen), 20ng/mL fibroblast growth factor (bFGF, invitrogen, USA), 0.4% albumin from bovine serum (BSA, sigma-Aldrich, USA), 2 μg/mL heparin (Sigma-Aldrich, USA) and insulin (Invitrogen, USA). Mammary gland microspheres were cultured at 37 ℃ and 5% co2, passaged every 7 days. To test for self-renewability, the number and size (diameter of the pellets were counted manually >50 μm) and representative images were obtained using an OLYMPUS IX70 microscope (Tokyo, japan). The percentage of mammary Microsphere Formation (MFE) was calculated using the following formula: (number of microspheres per well/number of cells seeded per well). Times.100. And the average size of 30 microspheres was calculated. All experiments were performedSecond generation microspheres and each experiment was performed at least three times.
In the embodiment of the invention, the method for RNA Fluorescence In Situ Hybridization (FISH) experiment comprises the following steps: for in situ detection of lncap in breast cancer cells or tumor tissue, cy3 labeled lncap probes, U6 probes and 18S probes were designed and synthesized using RiboBio (China) company. Cell or tissue FISH analysis was performed using a fluorescent in situ hybridization kit (RiboBio, china) according to the manufacturer's instructions. And the images were observed using a confocal laser scanning microscope (Leica, germany).
In the embodiment of the invention, the subcellular separation experiment method comprises the following steps: to determine cellular localization of lncrpm, cytoplasmic and nuclear RNAs were isolated using the pakes kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The extracted RNA was immediately followed by reverse transcription and analysis of gene expression by qRT-PCR. With U6 RNA as a nuclear control and GAPDH mRNA as a cytoplasmic control.
In the embodiment of the invention, the method for establishing the cell line for stably expressing the genes comprises the following steps: to knock down lncrop and PLA2G16 genes, specific shRNA synthesized by GenePharma (Shanghai, china) and control shRNA were inserted into the lentiviral expression vector pGLV3/H1/GFP/Puro of GenePharma (Shanghai, china), respectively. The detailed shRNA sequences are listed in supplementary table 1. For over-expression of the lncrpm and PLA2G16 genes, lncrpm and PLA2G16 sequences synthesized by GenePharma (Shanghai, china) were cloned into the lentiviral expression vector LV5/EF-1aF/GFP/Puro of GenePharma (Shanghai, china). The packaged lentiviruses were used to infect breast cancer cells according to the manufacturer's instructions. To establish a stably expressed cell line, the infected cells were treated with 1. Mu.g/ml puromycin (Gibco, USA) for two weeks.
In the embodiment of the invention, the Western blotting method comprises the following steps: breast cancer cells were collected, washed and lysed in RIPA buffer (Beyotime, china) containing a mixture of protease inhibitors (Beyotime, china). Total protein was extracted and quantified with BCA protein assay kit (Beyotime, china). Equivalent amounts of protein were separated on an 8% -12% SDS-PAGE gel and transferred to PVDF membrane (Bio-Rad, USA). Membranes were blocked in 5% skim milk (BOSTER, china) for 2h and incubated with primary antibody overnight at 4 ℃. After washing with TBST, the membranes were incubated with appropriate horseradish peroxidase (HRP) conjugated anti-mouse or rabbit IgG secondary antibodies (ZSGB-BIO, china) for 1 hour at room temperature. The protein bands were visualized by a chemiluminescent enhancement system (Bio-Rad, hercules, EDA USA). Images were captured using Scion image software and beta-actin protein was used as an internal control. The following antibodies were used in the present invention: KLF4 (1:500, 1880-1-AP, proteintech), SOX2 (1:500, ab97959, abcam), OCT4 (1:2000, 60242-1-Ig, proteintech), PLA2G16 (1:500, A16018, ABclonal), akt (1:500, 10176-2-AP, proteintech), p-Akt (Ser 473) (1:500, 66444-1-Ig, proteintech), PI3K (1:500, CY5355, abwax), p-PI3K (Tyr 607) (1:500, CY6427, abwax), YAP1 (1:500, CY5381, abwax), p-YAP1 (S127) (1:500, CY5743, abwax), β -catenin (1:500, A5038, bike), bi1 (1:51500, biA, bi500), bimaK (BimaK 1:500, CY5355, abwax), p-PI3K (1:35, bimak), bimak (1:35, bimak 1, bimak).
In the embodiment of the invention, the flow cytometer analysis method comprises the following steps: in order to detect breast cancer stem cell subpopulations, the present invention uses the following antibodies: APC-anti-CD44 (eBioscience, 17-0441, 1:167edition), FITC-anti-CD24 (eBioscience, 11-0247,1:20 edition), APC-rat isotype control (eBioscience, 174031, 1:167edition), FITC-mouse isotype control (eBioscience, 11-4714,1:20 edition). Will be 1X 10 6 The individual cells were incubated with the antibody in darkness for 30 minutes at 4℃and then the cells were washed and resuspended in 100. Mu.l FACS buffer and assayed using a flow cytometer (Beckman Coulter, high Wycombe, UK).
In the embodiment of the invention, the in-vitro limiting dilution experimental method comprises the following steps: cells were seeded into 96-well ultra-low attachment dishes at doses of 1000, 100 and 10 cells per well, and cultured in stem cell medium for 7 days, 24 times in duplicate. Counting wells containing microspheres; data were calculated and compared using limiting dilution analysis (http:// bioif. Wehi. Au/software/elda /).
