AU2021100731A4 - A Use of PIWI interacting RNA piR-hsa-211106 - Google Patents

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a new use of PIWI interacting RNA piR-hsa-211106, which is used to prepare targeted therapeutic drugs for inhibiting the proliferation of lung adenocarcinoma cells. Its mechanism of work is: The RNA piR-hsa-211106 sequence is prepared into an agomir or transformant that down-regulated the expression of Pyruvate carboxylase (PC), which inhibits the tricarboxylic acid cyclic process and makes energy metabolism affected, promoting the death of lung adenocarcinoma cells, and then achieving the purpose of inhibiting the growth of lung adenocarcinoma. It directly acts on the target site without causing toxic side effects and off-target phenomena. A large number of analyses and in vivo and in vitro experiments have confirmed that it has high credibility and significant therapeutic effects, providing a new research direction for anti-tumor treatment of lung adenocarcinoma. 1.5- 1.5 ' 1.0 - Z 1.0 I4 0.5 - 0.5 a a 0.0 0.0 0 11 1f 1? oCo oC A549 HCC2279 Figure 12 Experiment group PDD treated Control 1 Experiment group NS treated Control group Figure 13 5/6

Description

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Descriptions

A Use of PIWI interacting RNA piR-hsa-211106 Technical field: The invention belongs to the field of biomedicine technology, and specifically relates to the use of PIWI interacting RNA piR-hsa-211106 for inhibiting the proliferation of lung adenocarcinoma cells and preparing targeted therapy drugs for lung adenocarcinoma. Background technique: Lung cancer is the most common malignant cancer. According to GLOBOCAN data, in 2018, 2.1 million new lung cancers and 1.8 million deaths occurred worldwide, respectively accounting for 11.6% of all new tumor cases (ranking first) and accounting for 18.4% of all tumor deaths (ranking first) . Lung cancer includes small cell lung cancer and non-small cell lung cancer. Among them, non-small cell lung cancer accounts for about 85% of all lung cancers, and lung adenocarcinoma is the most common histological type of non-small cell lung cancer, accounting for about 40% of primary lung cancers. The majority of the 50% of the population originates from bronchial mucosal epithelium, mainly peripheral lung cancer. In recent years, the incidence of lung adenocarcinoma has been on the rise. Depending on the location and size of the tumor, the degree of invasion and compression of the adjacent organs, and the presence or absence of metastasis, the patient may present various symptoms, such as cough, bloody sputum, chest pain, local wheezing, fever and shortness of breath. Most patients with lung adenocarcinoma in the early stage have no symptoms, while in the late stage, there are symptoms such as wasting, fatigue, loss of appetite, hoarseness and esophageal compression, which cause great burden to the patients' body, family and society, and have become a major disease endangering human health that needs to be solved urgently. The pathogenesis of lung adenocarcinoma is still unclear. Studies have shown that the occurrence of lung adenocarcinoma is mostly related to benzopyrene. Surgical excision, chemoradiotherapy, molecular targeted therapy and traditional Chinese medicine are commonly used in the treatment of lung adenocarcinoma. Surgery is the preferred treatment for lung adenocarcinoma. However, about 75% of patients with lung adenocarcinoma are considered to be advanced once they are found clinically, and surgical treatment is no longer feasible. They mainly rely on radiotherapy and chemotherapy to delay the spread of the lesions. The overall efficacy and prognosis are poor and the 5-year survival rate is less than 20%. Molecular targeted therapies for lung adenocarcinoma include bevacizumab and COX2 inhibitors targeted at angiogenesis, as well as geffitinib, Erlotinib and HER2 inhibitors targeted at epidermal growth factor, etc. However, molecular targeted therapies are only effective for primary tumors and ineffective for metastatic tumors, which are expensive and of low prevalence. About 98% of