In the embodiment of the invention, the method for measuring the cell viability comprises the following steps: cell viability was measured using the CCK-8 kit (CCK 8, dojindo, japan) following the experimental procedure given by the manufacturer, i.e. seeding 96-well plates at a density of 2000 cells per well overnight at 37 ℃. Subsequently, after cells were treated with different concentrations of cisplatin, doxorubicin or tamoxifen for 48 hours, 10 μl of CCK-8 reagent was added to each well in which the cells were cultured. After 3 hours of incubation, plates were read at 450nm using a microplate reader (BioTek, winioski, vermont, USA)). All experiments were repeated three times.
In the embodiment of the invention, the method for analyzing the RNA stability comprises the following steps: cells were exposed to 2 μg/ml of the transcription inhibitor actinomycin D (Cayman Chemical, USA) for 0, 2, 4, 6 or 8 hours, respectively, to block transcription and DMSO (Sigma-Aldrich, USA) was used as control reagent. Cell samples were collected at designated time points followed by RNA extraction and subsequently qRT-PCR experiments to analyze PLA2G16 mRNA expression levels. The amount of PLA2G16 RNA remaining in each time point was quantified with respect to the PLA2G16 RNA content at the 0 hour time point as standard.
In the embodiment of the invention, the RNA pull-down method comprises the following steps: according to the manufacturer's instructions, pierce was used TM Magnetic RNA-protein pulldown kit (Thermo Fisher Scientific) RNA pulldown experiments were performed. I.e., desulphated RNA is incubated with cell lysates incubated with streptavidin magnetic beads. Bound RNA was detected by qRT-PCR experiments. The biotin-labeled RNA probes are listed in supplementary Table 1. All processes were performed in the absence of RNase.
In the embodiment of the invention, the method for luciferase reporter gene experiment comprises the following steps: the PLA2G16 3'-UTR sequence was inserted into the PMIR-Reporter vector, called PMIR-PLA2G16-3' -UTR. PMIR-PLA2G16-3' -UTR and control vector pRL-TK were then transfected into lncROPM-silenced breast cancer stem cells and lncROPM-overexpressed breast cancer non-stem cells, respectively, using lipofectamine 2000 (Invitrogen, USA). After 36 hours of co-cultivation, luciferase activity was detected using a dual luciferase reporter system (Promega, USA).
In the embodiment of the invention, the method for extracting the lipid comprises the following steps: lipids are extracted from cells according to the method of Rahul Vijay Kapoore et al. I.e., cells (collected for each experimental group) were washed twice with phosphate buffered saline (PBS, pH 7.4) followed by rapid quenching of the cells by the addition of five volumes of pre-chilled 60% aqueous methanol and 0.85% (w/v) ammonium bicarbonate; the quenched biomass was centrifuged at 2500 μg for 5min at-9 ℃; the supernatant was then removed, the cell pellet was collected, snap frozen in liquid nitrogen and stored at-80℃for further analysis.
In the embodiment of the invention, the method for analyzing the lipid by UHPLC-QTOFMS comprises the following steps: the extracted lipids were detected by ultra-high performance liquid tandem chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS, agilent 1290UHPLC+AB Triple TOF 6600 +) and a C18 chromatographic column from Kinetex (Phenomenex, USA; 2.1X100 mm,1.7 μm). The mobile phase conditions were: a:10mM HCOONH4 in ACN/H2O (v/v, 6:4), B:10mM HCOONH4 in ACN/IPA (v/v, 1:9); the program is set as follows: 0-12.0min,300 mu L/min,40% -100% B;12.0-13.5min,300 μL/min,100% B;13.5-13.7min,300 mu L/min,100% -40% B;13.7-18.0min, 300. Mu.L/min, 40% B; the MS parameters are: gas1:60psi; gas2:60psi; curtainGas, 30psi; temperature: 600 ℃; ion spray voltage: TOF mass (Da): 5000V; TOF Masses (Da): min=200.0000, max= 1200.0000. Lipids were identified by accurate mass of the metabolite and MS/MS spectra based on data in an internal database.
In the embodiment of the invention, the immunohistochemical staining method comprises the following steps: paraffin-embedded sections were dewaxed, rehydrated and blocked for endogenous peroxidases and non-specific binding sites. The sections were then incubated with polyclonal antibodies PLA2G16 (1:100,Sangon Biotech), KLF4 (1:200, proteintech), respectively, overnight at 4 ℃. Subsequently, the sections were incubated with peroxidase anti-rabbit IgG (ZSBC-BIO, china) for 30 minutes at 37℃and stained with diaminobenzidine. After final counterstaining with hematoxylin and sealing, images were captured using a Nikon Eclipse 80i microscope (Tokyo, japan).
In the embodiment of the invention, the method for establishing the breast microsphere xenograft model comprises the following steps: animal experiments have obtained Chongqing university of medical scienceAnimal care and ethics committee approval. Future 1×10 5 Microsphere cells from BT-549/shCtrl and BT-549/shlncROPM were inoculated subcutaneously into mammary fat pads of 5 week old female nude mice (five mice per group). When the tumor volume reaches 100mm 3 On the left or right, mice were given either 4mg/kg of doxorubicin intraperitoneally every 5 days (group 1) or 7.5mg/kg of gellan pa once daily (group 1) or doxorubicin in combination with gellan pa (group 1). Control mice were treated with sterile saline (group 1). Tumor size was measured every 5 days using a digital caliper and tumor volume was calculated using the following formula: volume= (length×width) 2 )/2. Three weeks after drug injection, mice were euthanized and tumors were isolated, weighed and photographed. Subsequently, tumor tissues were fixed in 10% formalin and then embedded in paraffin, and cut into 4 μm sections for immunohistochemical analysis.
In the embodiment of the invention, statistical analysis is carried out by using GraphPad Prism 7.0 software (San Diego, calif., USA), all experiments are repeated for at least three times, and the data are expressed as mean value plus or minus standard deviation; the statistical significance between the two groups is evaluated by adopting a two-tailed student t test, and the difference between the different groups is analyzed by adopting one-way analysis of variance (ANOVA); analysis of correlations between variables using Pearson correlation analysis, P <0.05 was considered statistically significant.
In the examples of the present invention, the primers used are shown in Table 1 below:
TABLE 1 summary of primer sequences used in the examples
Example 1 lncROPM screening, positioning and coding evaluation
The examples of the present invention investigated which lncRNAs play an important role in the regulation of breast cancer stem cell (CD 44 + CD24 -/low Microspheres) and non-stem cells (CD 44 -/low CD24 + ) The results of the differentially expressed lncRNAs in (2) are shown in FIG. 1 There were a total of 585 differentially expressed lncRNA transcripts (fold change>2，P<0.05 Of which 349 lncRNAs were up-regulated and 236 were down-regulated.
Metabolic remodeling has been reported to play an important role in regulating stem cell fate and biological behavior, and 23 lncRNAs associated with metabolic pathways in breast cancer stem cells were screened using bioinformatic analysis, as shown in fig. 2, in the examples of the present invention. Wherein, one of them is highly expressed in breast cancer stem cells, is located on human 11q12.3-q13.1 chromosome, is adjacent to long-chain non-coding RNA named lncROPM of PLA2G16 gene, and as shown in FIG. 3, lncROPM is composed of two exons connected, and has a length of 573bp.
The embodiment of the invention also uses CPAT and CPC system to evaluate the coding potential of lncROPM, and the result shows that lncROPM has no coding capability, and the result is shown in table 2 and table 3.
TABLE 2 CPAT coding potential evaluation results
TABLE 3 CPCT coding potential evaluation results
The present example also uses qRT-PCR (quantitative fluorescent PCR) to detect the expression of lncROPM in cancer stem cells and non-stem cells from various breast cancer cell lines (BT 549, hs578T, MDA231, MDA468, MDA436, MCF7, T47D), and the results show that lncROPM is highly expressed in cancer stem cells of various breast cancer cell lines compared with non-stem cells in FIG. 4.
In addition, the embodiment of the invention also uses an RNA Fish experiment and a subcellular separation experiment to locate the lncROPM, and the result shows that the lncROPM is mainly located in the cytoplasm of the breast cancer stem cells (as shown in fig. 5 and 6).
Example 2lncROPM promotes verification of breast cancer or other tumor progression
The present examples used the expression of lncrpm in patients retrieved from tanrc database (TCGA-BRCA, RNAseq data), and the results are shown in fig. 7 and 8, and the average expression level of lncrpm is highly expressed in patients dying from breast cancer (p < 0.05) compared to surviving breast cancer patients (as shown in fig. 7); the three year overall survival analysis results showed that high expression of lncrpm was significantly correlated with poor prognosis in breast cancer patients (n=607, p=0.012) (as shown in fig. 8). Moreover, lncrpm expression is inversely related to overall survival in treated ER-PR-Her 2-and er+pr+her 2-breast cancer patients, i.e., lncrpm may be related to drug tolerance.
The embodiment of the invention also detects the expression of lncrpm in 50 groups of breast cancer tissues and paired adjacent normal breast tissues by using qRT-PCR experiments, and the result is shown in fig. 9, and compared with the adjacent normal breast tissues, lncrpm is remarkably high expressed in the breast cancer tissues.
The invention also collects 130 clinical breast cancer patient specimens, analyzes the correlation between the expression of lncROPM and clinical pathological factors, and the clinical pathological characteristics of the specimens are shown in table 4. Representative images were obtained using an OLYMPUS IX70 microscope, and as shown in fig. 10, expression of lncROPM was closely related to tumor size (p=0.016), TNM staging (p=0.002), tissue staging (p=0.019), lymph node metastasis (p=0.042), tumor recurrence (p=0.001).
Table 4 clinical pathological characteristics of the specimens
In table 4: the differences between experimental groups were tested using Mann-Whitney, the data in the table being mean ± standard deviation, wherein p <0.05; * P <0.01.
The present examples also analyzed the correlation of lncrpm expression with expression of the dryness markers in these tissues using Pearson correlation coefficients, and the results were shown in fig. 11 and 12, in which lncrpm was positively correlated with expression of the dryness markers SOX2 and OCT4 in breast cancer tissues. I.e. the tissue grade of the tumor reflects the degree of dedifferentiation of the tumor, i.e. the dry state.
In the embodiment of the invention, qRT-PCR (quantitative reverse transcription-polymerase chain reaction) experiments are also used for detecting the expression condition of lncROPM in breast cancer stem cells and non-stem cells separated from primary breast cancer samples, and the result is shown in figure 13, and compared with the non-stem cells, lncROPM is highly expressed in the breast cancer stem cells.
The example of the invention also collects qRT-PCR experiments on breast cancer tissues which are tolerant to the chemotherapeutic drug and intolerant, and the results are shown in figure 14, which show that lncROPM is remarkably high expressed in breast cancer tissues which are tolerant to the chemotherapeutic drug compared with breast cancer tissues which are intolerant.