RNA are non-coding RNAs, which do not encode proteins, but directly function at the RNA level, including microRNAs ( miRNAs), long non-coding RNAs (lncRNAs) and PIWI interacting RNA(,piRNA), etc. piRNA is a small class of non-coding RNA first identified in male germ cells in 2006, consisting of 26 to 31 nucleotides, with 5' -enduridine modification or adenosine 10th bias, and without significant secondary structural regions. The sequence of piRNAs is not conserved, but the gene clusters encoding piRNAs are very conserved among species. piRNAs often form piRNA/PIWI complexes with PIWI proteins, and play an important role in the occurrence and development of many diseases, such as causing methylation of specific loci, phosphorylation of regulatory proteins, and silencing transcription products. Since many piRNAs are organized and staged expressed in a specific way, so their disorder can dynamically reflect the disease state. Recent studies have shown that some piRNAs in tumor cells can enter into circulation through cell membranes and become new tumor markers in body fluids for early diagnosis and prognostic monitoring of lung adenocarcinoma. At present, there are no literatures or reports about piRNA regulating the growth process of lung adenocarcinoma in vivo or using piRNA regulating the growth of lung adenocarcinoma in the treatment of lung adenocarcinoma. Based on the short molecular sequence of piRNAs, the full-length nucleic acid sequence is its functional motif, with strong specificity and high targeting rate, which is different from other long non-coding RNAs. Therefore, the application of piRNAs in the treatment of diseases can reduce the unpredictable off-target effect and toxic and side effects, and promote the clinical transformation of piRNAs drugs. Summary of the invention: The purpose of the present invention is to seek to design a PIWI interacting RNA piR-hsa-211106, which is used to prepare targeted therapeutic drugs for inhibiting the proliferation of lung adenocarcinoma cells to inhibit lung adenocarcinoma tumor growth. In order to achieve the above-mentioned purpose, the PIWI interacting RNA piR-hsa-211106 of the present invention is used to prepare targeted therapeutic drugs for inhibiting the proliferation of lung adenocarcinoma cells. The PIWI interacting RNA piR-hsa-211106 involved in the present invention is an agomir or a transformant of one of adenovirus, lentivirus and adeno-associated virus, and contains the nucleotide sequence fragment shown in SEQ ID NO.1 or contains a fragment of a nucleotide sequence with a sequence similarity greater than

%. The mechanism of PIWI interacting RNA piR-hsa-211106 inhibiting the proliferation of lung adenocarcinoma cells is as follows: After the PIWI interacting RNA piR-hsa-211106 was constructed as an agomir or transformant, the expression of pyruvate carboxylase (PC) was down-regulated to inhibit the tricarboxylic acid cycle, which inhibited the energy metabolism and promoted the death of lung adenocarcinoma cells, so as to realize the targeted therapy for lung adenocarcinoma. The PIWI interacting RNA piR-hsa-211106 (SEQ ID NO.1) involved in the present invention is a fragment of nucleotide sequence: TCCGGCTCGAAGGACTTCGTCTGTAATTTT. The primer is piR-hsa-211106, and the upstream primer sequence is piR-hsa-211106-F is: GGCTCGAAGGACTTCGTCTGT, and the downstream primer sequence piR-hsa-211106-R is: AGTGCAGGGTCCGAGGTATT. The present invention relates to PIWI interacting RNA piR-hsa-211106, whose targeting effect is detected as follows: PIWI interacting RNA piR-hsa-211106 was transfected into lung adenocarcinoma cells A549 and HCC2279, and the proliferation indicators of lung adenocarcinoma cells were detected by CCK8 kit and Edu staining to determine the effect of PIWI interacting RNA piR-hsa-211106 on cell proliferation activity. The effect of PIWI interacting RNA piR-hsa-211106 on cell migration ability was determined by Transwell chamber and Wound healing assay to detect migration indicators of lung adenocarcinoma cells. The effect of PIWI interacting RNA piR-hsa-211106 on apoptosis was determined by flow cytometry. The