The embodiment of the invention also finds that lncrpm has important functions in other types of tumors through analysis of a TANRIC database, and the result shows that in the renal clear cell carcinoma, the high-expression lncrpm is closely related to the poor prognosis of patients (n=515, p=0.0030); in squamous cell carcinoma of head and neck and ovarian serous cystic adenocarcinoma, lncrpm expression also increased significantly with increasing tissue classification (as shown in fig. 15 and 16).
In the embodiment of the invention, qRT-PCR is also used for detecting the expression of lncROPM in stem cells from renal clear cell carcinoma, head and neck squamous cell carcinoma, ovarian serous cyst adenocarcinoma and non-stem cells, and the results are shown in figures 17, 18 and 19, which indicate that compared with the non-stem cells, lncROPM is highly expressed in the stem cells of renal clear cell carcinoma (shown in figure 17), head and neck squamous cell carcinoma (shown in figure 18) and ovarian serous cyst adenocarcinoma (shown in figure 19).
Taken together, the examples of the present invention demonstrate that lncrpm is an important oncogene that promotes the development of breast cancer or other neoplasms.
Example 3 lncROPM promotes stem characterization of breast cancer stem cells
The embodiments of the present invention explore the function of lncrpm in breast cancer stem cells, in which lncrpm was silenced with two lentivirus-mediated short hairpin RNAs (shRNAs) (as shown in fig. 20); meanwhile, lncrpm was overexpressed in non-stem cells of breast cancer with lentiviral vectors (as shown in fig. 21). Effective knockdown of lncrpm in breast cancer stem cells can significantly reduce expression of stem-related genes (SOX 2, nanog, OCT4, KLF 4) (as shown in fig. 22), stem cell spheroidization rate (as shown in fig. 23), and stem cell size (as shown in fig. 24); whereas overexpression of lncROPM in non-stem cells significantly promoted expression of the stem-associated genes (SOX 2, nanog, OCT4, KLF 4) (as shown in fig. 25), stem cell pelleting rate (as shown in fig. 26), and stem cell size (as shown in fig. 27).
The effect of lncROPM on tumor initiation frequency was also examined in vivo and in vitro by limiting dilution experiments in the examples of the present invention, and the results are shown in fig. 28, 29, 30, 31. In stem cells, in vivo experimental results show that lncROPM deletion significantly reduces the initiation frequency of breast tumors (as shown in fig. 28); the in vitro experimental results show that lncROPM deletion significantly reduced the initiation frequency of breast tumors (as shown in fig. 29). Whereas in non-stem cells, in vivo experimental results showed that overexpression of lncrpm significantly increased the initiation frequency of breast tumors (see fig. 30); the in vitro experimental results show that overexpression of lncrpm significantly increases the initiation frequency of breast tumors (as shown in fig. 31).
Taken together, the examples of the present invention demonstrate that lncrpm promotes the stem characteristics of breast cancer stem cells.
Example 4 PLA2G16 is a target gene for lncROPM and promotes development and drug resistance of breast cancer
The embodiment of the invention researches the potential mechanism of lncap for regulating the stem cell stem property of the breast cancer, predicts the potential target gene of lncap by using a bioinformatics method and discovers that the adjacent gene PLA2G16 can be the potential target gene of the lncap. In order to detect whether lncrpm can affect the expression of PLA2G16, the change of target gene PLA2G16 after knockdown and overexpression of lncrpm was detected using qRT-PCR experiments and western blotting experiments. The experimental results show that the target gene PLA2G16 was down-regulated at both mRNA and protein levels after knockdown of lncrpm in breast cancer stem cells (as shown in fig. 32, 33). Whereas the target gene PLA2G16 increased both at mRNA and protein levels after overexpression of lncrpm in non-stem cells (as shown in fig. 34, 35).
The example of the present invention also uses Pearson correlation coefficient to analyze the collected breast cancer tissue samples, and the results are shown in fig. 36, which indicate that the expression of lncrpm in the collected breast cancer tissue samples is positively correlated with the expression of PLA2G 16. Highly expressed PLA2G16 was closely related to advanced TNM stage (p=0.004), high tissue grade (p=0.026), lymph node metastasis (p=0.039), tumor recurrence (p=0.005), representative images of which were acquired using an OLYMPUS IX70 microscope, as shown in fig. 37. Similarly, PLA2G16 was highly expressed in breast cancer tissues of tolerizing patients compared to breast cancer tissues sensitive to chemotherapeutic drugs (as shown in fig. 38).
The results of further searching using GEO database in the examples of the present invention are shown in fig. 39, where PLA2G16 is highly expressed in tumor tissues of breast cancer patients with only partial response (pPR) to neoadjuvant paclitaxel/radiation therapy, compared to patients with pathological complete response (pCR) to neoadjuvant therapy, indicating that PLA2G16 is closely related to poor prognosis and drug resistance of breast cancer patients.
Taken together, the examples of the present invention demonstrate that PLA2G16 is an important target gene for lncrpm and promotes the development and resistance of breast cancer.
Example 5 LncROPM demonstrates that expression is regulated by increasing the stability of PLA2G16 mRNA
The regulatory mechanism of LncRNA depends on its localization in cells, and example 1 of the present invention has verified that lncrpm is primarily localized to the cytosol, so it was first assumed that lncrpm might regulate PLA2G16 expression at post-transcriptional levels, and then verified this hypothesis.
(1) Expression levels of PLA2G16 pre-mRNA and mature mRNA after knocking down lncROPM in MCF7/BT549 breast cancer stem cells were first tested by qRT-PCR experiments. Results as shown in fig. 40, 41, and 42, the expression level of mature mRNA (including 3'-UTR region, CDS region, and 5' -UTR region) of PLA2G16 was significantly reduced after lncrpm knockdown, while the levels of pre-mRNA (including intron 1, intron 2, and intron 3) of PLA2G16 remained unchanged.
Subsequently, by sequence alignment analysis, the results of the analysis are shown in table 5 and fig. 43 below, and it was found that lncrpm may directly bind to the 3' -UTR region of PLA2G16 mRNA (energy= -17.86 kcal/mol).
TABLE 5 lncROPM binding to the 3' -UTR region of PLA2G16 mRNA
(2) Further, RNA-pull-down experiments were performed to examine the binding capacity between lncrpm and PLA2G16, and as a result, it was found that the use of lncrpm probe significantly increased enrichment of the PLA2G16 '-UTR region, rather than the 5' -UTR region or CDS region, compared to the control group probe. Conversely, dividing the PLA2G16 3' -UTR into two parts and performing an RNA-pull-down experiment using probes for both parts, the results are shown in Table 6 and FIG. 44, indicating that the 3' -UTR-1 region of PLA2G16, but not 3' -UTR-2, can specifically bind to lncROPM.
TABLE 6 PLA2G16' -UTR RNA-pull-Down experimental conditions
The results of the luciferase reporter experiments performed in the examples of the invention are shown in FIG. 45, which demonstrates that knocking down lncROPM significantly reduces the luciferase activity of the construct containing the PLA2G16 '-UTR region, while over-expressing lncROPM significantly increases the luciferase activity of the construct containing the PLA2G 16' -UTR region.
The 3' UTR region has been reported to be important for mRNA stability, and thus, in the examples of the present invention, the effect of the transcription inhibitor actinomycin D on PLA2G16 mRNA stability was also examined, and as a result, as shown in FIG. 46, the half-life of PLA2G16 mRNA was rapidly decreased in the breast cancer stem cells lacking lncROPM, while the half-life of PLA2G16 mRNA was significantly increased in the breast cancer non-stem cells overexpressing lncROPM.
Taken together, the examples of the present invention demonstrate that lncrpm can bind to the 3' utr region of PLA2G16 mRNA to enhance its stability, thereby increasing the expression level of PLA2G 16.
Example 6 validation of PLA2G16 on lncROPM mediated promotion of breast cancer Stem cell Stem Regulation
In order to know whether PLA2G16 regulates the stem cell dryness of breast cancer, the qRT-PCR and Western blotting experiments are utilized to detect the expression of PLA2G16 in different cell lines of breast cancer and cancer stem cells and non-stem cells from clinical tumor sources, and the results are shown in FIG. 47, which shows that the PLA2G16 is obviously highly expressed in the breast cancer stem cells compared with the non-stem cells.
The embodiment of the invention also utilizes Pearson correlation coefficient analysis, and the analysis results are shown as 48 and fig. 49, which show that the expression of PLA2G16 in the breast cancer tissue samples collected by the invention and the expression of the dry correlation genes (such as SOX2 and OCT 4) are positively correlated.
The present examples also used two lentivirus-mediated short hairpin RNAs (shRNAs) to construct a cell line that stably knocked down PLA2G16 in breast cancer stem cells (as shown in fig. 50, 51) and a lentivirus over-expression vector carrying the PLA2G16 gene to construct a cell line that stably over-expressed PLA2G16 in breast cancer non-stem cells (as shown in fig. 52, 53). Gelopadine, an inhibitor of cytoplasmic phospholipase A2, was also used to inhibit the enzymatic activity of PLA2G16 localized to the cytosol of the cell (as shown in FIG. 54). Then analyzing the relation with the dryness factor, and the result shows that the expression of the dryness related genes (SOX 2, OCT4 and KLF 4) is obviously reduced by effectively knocking down the endogenous PLA2G16 in the breast cancer stem cells or using Jilipa (shown in figure 55), the balling rate of the stem cells (shown in figures 56 and 57) and the size of the stem cells (shown in figure 58); whereas overexpression of PLA2G16 in non-stem cells promoted stem cell balling (as shown in FIGS. 59, 60, 62), expression of the stem-associated gene (SOX 2, OCT4, KLF4, beta-action) (as shown in FIG. 61). These data indicate that PLA2G16 promotes the stem characteristics of breast cancer stem cells.
Example of the invention to further demonstrate the role of PLA2G16 in stem cells, highly expressed PLA2G16 (PLA 2G 16) was isolated from breast cancer cells by flow cytometry +/high ) And low expression of PLA2G16 (PLA 2G 16) -/low ) The results are shown in FIG. 63 and FIG. 64, which show that CD44 is higher than that of cells in which PLA2G16 is expressed under low levels + /CD24 -/low The stem cell subset was significantly highly expressed in cells that highly expressed PLA2G16 (as shown in fig. 63); compared with cells with low expression of PLA2G16, the genes related to dryness (OCT 4, SOX2, KLF4,Beta-action) was significantly highly expressed in cells that highly expressed PLA2G16 (as shown in fig. 64).
Further analysis using GEO database in the examples of the present invention showed that PLA2G16 was most expressed in embryonic stem cells (fig. 65, 66); similarly, PLA2G16 was most expressed in liver stem cells in liver compared to cells of differentiated cells (as shown in fig. 67); in addition, PLA2G16 was significantly up-regulated in bone marrow-derived mast cells upon stimulation with stem cell factor (as shown in fig. 68).