PIWI interacting RNA piR-hsa-211106 involved in the present invention inhibits the proliferation and migration of lung adenocarcinoma cells and promotes apoptosis of lung adenocarcinoma cells. The molecular mechanism is experimentally verified by RNA pull-down protein technology and mass spectrometry analysis technology. The results have shown that PIWI interacting RNA piR-hsa-211106 can not only inhibit the expression of pyruvate carboxylase (PC), but also can bind to PC to inhibit the regulation of PC on cells, and PC is known to control one of the most important molecular cell energy metabolism pathways. The PIWI interacting RNA piR-hsa-211106 involved in the present invention was used to evaluate its inhibitory effect on lung adenocarcinoma tumor growth and targeted therapy effects, we constructed a nude mouse axillary percutaneous tumor model. The PIWI interacting RNA piR-hsa-211106 was injected into nude mice in vivo tumor, and to set up the negative and positive control group at the same time. The tumor volume of nude mice was measured regularly. It is found that that the tumor volume and weight of the experimental group were smaller than those of the control group, and the therapeutic effect was equivalent to that of the positive control cisplatin treatment group, indicating that PIWI interacting RNA piR-hsa-211106 has a targeted inhibitory effect on nude mouse tumor models. Compared with the existing technology, the PIWI interacting RNA piR-hsa-211106 is used to prepare targeted therapeutic drugs to inhibit the proliferation of lung adenocarcinoma cells, providing a new research direction for the prevention and treatment of abnormal activation of cellular energy metabolism pathway of lung adenocarcinoma. PIWI interacting RNA piR-hsa-211106 inhibits the tricarboxylic acid cycle by inhibiting the content of pyruvate carboxylase (PC), significantly inhibits energy metabolism and the proliferation and migration ability of lung adenocarcinoma cells, thereby promoting the growth of lung adenocarcinoma cells Apoptosis, which directly acts on the target site, does not produce toxic side effects and off-target phenomena. Through high-throughput sequencing analysis, mass spectrometry analysis technology, and in vivo and in vitro experiments, it is confirmed that PIWI interacting RNA piR-hsa-211106 has strong specificity, high targeting rate, low off-target effects and toxic side effects. Its application mechanism is scientific, the application method has high reliability and the treatment effect is remarkable. Description of the figures: Figure 1 is a schematic diagram of the effect of the agomir detected by the CCK8 kit in Example 3 of the present invention on the proliferation activity of lung adenocarcinoma cells A549 and HCC2279. Figure 2 is a schematic diagram of the effect of the agomir detected by EdU in Example 4 of the present invention on the proliferation activity of lung adenocarcinoma cells A549 and HCC2279. Figure 3 is a schematic diagram of the statistical and quantitative analysis results of the agomir detected by EdU in Example 4 of the present invention on the proliferation activity of lung adenocarcinoma cells A549 and HCC2279. Figure 4 is a schematic diagram of the effect of the agomir detected by the Transwell chamber in Example 5 of the present invention on the migration ability of lung adenocarcinoma cells A549 and HCC2279. Figure 5 is a schematic diagram of the statistical and quantitative analysis results of the migration ability of lung adenocarcinoma cells A549 and HCC2279 by the agomir detected by the Transwell chamber according to Example 5 of the present invention. Figure 6 is a schematic diagram of the effect of the agomir detected by the wound healing assay in Example 6 of the present invention on the migration ability of lung adenocarcinoma cells A549 and HCC2279. Figure 7 is a schematic diagram of the statistical and quantitative analysis results of the migration ability of the lung adenocarcinoma A549 and HCC2279 cells of the agomir detected by the wound healing assay involved in Example 6 of the present invention. Figure 8 is a schematic diagram