In order to investigate whether lncROPM was modulating the stem characteristics of breast cancer stem cells in a PLA2G16 dependent manner, a reversion experiment was performed. Experimental results show that the overexpression of PLA2G16 in breast cancer stem cells can partially recover the reduction of the expression of the stem related genes and the reduction of the balling rate of the stem cells caused by the deletion of lncROPM; whereas knockdown of PLA2G16 in non-stem cells can down-regulate the increase in stem-related gene expression and increase in stem cell balling rate due to overexpression of lncrpm.
Taken together, the verification of the examples of the present invention shows that PLA2G16 is involved in regulating stem cell viability and may become a marker for breast cancer stem cells and even other types of stem cells, i.e., PLA2G16 may promote self-renewal and proliferation of stem cells.
Example 7 LncROPM promotes stem cell stem verification of breast cancer by modulating PLA2G 16-mediated phospholipid metabolism
PLA2G16 is reported to be involved in phospholipid metabolism. The embodiment of the invention explores whether lncap is used for regulating the stem cell dryness of the breast cancer by influencing PLA2G16 mediated phospholipid metabolism, and which metabolites play a key role in regulating the stem cell dryness of the breast cancer, and a UHPLC-QTOFMS system is used for detecting the change of lipid metabonomics after the lncap is knocked down in the stem cell of the breast cancer and after the lncap is over-expressed in non-stem cells. The results show that the content of the substrates PC (phosphatidylcholine) and PG (glycerophosphate) of PLA2G16 is obviously increased after the lncROPM is knocked down in the breast cancer stem cells, and the content of the PC and PG is obviously reduced after the lncROPM is overexpressed; whereas the representative products Cer (ceramide) and FFA (free fatty acid) content were significantly reduced after knocking down lncrpm, the Cer and FFA content were significantly increased after over-expressing lncrpm, indicating that Cer and FFA may be key metabolites of lncrpm-PLA 2G16 driving the stem cell stem characteristics of breast cancer. Since the fold change of FFA is the greatest in the present invention, in order to explore the effect of metabolites on the characteristics of breast cancer stem cells in depth, the present examples will further study FFA. Among FFAs, the metabolite arachidonic acid (arachidonic acid) is the most varied metabolite (as shown in tables 7 and 8).
TABLE 7 shROPM vs shCtrl metabolite status
Name Log 2 (FC)
Arachidonic Acid -1.764
Mycolipanolic acid -1.622
Byrsonic acid -1.504
Tetracosatetraenoic acid n-6 -1.276
Ambrettolic acid -1.247
TABLE 8 LV-ROPM vs LEV metabolite case
Name Log 2 (FC)
Arachidonic Acid 2.089
Palmitic acid 1.358
Corynomycolic acid 1.139
Mycolipanolic acid 1.053
Hexatriacontylic acid 0.710
In order to confirm the relation between the arachidonic acid and the PLA2G16, the content of the arachidonic acid after knocking down the PLA2G16 in breast cancer stem cells or inhibiting the activity of the PLA2G16 enzyme by using the Jilipadi and after over-expressing the PLA2G16 in non-stem cells is also measured, and the results are shown in fig. 69 and 70, and the results show that the change of the content of the arachidonic acid is regulated by the PLA2G16, and the more active the expression of the PLA2G16, the higher the content of the arachidonic acid; meanwhile, arachidonic acid is significantly enriched in breast cancer stem cells compared to non-stem cells (as shown in fig. 71); in addition, the embodiment of the invention also finds that the arachidonic acid is obviously enriched in late-stage tumor tissues, recurrent tumor tissues and drug-resistant tissues (shown in figure 72). Arachidonic acid was validated as critical for maintaining breast cancer stem cell and may be clinically used as a biomarker for malignant progression of breast cancer.
Examples in order to further verify the effect of arachidonic acid on breast cancer stem cells, arachidonic acid was exogenously added to investigate its effect. The exogenous addition of arachidonic acid restored the expression of the suppressed stem-associated genes (SOX 2, OCT4 and KLF 4) and stem cell spheroidization rate (as shown in fig. 73) due to knockdown of lncrpm or PLA2G16 (as shown in fig. 74, 75).
The examples of the present invention also examined the change of stem cell classical related signaling pathway (Wnt/beta-catenin, notch, hedgehog, hippo/YAP, PI 3K/AKT), and the results show that arachidonic acid can activate PI3K/AKT, wnt/beta-catenin and Hippo/YAP signaling pathway (as shown in FIG. 76 and FIG. 77).
In summary, the embodiment of the invention verifies that the lncROPM-PLA2G16 signal axis regulates phospholipid metabolism and promotes the production of the metabolite arachidonic acid, thereby activating PI3K/AKT, wnt/beta-catenin and Hippo/YAP signal channels and promoting the dryness of breast cancer stem cells.
Example 8 validation of effective elimination of breast cancer Stem cells with Lipatadine in combination with chemotherapeutic drugs
Fig. 14 and 38 of example 2 of the present invention show that lncrpm and PLA2G16 are highly expressed in breast cancer resistant tissues.