of the effect of the agomir detected by the flow cytometry in Example 7 of the present invention on the apoptosis of lung adenocarcinoma cells A549 and HCC2279. Figure 9 is a schematic diagram of the statistical and quantitative analysis results of the apoptosis of lung adenocarcinoma cells A549 and HCC2279 by the agomir detected by flow cytometry according to Example 7 of the present invention. Figure 10 is a schematic diagram of Western Blot (WB) results of the binding of PIWI interacting RNA piR-hsa-211106 and pyruvate carboxylase(PC) involved in Example 8 of the present invention. Figure 11 is a schematic diagram of the qRT-PCR analysis result of the PIWI interacting RNA piR-hsa-211106 regulating the expression of PC at RNA level involved in Example 9 of the present invention. Figure 12 is a schematic diagram of the WB analysis result of the PIWI interacting RNA piR-hsa-211106 regulating the expression of PC at protein level involved in Example 10 of the present invention. Figure 13 is a schematic diagram of the tumor after treatment of the tumor-bearing nude mice with the PIWI interacting RNA piR-hsa-211106 involved in Example 12 of the present invention. Figure 14 is a statistical diagram of tumor volume after treatment of the tumor-bearing nude mice with PIWI interacting RNA piR-hsa-211106 involved in Example 12 of the present invention. Figure 15 is a statistical diagram of tumor weight after treatment of the tumor-bearing nude mice with the PIWI interacting RNA piR-hsa-211106 involved in Example 12 of the present invention. Detailed methods: The present invention will be further described below through implementation examples in conjunction with the drawings. Example 1: This example relates to the process of constructing an agomir with PIWI interacting RNA piR-hsa-211106 as follows. Using PIWI interacting RNA piR-hsa-211106 gene as a template, PCR amplification of piR-hsa-211106 is performed using primers to generate double chain PIWI interacting RNA piR-hsa-211106. The antisense strand of PIWI interacting RNA piR-hsa-211106 undergoes cholesterol modification and 4 thio skeleton modification at 3'end, 2 thio skeleton modification at 5'end, and full chain methoxy modification to complete the construction of agomir((Shanghai Jima Pharmaceutical Technology Co., Ltd.)). Example 2: This example relates to the process of transfection of agomir to lung adenocarcinoma cells as follows. The lung adenocarcinoma cells are passaged until the cell density reaches about 30-50%, and lipo3000 is used to transfect the agomir to lung adenocarcinoma cells. Take 5[1 (100pmol) of the agomir and add it to the EP tube containing 95d1DMEM medium by pipetting and mixing, then add 5[1 lipo3000 to the EP tube containing 951 DMEM medium by pipetting and mixing, subsequently mix the above two in a 1:1 volume. The transfection mixture is formed and placed at room temperature for 20 minutes, then was added to the culture plate. After 48 hours, the gene and protein level will be analyzed. Example 3: This example relates to the CCK8 proliferation viability analysis process of lung adenocarcinoma cells A549 and HCC2279 as follows. Lung adenocarcinoma cells were cultured, and the experimental group was transfected with PIWI interacting RNA piR-hsa-211106. In transfection 0, 1, 2, 3, 4 days later, the experimental group and control group respectively abandon the original medium absorption, flush lung adenocarcinoma cancer cell with phosphate buffer solution (PBS) twice, then add

[ fresh medium and 10 1 CCK8 reagent (Nanjing Vazyme Biotechnology Co., Ltd.) treating for 1 hour. OD value at 450 nm absorbance which represents the cell density and reflect the cell proliferation activity was detected. The result in figure 1 is shown that the agomir constructed by PIWI interacting RNA piR-hsa-211106 could significantly inhibit the proliferation of lung adenocarcinoma cells A549 and HCC2279. Example 4: This example relates to the process of Edu staining analysis of the proliferation activity of lung adenocarcinoma cells A549 and HCC2279 as follows. Lung adenocarcinoma cells were cultured. The experimental group