In order to further verify the effect of lncROPM and PLA2G16 on drug tolerance of breast cancer cells, the embodiment of the invention detects the survival of breast cancer cells when treated with chemotherapeutic drugs. The results indicate that knocking down lncrpm or PLA2G16 increases the sensitivity of breast cancer stem cells to chemotherapeutic drugs (Doxorubicin, cisplatin, tamoxifen), e.g., knocking down lncrpm or PLA2G16 increases the drug sensitivity of BT549 breast cancer stem cells to Doxorubicin (Doxorubicin) (shown in fig. 78); knocking down lncrpm or PLA2G16 increases drug sensitivity of BT549 breast cancer stem cells to Cisplatin (cispratin) (as shown in fig. 79); knocking down lncrpm or PLA2G16 increased the drug sensitivity of BT549 breast cancer stem cells to Tamoxifen (Tamoxifen) (as shown in figure 80). Also, treatment of breast cancer stem cells with gellan Pade had similar results.
According to the embodiment of the invention, the exogenous addition of arachidonic acid to breast cancer stem cells treated by the deletion of lncrpm or PLA2G16 and Ji Lipa can restore the drug resistance of the stem cells to drugs, for example, after the addition of arachidonic acid to BT549 breast cancer stem cells treated by the deletion of lncrpm or PLA2G16 and Ji Lipa, the drug resistance of the stem cells to Doxorubicin (Doxorubicin) can be restored (shown in figure 81); the drug resistance of stem cells to Cisplatin (Cisplatin) can be recovered after arachidonic acid is added to BT549 breast cancer stem cells treated by deleting lncROPM or PLA2G16 and Ji Lipa (shown in figure 82); the addition of arachidonic acid to MCF7 breast cancer stem cells treated with the deletion of lncrpm or PLA2G16, ji Lipa restored the drug resistance of the stem cells to Tamoxifen (Tamoxifen) (see figure 83).
In the embodiment of the invention, the sensitivity of the cells to chemotherapeutic drugs is reduced by over-expressing lncrpm or PLA2G16 in non-stem cells, for example, the sensitivity of the cells to Doxorubicin (Doxorubicin) is reduced after over-expressing lncrpm or PLA2G16 in BT549 breast cancer non-stem cells (as shown in fig. 84); drug sensitivity of cells to Cisplatin (cispratin) was reduced following overexpression of lncrpm or PLA2G16 in BT549 breast cancer non-stem cells (as shown in figure 85); over-expression of lncrpm or PLA2G16 in MCF7 breast cancer non-stem cells reduced the drug sensitivity of the cells to Tamoxifen (Tamoxifen).
The embodiment of the invention also detects the expression of lncrpm and PLA2G16 in stem cells derived from MCF7 cells resistant to tamoxifen, MDA-MB-231 cells resistant to cisplatin, and BT549 cells resistant to doxorubicin and their parent cells. The results are shown in FIGS. 87 and 88, in which lncROPM and PLA2G16 were significantly expressed in these drug-resistant cell-derived stem cells having strong stem properties, as compared to the parental cells.
The above examples demonstrate that lncROPM, PLA2G16 and arachidonic acid promote the tolerance of breast cancer stem cells to chemotherapeutic agents, and that jilipadi can inhibit the resistance of breast cancer stem cells by inhibiting the enzymatic activity of PLA2G 16.
Thus, embodiments of the present invention also use gemfibrozil in combination with a clinical drug (doxorubicin, cisplatin, or tamoxifen) to eliminate breast cancer stem cells. The combination of gellan gum with clinical drugs was shown to be more effective in reducing the number of breast cancer stem cells than single drug treatment alone by microsphere formation experiments, for example, the combination of gellan gum (Giripladb) with Doxorubicin (Doxorubicin) had superior balling inhibition on BT549/Hs578T breast cancer stem cells than alone (as shown in FIGS. 89, 90); the combined use of Gilipadib and Cisplatin (Cisplatin) has better balling inhibition on BT549/Hs578T breast cancer stem cells than that of the single use (shown in figures 91 and 92); gilipadib (Gilipladib) was used in combination with Tamoxifen (Tamoxifen) to provide superior balling inhibition on MCF7 breast cancer stem cells than alone (as shown in FIGS. 93, 94).
In vivo experiments are carried out in the embodiment of the invention, and a xenograft model is constructed in a nude mouse for verification. Consistently, the combined chemotherapy drug of gemfibrozil (doxorubicin) significantly reduced tumorigenesis and inhibited tumor growth compared to the monotherapy group (as shown in fig. 95, 96). And by Immunohistochemical (IHC) analysis, the expression of stem cell-associated transcription factor KLF4 was found to be significantly reduced in tumors receiving the combination treatment (as shown in fig. 97).