was transfected with PIWI interacting RNA piR-hsa-211106. Two days after transfection, Edu staining (Shanghai Yeasen Biotechnology Co., Ltd.) was performed to analyze cell proliferation: (1) Edu labeled cells: Incubate the cells with 10M Edu working solution at 37°C in the dark for 2 hours; (2) Cell fixation and permeation promotion: After the incubation is completed, remove the medium, add neutral paraformaldehyde with a concentration of 4% by mass, and fix for 15-30 minutes at room temperature. Then remove the fixative and add 0.5% Triton X-100 in PBS by mass, incubate at room temperature for 20 minutes to promote penetration;

(3) EdU detection: Add the Click-iT reaction mixture, shake the culture plate briefly to ensure that the reaction mixture can evenly cover the cells, and incubate at room temperature for 30 minutes in the dark; (4) DNA counterstaining: 5 g/mL Hoechst 33342 solution was used to incubate cells for 15-30 minutes in the dark at room temperature to stain the nucleus; (5) Wash the cells twice with PBS, take pictures with a fluorescence microscope, and analyze the proportion of lung adenocarcinoma cells that are proliferating; The results are shown in Figures 2 and 3, the PIWI interacting RNA piR-hsa-211106 constructed agomir can significantly inhibit the growth of lung adenocarcinoma cells. Example 5: This example relates the analysis process of lung adenocarcinoma cell A549 and HCC2279 Transwell cell migration ability as follows. Lung adenocarcinoma cells were cultured. The experimental group was transfected with PIWI interacting RNA piR-hsa-211106. Two days after transfection, migration assay was detected in the Millicell chamber in 24 wells plate: 200[ 1of cells in serum-free medium (2x104) was added to the coated filter membrane, and 500[ 1of medium containing 20% fetal bovine serum was added to the lower chamber as a chemoattractant. After being placed in a 37°C incubator for 24 hours, the cells that migrated through the filter were fixed with methanol, stained with 0.5% crystal violet, and the number of cells was photographed and counted under a microscope. The result in Figure 4 and Figure 5 is shown that the agomir constructed by PIWI interacting RNA piR-hsa-211106 can significantly inhibit the migration ability of lung adenocarcinoma cells. Example 6: This example relates to the process of analyzing the migration ability of lung adenocarcinoma cells A549 and HCC2279 by the wound healing assay as follows. Lung adenocarcinoma cells were cultured in a six-well plate. The experimental group was transfected with PIWI interacting RNA piR-hsa-211106. Two days after transfection, scrape the cell monolayer with a 200ul pipette tip, capture a representative image of cell migration by taking a 1Ox high-power field at Oh and 24h after the scratch, measure the reduced distance across the induced damage area, and compare it Normalized to 0 h control, expressed as relative migration rate. As shown in Figure 6 and Figure 7, the agomir constructed by PIWI interacting RNA piR-hsa-211106 can significantly inhibit the migration ability of lung adenocarcinoma cells. Example 7: This example relates to the flow cytometry detection process of lung adenocarcinoma cells A549 and HCC2279 as folloes. Lung adenocarcinoma cells were cultured. The experimental group is transfected with PIWI interacting RNA piR-hsa-211106. Two days after transfection, perform Annexin V-FITC/PI (Shanghai Yeasen Biotechnology Co., Ltd.) cell apoptosis detection: (1) After digestion of lung adenocarcinoma cells with trypsin( without EDTA), the cells were collected by centrifugation at 300g for 5 min at 4°C; (2) Wash the cells twice with pre-cooled PBS, 300 g each time, centrifuge at 4°C for 5 min and collect 1-5x105 cells; (3) Aspirate and discard PBS, add 1xBinding Buffer to re-suspend the cells; (4) Add Annexin V-FITC and PI Staining Solution, mix gently and react at room temperature in dark for 10-15 minutes; (5) Add 1xBinding Buffer, mix well, place on ice, and detect with flow cytometer within 1 hour; The results are shown in Figures 8 and 9, the agomir constructed by PIWI interacting RNA piR-hsa-211106 