In summary, the embodiment of the invention shows that lncROPM, PLA2G16 and arachidonic acid promote drug resistance of breast cancer stem cells, and the synergistic effect of gellanpatadine and chemotherapeutic drugs can effectively eliminate breast cancer stem cells by inhibiting the activity of PLA2G 16.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Sequence listing
<110> university of Chongqing medical science
<120> non-coding RNA, RNA sequence comprising non-coding RNA and use thereof
<130> 2021-4-16
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 573
<212> DNA
<213> Artificial Sequence
<400> 1
atttttttag attccatata taagtgagat cacaaagtat gtgtctttct gtgcctggct 60
tatttcactg aacgtaatgt tctccaggaa aataactgtt atctgggaaa ctgcatttaa 120
agggaagctg tttgcaatgc ttctttgcac acaaggaaca gaatccacaa agttcacaac 180
aaggaacaga aaccacaaat ttcacaacat gacaacattc aaaatgggag tatgtctcaa 240
gtgctccacc agccagctga atgcagggtt ttcacaggtg taaaacatgt gactatgcca 300
attttgatag tttcctctga ccagaaaaat caggctttta tgaaagaaac caaaagtgaa 360
gtataacttc cttctgaatc ccctttgaaa cccaagtcct actgaagagg aaagagaaca 420
aatccctatt gaaatgccct gaagagcttc cactgcctaa atgtgcagca taaccatctc 480
taccttctct ttaaaaaaaa cattctagag gctgggcacg gtggctcacg cctgtaatcc 540
cagcactttg ggaggctgag gcgggcggat cac 573
<210> 2
<211> 849
<212> DNA
<213> Artificial Sequence
<400> 2
ctgaaaaaga ctgtcctgtc agcgatgact ttatacatca agggggtctt gttttgctag 60
agagtttggg gtttggtttg tggatttcat tgtgatttat aataaggctt attttcacag 120
aataaaataa agcaaaacga gggaggattt tattggggga agtgcagcaa gaactgcctg 180
gtgtgaagtc tgttcaggga acagccgggc tgtcttcctg gtcagggatt gtctttcact 240
ttctttttgc gtgctgtgtt ctttgcattg caggaggcag agtccagctc cagttatctc 300
aagtagaggg cgtttctggt cgtcaggata catgcggggc ggaagggaga cagaaagaca 360
taatacacaa gaacagaaag cagcagacag ccaggaccct tggcccggaa ctagatctag 420
gatggcctct gagactcagc agctggagtc tgtagatgtt cagctgagtt gaaaaaaaaa 480
aagtcctcgg ctataaggaa atttagtata gatgcttgcc aatcttacta aaatttccct 540
tattttcctt tgctctacac gtctttcctt tgccttgttg cttccatctc cttggcactc 600
ctgcttcctc actgcttctg cttgccatga tccccaagtc tctgacccac cttcctgcct 660
gctcctctcc tcccacattg gctcagattc tttccccgct gtctgtgggt ccacactccc 720
agtggcacct ccaggagaga atctgattgg ctcagttcgc cagataactc aactttccca 780
ttggctacct ttgggtcagg tgatctccac tagacctatc gcctatgcct gatggtgggt 840
cacatggtg 849

Claims (8)

1. A non-coding RNA, wherein the nucleic acid Sequence of the non-coding RNA is as shown in Sequence No. 1.
Rna, characterized in that it comprises: an mRNA of a PLA2G16 gene and the non-coding RNA of claim 1, wherein the Sequence of the RNA is such that the non-coding RNA binds to the 3'-UTR-1 region of the mRNA of the PLA2G16 gene, and the Sequence of the 3' -UTR-1 region of the mRNA of the PLA2G16 gene is shown in Sequence No. 2.
3. Use of the non-coding RNA of claim 1 for the preparation of an agent for enhancing expression of the PLA2G16 gene, wherein the non-coding RNA of claim 1 is bound to the 3'-UTR-1 region of the mRNA of PLA2G16, and the Sequence of the 3' -UTR-1 region of the mRNA of the PLA2G16 gene is as shown in Sequence No. 2.
4. A method of modulating the stem characteristics of a breast cancer stem cell/non-stem cell, said method being an in vitro method, characterized by a) knocking out the non-coding RNA of claim 1 or inhibiting the expression of PLA2G16 gene in said breast cancer stem cell/non-stem cell, reducing the stem characteristics; or b) overexpressing the non-coding RNA of claim 1 or overexpressing the PLA2G16 gene in breast cancer stem cells/non-stem cells, promoting a drying profile; the stem characteristics include expression of a stem-related gene and/or a balling rate of breast cancer stem cells and/or a breast cancer stem cell size and/or a CD44+/CD24-/low stem cell subset.
5. A method of activating/inhibiting PI3K/AKT, wnt/β -catenin and Hippo/YAP signaling pathways in breast cancer stem cells/non-stem cells, said method being an in vitro method, characterized in that a) the non-coding RNA of claim 1 is overexpressed or PLA2G16 gene is overexpressed, and PI3K/AKT, wnt/β -catenin and Hippo/YAP signaling pathways are activated; or b) knocking out the non-coding RNA according to claim 1 or inhibiting PLA2G16 gene expression, and inhibiting PI3K/AKT, wnt/beta-catenin and Hippo/YAP signaling pathways.
6. A method of modulating the sensitivity of a breast cancer stem cell/non-stem cell to a drug, said method being an in vitro method, characterized in that a) knockdown of the non-coding RNA of claim 1 and/or inhibition of PLA2G16 gene expression increases the sensitivity of a breast cancer stem cell to a drug; or b) overexpressing the non-coding RNA of claim 1 and/or overexpressing the PLA2G16 gene reduces the sensitivity of breast cancer non-stem cells to drugs.
7. A method of inhibiting the development/progression of breast cancer stem cells/tumors in vitro comprising: the use of non-coding RNA of claim 1 or using gemfibrozil in combination with doxorubicin, cisplatin, tamoxifen, respectively, in a cell knockdown of the tumor inhibits the expression of the non-coding RNA of claim 1 with the PLA2G16 gene, thereby inhibiting the occurrence/progression of breast cancer stem cells/tumor.
8. The method according to claim 7, comprising: a) The combined use of gellan pa and doxorubicin inhibits the occurrence/development of BT549/Hs578T breast cancer stem cells/tumors; or b) inhibition of the occurrence/progression of BT549/Hs578T breast cancer stem cells/tumors in combination with cisplatin; or c) the use of gelipapide in combination with tamoxifen inhibits the occurrence/progression of MCF7 breast cancer stem cells/tumors.
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