can obviously cause apoptosis of lung adenocarcinoma cells A549 and HCC2279. Example 8: This example relates to the biotin-coupled probes RNA pull-down assay process as follows. Harvest, lyse and sonicate 1x107 lung adenocarcinoma cells, and biotinylated piR-hsa-211106 probe (Shanghai Jima Pharmaceutical Technology Co., Ltd.) and probe-M280 streptavidin sepharose beads (Invitrogen) were incubated at °C for 2 hours to generate probe-coated magnetic beads. The cell lysate and probe-coated magnetic beads were mixed and incubated overnight at 4°C. The RNA complexes bound to the beads are eluted with washing buffer, and purified with Trizol reagent (Bao Bioengineering (Dalian) Co., Ltd.) for Western Blot (WB) analysis. As shown in Figure 10, piR-hsa-211106 can bind to pyruvate carboxylase. Example 9: This example relates to the process of RNA extraction of lung adenocarcinoma cells and gene expression level analysis as follows. (1) Add 1 ml Trizol to each well of the 6-well plate to lyse the cells and pipette repeatedly with a pipette tip. After 5 minutes at room temperature, transfer the lysate to a 1.5 mL centrifuge tube, add 200ul chloroform and cover the tube tightly, vortex and mix and let stand for 3 minutes at room temperature. Centrifuge for 15 minutes at 12000g/min at 4°C; (2) Transfer the upper aqueous phase liquid to a new 1.5ml centrifuge tube. Add 500uL isopropanol, vortex and mix well, let stand for 10 minutes at room temperature, and centrifuge at 12000g/min at 4°C for 10 minutes; (3) Discard the supernatant, save the bottom white precipitate. Add 1ml 75% ethanol (prepared with DEPC water), gently lift the white precipitate by hand, and then centrifuge at 7500g/min for 5 minutes at 4°C; (4) Discard the supernatant, dry at room temperature for 10-15 minutes. Add -40 1 DEPC water to re-dissolve, store the RNA in a -80C refrigerator or use it directly after detecting the RNA concentration. (5) According to the operation process of the reverse transcription kit (Bao Biological Engineering (Dalian) Co., Ltd.), reverse transcription of the extracted total RNA into cDNA. Use real-time fluorescent quantitative PCR technology to detect gene expression levels refer to the SYBR kit instructions (Bao Biological Engineering (Dalian) Co., Ltd.) ; The results in Figure 11 are shown that the agomir constructed by PIWI interacting RNA piR-hsa-211106 can significantly inhibit the expression of pyruvate carboxylase at mRNA level. Example 10: This example relates to the process of analyzing the protein expression level of lung adenocarcinoma cells as follpws. (1) Use Radio Immunoprecipitation Assay (Dalian Meilune Biochemical Technology Co., Ltd.) to extract total cells proteins, and BCA colorimetric method to adjust the protein concentration to a consistent level; (2) Separate the protein samples by SDS-PAGE electrophoresis. After transfer to PVDF membrane, block with 5% skimmed milk powder for 1 hour. Then wash the PVDF membrane ans incubate the primary antibody (PC, GAPDH) overnight in the refrigerator at 4°C. Subsequently wash the PVDF membrane and incubate the secondary antibody at room temperature for 1 hour. Wash the PVDF membrane and use ECL chemiluminescence method to develop the target strip. Use Quantity One software to scan the strip with gray scale, and calibrate it with GAPDH internal reference; The results in Figure 12 are shown that the agomir constructed by PIWI interacting RNA piR-hsa-211106 can down-regulate the expression of pyruvate carboxylase at the protein level, indicating that PIWI interacting RNA piR-hsa-211106 inhibits the energy metabolic pathway in lung adenocarcinoma cells ,which promotes the apoptosis of lung adenocarcinoma cells. Example 11: This example relates to the process of constructing a nude mouse tumor-bearing model as follows. Take 3-4 weeks old BALB/c nude mice (Beijing Spacef Laboratory Animal Technology Company), and use them for experiments after 1 week of adaptation to local conditions. 1x107 A549 cells were injected subcutaneously into the left and right armpits of the mouse. When the implanted tumor reached 50mm3 , the drug was treated.

Example 12: This example relates to the drug-targeted treatment process of the nude mouse tumor-bearing modelas follows. (1) Divide nude mice into cisplatin experimental group and saline control group, each with 5 nude mice; (2) Intraperitoneal injection of cisplatin (5 mg/kg body weight/week) into the experimental group of nude mice, and 50uM PIWI interacting RNA piR-hsa-211106 agomir was injected directly into the tumor on the right side, and piR-NC (Shanghai Jima Pharmaceutical Technology Co., Ltd.) injected into the left side tumor was used as the control group of the cisplatin treatment group; (3) Intraperitoneal injection of an equal volume of normal saline into the control group of nude mice, the PIWI interacting RNA piR-hsa-211106 agomir treated tumors as above. The PIWI interacting RNA piR-hsa-211106 agomir was treated every other day for 28 days and tumor volume was measured once a week. the mice were sacrificed at the end of the experiment and the xenografts were peeled off and photographed. The results are shown in Figure 13, Figure 14 and Figure 15. The therapeutic effect of PIWI interacting RNA piR-hsa-211106 is comparable to the positive control drug cisplatin (PDD), and the PIWI interacting RNA piR-hsa-211106 increases the chemical sensitivity of the chemotherapeutic drug PDD, and there is a synergistic effect between the two. The above indicates that PIWI interaction RNA piR-hsa-211106 can inhibit the growth of lung adenocarcinoma tumors in vivo. Abstract The present invention belongs to the field of biomedicine, and specifically relates to a new use of PIWI interacting RNA piR-hsa-211106, which is used to prepare targeted therapeutic drugs for inhibiting the proliferation of lung adenocarcinoma cells. Its mechanism of work is: The RNA piR-hsa-211106 sequence is prepared into an agomir or transformant that down-regulated the expression of Pyruvate carboxylase (PC), which inhibits the tricarboxylic acid cyclic process and makes energy metabolism affected, promoting the death of lung adenocarcinoma cells, and then achieving the purpose of inhibiting the growth of lung adenocarcinoma. It directly acts on the target site without causing toxic side effects and off-target phenomena. A large number of analyses and in vivo and in vitro experiments have confirmed that it has high credibility and significant therapeutic effects, providing a new research direction for anti-tumor treatment of lung adenocarcinoma.

Claims (7)

1. Claims 1. The use of PIWI-interacting RNA piR-hsa-211106, which is characterized in preparing a targeted therapy drug for inhibiting the proliferation of lung adenocarcinomacells.
2. 2. The use of PIWI-interacting RNA piR-hsa-211106 according to claim 1, characterized in that the PIWI-interacting RNA piR-hsa-211106 is an agomir or one transformant of adenovirus, lentivirus and adeno-associated virus, containing a nucleotide sequence fragment shown in SEQ ID NO. 1 or a nucleotide sequence fragment whose sequence similarity is greater than 80%.
3. 3. The use of PIWI-interacting RNA piR-hsa-211106 according to claim 2, characterized in that the nucleotide sequence fragment shown in SEQ ID NO. 1 is : TCCGGCTCGAAGGACTTCGTCTGTAATTTT; the primer is piR-hsa-211106, whose forward primer sequence piR-hsa-211106-F is GGCTCGAAGGACTTCGTCTGT, and reverse primer sequence piR-hsa-211106-R is: AGTGCAGGGTCCGAGGTATT.
4. 4. The use of PIWI-interacting RNA piR-hsa-211106 according to any one of claims 1-3, characterized in that the regulatory mechanism of PIWI-interacting RNA piR-hsa-211106 on inhibiting the proliferation of lung adenocarcinoma cells is : after constructing PIWI-interacting RNA piR-hsa-211106 as an agomir or transformant, it can inhibit the tricarboxylic acid cycle process through down-regulating the expression of pyruvate carboxylase, which inhibit energy metabolism, and promote lung adenocarcinoma cells death, achieving the purpose of lung adenocarcinoma targeted therapy.
5. 5. The use of PIWI-interacting RNA piR-hsa-211106 according to claim 4, characterized in that the molecular mechanism of inhibiting the proliferation and migration of lung adenocarcinoma cells and promoting apoptosis of lung adenocarcinoma cells is experimentally verified through RNA pull-down protein assay and mass spectrometry analysis technology.
6. 6. The use of PIWI-interacting RNA piR-hsa-211106 according to claim 4, characterized in that the PIWI-interacting RNA piR-hsa-211106 is transfected into lung adenocarcinoma cells A549 and HCC2279, the detection of lung adenocarcinoma cells proliferation indicators by CCK8 kit and Edu staining were used to determine the effect of PIWI-interacting RNA piR-hsa-211106 on cell proliferation .
7. 7. The use of PIWI-interacting RNA piR-hsa-211106 according to claim 4, characterized in that the detection of lung adenocarcinoma cells migration indicators by Trannswell chamber and wound healing assays were used to determine the effect of PIWI-interacting RNA piR-hsa-211106 on cell migration ability.